Official
Official Journal
Journal ofof thethe Romanian
Romanian Society
Society ofof Physiological
Physiological Sciences
Sciences

CHIEF EDITOR
CO-CHIEF EDITORSASSOCIATE EDITORSEXECUTIVE EDITORSFRANCISC SCHNEIDERIOANA SISKACARMEN TATUMIHAI NECHIFORSORIN RIGA
FLORINA BOJINGABRIELA TANASIEDACIANA NISTORCALIN MUNTEANEDITORIAL BOARD
ARDELEAN
ARDELEAN AUREL
AUREL
BADIU
BADIU GHEORGHE
GHEORGHE
BĂDĂRĂU
BĂDĂRĂU ANCA
ANCA
BENEDEKBENEDEK GYÖRGYGYÖRGYBENGA
BENGA GHEORGHE
GHEORGHE
BUNU
BUNU CARMENCARMEN
COJOCARU
COJOCARU MANOLEMANOLE
CUPARENCU
CUPARENCU BARBUBARBUCONSTANTINCONSTANTIN NICOLAE
NICOLAEHAULICĂHAULICĂ ION
ION
IANCĂUIANCĂU MARIAMARIAMIHALAŞ
MIHALAŞ GEORGETAGEORGETAMUNTEANMUNTEAN DANINADANINAMUREŞANMUREŞAN ADRIANAADRIANANESTIANUNESTIANU VALERIU
VALERIU
OPREA
OPREA TUDOR
TUDOR(Arad)(Arad)(Constanţa)(Constanţa)(Bucureşti)(Bucureşti)(Szeged)(Szeged)(Cluj)(Cluj)(Timişoara)(Timişoara)(Bucureşti)(Bucureşti)(Oradea)(Oradea)(Bucureşti)(Bucureşti)(Iaşi)(Iaşi)(Craiova)(Craiova)(Timişoara)(Timişoara)(Timişoara)(Timişoara)(Cluj)(Cluj)(Craiova)(Craiova)(New(New Mexico)
Mexico)PĂUNESCU
PĂUNESCU VIRGIL
VIRGILPETROIUPETROIU ANA
ANAPOPESCU
POPESCU LAURENŢIU
LAURENŢIU
RÁCZRÁCZ OLIVER
OLIVERRIGA
RIGA DAN
DANSABĂUSABĂU MARIUS
MARIUSSIMIONESCUSIMIONESCU MAIA
MAIA
SIMON
SIMON ZENOZENOSAULEASAULEA I.I. AUREL
AUREL
SWYNGHEDAUWSWYNGHEDAUW BERNARDBERNARDTANGUAYTANGUAY M.
M. ROBERTROBERTTATUTATU FABIANFABIAN ROMULUS
ROMULUSVLAD
VLAD AURELIANAURELIAN
VOICU
VOICU VICTOR
VICTORZĂGREANZĂGREAN LEON
LEON

(Timişoara)(Timişoara)(Timişoara)(Timişoara)(Bucureşti)(Bucureşti)(Košice)(Košice)(Bucureşti)(Bucureşti)(Tg.
(Tg. Mureş)Mureş)(Bucureşti)(Bucureşti)(Timişoara)(Timişoara)(Chişinău)(Chişinău)(Paris)(Paris)(Canada)(Canada)(Timişoara)(Timişoara)(Timişoara)(Timişoara)(Bucureşti)(Bucureşti)(Bucureşti)(Bucureşti)ACCREDITEDACCREDITED BY
BY CNCSISCNCSIS -- B
B+CATEGORYCATEGORY -- CODE
CODE 240
240
http://journals.indexcopernicus.com/karta.php?action=masterlist&id=4929
http://journals.indexcopernicus.com/karta.php?action=masterlist&id=4929
http://www.ebscohost.com/titleLists/a9h-journals.pdf
http://www.ebscohost.com/titleLists/a9h-journals.pdf
Publication
Publication data:
data: Fiziologia
Fiziologia (Physiology)(Physiology) isis issued
issued quarterly
quarterly
Subscription
Subscription rates:
rates: Subscriptions
Subscriptions run
run aa full
full calendar
calendar year.
year. Prices
Pricesareare give
give perper volume,
volume, surface
surface postage
postage included.
included.
Personal
Personal subscription:
subscription: Romania
Romania -- 100
100 RON,
RON, Outside
OutsideRomania
Romania -- 35$
35$ (must(must bebe inin thethe namename of,of, billed
billed to,to, andand paid
paid byby anan
individual.
individual. Order
Order must
must bebe marked
marked “personal“personal subscription”)
subscription”)
Institutional
Institutional subscription:
subscription: 50$
50$ (regular(regular rate)
rate)
SingleSingle issues
issues andand backback volumes:
volumes: Information
Information on
on availability
availability
andand prices
prices cancan bebe obtained
obtained throughthrough thethe Publisher.
Publisher.
ChangeChange ofof address:
address: Both
Both old
old andand new
new address
address should
should be
be statedstatedandand send
send toto thethe subscription
subscription source.
source.
Bibliographic
Bibliographicindices:
indices:We
Wehopehopethis
thisjournal
journalwill
willbeberegularly
regularlylisted
listedinin bibliographic
bibliographic services,
services, including
including “Current“Current Contents”
Contents”..
Book
Book Reviews:
Reviews: Books
Books areare accepted
accepted forfor review
review byby special
specialagreement.
agreement.
Advertising:Advertising: Correspondence
Correspondence andand rate
rate requests
requests should
should bebe
addressed
addressed toto thethe Publisher.
Publisher.

Fiziologia-- Physiologysupplement
Fiziologia
Physiology 20102010
supplement
2011.21.3(71)
 Fiziologia- Physiology

1.
1. FORFOR SUBSCRIPTIONSUBSCRIPTION ADDRESSADDRESSHVB
HVB Bank
Bank TIMISOARA
TIMISOARA
RO
RO 21
21 BACXBACX 0000000218508250
0000000218508250
TIMISOARATIMISOARA –– ROMANIA
ROMANIAPENTRUPENTRU REVISTAREVISTA„FIZIOLOGIA„FIZIOLOGIA –– PHYSIOLOGY”
PHYSIOLOGY”
2.
2. CORRESPONDENCE
CORRESPONDENCE SHOULDSHOULD BE
BE
ADDRESSEDADDRESSED TO
TO THETHE CHIEF
CHIEF EDITOREDITOR
PROF.
PROF. DR.
DR. FRANCISC
FRANCISC SCHNEIDER
SCHNEIDERPO
PO BOX
BOX 135135
300024300024 –– TIMISOARA
TIMISOARA –– ROMANIA
ROMANIAe-mail:e-mail: carmen.tatu@umft.rocarmen.tatu@umft.ro
EdituraEditura EUROSTAMPAEUROSTAMPA
Tel./fax:
Tel./fax: 0256-204816
0256-204816
ISSN
ISSN 1223
1223 –– 2076
2076

111

Official Journal of the Romanian Society of Physiological Sciences

Instructions to Authors
Submission: Only original papers in English areconsidered and should be sent to:Prof. dr. Francisc Schneider
Chief Editor of “Fiziologia”
PO Box 135
300024, TIMISOARA, ROMANIA
Tel./Fax: 40-256/490507
Manuscripts should be submitted in triplicate setsof illustrations (of which one is an original), typewritten
doublespaced on one side of the paper, with a wide margin.
Conditions: All manuscripts are subject to editorial review.
Manuscripts are received with the explicit understanding thatthey are not under simultaneous consideration by any otherpublication. Submission of an article for publication implies thetransfer of the copyright from the author to the publisher upon
acceptance. Accepted papers become the permanent propertyof “Fiziologia” (Physiology) and may not be reproduced by anymeans, in whole or in part, without the written consent of the
publisher. It is the author’s responsibility to obtain permission to
reproduce illustrations, tables, etc. from other publications.
Arrangement:
Title page: The first of each paper should indicate the title(main title underlined), the authors’ names, and the institutewhere the work was conducted. A short title for use as runninghead is also required.
Keywords: for indexing purposes, a list of 3-10 keywords
in English and Romanian is essential.
Abstract: Each paper needs abstract and title in Romanianand English language, fonts size 9, Arial Narrow.
Bady text: fonts size 10, Arial Narrow.
Small type: Paragraphs which can or must be set in smaller
type (case histories, test methods, etc.) should be indicated witha „p” (petit) in the margin on the left-hand side.
Footnotes: Avoid footnotes. When essential, they are
numbered consecutively and typed at the foot of the appropriate
page, fonts size 8, Arial Narrow.
Tables and illustrations: Tables (numbered in Roman
numerals) and illustrations (numbered in Arabic numerals)
should be prepared on separate sheets, fonts size 9, Arial Narrow.
Tables require a heading, and figures a legend, also prepared ona separate sheet. For the reproduction of illustrations, only good
drawings and original photographs can be accepted; negatives
or photocopies cannot be used. When possible, group several
illustrations on one block for reproduction (max. size 140x188mm) or provide crop marks. On the back of each illustration
indicate its number, the author’s name, and article title. Colour

2

illustration are reproduced at the author’s expense.
References: In the text identify references by Arabic
figures, (in brackets), fonts size 9, Arial Narrow. Material
submitted for publication but not yet accepted should be
noted as “unpublished data” and not be included in thereference list. The list of references should include only thosepublications which are cited in the text. The references should
be numbered and arranged alphabetically by the authors’names. The surnames of the authors followed by initials
should be given. There should be no punctuation signs otherthan a comma to separate the authors. When there are morethan 3 authors, the names of the 3 only are used, followedby “et al”. abbreviate journal names according to the Index
Medicus system. (also see International Committee of Medical
Journal Editors: Uniform Requirements for manuscripts
submitted to biomedical journals. Ann Intern Med 1982; 96:766 – 771).

Examples:(a) Papers published in periodicals: Kauffman HF, van der
Heide S, Beaumont F, et al: Class-apecific antibody determination
against Aspergillus fumigatus by mean of the enzyme-linked
immunosorbent assay. III. Comparative study: IgG, IgA, IgM, ELISA
titers, precipitating antibodies and IGE biding after fractionationof the antigen. Int Arch Allergy Appl Immunol 1986; 80: 300 –306.

(b) Monographs; Matthews DE, Farewell VT: Using andUnderstanding Medical Statistics. Basel, Karger, 1985.
(c) Edited books: Hardy WD Jr, Essex M: FeLV-inducted felineacquired immune deficiency syndrome: A model for human
AIDS; in Klein E(ed): Acquired Immunodeficiency Syndrome. Prog Allergy,
Busel, Karger, 1986, vol 37, 353 – 376.
Full address: The exact postal address complete withpostal code of the senior author must be given; if correspondenceis handled by someone else, indicate this accordingly. Add theE-mail address if possible.
Page charges: There is no page charge for papers of
4 or fewer printed pages (including tables, illustrations andreferences).
Galley proofs: unless indicated otherwise, galley proofs
are sent to the first-named author and should be returned withthe least possible delay. Alternations made in galley proofs, otherthan the corrections of printer’s errors, are charged to the author.
No page proofs are supplied.
Reprints: Order forms and a price list are sent with thegalley proofs. Orders submitted after the issue is printed are
subject to considerably higher prices. Allow five weeks fromdate of publication for delivery of reprints.

Fiziologia- Physiology 2011.21.3(71)
Fiziologia- Physiology 2010supplement

CONTENTS
1. Silencing of the Her2 Gene by RNA Interference Inhibits Proliferation of Breast Cancer Cell Line SK-BR3............................................................................................................................................. 4
Oana I. Gavriliuc, Florina M. Bojin, Adriana Rosca, Calin A. Tatu, Virgil Paunescu
2. Biogerontology Today. Part I. 61st Annual Scientific Meeting of the British Society for Research on Ageing - BSRA & 14th Congress of the International Association of Biomedical Gerontology - IABG ........ 10
Dan Riga, Sorin Riga, Aurel Ardelean, Daniela Motoc, Francisc Schneider
3. Evaluation of Skin Parameters Changes in Chemical and Photochemical Initiated Tumors on SKH1 Mice .........................................................................................................................................18
Sorina Ciurlea, Maria-Florina Bojin, Erzebet Csanyi, Daniela Ionescu, Florin Borcan, Atena Galuscan, Cristina A. Dehelean
4. On the Enteric Nervous System’s Role in Physiological Activities of the Gastrointestinal Tract.............................................................................................................................................................23
Alina Sisu, Codruta Petrescu, Carmen Tatu, Loredana Stana, R.F.Tatu, A. Motoc
5. Ambulatory Systolic Augmentation Index-A New Arterial Rigidity Marker for Cardiovascular Risk Stratification..............................................................................................................................27
Ionescu V.A, Georgiana Ionescu, Dana Bâibâţã, Silvia Mancaş, Şt. I. Drăgulescu
6. The Relationship between Hypertensive Retinopathy and Coronary Artery Disease............................................................................................................................................................................33
Ovidiu Boruga, Ioana Zolog
7. REVIEW - Potential Circulating Markers of Preeclampsia.......................................................................................................................................................................................................................38
Dana Carmen Zaha
8. New Approaches for the Dried Blood Spot Analysis...............................................................................................................................................................................................................................43
Ileana Scurtu, Alexandra Gruia, Valentin Ordodi, Elena Ciurariu, Felix Mic, Calin Tatu, Victor Dumitrascu

CUPRINS
1. Inhibitia expresiei genei Her2 prin interferenta ARN blocheaza proliferarea liniei tumorale mamare SK-BR3...................................................................................................................................... 4
Oana I. Gavriliuc, Florina M. Bojin, Adriana Rosca, Calin A. Tatu, Virgil Paunescu
2. Biogerontologia astazi. Partea I. A 61-a Conferinţă Ştiinţifică Anuală a Societăţii Engleze de Cercetare a Îmbătrânirii - BSRA & Al 14-lea Congres al Asociaţiei Internaţionale de Gerontologie
Biomedicală - IABG......................................................................................................................................................................................................................................................................................10
Dan Riga, Sorin Riga, Aurel Ardelean, Daniela Motoc, Francisc Schneider
3. Evaluarea modificărilor parametrilor tegumentari in tumorile iniţiate chimic şi fotochimic la linia murină SKH1..............................................................................................................................18
Sorina Ciurlea, Maria-Florina Bojin, Erzebet Csanyi, Daniela Ionescu, Florin Borcan, Atena Galuscan, Cristina A. Dehelean
4. Rolul sistemului nervos enteric in activitatile fiziologice ale tractului gastrointestinal.........................................................................................................................................................................23
Alina Sisu, Codruta Petrescu, Carmen Tatu, Loredana Stana, R.F.Tatu, A. Motoc
5. Indicele ambulatoriu de augmentare sistolică - un nou marker de rigiditate arterială folosit pentru stratificarea riscului cardiovascular.........................................................................................27
Ionescu V.A, Georgiana Ionescu, Dana Bâibâţã, Silvia Mancaş, Şt. I. Drăgulescu
6. Relatia dintre retinopatia hipertensiva si boala coronariana..................................................................................................................................................................................................................33
Ovidiu Boruga, Ioana Zolog
7. Markeri potentiali circulanti ai preeclampsiei.........................................................................................................................................................................................................................................38
Dana Carmen Zaha
8. Noi abordari pentru analiza spotului de sange uscat.............................................................................................................................................................................................................................43
Ileana Scurtu, Alexandra Gruia, Valentin Ordodi, Elena Ciurariu, Felix Mic, Calin Tatu, Victor Dumitrascu
2011.21.3 (71)  Fiziologia - Physiology

3

SILENCING OF THE HER2 GENE BY RNA INTERFERENCEINHIBITS PROLIFERATION OF BREAST CANCER CELL LINE
SK-BR-3
OANA I. GAVRILIUC1, FLORINA M. BOJIN1, ADRIANA ROSCA2, CALIN A. TATU2, VIRGIL
PAUNESCU1
1
Physiology-Immunology Department, “Victor Babes”University of Medicine and PharmacyTimisoara
2
Regional Center for Transplant Immunology, Emergency Clinical County Hospital Timisoara

ABSTRACTIntroduction: HER2 overexpression has been reported in approximately 25% of invasive breast cancers, with or without genomic
amplification of the gene and is associated with poor disease survival and resistance to chemotherapeutic agents. In this study, weinvestigated the effect the silencing of c-erbB2 gene expression by RNA interference would have on the proliferation and viability ofbreast cancer cell line SK-BR-3, known to express the HER2neu protein at very high levels.
Materials and methods: siRNA were delivered into the target cells using the Lipofectamine 2000 reagent. Total RNA was extractedwith TRIzol reagent, reverse transcribed to cDNA and cDNA samples were analyzed by qPCR for HER2 and VEGF expression
following lipofection. HER2 protein expression was assessed by immunohistochemistry, while VEGF presence in the cell culture
supernatants was documented by ELISA. siRNA treated and untreated SK-BR-3 cells were also assayed for cell cycle distribution
and induction of apoptosis.
Results: An inhibition of ~ 80% in the expression of the oncogene inhibited the proliferation of breast cancer cells, by blocking theircell cycle in the G0/G1 phase and also resulted in a reduction of VEGF secretion by half. However, we did not observe an increased
apoptosis in the siRNA treated breast cancer cells when compared to control untreated cells.
Conclusion: The complex mechanism by which c-erbB2 stimulates cancer cell growth and resistance to chemotherapy has not been
completely defined and there certainly exist redundant pathways sustaining the proliferation of neoplastic cells.
Keywords: HER2/neu, RNA interference, breast cancer, SK-BR-3

INTRODUCTION
Human tumors demonstrate unique profiles of gene expression, different from normal tissue and from other tumors,resulting in a distinct rewiring of protein pathways which drive
the abnormal proliferation of cancer cells. In turn, the malignant
cells will become highly dependent on the rewired pathway that
ensures their survival and robustness to certain types of therapeutic attacks. Therefore, a targeted knockdown of an oncogenic
pathway should theoretically lead to cell death in malignant cells
without affecting the normal tissues (1).
A particularly efficient posttranscriptional silencing of a geneof interest can be achieved by RNA interference (RNAi), which
represents a sequence-specific, naturally occurring mechanismof cellular defense and gene regulation that has been converted
to a key laboratory technique over the last decade. RNAi relies on
the cleavage of a long double-stranded RNA (dsRNA) into 21-23
base pair molecules termed small interfering RNAs (siRNA) by
an intracellular endonuclease called Dicer before being loaded
into RISC and targeting the specific degradation of an homologous mRNA (2). In vitro, RNAi can be induced either by directtransfection of small interfering RNA duplexes (3), which bypass
the defense mechanisms against dsRNAs, by endogenously
produced noncoding microRNAs, or by introduction of a vector
driving the expression of short hairpin RNA (shRNA) that are

further processed into siRNA by Dicer. Although the actual useof siRNA for in vivo human therapy is still plagued by the lackof a safe and efficient method of delivery to the target tissues
and cells, a number of preclinical studies and early clinical studies have established the proof of concept that RNAi could be
potentially used in the treatment of a variety of disease, among
which cancer is of leading importance (4).
In this report, we addressed a gene that has been shown
previously to be relevant or rate-limiting for tumor growth: c-erbB2.
The c-erbB2 proto-oncogene encodes the receptor tyrosine kinase
HER2neu which is responsible for increased proliferation andsurvival of the primary tumor, but also plays a role in the motilityof intravasating and extravasating tumor cells (5). HER2 overexpression has been reported in approximately 25% of invasive
breast cancers, with or without genomic amplification of the gene(6) and is associated with poor disease survival and resistance tochemotherapeutic agents (7,8). Monoclonal antibodies designed
to dowregulate HER2 expression in cancer cells, currently used inclinical therapy, have shown a number of limitations in their ability tomodulate HER2 function (9), while employing antisense oligonucleotides for RNAi-mediated inhibition of HER2neu demonstrated
better regulation of HER2 expression and induction of apoptosis
in cancer cells (10,11). We investigated the effect a silencing of
c-erbB2 gene expression would have on the proliferation andviability of breast cancer cell line SK-BR-3, known to express theReceived August 12th 2011. Accepted September 15th 2011. Address for correspondence: Oana Gavriliuc, PhD, Immunology Department, “Victor Babes” University of
Medicine and Pharmacy Timisoara, Spl. Tudor Vladimirescu no. 14A, phone/fax: +40256490507, e-mail: oana_gav@yahoo.com

4

Fiziologia - Physiology  2011.21.3 (71)HER2neu protein at very high levels.
MATERIALS AND METHODS
Cell culture
Breast cancer cell line SK-BR-3 was obtained from American
Type Culture Collection (Manassas, VA). Media and FCS werepurchased from Sigma (St. Louis, MO, USA) and Gibco-BRL(Invitrogen, Carlsbad, CA, USA), respectively. The cell line wasmaintained in culture according to supplier’s instructions, in McCoy’s 5a medium with 1.5 mM L-glutamine, 3.0 g/L glucose, 2.2
g/L sodium bicarbonate and 10% FCS.
siRNA
siRNA were designed according to Elbashir et al. [12] andpurchased from Eurogentec (Seraing, Belgium). Three different
regions within the c-erbB2 (NCBI accession no. NM_001005862)
gene were used: siR_1H (positions 861-879), siR_2H (positions 2678-2696), siR_3H (positions 3136-3154). A proprietary
Her2-targeted siRNA and fluorescent control siRNA were obtained from SCBT (Santa Cruz, CA). All siRNA contain a 19-bp
double-stranded (ds) sequence and symmetric 3′ overhangs of
two deoxythymidines.
In vitro transfection of siRNA
siRNA were delivered into the target cells using the Lipofectamine 2000 reagent (Invitrogen). Prior to the transfection,
the cells were permitted to reach 60-70% confluence, and thecomplete culture medium was changed to medium without antibiotics 30 minutes before lipofection. Alternatively, cells weretransfected in suspension, according to the manufacturer’s
protocol. siRNA were diluted in serum-free OptiMEM so that the
final concentration when added to the cells would be 100 nM.
Lipofectamine 2000 was mixed and diluted at the appropriate
amount in serum-free OptiMEM. The diluted DNA was mixed
with Lipofectamine 2000 in a ratio of 1:1, and the liposomal-DNA
complexes formed after a 20- minute incubation at room temperature were added to the cells. We used as a positive control
for assessing transfection rate the control siRNA (Fluorescein
Conjugate)-A (SCBT). The transfection medium was changed tocomplete growth medium after 16-20 hours and gene expression
was evaluated at different time points. All transfection reagentswere purchased from Invitrogen.
qRT-PCR assay of gene expression levels
All RNA samples used in this study were isolated in ourlaboratory from breast cancer cell lines MDA-MB-231 andMCF-7, mammary tumor and normal tissue, tumor-associatedfibroblasts (TAF) and mesenchymal stem cells (MSC). Total RNA
was extracted with TRIzol reagent (Invitrogen) following the supplier’s instructions. RNA concentration was determined with the
ND-1000 spectrophotometer. Sample purity, as indicated by the
260/280 absorbance ratio, was 1.9-2.02. We used 1 ug total RNA
for every 20 ul reverse transcription reaction performed with the
AccuScript High Fidelity 1st Strand cDNA Synthesis Kit (Agilent
2011.21.3 (71)  Fiziologia - Physiology

Technologies, Palo Alto, CA). cDNA samples were analyzed byquantitative real-time PCR, using the LightCycler 480 SYBRGreen I Master (Roche, Florence, SC, USA) and the following
primers: human erbB2 forward 5’-CTGGTGACACAGCTTATGCCCT-3’; reverse 5’- ATCCCCTTGGCAATCTGCA-3’; human
VEGF forward 5’-CTACCTCCACCATGCC-AAGT-3’ and reverse
5’-TGGTGAGAGATCTGGTTCCC. HPRT1 (hypoxanthine phosphoribosyltransferase 1) was chosen as a suitable reference
gene; the primers were HPRT1 forward 5’-CCTGGCGTCGTGATTAGTGAT-3’ and reverse 5’-AGACGTTCA-GTCCTGTCCATAA-3’ We performed a relative basic quantitation based on
the ΔΔCt method with the LightCycler480 Software.
Cell cycle analysis and apoptosis assay
The cell cycle distribution of erbB2 siRNA treated anduntreated SK-BR-3 cells was determined by flow cytometry
and measured as propidium iodide (PI) fluorescence intensity
on a FACSCalibur (BD Bioscience, Heidelberg, Germany). At
the specified time following siRNA transfection, cells were harvested and stained using the CycleTEST PLUS DNA reagent
kit (BD Bioscience). Analysis was performed on 10 000 eventsfor each sample. The percentages of cells within the G0/G1, S
and G2/M phases of the cell cycle were determined using the
CellQuest Pro software.
To assay for apoptosis induction following the inhibition of erbB2
in the breast cancer cell line SK-BR-3, cells were stained with 10
uL FITC-conjugated Annexin V (Miltenyi Biotec, Bergisch Gladbach,
Germany) and 10 uL propidium iodide (PI, 1 ug/mL; BD Bioscience).
The cells were subsequently analyzed by flow cytometry.
Her2/neu protein expression
To determine inhibition of the protein product of the erbB2
gene following RNAi, Her2/neu expression on cell membrane wasquantified by flow cytometry. Cells were harvested by trypsinization
at the specified time points, washed twice with PBS and stained for
Her2/neu with a fluorescein-conjugated anti-erbB2 Affibody Molecule (Abcam, Cambridge, MA) directed against the extracellulardomain of human erbB2. At least 20,000 cells from each staining
were analyzed on a FACSCalibur flowcytometer (BD Bioscience)equipped with the CellQuest Pro software.
We also assessed the Her2/neu protein level in SK-BR-3
cells by immunohistochemical staining. Transfected cells werespotted on CytoSpin coated slides (Thermo Scientific, Bremen,
Germany) by cytocentrifugation, fixed with 4% paraformaldehyde,
and permeabilized with 0.1% Triton X-100. The primary polyclonal
rabbit anti-human c-erbB2 antibody was purchased from DakoCytomation (Glostrup, Denmark) and was tested for human specificity
and cross-reactivity. Staining protocol continued with secondary
biotinylated antibody binding, fuchsin substrate addition, and hematoxylin counterstaining of the nuclei (LSAB+ System-AP, Dako)following the manufacturer’s procedures. Microscopy analysis was
performed on a Nikon Eclipse E800 microscope.
VEGF ELISATo determine VEGF secretion in the cell culture medium,5

we collected culture supernatants at 48 hours following transfection, filtered through 0.22 µm strainers and stored them at-80°C. The undiluted samples were analyzed with the Quantikine
HUman VEGF Immunoassay Kit (R&D Systems, Minneapolis,
MN, USA), as per manufacturer’s instructions. We comparedsupernatants collected from equal numbers of cells which were
lipofected and VEGF concentration in the supernatant was
expressed as pg/mL.
Statistical analysis
All statistical tests and values were calculated with GraphPad Prism 4 (GraphPad Software, San Diego, CA, USA). Data
were expressed as mean ± s.e.m. Groups were comparedusing one-way ANOVA for normally-distributed data or the
Mann-Whitney U test for nonparametric values. P<0.05 wasconsidered to be significant.
RESULTS
Expression of c-erbB2 mRNA and protein in breast
cancer cell line SK-BR-3
We documented c-erbB2 mRNA expression by quantitative
RT-PCR in three breast cancer cell lines, mammary tumor tissue
and tumor-associated fibroblasts (TAFs), as well as mesenchymal stem cells (MSCs) as normal cell culture counterparts, andnormalized the results to c-erbB2 expression in normal tissue
samples (Figure 1 A). As previously reported in the literature(13,14), HER2/neu expression is highest for SK-BR-3 cells,
while MCF-7 and MDA-MB-231 expressed less to no HER2/
neu protein. Mesenchymal stem cells expressed almost undetectable levels of c-erbB2 mRNA, when mammary tumor tissue
and TAFs showed somewhat increased expression of c-erbB2.
High levels of HER2/neu transcripts in the breast cancer line
SK-BR-3 were correlated with an abundant expression of theprotein on cell membrane, as revealed by immunocytochemistry(Figure 1 B).

Fig.1. Expression of c-erbB2 mRNA in breast cancer cell lines MCF-7, MDAMB-231 and SK-BR-3, mammary tumor tissue, TAFs and MSCs. Normalized
to normal tissue RNA. Breast cancer cells SK-BR-3 overexpress c-erbB2
by more than 30-fold (A, quantitative PCR analysis). B Positive immunocytochemical staining for c-erbB2 in SK-BR-3 cell line. Optical microscopy, 100x
magnification.

Inhibition of c-erbB2 mRNA and protein expression
by specific siRNA
Three siRNA duplexes were designed against the c-erbB2
oncogene and we examined the ability of the constructs, individually or combined in equimolar amounts, to downregulate
HER2/neu transcription in the HER2-overexpressing breast
carcinoma SK-BR-3. Alternatively, we purchased an siRNA
6

duplex targeting HER2/neu from SCBT. We transfected thecells with the Lipofectamine 2000 reagent and 100 nM siRNA
duplexes and determined oncogene expression by quantitative
real-time PCR at 24 hours following lipofection. Cells transfectedwith fluorescein-conjugated control siRNA served as both control
for lipofection and for inhibition.
Although all siRNA duplexes resulted in some level ofinhibition ranging from 21% to 82% (Figure 2 A) compared tooncogene expression in the mock transfected cells, the mostpronounced reduction of mRNA levels were observed for siR_1Hagainst Her2/neu. Simultaneous delivery of a pool of all threesiRNA did not improve efficiency of inhibition significantly. Consequently, for all further inhibition experiments, we employedsiR_1H siRNA.

Fig. 2. A Expression of c-erbB2 mRNA at 24 hours from siRNA lipofection of
SK-BR-3 c-erbB2 overexpressing breast cancer cells. The highest efficiency
of mRNA inhibition was observed for siR_1H , when c-erbB2 expression was
reduced by 77% . B mRNA c-erbB2 expression was determined and plotted
every 24 hours post-silencing, showing the step-wise return to previously
detected levels. siRNA lipofection of either adherent or trypsinized SK-BR-3
cells did not influence treatment outcome. C Flow cytometry analysis of
HER2neu expression at 48, 96 hours and 8 days in siRNA treated and control
SK-BR-3 cells.

Next, we assessed the time frame for which the siRNA
siR_1H is capable of downregulating c-erbB2 mRNA and protein
expression to therapeutically useful levels and documented thereturn of c-erbB2 expression to the levels detected before RNAi.
Transcription of c-erbB2 was maintained at below 50% of that in
untreated cells for the first 48 hours, quickly increasing afterwards
to previous expression levels at 6 days and even overcompensating with higher levels than before transfection when measured
at 8 days post-silencing (Figure 2 B). Flow cytometry analysis of
HER2 levels in transfected SK-BR-3 showed decreased protein
levels to less than 50% at 48 hours following inhibition. The levelof HER2 protein inhibition regressed slower than mRNA levels,with 61.6% of the transfected cells still expressing significantly
low HER2 96 hours post-treatment (Figure 2 C). At the last time
point considered, 8 days, there was still a small population ofcells that did not regain previous HER2 expression.
Furthermore, we examined by immunocytochemistry andconfirmed c-erbB2 protein knockdown at 48 hours after siR_1H
delivery in the SK-BR-3 line (Figure 3).
Fiziologia - Physiology  2011.21.3 (71)

staining, respectively, did not differ significantly from controls.

Fig. 4. A VEGF presence in SK-BR-3 cell culture supernatant (pg/mL; left
y-axis, connecting dots plot), determined by ELISA immunoassay, following
siRNA inhibition of c-erbB2. Supernatants were collected at 48 hours fromtransfection, from identical numbers of cells, incubated for 24 hours in normal
cell growth medium. VEGF transcription, detected by qRT-PCR (right y-axis,
bar plot). B Cell cycle distribution determined by flow cytometry analysis ofcells treated with propidium iodide (PI). Sub-G0 phase indicates apoptotic
cells. C Quantitative analysis of apoptosis by Annexin V and propidium iodide(PI) staining.

Fig. 3. Immunocytochemical analysis of SK-BR-3 cells of Her2/neu protein.
Untreated control cells (A) express the protein at very high levels (3+), whilecells treated with 100 nM siR_1H (B), still express HER2/neu ubiquitously, but
at significantly lower levels. Optical microscopy, magnification 400x.

Her2/neu inhibition reduces VEGF secretion in breast
cancer cell line SK-BR-3
We determined the presence of pro-angiogenic vascularendothelial growth factor (VEGF) in the cell culture medium of
SK-BR-3 cells after siRNA inhibition of the oncogene of interest.
A significantly lower concentration of VEGF was correlated withc-erbB2 inhibition and downregulation of VEGF mRNA expression, as detected by quantitative RT-PCR (Figure 4 A).
Cell cycle and viability of HER2neu-silenced SK-BR-3
cells
SK-BR-3 cells treated with c-erbB2 siRNA showed growth
inhibition, morphological changes and an inability to form a confluent monolayer when examined under an inverted microscope(data not shown). When stained with PI and analyzed for cell cycle
distribution, HER2-silenced cells displayed gowth arrest and an
accumulation of cells in the G0/G1 phase of the cell cycle (Figure4 B) at 48 and 96 hours post-transfection when compared with theuntreated control cells. However, at 8 days following inhibition, thecell cycle distribution was similar to control cells.
We tried to determine whether there was also an increase in
the percentage of apoptotic cell in the SK-BR-3 cells transfectedwith HER2neu siRNA, but that was not case as the apoptotic
and dead cell populations recognized by characteristic Annexin
V- positive, PI negative and Annexin V - positive, PI positive
2011.21.3 (71)  Fiziologia - Physiology

DISCUSSION
We studied the effects of HER2 siRNA transfection on
SK-BR-3 cells, a HER2/neu overexpressing breast adenocarcinoma cell line. Previously published experiments using RNAinterference for silencing c-erbB2 expression in human breast
cancer cells reported a dose-dependent suppression of HER2
levels and inhibition of growth or induction of apoptosis in thecancer cell lines employed in the study (9-11,15,16). Testing 4
different siRNAs directed against different parts of the -erbB2mRNA sequence, as well as an equimolar mix of these siRNAs, we were able to obtain a significant knockdown of HER2transcription ranging from 15 to 85%, correlated to a morethan 50% decrease of surface protein expression. Three of thesiRNA sequences were not published before, while the fourth
was purchased from SCBT. We obtained the highest inhibition
efficiency in the case of siR_H1, one of our custom designed
sequences, which we proceeded to use for all subsequent
experiments. As expected in the case of lipofection, a transienttransfection method, HER2 levels of gene expression returned
to normal in approximately 6 days, with a slight increase above
the starting level of transcription at 8 days following the silencing procedure, but we could not find any data in the literature tocompare the kinetics of inhibition specifically for the SK-BR-3cell line for a period longer than 96 hours. The effect of siRNAtransfection on HER2 protein presence on the cell surface wasmore long-lasting, the cells recovering to 90% of the initial protein level at 8 days after inhibition. Although the silencing of thetarget gene by RNAi was not permanent, one single transfection
resulted in levels of inhibition of the oncogene much higher thanthose reported in the case of monoclonal antibodies (17,18) orantisense oligonucleotides (19).
7Breast cancer cells that overexpress HER2 have beenshown to produce high levels of the potent pro-angiogenic
vascular endothelial growth factor VEGF (20,21). In our present
study, we also observed a marked decrease of VEGF level after
silencing c-erbB2 expression by more than half, compared tountreated SK-BR-3 cells, a very important effect of HER2 RNAi
resulting in a multi-targeted tumor control.
One of the most significant physiological impacts of HER2/
neu inhibition on SK-BR-3 cells was an increased arrest of thesiRNA treated cells in the G0/G1 phase of the cell cycle. Thisfinding is consistent with other reports in the literature (10,11),while there is also one study that presented growth arrest at
G2/M (9). Down-regulation of c-erbB2 would result to decreased
phosphorylated Akt, which leads to decreased expression of thecyclin D1 involved in the regulation of G0/G1 cell cycle arrest. Another possible way of explaining G1 arrest would be a decreaseof CDK inhibitor p27Kip1 phosphorylation, another downstream
target of Akt (22,23). We did not however observe an increased
apoptosis in the siRNA treated breast cancer cells when compared to control untreated cells. This might be explained by thevery high initial level of HER2 expression in SK-BR-3 cells witheven 20 percent of remaining oncogene expression accounting
for continued survival of tumor cells. The complex mechanism
by which c-erbB2 stimulates cancer cell growth and resistanceto chemotherapy has not been completely defined and there
certainly exist redundant pathways conveying a proliferation
stimulus in the neoplastic cells which converge. We intend toinvestigate next the effects of a longer inhibition of HER2 in thebreast cancer cell line SK-BR-3 either by repeated transfection
or by using a viral construct capable of long-term silencing of thetarget gene, in which experimental setting the network of HER2
targets might be more apparent.
Acknowledgements
This work was supported by CNCSIS – UEFISCSU,
project number PNII – IDEI 1748/2008 awarded to VP and
POSDRU/89/1.5/S/62371 awarded to OIG.
REFERENCES
1. Phalon C, Rao DD, Nemunaitis J. Potential use of RNA interferencein cancer therapy. Expert Rev Mol Med 2010; 12e26.
2. Latterich M. Rnai advanced methods. illustrated ed. Taylor & Francis;
2008.
3. Elbashir SM, Lendeckel W, Tuschl T. RNA interference is mediatedby 21- and 22-nucleotide rnas. Genes Dev 2001; 15(2): 188-200.
4. Manjunath N, Dykxhoorn DM. Advances in synthetic sirna delivery.
Discov Med 2010; 9(48): 418-30.
5. Freudenberg JA, Wang Q, Katsumata M, et al. The role of HER2 inearly breast cancer metastasis and the origins of resistance to her2targeted therapies. Exp Mol Pathol 2009; 87(1): 1-11.
6. Press MF, Slamon DJ, Flom KJ, et al. Evaluation of HER-2/neu
gene amplification and overexpression: Comparison of frequently used

8

assay methods in a molecularly characterized cohort of breast cancer
specimens. J Clin Oncol 2002; 20(14): 3095-105.
7. Esteva FJ, Cheli CD, Fritsche H, et al. Clinical utility of serum HER2/
neu in monitoring and prediction of progression-free survival in metastatic breast cancer patients treated with trastuzumab-based therapies.
Breast Cancer Res 2005; 7(4): R436-43.
8. Morrow PK, Zambrana F, Esteva FJ. Recent advances in systemic
therapy: Advances in systemic therapy for her2-positive metastatic
breast cancer. Breast Cancer Res 2009; 11(4): 207.
9. Choudhury A, Charo J, Parapuram SK, et al. Small interfering RNA(sirna) inhibits the expression of the her2/neu gene, upregulates HLAclass I and induces apoptosis of her2/neu positive tumor cell lines. Int
J Cancer 2004; 108(1): 71-7.
10. Faltus T, Yuan J, Zimmer B, et al. Silencing of the HER2/neu geneby sirna inhibits proliferation and induces apoptosis in HER2/neuoverexpressing breast cancer cells. Neoplasia 2004; 6(6): 786-95.
11. Yang G, Cai KQ, Thompson-Lanza JA, et al. Inhibition of breastand ovarian tumor growth through multiple signaling pathways by using retrovirus-mediated small interfering RNA against her-2/neu gene
expression. J Biol Chem 2004; 279(6): 4339-45.
12. Elbashir SM, Harborth J, Lendeckel W, et al. Duplexes of 21-nucleotide rnas mediate RNA interference in cultured mammalian cells. Nature
2001; 411(6836): 494-8.
13. Hartsuiker L, Zeijen NJ, Terstappen LW, et al. A comparisonof breast cancer tumor cells with varying expression of the her2/
neu receptor by raman microspectroscopic imaging. Analyst 2010;
135(12): 3220-6.
14. Xiao Y, Gao X, Maragh S, et al. Cell lines as candidate referencematerials for quality control of ERBB2 amplification and expression
assays in breast cancer. Clin Chem 2009; 55(7): 1307-15.
15. Colomer R, Lupu R, Bacus SS, et al. Erbb-2 antisense oligonucleotides inhibit the proliferation of breast carcinoma cells with erbb-2
oncogene amplification. Br J Cancer 1994; 70(5): 819-25.
16. Roh H, Pippin JA, Green DW, et al. HER2/neu antisense targetingof human breast carcinoma. Oncogene 2000; 19(53): 6138-43.
17. Brodowicz T, Wiltschke C, Budinsky AC, et al. Soluble HER-2/neu
neutralizes biologic effects of anti-her-2/neu antibody on breast cancer
cells in vitro. Int J Cancer 1997; 73(6): 875-9.
18. Drebin JA, Link VC, Weinberg RA, et al. Inhibition of tumor growth
by a monoclonal antibody reactive with an oncogene-encoded tumor
antigen. Proc Natl Acad Sci U S A 1986; 83(23): 9129-33.
19. Roh H, Pippin J, Drebin JA. Down-Regulation of HER2/neu expression induces apoptosis in human cancer cells that overexpress HER2/
neu. Cancer Res 2000; 60(3): 560-5.
20. Le XF, Mao W, Lu C, et al. Specific blockade of VEGF and HER2
pathways results in greater growth inhibition of breast cancer xenografts
that overexpress HER2. Cell Cycle 2008; 7(23): 3747-58.
21. Loureiro RM, Maharaj AS, Dankort D, et al. Erbb2 overexpression in
mammary cells upregulates VEGF through the core promoter. Biochem
Biophys Res Commun 2005; 326(2): 455-65.
22. Liang J, Zubovitz J, Petrocelli T, et al. PKB/akt phosphorylates p27,
impairs nuclear import of p27 and opposes p27-mediated G1 arrest.
Nat Med 2002; 8(10): 1153-60.
23. Zhuang Y, Miskimins WK. Cell cycle arrest in metformin treatedbreast cancer cells involves activation of AMPK, downregulation of cyclin
D1, and requires p27kip1 or p21cip1. J Mol Signal 2008; 318.

Fiziologia - Physiology  2011.21.3 (71)INHIBITIA EXPRESIEI GENEI HER2 PRIN INTERFERENTA ARN
BLOCHEAZA PROLIFERAREA LINIEI TUMORALE MAMARE SK-BR-3
REZUMAT

Introducere: Supraexpresia genei HER2, cu sau fara amplificare genomica, a fost documentata in aproximativ 25% din cazurile decancer mamar si a fost asociata cu un prognostic nefavorabil si rezistenta la chemoterapie. In acest studiu, am investigat efectul
inhibitiei expresiei protooncogenei c-erbB2 prin interferenta ARN aupra proliferarii si viabilitatii liniei tumorale mamare SK-BR-3, care
exprima HER2neu la un nivel ridicat.
Materiale si metode: Moleculele de siRNA au transfectate cu ajutorul reactivului Lipofectamine 2000. ARN-ul total a fost extras cu
TRIzol, revers-transcris sub forma de ADNc, iar probele de ADNc au fost analizate prin metoda qPCR pentru a determina nivelul detranscriptie a genelor HER2 si VEGF in urma inhibitiei. Expresia proteica de HER2 a fost pusa in evidenta prin colorare imunohistochimica, in timp ce prezenta VEGF in supernatant a fost documentata prin ELISA. Am determinat, de asemenea, distributia ciclului
celular si gradul de apoptoza in celulele SK-BR-3 tratate cu siRNA, comparativ cu un control.
Rezultate: O inhibitie de ~80% a expresiei oncogenei a oprit proliferarea celulelor tumorale prin blocarea ciclului celular in faza
G0/G1 si a rezultat intr-o reducere accentuata a secretiei de VEGF. Nu am observat, insa, un nivel crescut de apotoza in celuletumorale tratate cu siRNA.
Concluzii: Mecanismul complex prin care gena c-erbB2 stimuleaza proliferarea tumorala si rezistenta la chemoterapie nu a fostcomplet elucidat si, in mod cert, exista cai de semnalizare redundante care sustin proliferarea neoplastica in absenta HER2.
Cuvinte-cheie: HER2/neu, interferenta ARN, cancer mamar, SK-BR-3

2011.21.3 (71)  Fiziologia - Physiology

9BIOGERONTOLOGY TODAY. PART I. 61ST ANNUAL SCIENTIFICMEETING OF THE BRITISH SOCIETY FOR RESEARCH ON
AGEING - BSRA & 14TH CONGRESS OF THE INTERNATIONALASSOCIATION OF BIOMEDICAL GERONTOLOGY - IABG,
BRIGHTON, UK, 11TH - 14 JULY 2011
DAN RIGA1, SORIN RIGA1, AUREL ARDELEAN2, DANIELA MOTOC2, FRANCISC SCHNEIDER2
Department of Stress Research & Prophylaxis, Al. Obregia Clinical Hospital of Psychiatry, Bucharest, Romania
2
Vasile Goldis Western University, Arad, Romania

1

ABSTRACTThe presentation, analysis and summary of the 61st Annual Scientific Meeting of the British Society for Research on Ageing – BSRA
and of the 14th Congress of the International Association of Biomedical Gerontology – IABG are the alignment to the current stage
and, especially, to future directions of transforming present day reality of an aging people with disabilities into long-lived, competitive
psychologically and physically active human beings, with much longer ontogenetic periods. The focus on certain areas of research
becomes essential for the speedier discovery and introduction of prophylactic, therapeutic and recovery solutions for human senescence and its transformation into human longevity.
Keywords: 61st Annual Scientific Meeting of the British Society for Research on Ageing - BSRA; 14th Congress of the International
Association of Biomedical Gerontology - IABG; Science of aging; Biogerontology; Present period; Future directions.

MODEL OF MEETINGS FOR THE SCIENCE OF AGING
These two international congresses reunited under thetitle The Science of Ageing. Global Progress and connected
meetings, held in Brighton, UK, 2011 (3, 10), represent a scientometric pattern to establish the present and future directionsof a determinant scientific field for today - biogerontology. Thisscience - the study of the biological basis of aging - has important significations in biomedical research, aging and healtheducation, prophylaxis, therapy and recovery of senescencewith age-related diseases, as well as in general and specifichealth care systems.
Adjacent events included:- the BSRA Annual General Meeting and BSRA Executive
Committee Meeting;- Award of the Lord Cohen Medal to Prof. Dr. Suresh I. S.
Rattan, at the Department of Molecular Biology, Aarhus University, Aarhus, DK, for his contribution to the science of gerontology.
This medal is the highest honour the BSRA can bestow and its
previous recipients include Robin Holliday, Leonard Hayflickand Tom Kirkwood;- Biogerontology, Springer Editorial Board Meeting (Suresh
I. S. Rattan, Editor-in Chief);- Public Round Table Discussion by the Honorary Life
Members of the BSRA to discuss Physiological and ethicalaspects of aging research (Chair Prof. Alan Maryon-Davis; andProf. Leonard Hayflick, Prof. Dame Linda Partridge, Prof. Tom
Kirkwood, Prof. Arlan Richardson, Prof. Sydney Shall, Mrs.

Elizabeth Mills);- Funders Round Table Discussion - Chair Prof. R. Faragher
and Representatives from National Institute on Aging-National
Institutes of Health (NIA-NIH, USA), Medical Research Council(MRC, UK), Biotechnology and Biological Sciences ResearchCouncil (BBSRC, UK), Australian Research Council (ARC, AU),Age UK (the previously separate charities Age Concern and Help
the Aged, which now together form the UK’s largest charity forthe elderly and British Council - Research Council (UK);- NIA-BBSRC Joint Transatlantic Symposium and Workshop(Chair Prof. R. Faragher);- PROTEOMAGE Session, UE (Chair Prof. Brian Clark);- American Aging Association (AAA) Symposium (Chair Prof.
Janko Nicolich-Zugich);- Chemistry Central Journal Meeting for Chemistry of Ageing
Special Issue (Section Editor Elisabeth Ostler).
Historical landmarks
Chair, Prof. Dr. Richard Faragher, University of Brighton,
UK opened this local and international joint meeting emphasizing the importance of the British Society for Research on
Ageing - BSRA (1, 5) and of the International Association of
Biomedical Gerontology - IABG in the evolution and development of aging sciences as worldwide global progress. Also,
historical landmarks are very significant because the BSRA was
one of the pioneers of aging investigation, which grew out of the
British Club for Research on Ageing founded by Dr. Vladimir

Received August 20th. Accepted September 21st. Address for correspondence: Sorin Riga, Department of Stress Research and Prophylaxis, ”Al. Obregia” Clinical Hospitalof Psychiatry, 10 Berceni Rd., 041914 Bucharest 8, Romania, Phone: +40 21 334 3008, Fax +40 21 230 95 79, e-mail: D_S_Riga@yahoo.com

10

Fiziologia - Physiology  2011.21.3 (71)Korenchevsky (1880-1959) in Oxford, in 1945, and a leadingorganization in the creation of the International Association ofGerontological Societies in Liège, BE, in 1950 (5).
Congress themes
Up-to-date lectures, poster presentations and sessions, aswell as interesting discussions focused on:- the present and future directions of aging research andbiogerontology;- basic and fundamental molecular and cellular mechanisms of aging processes;- immune system in aging, immune risk profile and experimental and medical immunological researches;- Werner syndrome and its correlation with premature andaccelerated aging;- fundamental mechanisms of brain aging and its connection with neuropathology (as part of neurosciences), as well as
muscle aging mechanisms and interventions;- PROTEOMAGE session (Chair: Prof. Brian Clark);- nutrition, nutrigenomics, calorie restriction mimetics; and- exceptional longevity.
SENESCENCE INVESTIGATION: PRESENT AND FUTURE
The present and future directions of aging research andbiogerontology was one of the main objectives of these twocongresses:- The need for, and promise of, research on aging (Elisabeth
Mills, UK);
- Developing knowledge management systems for aging
research (Alex Zhavoronkov, RU), a flexible open-accessknowledge management system for aging research - theInternational Aging Research Portfolio (IARP) accessible via
www.agingporfolio.org;- Aging - from here to where? (Suresh Rattan, DK);
- Health-longevity pyramid in the anti-aging global progress (Sorin Riga, Dan Riga et al., RO), (9);- Ethical, social & cultural aspects of anti-aging science,biomedicine and older people (Joanna Latimer, UK).
Elisabeth Mills, UK (The need for, and promise of, research on aging) pointed out ways to transform unsuccessful
aging, a complex mixture of morbidity, social isolation, poverty
and invisibility into a successful period of life. Research efforts to understand the biology of aging, which is the basis ofsenescence, should better coordinate with government andthe third sector structures, measures and funds.
Alex Zhavoronkov, RU (Developing knowledge management systems for aging research) addressed a flexible openaccess knowledge management system for aging research- the International Aging Research Portfolio (IARP) accessible
via www.agingporfolio.org. The IARP system, highly modular
and portable, may be used as a platform for developing otherknowledge management systems for aging research.
Suresh Rattan, DK (Aging - from here to where?)
2011.21.3 (71)  Fiziologia - Physiology

presented the goal to extend health-span by elucidation andexploration of the successful and dynamic interactions among
biological, psycho-social and environmental factors, because
aging can be understood at various levels, from evolutionary
and biological levels to psychological and sociological ones.
At the molecular biological basal level, aging is characterized
by the stochastic occurrence and progressive accumulationof molecular damages, with inefficiency and imperfection of
the maintenance and repair systems, and finally failure of
homeodynamics. The holistic view of senescence facilitates
the setting up of a framework for understanding, researching and developing effective and realistic strategies for aging
interventions.
Sorin Riga, Dan Riga et al., RO (Health-longevity
pyramid in the anti-aging global progress) emphasized theconstruction of human health-longevity as a global and holistic
progress, because man is a bio-psycho-social being. Consequently, future medicine will be and must be the medicine ofhealth, mainly the planning of personalized and public health,
together with the strategies of longevity, somatic and mental
health (9).
Molecular and cellular mechanisms of senescence
Basic and fundamental molecular and cellular mechanisms of aging processes were largely presented anddebated:- Cell senescence is a cause of aging (Thomas von Zglinicki, UK);
- Sources, transmission and effects of transcriptional noisein C. Elegans aging (Hang Lu, USA, Quee Lim Ch’ng, UK);
- Mechanism and functions of epigenetic reprogramming in
mammalian development (W. Reik, UK);
- Epigenomics: understanding how nutrition modulates theaging process (John Mathers, UK);
- Maternal diet, epigenetics and programming of adult health(Susan Ozanne, UK);
- Induced pluripotent stem cells in aging research (Alexandra
Stolzing, DE);- Wine yeast as a novel model system to study chronologicallife span (Augustin Aranda, SP);
- Probing fundamental aging biology with model systems(David Gems, UK);
- Stress response profiles in aging human cells (Dino
Demirovic and Suresh Rattan, DK);- Application of cross species analysis of conserved pathways to the study of cellular aging (João F. Passos et al., UK);
- Identification of major biomarkers of aging (Shiva Marthandan et al., DE);
- A compartmentalized protein-protein interaction network
as a novel model of aging (Daniel V. Veres and Peter Csermely,
HU).
Thomas von Zglinicki, UK (Cell senescence is a cause of
aging) pointed out the adverse effects of cell senescence, mainly
the pro-oxidant and inflammatory cellular phenotype, which isa delayed consequence of a DNA damage response (DDR),
11dependent on signalling through p21 (CDKN1A), p38MAPK
and (for the inflammatory phenotype) NF-kB. Adult peripheral
and brain neurons also present a senescent phenotype, as
characterized by DDR, p38MAPK activation, oxidative damage
and interleukin production. nfkb1-/- mice show a constitutively
activated inflammatory phenotype due to increased formationof p65 homodimers.
Hang Lu, USA, Quee Lim Ch’ng, UK (Sources, transmission and effects of transcriptional noise in C. Elegans aging)presented the beginning of a collaborative project with aims todetermine how transcriptional noises in environmental responses
translate to intoheterogeneity in lifespan and aging in nematodeworm C. Elegans and to define environmental and genetic factorsthat contribute to these sources of noise. They have developed
microfluidic and automated microscopy systems capable ofcollecting large scale data necessary for their analysis, as wellas generating a molecular genetic pipeline for single copy insertions of transcriptional markers. In addition, they developed anarray of devices for individual-animal long-term culture to assay
aging phenotypes.
W. Reik et al., UK (Mechanism and functions of epigenetic
reprogramming in mammalian development) spoke about epigenetic information in the genome, which is relatively stable insomatic cells, but is reprogrammed on a genome wide level ingerm cells and early embryos. A key component of reprogramming is the erasure of DNA methylation, which may occur by passive (replication dependent) or active mechanisms. They used
genome-wide profiling methods based on bisulfite sequencing(BS-Seq) or antibody pulldown sequencing (MeDIP-Seq) in orderto understand better the dynamics of erasure of DNA methylation. The study of genome-wide BS-Seq in primordial germ cells
has revealed the extent of reprogramming on an unprecedented
scale, and has shown that the activated-induced cytidine
deaminase (AID) is implicated in epigenetic reprogramming andpotentially in active demethylation of DNA. Also, the distribution
in the genome of methylation (5mC) and hydroxymethylation(5hmC) was investigated in (embryonic stem (ES) cells andduring their differentiation, using MeDIP-Seq and hMeDIP-Seq.
5hmC is enriched in euchromatic parts of the genome, including
in promoters of some pluripotency-related genes, where it isassociated with transcription. There is a considerable turnoverof methylation by hydroxymethylation in ES cells, which helps
to maintain their plasticity and pluripotency.
João F. Passos et al., UK (Application of cross species
analysis of conserved pathways to the study of cellular aging)developed the Cross-species Interactome Database (CID), an
integrated resource for the analysis and comparison of interaction
networks across different species. The CID database and thedescribed computational methods are made available through a
newly developed plugin for Cytoscape, an open source software
platform for visualising and analysing molecular interaction
networks.
Shiva Marthandan et al., DE (Identification of major
biomarkers of aging) presented their research program, partof the JenAge project, with the aim to elucidate the molecular
12

mechanisms and cellular pathways that drive cellular senescencein primary cell culture, mainly human fibroblasts.
Alexandra Stolzing, DE (iPS-Induced pluripotent stem cells
in aging research) discussed about first iPs cell lines, recently
created from donors with aging-accelerating genetic disorders(neurodegenerative disease or mitochondrial mutations), toelucidate the role of these mutations in aging processes.
David Gems, UK (Probing fundamental aging biology withmodel systems) in his closing keynote lecture of the congressaddressed the accumulation of molecular damages in C. Elegans
worm senescence as cause or not of the aging process.
Proteomage project
PROTEOMAGE Session (Chair: Prof. Brian Clark) presented this integrated EU-project (2006-2011), which used state-ofthe-art proteomic technology to study molecular mechanisms ofaging in various model organisms ranging from yeast to humans,
as well as significant results with this topic:- From the stability of living cells to cellular stress-inducedpremature senescence and geriatric diseases (Olivier Toussaint, BE);- Oxidative proteome alteration and protein maintenanceduring aging and upon oxidative stress (Bertrand Friguet, FR);- The role of secreted proteins in cellular senescence andaging (Pidder Jansen-Duerr, AT).
IMMUNE SYSTEM IN AGING
Immune system in aging, immune risk profile and experimental and medical immuno-logical researches were an
important pillar of these traditional scientific meetings:- The immune system in aging (Graham Pawelec, DE);
- Steroid receptors and the control of thymic involution(Nancy Manley, USA);- Mechanism of reduced T cell immunity in older adults(Arne Akbar, USA);- Rejuvenation strategies to rebalance the aging T-cell
repertoire (Megan Smithey, USA);- A novel high-throughput autophagy assay for primarycells: senescent T cells show decreased levels of autophagy(Kanchan Phadwal, UK);
- Preparation of integral membrane proteins in order toevaluate cell surface protein expression in human CD4+ Tlymphocytes from young and mid-life adults (S. J. Bennett etal. UK);- The role of peroxiredoxin antioxidant enzymes in stressresistance, aging and innate immunity (E. L. Button et al., UK);
- The effect of aging on regulatory B cells (Niharika A. Duggal
and Janet M. Lord, UK);
- Immune system aging in heterochronic parabiosis: starting mechanism and impact on life expectancy (I. Pishel et al.,Ukraine);- Reversible senescence in human CD4+ CD45RA+CD27- memory T cells (D. Mitri et al., IT).
- The impact of lifelong persistent viral infections on naïve
and memory T-cell receptor Vb repertoire diversity (P. Samadder,
Fiziologia - Physiology  2011.21.3 (71)USA);- Peripheral lymphocyte activation and differentiation in oldmelanoma patients (J. A. Seidel, UK);
- Impaired neutrophil extracellular trap (NET) formation: anovel defect in the innate immune system of aged individuals(J. Hazeldine et al., UK);
- Splenic architecture and cellularity is perturbed in aging
mice and exhibit altered immune responses to T-independent
and T-dependent antigens (D. Aw et al., UK).
Graham Pawelec, DE (The immune system in aging) spoke
about immune system in the elderly, which paradoxically suffer
both from failing immunity resulting in increased susceptibility toinfections, and decreased responsiveness to vaccination, andat the same time increased immunopathology accompanying
immune responses. Limited longitudinal studies have begun toreveal biomarkers of immune aging increasingly recognized asan ”immune risk profile” (IRP) predicting mortality in the very
elderly. Usually asymptomatic infection with the widespreadpersistent β-herpesvirus HHV5 (Cytomegalovirus, CMV) has
an enormous impact on these immune biomarkers. The prevalence of CMV infection in the population increases with age,
and within individuals, the degree of immune commitment also
increases with age. This may cause pathology by maintaining
higher systemic levels of inflammatory mediators and decreasing the ”immunological space” available for immune cells withother specificities.
Nancy Manley et al., USA (Steroid receptors and thecontrol of thymic involution) emphasized the role of Androgen
Receptor (AR) in postnatal involution of the thymus with age, andpresented their initial findings on the expression and localizationof AR in the thymus, the thymus phenotypes in ArTfm/Y mutant
mice and on TEC-specific deletion of the AR.
Megan Smithey, USA (Rejuvenation strategies to rebalance
the aging T-cell repertoire) covered the topic of immunosenescence in individuals with lifelong persistent viral infections,particularly with the Herpesvirus family members CMV andherpes simplex virus (HSV). In both mouse and human, repeatedinteractions between these reactivating viruses and antiviral
T cells leads to memory T-cell inflation (MI), with increasing
accumulation of these cells over life span. Her research teamhypothesized and tested that MI carries a price for the immune
system, namely competition between memory and naïve T-cells
for survival signals may impair the maintenance of the diversenaïve T-cell pool, consequently leaving the individual at a disadvantage when exposed to a new pathogen.
D. Aw et al., UK presented Splenic architecture and cellularity is perturbed in aging mice and exhibit altered immune
responses to T-independent and T-dependent antigens. Indeed,
these age-related changes within the spleen could potentiallycontribute to the age-dependent deficiencies in functional immunity.
CEREBRAL AGING
Fundamental mechanisms of brain aging and its connection with neuropathology (as part of neurosciences) are
2011.21.3 (71)  Fiziologia - Physiology

presented at length:- A RNA-seq analysis of brain aging in the Brown rat (Shona
Wood, UK);
- A senescence-like phenotype in postmitotic neurons (C.
Wang et al., UK);
- How well do sleep and activity behaviours predict lifespanin Drosophila? (S. Koudounas et al., UK);
- Upregulation of the synaptic plasticity regulating major
histocompatibility complex I pathway in the hippocampus withaging and cognitive decline (C. Van Kirk et al., USA);- A new Caenorhabditis elegans model of age-dependentneurodegeneration (Sudhanva Kashyap, UK);- A complex dietary supplement prevents age-related cognitive and motor declines and offsets cellular biomarkers of aging
in mice (Vadim Aksenov, CA);- Supplementation of tocotrienol rich fraction (TRF) improved cognitive functions in aged rats (M. T. Nursiati et al.,
Malaysia);- Perceptual plasticity in the peripheral visual field of older
adults (A. Blighe et al., UK);
- Senile local eye amyloidosis risk factor for age-related
macular degeneration (V. V. Ermilov and O. V. Makhonina,
RU);- Upregulation of calpastatin may protect motor neuron
death during the development amyothrophic lateral sclerosis(Liang Li and Adu Jimi, UK);- Pharmacological modulation of the abnormal oscillatory
profile in Parkinson’s disease (K. Ronnqvist et al., UK);
- Aβ and oxidative stress in the brain of the longest-living
rodents (Y. Edrey et al., USA);- Brain lipopigments - link between aging and Alzheimer
disease (Dan Riga, Sorin Riga et al., RO), (8);- Saffron (Crocus sativus L.) extract prevents and improves
the Alzheimer’s disease in mice (M. H. Dashti et al., Iran);
Shona Wood and co-workers, UK (A RNA-seq analysisof brain aging in the Brown rat) used RNA-seq examination (amore accurate method than mRNA microarrays) to sequence thecerebral cortex transcriptome in young, middle age and old rats.
This analysis allows non-coding RNA and splice variant detection/comparison across phenotypes. The study showed that there
is a relatively large amount of non-coding RNAs differentiallyexpressed with aging in the cerebral cortex. They have a regulatory role in gene expression and splice variant selection.
C. Wang et al., UK (A senescence-like phenotype in postmitotic neurons) presented (for the first time) that DNA damage
triggers a senescent-like phenotype, characterized by activationof p38 MAPK, production of reactive oxygen species (ROS) andpro-inflammatory cytokines and increased levels of senescentassociated β-galactosidase in brain and peripheral neurons.
Sudhanva Kashyap and collab., UK (A new Caenorhabditis elegans model of age-dependent neurodegeneration)
described this type of worm produced by mutation of the dnj-14
gene. DNJ-14 is the worm homologue of cysteine string protein(CSP), a neuronal protein that prevents the misfolding of presynaptic proteins. Besides, CSP knockout mice have a short lifespan13and exhibit progressive age-related neurotransmission defects,
sensiomotor dysfunction and pre-synaptic neurodegeneration.
Y.H. Edrey et al., USA (Aβ and oxidative stress in the brainof the longest-living rodents) used Naked Mole-Rats (NMRs,
Heterocephalus glaber), subterranean rodents, evolved in a
hypoxic habitat, naturally vitamin D deficient, with a maximum
lifespan of 32 yrs. In captivity they incur high levels of oxidative
damage evident even at a young age. Their sequence of Aβ
peptide was more similar to the human peptide, than to that ofits mouse counterpart. NMRs brains had a detectable amount ofboth soluble and insoluble fractions of Aβ, even at a young age
and showed significantly higher levels than young adult humans
or transgenic mice that express human Aβ.
Dan Riga, Sorin Riga and co-workers, RO (Brain lipopigments - link between aging and Alzheimer disease) studied
cerebral lipopigments - LPs (lipofuscin and ceroid) in normal
aging in comparison with Alzheimer disease (AD). In AD, theLPs become a constancy and are associated with specific neurodegenerative changes. Moreover, LP storages are constitutive parts of amyloid (senile, neuritic, argyrophilic) plaques andneurofibrillary tangles (8).
SENESCENCE OF MUSCULAR SYSTEM
The Muscle aging session, with constant lectures at every
biogerontology meeting, presented the mechanisms of sarcopenia and interventions to maintain muscle mass and function:- Can exercise/physical activity prevent loss of motor units?
(Susan Brooks, USA);- Aging, inflammation, and weakness (Mike Reid, USA);- Do oxidative damage or defective redox signalling
contribute to age-related loss of skeletal muscle? ( Malcolm
Jackson, UK);
- Changes in muscle protein homeostasis with aging (Marco
Sandri, IT);
- Overexpression of HSP10 in transgenic mice protects
skeletal muscle against endotoxic shock-induced loss of force
generation (Anna Kayani et al., UK);
- Insulin/IGF-like signalling and aging-related locomotor
decline in Drosophila melanogaster (Sue Broughton, UK);
- Losartan restores skeletal muscle remodeling and protects
against disuse atrophy in sarcopenia (T. N. Burks et al., USA);- Life events stress and physical activity are associated withthe diurnal rhythm of dehydroepiandrosterone and the cortisol:
DHEA ratio in older adults (J. Heaney et al., UK);
- Metabolic equivalent of a one-mile walk by older adults:
implications for health promotion (M. Gault et al., UK);
- ROS generation during myogenesis: role of altered ROS
generation in failed regeneration of old muscle (A. Vasilaki etal., UK).
In Aging, inflammation, and weakness, Mike Reid, USA
presented correlations between these concepts. As such,ceramide levels and sphingomyelinase activity are elevated insenescence, and also depress specific force. Cytosolic oxidants
preferentiallyact on myofilrillary proteins, decreasing specific force,14

without altering calcium regulation or other aspects of myofibrillar mechanics.
Marco Sandri, IT (Changes in muscle protein homeostasiswith aging) emphasized his recent data that autophagy-lysosome
system is critical to maintain muscle mass, and its alteration
leads to muscle atrophy, weakness and to several features thatare present in aging sarcopenia.
T. N. Burks et al., USA (Losartan restores skeletal muscle
remodeling and protects against disuse atrophy in sarcopenia)
studied the increased TGF-β signalling, which contributes
to impaired satellite cell function and muscle repair in aged
skeletal muscle. They evaluated whether antagonism of TGF-βsignalling via losartan, an angiotensin II receptor antagonist
commonly used to treat high blood pressure, had a beneficial
impact on the muscle remodelling process of sarcopenic mice.
They demonstrated that mice treated with losartan developed
significantly less fibrotic tissue and exhibited improved in vivo
muscle function after cardiotoxin-induced injury.
Efficient therapies and strategies to preserve mobility andmovements in the elderly become very useful.
WERNER SYNDROME - WS
Werner syndrome - WS (described by Otto C. W. Werner,
1879-1836, German physician) in the young adult (child’s equivalent disease - Hutchinson-Gilford, individualized by Jonathan
Hutchinson, 1828-1913, English surgeon and Hastings Gilford,
1861-1941, English physician) was debated in its connexion
with premature and accelerated aging:- Towards a cure for adult progeria (David Kipling et. al.,UK);
- Using phage display to screen for protein partners of WRN,
implicated in premature aging Werner syndrome (Lynne S. Cox
and Ivan Boubriak, UK);
- Assessing the role of p38 MAP kinase activity in replicative
senescence in fibroblasts from progeroid syndromes (Terence
Davis et al., UK);
- Exploring the function of progeroid WRN gene homologues
in Caenorhabditis elegans (H. Lees et al., UK).
David Kipling et. al., UK (Towards a cure for adult progeria) focused their research on p38 activation in WS, which upregulate immunomodulatory molecules such as pro-inflammatory
cytokines. The premature aging in WS may be explained exclusively by the accumulation of senescent cells in certain lineages, exclusively as a result of p38-driven ”inflamm-aging”, or a
combination of both. p38 inhibitors are being actively developedby pharmaceutical groups, raising opportunities to test potential
therapeutic approaches in vivo.
Lynne S. Cox and Ivan Boubriak, UK (Using phage display
to screen for protein partners of WRN, implicated in prematureaging Werner syndrome) insisted on WS as premature humanaging model. WS is result from loss of functional WRN, a large
protein with multiple active domains including a DNA helicase anda 3´-5´exonuclease. This protein is implicated in many aspects
of DNA metabolism comprising DNA replication, DNA repair andhomologous recombination. They recently developed a fly model
Fiziologia - Physiology  2011.21.3 (71)

in which the exonuclease component of WRN, DmWRNexo is
expressed at extremely low levels. Such flies show WS-like phenotypes of sensitivity to the topoisomerase poison camptothecin,
and very high rates of DNA recombination. Patients with WS are
essentially null for both the helicase and exonuclease activities
of WRN, and show elevated rates of DNA recombination.
H. Lees et al., UK (Exploring the function of progeroid
WRN gene homologues in Caenorhabditis elegans) discussed
the correlation between the WRN human gene, encoding bothhelicase and exonuclease activities and progeroid WRN gene
homologs in C. Elegans. Their comparative genomic analyses
in C. Elegans suggested that wrn-1 is the closest homologueof the human WRN helicase domain, and mut-7 is the closest
homologue of the human WRN exonuclease domain. Therefore,
this worm model can be used to dissect at the molecular level
the relative impact of the WRN helicase and exonuclease to DNA
damage responses and genome stability.
EXCEPTIONAL LONGEVITY
Exceptional longevity, in animals and humans, represents
an important concept and tool for understanding aging mechanisms. Therefore, this subject was present at each biogerontology meeting:- Mechanisms of exceptional longevity in the world’s longest
lived animal (Iain Ridgway, USA);- Hearty healthspan and lengthened lifespan: the contribution of cytoprotection in the extraordinarily long-lived naked
mole-rat (Kaitlyn Lewis, USA);- Proteome stability and exceptional longevity in bivalve
molluscs (S. Treaster, USA);- Transcriptome analysis of the long lived bivalve Artica
islandica (E. Philipp, DE);
- Extreme life histories: the long-lived ocean quahog Artica
islandica (R. Schaible, DE);- Comparing gene expression between octo/nonagenarians
and offspring to elucidate biological processes involved in aging(A. Abdul Rahman, Malaysia);- Antioxidant profiles do not explain the decreased susceptibility to oxidative stress in Malaysian octo/nonagenarians(Yasmin Anum Mohd Yusof, Malaysia).
Iain Ridgway and co-workers, USA, UK and DE(Mechanisms of exceptional longevity in the world’s longest
lived animal) investigated the uncommon longevity exhibitedby the ocean aquatic molluscs (Phylum Mollusca) of the class
Bivalvia (long-lived Artica islandica, the world’s longest living noncolonial animal, in comparison with the shorter-lived Mercenaria
mercenaria). First, they comparatively investigated biochemical profiles, age related change responses to exposure to theoxidative stressor Tert-Butyl HydroPeroxide (TBHP). Following
TBHP exposure, an association between longevity and resistance to oxidative stress-induced mortality, and also a marked
resistance to oxidative stress-induced cell death was observedin A. islandica compared with M. mercenaria. Second, they havedeveloped new approaches for investigation of proteome stability,
which included assessment of cytosolic carbonyls, total insolu2011.21.3 (71)  Fiziologia - Physiology

ble carbonyls, insoluble carbonyls localized to either nuclear,microsomal or mitochondrial fractions, and protein structural
stability in the face of unfolding stress. The pattern of proteome
homeostasis and stability are different between long-lived andshort-lived species.
In addition, very promising research performed by E.
Philipp, R. Schaible et al., German team from Kiel and Rostock, DE (Extreme life histories: the long-lived ocean quahog Artica islandica and Transcriptome analysis of the long lived bivalve
Artica islandica), identified extreme differences in lifespan among
Artica islandica populations: short-lived Baltic Sea population,with max. lifespan < 50yrs., and extremely long-lived Icelandic
population, with max. lifespan of > 400 yrs.
Kaitlyn Lewis, USA (Hearty healthspan and lengthened
lifespan: the contribution of cytoprotection in the extraordinarily
long-lived naked mole-rat) described investigations on naked
mole rat (Heterocephalus glaber, Rüppel, 1842), a burrowing
rodent native to parts of East Africa, which holds the record
for the longest living rodent, up to 32 yrs. (maximum lifespan).
There is a positive correlation between the transcription factor
Nrf2 (nuclear factor-erythroid 2-releated factor-2) signalling activity and species longevity. Upregulation of the Nrf2- signalling
pathway may be a conserved mechanism contributing to hearty
healthspan and lengthened lifespan. It is important to mention
that Nrf2 regulates the transcription of over 200 cytoprotective
genes with key roles in protection against aging, toxins, inflammation, neurodegeneration and cancer, and is also implicatedin aging and longevity.
NUTRITION IN AGING
Nutrition, nutrigenomics, calorie restriction mimetics
also pointed out the importance in ontogenesis of food normality
and physiological good lifestyles:- Maternal diet affects the aging trajectory (S. Barnes,
UK);- The influence of early nutrition on splenic aging (C. Heppolette, UK);
- Does atypical methionine metabolism contribute to longevity? (Holly Brown Borg, USA);- Depletion of mTOR and mLST8 uncouples longevity fromrapamycin-induced changes in glucose homeostasis (Dudley
W. Lamming, UK);
- Is rapamycin an anti-aging drug? (Arlan Richardson,
USA);- Caloric restriction mimetics: state of flux (Don Ingram,
USA).
John Mathers, UK (Epigenomics: understanding how nutrition modulates the aging process) presented two separate approaches to investigate nutrition mechanisms influence and thebiological processes responsible for the senescence phenotype.
For the discovery of genes the expression of which is epigenetically regulated and which are susceptible to modification bydietary factors he used a combination of genome-wide analysis
of gene expression and of genome-wide methylation of CpG
islands and gene promoters. Moreover, he studied nutritional
15

modulation of brain aging, focused on DNA mechanisms in amouse model exposed to nutritional insults in early life.
Susan Ozanne and collab., UK (Maternal diet, epigenetics and programming of adult health) demonstrated within theframework of ”early life programming” concept how maternal dietcan mediate its effects on subsequent age-associated diseases.
Their findings pointed out that expression of HNF4 alpha, a key
developmental transcription factor previously implicated in therisk of diabetes, was reduced in pancreatic islets following suboptimal nutrition in early life through epigenetic mechanisms.
S. Barnes et al., UK (Maternal diet affects the aging trajectory. A microarray approach)emphasized that maternal diet modulates age-associatedchanges in renal gene expression, that may contribute to its
effects on kidney function and ultimately lifespan.
C. Heppolette and co-workers, UK (The influence ofearly nutrition on splenic aging) through their research argue
the Developmental Origin of Health and Disease Hypothesis,
which proposes that environmental factors such as nutrition
during early life influence long-term health. The spleen, the major
secondary lymph organ and site of peripheral antigen recognition, was investigated. They used the low protein mouse modelof nutritionally-induced foetal growth restriction and postnatal
catch-up growth (recuperated) which results in reduced lifespan,and demonstrated that low birth weight and catch-up growthleads to accelerated aging of the spleen.
Arlan Richardson and collab., USA (Is rapamycin ananti-aging drug?) brought out the effective anti-aging effectsof rapamycin, an FDA approved drug for human use for tissuetransplant therapy and treating certain types of cancer. Using
two transgenic mouse models of Alzheimer disease (AD), they
showed that feeding mice rapamycin at a level that increased
lifespan rescued cognitive deficits and ameliorated Aβ and tau
pathology, by autophagy. In ApoE-/- mice fed a high fat diet, feeding rapamycin attenuated the development and progression ofatherosclerotic lesions. In addition, feeding rapamycin prevented
the development of colon cancer in a transgenic model (ApcMin/+)of adenomatous polyposis.
Don Ingram, USA (Caloric restriction mimetics: state offlux) emphasized the importance of Calorie Restriction (CR) for
anti-aging and health-promoting benefits, practically the mostrobust means to retard aging in numerous species. But, despite
its strong beneficial effects in humans, it is difficult to apply andto be accepted by individuals, due to their nutritional habits andwrong lifestyles. Therefore, the CR mimetics (CRM) has been
introduced to obtain the same positive effects, without reducing
food intake. Several candidate CRM compounds have been
proposed (sirtuin activator, resveratrol, metformin, rapamycin),but only the latter has produced consistent prolongevity effects
in mice with normal diet.
CONTINUITY OF THE IABG CONGRESSESThe International Association of Biomedical Gerontology(IABG) was founded by Denham Harman in 1985. We mustmention the importance of previous IABG congresses, where
16

Prof. Denham Harman (originator of the free radical theory of
aging and disease) was very active, as communicator, session
chairman, organizer, and editor (4, 12).
In addition, the 10th Congress of IABG, held on September
19-23, 2003 in Cambridge, United Kingdom (7), was the hallmark
in the conceptual evolution and development of bio-gerontology,
aging control and prolongevity. The meeting papers were published as volume, Editor Aubrey D. N. J. de Grey, Strategies
for Engineered Negligible Senescence - SENS. Why Genuine
Control of Aging May Be Foreseeable, Annals of the New York
Academy of Sciences, vol. 1019, New York Academy of Sciences, New York, NY, June 2004 (2).
Practically, the next IABG congresses - Aarhus, DK, 2005
(6); Spetses Island, GR, 2007; Quebec, CA, 2009 (11); and this in
Brighton, UK, 2011 (3) - had the same topics and directions, but
up-to-date and with relevance to new projects, search groups andconsortia of aging investigation and rejuvenation research.
As previous scientific meetings, these two congresses
from Brighton 2011 were models for integration of aging
sciences: from genomics, proteomics, transcriptomics tocytomics and epigenomics, and from fundamental studies totranslational medicine and clinical applications.
REFERENCES

1. Bowman C, Dubbey R, Ford P et al: Standards of medical care forolder people. Expectations and recommendation. London, UK, British
Geriatric Society, 1997.
2. de Grey ADNJ (ed): Strategies for Engineered Negligible Senescence- SENS. Why Genuine Control of Aging May Be Foreseeable. Annalsof the New York Academy of Sciences, vol. 1019. New York, NY, NewYork Academy of Sciences, 2004.
3. Faragher R: The Science of Ageing. Global progress. Brighton, UK,
University of Brighton, 2011.
4. Harman D, Holliday R, Meydani M (eds): Towards Prolongation of
the Healthy Life Span. Practical Approaches to Intervention. Annals of
the New York Academy of Sciences, vol. 854. New York, NY, New York
Academy of Sciences, 1998.
5. Phillips J (ed): British gerontology and geriatrics: experience andinnovation. London, UK, British Geriatric Society, 1997.
6. Rattan SIS, Kristensen P, Clark BFC (eds): Undestanding andModulating Aging. Annals of the New York Academy of Sciences, vol.
1067. Boston, MA, Blackwell 2006.
7. Riga D: SENS acquires SENSe: present and future anti-aging strategies, 10th Congress IABG - International Association of Biomedical
Gerontology, Queens’ College, Cambridge, UK, September 19-23, 2003.
Journal of Anti-Aging Medicine 2003; 6(3): 231-236.
8. Riga D, Riga S, Ardelean A, Pribac G, Motoc D, Schneider F: Brainlipopigments - link between aging and Alzheimer disease, 61st Annual
Scientific Meeting of the British Society for Research on Ageing - BSRA
& 14th Congress of the International Association of Biomedical Gerontology - IABG, Brighton, UK, 11th - 14 July 2011.
9. Riga S, Riga D, Ghinescu M, Mihăilescu A, Motoc D, Geacăr S:
Health-longevity pyramid in the anti-aging global progress. 61st Annual
Scientific Meeting of the British Society for Research on Ageing - BSRA
& 14th Congress of the International Association of Biomedical Gerontology - IABG, Brighton, UK, 11th - 14 July 2011.
10. Riga D, Riga S: The science of aging: global progress, Rejuvenation
Research 2011; 14(5): in press.

Fiziologia - Physiology  2011.21.3 (71)

11. Tanguay RM (ed): Aging, Cancer, and Age-Related Diseases.
Annals of the New York Academy of Sciences, vol. 1197. Boston, MA,
Blackwell, 2010.
12. Zs.-Nagy I, Harman D, Kitani K (eds): Pharmacology of Aging

Processes. Methods of
13. Assessment and Potential Interventions. Annals of the New York
Academy of Sciences, vol. 717. New York, NY, New York Academy of
Sciences, 1994.

BIOGERONTOLOGIA ASTAZI. PARTEA I. A 61-A CONFERINŢĂ ŞTIINŢIFICĂANUALĂ A SOCIETĂŢII ENGLEZE DE CERCETARE A ÎMBĂTRÂNIRII BSRA & AL 14-LEA CONGRES AL ASOCIAŢIEI INTERNAŢIONALE DEGERONTOLOGIE BIOMEDICALĂ - IABG, BRIGHTON, ANGLIA, 11 - 14
IULIE 2011
REZUMAT
Prezentarea, analiza şi sinteza celei de a 61a Sesiune Ştiinţifică Anuală a Societăţii Britanice pentru Cercetarea Îmbătrânirii - BRSA
şi celui de al 14lea Congres al Asociaţiei Internaţionale de Gerontologie Biomedicală - IABG reprezintă înscrierea stadiului prezent
cât, mai ales, a direcţiilor viitoare de a transforma umanitatea în curs de îmbătrânire cu dizabilităţi, în fiinţe umane longevive cu osănătate optimă, competitivă şi active fizic şi psihic perioade ontogenetice mult mai lungi. Concentrarea pe anumite direcţii de cercetare devine esenţială pentru găsirea şi introducerea mai rapidă a soluţiilor profilactice, terapeutice şi de recuperare în senescenţa
umană şi trensformarea ei în longevitate umană.
Cuvinte cheie: A 61a Sesiune Ştiinţifică Anuală a Societăţii Britanice pentru Cercetarea Îmbătrânirii - BRSA; Al 14lea Congres
al Asociaţiei Internaţionale de Gerontologie Biomedicală - IABG; Ştiinţa îmbătrânirii; Biogerontologie; Stadiul prezent; Direcţii
viitoare.

2011.21.3 (71)  Fiziologia - Physiology

17

EVALUATION OF SKIN PARAMETERS CHANGES INCHEMICAL AND PHOTOCHEMICAL INITIATED TUMORSON SKH1 MICESORINA CIURLEA1, MARIA-FLORINA BOJIN2, ERZEBET CSANYI3, DANIELA IONESCU1,FLORIN BORCAN4, ATENA GALUSCAN5, CRISTINA A. DEHELEAN1*
1
University of Medicine and Pharmacy “Victor Babes” Timisoara, Faculty of Pharmacy, Toxicology Dept., 2nd Eftimie Murgu Sq., Timisoara-300041, Romania
2
University of Medicine and Pharmacy “Victor Babes” Timisoara, Faculty of Medicine, Physiology Dept., 2nd Eftimie Murgu Sq., Timisoara-300041, Romania
3
University of Szeged, Faculty of Pharmacy, Pharmaceutical Technology Dept. Szeged, Hungary
4
University of Medicine and Pharmacy “Victor Babes” Timisoara, Faculty of Medicine, Biochemistry Dept., 2nd Eftimie Murgu Sq.,
Timisoara-300041, Romania
5
University of Medicine and Pharmacy “Victor Babes” Timisoara, Faculty of Dentistry, 2nd Eftimie Murgu Sq., Timisoara-300041, Romania

ABSTRACTUltraviolet B (UVB) or medium wave irradiation, with a wavelength in the range 315-280 nm and a energy per photon between 3.944.43 eV, regulates ultraviolet-responsive genes, including matrix metalloproteinases. Moreover, it causes connective tissue damage
and the skin to become wrinkled and aged. The aim of this study was to investigate the evolution of the mouse skin papillomas initiated by 7,12-dimethylbenzanthracene (DMBA) and promoted by 12-O-decanoylphorbol-13-acetate (TPA), which progressed more
rapidly to carcinoma on mice exposed to UVB. The biological effects of these two tumors (chemically and photochemically initiated)were evaluated for twelve weeks on the SKH1 hairless mice skin by using of non-invasive techniques (tewameter, skin-pH-meter,melanin and erythema measurements). It was found that UVB irradiation affects very much the parameters which were monitored
and which characterizing the skin condition.
Keywords: tumor, DMBA, TPA, UVB, SKH1 mouse, non-invasive technique

INTRODUCTION
Skin cancer, including basal cell carcinoma (BCC) andsquamous cell carcinoma (SCC) are the most common malignant
neoplasms in humans (1,2). It has been estimated that casesof BCC and SCC are diagnosed each year and are increasing
dramatically. Also this is equivalent with the incidence of malignancies in all other organs combined (3,4).
Chronic exposure to ultraviolet (UV) radiation is responsible
for about 90% of human skin cancers and is a cancer promotor(5). Solar UV radiation is conventionally divided into UVA (320400 nm), UVB (280-320 nm) and UVC (200-280 nm). The UV
light that reaches the earth is composed of about 90% UVA and
10% UVB (6). Both UVA and UVB are being studied for their skincancer-causing potential, but currently UVB is thought to be themost important etiologic factor and UVB is the most frequently
used photocarcinogen in animal studies (7, 8)
Hairless mice are valuable for experimental carcinogenesis
and more specific skin carcinogenesis studies. Carcinogens
as initiators or promoters, chemopreventive agents, and chemotherapeutic compounds are readily applied to unperturbed
hairless skin (9). SKH1 mice are highly susceptible to UVRinduced skin cancer (10) this susceptibility may be a function of
the hairless gene itself, as C3H hairless mice. They are more
susceptible to UVR carcinogenesis than haired mice even it is thesame strain (11). A classification scheme for UVR-induced skintumors in SKH1 mice accurately discriminates multiple stages
in the carcinogenic stages of evolution (12).
There are some reports that suggest that 12-O-tetradecanoylphorbol-13-acetate (TPA) promotes 7,12-dimethylbenz[a]anthracene (DMBA)-initiated mouse skin carcinogenesis, which
is closely related to the inflammatory responses and also tocarcinoma.
In the present study we examined the tumor-promoting
activity of UVR, DMBA and TPA-induced acute inflammation in a
multi-stage mouse skin carcinogenesis model, an ideal system tostudy a number of biochemical alterations in the different stagesof chemical carcinogenesis (13).
MATERIALS AND METHODS
Animals
SKH1 mice of eight weeks were purchased from Charles
River (Sulzfeld, Germany), female, 8 weeks age. The work protocol followed all NIAH - National Institute of Animal Health rules:
animals were maintained during the experiment in standard
conditions offered by the Biobase of University of Medicine andPharmacy Timisoara: 12 hours light-dark cycle, food and water

Received June 14th 2011. Accepted August 15th 2011. Address for correspondence: Cristina-Adriana DEHELEAN, Department of Toxicology, Faculty of Pharmacy,
University of Medicine and Pharmacy “Victor Babes” Timisoara, 2nd, E. Murgu Sq. 300041 Timisoara, RO, E-mail: cadehelean@umft.ro18

Fiziologia - Physiology  2011.21.3 (71)ad libidum, temperature 24 oC, humidity above 55%. 11 mice
were taken into study and were divided in three groups (three
for blank and four for each tumor model). It was obtained thepermission from the Ethical Commission on Animal Experimentsof the University of Medicine and Pharmacy Timisoara.
Chemical treatment
7,12-Dimethylbenz(a)anthracene (DMBA) and 12-Otetradecanoylphorbol-13-acetate (TPA) were purchased from
Sigma (Germany). Mice were topically treated once in the firstexperimental week with DMBA solution (25 nmol in 200 µl acetone) and in the following weeks the DMBA was replaced by
TPA solution, twice weekly (6.8 nmol in 200 µl acetone)(14).
UVB exposure
For UVB exposure, cages were placed in an automaticallytime-switched irradiation setup. For UVB exposure, cages wereplaced in an automatically time-switched irradiation setup. In theexperiment, VL-6.M/6W (312 nm wavelength and 680 µW/cm2
intensity at 15 cm) tubes (Vilber Lourmat, France) were used.
Under the lamps the minimal erythema dose (MED) of hairless
SKH-1 mice, was ≈300 J/m2. The irradiation was twice weekly, 5
minutes/day, 2 times/week, hairless SKH-1 mice were exposed
to a total dose arround 200 J/m2 UVB radiation.
Characterization of skin parameters
Each skin parameter determination was performed before
any application or UVB exposure. All the measurements on themice skin were carried out with a Multiprobe Adapter System(MPA5) from Courage&Khazaka Electronics, Germany, equippedwith a Tewameter® TM300 probe for measurements of transepidermal waterloss (TEWL), a Skin-pH-meter® PH905 probe anda Mexameter® MX18 probe for determinations of melanin anderythema.

Fig.1. The mascroscopic images of SKH1 mice on week 12 of experiment A.
application of DMBA/TPA and B. Application of DMBA, TPA and UVB radiation

The non-invasive methods can offer quantitative data andthe image of pathology evolution in experimental models (Fig
2, 3, 4, 5).

Statistics
Data are presented as mean + SEM and were analysedusing one-way ANOVA and Tukey’s post hoc analysis.
RESULTSIn vivo models like mouse or rats are important and highly
adapted methods for studying cancer development and treatment surveillance and easy to reproduce (14, 15). The models
that included chemical initiators and promoters like polycyclicaromatic hydrocarbures and phorbol esters + other factors likeUVB are frequent use for studying skin carcinoma and melanoma
development. The two models of skin tumor initiation, a single application of the chemical initiator mutagen 7,12-dimethylbenz[a]anthracene (DMBA), followed by repeated applications of apro-inflammatory phorbol ester 12-O-tetradecanoylphorbol
13-acetate (TPA) is an easy adaptable one. It can be applied
on SKH1 mice also with skin tumor development (16, 17). The
results are also easy observable (Fig 1).

2011.21.3 (71)  Fiziologia - Physiology

Fig. 2. The TEWL evaluation for UVB group comparing to group treated withcarcinogens (DMBA/TPA) and blank group(data are expressed as mean ± SEM, p is UVB vs. DMBA/TPA)The TEWL measurements indicate a very large increaseof transepidermal waterloss after the first week of applications.
It is well-known that values of the transepidermal water loss
under 10 g/h/m2 are characteristic of a skin in good condition(see the blank group from fig 2) and over 25 g/h/m2 correspondto a skin in bad and very bad condition. So, the application of
DMBA in this week changed a lot the skin condition. The loss inthe coming weeks is not so pronounced and the skin condition
begins to recover.
19

The evolutions of erythema average values show the very
toxicological effect of DMBA and the aggressive effect of UVB
on the mice skin. The UVB group record a continued increaseduring the twelve weeks.

Fig. 3. The skin-pH evaluation for UVB group comparing to group treated withcarcinogens (DMBA/TPA) and blank group (data are expressed as mean ±SEM, p is UVB vs. DMBA/TPA)

The pH evolutions are chaotics. The big values for standard
errors of the mean and p are sufficient grounds to consider thatthese tests on the SKH1 mice skin does not affect the theirskin-pH values (Fig 3).

Fig. 4. The melanin skin content evaluation for UVB group comparing togroup treated with carcinogens (DMBA/TPA) and blank group (data areexpressed as mean ± SEM, p is UVB vs. DMBA/TPA)

The melanin measurements indicate a very large decrease,for the groups with carcinogens and UVB exposure, in the second
week which is an extra argument to the TEWL modification due
to the toxicological DMBA effect.

Fig. 5. The erythema evaluation for UVB group comparing to group treated withcarcinogens (DMBA/TPA) and blank group (data are expressed as mean ±SEM, p is UVB vs. DMBA/TPA)

20

DISCUSSIONS
Over-exposure to UV from the sun can cause sunburn,skin damage and in the final steps skin cancer. UVB can act
in mouse skin models as a complete carcinogen, meaning thatUVB can function as an initiator as well as a promoter (18, 19)
and for this reason the combination of chemical carcinogens withUVB determine intense skin damages. Tumors and skin damages are easily identified and observable (Fig1). Each mouse
develops multiple independent skin tumors, which may exhibit
significant differences in rate of development and aggressiveness. Skin tumors progress from area of epithelial hyperplasia
to papillomas and ultimately into squamous cell and spindle cell
carcinomas and the evolution is in weeks (9). In first 12 weeks
lesions, skin physiology and inflammation seems to be a generalcharacteristic.
The SKH1 hairless mouse develops lesions resembling
UVR-induced tumors and it is widely considered to be the mostsuitable mouse model for studies of UVR carcinogenesis (20).
Our study confirms this general aspect.
The first responses after carcinogens application include thedevelopment of edema, hyperplasia, induction of pro-inflammatory cytokine interleukin-1a, enhanced release of reactive oxygen
species, induction of epidermal ornithine decarboxylase (ODC)
and over-expression of cyclooxygenase-2 (COX-2) and inducible
nitric oxide synthase (iNOS) proteins and are related to initial
step of a carcinogenic process (21, 22). Increased expressionof COX-2 and/or iNOS was observed in many different types oftumors and transformed cells (23, 24). Besides its ability to act
as an initiator of carcinogenesis, nitric oxide is also involvedin the promotional stage of tumorigenesis (25) and in tumor
progression by regulating angiogenesis (26).
Tumors are graded as papillomas microinvasive squamouscell carcinomas or fully invasive SCCs (12). This aspect isobservable by transepidermal waterloos and also by erythemal
evolution. The other parameters confirm the evolution of skinpathology.
These measurements indicated important changes from thefirst weeks of experiments and also the most increased process
after 10 weeks. The erythemal values and changes in skin watercontent are obvious more increased in case of the UVB adding.
Melanin is not very much influenced in case of SKH1 mice on
the first weeks of experiment.
CONCLUSIONS
Experimental models on sensitive specific animals areapplicable in cancer research and also in models like skincarcinoma. Increasing the number of carcinogenic factors,
such as chemicals and UV radiation, leads to a faster evolution of pathology and to important skin damages. The detailed
observation of these aspects can be performed by application
Fiziologia - Physiology  2011.21.3 (71)

of non-invasive methods that establish important changes inskin physiology during pathology evolution. After a few weeks
of exposure to initiators and promoters mouse skin suffer
important changes and the major damages are in a started
process.
REFERENCES
1. Dehelean CA. Toxicologie. Notiuni generale de toxicologie.
Ed. Mirton, Timisoara, 2008: 7-16.
2. Mantena SK, Sharma SD, Katiyar SK. Berberine inhibits
growth, induces G1 arrest and apoptosis in human epidermoid
carcinoma A431 cells by regulating Cdki-Cdk-cyclin cascade,disruption of mitochondrial membrane potential and cleavage of
caspase 3 and PARP. Carcinogenesis 2006; 27:2018-2027.
3. Naylor MF, Boyd A, Smith DW, et al. High sunprotection
factor sunscreens in the uppression of actinic neoplasia. Arch.
Dermatol. 1995; 131:170-175.
4. Yusuf N, Irby C, Katiyar SK, et al. Photoprotective effects ofgreen tea polyphenols. Photodermatol. Photoimmunol. Photomed. 2007; 23: 48-56.
5. Koh HK. Preventive strategies and research for ultraviolet-associated cancer. Environ. HealthPerspect. 1995;103: 255-257.
6. Learn DB, Beasley DG, Giddens LD, et al. Minimum doses ofultraviolet radiation required to induce murine skin edema andimmunosuppression are different and depend on the ultraviolet
emission spectrum of the source. Photochem. Photobiol.1995;
62: 1066-1075.
7. De Gruijl FR, Forbes PD. UV-induced skin cancer in a hairlessmouse model. BioEssays 1995; 17: 651-660.
8. Forbes PD. Relevance of animal models of photocarcinogenesis to humans. Photochem.Photobiol. 1996; 63:
357-362.
9. Slaga TJ, Budunova IV, Gimenez-Conti IB, Aldaz CM. The
mouse skin carcinogenesis model. J Investig Dermatol Symp
Proc 1996;1:151-6.
10. Berton TR, Fischer SM, Conti CJ, Locniskar MF. Comparisonof ultraviolet lightinduced skin carcinogenesis and ornithine
decarboxylase activity in sencar and hairless SKH-1 mice fed
a constant level of dietary lipid varying in corn and coconut oil.
Nutr Cancer 1996;26:353-63.
11. Davies RE, Forbes PD. Effect of UV radiation on survival of
non-haired mice. Photochem Photobiol 1986;43:267-74.
12. Thomas-Ahner JM, Wulff BC, Tober KL, Kusewitt DF, Riggenbach JA, Oberyszyn TM. Gender differences in UVB-induced
skin carcinogenesis, inflammation, and DNA damage. Cancer
Res 2007;67:3468-74.
13. Slaga TJ, et al. Studies on the mechanisms involved in
multistage carcinogenesis in mouse skin. J. Cell. Biochem.
1982;18:99-119.

2011.21.3 (71)  Fiziologia - Physiology

14. Hara-Chikuma M, Verkman AS. Prevention of skin tumorigenesis and impairment of epidermal cell proliferation by targeted
aquaporin-3 gene disruption. Molecular and Cellular Biology
2008;28(1): 326-332.
15. Ciurlea S, Dehelean CA, Ionescu D, Berko S, Csanyi
Erszebet, Hadaruga N, Srinivas Ganta, Mansoor M. Amiji. A
comparative study regarding melanoma activity of Betulinic acidon topical ointment vs. Systemic nanoemulsion delivery systems.
Journal of Agroalimentary Processes and Technologies, 2010,
16(4), 420-426.
16. Kundoor V, Zhang, X, Bommareddy A, et al. Chemopreventive effects of sarcotriol on ultraviolet B-induced skin tumor development in SKH-1 hairless mice. Mar. Drugs 2007; 5:197-207.
17. Dwivedi C, Valluri HB, Guan X, Agarwal R. Chemopreventive effects of santalol on ultraviolet B radiation-induced skintumor development in SKH-1 hairless mice. Carcinogenesis
2006; 27:1917-1922.
18. Willis I, Menter JM, Whyte HJ. The rapid induction of cancers
in the hairless mouse utilizing the principle of photoaugmentation.
J. Invest. Dematol. 1981; 76: 404-408.
19. Bowden GT. Prevention of non-melanoma skin cancer bytargeting ultraviolet-B-light signaling.Nat. Rev. Cancer 2004;
4:23-35.
20. de Gruijl FR, Forbes PD. UV-induced skin cancer in a hairlessmouse model. Bioessays 1995;17:651–60.
21. Murakami A, et al. Inhibitory effect of citrus nobiletin on phorbol ester-induced skin inflammation, oxidative stress, and tumor
promotion in mice. Cancer Res. 2000; 60: 5059-5066.
22. Seo HJ, et al. Inhibitory effects of the standardized extract(DA-9601) of Artemisia asiatica Nakai on phorbol ester-induced
ornithine decarboxylase activity, papilloma formation, cyclooxygenase-2 expression, inducible nitric oxide synthase expression
and nuclear transcription factor kappa B activation in mouse skin.
Int. J. Cancer 2002; 100: 456-462.
23. Franco L, Doria D, Bertazzoni E, et al. Increased expression of inducible nitric oxide synthase and cyclooxygenase-2
in pancreatic cancer. Prostaglandins and Other Lipid Mediat
2004; 73: 51-58.
24. Li W, Xu R, Jiang L, et al. Expression of cyclooxygenase-2
and inducible nitric oxide synthase correlates with tumor angiogenesis in endometrial carcinoma. Med. Oncol. 2005;22:63-70.
25. Mordan LJ, Burnett TS, Zhang LX, et al. Inhibitors of endogenous nitrogen oxide formation block the promotion of neoplastictransformation in C3H10T1/2 fibroblasts. Carcinogenesis 1993;
14: 1555-1559.
26. Chin K, Kurashima Y, Ogura T, et al. Induction of vascular endothelial growth factor by nitric oxide in human glioblastoma andhepatocellular carcinoma cells. Oncogene 1997; 15: 437-442.

21

EVALUAREA MODIFICĂRILOR PARAMETRILOR TEGUMENTARI IN TUMORILE
INIŢIATE CHIMIC ŞI FOTOCHIMIC LA LINIA MURINĂ SKH1
REZUMAT
Radiaţia medie sau ultravioletă B (UVB) cu o lungime de undă cuprinsă între 315-280 nm şi o energie per foton între 3.94-4.43 eV
reglează răspunsul genetic la ultraviolete incluzând matricea metaloproteinazelor. Mai mult decât atât, acestea provoacă leziuni aleţesutului conjunctiv iar pielea devine ridată şi îmbătrânită. Scopul acestui studiu a fost de a investiga evoluţia papiloamelor pe pieleaşoarecelui la care s-a realizat iniţierea cu 7,12-dimethylbenzanthracene (DMBA) şi promovarea cu 12-O-decanoylphorbol-13-acetat(TPA), papiloame care au progresat mai rapid spre carcinom la şoarecii expuşi la UVB. Efectele biologice ale acestor doua tipuri
de tumori (iniţiate chimic şi fotochimic) au fost evaluate timp de douăsprezece săptămâni pe şoareci SKH1 fără piele prin utilizarea
unor tehnici non-invazive (tewameter, pH-metru tegumentar, măsurători privind melanina şi eritemul). S-a constatat că iradierea cu
UVB afectează foarte mult parametrii care monitorizează şi caracterizează starea/condiţia pielii.
Cuvinte cheie: tumora, DMBA, TPA, UVB, şoareci SKH1, tehnică non-invazivă

22

Fiziologia - Physiology  2011.21.3 (71)ON THE ENTERIC NERVOUS SYSTEM’S ROLE IN PHYSIOLOGICAL ACTIVITIES OF THE GASTROINTESTINAL TRACT
ALINA SISU1, CODRUTA PETRESCU1, CARMEN TATU2, LOREDANA STANA1, R.F.TATU3, A.
MOTOC1
1
Department of Anatomy and Embryology, University of Medicine and Pharmacy „Victor Babes” Timisoara
2
Department of Functional Sciences, University of Medicine and Pharmacy „Victor Babes” Timisoara
3
Department of Orthopaedics, Traumatology, Urology and Imagistics, University of Medicine and Pharmacy „Victor Babes” Timisoara

ABSTRACT
Intestinal smooth muscle is served by two types of intrinsic motor neurons, enteric excitatory neurons and inhibitors enteric neurons.
Chemical mediators of the enteric nervous system were initially considered to be just as neurotransmitters acetylcholine and serotonin. Subsequent studies have added to the list purines such as ATP (adenosine triphosphate), peptides and amino acids such as
GABA and VIP. More recently, NO was considered a neurotransmitter in the enteric nervous system, as in the CNS. The study was
performed on 12 foetuses. There were made microscopic samples using trichrome Masson and Nissl stains. Submucous plexus,
located in the submucosa, between circular muscle layer and mucosa muscle, is best developed in the small intestine where it plays
an important role in controlling secretion. Keywords: neurotransmitters, foetuses, chemical mediators.

INTRODUCTIONEnteric nervous system is unusual in keeping its functions
after central connections are removed. A variety of neuronalmessengers (classical transmitters, gaseous messengers, aminoacids and neuropeptide transmitters) are able to mediate andmodulate gastric functions. Gastric nerves are either intrinsic
to the gastric wall (have cellular corpus in intramural ganglia
and thus belong to the enteric nervous system) or go to thestomach from the outside (extrinsic nerves), originating in the
medulla oblongata, sympathetic ganglia and sensory ganglia.
The endocrine and paracrine cells lining the stomach’s mucosae(gastrin-secreting andsomatostatin cells), exocrine cells (parietal,
mucous), smooth muscle cells and stromal cells are coordinatedby neuronal messengers.
MATERIAL AND METHOD
The study was performed on 12 foetuses. There weremade microscopic samples using trichrome Masson and Nissl
stains.
RESULTS AND DISSCUTIONS
Although the enteric nervous system can operate independently of the central nervous system, the latter has an importantrole in coordinating the various functions of the enteric nervous
system. Enteric nervous system is well connected to central
autonomous network of CNS through both, motor and sensory
sympathetic and parasympathetic pathways systems (Table I).

Table I: ENS role in physiological activities

Motility
Tonic inhibition
Segmental contraction

suppression of spontaneous miogenic contractions by VIP or nitroxidergic neurons
mixture of lumen’substances of SI; suppression of inhibitors neurons may be involved in
specific intestinal areas

series of cyclical contractions, each 1-2 hours,
whose frequency is determined by the slow
Spreading the electrical wave generated by interstitial cells of
motor complex Cajal; Cleansing interprandial and is initiatedby motilin, somatostatin, prokinetic agents andopioids (Soudah et al., 1991) (1)sequence of esophageal contractions central
mediated and coordinated by nervous stimuPrimary oesophalants and inhibitors of the mienteric plexus andgeal peristaltic
started by swallowing (Goyal and Paterson,
1989, Yamato et al., 1992b) (2,3)
motor sequence consisting of proximal contraction and relaxation distal to an intestinal
distension stimulus; peristaltic movement is
Peristaltic by local
achieved by anal continuous displacement ofreflexthe stimulus, being activated by the primary
intrinsic afferent neurons containing 5HT (serotonin) (Furness and Bornstein, 1995)(4)Received May 15th 2011. Accepted July 20th 2011. Address for correspondence: Assistant Professor Alina Sisu, MD, Department of Anatomy andEmbryology, University of Medicine and Pharmacy “V. Babes” Timisoara, phone: 0724361671, e-mail: alinasisu@gmail.com; alinasisu@umft.ro2011.21.3 (71)  Fiziologia - Physiology

23

Huge peristaltic
contraction

contraction of high amplitude, duration,
representing a sustained peristaltic reflex,
induced by cholera toxin and found in patientswith spastic bowel syndrome; the electricalcorrelation is represented by the migration ofaction potential (Sarna, 1991, Mathias et al.,1976) (5,6)

involved in retrograde propulsion as in vomitRetrograd periing; neural circuit is unknown.
staltic contraction
Sphincterial
function
Secretion
Gastric acidsecretion

Pancreatic enzyme secretion
Intestinal secretion

In submucosa are seen in dense groups of 3-10 nervous cells,
evenly spaced on the circumference corresponding to Meissner- Henle submucos ganglia (black arrows). There are also seen
rarified group of nervous cells but better represented numerically(blue border). Muscle motor neurons are located fully in themienteric plexus, and most mucous neurons – in the submucous
plexus (Furness and Costa, 1987) (15). Normal motility andsecretory functions are dependent on the anatomical, chemicaland functional integrity of this network that forms the entericnervous system plexuses (Figure 1).

sphincters have intrinsic tonus by miogenic
origin; relaxation is produced by or VIP and nitroxidergic neurons, contraction is provided bycholinergic neurons (Yamato et al., 1992a) (7)stimulated by vagal activation of parietal cells
and cells containing histamine or gastrin;
inhibited by vagal activation of cells containing
somatostatin
stimulated by activation colecistokinin inducedof vagal primary afferent neurons and vagus
vagal reflexes (Li and Owyang, 1994) (8)peristaltic activated, propagated complex motor
and giant peristaltic contractions

efferent sympathetic neurons produce ATPmediated vasoconstriction; submucous intrinsic
secretory neurons and axon reflexes cause
Microcirculation vasodilation (Lundgren et al., 1989, Vannerand Surprenant, 1991, Evans and Surprenant,1992) (9,10,11)
splanhnic primary afferent neurons innervate
mucosal mast cells that degranulating and
Immune and inreleasing inflammatory mediators and (Krumins
flammatory reet al., 1992, Stead, 1992, Shanahan, 1994)
sponses(12,13,14)

Fig. 1. Cross section through stomach; 20 cm human foetus. Trichrome Masson stain , x200. well-configured submucous gangllia (21 weeks)

Gastric wall layers contain, in humans, a rich cholinergic
innervation: in the mucosa acetylcholinesterase positive fibers,around gastric glands and between the muscle fibers; in the
submucosa are interconnected fibers and dispersed ganglioniccells, with strong positive reaction. The muscular layer contains
the highest number of cholinergic fibers, grouped in bundles, andAuerbach plexus has the most intense positive reaction at thecellular level. Subserous layer contains a plexus having a positive
reaction, and the external layer of vessels is rich in cholinergic
fibers (Onicescu et al., 1975) (16) (Figure 2).

Sympathetic fibers arrived in the gut are adrenergic – postganglia fibers having the cellular corpus prevertebral ganglia.
They have at least four targets in the gut:• secretomotori neurons containing VIP;
• cholinergic presynaptic nervous endings;• submucous blood vessels;• gastrointestinal sphincters.
Cross section of the gastric body wall shows a magnification
factor of 200 parietal layers from outside to inside (left> right)
serous (with longitudinal vessels with erythrocytes in lumen
caliber, on the cross section), muscle layers (longitudinal andcircular, separated by a connective space and occupied byneuronal cell groups), submucosa, muscle and mucosae muscle.
24

Fig.2. Oblique section through stomach; 18 cm human foetus; Nissl stain,
x630. morphologic polimorphism on the gastric submucous plexus (19 weeks)

Fiziologia - Physiology  2011.21.3 (71)Details of submucous glands on 5 months foetus show
submucous dense units (A) composed of variable individual
cell-sized, which correlated directly with the amount of individual
cytoplasm. These cells are emitting extra fine, short extensions,and long extensions that make up the periphery of a polygonal
plexus. Internal area of the submucous layer (B) shows a deficitof morphological grouping on nerve cells that appear dispersed
and with variable size, generally small (primitive neurons),
confounding by fibroblasts and collagen fibers of submucoasae.
Rudimentary plexuses are most obvious near the nervous cells(Figure 3).

Fig. 3. Cross section through stomach; 20 cm human foetus; Trichrome Masson stain, x1000. submucous ganglia:
A: external layer; B: internal layer.

CONCLUSIONS
1. Vagal primary afferent neurons distributed in smoothmuscle layer are sensitive to mechanical distension of the bowel,
and lead information about certain motor intestinal physiologicalactivities (Sengupta and Gebhart, 1994) (17).
2. Some vagal primary afferent neurons are sensitive toconcentrations of glucose lights, amino acids or long chain fatty
acids (Mei, 1985) (18), and others respond to a wide variety ofmechanical and chemical stimuli.
3. Enteric glial cells produce interleukins and express MHC
antigens / class II in response to cytokine stimulation (Ruhl andCollins, 1995) (19). They suggest an important role of enteric glial
cells in modulating the inflammatory response to the gut.
4. Postnatal the central nervous system neuronal cellnumber will increase (rat, Altman and Das, 1966) (20) but the
number of neurons decreases in visceral nervous system (Gabella, 1971) (21).
5. Although up to eight morphological types of neurons wereidentified in the enteric nervous system, there are two main types(Furness and Costa, 1987) (22,23,24,25):• Type I neurons, having many processes in the form of cluband one long thin process;• Type II neurons, are multipolar and have many long andsmooth processes.
6. Also it is considered that Cajal’s interstitial cells acting
like pacemakers, imposing the rate of intestinal contractions by
2011.21.3 (71)  Fiziologia - Physiology

affecting the electrical slow waves (Rich et al., 2002) (26); thisassumption is supported by;• ICC localization in smooth muscle layers where slow
waves are generated;• spontaneous activity of pacemaker type that has isolated
ICC in the colon;• disappearance of interruptions or slow electrical waves
when ICC are removed or decoupled from intestinal smoothmuscle (27,28,29).
REFERENCES
1. Soudah HC, Hasler WL, Owyang C. Effect of octreotide on
intestinal motility and bacterial overgrowth in scleroderma. N
Engl J Med 1991; 325: 1461-1467.
2. Goyal RK, Paterson WG. Esophageal motility. In: Schultz SG,
ed. Handbook of physiology: a critical, comprehensive presentation of physiological knowledge and concepts. Sect. 6. The
gastrointestinal system. Vol. 1. Motility and circulation. Bethesda,
Md.: American Physiological Society 1989: 865-908.
3. Yamato S, Saha JK, Goyal RK. Role of nitric oxide in lower
esophageal sphincter relaxation to swallowing. Life Sci 1992;
50:1263-1272.
4. Furness J B, Bornstein J C, Murphy R, Pompolo S. Roles of
peptides in transmission in the enteric nervous system. Trends
Neurosci 1992; 15: 66-71.
5. Sarna SK. Physiology and pathophysiology of colonic motor
activity. Dig Dis Sci 1991; 36:998-1018.
6. Mathias JR, Carlson GM, DiMarino AJ, Bertiger G, Morton
HE, Cohen S. Intestinal myoelectric activity in response to live
Vibrio cholerae and cholera enterotoxin. J Clin Invest 1976;
58:91-96.
7. Yamato S, Spechler SJ, Goyal RK. Role of nitric oxide inesophageal peristalsis in the opossum. Gastroenterology 1992;
103:197-204.
8. Li Y, Owyang C. Endogenous cholecystokinin stimulates
pancreatic enzyme secretion via vagal afferent pathway in rats.
Gastroenterology 1994; 107:525-531.
9. Lundgren O, Svanvik J, Jivegard L. Enteric nervous system.
I. Physiology and pathophysiology of the intestinal tract. Dig Dis
Sci 1989; 34: 264-283.
10. Vanner S, Surprenant A. Cholinergic and noncholinergic
submucosal neurons dilate arterioles in guinea pig colon. Am J
Physiol 1991; 261:G136-G144.
11. Evans RJ, Surprenant A. Vasoconstriction of guinea-pig
submucosal arterioles following sympathetic nerve stimulation is mediated by the release of ATP. Br J Pharmacol 1992;
106:242-249.
12. Krumins SA, Broomfield CA. Evidence of NK1 and NK2tachykinin receptors and their involvement in histamine releasein a murine mast cell line. Neuropeptides 1992; 21:65-72.
13. Stead RH. Nerve remodelling during intestinal inflammation.
Ann N Y Acad Sci 1992; 664:443-455.
14. Shanahan F. The intestinal immune system. În: Johnson LR,
ed. Physiology of the gastrointestinal tract. 3rd ed. New York:
Raven Press, 1994:643-84.
25

15. Furness JB, Costa M. The enteric nervous system. New York:
Churchill Livingstone, 1987.
16. Onicescu D, Cuida I, Popescu M. Cholinergic innervation of
the human stomach – Morphol Embryol (Bucur). 1975;21(2):937.
17. Sengupta JN, Gebhart GF. Gastrointestinal afferent fibers andsensation. In: Johnson LR, ed. Physiology of the gastrointestinal
tract. 3rd ed. New York: Raven Press, 1994: 483-520.
18. Mei N. Intestinal chemosensitivity. Physiol Rev 1985;
65:211-237.
19. Rühl A, Collins SM. Enteroglial cells (EGC) are an integral
part of the neuroimmune axis in the enteric nervous system(ENS). Gastroenterology 1995;108: Suppl:A680-A680.
20. Altman J, Das GD. Autoradiographic histological studies
of postnatal neurogenesis. I. A longitudinal investigation of
the kinetics, migration and transformation of cells incoporating
tritiated thymidine in neonate rats, with special reference topostnatal neurogenesis in some brain regions. J Comp Neurol
1996; 126: 337-390.
21. Gabella G. Neuron size and number in the myenteric plexusof the newborn and adult rat. J Anat 1971; 109: 81-95
22. Furness JB. Types of neurons in the enteric nervous system.
J. Autonom Nervous System 2000; 81, 87- 96.

23. Furness JB, Costa M. Types of nerves in the enteric nervous
system. Neurosci 1980; 5: 1-20.
24. Furness JB, Bornstein JC. The enteric nervous system
and its extrinsic connections. In: Yamada T, ed. Textbook ofgastroenterology. 2nd ed. Vol. 1. Philadelphia: J.B. Lippincott,
1995:2-24.
25. Furness JB, Kunze WA, Clere N. Nutrient tasting andsignaling mechanisms in the gut. II. The intestine as a sensory
organ: neural, endocrine and immuno responses. Am J Physiol
1999;277:G922-8.
26. Rich A, Hanani M, Ermilov LG, Malaysz J, Belzer V, Szurszewski JH, Farrugia G. Physiological study of interstitial cells of
Cajal identified by vital staining. Neurogastroenterology andMotility 2002; 14:2-189.
27. Gabella G. Structure of muscles and nerves in the gastrointestinal tract. In: Johnson LR, ed. Physiology of the gastrointestinal tract. 3rd ed. New York: Raven Press, 1994:751-94.
28. Gabella G. Fine structure of the myenteric plexus in theguinea-pig ileum. J Anat 1972; 111: 69-97.
29. Costa M, Brookes SJ. The enteric nervous system. Am J
Gastroenterol 1994; 89:Suppl:S129-S137.

EFECTELE HIPOXIEI ASUPRA PROLIFERARII SI APOPTOZEI CULTURILORHUVEC IN VITROREZUMATMuşchiul neted intestinal este deservit de două tipuri de neuroni motori intrinseci, neuroni enterici excitatori şi neuroni enterici
inhibitori, care în general proiectează oral şi anal, respectiv. Mediatorii chimici ai sistemului nervos enteric au fost consideraţi iniţial afi doar neurotransmiţători precum acetilcolina şi serotonina. Studiile ulterioare au adăugat purinele pe listă, precum ATP, aminoacizii
precum GABA şi peptidele precum VIP. Mai recent, NO a fost considerat drept neurotransmiţător în sistemul nervos enteric, precumîn SNC. Studiul a fost realizat pe 12 feti. Au fost realizate preparate microscopice utilizand tehnicile de colorare tricrom Masson si
Nissl. Plexul submucos, localizat în submucoasă, între stratul muscular circular şi musculara mucoasei este cel mai bine dezvoltat
în intestinul subţire unde joacă un rol important în controlul secreţiei.
Cuvinte cheie: neurotransmitatori, feti, mediatori chimici.

26

Fiziologia - Physiology  2011.21.3 (71)AMBULATORY SYSTOLIC AUGMENTATION INDEX-A NEW
ARTERIAL RIGIDITY MARKER FOR CARDIOVASCULAR RISKSTRATIFICATIONIONESCU V.A, GEORGIANA IONESCU, DANA BÂIBÂŢÃ, SILVIA MANCAŞ, ŞT. I.
DRĂGULESCUUniversity of Medicine and Pharmacy “Victor Babeş” Timişoara

ABSTRACT
Ambulatory augmentation systolic index (AASI) derived from the 24h blood pressure monitoring is one of the new markers of arterial stiffness. Our study aims to analyze possible associations between AASI and global cardiovascular risk. Means and method:
we included 130 asymptomatic subjects that were evaluated through several perspectives: global cardiovascular risk - SCORE risk
charts; cardio-metabolic risk: traditional cardiovascular risk factors, metabolic syndrome defined by the IDF criteria; hemodynamic
profile: 24h blood pressure monitoring with the derived data - pulsed pressure (PP); arterial wall profile: the carotid-femoral artery
pulsed wave velocity (C-F PWV by the Complior method) and AASI, derived from 24h blood pressure monitoring. Results: we foundstatistically signifficant correlations between AASI and 24h-PP: r = 0.46, p < 0.001; we also obtained signifficant differences of mean
AASI in subjects with and without metabolic syndrome, t(128) = -2.63, p<0.01. Mean AASI variations according to the number ofcardiometabolic risk factors were not signifficant. We found a strong corelation between C-F PWV and SCORE: r=0.404, p < 0.001,
but not between AASI and SCORE. There also was no significant correlation between AASI and C-F PWV. Conclusions: we appreciated the vascular remodelling of asymptomatic patients at risk by using non invasive methods of evaluating arterial stiffness: C-F
PWV and AASI. AASI behaviour related to the hemodynamic variable of the cardivascular risk, but not to the cardiometabolic profile.
As there are no corelations found between C-F PWV and AASI, the two markers can not substitute one another; AASI identifies thehemodynamic risk, whereas PWV gives us extra data regarding the contribuiton of metabolic syndrome on the vascular remodelling.
In light of this data, it becomes clear that SCORE charts are no longer sufficient for risk stratification, and other parameters should
also be taken into account.
Key words: risk stratification, asymptomatics at risk, arterial stiffness, ambulatory augmentation systolic index, carotid-femoral
pulsed wave velocity, pulsed pressure.

THEORETICAL ASPECTS
Over the past 5-6 years, the systolic augmentation index
derived from 24 h monitoring of blood pressure begun to beinterpreted as a new marker of arterial rigidity. The relationshipbetween the index and arterial stiffness is based on following
observations:First of all, Li Y et al (1), after analyzing 166 normotensive
Asiatic subjects, proved a direct and statistically significant
relationship between systolic augmentation index and carotidfemoral pulsed pressure.
Second of all, it seems that this index is somewhat related
to the degree of nocturnal reduction in blood pressure. Schillaci
et al (2) has proved that the systolic augmentation index in
hypertensive patients included in the non-dipper category wassignificantly higher than in patients with dipper hypertensive
profile: 0.44 ± 0.20 vs. 0.29 ± 0.15; p<0.001.
The significant association between the systolic augmentation index and arterial rigidity might be explained by the connection with the same variables: age, arterial distension pressure,heart rate and nocturnal reduction in blood pressure. If this iscorrect, could the degree of nocturnal drop in blood pressure be
regarded as a marker of arterial rigidity? In Schillaci’s study (2),the non-dipper hypertensive patients have had higher velocitiesof the carotid-femoral pulsed wave than the dippers, but the difference was not statistically significant after age adjusting: 9.5± 2 m/s vs. 9.2 ± 2 m/s, p=0.30.
Third of all, the variance of arterial rigidity depending on thearterial distension pressure is a non-linear curve. This means
that, both arterial rigidity and pulsed wave velocity comport great
variations throughout the cardiac cycle. Therefore, the use of thesystolic/diastolic regression slope in expressing the ambulatory
systolic augmentation index becomes an independent parameter:
non-dependent on the pressure. In order to sustain this theory,
Gavish B et al (3) have proved that this systole-diastolic slope
quantitatively expresses the relative increase of arterial stiffness
throughout the systole and it is independent of the pressure
within the vessel.
Is it possible then, that the two markers of arterial rigidity
express different aspects? It seems so: the pulsed wave velocity
estimates the arterial rigidity at one point on the pressure-volume
curve, whereas the ambulatory systolic augmentation index
estimates intrinsic arterial rigidity.
Given the scientific context, we have designed this transversal study regarding the practical use of the systolic augmentationindex for the characterization of the arterial function in high risk
asymptomatic patients.

Received August 16th 2011. Accepted September 15th 2011. Address for correspondence: Ionescu Vlad Alexandru, MD, “Victor Babes” University of Medicine andPharmacy Timisoara, Eftimie Murgu Square No. 2A, 300041, phone/fax: +40256220479, phone: +4 0722221802, e-mail: i_andru@yahoo.com

2011.21.3 (71)  Fiziologia - Physiology

27

Hypothesis of the study: In order to be able to evaluate theimplication of a new parameter in cardiovascular risk stratificationof asymptomatic patients, we issued the hypothesis that ambulatory systolic augmentation index might reflect arterial stiffness,a condition associated with cardiovascular events.
The main objective of the study was to analyze possible
associations between ambulatory systolic augmentation index
and global cardiovascular risk, with serious implication in the
primary prophylaxis of cardiovascular disease.
MEANS AND METHOD
We included in our study high risk asymptomatic subjects,
assessed according to the cardiovascular risk factors. The
cases were stocked in the archive of the Preventive Cardiology
and Cardiovascular Rehabilitation Clinic of the Cardiovascular
Disease Institute, Timisoara. The including and excluding criteria
are presented in Table I. All patients signed the informed consent
and the study was approved by The Ethic’s Committee at theCardiovascular Disease Institute, Timisoara.
Table I: Inclusion and exclusion criteria
Inclusion criteria

Exclusion criteria• Asymptomatic subject acc. to SEC • Signifficant valvular heart disease• Synus rythm• Arrhythmias• Preserved ejection fraction• Perripheral obstructive arterial disease• Normotensive/ dipper hypertensive profile • Congenital heart disease

Data collection: we used the clinical charts for data collection; the analyzed parameters included demographic data,
anthropometric data, anamnesis, complete clinical examinationand paraclinical evaluation.
The evaluation protocol was designed in order to define theglobal cardiovascular risk of the asymptomatic subject and tocharacterize their arterial function.
A. The risk for fatal cardiovascular event within the next 10
years: we used the SCORE chart- the high risk European population chart. The quantified data were: age, sex, systolic bloodpressure (mmHg), smoking, total cholesterol (mg/dl). According
to the SCORE risk, the asymptomatic subjects were classified
as: SCORE<5% and SCORE>5%.
B. Cardiometabolic risk: for the cardiometabolic risk stratification we included traditional cardiovascular risk factor assessment, as well as the basal characteristics of the group: age, sex,
waist circumference, body mass index (kg/m2).
Arterial hypertension was defined according to The 2007
Guidelines for Management of Arterial Hypertension (4): SBP≥
140 mmHg and/or DBP≥ 90 mmHg or antihypertensive treatment; this corresponds to a 24 h mean arterial blood pressure> 120/80 mmHg.
Smoking status: number of cigarettes smoked daily. Nonsmoking: at least 4 weeks from smoking cessation.
Type II diabetes mellitus was defined according to a fasting
plasma glucose ≥ 126 mg/dl or andt-diabetic therapy.
28

Hypercholesterolemia, according to the National Cholesterol
Education Program-Adult Treatment Panel III 2001 (5) as defined for total cholesterol values higher than 200mg/dl. Because
target level of total cholesterol in primary prophylaxis is below
190mg/dl, all values above this were considered a major risk
factor. Furthermore, hypolipemiant therapy was also considereddiagnostic criteria for hypercholesterolemia.
The metabolic profile: pro-atherogenic dyslipidemia wasdefined by NCEP-ATP III (5). We took into account the recommendations of The Guidelines on Cardiovascular Disease
Prevention (6) in order to characterize, in the view of primary prophylaxis, the “healthy” condition: LDLc<115mg/dl, HDLc>40mg/
dl, TG<150mg/dl. Metabolic syndrome was defined according to
the IDF 2005 criteria (Table II).
Table II: Metabolic syndrome: definition criteria
CRITERIA
NCEP-ATP III
IDF
Abdominal>88 (F),>102 (M)>80 (F),>94 (M)*circumference(cm)Arterial blood pressure ≥ 130/80≥ 130/80(mmHg)
Fasting plasma glucose > 110> 100(mg/dl)Triglyceride (mg/dl)
>150
>150
HDL-c (mg/dl)<50(F),<40(M)<50(F),<40(M)* mandatory criteria for IDF classiffication
Note: if IMC ≥ 30 kg/m2, waist measurement is no longer
necessary according to the IDF

C. Hemodynamic risk: measurement of arterial blood pressure was done at the brachial artery site, bilateral, in respect tothe recommendations of The European Society of Hypertension
Guideline (7): sitting position, after 5 minutes of rest. We used
the media from 3 consecutive measurements.
Ambulatory monitoring of the arterial blood pressure: thedata regarding the 24h blood pressure profile was collectedusing the BTL-08 ABPM program automatic readings of theblood pressure were performed every 15 minutes during the day(7am-11pm) and every 30 minutes during the night (11pm-7am).
The monitoring was repeated if it experienced more than 30%
artefacts. Throughout the entire monitoring period, all medication
was withdrawn. From the ambulatory blood pressure monitoring
protocol, we used the following parameters: 24h SBP, 24h PP,
24h MABP. We considered as cut off value for nocturnal reductionof blood pressure 10%. Those with more than 10% nocturnal
reduction in blood pressure were considered as dippers, thus
becoming possible candidates for our study.
D. Arterial function was appreciated through the following
measurements:- Carotid-femoral pulsed wave velocity: C-F PWV-Ambulatory systolic augmentation index: AASIFor determining the C-F PWV we used the Complior SP
System. PWV represents the pulsed wave generated by eachventricular ejection; it is then propagated along the arterial tree,according to the Moens-Korteweg equation, PWV² = (E x h)/(2r xrho, where E=the elasticity module of the arterial wall, h=arterial
Fiziologia - Physiology  2011.21.3 (71)

stiffness, r=blood density, rho=the internal diameter of the femoral
artery. In normal physiologic conditions, the h/r ratio is constant;
therefore, PWV is directly related with the elasticity module of
the arterial wall.
C-F PWV measurement was done by simultaneous registration of the 2 wave forms: carotid and femoral wave forms. C-F
PWV was calculated as the ratio between the distance traveled
between 2 selected points and the time difference between the
2 wave forms: c-f PWV(m/sec) = d/ Δ t.
Note: the C-F PWV measurement was obtained after at
least 24 hours cessation of alcohol and coffee intake; the last
pharmacological treatment was administrated 12h prior to theexam.
The AASI was calculated using the linear regression, asa ponderate function of successive evaluation of arterial bloodpressure within a 24 hours interval (1). For each subject, weanalyzed the regression slope of the diastolic blood pressure
in relation to the systolic blood pressure. AASI was calculated
after the formula (8):AASI = 1 – regresion slope of DBP/SBPThe more rigid the arterial wall, the more tendency of theregression slope towards 0, and of AASI towards 1 (9).
STATISTICAL ANALYSISThe statistical analysis performed used the EPIINFO
program, 6.0, 2001 version; data was analyzed using the SPSS
program, the 2010 18th version; it was electronically stocked using
the 97-03 version of The Microsoft Excel program and the PASW
program, 18th version 2010. The cut off value for the statisticalsignificance was p<0.05; a very strong statistical significancy
was considered for a p<0.01.
RESULTSWe included 214 consecutive patients evaluated in thePreventive Cardiology and Cardiac Rehabilitation Clinic of the
Cardiovascular Disease Institute Timisoara between May 2009May 2011: 109 females, 105 males, and 1.03 to 1 ratio. From this214 group of patients, 130 were asymptomatic subjects (meanage 58.62±8.39 years old, 36.15 males) at cardiovascular risk,
according to the criteria mentioned above, who signed the approval for the study. 66.15% of them had a high SCORE ≥ 5%.
The prevalence of traditional cardiovascular risk factors was:
 Essential hypertension 88.5 % (n=115 subjects)
 Type II diabetes mellitus 23.1 % (n= 30 subjects)
 Hypercholesterolemia 60.0 % (n= 78 subjects)
 Smokers 46.2 % (n= 60 subjects)
 Family history ofpremature CV disease 56.2 % (n= 78 subjects)
The metabolic profile of the group consisted of these mean
(mg/dl): Total cholesterol 223.86 ± 55.16 mg/dl
 Triglycerides 184.62 ± 94.61 mg/dl
 LDLc 139.73 ± 50.59 mg/dl
 HDLc 46.48 ± 13.39 mg/dl
2011.21.3 (71)  Fiziologia - Physiology

 Fasting plasma glucose 107.42 ± 25.34 mg/dl
Metabolic syndrome defined according to the IDF criteria
was recorded in 52.3% of the subjects. According to the protocol,
the entire lot had 24h blood pressure monitoring. Mean SBP was
151.48± 12.16 mmHg. Mean PP was 63.85 ± 10.72 mmHg.
The relationship between AASI and the hemodynamic
profile: mean AASI was 0.58 ± 0.12, with a maximum of 0.92 anda minimum of 0.36. Individual value had a Gaussian-like distribution. We found statistically significant correlations between AASI
and 24h-PP: r = 0.46, p < 0.001; the association size between
the 2 variables was 21.9% (Figure1).

Fig.1. Correlation graphic between AASI and PP

Fig.2. AASI and metabolic syndrome

The relation between the cardiometabolic risk scoreand AASI
a. The relation between AASI and metabolic syndrome: weobtained significant differences of the mean AASI in subjects
with and without metabolic syndrome, t(128)=-2.63, p<0.01(Fig. 2).
b. The analysis of AASI behavior and the cardiometabolic
29

SCORE risk and the behavior of arterial stiffness markers: PWN and AASI. Mean C-F PWV was 10.63± 2.12 m/sec.
We found a strong significant correlation between c-f PWV and
SCORE: r=0404, p < 0.001 (Figure 3).

risk factors: SCORE≥ 5%, LDLc ≥ 115 mg/dl, TG ≥ 150 mg/dl,
type II diabetes mellitus. The highest frequency of the cases
had 2 (33.8%), respectively 3 of these criteria (30.8%). MeanAASI ranged between 0.45 and 0.59 (Table III). We applied
the ANOVA test to see if there are any statistically significant
differences of the mean AASI according to the number of present
criteria; we found none, p>0.05 (Table IV).
Study of the AASI variation according to the global highcardiovascular risk: SCORE> 5%, LDL > 115 mg/dl, TG > 150
mg/dl and type II diabetes mellitus proved no statistical significant
difference (p>0.05). The linear regression model used to provethat that these 4 variables are predictors of the AASI evolution
failed (Table V).

25
y = 0,7394x - 1,3161
R2 = 0,1629

EUROSCORE

20
15
10
5
0
0

Standard
Mean deviation Minimum Maximum

0

4

.45

.08 2

.36

.56

1

21

.60

.12

.41

.78

2

44

.59

.12

.39

.91

3

40

.59

.12

.39

.92

4

21

.57

.10

.43

.80

Total

130 .58

.12

.36

.92

4

6

8

10

12

14

16

PWV_CF

Table III. Mean AASI according to the number of cardiometabolic risk factors

No. ofcriteria No

2

Fig. 3. C-F PWV and SCORE risk: corelation graphic

Mean AASI was 0.58 ± 0.12. The association between AASI
and the SCORE risk was not statistically significant: 0.59 ± 0.14
in subjects with SCORE<5% and 0.58 ± 0.11 in subjects withSCORE≥ 5%, p>0.05 (Figure 4).

Table IV. Comparing mean AASI according to the numberof cardiometabolic risk factors

Criteria
AASI

>= 2
<2

Criteria
>= 3
<3

AASI

No

Mean

Standard
deviation

P

105

.58

.12

>0.05

25

.58

.12

No

Mean

Standard
deviation

P

61

.58

.11

>0.05

69

.59

.12

Fig.4. Relation between AASI and SCORE risk

The correlation between the 2 markers of arterial rigidity
AASI and c-f PWV was not significant, p>0.05.
DISCUSSION
In 1914, Mac William and Melvin proved that the reductionin arterial elasticity influenced DBP behavior and the relation with

Table V. Liniar regression model for AASI

Model Summary

Model R
1
2

.141a
.135b

Adjusted R
R Square Square
.020
-.011
.018
-.005

Change Statistics
Std. Error of the R Square
Estimate
ChangeF Change df1.1224664
.020
.634
4
.1220759
-.002
.198
1

df2 Sig. F Change
125 .639
125 .657

3
4
5

.124c
.099d
.000e

.015
.010
.000

.1217770.1216463.1217698

126
127
128

.000
.002
.000

b. Predictors: (Constant), FR_DZ, TG150, EUROSCORE5
c. Predictors: (Constant), FR_DZ, TG150
d. Predictors: (Constant), FR_DZ

30

-.003
-.006
-.010

.379
.725
1.262

1
1
1

.539
.396
a. Predictors: (Constant), FR_DZ,
TG150, riscSCORE5, LDL115

Fiziologia - Physiology  2011.21.3 (71)

SBP (10). Considering this, it means that AASI refect the distinct
relation between DBP and SBP, during the night.
However, the connection between AASI and the mechanicalproperties of the arterial wall remains controversial.
Our study clearly proved the association relation between
AASI and 24h PP, among a heterogenous group of asymptomatic
subjects with cardiovascular risk, including both normotensive
as well as hypertensive subjects.
Therefore, from the 2 methods of evaluating arterial stiffness, which one is the most accurate in determining the risk andprognosis of these subjects? Dublin Outcome Study (8) shades
light onto some of the aspects:- AASI was a better predictor of cardiovascular mortality
than PP in normotensive patients compared to hypertensive
patients, but,
- PP was a prognostic factor among hypertensive patients.
Elizabeth S. Muxfeldt (11) has studied the arterial wall
rigidity in a group of 391 hypertensive patients (resistant hypertension), using 3 methods: PP, AASI and PWV (the Compliormethod). PP pressure was best correlated with arterial wall
stiffness in these patients.
Recent data talks about the correlation between C-F PWV,AASI and PP, thus justifying the evaluation of arterial function
even among adolescents (12). The best evidence was obtainedfor 24h PP, which was correlated with: arterial stiffness measured
by PWV, target organ damage (left ventricular mass) and thepresence of hypertension.
It is important to underline that some aspects coming from
3 cohort prospective studies regarding the significant association
between AASI and fatal/nonfatal cardiovascular events (8,13,14):
the relation between AASI and cardiovascular mortality is of aU-curve type; furthermore, AASI was proved to have a predictive
value for cerebrovascular event in normotensive patients.
Another important issue worth discussing is that thispredictive value of AASI for cerebrovascular events should not
be regarded only through the hemodynamic perspective, butalso through the cardiometabolic one. Leoncini G et al (15), by
analyzing the association between AASI and metabolic profile
in 156 hypertensive non-diabetic patients, has reached the following conclusions:- the prevalence of high AASI was significantly higher among
patients with metabolic syndrome (p=0.02)- the presence of metabolic syndrome doubles the risk for
AASI augmentation.
Our study has found significantly higher values for AASI
among the asymptomatics with metabolic syndrome definedby the IDF criteria, but not in asymptomatics with metabolic
syndrome defined by the ATP III criteria. The mandatory criterion for the IDF is the waist circumference or a BMI≥30 kg/m2.
In our study, we obtained a positive and significant correlation
between PWV, as a marker of arterial stiffness, and BMI. Ourmodel though, designed in order to analyze the AASI variancefrom the cardiometabolic perspective, was not valid. This is why
we could use AASI and 24 h PP in our algorhythm to evaluate
2011.21.3 (71)  Fiziologia - Physiology

arterial function in asymptomatic subjects with hemodynamic
risk: associated cardiometabolic risk does not influence in any
was AASI behavior.
We want to underline that in our study the statistical procedure has discarded age from the equation analysis of arterial
wall rigidity. This because mean age was 58.62 years old, an age
when there is a normal remodelling of the arterial wall.
The PROOF study (16) was centred on the influence of age
and blood pressure on cardiovascular events related to increased
arterial stiffness; it only enrolled subjects over 65 years of age.
At this age, the conclusions were that AASI varied significantly
according to traditional risk factors (hypertension, obesity, hypercholesterolemia and diabetes); there was a negative correlation
between AASI and HDLc; there were no correlations foundbetween AASI and C reactive protein. However, the multivariate
analysis showed that only hypertension and diabetes mellitus
contributed to AASI augmentation in subjects older than 65. The
2 years follow up showed that, even in normotensive patients,
AASI augment with age. This process is not so obvious among
hypertensive patients under optimal management of 24h bloodpressure.
Study limitations: the design of our observational crosssectional study gives us the results of an analysis at one point
in time, as any such study. Obviously, longitudinal follow-up
over several years of the interactions between different indicesof arterial stiffness would bring more information in regards ofwhat the arterial wall dysfunction really means.
CONCLUSIONS
1. We appreciated the vascular remodelling of asymptomaticpatients at risk by use of 2 non invasive methods of evaluatingarterial stiffness: c-f PWV and AASI.
2. AASI behavior related with the hemodynamic variable(24h PP) of the cardiovascular risk.
3. AASI variance was not influenced by the cardiometabolicrisk profile.
4. As there are no correlations between c-f PWV and AASI,
the two markers can not substitute one another.
5. AASI identifies the hemodynamic risk, whereas PWV
gives us extra data regarding the contribution of metabolic syndrome at the vascular remodelling of the asymptomatic patientwith traditional cardiovascular risk factors.
6. Cardiovascular risk stratification based only on the
SCORE charts is insufficient
7. The use of biomarkers for arterial stiffness allows not
only a more accurate characterization of the risk profile in asymptomatic subjects, but also a more specific approach of therisk management.
REFERENCES

1. Li Y, et al. Ambulatory arterial stiffness index derived from 24 hour ambulatory blood pressure monitoring. Hypertension, 2006; 47: 359-64.
2. Schilacci G, et al. Ambulatory arterial stiffness index is not a specific marker of reduced arterial compliance. Hypertension, 2007; 49:
986-91.

31

3. Gavish B, et al. What Arterial Property Is Expressed By the
Systolic-Diastolic Pressures Relationship? A Model View: Pp.14.41.
Journal of Hypertension. 28:e260, June 2010.
4. Mancia G, et al. The 2007 Guidelines for the Management of Arterial Hypertension. Europ.Heart J, 2007; 28: 1462-536.
5. Scott GM, et al. Implications of Recent Clinical Trials for the
National Cholesterol Education Program Adult Treatment Panel
Guidelines. Circulation, 2004; 110: 227-39.
6. Ian Grahan, et al. The Guidelines on Cardiovascular Disease
Prevention. European J. Cardiovascular Prevention and Rehabilitation, 2007; 4 (2).
7. Mancia G, et al. Ghidul Societătii Europene de HTA pentru evaluarea si tratamentul HTA. Rev. Română Cardiologie XVIII, 2003; 3:1-42.
8. Dolan E et all- Ambulatory arterial stiffness index as a predictor of
cardiovascular mortality in the Dublin Outcome Study. Hypertension
2006,47,3,365-70
9. Adiyaman A, et al. Determinants of the ambulatory arterial stiffness index in 7604 subjects from 6 populations. Hypertension 2008;

52(10): 38-44.
10. Mac William, et al. Systolic and diastolic blood pressure estimation with special reference to the auditory method. BMJ, 1994; 2:693.
11. Muxfeldt ES, et al. Ambulatory arterial stiffness or pulse pressure? Hypertension Research 2008; 31: 607-13.
12. Stergiou GS, et al. Ambulatory arterial stiffness index, pulse pressure and pulse wave velocty in children and adolescents. Hypertension Research, 2010; 33: 1272-77.
13. Hansen TW, et al. Ambulatory arterial stiffness index predicts
stroke in a general population. J. Hypertension, 2006; 24: 2247-53.
14. Kikuya M, et al. Ambulatory arterial stiffness index and 24h ambulatory pulse pressure as predictors of mortality in Ohasama. Japan.
Stroke, 2007; 38: 1161-66.
15. Leoncini G, et al. Metabolic syndrome and ambulatory arterial
stiffness index in non-diabetic patients with primary hypertension.
Journal of Human Hypertension, 2007; 21: 802-07.
16. Gosse P, et al. Components of arterial stiffness in a population of
65 year-old subjects: PROOF study. J.Hypertens, 2008; 26:1138-46.

INDICELE AMBULATORIU DE AUGMENTARE SISTOLICĂ - UN NOUMARKER DE RIGIDITATE ARTERIALĂ FOLOSIT PENTRU STRATIFICAREA
RISCULUI CARDIOVASCULARREZUMAT

Indicele ambulatoriu de augmentare sistolica (AASI), derivat din monitorizarea Holter 24 de ore a tensiunii arteriale este un nou
marker al rigiditatii arteriale. Studiul nostru are ca scop analiza unor posibile asocieri intre AASI si riscul cardiovascular total. Material
si metoda: am inclus in studiu 130 de subiecti asimptomatici evaluati din mai multe perspective: risc cardiovascular global - riscul
SCORE; riscul cardio-metabolic - factorii de risc cardiovascular traditionali, sindromul metabolic definit dupa criteriile IDF; profilul
hemodinamic: monitorizare Holter 24 de ore a tensiunii arteriale si presiunii pulsate; profilul peretelui arterial: viteza undei pulsate
carotida-femurala (PWV C-F, metoda Complior) si AASI, derivat din monitorizare Holter 24 de ore. Rezultate: am gasit corelatii semnificative statistic intre AASI si PP: r=0,46; p<0,001; de asemenea, diferente semnificative au fost demonstrate si intre AASI la subiectii
cu sindrom metabolic vs AASI la cei fara sindrom metabolic, t(128)=-2.63, p<0.01. Variatiile mediei AASI in functie de numarul defactori de risc cardiometabolic nu au fost semnificative. Am dmonstrat o corelatie puternica intre PWV C-F si riscul SCORE: r=0.404,
p < 0.001, dar nu si intre AASI si SCORE. De asemenea, nici asocierea AASI - PWV nu a fost semnificativa. Concluzii: folosind
metode noninvazive - AASI, PWV C-F, am apreciat afectarea arteriala la asimptomaticii la risc. Comportamentul AASI s-a relationat
cu componenta hemodinamica a riscului cardiovascular, dar nu si cu componenta cardio-metabolica. In plus, deoarece nu exista
corelatii intre AASI si PWV, cei doi trebuie priviti ca doi markeri independenti, care nu se pot substitui unul cu celalalt; AASI defineste
riscul hemodinamic, in timp ce PWV ne ofera informatii cu privire la impactul vascular al componentei metabolice a riscului. Astfel,
devine evidenta concluzia ca riscul SCORE este deja insuficient pentru stratificarea fina a riscului cardiovascular, si ca este nevoie
si de alte metode complementare pentru o mai buna intelegere a riscului global al asimptomaticilor.
Cuvinte cheie: stratificarea riscului, asimptomatic la risc, rigidiatate arteriala, indicele ambulatoriu de augmentare sistolică, velocitatea
undei pulsate carotida-femurala, presiune pulsata.

32

Fiziologia - Physiology  2011.21.3 (71)THE RELATIONSHIP BETWEEN HYPERTENSIVE RETINOPATHY
AND CORONARY ARTERY DISEASEOVIDIU BORUGA, IOANA ZOLOG
Victor Babes University of Medicine and Pharmacy Timisoara, Ophthalmology Clinic Timisoara

ABSTRACT
Aim of study. To assess the relationship between hypertensive retinopathy and coronary artery disease in a population from Timis
County, Romania.
Methods. We conducted an observational study that included 600 patients (382 men and 218 women), aged 40-65 years who were
examined for retinal microvascular changes by retinal photographs within the Ophthalmology Clinic Timisoara between March 2008
and May 2011. We included in our study patients with hypertension on the basis of systolic and diastolic blood pressure measured
in three consecutive visits (more than 140 systolic or/and 90 diastolic mmHg, measure in a rest condition with a mercury sphygmomanometer).
Results. After initial evaluation of blood pressure 360 patients (60%) had first stage hypertension, 180 patients (30%) had second stage
hypertension and 60 patients (10%) had third stage hypertension. No patient had malignant hypertension at initial evaluation.
We found hypertensive retinopathy in 210 patients (35%): focal and general arteriolar narrowing in 108 patients (18%); arteriovenous
nicking in 60 patients (10%); microaneurysms, haemorrhages and exudates (retinopathy) in 42 patients (7%). No patient had discswelling or optic nerve oedema. The presence of any retinopathy sign predicted a three fold higher risk for coronary artery disease(RR 3.71; 95% CI 2.76 to 4.98), with higher risk for patients with retinopathy (RR 4.19; 95% CI 3.41 to 5.15) as compared to AV nicking(RR 2.25; 95% CI 1.69 to 2.97) and arteriolar narrowing (RR 1.23; 95% CI 0.88 to 1.71). The incidence of coronary artery disease
was higher in patients with second and third stage hypertension, in patients with a poor control of blood pressure, despite treatment.
Incidence of hypertensive retinopathy was higher in women, while the incidence of coronary artery disease was higher in men.
Conclusion. Our study showed that evaluation of hypertensive retinopathy is important in hypertensive patients because the presence of any modification (arteriolar narrowing, arteriovenous nicking and retinopathy) is associated with three fold higher risk ofdeveloping coronary artery disease. The incidence of hypertensive retinopathy is higher in women, but men had higher incidence ofcoronary artery disease. Patients with retinopathy (microaneurysms, hemorrhages and/or exudates) are at highest risk. It is therefore
important to undergo adequate control of blood pressure and other risk factor.
Key words: hypertensive retinopathy, coronary artery diseases

INTRODUCTION
Keith, Wagener, and Barker classification has been used
to assess retinal changes associated with hypertension since
1939 (1). The recent guidelines of Joint National Committee
and European Society of Hypertension still recommend routine
ophthalmoscopic examination to detect signs of retinopathy inpeople with hypertension for the purposes of risk stratificationand treatment decisions, although its value is being questioned.
The guidelines emphasize that hypertensive retinopathy, together
with left ventricular hypertrophy and renal impairment may beconsidered an indicator of target organ damage, suggestingthat physicians should consider a more aggressive approach inmanaging these patients (2, 3).
Hypertension has profound effects on the structure andfunction of the eye. First, the retinal, choroidal, and optic nerve
circulations undergo a series of pathophysiological changes in
response to raised blood pressure, resulting in a range of clinical signs referred to as hypertensive retinopathy, hypertensive
choroidopathy, and hypertensive optic neuropathy. Second,
hypertension is an important risk factor for the development ofpotentially blinding vascular diseases of the eye, including retinal
vein and artery occlusion, retinal-arteriolar emboli, and diabetic

retinopathy. Finally, hypertension might be a pathogenic factor for
non-vascular ocular diseases, including two of the leading causesof blindness-glaucoma and age-related macular degeneration.
Hypertensive retinopathy refers to retinal microvascular signs
related to increased blood pressure (4, 5).
Hypertensive retinal vascular signs can be broadly classified
into arteriolar changes (generalized arteriolar narrowing, focal
arteriolar narrowing, arteriovenous nicking and arteriolar wall
opacification) and more advanced retinopathy lesions (microaneurysms, blot and flame-shaped hemorrhages, cotton-wool
spots, hard exudates and optic disk swelling). With the exceptionof disk swelling, these signs can be detected fairly frequently inadult populations, even in persons without a known history of
hypertension (4).
Many studies have reported a strong association between
various hypertensive retinopathy signs and both subclinicaland clinical cerebrovascular disease (6-14) and stroke mortality (15).
We conducted an observational study to asses the relationship between hypertensive retinopathy and coronary arterydisease in a population from Timis County, Romania.

Received 15th August. Accepted 15th September. Address for correspondence: Ovidiu Boruga, MD, Ophthalmology Department, University of Medicine and Pharmacy“Victor Babes” Timisoara, Eftimie Murgu Sq. No. 2A, 300041 Timisoara, RO, E-mail: ileanazolog@yahoo.com

2011.21.3 (71)  Fiziologia - Physiology

33

METHODS
Study population
Our study included 600 patients (382 men and 218 women),
aged 40-65 years who were examined for retinal microvascularchanges by retinal photographs in Ophthalmology Clinic Timisoara between March 2008 and May 2011. We included in ourstudy patients with hypertension on the basis of systolic anddiastolic blood pressure measured at three consecutive visits(more than 140 systolic or/and 90 diastolic mmHg, measure in a
rest condition with a mercury sphygmomanometer).The diagnoseof hypertension was confirmed by a cardiologist.
We excluded from our study patients with history of stroke ortransient ischemic attack; significant peripheral vascular disease(previous surgery or symptoms of intermittent claudication);
aortic aneurysm; diabetes mellitus (fasting glucose > 126 mg/dl
or receiving insulin or oral hypoglycemic treatment); thyroid disease; nephrotic syndrome or patients with prior coronary arterydiseases (history of angina, previous myocardial infarction).
The characteristics of study population are shown in Table
I.
Table I. Baseline characteristics

Age
Systolic blood pressure
Diastolic blood pressureMen, %
Total cholesterol
LDLc
Smokers, %Fasting glucose
Serum creatinine level

Mean ± SD
52±8 years
149±8 mm Hg
96±5 mmHg
382, 64%216±72 mg%
184±64 mg%
420, 70%86±12 mg%
0.94 ±0.16 mg%

The study was approved by local Ethics Committee andinformed consent was obtained from all participants.
Procedures
At the baseline examinations, blood pressure (BP) was
measured three different times, using a single mercury sphygmomanometer with an appropriate cuff size, after participants
had been seated for at least 5 minutes. Systolic and diastolicblood pressures (SBP and DBP) were recorded from the firstand fifth Korotkoff sounds. Hypertensive subjects were divided
into 3 groups: controlled (using medication, normal BP at examination), uncontrolled (using medication, elevated BP at
examination), and untreated (elevated BP at examination, but
not using medication). The complete history of cardiovasculardiseases was taken and an ECG was performed and interpreted
by a cardiologist; complete blood analysis (including fasting
glycemia, serum creatinine, TSH, FT3, FT4) was done before
inclusion in our study. The photographs were read in a masked
manner by 2 trained graders, and the presence of any retinal
changes was recorded.
All participants underwent a detailed ocular examination,
after pupillary dilation, 30° stereoscopic retinal photographs of34

the macula, optic disc, and other retinal fields of both eyes weretaken, using a fundus camera (model FF3; Carl Zeiss Meditech,
Oberkochen, Germany).
We use the term hypertensive retinopathy to describe all
retinal changes included in the Keith, Wagener, and Barker
classification (Table II).
Table II. Keith, Wagener, and Baker classification of hypertensive retinopathy

Grade I
Grade II

Grade III
Grade IVslight or modest narrowing of the retinal
arterioles, with arteriovenous ratio ≥ 1:2
modest to severe narrowing of retinal
arterioles (focal or generalized), witharteriovenous ratio < 1:2 or arteriovenous nicking
bilateral soft exudates or flame-shaped
hemorrhages
bilateral optic nerve oedema

We have documented the following individual lesions aspresent versus absent: generalized and focal arteriolar narrowing, arteriovenous nicking, retinal hemorrhage and exudates,
microaneurysms and disc swelling. Hypertensive retinopathy
was defined to include the presence of any one of the above
lesions.
The presence of coronary artery diseases was definedby history of pectoris angina (with one of the following: specific ischemic changes on ECG, positive effort test, positive
coronarography) and also acute myocardial infarction (typicalchest pain, specific rise and fall of cardiac enzyme and ST
elevation on ECG). The diagnostic of coronary artery disease
was made by a cardiologist from Cardiovascular Disease
Institute Timisoara.
Statistical analysis
We used the MedCalc Software for calculating the relativerisk, confidence interval and prevalence proportion. The results
for continuous variables were given as mean ± SD and for categorical variables as percentage. The limit of P value of statisticalsignificance was considered 0.05.
RESULTSWe included in our study 600 patients (382 men, 218
women), mean age 50 ± 12 years. The mean follow-up period
was 26±12 months.
After initial evaluation of blood pressure 360 patients (60%)
had first stage hypertension, 180 patients (30%) had second
stage hypertension and 60 patients (10%) had third stage
hypertension. No patient had malignant hypertension at initial
evaluation.
We found hypertensive retinopathy in 210 patients (35%):
focal and general arteriolar narrowing in 108 patients (18%);
arteriovenous nicking in 60 patients (10%); microaneurysms,
hemorrhages and exudates (retinopathy) in 42 patients (7%).
No patient had disc swelling or optic nerve oedema. The
characteristic lesions according to the type of hypertension are
described in Table III.
Fiziologia - Physiology  2011.21.3 (71)Table III. Characteristic lesion according to hypertension type

LesionHypertension

Arteriolar
narrowing
72 (12%)

AV nicking

Retinopathy
3 (0.5%)Treated and
10 (1.6%)
controlled
Treated and26 (4.3%)
33 (5.5%)
12 (2%)
uncontrolled
Untreated
10 (1.6%)
17 (2.83%)
27 (4.5%)
Retinopathy: microaneurysms, hemorrhages and/or exudates

During follow-up 150 patients were diagnosed with coronary
artery disease (110 patients with stable angina, 24 patients withunstable angina, and 16 patients with acute ST elevation myocardial infarction). Out of them 100 patients had hypertensive
retinopathy (38 patients retinopathy, 32 patients AV nicking and
30 patients arteriolar narrowing).
The presence of any retinopathy sign predicted a threefold higher risk for coronary artery disease (RR 3.71; 95% CI
2.76 to 4.98), with higher risk for patients with retinopathy (RR
4.19; 95% CI 3.41 to 5.15) as compared to AV nicking (RR 2.25;
95% CI 1.69 to 2.97) and arteriolar narrowing (RR 1.23; 95%
CI 0.88 to 1.71).
The incidence of coronary artery disease was higher in patients with second and third stage hypertension, in patients witha poor control of blood pressure, despite treatment. Incidence ofhypertensive retinopathy was higher in women, but the incidenceof coronary artery disease was higher in men.
We didn’t use in our study correlation with other risk factor
such as smoking or high cholesterol level.
DISCUSSION
Poorly controlled systemic hypertension causes damage tothe retinal microcirculation, so that recognition of hypertensive
retinopathy may be important in cardiovascular risk stratificationof hypertensive patients (16).
Several recent studies have shown that retinal microvascular changes can be reliably documented by retinal photographs.
In general, reproducibility from photographs has been found to
be excellent for well defined retinopathy signs (17-21).
Data from population-based studies indicate that hypertensive retinopathy signs, defined from retinal photographs,
are seen in 3–14% of adult individuals aged ≥40 years (1, 19,
20, 21-26).
In our study we found hypertensive retinopathy in 210
patients (35%): focal and general arteriolar narrowing in 108
patients (18%); arteriovenous nicking in 60 patients (10%);
microaneurysms, hemorrhages and exudates (retinopathy) in
42 patients (7%).
In the Beaver Dam Eye Study they find signs of retinopathyin 11% of subjects, arteriolar narrowing in 19% of subjects,
and arteriovenous nicking in 3% of subjects in the nondiabetic
population with systemic hypertension.
In two other population-based studies, both of which included subjects with diabetes, the relationship of blood pressure
with retinopathy signs, arteriolar narrowing, and arteriovenous
2011.21.3 (71)  Fiziologia - Physiology

nicking was studied. Signs of retinopathy (exudates and flameshaped hemorrhages) were detected with ophthalmoscopy in
0.8% of white males and 2.3% of white females, and arteriovenous nicking was detected in 11.5% of white males and 14.2%
of white females in a population-based study in Evans County,
Georgia (27). In that study, retinopathy was present in 2.3%
of white males and 4.9% of white females, and arteriovenous
nicking was present in 15.5% of white males and 23.2% of whitefemales whose diastolic BP was greater than 100 mm Hg. In a
population-based study of 855 men aged 50 years in Gothenburg,
Sweden, in which both ophthalmoscopy and fundus photographs
were used to detect retinopathy, hemorrhages were found in
0.4% of subjects, arteriolar narrowing in 6.0% of subjects, andarteriovenous nicking in 8.9% of subjects (28).
In our study the presence of hypertensive retinopathy was asdetected using stereoscopic retinal photographs of the macula,
optic disc, and other retinal fields of both eyes and interpretedby two experimented non-ophthalmologists. The incidence ofretinopathy was higher in women, data similar with other study(23). Hypertensive retinopathy was associated with a three foldhigher risk of coronary artery disease. We included in our study
both men and women. Similar data are shown in a study that
included 560 only men at high risk (29). Hypertensive retinopathy
signs have been linked with both subclinical and clinical coronary heart disease and congestive heart failure. For example,
various hypertensive retinopathy signs have been associatedwith ischemic changes on electrocardiogram (30), severityof coronary artery stenosis on angiography (31) and incident
coronary heart disease and myocardial infarction in men (29)and women (32).
Low sensitivity of retinal abnormalities associated withhypertension indicates that hypertensive retinopathy is notcommon in hypertensive people. Less than half of the retinal
changes associated with hypertension cannot be explainedby high blood pressure (low positive predictive value). In boththe Beaver Dam eye study and the Blue Mountains eye study
little difference was found in the presence of hemorrhages andexudates between normotensive and hypertensive people aged
over 65. Various other conditions have been associated with hypertensive retinopathy, such as ethnicity, smoking, intima-media
thickness, carotid plaque score, carotid artery stiffness, serum
cholesterol concentration, diabetes, and body mass index. The
high specificity indicates that hypertensive retinopathy is rarein patients with normal blood pressure. Half the people without
hypertensive retinopathy, however, still have hypertension (lownegative predictive value).
A number of important limitations of this study should be
mentioned. The people in our study were white Caucasians aged
between 40 and 65 years. We didn’t consider other importantrisk factors for coronary artery disease such as smoking, highcholesterol level, body mass index. Thus, caution should betaken when extending these findings to other segments of the
population -older and younger age groups, other ethnic groups,
etc.
35CONCLUSION
Our study showed that evaluation of hypertensive retinopathy is important in hypertensive patients because the presenceof any modification (arteriolar narrowing, arteriovenous nicking
and retinopathy) is associated with three fold higher risk of developing coronary artery disease. The incidence of hypertensive
retinopathy is higher in women, but still the men had a higher
incidence of coronary artery disease. Patients with retinopathy(microaneurysms, hemorrhages and/or exudates) are at highest
risk and it is important to have adequate control of blood pressureand other risk factors. Other studies are necessary to assess
the correlation with cardiovascular risk factors, hypertensive
retinopathy and cardiovascular mortality.
REFERENCES

1. Klein R, Klein BE, Moss SE, Wang Q. Hypertension and retinopathy,
arteriolar narrowing, and arteriovenous nicking in a population. Arch
Ophthalmol 1994; 112: 92-8.
2. Chobanian AV, Bakris GL, Black HR, Cushman WC, Green LA,
Izzo JL Jr. et al. The seventh report of the joint national committee on
prevention, detection, evaluation, and treatment of high blood pressure:
the JNC 7 report. JAMA 2003; 289: 2560-72.
3. Cifkova R, Erdine S, Fagard R, Farsang C, Heagerty AM, Kiowski W
et al Practice guidelines for primary care physicians: 2003 ESH/ESC
hypertension guidelines. J Hypertens 2003; 21: 1779-86.
4. Wong TY, Klein R, Klein BE, Tielsch JM, Hubbard L, Nieto FJ.Retinal
microvascular abnormalities and their relationship with hypertension,
cardiovascular disease, and mortality. Surv Ophthalmol 2001; 46:
59-80.
5. Tso MO, Jampol LM. Pathophysiology of hypertensive retinopathy.
Ophthalmol 1982; 89: 1132–45.
6. Keith NM, Wagener HP, Barker NW. Some different types of essential hypertension: their course and prognosis. Am J Med Sci
1939;197:332-43.
7. Ralph RA. Prediction of cardiovascular status from arteriovenous
crossing phenomena. Ann Ophthalmol 1974;6: 323-610.
8. Svardsudd K, Wedel H, Aurell E, et al. Hypertensive eye groundchanges: prevalence, relation to blood pressure and prognostic importance. Acta Med Scand 1978;204: 159-67.
9. Palmer RS, Loofbourow D, Doering CR. Prognosis in essential hypertension: eight-year follow-up study of 430 patients on conventional
medical treatment.N Engl Med J 1948;239:990–4.
10. Oglesby P, Lepper MH, Phelan WH, et al. A longitudinal study ofcoronary heart disease.Circulation 1963;28:20–31.
11. Breslin DJ, Gifford RW Jr, Fairbairn JF II, et al. Prognostic importance of ophthalmoscopic findings in essential hypertension. JAMA
1966;195:335-8.
12. Breslin DJ, Gifford RW Jr, Fairbairn JF II. Essential hypertension: atwenty year follow-up study. Circulation 1966;33:87-97.
13. Tanaka H, Hayashi M, Date C et al. Epidemiologic studies of strokein Shibata, a Japanese provincial city: preliminary report on risk factors
for cerebral infarction. Stroke 1997; 16, 773-80.
14. Nakayama T, Date C, Yokoyama T, Yoshiike N, Yamaguchi M, Tanaka
H (1997) A 15.5-year follow-up study of stroke in a Japanese provincial

36

city. The Shibata Study. Stroke 1997; 28, 45-52.
15. Wong TY, Klein R, Nieto FJ et al. Retinal microvascular abnormalities
and ten-year cardiovascular mortality. A population-based case-control
study. Ophthalmology 2003; 110, 933-40.
16. Chatterjee S, Chattopadhya S, Hope-Ross M, et al. Hypertension
and the eye: changing perspectives. J Human Hypertens 2002;16:667-75.
17. Sharrett AR, Hubbard LD, Cooper, et al. Retinal arteriolar diametersand elevated blood pressure. The Atherosclerosis Risk in Communities
Study.Am J Epidemiol 1999;150:263-70.
18. Hubbard L, Brothers RJ, King WL, et al. Methods for evaluation ofretinal microvascular abnormalities associated with hypertension/sclerosis in the Atherosclerosis Risk in Communities Study. Ophthalmology
1999; 106:2269-80.
19. Klein R, Klein BEK, Moss SE. The relation of systemic hypertension
to changes in the retinal vasculature: the Beaver Dam Eye Study. TransAm Ophthalmol Soc 1997; 95: 329-48.
20. Wang JJ, Mitchell P, Leung H, et al. Hypertensive retinal wall signs
in a general older population: the Blue Mountains Eye Study. Hypertension 2003; 42: 534-41.
21. Wong TY, Klein R, Klein BE, et al. Retinal vessel diameters andtheir associations with age and blood pressure. Invest Ophthalmol Vis
Sci 2003; 44: 4644-50.
22. Klein R, Sharrett AR, Klein BEK et al. Are retinal arteriolar abnormalities related to atherosclerosis? The Atherosclerosis in Communities
Study. Arterioscler Thromb Vasc Biol, 20, 1644-50.
23. Sharp PS, Chaturvedi N, Wormald R et al. Hypertensive retinopathyin Afro-Caribbeans and Europeans. Prevalence and risk factor relationships. Hypertension 1995; 25: 1322-5.
24. Stolk RP, Vingerling JR, de Jong PT et al. Retinopathy, glucose,and insulin in an elderly population. The Rotterdam Study. Diabetes
44: 11-15.
25. Wong TY, Klein R, Sharret AR et al. The prevalence and risk factorsof retinal microvascular abnormalities in older people. The Cardiovascular Health Study. Ophthalmology 2003; 110: 658-66.
26. Yu T, Mitchell P, Berry G, Li W, Wang JJ. Retinopathy in older
persons without diabetes and its relationship to hypertension. Arch
Ophthalmol 1998; 116: 83-9.
27. McDonough JR, Garrison GE, Hames CG. Blood pressure andhypertensive disease among Negroes and whites: a study in Evans
County, Georgia. Ann Intern Med. 1964; 61: 208-28.
28. Svardsudd K, Wedel H, Aurell E, Tibber G. Hypertensive eye
ground changes:prevalence, relation to blood pressure and prognostic
importance. Acta Med Scand. 1978; 204:159-167.
29. Duncan BB, Wong TY, Tyroler HA, Davis CE, Fuchs FD. Hypertensive retinopathy and incident coronary heart disease in high risk men.
Br J Ophthalmol. 2002; 86(9): 1002-6.
30. Breslin DJ, Gifford RWJ, Fairbairn JFI. Essential hypertension: atwenty year follow-up study. Circulation 1996; 33: 87-97.
31. Michelson EL, Morganroth J, Nichols CW, MacVaugh H. Retinal
arteriolar changes as an indicator of coronary artery disease. Arch
Intern Med 1997: 139: 1139-41.
Wong TY, Klein R, Sharret AR et al. Retinal arteriolar narrowing andincident coronary heart disease in men and women: The Atherosclerosis Risk in the Communities Study. JAMA 2002; 287: 1153-9.

Fiziologia - Physiology  2011.21.3 (71)RELATIA DINTRE RETINOPATIA HIPERTENSIVA SI BOALA CORONARIANAREZUMAT
Scopul studiului. Evidentierea relatiei dintre retinopatia diabetica si boala coronariana la populatia din judetul Timis, Romania.
Metode. Am efectuat un studiu observational care a inclus 600 de pacienti (382 barbati si 218 femei), cu varste cuprinse intre 40
si 60 de ani, care au fost examinati in vederea evidentierii modificarilor microvasculare prin fotografii retiniene, in cadrul Clinicii de
Oftalmologie Timisoara, in perioada martie 2008, mai 2011. In studiul nostru au fost inclusi pacienti cu hipertensiune, pe baza valorilor
tensiunii arteriale sistolice si diastolice determinate la 3 intervale succesive (valori mai mari de 140 mmHg ale TAS si/sau mai maride 90 mmHg ale TAD, determinate in conditii de repaus, cu ajutorul unui sfigmomanometru cu mercur).
Rezultate. In urma evaluarii initiale a tensiunii arteriale, 360 de pacienti (60%) au prezentat hipertensiune arteriala stadiul I, 180
pacienti (30%) au prezentat HTA stadiul II, iar 60 de pacienti (10%) au fost detectati cu HTA stadiu III. La evaluarea initiala nici unul
dintre pacienti nu a prezentat hipertensiune maligna.
Retinopatia diabetica a fost detectata la 210 pacienti (35%): ingustare arteriolara focala si generala la 108 pacienti (18%); constrictie
arterio-venoasa la 60 de pacienti (10%); microanevrisme, hemoragii si exudate (retinopatie) la 42 de pacienti (7%). Nici unul dintre
pacienti nu a prezentat exudate ale discului optic sau edeme ale nervului optic. Prezenta semnelor de retinopatie este predictiva
pentru un risc de trei ori mai crecut de a dezvolta boala coronariana (RR 3,71; 95% CI 2,76 to 4,98), cu risc crescut pentru pacientii
cu retinopatie (RR 4,19; 95% CI 3,41 to 5,15) comparativ cu constrictia AV (RR 2,25; 95% CI 1,69 to 2,97) si ingustarea arteriolara(RR 1,23; 95% CI 0,88 to 1,71). Incidenta bolii coronariene a fost mai crescuta la pacientii cu hipertensiune arteriala stadiile II si
III, la pacientii cu control precar al tensiunii arteriale, in ciuda tratamentului recomandat. Incidenta retinopatiei hipertensive a fostmai crescuta in cazul pacientilor de sex feminin, in timp ce incidenta afectiunilor coronariene a fost mai crescuta pentru pacientii desex masculin.
Concluzii. Studiul nostru demonstreaza ca evaluarea retinopatiei hipertensive este importanta in cazul pacientilor hipertensivi deoarece
prezenta oricaror modificari (ingustare arteriolara, constrictie arterio-venoasa si retinopatie) este asociata cu un grad crescut de a
dezvolta boala arteriala coronariana. Incidenta retinopatiei hipertensive este mai mare la persoanele de sex feminin, dar pacientii desex masculin prezinta o incidenta mai crescuta a bolii coronariene. Pacientii cu retinopatie (microanevrisme, hemoragii si/sau exudate)prezinta un risc crescut. De aceea, este importanta efectuarea unui control adecvat al tensiunii arteriale si a altor factori de risc.
Cuvinte cheie: retinopatie hipertensiva, boala coronariana

2011.21.3 (71)  Fiziologia - Physiology

37

REVIEWPOTENTIAL CIRCULATING MARKERS OF PREECLAMPSIADANA CARMEN ZAHA
Preclinical Department, University of Oradea, Faculty of Medicine and Pharmacy

ABSTRACTPreeclampsia and eclampsia are estimated to be responsible up to 20% of maternal deaths per year. Preeclampsia is a multisystemdisorder of pregnancy which is characterized by new onset hypertension and proteinuria that develop after 20 weeks of gestation inpreviously normotensive women and several other symptoms, such as edema, disturbance of hemostasis, renal or liver failure andthe HELLP syndrome that may also complicate the clinical picture. The etiology remains insufficiently elucidated; a certainty is thecentral role played by the placenta in its pathology and the general consensus is that preeclampsia is an endothelial cell disorder.
As research in the field of preeclampsia progresses, much of the attention in recent years has been focused on peptides related toangiogenesis. Recent data suggest that circulating factors that interfere with the action of vascular endothelial growth factor (VEGF)
and placental growth factor (PlGF) play a major role in maternal manifestation of the disorder. Similarly, maternal plasma levels of
PlGF have been shown to be significantly reduced in the second trimester in women who went to develop preeclampsia compared
to controls. Many recent studies have therefore concentrated on factors which antagonize VEGF and PlGF to assess their role in thedevelopment of preeclampsia. Two of the most studied peptides, which are produced by the placenta, are soluble FMS-like tyrosine
kinase (sFLT-1) and soluble endoglin.
This paper presents current knowledge on the biology of preeclampsia and review several biochemical markers which may be used
to early diagnosis and monitor preeclampsia.
Key words: preeclampsia, markers, angiogenesis.

INTRODUCTION
Worldwide pregnancy-induced hypertension affects 10%of pregnancies and pre-eclampsia complicates about 5% ofpregnancies; the range is 5-10% (1). Eclampsia occurs in about
1/2000 deliveries in resource-rich countries (2). In resource-poor
countries, estimates of the incidence of eclampsia vary from1/100 to 1/1700 (3,4). Although eclampsia is a rare complication of pregnancy, approximately 50,000 women worldwide are
estimated to die annually because of eclampsia. The reported
maternal mortality rate ranges from 1-20%. The perinatal mortality rate of neonates born to eclamptic mothers ranges from
1.3-3% (5).
Preeclampsia is a multi-system disorder of pregnancy,
which is characterized by new onset hypertension (systolic anddiastolic blood pressure of ≥ 140 and 90 mm Hg, respectively,on two occasions, at least 6 hours apart) and proteinuria (protein
excretion of ≥ 300 mg in a 24 h urine collection or a dipstick ≥2+), that develop after 20 weeks of gestation in previously normotensive women. Edema, disturbance of hemostasis, renal orliver failure and the HELLP syndrome also complicate the clinicalpicture. Preeclampsia can show mild or severe symptoms andcan have an early onset (preeclampsia starting before 34 weeks
of gestation) or late onset (preeclampsia starting after 34 weeks
of gestation). It can manifest as a maternal disorder only, with
an appropriate fetal growing or it can present itself with a growth
restricted fetus (in utero growth restriction IUGR) or sudden fetal
distress. The maternal risks must be carefully weighed against
the possible fetal benefits in temporizing management, as therisk of fatal deterioration of the maternal and/or fetal healthcondition is high.
Resolution of preeclampsia is the removal of the placenta
and prematurity, with the adverse consequence of delivering a
pre-term baby. Anyway, preeclampsia, with or without IUGR,
remains a major cause of maternal and neonatal mortality
and morbidity worldwide. Several prophylactic therapies likeanti-oxidant vitamins, calcium or folic acid supplementation,
Aspirin have so far failed to prove efficacious in the preventionof pre-eclampsia, although some benefit has been shown inhigh risk group: previous episodes of pre-eclampsia, obesity,
black race, diabetes or insulin resistance, collagen vasculardisease, thrombophilias, multiple gestation, molar pregnancy
and extremes of age (<20 or >40 years) increase the risk forpre-eclampsia (6,7).
The etiology remains insufficiently elucidated; it is believed
to be multifactorial and a certainty is the central role played by theplacenta in its pathology. An imbalance between vasoconstrictor
and vaso-relaxant factors was proposed as a possible cause of
the hypertension accompanying pre-eclampsia. It was reported
a relative decrease in the concentration of the circulating vasodilator angiotensin (1–7), compared with circulating levels of thevasoconstrictor angiotensin II (8). There also appears to be anincreased sensitivity to Ang II in women with pre-eclampsia (9).
The vasodilator prostaglandin I2 is elevated in normal pregnancy,
but its production is reduced in pre-eclampsia. The vasoconstrictor thromboxane A2 is increased out of proportion to prostaglandin
I2 in pre-eclampsia but administration of aspirin, a thromboxane
inhibitor, does not consistently prevent pre-eclampsia in pregnant
women (10,11).

Received August 20th 2011. Accepted September 10th 2011. Address for correspondence: Dana Carmen Zaha, MD, PhD, Preclinical Department, Faculty of Medicine andPharmacy, University of Oradea, 1 decembrie Square No. 10 Oradea, Romania, phone: +40723 604496, e-mail: danazaha@rdslink.ro38

Fiziologia - Physiology  2011.21.3 (71)The general consensus is that preeclampsia is an endothelial cell disorder resulting in a microangiopathy of target
organs such as brain, liver, kidney and placenta. Evidence todate suggests that oxidative stress, circulatory maladaptation,
inflammation, humoral, mineral and metabolic abnormalities
may all contribute to endothelial dysfunction and pathogenesisof preeclampsia (12,13,14,15). Recently, it was proposed instead that intrinsic failure in trophoblast differentiation, but theorigin of preeclampsia might not be restricted to an alteration oftrophoblast differentiation.
Endothelin, cellular fibronectin, plasminogen activator
inhibitor-1and altered prostacyclin / thromboxane profile havebeen shown to be elevated in women who develop preeclampsia
before they became symptomatic (16). Recent data suggestthat circulating factors that interfere with the action of vascularendothelial growth factor (VEGF) and placental growth factor(PlGF) play a major role in this disorder. This is the why muchof the attention in recent years has been focused on peptides
related to angiogenesis.
ANGIOGENIC AND ANTIANGIOGENIC FACTORS
Angiogenesis represent the development of new bloodvessels from existing endothelium and is essential for normal
placental development. Angiogenesis requires the complex
interplay between the pro-angiogenic factors like vascular endothelial growth factor VEGF and placental growth factor PlGF)
with their receptors VEGF receptor-1 (VEGFR-1) and VEGFR-2
and these factors have been shown to be key components inregulating trophoblast cell survival and function (17, 18, 19, 20).
As normal pregnancy progresses, maternal VEGF and PlGF
circulate in high concentration (21). Placental levels of mRNAencoding VEGF have been shown to be much lower in womenwith preeclampsia compared to controls and maternal plasma
levels of PlGF have been shown to be significantly reduced in thesecond trimester in women who went on to develop preeclampsia
compared to controls (22,23). The use of anti-VEGF antibodies
for systemic treatment in cancer has shown a dose dependantassociation with hypertension and proteinuria (24), which may
indicate that these factors have a role in the development ofpreeclampsia.
Many recent studies have concentrated on factors whichantagonize VEGF and PlGF to assess their role in the development of preeclampsia. Two of the most extensively studied peptides, which are produced by the placenta, are soluble FMS-liketyrosine kinase (sFLT-1) and soluble endoglin.
Soluble FLT-1 (or soluble VEGF receptor 1 or sFLT-1) binds
to and neutralizes the angiogenic actions of VEGF and PlGF andis thought to be one of the key peptides involved in the development of preeclampsia (25). Maternal serum levels of sFLT-1
have been shown to be elevated in women with preeclampsia
compared to controls (26, 27), to correlate with disease severity(28) and to decrease markedly following delivery (26). Levels ofsFLT-1 are increased in nulliparity when compared to multiparous
women. Others researchers found that although fetal levels ofsFLT-1, measured in cord blood are elevated in preeclampsia.
2011.21.3 (71)  Fiziologia - Physiology

Alterations in sflt-1 and PlGF are also more accentuated inearly onset in comparison to late onset preeclampsia (29). It
was also noticed that increased levels of sflt-1 were associatedwith IUGR (30).
Another peptide that has been implicated in the pathogenesis of pre-eclampsia is soluble endoglin. Soluble endoglin(Eng) is a co-receptor for transforming growth factor (TGF)-β1
and TGF-β3 that is highly expressed on cellular membranesof the vascular endothelium and on the syncytiotrophoblast. It
functions as a modulator of TGF-β signaling and is involved inangiogenesis and the regulation of the vascular tone (31,32).
The circulatory form of endoglin has been identified in normal
pregnancy and in preeclampsia. In vitro, sEng acts as a negative
regulator of angiogenesis, thereby impairing capillary formation
by endothelial cells. Soluble Eng level rises up in preeclamptic
patients compared to normotensive controls and its concentrations appear to grow higher with the severity of the symptoms
and are the highest in preeclampsia complicated by the HELLP
symptom (32,33). Pregnancies with IUGR without the maternal
syndrome may also be particularized by high levels of sEng,
suggesting that this factor is not specific for preeclampsia, but
may be a marker for clinical conditions associated with an underlying placental pathology (34). However, these results remain
conflicting as others demonstrated no association between IUGR
and the levels of sEng. Another study has suggested that sEng
may prove useful in differentiating preeclampsia from otherhypertensive diseases of pregnancy, such as gestational or
chronic hypertension. It had demonstrated that circulating sEng
levels increase earlier and to a significantly greater degree inwomen who eventually develop pre-eclampsia. This process
begins at 17–20 weeks in women who develop preterm preeclampsia and at 25–28 weeks in women who develop term
pre-eclampsia (35,36).
It has therefore been proposed that the maternal endothelial
dysfunction in preeclampsia was caused by the imbalance ofthe levels of circulatory angiogenic factors. Amounts of sflt-1
are apparent in second trimester-, but not first trimester blood,in women destined to develop preeclampsia, whereas PlGF
and VEGF levels already show alterations at the end of thefirst trimester of pregnancy in these patients. In normotensive
pregnancies, sflt-1 levels remain relatively stable until the last 2
months of gestation when they steadily increase. This increase ismuch more pronounced in pregnancies ending with preeclampsia
and can discriminate this condition beginning approximately 5
to 8 weeks before the symptoms arise, in particular in caseswith preterm (<37 weeks) symptoms. In contrary to sflt-1, thelevels of circulatory PlGF increase gradually and peak at mid
gestation before declining again in uneventful pregnancies. PlGF
concentration profile follows a similar pattern in women who later
developed preeclampsia, however with decreased amplitude.
PlGF concentrations are already significantly reduced at theend of the first trimester and remain lower throughout pregnancy
and the difference in circulatory PlGF between normotensive
pregnancies and those affected by preeclampsia is the highest
within weeks of the onset of the clinical symptoms. Urinary PlGF
39

is likewise lower in preeclamptic patients before and at the time ofsymptoms (37, 38). On the other hand, it has demonstrated thatthe angiogenic factor PIGF is reduced in trophoblast cells fromtubal ectopic pregnancies when compared to that of intrauterine
pregnancies. In addition, it shows that this difference can beassessed systemically and that serum PIGF levels are reducedin tubal ectopic pregnancy and miscarriage (39).
According to some studies, the presymptomatic alterationsin sflt-1 levels appeared to be specific for preeclampsia as nochanges are detected in women who later deliver SGA neonate
or whose pregnancies are complicated by IUGR, compared to
women with normal pregnancy outcome (35,37). However, others
found that in a selected group of patients with abnormal uterine
perfusion, similar alterations in sflt-1 and PlGF levels could be
detected during the second trimester in cases with subsequentIUGR. Levels of sEng and sFLT-1 have been elevated in the serumof preeclamptic women at 17-20 weeks gestation when compared
to controls. The level at 11-13 weeks was similar between casesand control (35). Both sEng and sFLT-1 appear to be important
peptides in the pathogenesis of preeclampsia but when used alone,
do not appear to have a sufficiently high positive predictive value
to be translated into routine clinical practice.
Others studies have therefore evaluated the potential of
combination between the pro- and anti-angiogenic factors PlGF
and sflt-1 for the prediction of preeclampsia. The studies reported
that the pattern of changes in the ratio of different combinationsof these factors (PlGF/sEng; (sflt-1+sEng)/PlGF; etc), collected
at 13 weeks and around 20 weeks, was more informative than
the individual biomarkers at single time-point screening.
This highlights that the differential secretion of angiogenic
molecules associated with embryo implantation could be used
to define a diagnostic biomarker strategy.
CURRENT CANDIDATES FOR EARLIER DETECTION
Placental protein 13 (PP-13) is a small protein with 139 aminoacids which is highly homologous (69%) to the human eosinophil
Charcot-Leyden Crystal protein. The homodimer which is linked bydisulfide bonds probably has special haemostatic and immunobiological functions at the feto-maternal interface or a developmentalrole in the placenta.PP-13 may interfere with the balance between
vasoconstrictors and vaso-relaxants especially by interfering witharachidonic acid metabolites on tropfoblastic cells (42). Levels of
PP-13 slowly increase during a normal pregnancy but abnormally
low levels of PP-13 were detected in first trimester serum samples
of women subsequently developing fetal growth restriction andpreeclampsia (40-43). Elevated serum concentrations of PP-13
have been found in the second and third trimester in women withpreeclampsia, IUGR and in preterm delivery (43). Other researchers
demonstrated no statistical significance between early PP-13 levels
in complicated pregnancies by SGA, IUGR.
The combination of several diagnostic tools results in improved predictive power as was shown by combined measuringof first trimester serum PP-13 levels and median uterine artery
pulsatility index by ultrasound. This combination achieved a40

detection rate for preeclampsia of 90% with a false positive rateof 6% (45). However, this combination of serum PP-13 levels
and uterine artery pulsatility index loses its predictive power
when late second trimester (22–24 weeks of gestation) serum
is analyzed (42).
Pentraxin 3 (PTX3, tumor necrosis factor stimulated gene14) belongs to the same family as C-reactive protein (CRP) or
serum amyloid P component and consists of 381 amino acids.
PTX3 then interacts with several growth factors, extracellularmatrix components and certain pathogens but is also involved in
the activation of the complement system and facilitates pathogen
recognition by phagocytes. It has been proposed as a marker
of endothelial dysfunction and inflammation in pre-eclampsia.
During pregnancy, PTX3 is increasingly expressed in amniotic
epithelium, chorionic mesoderm, trophoblast terminal villi, andperivascular stroma of placentae. Some studies showed that incase of a future preeclampsia and IUGR the PTX3 plasma levels
are even more increased in all three trimesters (45).
P-Selectin is a cell surface adhesion molecules expressed byplatelets and endothelial cells upon activation and plays importantroles in inflammatory reactions and in coagulation. P-selectin is
rapidly shed from the cellular membrane of activated platelets andthis release is suggested to contribute to most of the soluble isoform of the molecule that is found in the plasma (46). Preeclampsia
is associated with extensive platelet activation (47-49). P-selectinexposing microparticles with procoagulant activity, released from
activated platelets, have been detected in the peripheral bloodof preeclamptic women (50,51). In addition, soluble P-selectinhas been repeatedly, though not constantly, observed in higher
amounts in serum or plasma of patients with this disorder (52-55).
It has recently been shown that alterations in the levels of soluble
P-selectin before 20 weeks of gestation antedate the symptoms(56-58). This early up-regulation of soluble P-selectin has beensuggested to reflect the early but still asymptomatic disturbancesof the maternal vascular system. In one of these studies, P-selectin
was identified as the marker with the highest discriminatory ability
among three biomolecules evaluated between gestational weeks
11 to 15 (59). However, the combination of P-selectin with the twoother markers showed a detection rate of only 59% (with a falsepositive rate of 5%), which is not sufficient for a possible routine
clinical implementation as a screening test.
PAPP-A (pappalysin 1, pregnancy-associated plasma
protein A) is a disulfide bond linked homodimeric peptidase of
1628 amino acids and a mass of 400 kDa. It can be detectedduring pregnancy in maternal circulation mainly as a complex
with the proform of the eosinophil major basic protein, an inhibitor of PAPP-A (60). Recent studies have shown that although
reduced in all trimester serum levels of PAPP-A are associatedwith preeclampsia, levels are also low in other complications ofpregnancy. It was described a small increase in likelihood ratio
of developing preeclampsia with decreasing levels of PAPP-A.
Although PAPP-A alone was not a good predictor for preeclampsia, they felt, similarly to PP-13, that sensitivity could be improvedby combining with uterine artery Doppler studies (59).
Fiziologia - Physiology  2011.21.3 (71)CONCLUSION
Despite there exists many different potential markers forpreeclampsia, the reliability of these markers in predicting
preeclampsia has been inconsistent between different studies.
Whereby preeclampsia is a multifaceted disorder, there is a needfor high quality, large scale multi-center trials which enroll patientswith different risks of developing the syndrome and throughout
multi-ethnical background, in order to assess the predictive
value of different markers and finally propose the best marker
combination for a routine use in clinical settings.
Preeclampsia is an appropriate disease to screen and ispossible to decreases both maternal mortality and perinatal
mortality. Currently, the clinical value of an accurate predictive
test for preeclampsia is not clear since we lack prevention.
Intensive monitoring in women, who are at increased risk ofdeveloping preeclampsia, when identified by a predictive test,
may lower the incidence of adverse outcome for both mother andthe neonate. However, the effectiveness of such a strategy mustbe rigorously investigated. The implementation of clinical tests
will require close collaboration between the medical institutions
together with the pharmaceutical industry in order to developfunctional and, as best as possible, affordable tests which could
profit to the pregnant women worldwide. Further studies withlarge number of patients will be required to confirm these very
promising preliminary results and assess the utility of analyzingthese biomarkers in the clinical routine.
REFERENCES

1. WHO International Collaborative Study of Hypertensive Disordersof Pregnancy. Geographic variation in the incidence of hypertension inpregnancy. Am J Obstet Gynecol 1988; 158:80–83.
2.Douglas K, Redman C. Eclampsia in the United Kingdom. BMJ 1994;
309:1395–1400.
3.Crowther CA. Eclampsia at Harare maternity hospital. An epidemiological study. S Afr Med J 1985; 68:927–929.
4.Bergstrom S, Povey G, Songane F, et al: Seasonal incidence ofeclampsia and its relationship to meteorological data in Mozambique.
J Perinat Med 1992; 20:153–158.
5.WHO, 2004. Bethesda, MD. Global Burden of Disease for the Year
2001 by World Bank Region, for Use in Disease Control Priorities in
Developing Countries, National Institutes of Health: WHO. Make every
mother and child count. World Health Report, 2005, Geneva:World
Health Organization, 2005. 2nd ed.
6.Karumanchi SA, Maynard SE, Stillman IE, et al: Preeclampsia: A
renal perspective. Kidney Int. 2005; 67: 2101–2113.
7.Lain KY, Roberts JM. Contemporary concepts of the pathogenesis andmanagement of preeclampsia JAMA. 2002; 287:3183–3186.
8.Brosnihan KB, Neves LA, Anton L, et al: Enhanced expression of Ang(1–7) during pregnancy. Braz J Med Biol Res. 2004; 37:1255–1262.
9.AbdAlla S, Luther H, Massiery A, et al: Increased AT (1) receptor
heterodimers in preeclampsia mediate enhance angiotensin II responsiveness. Nat Med. 2001;7: 1003–1009.
10.Mills JL, DerSimonian R, Raymond E, et al: Prostacyclin andthromboxane changes predating clinical onset of preeclampsia. JAMA.
1999; 282: 356–362.
11.Caritis S, Sibai B, Hauth J, et al: Low-dose aspirin to prevent preeclampsia in women at high risk. N Engl J Med. 1998; 338:701–705.

2011.21.3 (71)  Fiziologia - Physiology

12.Hahn S, Gupta AK, Troeger C, et al: Disturbances in placental
immunology: ready for therapeutic interventions? Springer Semin Immunopathol. 2006; 27:477–493.
13.Huppertz B. The feto-maternal interface: setting the stage for potential immune interactions. Semin Immunopathol. 2007; 29:83–94.
14.Jauniaux E, Poston L, Burton GJ. Placental-related diseases ofpregnancy: Involvement of oxidative stress and implications in human
evolution. Hum Reprod Update. 2006;12:747–755.
15.Hung TH, Skepper JN, Charnock-Jones DS, et al: Hypoxiareoxygenation: a potent inducer of apoptotic changes in the humanplacenta and possible etiological factor in preeclampsia. Circ Res.
2002; 90:1274–1281.
16.Taylor RN, de Groot CJ, Cho YK, et al: Circulating factors as markers
and mediators of endothelial cell dysfunction in preeclampsia. Semin
Reprod Endocrinol.1998; 16(1): 17-31.
17.Khaliq A, Li XF, Shams M, et al: Localisation of placenta growth factor(PIGF) in human term placenta. Growth Factors. 1996; 13:243–50.
18.Crocker IP, Strachan BK, Lash GE, et al: Vascular endothelial growth
factor but not placental growth factor promotes trophoblast syncytialization in vitro. J Soc Gynecol Investig. 2001;8:341–346.
19.Desai J, Holt-Shore V, Torry RJ, et al: Signal transduction and biological function of placenta growth factor in primary human trophoblast.
Biol Reprod. 1999; 60:887–892.
20.Shore VH, Wang TH, Wang CL, et al: Vascular endothelial growth
factor, placenta growth factor and their receptors in isolated humantrophoblast. Placenta. 1997;18:657–665.
21.Ong S., Lash G., Baker P.N. Angiogenesis and placental growthin normal and compromised pregnancies. Best Pract Res Clin Obstet
Gynaecol. 2000; 14:969–980.
22.Cooper J.C., Sharkey A.M., Charnock-Jones D.S. VEGF mRNAlevels in placentae from pregnancies complicated by pre-eclampsia.
Br J Obstet Gynaecol. 1996;103:1191–1196.
23.Kim S.Y., Ryu H.M., Yang J.H. Maternal serum and amniotic fluid
inhibin A levels in women who subsequently develop severe preeclampsia. J Korean Med Sci. 2006; 21:452–456.
24.Ostendorf T., De Vriese A.S., Floege J. Renal side effects of antiVEGF therapy in man: a new test system. Nephrol Dial Transplant.
2007; 22:2778–2780.
25.Venkatesha et al: Soluble endoglin contributes to the pathogenesisof preeclampsia. Nature Med. 2006;12:642–649.
26.Salahuddin S., Lee Y., Vadnais M. Diagnostic utility of soluble fmslike tyrosine kinase 1 and soluble endoglin in hypertensive diseases ofpregnancy. Am J Obstet Gynecol. 2007;197:28.e1–28.e6.
27.Chaiworapongsa T, Romero R, Kim YM, et al: Plasma solublevascular endothelial growth factor receptor-1 concentration is elevatedprior to the clinical diagnosis of pre-eclampsia. J Matern Fetal Neonatal
Med. 2005;17:3–18.
28.Wolf M., Sandler L., Munoz K. First trimester insulin resistanceand subsequent preeclampsia: A prospective study. J Clin Endocrinol
Metabol. 2002;87:1563–1568.
29.Barton JR, Witlin AG, Sibai BM. Management of mild preeclampsia. Clin Obstet Gynecol. Sep 1999;42(3):455-69.
30.Stepan H, Kramer T, Faber R: Maternal plasma concentrations ofsoluble endoglin in pregnancies with intrauterine growth restriction. J
Clin Endocrinol Metab 2007, 92:2831-2834.
31.St-Jacques S, Forte M, Lye SJ, Letarte M: Localization of endoglin,a transforming growth factor-beta binding protein, and of CD44 andintegrins in placenta during the first trimester of pregnancy. Biol Reprod
1994, 51:405-413.
32.Duff SE, Li C, Garland JM, et al: CD105 is important for angiogenesis:
evidence and potential applications. FASEB J 2003, 17:984-992.

41

33.Jeyabalan A, McGonigal S, Gilmour C, et al: Circulating and placental
endoglin concentrations in pregnancies complicated by intrauterine
growth restriction and preeclampsia. Placenta 2008, 29:555-563.
34.Levine RJ, Maynard SE, Qian C, et al: Circulating angiogenic factors
and the risk of preeclampsia. N Engl J Med. 2004; 350:672–683.
35.Levine RJ, Lam C, Qian C, et al: Soluble endoglin and other circulating antiangiogenic factors in preeclampsia. N Engl J Med. 2006; 355: 992–1005.
36.Rana S., Karumanchi S.A., Levine R.J. Sequential changes in antiangiogenic factors in early pregnancy and risk of developing preeclampsia.
Hypertension. 2007;50:137–142.
37.Aggarwal PK, Jain V, Sakhuja V, et al: Low urinary placental growth
factor is a marker of pre-eclampsia. Kidney Int 2006, 69:621-624.
38.Levine RJ, Thadhani R, Qian C, et al: Urinary placental growth factor
and risk of preeclampsia. JAMA 2005, 293:77-85.
39.Nancy Fowler Larson, Novel Biomarker May More Quickly Confirm
Ectopic Pregnancy, J Clin Endocrinol Metabol. November 3, 2010.
40.Huppertz B, Sammar M, Chefetz I, et al: Longitudinal determinationof serum placental protein 13 during development of preeclampsia. Fetal
Diagn Ther 2008, 24:230-236.
41.Chafetz I, Kuhnreich I, Sammar M, et al: First-trimester placental
protein 13 screening for preeclampsia and intrauterine growth restriction.
Am J Obstet Gynecol 2007, 197:35-37.
42.Spencer K, Cowans NJ, Chefetz I ,et al: First-trimester maternal
serum PP-13, PAPP-A and second-trimester uterine artery Doppler
pulsatility index as markers of preeclampsia. Ultrasound Obstet Gynecol
2007, 29:128-134.
43.Burger O, Pick E, Zwickel J, et al: Placental protein 13 (PP-13):
effects on cultured trophoblasts, and its detection in human body fluids
in normal and pathological pregnancies. Placenta 2004, 25:608-622.
44.Khalil A, Cowans NJ, Spencer K, et al: First trimester maternal serum
placental protein 13 for the prediction of pre-eclampsia in women witha priori high risk. Prenat Diagn 2009
45.Nicolaides K.H., Bindra R., Turan O.M. A novel approach to firsttrimester screening for early pre-eclampsia combining serum PP-13 andDoppler ultrasound. Ultrasound Obstet Gynecol. 2006;27:13–17.
46.Rovere-Querini P, Antonacci S, Dell'Antonio G, et al: Plasma andtissue expression of the long pentraxin 3 during normal pregnancy andpreeclampsia.Obstet Gynecol 2006, 108:148-155.
47.Dunlop LC, Skinner MP, Bendall LJ, et al: Characterization of GMP
140 (P-selectin) as a circulating plasma protein. J Exp Med 1992,
175:1147-1150.
48.Holthe MR, Staff AC, Berge LN, Lyberg T. Different levels of plateletactivation in preeclamptic, normotensive pregnant, and nonpregnant
women. Am J Obstet Gynecol 2004, 190:1128-1134.
49.Konijnenberg A, Stokkers EW, Post JA van der, et al: Extensive
platelet activation in preeclampsia compared with normal pregnancy:enhanced expression of cell adhesion molecules. Am J Obstet Gynecol
1997, 176:461-469.
50.Yoneyama Y, Suzuki S, Sawa R, et al: Plasma adenosine levels
and P-selectin expression on platelets in preeclampsia. Obstet Gynecol
2001, 97:366-370.
51.Bretelle F, Sabatier F, Desprez D, et al: Circulating microparticles:
a marker of procoagulant state in normal pregnancy and pregnancy
complicated by preeclampsia or intrauterine growth restriction. Thromb
Haemost 2003, 89:486-492.
52.Lok CA, Nieuwland R, Sturk A, et al: Microparticle-associatedP-selectin reflects platelet activation in preeclampsia. Platelets 2007,
18:68-72.
53.Aksoy H, Kumtepe Y, Akcay F, et al: Correlation of P selectin andlipoprotein(a), and other lipid parameters in preeclampsia. Clin Exp
Med 2002, 2:39-43.
54.Chaiworapongsa T, Romero R, Yoshimatsu J, et al: Soluble adhesion
molecule profile in normal pregnancy and pre-eclampsia. J Matern Fetal
Neonatal Med 2002, 12:19-27.
55.Halim A, Kanayama N, Nakashima A, et al: Plasma P selectin (GMP140) and glycocalicin are elevated in preeclampsia and eclampsia: theirsignificances. Am J Obstet Gynecol 1996, 174:272-277.
56.Heyl W, Handt S, Reister F, et al: Elevated soluble adhesion
molecules in women with pre-eclampsia. Do cytokines like tumour
necrosis factor alpha and interleukin-1beta cause endothelial activation.
Eur J Obstet Gynecol Reprod Biol 1999, 86:35-41.
57.Banzola I, Farina A, Concu M, et al: Performance of a panel ofmaternal serum markers in predicting preeclampsia at 11–15 weeks'
gestation. Prenat Diagn 2007, 27:1005-1010.
58.Bosio PM, Cannon S, McKenna PJ, O'Herlihy C, et al: Plasma
P-selectin is elevated in the first trimester in women who subsequently
develop pre-eclampsia. BJOG 2001, 108:709-715.
59.Chavarria ME, Lara-Gonzalez L, Garcia-Paleta Y, et al: Adhesion
molecules changes at 20 gestation weeks in pregnancies complicated by
preeclampsia. Eur J Obstet Gynecol Reprod Biol 2008, 137:157-164.
60. Spencer K., Cowans N.J., Nicolaides K.H. Low levels of maternal
serum PAPP-A in the first trimester and the risk of pre-eclampsia. Prenat
Diagn. 2008;28:7–10.

MARKERI POTENTIALI CIRCULANTI AI PREECLAMPSIEIREZUMAT
Preeclampsia şi eclampsia sunt estimate a fi responsabile pentru până la 20% din decesele materne anuale. Preeclampsia este otulburare specifică sarcinii caracterizată prin hipertensiune şi proteinurie care se dezvoltă după săptămâna 20-a de sarcină la femei
normotensive anterior. Edemul, tulburările hemostazei, ale funcţiilor renale şi hepatice, precum şi sindromul Hellp pot complica
tabloul clinic. Etiologia rămâne insuficient cunoscută; cert este rolul central pe care îl joacă placenta, iar consensul general estecă, preeclampsia este o tulburare a celulelor endoteliale. Progresele în cercetările asupra preeclampsiei au fost în ultimii ani maiales îndreptate asupra peptidelor implicate în angiogeneză. Date recente sugerează că, factori circulanţi care interferă cu acţiunea
factorului de creştere endothelial (VEGF) şi factorul de creştere placental (PlGF) joacă un rol important în manifestarea maternă aacestei tulburări. Astfel, nivelul plasmatic matern al PlGF a fost semnificativ redus în al doilea trimestru la femei care au dezvoltat
preeclampsie comparativ cu grupul de control. Alte studii recente şi-au concentrat interesul asupra factorilor care se opun acţiunii
VEGF şi PlGF pentru a determina rolul lor în dezvoltarea preeclampsiei. Două dintre cele mai studiate asemenea peptide produsede placentă sunt: tirozin kinaza FMS-like solubilă (sFLT-1) şi endoglina solubilă.
Acest articol îsi propune să prezinte cunostintele de actualitate referitoare la biologia preeclampsiei si revizuirea unor markeri biochimici care pot fi utilizati în diagnosticul precoce si monitorizarea preeclampsiei.
Cuvinte cheie: preeclampsia, markeri, angiogeneza42

Fiziologia - Physiology  2011.21.3 (71)NEW APPROACHES FOR THE DRIED BLOOD SPOT ANALYSISILEANA SCURTU1, ALEXANDRA GRUIA2, VALENTIN ORDODI1, 2, ELENA CIURARIU1, FELIX
MIC2, CALIN TATU1, 2, VICTOR DUMITRASCU1, 21
1Victor Babes University of Medicine and Pharmacy, Timisoara
2
Emergency Clinical County Hospital, Timisoara

ABSTRACTThe recent recognition of dried blood spot (DBS) technology, as an alternative to plasma as the matrix for systemic
measurement of drug analytes in pharmacokinetic (PK) and toxicokinetic (TK) studies. This article is a review of various
types of analysis of dried blood spot, from off-line extraction to on-line extraction, and various applications in areas as
genetics, virology, pharmacology, toxicology. This technique permits the decrease of blood volume which is necessary
to analysis, making possible pharmacokinetics studies on small lab animals. In the last few years new concepts of extraction have emerged, thus there is developed an in line extraction method of pharmaceutical compounds from dried
blood spot prior chromatography analysis.
Keys words: dried blood spot, paper DBS, pharmacokinetic, toxicology

INTRODUCTION
Dried blood spot (DBS) analysis is a blood samples preparation technique by liquid-solid extraction. The preparation ofsamples is one of the most important parts of the analyticalprocess, because influences the detection, the repeatability, andthe reproducibility of the analysis.
This technique of dried blood spot (DBS) was first describedin 1913 by Bang (1) in the estimation of blood glucose concentration. However, the use of dried blood spots dates to the early
1960s when Dr. Robert Guthrie developed an assay for the detection of phenylketonuria (2). His application of collecting blood on
filter paper led to the population screening of newborns for thedetection of inherited metabolic diseases. In recent years, thecollection of blood on filter paper has become a significant tool
for screening individuals for clinical purposes, for animal testingfor preclinical purposes, for therapeutic and illicit drug monitoring,and for the application to routine drug development. Nowadays,pressure in the pharmaceutical industry to deliver high-quality
pharmacokinetic data in the shortest possible timeframe is requiring workers in the field to accurately and precisely collect, share,
store, and analyze thousands of biological samples. In addition
the movement to reduce, refine, and replace the use of animals
in drug development is challenging analysts to collect adequate
samples while remaining within acceptable total blood collection
volumes and avoiding excess animal usage. Thus DBS analysis
is a way to address these challenges in the pharmaceutical industry. In addition, the use of techniques picked up in performing
DBS analysis is extending this approach to other fields.
Recent advances such as the production of monoclonal
antibodies, expression of synthetic proteins, and the introductionof the polymerase chain reaction have overcome many of these
problems. This type of blood testing is now available for uses

at home by consumers in the U.S. Available blood tests include
Vitamin D, estrogen, testosterone, cortisol, TSH and lipids. New
York is the only state that prohibits home blood spot testing (3)1. Papers for DBS analysis
Schleicher & Scheull 903 (S&S 903, Whatman 903 or
903®) paper (Fig.1), manufactured from 100% pure cotton
linters with no wet-strength additives added, is an FDA listedclass II medical device.

Fig.1. Commercially Available DBS card

The paper has been extensively used in newborn screening
and other applications worldwide. Some other cards/papers, e.g.
no. 545 paper (Advantec, Toyo, Tokyo), have also been used. The
DBS paper/card might be further treated via impregnation for an
improved extraction recovery (van der Heijden et al., 2009). In addition to particle retention, pore size and thickness that determine
the loading capacity and spreadability of blood sample onto the
DBS paper/card, uniformity and absorption characteristics of theReceived June 24th 2011. Accepted September 1st 2011. Address for correspondence: Ileana Scurtu, PhD, “Victor Babes” University of Medicine and Pharmacy
Timisoara, Eftimie Murgu Square No. 2A, 300041, Timisoara, Romania, phone/fax: +40256490507, e-mail: ileana.scurtu@gmail.com

2011.21.3 (71)  Fiziologia - Physiology

43

card/paper from lot to lot are important. In neonatal screening, theperformance of DBS paper is monitored by the Newborn Screening Quality Assurance Program (NSQAP) to ensure that any newpaper lots are consistent with the established guidelines and that
performance is consistent from lot to lot. NSQAP has developed aprocedure to evaluate the paper’s uniformity and absorption characteristics. The procedure involves mixing washed red blood cellswith plasma or serum to give an adjusted hematocrit of 0.55±0.01.
The ‘whole blood’ is then enriched with 125I-l-thyroxine, and a 100
μL volume of the blood is applied onto the paper lots to be tested.
The absorption time and the diameter of spots are then measured,and the blood volume for 3.2 mm disks is determined by measuring
and comparing the total counts using a gamma counter. FTA andFTA Elute cards, also manufactured by Whatman (now part of GE
Healthcare), are designed for nucleic acid analysis.
According to the manufacturer, both cards are chemicallytreated with proprietary reagents that, upon contact, lyses cells,
denature proteins and prevent the growth of bacteria and othermicroorganisms. Recently, both cards have been employed in
combination with LC-MS/MS for the quantitative analysis ofsmall drug molecules and their metabolites in preclinical andclinical studies (4).
2. DBS sample collection
Two methods are commonly used to obtain capillary whole
blood on a filter paper. The drop of blood can be collected directlyon the filter paper or with the aid of a precision capillary. As thecomplete drop can be excised instead of punching out a disk
from it, the influence of sample volume, hematocrit and samplingtechnique is minimized.
In the procedure to obtain a DBS, the hand is first cleaned
and held down or warmed for a few minutes. With the help of an
automatic lancet, the fingertip is pricked. While the first drop is
wiped off with a sterile piece of cloth because of the presence oftissue fluid, the following drops are collected in a 50-μl precisioncapillary. Then, once completely filled, the entire capillary is placedin the centre of two concentric circles pre-printed on a Whatman
903 filter paper. The inner circle (10 mm diameter) must be entirely
filled with blood, but blood may not pass the outer circle line (15mm diameter), which was used for excision of the DBS. Although
the blood is spot on just one side, both sides of the filter paper must
be coloured. After visual inspection of the DBS analyze can start.
The DBS are dried for minimum 4 hours at ambient temperatureand subsequently analyzed or preserved in a sealable plastic bag
at room temperature or -20 °C until analysis (Fig.2) (5).

Fig. 2. DBS blood sample collection technique44

3. Drying, Storage, Transportation and Punching
It is very important to dry blood spots completely before
storage or transportation. In order to be transported DBS
samples are kept in plastic bags containing desiccants and ahumidity indicator and portable refrigerators at -20°C. However,the drying time depends on the type of paper/card and the blood
volume applied. In general, DBS samples are drying 2-3 h atroom temperature, or room temperature 3-4 hours in horizontal
position, dried at room temperature and stored at 4°C in zip-lockpoly bags (Fig.3.a and 3.b). However, samples that contain
unstable compounds should be stored at a lower temperature(e.g. 2–8°C, ≤−15°C or ≤−60°C) in order to enhance stability.
DBS samples that have been packed as described above canbe transported through the mail in a high-quality bond envelope(6).

Fig. 3a. Drying of DBS samples

Fig. 3b. Storage of DBS samples

Manual punching can be a laborious and repetitive process.
Typical punch size is 3–4 mm, but available sizes range from 0.35
to 8 mm. Punch size can be optimized based on the required
limit of detection (i.e., a larger punch size leads to an increased
signal) and available spotted blood volume. This can result in
hand and thumb fatigue, especially when a large number of spots
are punched. In an attempt to alleviate this, GSK–Research
Triangle Park developed the prototype automated hand puncher(Figure 3), which incorporates a spinning core punch that helps to
cut the DBS disk from the paper. It has been shown to decrease
hand fatigue, due to less external force needed to punch the disk,
Fiziologia - Physiology  2011.21.3 (71)but still has the same time constraints as a single punch. Manual
punch times typically vary by analyst, but are within 10–15 min
per 96 samples, enables clot detection, and a proper liquidsensing program are critical for accurate pipetting (6).
4. DBS sample processing and analysis
Five microliters of real or spiked whole blood were spotted on a filter paper card using a volumetric micropipette. The
blood spots are allowed to dry at room temperature for 2 h andthen packed in a sealable plastic bag containing desiccant until
analysis. They were stored in the dark at ambient temperature
except for short-term stability experiments, in which a variety oftemperatures were tested. Before analysis, a disc (i.d. 10 mm)
containing the whole DBS was manually punched out and directly
introduced into the desired position of the DBS prototype (Fig.4).
Then, a volume of internal standard was added to the disc using
the same volumetric micropipette used above (7).
On-line desorption of dried blood spots (on-line DBS) (Fig.4),
allowing the direct analysis of a dried blood spot coupled to liquid
chromatography mass spectrometry device. The system is basedon an inox cell which can receive a blood sample (10 μL) previously
spotted on a filter paper. The cell is integrated into LC/MS system
where the analytes are desorbed out of the paper towards a column
switching system ensuring the purification and separation of thecompounds before their detection on a single quadrupole MS
coupled to atmospheric pressure chemical ionisation source. The
described procedure implies that no pretreatment is necessary inspite the analysis is based on whole blood sample. To ensure theapplicability of the concept couple of drugs were chosen. Despite
the use of a small sampling volume and a single quadrupole detector, on-line DBS allowed the analyses of the compounds over their
therapeutic concentrations. The method showed good repeatability
with relative standard deviation (RSD) lower than 15% based on
two levels of concentration (low and high). Function responses
were found to be linear over the therapeutic concentration foreach compound and were used to determine the concentrationsof real patient samples. Comparison of the founded values withthose of a validated method used routinely in a reference laboratory showed a good correlation between the two methods. Good
selectivity was observed ensuring that no endogenous or chemicalcomponents interfered with the quantitation of the analytes. This
work demonstrates the feasibility and applicability of the on-line
DBS procedure for bioanalysis (7).

5. Applicationsa) Genetics
The stability of genomic DNA stored in dried blood specimens is a major concern for genetic studies. The dried blood
specimens have been retained for a long period based on thestalability of DNA is the most stable analyte in dried blood andan important tool for population-based genetic studies. Casoleand colleages showed that the maximum length of stability of HIVproviral DNA in dried blood spots kept at ambient temperatureand dessicated at -20ºC was about 3.5 months. Beta-globin DNA
has been detected in dried blood stored at ambient temperature
for about 1 year (8).
b) Virology
Quantification of HIV-RNA from dried blood spots (DBS) has
been evaluated in several studies using different methodologies(9,10). Overall, the results have shown that determination ofviral load in DBS is well correlated to plasma levels, especiallywhen HIV-RNA is above a certain threshold (generally ~3.5 log
copies/mL), and its clinical utility to detect virological failure inresource-limited settings has been confirmed (11). Particularly
good results have been obtained with new methodologies for
HIV-RNA quantification based on real-time PCR technology(12,13.14).
Previous studies (9,14) have suggested that the presenceof proviral HIV-DNA could affect the results obtained from DBS.
Viljoen et al.(14), testing DBS with and without DNase pretreatment, concluded that a prior DNA treatment step was aprerequisite for accurate monitoring. In other study was found that
~80% of the HIV-RNA values obtained from DBS were lower thanthose obtained from plasma, although this proportion was lower
in samples with <5000 copies/mL, suggesting that the possible
DNA contamination may play a role in samples with low levelsof HIV-RNA. Some results suggested that a further step in DBS
processing could be avoided in order to make the procedureeasier and feasible in resource-limited settings.
Other previous studies have evaluated the stability over timeof DBS for viral load quantification. Some of them (9,12,14) haveshown significant stability over 3–12 months of storage at room
temperature. Others (16,17) have suggested slight to significant
decreases. In other studies (18), 7 months of storage at room
temperature did actually cause a change in quantification, witha significantly higher mean difference between plasma and DBS
HIV-RNA values. The most important differences were observed
for the lowest viral loads (and indeed a higher variability for low
copy samples was also observed in the first series), suggestingthat the use of DBS stored for several months, although notrecommended, could still be considered in specific circumstances
to detect virological failure (17).
In conclusion, HIV-RNA quantified from DBS was strongly
correlated with that measured in plasma. The detection rate in
DBS was 100% when the plasma level was ≥3.0 log. Quantification of HIV-RNA from DBS by the VERSANT assay can be used
to diagnose virological failure in patients with HIV (17).

Fig. 4. On-line DBS prototype

2011.21.3 (71)  Fiziologia - Physiology

45c) Newborn screening
Newborn screening refers to the process where babies are
given a simple blood test a few days after birth to see if they havea rare genetic or metabolic condition. The conditions screened
for in Newborn Screening (NBS) may be life threatening and/or
cause intellectual disability.
These conditions are often referred to as ‘inborn errors of
metabolism’.
- Metabolism is the chemical process by which food is broken down to make energy available for the normal functioning,
growth, and development of the body;- Enzymes are proteins that are used by the cells to break
down food into a form that can be used;- Errors in metabolism occur when the essential enzymes
are absent or malfunction;
The aim of NBS is to detect the conditions before the onsetof symptoms so treatment can be started early to reduce theeffect of the condition.
This form of testing is known as screening because it involves testing a whole population - in this case, newborn babies.
All babies are tested even if they do not have any obvious signsof a condition that affects their metabolism.
Over 30 rare conditions can now be detected by the newborn
screening test. Such early detection enables treatment to beundertaken as soon as possible if a baby is shown to have thecondition for which testing is done (7).
d) Toxicology
DBS technique is used also in toxicology field. Analytes
measured in toxicological applications of DBS are: elements (e.g.
Pb); environmental pollutants; therapeutic drugs; drugs of abuse(e.g. phosphatidyl-ethanol, cotinine, benzodiazepines, morphine,
methadone, fentanyl, cocaine, gamma-hydroxybutyric acid, etc.).
These compounds should be derivatized “on spot” in order to be
LC/MS, LC-MS/MS or GC/MS analyzed (7).
e) Pharmacokinetics
For drugs showing little variability in the fraction unboundin plasma (fu) and the blood cell-unbound plasma concentration ratio (ρ) under conditions of study, there is little concern inthe use of DBS as an alternative to plasma. However, caution
should be exercised when variability in either fu or ρ is large.
Then it is important to identify, quantify, and, where possible,
correct for, or accommodate, the variability, especially wheninterpreting pharmacokinetics data or linking pharmacokinetics
to pharmacodynamics. For drugs with a blood-to-plasma ratio
approaching the lower limit of 0.55, indicating minimal blood
cell uptake, general concern is with variability in fu (about whichmuch is known) when using DBS measurement as a surrogate
for unbound drug in plasma. For drugs with larger values ofthe blood-to-plasma ratio, especially >2, variability in blood cellaffinity becomes the critical factor. Fortunately, the magnitude
and determinants of much of the variability can be evaluated invitro using blood obtained from the study population of interest,
which should be undertaken prior to implementing the use of46

DBS in a drug development program. So too should the studyof the influence of drug concentration, temperature, and kineticsof blood cell distribution (1).
6. Advantages and Disadvantages of DBS
There are some advantages of DBS technique:• Small volume samples: great for pediatric studies; use one
animal in preclinical rodent studies for multiple draws – improves
reproducibility, and reduces cost;• Uses besides whole blood, also urine, synovial fluid, saliva,
lavage fluid, tears;• Easier patient recruitment – finger prick not venous
draw;• Ideal for Phase II/III where collection and storage ofsamples at non-ambient conditions not possible – no freezers,
no centrifuges, no thawing;• Advantages for ill patients (e.g. oncology);• Shipment of samples on cards deactivates HIV, HCV,etc.
• Compound stability is better than in solution phase.
There are some disadvantages of DBS technique:• Uses blood as matrix for exposure and pharmacokinetic
determinations – conventionally, plasma used for ease of handling, shipping, storing; FDA states blood is an acceptable matrix
for exposure and pharmacokinetic;• Not a pre-concentration technique – LLOQ of 1-10 ng/
mL typical (19);
FUTURE DIRECTION
Interest in DBS technology has gained momentum in thepast several years. Areas of improvement in the field of DBS
analysis include increased automation, ability to spray-depositinternal standard on the DBS card, specialty papers that include
stabilizing and/or derivatization reagents, and direct analysis
without the need for sample preparation (20).
CONCLUSIONS
DBS as a blood sampling technique for applications in drug
development is a recent phenomenon, spurred by financial andethical motivations. It is remarkable that the power of modern
analytical technology has allowed an old sampling technique tobecome prominent in a short period of time. In view of its advantages compared to traditional plasma-based strategies.
Expansion of dried spot sampling involves developing new
analytical strategies to be competitive with high-throughput
requirements. As described, the prototype presents novel opportunities in filter paper analysis by affording an efficient tool
for rapid and direct analyses of DBS specimens.
Automation and technological advances will help further
the implementation of DBS methods in the laboratory. Whilethe benefits of executing this technology in regard to the 3Rs(refinement, reduction, and replacement) are well understoodand documented, development and analysis of DBS methods arestill tedious and time consuming. Future automation and direct
analysis from the DBS can expedite this procedure.
Fiziologia - Physiology  2011.21.3 (71)

REFERENCES

1. Rowland M, Emmons GT. Use of Dried Blood Spots in Drug Development: Pharmacokinetic Considerations, The AAPS Journal, 2010;12(3):290-293.
2. Guthrie R and Susi A. Pediatrics, 1963; 32: 338-343.
3. Parker SP, Cubitt WD. The use of the dried blood spot sample inepidemiological studies. J. Clin. Pathol., 1999; 52 (9): 633-9.
4. http://www.whatman.com
5. Ingels A-S ME, Lambert WE, Stove CP. Determination of gammahydroxybutyric acid in dried blood spots using a simple GC-MS
method with direct “on spot” derivatization. Anal Bioanal Chem, 2010;
398:2173-2182.
6. Craft NE, Haitema T, Brindle LK, Yamini S, Humphrey JH, West KP Jr. Retinol Analysis in Dried Blood Spots by HPLC. J.Nutr., 2000; 130: 882-885.
7. Déglon J, Thomas A, Cataldo A, Mangin P, Staub C. On-line desorption of dried blood spot: A novel approach for the direct LC/MS
analysis of micro-whole blood samples. J Pharm Biomed Anal., 2009;49(4):1034-9.
8. Chaisomchit S, Wichajarn R, Janejai N, Chareonsiriwatana W.
Stability of Genomic DNA in Dried Blood Spots Stored on Filter Paper,
Southeast Asian J Trop Med Public Health, 2005; 36(1): 270-3.
9. Brambilla D, Jenning C, Aldrovrandi G et al. Multicenter evaluation ofuse of dried blood and plasma spot specimens in quantitative assays
for human immunodeficiency virus RNA: measurement, precision, andRNA stability. J Clin Microbiol 2003; 41: 1888-93.
10. Waters L, Kambugu A, Tibenderana H et al. Evaluation of filter papertransfer of whole-blood and plasma samples for quantifying HIV RNA
in subjects on antiretroviral therapy in Uganda. J Acquir Immune Defic
Syndr, 2007; 46: 590-3.
11. Johannessen A, Garrido C, Zahonero N et al. Dried blood spots
perform well in viral load monitoring of patients who receive antiretroviraltreatment in rural Tanzania. Clin Infect Dis, 2009; 49: 976-81.

12. Mbida AD, Sosso S, Fleri P et al. Measure of viral load by using the
Abbott Real-Time HIV-1 assay on dried blood and plasma spot specimens collected in 2 rural dispensaries in Cameroon. J Acquir Immune
Defic Syndr 2009; 52: 9-16.
13. Andreotti M, Pirillo M, Guidotti G et al. Correlation between HIV1 viral load quantification in plasma, dried blood spots, and driedplasma spots using the Roche COBAS Taqman assay. J Clin Virol,
2010; 1: 4-7.
14. van Deursen P, Oosterlaken T, Andre P et al. Measuring human immunodeficiency virus type 1 RNA loads in dried blood spot specimens
using NucliSENS EasyQ HIV-1 v2.0. J Clin Virol, 2010; 47: 120-5.
15. Viljoen J, Gampini S, Danaviah S et al. Dried blood spot HIV-1 RNA
quantification using open real-time systems in South Africa and Burkina
Faso. J Acquir Immune Defic Syndr, 2010; 55: 290-8.
16. Leelawiwat W, Young NL, Chaowanachan T et al. Dried blood
spots for the diagnosis and quantitation of HIV-1: stability studies andevaluation of sensitivity and specificity for the diagnosis of infant HIV-1
infection in Thailand. J Virol Methods, 2009; 155: 109-17.
17. Monleau M, Butel C, Delaporte E et al. Effect of storage conditionsof dried plasma and blood spots in HIV-1 RNA quantification and PCRamplification for drug resistance genotyping. J Antimicrob Chemother,
2010; 65: 1582-6.
18. Pirillo MF, Recordon-Pinson P, Andreotti M, Mancini MG, Amici R,
Giuliano M. Quantification of HIV-RNA from dried blood spots using
the Siemens VERSANTw HIV-1 RNA (kPCR) assay, J Antimicrob
Chemother, 2011; 66(12): 2823-6.
19. Wiseman JM, Evans CA, Bowen CL, Kennedy JH. Direct analysis
of dried blood spots utilizing desorption electrospray ionization (DESI)
mass spectrometry. Analyst, 2010; 135: 720-5.
20. Abu-Rabie P, Spooner N. Direct quantitative bioanalysis of drugs
in dried blood spot samples using a thin-layer chromatography mass
spectrometer interface. Anal. Chem., 2009; 81(10): 275-84.

NOI ABORDARI PENTRU ANALIZA SPOTULUI DE SANGE USCATREZUMAT
Tehnica spotului de sange uscat (DBS) este recunoscuta recent ca o metoda alternativa la analiza medicamentelor (analitilor) din
plasma in studii de farmacocinetica si toxicocinetica. Acest articol reprezinta un review despre diversele tipuri de analiza a picaturilorde sange uscat, pornind de la extractia off-line la cea on-line, precum si diversele sale aplicatii in domenii ca genetica, virusologie,
farmacologie, toxicologie. Aceasta tehnica permite micsorarea volumului de sange necesar pentru analiza, facand astfel posibile studiilede farmacocinetica pe animale mici de laborator. In ultimii ani s-au dezvoltat noi concepte de extractie, astfel s-a dezvoltat o metoda
de extractie in line pentru analiza compusilor farmaceutici din picaturile de sange uscat anterior unei analize cromatografice.
Cuvinte cheie : spotul de sange uscat, hartia DBS, farmacocinetica, toxicologie

2011.21.3 (71)  Fiziologia - Physiology

47

Physiology Department“Victor Babes” University of
Medicine and Pharmacy Timisoara

Romanian Society of Physiological
Sciences

PRELIMINARY ANNOUNCEMENT
Physiology of the XXIst century – from genotype to phenotype
The 11th National Congress of Romanian Society of Physiological Sciences
Timisoara, 10-12 May 2012
ORGANIZING COMMITTEE
President: Prof. Dr. Carmen Panaitescu
Secretaries: Asist. Univ. Dr. Daciana Nistor, Philologist Carmen Pitic
Prof. Dr. Smaranda Goţia
Conf. Dr. Gabriela Tănasie
Ş.l. Dr. Carmen Tatu
Ş.l. Dr. Lavinia Noveanu
Ş.l. Dr. Bojin Florina

Asist.Univ. Dr. Daniela Crîsnic
Asist.Univ. Dr. Adriana Gherbon
Asist.Univ. Dr. Laura Smaranda Goţia
Asist.Univ. Dr. Meda Deică
Asist.Univ. Dr. Sabine GrozaSCIENTIFIC COMMITTEE
Prof. Dr. Mureşan Adriana (UMF Iuliu Haţieganu Cluj Napoca)Preşedinte Societatea Română de Ştiinţe Fiziologice
Prof. Dr. Dobreanu Dan (UMF Tg. Mureş)Prof. Dr. Gusti Simona (UMF Craiova)
Prof. Dr. Iancău Maria (UMF Craiova)
Prof.Dr. Ion Ileana (UMF Ovidius Constanţa)Prof. Dr. Panaitescu Carmen (UMF Victor Babeş Timişoara)
Prof. Dr. Păunescu Virgil (UMF Victor Babeş Timişoara)
Prof. Dr. Sabău Marius (UMF Tg. Mureş)Prof. Dr. Saulea Aurel (USMF Chişinău)Prof. Dr. Schneider Francisc (UMF Arad)
Prof. Dr. Schneider Francisc (UMF Arad)

Prof. Dr. Zăgrean Leon (UMF Carol Davila Bucureşti)Conf. Dr. Bădărău Anca (UMF Carol Davila Bucureşti)Conf. Dr. Cismaş Petrişor (UMF Oradea)
Conf. Dr. Cismaş Petrişor (UMF Oradea)
Conf. Dr. Dragomir şerban (UMF Gr. T Popa Iaşi)Conf. Dr. Grama Maria (UMF Sibiu)
Conf. Dr. Motoc Daniela (UMF Arad)
Conf. Dr. Pumnea Manuela (UMF Sibiu)
Conf. Dr. Suciu şoimiţa (UMF Iuliu Haţieganu Cluj Napoca)
Conf. Dr. Tănasie Gabriela (UMF Victor Babeş Timişoara)
Ş.l. Dr. Bojin Florina (UMF Victor Babeş Timişoara)
Ş.l. Dr. Tatu Carmen (UMF Victor Babeş Timişoara)

You are welcomed with fundamental and applied researches on the following topics:
Inflammation-a double-edged sword
Respiration-from cellular mechanism to systemic process
Stem cells in the biotechnology era
Neurosciences-a multidisciplinary approach
Cardiovascular physiology-from experiment to clinical practicePhysiology curriculum-national harmonization and ECTS integration
The registration and abstract submission will be completed at http://www.umft.ro/newpage/cnf2012/index.htm before March 30th, 2012. The Congress is credited by Romanian College of Physicians.
48

Fiziologia - Physiology  2011.21.3 (71)