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 bOjIN
GAbRIELA 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)AccREDITED
AccREDITED By
By cNcSIS
cNcSIS -- 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
2013.23.1(77)
 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 2013.23.1(77)
Fiziologia‑ Physiology 2010supplement

CONTENTS
1. Review: Cancer-Associated Fibroblasts – Inhabitants of Tumor, However Obscure Underlying the Hallmarks of Cancer ....................................................................................4
Cernat L, Bojin F, Deica M, Tanasie G, Paunescu V, Panaitescu C
2. The Effects of Rutin and Omega-3 Polyunsaturated Fatty Acids on the Oxidant-Antioxidant Balance in a Rat Model of Adjuvant-Induced Arthritis .....................................13
Baltaru D, Chiş IC, Marton A, Socaciu C, Apostu D, Moldovan R, Vlad D, Mureşan A
3. Quercetin, Lycium Barbarum and Chitosan Reverse the Effects of Hypobaric Hypoxia and Exert Cardioprotective Effects in Rats ...................................................................18
Dumitrovici A, Chiş IC, Mureşan A, Marton A, Moldovan R, Vlad D, Borza G, Bolfa P
4. The Importance of Myo-Inositol in the Physiopathology of Hepatic Encephalopathy .........................................................................................................................................23
Scheau C, Lazăr M, Bunea M, Ciornei C, Papacocea R, Ion DA, Bădărău AI
5. Pulse Pressure: Prognostic Significance in the Stratification of Cardiovascular Risk .............................................................................................................................................26
Bâibâţa D, Ionescu G, Dragomir A, Mancaș S
6. Juvenile Nasopharyngeal Angiofibroma - Histological And Immunohistochemical Landmarks ........................................................................................................................31
Gidea DF, Iovanescu G, Ghiran RM, Domuta M, Cotulbea S, Horhat D
7. New Insight into the Pulmonary Vasculitis ............................................................................................................................................................................................................35
Cojocaru M, Rusu E, Vrabie CD, Cojocaru IM
8. Amiodarone Associated Optic Neuropathy, Is It a Real Thing? ..............................................................................................................................................................................38
Niculescu N, Muntean M, Zolog I

CUPRINS

1. Fibroblastele peritumorale – Componente tumorale cu rol definitoriu in cancer ..................................................................................................................................................4
Cernat L, Bojin F, Deica M, Tanasie G, Paunescu V, Panaitescu C
2. Efectul Rutinului şi al acizilor graşi polinesaturaţi Omega-3 asupra balanţei oxidanţi-antioxidanţi la şobolani cu artrită indusă de adjuvant ................................................13
Baltaru D, Chiş IC, Marton A, Socaciu C, Apostu D, Moldovan R, Vlad D, Mureşan A
3. Quercetinul, Lycium Barbarum şi Chitosanul contracarează efectele hipoxiei hipobare şi exercită un răspuns cardioprotector într-un model de şobolan .............................18
Dumitrovici A, Chiş IC, Mureşan A, Marton A, Moldovan R, Vlad D, Borza G, Bolfa P
4. Importanţa mio-inozitolului în fiziopatologia encefalopatiei hepatice ................................................................................................................................................................23
Scheau C, Lazăr M, Bunea M, Ciornei C, Papacocea R, Ion DA, Bădărău AI
5. Presiunea pulsului: semnificatia prognostica pentru stratificarea riscului cardiovascular ....................................................................................................................................26
Bâibâţa D, Ionescu G, Dragomir A, Mancaș S
6. Angiofibromul nasofaringian juvenil – caraceristici histologice şi imunohistochimice .......................................................................................................................................31
Gidea DF, Iovanescu G, Ghiran RM, Domuta M, Cotulbea S, Horhat D
7. Actualităţi în vasculita pulmonară .........................................................................................................................................................................................................................35
Cojocaru M, Rusu E, Vrabie CD, Cojocaru IM
8. Neuropatia asociată tratamentului cu amiodarona – este o realitate? .................................................................................................................................................................38
Niculescu N, Muntean M, Zolog I
2013.23.1 (77)  Fiziologia - Physiology

3

REVIEWCANCER-ASSOCIATED FIBROBLASTS – INHABITANTS
OF TUMOR, HOWEVER OBSCURE UNDERLYING THE
HALLMARKS OF CANCERLAURA CERNAT2, FLORINA BOJIN1, MEDA DEICA1, GABRIELA TANASIE1, VIRGIL PAUNESCU1, CARMEN PANAITESCU1
1
Department of Functional Sciences, “Victor Babes” University of Medicine and Pharmacy Timisoara
2
PhD student at “Victor Babes” University of Medicine and Pharmacy Timisoara

ABSTRACT

It becomes conspicuous the active role of stromal components in solid tumors proliferation, invasion and metastasis. Cancer-associatedfibroblasts (CAFs) or tumor-associated fibroblasts (TAFs) are the main stromal cells which compete towards the malignant properties.
It is undeniable that CAFs are distinguished from normal fibroblasts, epigenetically and perhaps genetically. Still, the origin and themolecular phenotype of CAFs are subjects of intensive debates. Any new findings regarding non-tumor elements and their interactions with the tumor cells could lead to new approaches to cancer therapy.
Key words: cancer-associated fibroblasts, tumor biology, stroma, cellular signaling, therapeutical targets

INTRODUCTION. HALLMARKS OF CANCER – INVETERATE AND NEW CONCEPTS
In a millenium issue article from January 2000, Hanahan
and Weinberg have been proposed a suite of six characteristicsof neoplastic disease, “hallmarks of cancer’, features which
consist of (1): self-sufficiency in growth signals; insensitivity to
anti-growth signals; evasion of apoptosis; limitless replicative potential; sustained angiogenesis; tissue invasion and metastasis.
Same authors return upon this concept and consider two
more hallmarks: reprogramming of energy metabolism andevade immune destruction (2).
Thinking about notable progress in cancer research, thetwo new traits are comprehensible and challenging for researchers. Past decades are extensive in understanding of molecularmechanisms, signaling pathways and recognition of active involvement of tumor microenvironment in neoplastic progression
with its consequences.
Alteration of signals involved in cellular growth regulation,
inactivation of tumor suppressors (such as RB protein (3) andTP53 protein), increase of antiapoptotic regulators expression(Bcl-2,Bcl-xL or of survival signals – Igf 1/2) (2), the significant
expression of telomerase, upregulation of VEGF and FGF familygene expression (4), downregulation or even mutational inactivation of E-cadherin (5), are only a few examples of multiple
mechanisms which are underlying the six hallmarks of cancer.

Acquiring by tumor cells a multitude of characteristics through
EMT (epithelial-mesenchymal transition) mechanism, leads
to ability of tumor cells to disseminate and to be resistant toapoptosis (6) (Figure 1).

Fig.1. The hallmarks of cancer (1)Emerging hallmarks proposed by Hanahan and Weinberg
are the reprogramming of cellular metabolism (so that the nurture
support will be assured) and the evasion of destructive activityof B cells, T cells, natural killer cells, macrophages that belong
to host (2). From these points will derive two more features: genomic instability and inflammation which maintain all neoplastic
characteristics (Figure 2).

Received January 4th 2013. Accepted February 20th 2013. Adress for correspondence: Laura Cernat, MD PhD student, “Victor Babes” University of Medicine andPharmacy Timisoara, Eftimie Murgu Square No. 2A, RO-3000041, Timisoara, phone/fax: +40256220479; e-mail: mlaura_73@yahoo.com

4

Fiziologia - Physiology  2013.23.1 (77)Fig.2. Emerging cancer hallmarks (2)These hallmarks of cancer and not only them ought to permit exploring of new therapeutic horizonts, as a possible broad
mapping could be assessed and the personalised medicine willbe the first therapeutic choice.
FIBROBLASTS VERSUS CAFS – CHARACTERISTICS,
ACTIVATION, PHENOTYPE
Beside alterations in cellular genome that develops theneoplastic potential of a tumor, recently data have revealed
the outstanding involvement of microenvironmental component(tumor stroma) in neoplastic initiation, progression, invasion andmetastasis (7,8).
Fibroblasts are elongated cells embedded in the connective
tissue and are responsible for its synthesis and maintenance (9).
They are non-vascular, non-epithelial and non-inflammatory cells
and form the essential cellular component of connective tissue.
Fibroblasts participate in the regulation of processes such as inflammation, wound healing and differentiation of their neighboring
epithelial cells (10). They synthesize fibrillar Extra Cellular Matrix(ECM) proteins, such as type I, III and V collagens and fibronectin.
At the same time, fibroblasts are also an important source of ECM
degrading proteases such as matrix metalloproteinases (MMPs)
which are involved in maintaining the ECM homeostasis. In
healthy state, the rate of proliferation and the metabolic capacityof fibroblasts are low. During the wound healing, fibroblasts acquire
secretory and contractile capacities and the index of proliferation
increases (11). At the same time, activated fibroblasts secretevarious chemokines, leading to recruitment of blood cells at thesite of injury. After the reparation of damaged tissue, fibroblasts
are returning to resting state (12). Main expression of fibroblast
in resting state and upon activation can be observed in Figure 3.
Fibroblasts display different phenotypic characteristics,
provided of anatomic region where they were isolated from(skin, articulation) (13). In addition they demonstrate diversity in
topographic expression for genes involved in extracellular matrix
synthesis, growth and differentiation, cell migration as well as
genes involved in genetic syndromes. Chipev and Simon (14)indicated that fibroblasts from different body sites differ in size,with palmar fibroblasts being smaller than non-glabrous derived
fibroblasts. In addition, growth kinetics and TGF (Transforming
Growth Factor) -β1 Receptor II expression as well as the ability
2013.23.1 (77)  Fiziologia - Physiology

to contract collagen lattices were found to differ with palmoplantar skin derived fibroblasts having lower receptor levels and anincreased mitotic rate. The authors speculated that this regional
diversity may in part account for localized susceptibility to disease
manifestation like scarring and keloid formation. Recent work on
oral mucosal fibroblasts has demonstrated the differences in thecapacity of these fibroblasts to reorganize collagen lattices byan increased matrix metalloproteinase (MMP)-2 expression ascompared to skin derived fibroblasts (15).
Additional patterns of fibroblasts heterogeneity have been
identified through the study of wounding and wound healing.
Wound healing proceeds in three interrelated phases (16):inflammation phase, proliferation phase and maturation phase.
Although fibroblasts have long been recognized as key cells
in those process, it is only recently that we have begun tounderstand the origin and phenotypic differences of fibroblasts
as related to the specific phases of wound healing. The initialinflammatory phase consists of an orderly extravasation of leucocytes like PMN and monocytes/macrophages into the wound.
It has been shown that a population of fibroblasts which arise
from blood-borne circulating progenitor cells migrate into thewound site and may account for as many as 10% of the cells
that infiltrate sites of acute tissue injury (17). These cells werealso termed fibrocytes and express markers for cells of both hematopoietic and mesenychmal origin. The collagen+/vimentin+/CD34+/CD13+/CD45+ positive fibrocyte cells may contributeto normal wound repair by antigen presentation but may also
participate in pathologic fibrotic responses (18).
In the final phase of wound healing, a specialized populationof differentiated fibroblasts occur (19). It has been demonstrated
that fibroblasts under mechanical tension and under the influenceof Platelet Derived Growth Factor (PDGF) express stress fibers.
This cell type is termed proto-myofibroblast. Additional tension,
TGF-β and a splice variant of fibronectin promote further differentiation into the myofibroblasts. These contractile myofibroblasts
express alpha smooth muscle actin and are involved in the production of extracellular matrix and tissue contraction. Provided
evidences showed that human wound fibroblasts represent afunctionally different population than fibroblasts isolated fromunwounded dermis (20). In their analysis, scientists concluded
that 30–40% of wound-derived fibroblasts were myofibroblasts
whereas only 1% of normal fibroblasts were alpha smoothmuscle actin positive. Early studies demonstrated that fibroblastsof high population doubling which have left the cell cycle cancarry out lattice contraction at least as effectively as cycling
cells from early passages, leading the authors to conclude thatthe contraction of wounds is not due to myofibroblasts. In vivo
data indicated that the number of myofibroblasts in the woundis dependent upon the amount of transplanted deeper dermal
portion (21). Unfortunately it remains unclear whether this effectis due to the matrix or the resident cell population. Regardlessof whether myofibroblasts are the true motile force for woundcontraction, there is general agreement that wound contraction
is an active cellular phenomenon depending on the activity of
viable fibroblasts. Biological control of these cells either by the5matrix or by surrounding cells may minimize scar contraction (22).
During tumor progression, tumorigenesis and stromagenesis occur as parallel processes. The normal stroma serves
as a natural tumorigenic barrier. During the initial stages oftumorigenesis, tumor cells induce normal stroma to become
primed and engage in a feedback loop between stroma andtumor, which further promotes both processes (23). Additional
alterations in the primed stroma will result in a series of gradualtransitions, which will culminate in a phenotypically identiﬁable
activated stroma (24) (25). Eventually, both stroma and tumor will
become independent of one another irreversibly committed to atransformed phenotype (26). The ‘point of no return’ manifested
either at the transition from primed to activated, or within theactivated stromagenic stage remains to be identiﬁed.
One promising candidate for a stroma-speciﬁc marker is theﬁbroblast activation protein (FAP), a dipeptidyl peptidase of theserine protease family (27,28). FAP displays a restricted pattern ofprotein expression and enzymatic activity since FAP is expressedby stromal ﬁbroblasts and ﬁbroblasts in granulation tissue duringwound healing, but not on ﬁbroblasts in normal stroma or byepithelial cells regardless of their transformation stage (29). The
selective expression of FAP postulates it as an attractive candidate
for early therapeutic intervention. FAP has been shown to induce
tumor growth in a murine animal model of tumorigenesis, andpretreatment with FAP antibodies attenuated FAP-dependent
tumor growth (28). Another protein whose expression was originally
thought to be restricted to the stroma is tenascin-C. Tenascin-C
was initially considered to be a stromal marker for malignancy,
since it was not detected in normal adult mammary tissue or its
expression was limited to ductile tissue (27). However, recent
studies have shown that tenascin-C is also expressed in breast
hyperplasia, dysplasia and benign breast neoplasia (30). Thus, therelevance of tenascin-C as a diagnostic marker for tumors may be
its enhanced levels of expression in primed and activated stroma.
On early stage of tumor, neoplastic cells form a lesion which
is surrounded and separated by the rest of tissue by a basement
membrane – carcinoma in situ (1). This membrane, along withcapillary vessels, imune cells, fibroblasts and ECM, is forming
the tumor reactive stroma (1,31). Due to an increased numberof fibroblasts and capillary vessels, an increased amount of typeI-collagen, it is assumed that this reactive stroma provides protumorigenic signals. Some studies carried out on cancer-prone
chickens are sustaining this assumption (32,33).

6

cmyk 6

Fig.3. Fibroblast in resting state (A) and activated fibroblast (B) (10)Fibroblasts are found in various proportions across the spectrum of carcinomas, constituting in many cases the preponderantcell population of the tumor stroma. The term ‘‘cancer-associatedﬁbroblast’’ includes at least two distinct cell types: (1) cells withsimilarities to the ﬁbroblasts that create the structural foundation
supporting most normal epithelial tissues and (2) myoﬁbroblasts,
whose biological roles and properties differ markedly from thoseof tissue-derived ﬁbroblasts. Myoﬁbroblasts are identiﬁable bytheir expression of α-smooth muscle actin (α-SMA). They arerare in most healthy epithelial tissues, although certain tissues,
such as the liver and pancreas, contain appreciable numbersof α-SMA-expressing cells. Myoﬁbroblasts transiently increasein abundance in wounds and areal so found in sites of chronic
inﬂammation. Although beneﬁcial to tissue repair, myoﬁbroblasts
are problematic in chronic inﬂammation, contributing to thepathological ﬁbrosis observed in tissues such as lung, kidney
and liver. Recruited myoﬁbroblasts and reprogrammed variants ofnormal tissue-derived ﬁbroblastic cells have been demonstrated
to enhance tumor phenotypes, notably cancer cell proliferation,
angiogenesis, and invasion and metastasis; their tumor promoting activities have largely been deﬁned by transplantation ofcancer-associated ﬁbroblasts ad mixed with cancer cells into
mice, and more recently by genetic and pharmacologic perturbation of their functions in tumor-prone mice (34-36).
Because they secrete a variety of extracellular matrix
components, cancer-associated ﬁbroblasts are implicated in
the formation of the desmoplastic stroma that characterizes
many advanced carcinomas. The full spectrum of functions
contributed by both subtypes of cancer-associated ﬁbroblasts
to tumor pathogenesis remains to be elucidated.
Fibroblasts communicate with cancer cells, resident epithelial cells, endothelial cells, pericytes and inflammatory cells
through the secretion of growth factors and chemokines. Throughthe increased deposition of collagen types I and III and de novo
expression of tenascin C they induce an altered extracellularmatrix microenvironment, which potentially provides additional
oncogenic signals, probably leading to accelerated cancer progression. Fibroblasts mediate the inflammatory response by
secreting chemokines such as monocyte chemotactic protein
1 (MCP1) and interleukins such as IL-1. Fibroblasts interact
with the microvasculature by secreting matrix metalloproteinFiziologia - Physiology  2013.23.1 (77)

ases (MMPs) and vascular endothelial growth factor (VEGF).
Fibroblasts also provide potentially oncogenic signals such astransforming growth factor-β (TGF-β) and hepatocyte growth factor (HGF) to resident epithelia, and directly stimulate cancer-cellproliferation and invasion by secreting growth factors such as
TGF-β and stromal-cell-derived factor 1 (SDF1) (10).
Once activated to differentiate, myoﬁbroblasts produce
a myriad of mediators, including TGF-β, VEGF, bFGF, hepatocyte growth factor and the insulin-like growth factor – 1(IGF-1) that contribute to the proliferation and expansion of thetumor cell clone. Additionally, TGF-β activation induces stromal
myoﬁbroblasts to overproduce various ECM components, including collagen I (27,37), and increased amounts of collagen types
VI, XV, and XVIII, tenascin-C, plasminogen activator inhibitor-1(PAI-1), and various growth factors are deposited in the ECM
of human skin (38). During tumorigenesis, changes in the ECM
microenvironment induce stromal ﬁbroblasts and tumor cells tomodify their complex network of signal transduction pathways(27,39). Although signaling pathways affected by TGF-β treatment comprise Smad pathways, as well as non-Smad signaling
events including the mitogen activated protein kinase pathways(24,40), additional work remains to clarify signaling cascades
downstreamof TGF-β stimulation.
B
Next to these molecular markers of fibroblasts activation,some other proteins expressed by stromal fibroblasts are recognised to have a prognostic value for solid tumors. In particular, apoor prognosis has been associated with expression in CAFs of
the hypoxia marker carbonic anhidrase IX in human lung adenocarcinoma (41), or periostin in cholangiocarcinoma (42), or p53
tumor suppressor in ductal carcinoma (43). On the other side,
expression in CAFs of Caveolin-1, PTEN or podoplanin correlateswith a favourable prognosis for several carcinomas (44-46).
In addition, genetic studies mainly carried out in breast
cancers reported CAF somatic mutations in TP53 and PTEN,
as well as gene copy number alteration at other loci in tumor
stroma (47). In keeping with this idea, p53 inactivation in stromal
fibroblasts, as well as that genetic inactivation of PTEN in CAFs
enhances tumor progression in breast carcinoma models (48)(44). These studies collectively evoke the idea that the tumor
promoting activity of CAFs may be, above all, founded on thesesomatic mutations in key tumor suppressor genes. In addition,
somatic alterations were consistently observed at a high frequency (>30%) in tumor adjacent fibroblasts (49).
Oppositely, more recent studies have alleged that genetic
alterations were detected only in cancer epithelial cells and notin the stroma (50), and copy number and loss of heterozygosity
analysis of CAFs derived from breast and ovarian carcinomas
showed that somatic genetic alterations in CAFs are extremelyrare and cannot be the basis of the carcinoma-promoting phenotypes of these cancers (51).
SIGNALING PATHWAYS BETWEEN CAFS AND TUMORCELLS – A GENUINELY MAZE
Cancer cells have the ability to divide indefinitely and2013.23.1 (77)  Fiziologia - Physiology

spread to different parts of the body during metastasis. In thepast decade, many of the molecular mechanisms underlying
tumorigenesis in cancer have been elucidated. JAK/STAT, Notch,
MAPK/ERK, PI3K/AKT, NF-κB, Wnt and TGF-β pathways are
involved in cancer progression. The genetic elements that arenecessary for tumorigenesis have been well characterized in thepast 20 years. Human somatic cells can become cancerous by
activation of defined genetic elements such as introduction of
oncogenic RAS, blockage of the p53/Rb pathway and activationof telomerase (52,53).
The JAK/STAT pathway is intimately linked to growth factor
signaling, apoptosis and the cellular immune response. Deregulated JAK/STAT signaling can contribute directly and indirectly
to tumorigenesis (54). Mutations, fusions, and/or amplificationof JAK/STAT signaling components, such as the HER2/neu- in
mammary and stomach carcinomas, or Epidermal Growth
Factor-Receptor (EGF-R) in breast, brain and stomach tumors,can confer hypersensitivity to mitogenic signals and promote
proliferation (55,56).
In some animal models, Notch plays a crucial role in theearliest stages of T-cell development and is dispensable forfurther differentiation to intrathymic T-cells. These results suggest that neoplasias could occur when Notch signaling remains
active beyond normal development (57). Recent studies have
implicated aberrant Notch signaling in human breast tumors,melanoma progression, medullablastoma and ovarian cancers.
In the case of human breast tumors, amplification of Notch receptors and the presence of ligands, such as Jagged-1 correlate witha more aggressive disease phenotype. This phenotype can be
relapsed by the expression of NUMB, a Notch antagonist (58).
RAS mutations are found in ∼45% of colon cancers and∼90% of pancreatic cancers (59). RAF mutations, in turn, arefound in just about two thirds of all melanoma (60). Disregulated
PI3K/AKT signaling has been observed in various cancers. Mutations in the PI3K/AKT pathway inhibitor and tumor suppressor
PTEN has been found in glioblastomas, lung carcinomas andmelanomas whereas AKT overexpression or overactivation has
been found in breast, ovarian, thyroid and other cancers (61).
In conclusion, the PI3K/AKT pathway is constitutively active
in numerous human cancers. Activation of this pathway canpromote cell survival and proliferation.
Mutations and miss-regulation of NF-κB signaling has
been involved in a variety of cancers (such as human B-cellmalignancies).
Wnt-signaling is active in the cycling stem cells that reside in
the bottom layer and turned off upon differentiation and migration
to the top layer. In a majority of colon cancers (90%), APC protein,which under normal states targets β-catenin for degradation, ismutated condition producing β-catenin stabilization and initiationof the Tcf/Lef transcriptional program (62). Accordingly, APC
deficient progenitor cells keep replicating, remain within the tissue, and increase the risk of gaining further mutations that alterthem and become malignant (63).
Mutations or downregulation of TGF-β receptors, inactivation of SMAD4 or p15 INK4B can be found in a variety of cancers.
7

Primarily, SMAD4 inactivation arises in 53% of human pancreatic
ductal adenocarcinomas (PDAC) (64) (65). Moreover, BMP2
is dramatically overexpressed in 98% of lung carcinomas (66)(67). TGF-β signaling can also amplify malignancy of epithelial
tumors by stimulating metastasis: heterogeneous processes
during which a cell disintegrates, develops increased motility
and invasiveness to enter the circulatory system and settles a
new niche within the body. In varoius aspects, metastasis resembles the highly coordinated cell movements that occur during
embryonic development. EMT during metastasis causes cellularadhesion molecules, such as E-cadherins and cytokeratins, tobe replaced by mesenchymal proteins N-cadherin and vimentin(68). Surprisingly, pathways and genes involved in EMT during
development such as TGF-β, GSC or TWIST, are re-expressed
during tumorigenesis and can initiate metastasis (69,70).
CAFs are actively implicated in communication with othercellular populations inside the tumor. In this condition, TGF-β,which is expressed by both cancer cells and CAFs, boosts tumorigenesis and causes the invasive phenotype and also avoiding
the immune defense (71,72). Hepatocyte growth factor (HGF),
also produced by CAFs, binds with high affinity to the receptor
c-Met. The overexpression of c-Met signaling induces tumor
proliferation, invasion and metastasis (73).
Figure 4 is illustrating the envolvement of CAFs in tumoral
signaling mechanisms.

Fig.4. Signaling with envolvement of CAFs (10)Many recent data reveal the essential role of WNT signaling
in extracelullar, cytoplasmic and nuclear key regulation, in orderto maintain tissues maintenance. WNT/PCP signaling pathway
is implicated in embryogenesis as well as in carcinogenesis.
Single nucleotide polymorphisms (SNPs) and copy number
polymorphisms (CNPs) of cancer associated genes are utilized
for the screening of cancer predisposed patients (74). Aberrant
activation of the WNT/PCP signaling pathway leads to abnormal
tissue polarity, which is one of characteristics of cancer cells.
SNPs and CNPs for WNT/PCP signaling molecules are importantcandidates for use in screening of cancer predisposition, especially for gastric cancer. The deviant activation of the WNT/PCP
signaling pathway also conducts to poor prognosis of human8

cancer patients via induction of invasion and metastasis. cDNA-PCR, microarray or ELISA reflecting the aberrant activationof WNT/PCP signaling pathway could be developed as novel
cancer prognostics (75).

Fig.5. WNT/PCP signaling pathway and human cancer (75). (A) Aberrant
activation of WNT/PCP signaling pathway leads to poor prognosis; (B) Clinicalapplications

EPITHELIAL - MESENCHYMAL TRANSITION (EMT) – ESSENTIAL IN BODY DEVELOPMENTAND POWERFUL “ALLY”
OF NEOPLASTIC THREAT
EMT possess an essential role in formation and differentiation of tissues and organs during the body development. Throughout the embryonic ontogeny, EMT induces intense phenotypicchanges that include the loss of cell-cell adhesion, the loss ofcell polarity, and the acquisition of migratory and invasive properties. During gastrulation, the embryonic epithelium undergoes
EMT to give rise to the mesoderm. During delamination of theneural crest, EMT is used to form a population of highly motile
cells that ultimately incorporate into many different tissues (76).
Induction of EMT alters cytoskeletal structure and leads to thebreakdown of interactions between cells, their neighbors, andthe underlying substratum. These phenotypic changes are
driven by alterations in the expression of many genes including
cytoskeletal components, transcription factors and enzymes. By
any chance the most well-studied EMT stimulus is transforming
growth factor (TGF)-β, which initiates and maintains EMT in avariety of biological systems (70).
Whereas embryonic EMT is normally a self-resolving process, EMT serving tissue repair or metastatic dissemination isoften correlated with concomitant inflammation and continues
until the provoking cause is eliminated (77). In this framework,
CAFs are active in sustaining a chronic pro-inflammatory drive,eliciting multiple effects concurring to induce/select cancer cell
phenotypes resistant to the hostile tumour microenvironment.
In incipient tumors, CAFs have been shown to orchestrate
macrophage recruitment and neovascularization in strict
dependence on nuclear factor-κB (NF-κB) activation (78). Furthermore, CAFs exert their propelling role for EMT by eliciting
similar pro-inflammatory pathways in metastatic cells, exploiting
oxidative stress and involving the activation of COX-2, NF-kB
and HIF-1 (79). The fact that cancer cells undergoing EMT in
response to CAF contact share with inflammatory cells thesame signals is in keeping with the idea that metastasis is aphenomenon reminiscent of the igratory/invasive behaviour of
inflammatory cells. Besides epigenetic mechanisms influencing
cross signalling between CAFs and cancer cells, malignancy may
Fiziologia - Physiology  2013.23.1 (77)

also be regulated by stromal mutations affecting the motility andaggressiveness of cancer cells.
Epithelial to mesenchymal transition (EMT) is thought to be
one of the sources of CAFs in tumors (77). Thus, epithelial cells,
through an EMT process, achieve mesenchymal characteristic
and become fibroblasts (80). This hypothesis arises from theevidence that epithelial cells exposed to MMP-driven oxidative
stress undergo DNA oxidation and experience mutations, thereby
undergoing to specialized EMT in which they transdifferentiate
into activated myofibroblasts (81,10). Studies found that whennormal fibroblasts were coinjected with cancer cells and thenisolated by flow cytometry from the resultant orthotopic tumors,fibroblasts expressed the characteristic proinflammatory CAF signature. It was previously shown that coculturing of pancreatic orlung cancer cells with stromal fibroblasts induced the expressionof COX-2 and IL-8 (82,83). The concept of in vivo education of
normal cells into tumor-promoting stromal cells was previously
described to the recruitment of monocyte progenitors and theirdifferentiation into tumor-associated macrophages, which arefunctionally distinct from resident tissue macrophages (84).
EMT allows cancer cells to adopt a mesenchymal cell phenotype, characterized by an enhanced migratory capacity andinvasiveness (77). EMT is induced by many factors (i.e. PDGF,
TGFβ, EGF, etc) and is mediated by the activation of typicaltranscription factors like Snail, Slug, Twist and FOXC2 (85).
Similarly, proliferating endothelial cells might contribute to CAF
via endothelial to mesenchymal transition (EndMT). This process
is characterized by the loss of endothelial markers like CD31, theexpression of mesenchymal markers like FSP-1 and SMA under
the stimulation of TGFβ , a growth factor abundantly present
within tumor microenvironment (77). Conversely to embryonic
EMT, EMT serving tissue repair or metastatic dissemination isoften correlated with tissue inflammation and persists until the
provoking stimulus is eliminated (77). In this context CAFs playa key role in sustaining a chronic pro-inflammatory stimulus,
giving rise to multiple effects and culminating in prompting theescape from the hostile tumor microenvironment.
Inducing the EMT of tumor cells, CAFs contribute to theepigenetic programme leading to invasive process engaging amesenchymal, motile and proteolytic phenotype (77) (6). In addition to the pro-migratory stimulus, EMT has also been correlatedwith the induction of a cancer stem cell phenotype. Indeed, inbreast cancer cells, EMT, due to the overexpression of Snail or
Twist transcription factors, is correlated with increased CD44/
CD24 ratio as well as with the generation of tumor initiating cells
which spread metastases (86,87). Congruent with this, Giannoni(79) reported that CAFs isolated from prostate carcinoma specimens, by means of activating the EMT epigenetic programme,
promote/select the generation of cancer stem cells. Indeed,
CAFs affect the self-renewal ability of carcinoma cells andenhance their expression of cancer stem cell markers (CD133+
and high CD44/CD24 ratio) and the formation of non-adherent
prostaspheres, a property associated with prostate stem cells(86-88). Analysis of tumor-forming ability and spontaneous lungmetastasis formation after contact with CAFs revealed that the2013.23.1 (77)  Fiziologia - Physiology

activated stroma contributes to generate a population of prostatecancer stem cells with defined ability to form primary tumorsand distant metastases (88). In keeping with this, in a onditional
PTEN deletion mouse model of prostate adenocarcinoma, CAFs
confirm their key role in the regulation of stemness properties
as they enhance spheroid formation and prostate glandular
structures with lesions, high proliferative index and tumor-like
histopathology (89).
Mutations in stromal cells have been attributed to several
reasons. First, EMT induced by CAFs in cancer cells may
be directly responsible for the accumulation of mutations bytumor or stromal cells. Indeed, stromal-derived MMP-3, whichis frequently upregulated in breast cancers, induces genomic
instability through the upregulation of ROS (81). Alternatively,
mutated stromal cells might directly derive from cancer cells thathave undergone EMT and achieve the characteristics of CAFs
(90). Finally, in mouse prostate carcinoma cells, a paracrine
mechanism determines a selective pressure for the expansionof a p53-lacking subpopulation of CAFs. These CAFs lacking
p53 contribute to cancer invasion and to the eventual loss of
p53in the epithelium. Furthermore, p53 mutations in CAFs arealso significantly associated with lymph node metastases inseveral sporadic breast cancers (91). Whatever their origin,these mutated CAFs show a profoundly altered behaviour andconcur to tumor malignancy.
Because the EMT process generates cells of mesenchymal,
migratory, contractile phenotype, these signals may cause bothfeed-back and feed-forward loops that dynamically impact thepatterning of the tissues. Biological systems generate, transmit
and concentrate mechanical stress into spatial patterns; these
gradients in mechanical stress may serve to spatially patterndevelopmental and pathologic EMTs (92). Future studies investigating the signaling and mechanical regulatory networks
within tissues undergoing EMT may reveal points and targets
to augment the process (in the case of wound healing) or cut it
short (in the case of metastasis).
CONCLUSIONS. FUTURE INSIGHTSThe stroma is generally made up of other cell types, extracellular matrix, and the vasculature and lymphatic systems
feeding the tumors. Cell types in the tumor stroma include
fibroblasts, myofibroblasts, granulocytes, macrophages, mesenchymal stem cells, and lymphocytes (93). In this context of thetumor microenvironment, some of these cell types additionallychange phenotype and become accessories to tumorigenesis
and metastasis. Hypoxic stress also promotes angiogenesis,
lymphangiogenesis, and inflammation, leading to recruitmentof new nutrient supplies and inflammatory cells that can thenfurther potentiate the metastatic phenotype (94).
It is well known that origins of many human diseases, including cancer, diabetes, and neural disorders, are in the functioning(and malfunctioning) of signaling components. It is required to understand how individual components function within the context
of the entire tumoral system. Tumoral microenvironment, through9

its complicated and heterogenous mechanisms of maintaining
the tumor homeostasis, could comprise a considerable variantsof novel targets for anti-neoplastic therapeutics.
Nevertheless, there are enough obstacles which have to be
regarded: the cellular components (CAFs, inflamatory cells, ECs)
are not malignant cells, in consequence the therapeutic target
must be as high as specific in order to avoid normal cells to beaffected; there are a lot of mutual interactions between CAFs
and tumor cells, multiple signaling interactions, such that it ishighly difficult to establish a therapeutic target without the tumor
to find an escape way or to develop resistance; there is strong
evidence, in vitro and in vivo, demonstrating that EMT grants
dedifferentiated malignant epithelial cells with mesenchymal,
migratory and proteolytic properties that are required for local
tumor invasiveness, a mandatory to metastasis formation.
Research on the role of EMT in cancer progression, isfully ongoing. It is plausible that specific targeting of EMT could
potentially serve to decrease metastasis and overcome drug
resistance. At the same time, depicting the molecular crosstalk
between stromal cells and carcinoma cells may provide invaluable insights into the pathophysiological processes involvedin cancer. In this respect, the occurence and improvement ofvaried methods of investigation based on high-quality, basic andtranslational research (eg. development of new mouse models,
multiscale mathematical models, improved in vivo imaging
techniques and the identification of new molecules important inregulating stroma-tumor interactions, etc.)
AKNOWLEDGMENTS
This work was supported by the Sectorial Operational Programme for Human Resources Development, financed fromthe European Social Fund, FSE POSDRU 107/1.5/S/78702.
REFERENCES
1. Hanahan D, Weinberg RA. The hallmarks of cancer. Cell, 2000;
100: 57-70.
2. Hanahan D, Weinberg RA. Hallmarks of Cancer: The Next Generation. Cell, 2011; 144: 646-674.
3. Burkhart DL, Sage J. Cellular mechanisms of tumour suppression by
the retinoblastoma gene. Nat. Rev. Cancer, 2008; 8: 671-682.
4. Ferrara N. Vascular endothelial growth factor. Arterioscler. Thromb.
Vasc. Biol. 2009; 29: 789-791.
5. Berx G, van Roy F. Involvement of members of the cadherin superfamily in cancer. Cold Spring Harb. Perspect. Biol. 2009; 1, a003129.
6. Thiery JP, Acloque H, Huang RY, Nieto MA. Epithelial-mesenchymaltransitions in development and disease. Cell, 2009; 139: 871-890.
7. Qian BZ, et al. CCL2 recruits inflammatory monocytes to facilitate
breast-tumour metastasis. Nature, 2011; 475: 222-225.
8. Soria G, et al. Concomitant expression of the chemokines RANTES
and MCP-1 in human breast cancer: a basis for tumor-promotinginteractions. Cytokine 44, 191-200 (2008)
9. Gerber, P.A., Hippe, A., Buhren, B A., Muller, A. & Homey, B. Chemokines in tumor-associated angiogenesis. Biol Chem. 2009; 390:
1213-1223.
10. Kalluri R, Zeisberg M. Fibroblasts in cancer. Nat Rev Cancer,
2006; 6: 392-401.

10

11. Xouri G, Christian S. Origin and function of tumor stroma fibroblasts.
Seminars in Cell & Developmental Biology, 2010; 21: 40-46.
12. Tomasek JJ, Gabbiani G, Hinz B, Chaponnier C, Brown RA. Myofibroblasts and mechano-regulation of connective tissue remodelling.
Nat Rev Mol Cell Biol, 2002; 3:349-363.
13. Rinn JL, Wang JK, Liu H, Montgomery K, van de Rijn M, Chang
HY. A systems biology approach to anatomic diversity of skin. J Invest
Dermat, 2008; 128: 776-782.
14. Chipev CC, Simon M. Phenotypic differences between dermal
fibroblasts from different body sites determine their responses to tension
and TGFbeta1. BMC Dermatol, 2002; 2:13.
15. Stephens P, Davies KJ, Occleston N, Pleass RD, Kon C, Daniels
J, Khaw PT, Thomas DW. Skin and oral fibroblasts exhibit phenotypic
differences in extracellular matrix organization and matrix metalloproteinase activity. Br J Dermatol, 2001; 144: 229-237.
16. Clark RAF, Fitzpatrick TB, Eisen AZ, Wolff K, Freedberg IM, Austen
KF. Mechanisms of cutaneous wound repair; dermatology in general
medicine. McGraw Hill, New York, 1993: 473-486.
17. Bucala R, Spiegel LA, Chesney J, Hogan M, Cerami A. Circulating
fibrocytes define a new leukocyte subpopulations that mediates tissue
repair. Mol Med, 1994; 1: 71-81.
18. Chesney J, Bacher M, Bender A, Bucala R. The peripheral blood
fibrocyte is a potent antigen-presenting cell capable of priming naive T
cells in situ. Proc Natl Acad Sci USA 1997; 94:6307-12.
19. Gabbiani G. The myofibroblast in wound healing and fibrocontractive
diseases. J Pathol, 2003; 200:500-3.
20. Germain L, Jean A, Auger F, Garrel DR. Human wound healing
fibroblasts have greater contractile properties than dermal fibroblasts.
J Surg Res, 1994; 57: 268-273.
21. Rudolph R, Vande J, Berg G, Pierce F. Changing concept inmyofibroblast function and control. In: Janssen H, Rooman JIS (eds)
Wound healing. Wrightson Biomedical Publishing Ltd., Petersfield,
1991: 103-115.
22. Bell E. Strategy of the selection of scaffolds for tissue engineering.
Tissue Eng, 1995; 1:163-179.
23. Dvorak HF. Tumors: wounds that do not heal. Similarities between
tumor stroma generation and wound healing. N Engl J Med, 1986;
315: 1650-9.
24. De Wever O, Mareel M. Role of tissue stroma in cancer cell invasion.
J Pathol, 2003; 200: 429-47.
25. Weaver VM, Gilbert P. Watch thy neighbor: cancer is a communal
affair. J Cell Sci, 2004; 117: 1287-90.
26. Edlund M, Sung SY, Chung LW. Modulation of prostate cancer
growth in bone microenvironments. J Cell Biochem, 2004; 91: 686-705.
27. Kunz-Schughart LA, Knuechel R. Tumor-associated fibroblasts (partI): active stromal participants in tumor development and progression.
Histol Histopathol, 2002; 17: 599-621.
28. Cheng JD, Dunbrack Jr RL, Valianou M, Rogatko A, Alpaugh RK,
Weiner LM. Promotion of tumor growth by murine fibroblast activation
protein, a serine protease, in an animal model. Cancer Res, 2002;
62: 4767–72.
29. Huber MA, Kraut N, Park JE, Schubert RD, Rettig WJ, Peter RU,
et al. Fibroblast activation protein: differential expression and serine
protease activity in reactive stromal fibroblasts of melanocytic skintumors. J Invest Dermatol, 2003; 120: 182-8.
30. Moch H, Torhorst J, Durmuller U, Feichter GE, Sauter G, Gudat
F. Comparative analysis of the expression of tenascin and establishedprognostic factors in human breast cancer. Pathol Res Pract, 1993;
189: 510-4.
31. Ronnov-Jessen L, Petersen OW, Bissell MJ. Cellular changes
involved in conversion of normal to malignant breast: importance ofFiziologia - Physiology  2013.23.1 (77)the stromal reaction. Physiol. Rev., 1996; 76, 69-125.
32. Dolberg DS, Hollingsworth R, Hertle M, Bissell MJ. Wounding and
its role in RSV-mediated tumor formation. Science, 1985; 230: 676-678.
33. Sieweke MH, Thompson NL, Sporn MB, Bissell MJ. Mediation ofwound-related Rous sarcoma virus tumorigenesis by TGF-β. Science,
1990; 248: 1656-60.
34. Dirat B, Bochet L, Escourrou G, Valet P, Muller C. Unravelingthe
obesity and breast cancer links: a role for cancer-associated adipocytes?
Endocr. Dev. 2010; 19: 45-52.
35. Pietras K, Ostman A. Hallmarks of cancer: interactions with thetumor stroma. Exp. Cell Res., 2010; 316: 1324-31.
36. Shimoda M, Mellody KT, Orimo A. Carcinoma-associated fibroblasts
are a rate-limiting determinant for tumour progression. Semin. Cell Dev.
Biol. 2010; 21: 19-25.
37. Dugina V, Fontao L, Chaponnier C, Vasiliev J, Gabbiani G. Focal
adhesion features during myofibroblastic differentiation are controlled
by intracellular and extracellular factors. J Cell Sci, 2001; 114:3285-96.
38. Hsu MY, Meier F, Herlyn M. Melanoma development and progression: a conspiracy between tumor and host. Differentiation, 2002; 70:
522-36.
39. Cunha GR, Matrisian LM. It’s not my fault, blame it on my microenvironment. Differentiation 2002; 70: 469-72.
40. Leask A, Abraham DJ. TGF-beta signaling and the fibrotic response.
FASEB J, 2004; 18: 816-27.
41. Nakao M, Ishii G, Nagai K, Kawase A, Kenmotsu H, Kon-No H,
Hishida T, Nishimura M, Yoshida J, Ochiai A. Prognostic significanceof carbonic anhydrase IX expression by cancer-associated fibroblasts
in lung adenocarcinoma. Cancer, 2009; 115: 2732-43.
42. Utispan K, Thuwajit P, Abiko Y, Charngkaew K, Paupairoj A, Chau-in
S, and Thuwajit C. Gene expression profiling of cholangiocarcinomaderived fibroblast reveals alterations related to tumor progression andindicates periostin as a poor prognostic marker. Mol Cancer, 2010; 9: 13.
43. Hasebe T, Tamura N, Okada N, Hojo T, Akashi-Tanaka S, Shimizu
C, Tsuda H, Shibata T, Sasajima Y, Iwasaki M, and Kinoshita T. p53
expression in tumor-stromal fibroblasts is closely associated with thenodal metastasis and outcome of patients with invasive ductal carcinoma
who received neoadjuvant therapy. Hum Pathol, 2010; 41: 262-270.
44. Trimboli AJ, Cantemir-Stone CZ, Li F, Wallace JA, Merchant A, etal. Pten in stromal fibroblasts suppresses mammary epithelial tumours.
Nature 2009; 461: 1084-1091.
45. Witkiewicz AK, Dasgupta A, Sotgia F, Mercier I, Pestell RG, Sabel M,
Kleer CG, Brody JR, and Lisanti MP. An absence of stromal caveolin-1
expression predicts early tumor recurrence and poor clinical outcome
in human breast cancers. Am J Pathol 2009; 174: 2023-34.
46. Yamanashi T, Nakanishi Y, Fujii G, Akishima-Fukasawa Y, Moriya Y,
Kanai Y, Watanabe M, and Hirohashi S. Podoplanin expression identifiedin stromal fibroblasts as a favorable prognostic marker in patients withcolorectal carcinoma. Oncology 2009; 77: 53-62.
47. Tuhkanen H, Anttila M, Kosma VM, Yla-Herttuala S, Heinonen S,
Kuronen A, Juhola M, Tammi R, Tammi M, and Mannermaa A. Genetic
alterations in the peritumoral stromal cells of malignant and borderline
epithelial ovarian tumors as indicated by allelic imbalance on chromosome 3p. Int J Cancer 2004; 109: 247-252
48. Hill R, Song Y, Cardiff RD, and van DT. Selective evolution of stromal
mesenchyme with p53 loss in response to epithelial tumorigenesis. Cell
2005; 123: 1001-1011.
49. Kurose K, Gilley K, Matsumoto S, Watson PH, Zhou XP, and Eng C.
Frequent somatic mutations in PTEN and TP53 are mutually exclusive
in the stroma of breast carcinomas. Nat Genet 2002; 32: 355-357.
50. Allinen M, Beroukhim R, Cai L, Brennan C, Lahti-Domenici J, Huang
H, Porter D, Hu M, Chin L, Richardson A, Schnitt S, Sellers WR, and2013.23.1 (77)  Fiziologia - Physiology

Polyak K. Molecular characterization of the tumor microenvironment inbreast cancer. Cancer Cell 2004; 6: 17-32.
51. Qiu W, Hu M, Sridhar A, Opeskin K, Fox S, Shipitsin M, Trivett
M, Thompson ER, Ramakrishna M, Gorringe KL, Polyak K, Haviv I,
and Campbell IG. No evidence of clonal somatic genetic alterations incancer-associated fibroblasts. Nat Genet. 2008; 40(5): 650-5.
52. Hahn WC, Counter CM, Lundberg AS, Beijersbergen RL, Brooks
MW, Weinberg RA. Creation of human tumour cells with defined genetic
elements. Nature, 1999; 400: 464-468.
53. Gupta GP, Massague J. Cancer metastasis: Building a framework.
Cell, 2006; 127: 679-695.
54. Bromberg J. Stat proteins and oncogenesis. J Clin Invest, 2002;
109: 1139-42.
55. Slamon DJ, Clark GM, Wong SG, Levin WJ, Ullrich A, McGuire WL.
Human breast cancer: Correlation of relapse and survival with amplification of the HER-2/neu oncogene. Science, 19987; 235, 177-182.
56. Yarden Y, Ullrich A. Growth factor receptor tyrosine kinases. Annual
Review of Biochemistry, 1988; 57: 443-478.
57. Roy M, Pear WS, Aster JC. The multifaceted role of Notch in cancer.
Current Opinion in Genetics & Development, 2007; 17: 52-59.
58. Stylianou S, Clarke RB, Brennan K. Aberrant activation of notch
signaling in human breast cancer. Cancer Research, 2006; 66: 1517-25.
59. Katz M, Amit I, Yarden Y. Regulation of MAPKs by growth factors
and receptor tyrosine kinases. Biochimica et Biophysica Acta, 2007.
60. Sebolt-Leopold JS, Herrera R. Targeting the mitogen-activatedprotein kinase cascade to treat cancer. Nature Reviews. Cancer, 2004;
4: 937-947.
61. Vivanco I, Sawyers CL. The phosphatidylinositol 3-Kinase AKT
pathway in human cancer. Nature Reviews. Cancer, 2002; 2: 489-501.
62. Giles RH, van Es JH, Clevers H. Caught up in a Wnt storm: Wnt
signaling in cancer. Biochimica et Biophysica Acta, 2003; 1653: 1-24.
63. Reya T, Clevers H. Wnt signalling in stem cells and cancer. Nature,
2005; 434: 843-850.
64. Hansel DE, Kern SE, Hruban RH. Molecular pathogenesis ofpancreatic cancer. Annual Review of Genomics and Human Genetics,
2003; 4: 237-256.
65. Bardeesy N, Cheng KH, Berger JH, Chu GC, Pahler J, Olson P,
et al. Smad4 is dispensable for normal pancreas development yet
critical in progression and tumor biology of pancreas cancer. Genes &Development, 2006; 20: 3130-46.
66. Langenfeld EM, Calvano SE, Abou-Nukta F, Lowry SF, Amenta P,
Langenfeld J. The mature bone morphogenetic protein-2 is aberrantly
expressed in non-small cell lung carcinomas and stimulates tumor
growth of A549 cells. Carcinogenesis, 2003; 24; 1445-54.
67. Langenfeld EM, Kong Y, Langenfeld J. Bone morphogenetic protein 2 stimulation of tumor growth involves the activation of Smad-1/5.
Oncogene, 2006; 25; 685-692.
68. Thiery JP, Sleeman JP. Complex networks orchestrate epithelialmesenchymal transitions. Nature Reviews. Molecular Cell Biology,
2006; 7: 131-142.
69. Hartwell KA, Muir B, Reinhardt F, Carpenter AE, Sgroi DC, Weinberg RA. The Spemann organizer gene, Goosecoid, promotes tumor
metastasis. PNAS, 2006; 103: 18969-74.
70. Zavadil J, Bottinger EP. TGF-beta and epithelial-to mesenchymaltransitions. Oncogene, 2005; 24, 5764-74.
71. Siegel PM, Massague J. Cytostatic and apoptotic actions of TGFbeta in homeostasis and cancer. Nat Rev Cancer 2003;3:807– 821.
72. Gorelik L, Flavell RA. Immune-mediated eradication of tumors
through the blockade of transforming growth factor-beta signaling in T
cells. Nat Med 2001; 7: 1118-22.
73. Benvenuti S, Comoglio PM. The MET receptor tyrosine kinase in11

invasion and metastasis. J Cell Physiol 2007;213:316 –325.
74. Katoh M, Katoh M. Pharmacogenomics on gastric cancer. Cancer
Biol Ther, 2004; 3: 566-567.
75. Katoh M. WNT/PCP signaling pathway and human cancer. Oncology
Reports, 2005; 14: 1583-88.
76. Nieto MA. The early steps of neural crest development. Mech Dev,
2001; 105: 27-35.
77. Kalluri R. EMT: When epithelial cells decide to become mesenchymal-like cells. J Clin Invest., 2009; 119(6): 1417-19.
78. Wu Y, Deng J, Rychahou PG, Qiu S, Evers BM, Zhou BP. Stabilization of snail by NF-kappaB is required for inflammation-induced cell
migration and invasion. Cancer Cell, 2009; 15(5): 416-428.
79. Giannoni E, Bianchini F, Calorini L, Chiarugi P. Cancer associatedfibroblasts exploit reactive oxygen species through a pro-inflammatory
signature leading to epithelial mesenchymal transition and stemness.
Antioxidand & Redox Signaling, 2011; 14: 2361-71.
80. Radisky DC, Kenny PA, and Bissell MJ. Fibrosis and cancer: do
myofibroblasts come also from epithelial cells via EMT? J Cell Biochem
2007; 101: 830-839.
81. Radisky DC, Levy DD, Littlepage LE, Liu H, Nelson CM, Fata JE,
Leake D, Godden EL, Albertson DG, Nieto MA, Werb Z, and Bissell MJ.
Rac1b and reactive oxygen species mediate MMP-3-induced EMT andgenomic instability. Nature 2005; 436: 123-127.
82. Anderson IC, Mari SE, Broderick RJ, Mari BP, Shipp MA. The
angiogenic factor interleukin 8 is induced in non-small cell lung cancer/
pulmonary fibroblast cocultures. Cancer Res. 2000; 60: 269-272.
83. Sato N, Maehara N, Goggins M. Gene expression profiling oftumor-stromal interactions between pancreatic cancer cells and stromal
fibroblasts. Cancer Res. 2004; 64: 6950-56.
84. Pollard JW. Tumour-educated macrophages promote tumour progression and metastasis. Nat. Rev. Cancer, 2004; 4: 71-78.

85. Medici D, Hay ED, Olsen BR. Snail and Slug promote epithelialmesenchymal transition through beta-catenin-T-cell factor-4-dependent
expression of transforming growth factor-beta3. Mol Biol Cell 2008;
19: 4875-87.
86. Blick T, Hugo H, Widodo E, Waltham M, Pinto C, Mani SA, et al.
Epithelial mesenchymal transition traits in human breast cancer celllines parallel the CD44(hi/)CD24 (lo/-) stem cell phenotype in humanbreast cancer. Journal of Mammary Gland Biology and Neoplasia,
2010; 15(2): 235-252.
87. Mani SA, Guo W, Liao MJ, Eaton EN, Ayyanan A, Zhou AY, et al.
The epithelial–mesenchymal transition generates cells with propertiesof stem cells. Cell, 2008; 133(4): 704-715.
88. Giannoni E, Bianchini F, Masieri L, Serni S, Torre E, Calorini L, etal. Reciprocal activation of prostate cancer cells and cancer-associatedfibroblasts stimulates epithelial–mesenchymal transition and cancer
stemness. Cancer Research, 2010; 70(17): 6945-56.
89. Liao CP, Adisetiyo H, Liang M, Roy-Burman P. Cancer-associatedfibroblasts enhance the gland-forming capability of prostate cancer stem
cells. Cancer Research, 2010; 70(18): 7294-7303.
90. De WO, Pauwels P, De CB, Sabbah M, Emami S, Redeuilh G, etal. Molecular and pathological signatures of epithelial–mesenchymaltransitions at the cancer invasion front. Histochemistry and Cell Biology,
2008; 130(3): 481-494.
91. Patocs A, Zhang L, Xu Y, Weber F, Caldes T, Mutter GL, et al. Breastcancer stromal cells with TP53 mutations and nodal metastases. The
New England Journal of Medicine, 2007; 357(25): 2543-51.
92. Gjorevski N, et al. Regulation of Epithelial-Mesenchymal Transition by Transmission of Mechanical Stress through Epithelial Tissues.
Cancer Microenvironment, 2012; 5: 29-38.
93. Chaffer CL, Weinberg RA. A perspective on cancer cell metastasis.
Science 2011; 331: 1559-1564.

FIBROBLASTELE PERITUMORALE – COMPONENTE TUMORALE CU ROL
DEFINITORIU IN CANCERREZUMAT

Este evident rolul activ al componentei stromale in proliferarea, invazia si metastazarea tumorilor solide. Fibroblastele peri-tumorale
sunt principalele elemente celulare stromale care contribuie la dobandirea caracterelor maligne. Se cunoaste faptul ca fibroblastele
peri-tumorale difera de fibroblastele normale din punct de vedere epigenetic, posibil si genetic. Totusi, originea si caracterizarea
fenotipica moleculara sunt aspecte intens dezbatute. Elucidarea caracteristicilor si comportamentului celulelor non-tumorale precum
si interactiunea acestora cu celulele tumorale pot dezvalui noi tinte terapeutice in bolile neoplazice.
Cuvinte-cheie: fibroblaste peri-tumorale, biologia tumorii, stroma, semnalizare celulara, tinte terapeutice12

Fiziologia - Physiology  2013.23.1 (77)THE EFFECTS OF RUTIN AND OMEGA-3
POLYUNSATURATED FATTY ACIDS ON THEOXIDANT-ANTIOXIDANT BALANCE IN A RATMODEL OF ADJUVANT-INDUCED ARTHRITISDOINA BALTARU1†, IRINA C. CHIŞ2†, ADRIANA MARTON3, CARMEN SOCACIU4, DRAGOŞ
APOSTU5, REMUS MOLDOVAN2, DELIA VLAD6, ADRIANA MUREŞAN2
1”Constantin Papilian” Military Emergency Hospital, Cluj-Napoca, Romania
2
Department of Physiology, University of Medicine and Pharmacy “Iuliu Haţieganu”, Cluj-Napoca, Romania
3
Nephrology Clinic, Emergency County Hospital, Cluj-Napoca, Romania
4
Department of Agrifood Chemistry and Biochemistry, USAMV, Cluj-Napoca, Romania
5
Student, University of Medicine and Pharmacy “Iuliu Haţieganu”, Cluj-Napoca, Romania
6
Section of Cardiology, General Hospital Nikaia Pireus, Grecia†

Equal contributors

ABSTRACT

Oxidative stress plays an important role in the pathogenesis of rheumatoid arthritis (RA). Reactive oxygen species (ROS) produced
during cellular oxidative phosphorylation and during the oxidative burst by activated phagocytes exceed the physiological buffering
capacity and result in oxidative stress. The excessive production of ROS can damage proteins, lipids, nucleic acids and matrix components. Patients with RA have an altered antioxidant defense capacity. The purpose of this study was to observe and quantify the
antioxidant effects of rutin and omega-3 polyunsaturated fatty acids (PUFA) in a rat model of adjuvant-induced arthritis (AIA). Fivegroups of rats were compared: a non-arthritic control group and four AIA groups treated via an intragastric tube for 21 days, 7 days
after the induction of AIA, with: vehicle (saline solution), non-steroidal anti-inflammatory agents (indomethacin), rutin or omega-3(PUFA) respectively. The arthritic score was determined in AIA rats as an indicator of destructive arthritis-associated clinical changes.
After 28 days the serum levels of the malondialdehyde (MDA), carbonylated proteins (CP), sulfydryl groups (SH), hydrogen donor
ability (HD) and glutathione (GSH) were measured. The results indicated an increase in the oxidative stress as seen by the increasein free radical production, MDA and CP levels, and impaired antioxidant defense systems shown by HD, SH and GSH levels in AIA
rats. The MDA and CP levels were significantly decreased and HD, SH and GSH levels were significantly increased after the oral
administration of rutin. Omega-3 (PUFA) increased the MDA, CP, HD, SH levels and decreased the GSH level. This study suggeststhat rutin and omega-3 (PUFA) treatment can be beneficial in attenuating the oxidative stress associated with RA.
Key words: oxidative stress, adjuvant-induced arthritis, rutin, rats.

INTRODUCTION
Rheumatoid arthritis (RA) is a frequent chronic diseasecharacterized by persistent inflammation in multiple joints.
Rheumatoid arthritis is associated with a high cardio-vascularmortality, which exceeds that of the general population. Pain
and inflammation are the initial symptoms, followed by various
degrees of joint destruction. Prostaglandin production (PG) by
cyclooxygenase (COX) from the arachidonic acid’s metabolism
is one of the main pathways in the pathogenesis of acute inflammation. Non-steroidal anti-inflammatory drugs are used for thetreatment of RA but have many side effects because of their COX
inhibition (8). Therefore, the anti-inflammatory drugs available
nowadays are not a very good therapy option.
Recent research shows that oxidative stress plays an important role in the pathogenesis of RA (7). Reactive oxygen species(ROS) produced during the cellular oxidative phosphorylation andduring the oxidative burst by activated phagocytes exceed thephysiological buffering capacity and result in oxidative stress. The
excessive production of ROS can damage proteins, lipids, nucleic
acids and matrix components. Epidemiological studies showed
an inverse association between the antioxidant intake and theincidence of RA, and between the antioxidant level and inflammation (13, 14). During the lipid peroxidation, polyunsaturated fatty
acids are oxidized, generating peroxide radicals, which oxidize
other polyunsaturated fatty acids, in a chain reaction that affects
cellular membranes. The degradation of extracellular matrix bycytokine-stimulated chondrocytes is mainly due to lipid peroxides
and can be prevented by antioxidant intake (10).
Adjuvant-induced arthritis (AIA) is characterized by inflammation and aggressive pannus formation, which leads to cartilage
and bone degradation. AIA in rats is a model of inflammatory joint
disease associated with RA (8).

Received February 2nd 2013. Accepted March 10th 2013. Address for correspondence: Irina Camelia Chis, Lecturer, MD, PhD, Department of Physiology, 1-3,
Clinicilor Street, “Iuliu Hatieganu” University of Medicine and Pharmacy, RO 400023, Cluj-Napoca, Romania; Phone: +40-744-707080; Fax 0040-264-597257;
E-mail: irinnaus@yahoo.com

2013.23.1 (77)  Fiziologia - Physiology

13

Rutin is an important flavonoid due to its many biologicalproperties, including anti-carcinogenic, cytoprotective, antiplatelet, antithrombotic, vasoprotective and cardioprotective
effects, and, last but not least, it is a powerful anti-inflammatory
and a potent antioxidant polyphenol because it scavenges ROS
and free radicals directly (5, 21, 24, 25).
Alpha-linolenic acid (ALA) is an essential fatty acid from thehuman diet which can be metabolized into n-3 long-chain polyunsaturated fatty acid (n-3 LC-PUFA) in the mammalian tissues(23). The major bioactive n-3 LC-PUFAs are eicosapentaenoic acid
(EPA) and docosahexaenoic acid (DHA). Recent research showsthat omega-3 PUFA reduces the risk of cardiovascular disease(CVD), obesity, diabetes, inflammation, and several neurologicaldiseases (17). The anti-inflammatory effects of omega-3 PUFA
are due to its inhibiting the activation of the transcription factor
NF-κB with consequent inhibition of pro-inflammatory cytokine
production, such as IL-6 and TNFα (2, 9). Thus, new researchshows that omega-3 PUFA modulates the oxidative stress (9).
The purpose of this study was to evaluate the role of rutin
and omega-3 PUFA administration in attenuating the oxidative
stress in RA.

The total score was the cumulative value in each of the 4 paws,
with a maximum of 12 for each rat. Rats with a total score of >2
were considered to have arthritis.
After the last treatment (day 28), all the rats were fasted
overnight and sacrificed by cervical decapitation with sodium
pentobarbital (60 mg/rat ip). Venous blood samples werecollected from the rats’ retro-orbital sinuses. The serum wasseparated for the estimation of free radical production: malodialdehyde (MDA) (by fluorescein dosage, Conti method) (6)and carbonylated proteins (CP) levels (18). The results wereexpressed in mmol/mg protein. For the estimation of the defense
systems: the assessment of hydrogen donor ability (HD) was
measured by Janaszewska method and expressed in inhibition% (12); total SH groups of serum were assayed according to the
Hu method (11), the concentration was expressed in micromol/
ml and the serum gluthatione (GSH) level was measured using
the Ellman method and expressed in nmol/ml.
The experimental protocol has been approved by the
Ethics Committee of the University of Medicine and Pharmacy
Cluj-Napoca and conforms to the Guide for the care and useof laboratory animals NIH publication No. 85-23, revised 1996.

MATERIALS AND METHODS

Statistical analysis
The statistical analysis was carried out using the SPSS 17 Statistical software package (SPSS Inc, Chicago, Illinois, USA).
Data are expressed as the mean ± standard deviation. Statisticalsignificance was tested at a confidence interval of 95% (p <0.05).

Based on the results of the preliminary test, we establishedthe experimental design. The study was conducted on 35 adultmale Wistar rats, weighing 225± 25 g, divided into 5 groups (n=7).
Two control groups: group I (healthy, non-arthritic controlrats) and group II (arthritic rats) were treated with saline solution,
administered daily via an intragastric tube (0.6 ml/rat/day) for aperiod of 21 days. Three arthritic groups (group III, IV and V)
were treated with non-steroidal anti-inflammatory agents (IND,
indomethacin 2 mg/kg/day, dissolved in saline solution), rutin(RUT, 80 mg/kg/day, dissolved in saline solution) or omega-3
PUFA (10 mg/kg/day, dissolved in saline solution) administereddaily via an intragastric tube (0.6 ml/rat) for a period of 21 days.
Chronic AIA rats were used as a typical inflammatory arthritis
pain model (16). A total of 35 animals were used in the present
investigations, out of which 28 underwent chronic, unilateral
inflammation, the remaining seven served as untreated (noninflamed) control animals. Arthritis was induced by injection of
0.1 ml of Freund’s adjuvant (FA; Sigma, UK) into the left hindfootpad. AIA was confirmed 7 days after the injection by clinicalexamination. The rats were treated with indomethacin, rutin
and omega-3 PUFA for 21 days after the arthritis confirmation.
At the onset of arthritis (day 7) and on days 14, 21 and 28,
the severity of arthritis was assessed visually by two independent
observers who were unaware of the treatment protocol the rats
received. A modified macroscopic scoring system was used tomonitor the severity of arthritis, as described previously (15).
Briefly, the severity of inflammation in each paw graded on ascale from 0-4, where 0= no signs of arthritis, 1=detectable
swelling and/or redness of the paw or one digit, 2= two joints
involved with moderate swelling and redness, 3= more than two
joints involved, 4= severe arthritis of the entire paw and all digits.
14

RESULTS
Effects of indhomethacin, rutin and omega-3 PUFA on clinicalsigns of AIA
After 7 days, the rats began to show evidence of clinicalinflammation in one or both hind paws. The first manifestationof disease was erythema of one or more ankle joints, followed
by involvement of the metatarsal and interphalangeal joints.
The typical time course for the development and progressionof disease, as assessed by the mean arthritis severity scoreis shown in Figure 1. The disease was progressive, with joint
recruitment following: tarsal, metatarsophalangeal and theninterphalangeal articulations.

Fig.1. Effects of indhomethacin, rutin and omega-3 polyunsaturated fatty
acids on clinical signs of adjuvant-induced arthritis

Fiziologia - Physiology  2013.23.1 (77)In the non-treated arthritic group (group II) the incidenceof disease was 100% (all animals in the group were affected)
at day 7, and remained as such throughout the duration of theexperiment. In contrast, treatment with indomethacin, rutin andomega-3 PUFA exerted a significant attenuation in the incidenceof AIA: 60% with IND treatment (P<0.05) and 50% with RUT andomega-3 PUFA treatment (P<0.05).
The serum oxidant-antioxidant balanceMedian MDA levels were increased in the rats from groupII (2.76±0.55) versus the rats from control group I (1.33±0.28).
After the induction of AIA the MDA levels were very significantly(p<0.0001) increased compared to those of the control group(Figure 2). Median MDA levels were decreased after 21 days
supplementation with IND (group III) (2.05±0.50) or RUT (groupIV) (1.52±0.66) in the AIA rats. The difference was significant:p<0.05 and p<0.01 respectively versus the rats from group II.
After 21 days of omega-3 supplementation (group V) the median
MDA levels (3.28±0.57) were significantly increased (p<0.05)
versus those of the rats from group II.

Fig.3. Serum CP levels in tested groups and controls

HD median levels were decreased after the induced of AIA in
the rats from group II (26.10±2.75) versus the rats from the control group I (37.44±4.30). After the induction of AIA the HD levels
were very significantly decreased (p<0.001) compared to the HD
levels from the control group (Figure 4). After supplementation
with IND (group III) (28.27±4.52) the median HD levels were
not significantly increased versus those of the rats from group
II. Supplementation with RUT (group IV) (36.48±3.00) in the AIA
rats determined very significantly increased (p<0.0001) median
HD levels compared to group II, and omega-3 supplementation(group V) (31.79±4.44) in the AIA rats determined significantly
increased (p<0.0001) median HD levels.

Fig.2. Serum MDA levels in tested groups and controls

Similar results were obtained for the CP determination.
Statistical data showed a value of 2.62±0.16 in the rats fromgroup II and 1.15±0.22 in the rats from the control group I.
After the induction of AIA the CP levels were very significantly
increased (p<0.0001) compared to the control group (Figure3). After supplementation with IND (group III) (1.74±0.49) or
RUT (group IV) (1.37±0.28) the median CP levels were verysignificantly decreased (p<0.002) versus those from group II.
Supplementation with omega-3 (group V) (2.26±0.46) decreased
significantly (p<0.05) the median CP levels versus those fromgroup II, but significantly increased (p<0.05) versus those of therats from group I.

2013.23.1 (77)  Fiziologia - Physiology

Fig.4. Serum HD levels in tested groups and controls

Median serum SH was lower after the induction of AIA ingroup II (0.09±0.01) versus the control group I (0.13±0.03). Afterthe induction of AIA the SH levels were significantly decreased(p<0.01) compared to the SH levels in the control group (Figure5). After supplementation with IND (group III) (0.16±0.06), RUT(group IV) (0.20±0.03) or omega-3 (group V), the median SH
levels (0.15±0.02) in the AIA rats were significantly increased(p<0.01) or very significantly increased (p<0.0001) and (p<0.005)
15

respectively, versus those from group II.

Fig.5. Serum SH levels in tested groups and controls

The median level of GSH was higher after the induced of
AIA in group II (12.92±1.85) versus control group I (4.22±4.35).
After the induction of AIA the GSH levels were very significantly
increased (p<0.005) compared to the control group (Figure 6). After supplementation with IND (group III) (12.37±1.42) the median
GSH levels were not significantly decreased versus those fromgroup II. Supplementation with RUT (group IV) (16.66±3.58) in
the AIA rats determined significantly increased (p<0.05) median
GSH levels compared to group II, and omega-3 supplementation(group V) (6.77±0.92) in the AIA rats determined very significantly
decreased (p<0.0001) median GSH levels compared to thosefrom group II.

The anti-inflammatory effect of indhomethacin is accompanied
by a suppression of the oxidative burst of the stimulated blood
phagocytes in animals treated with indhomethacin (4).
One of the mechanisms involved in the pathogenesis of
RA is oxidative stress (10). A frequently used oxidative stressmarker is determining the lipid and protein peroxidation bymeasuring MDA and CP levels. Clinical and experimental studies have shown increased plasmatic levels of MDA and CP in
RA (3, 4, 20, 22).
The AIA model is robust, the incidence rate of the diseaseis 100%, and AIA rats share many features of RA with humans,such as in inflammation, marked bone resorption and periostal
bone proliferation (19). AIA in rats has been adopted as anexperimental paradigm for the pre-clinical screening of RA
treatments. In this model we measured RA markers. Our data
showed increased arthritis scores in AIA rats (Figure 1) andthe fact that oral treatment with indhomethacin ameliorates theclinical severity of arthritis (P>0.05). The oral administration ofrutin or omega-3 PUFA in AIA rats reduced the progression ofarthritis by inhibiting the arthritis score.
Our present study provides clear evidence of increasedoxidative stress status in an experimental rat model of RA.
These observations are in agreement with previous reported data
showing an increase of malondialdehyde and protein carbonylin the serum of AIA rats. In the current study, we demonstrated
that oral treatment with rutin reverses oxidative stress in AIA rats.
This effect is accompanied by a decreased ROS production inserum, shown by the decrease of MDA and CP, and increased
antioxidant status in serum as shown by HD levels, SH levels
and GSH levels.
Recent studies clearly showed that high levels of unsaturated fatty acids induce an increase of n-3 PUFA levels in thetissues, which may lead to increased susceptibility to lipid peroxidation and in turn, oxidative stress. In our present study theoral treatment with omega-3 PUFA increased lipid and protein
peroxidation (increased MDA and CP), increased the antioxidant
status in serum: HD levels and SH levels and very significantly
decreased the GSH levels.
CONCLUSION

Fig.6. Serum GSH levels in tested groups and controls

DISCUSSIONS
Rheumatoid arthritis is characterized by persistent synovitis,
systemic inflammation, and presence of auto-antibodies (7).
The analgesic and anti-inflammatory effects of indhomethacin,a non-steroidal anti-inflammatory drug often used for the treatment of RA, partly produced by COX inhibition, are well known.
16

Our results indicate that indhomethacin and rutin administration ameliorates the clinical severity of arthritis and decreases
the oxidative stress. Thus we can conclude that oxidative stressplays an important role in AIA and could be controlled by a suitable combination therapy of indhomethacin and an antioxidant
substance, as demonstrated for rutin. Omega-3 PUFA ameliorates the clinical severity of arthritis and moderately decreases
the oxidative stress, and its administration can be beneficial in
reducing the risk of CVD in RA.
REFERENCES
1. Ahmed AS, Li J, Ahmed M, Hua L, Yakovleva T, Ossipov MH, Bakalkin
G, Stark A. Attenuation of pain and inflammation in adjuvant-induced
arthritis by the proteasome inhibitor MG132. Arthritis Rheum, 2010;

Fiziologia - Physiology  2013.23.1 (77)

62(7): 2160-2169.
2. Arisue A, Shimojima N, Tomiya M, Shimizu T, Harada D et al. Effect of an omega-3 lipid emulsion in reducing oxidative stress in a rat
model of intestinal ischemia-reperfusion injury. Pediatr Surg Int, 2012;
28: 913-918
3. Baskol G, Demir H, Baskol M, Kilic E, Ates F, Karakukcu C, Ustdal M.
Investigation of protein oxidation and lipid peroxidation in patients withrheumatoid arthritis. Cell Biochem Func, 2006; 24: 307–311.
4. Bauerova K, Paulovičova E, Mihalova D, Drafi F, Ktrosova M, Mascia
C, Biasi F, Rovensky J, Kucharska J, Gvozdjakova A, Ponint S. (2010).
Combined methotrexate and coenzyme Q10 therapy in adjuvantinduced arthritis evaluated using parameters of infl ammation andoxidative stress. Acta Biochim Pol, 2010; 57: 347-354.
5. Chen S, Gong J, Liu F, Mohammed U. Naturally occuring polyphenolic antioxidants modulate IgE-mediated mast cell activtion. Immunology, 200; 100: 471-80.
Conti M, Morand PC, Levillain P et al. Improved Fluoromeric Determination of Malonaldehyde. Clin Chem, 1991; 37(7): 1273-1275.
6. Drafi F, Bauerova K, Kuncirova V, Ponist S. Pharmacological influence on processes of adjuvant arthritis: Effect of the combination ofan antioxidant active substance with methotrexate. Interdiscip Toxicol,
2012; 5(2): 84-91.
7. Egan CE, Lockhart JC, Ferrell WR. Pathophysiology of vascular
dysfunction in a rat model of chronic joint inflammation. J. Physiol,
2004; 2: 635-643.
8. Ferretti G, Bacchetti T, Masciangelo S, Saturni L. Celiac Disease,
Inflammation and Oxidative Damage: A Nutrigenetic Approach.
Nutrients, 2012; 4: 243-257.
9. Hitchon CA, El-Gabalarawy HS. Oxidation in rheumatoid sarthritis.
Arthritis Res Ther, 2004; 6: 265-278.
10. Hu ML. Measurement of protein thiol groups and gluthatione inplasma. Methods Enzymol, 1994; 233: 380-5.
11. Janaszewska A, Bartosz G. Assay of total antioxidant capacity:
comparison of four methods as applied to human blood plasma. Scan
J Clin Invest, 2002; 62(3): 231-236.
12. Jaswal S, Mehta HC, Sood AK, Kaur J. Antioxidant status in rheumatoid
arthritis and role of antioxidant therapy. Clin Chim Acta, 2003; 338: 123-129.
13. Kunsch CH, Sikorski JA, Sundell CL. Oxidative stress and the useof antioxidants for the treatment of rheumatoid arthritis. Cur Med Chem
Immunol Endocr Metab Agents, 2005; 5: 249-258.

14. Larsson P, Kleinau S, Holmadahl R, Klareskog L. Homologous
type II collagen-induced arthritis in rats: characterization of the disease
and demonstration of clinically distinct forms of arthritis in two strainsof rats after immunization with the same collagen preparation. Arthritis
Rheum, 1990; 33: 693-701.
15. McDougall JJ, Kamian SM, Ferrell WR. Prolonged alteration of vasoconstrictor and vasodilatator responses in rat knee joints by adjuvant
monoarthritis. Exp. Physiol, 1995; 80: 349-357.
16. Riediger ND, Othman RA, Suh M, Moghadasian MH. A systemic
review of the roles of n-3 fatty acids in health and disease. J Am Diet
Assoc, 2009, 109:668-679.
17. Reznick AZ, Packer L. Oxidative damage to proteins: Spectrophotometric method for carbonyl assay. Methods Enyymol, 1994; 233: 347-357.
18. Shi Q, Abusarah J, Baroudi G, Fernandes JC, Fahmi H, Benderdour M. Ramipril attenuates lipid peroxidation and cardiac fibrosis in
an experimental model of rheumatoid arthritis. Arthritis Research &Therapy, 2012; 14: 223.
19. Strosova M, Karlovska J, Spickett CM, Orszaghova Z, Ponist S,
Bauerova K, Mihalova D, Horakova L. Modulation of SERCA in thechronic phase of adjuvant arthritis as a possible adaptation mechanismof redox imbalance. Free Radic Res, 2009; 43: 852-864.
20. Tang DQ, Wei YQ, Gao YY, Yin XX, Yang DZ, Mou J et al. Protective eefects of rutin on rat glomerular mesangial cells cultured in highglucose conditions. Phytother Res, 2011; 25: 1640-7.
21. Tastekin N, Aydogdu N, Dokmeci D, Usta U, Birtane M, Erbas H,
Ture M. Protective effects of L-carnitine and alpha-lipoic acid in rats withadjuvant arthritis. Pharmacol Res, 2007; 56: 303-310.
22. Tou JC, Altman SN, Gigliotti JC, Benedito VA, Cordonier EL. Different sources of omega-3 polyunsaturated fatty acids affects apparent
digestibility, tissue deposition, and tissue oxidative stability in growing
female rats. Lipids in Health and Disease, 2011; 10:179.
23. Wang Shao-wei, Wang You Jiong, Su Ya-jing, Zhou Wei-wei, etal. Rutin inhibits beta-amyloid aggregation and cytotoxicity, attenuates
oxidative stress, and deaceses the production of nitric oxide an proinflammatory. NeuroToxicology, 2012; 33: 482-490.
24. Wu CH, Lin MC, Wang HC, Yang MY, Jou MJ, Wang CJ. Rutin
inhibits oleic acid induced lipid accumulation via reducing lipogenesis
and oxidative stress in hepatocarcinoma cells. J Food Sci, 2011; 76:
T65-72.

EFECTUL RUTINULUI ŞI AL ACIZILOR GRAŞI POLINESATURAŢI OMEGA-3
ASUPRA BALANŢEI OXIDANŢI-ANTIOXIDANŢI LA ŞOBOLANI CU ARTRITĂ
INDUSĂ DE ADJUVANTREZUMATStresul oxidativ joacă un rol important în patogeneza artritei reumatoide (AR). Speciile reactive ale oxigenului (SRO) produse în
cursul proceselor celulare de fosforilare oxidativă şi prin activarea celulelor fagocitare depăşesc capacitatea antioxidantă determinândapariţia stresului oxidativ. Producţia excesivă de SRO atacă proteinele, lipidele, acizii nucleici şi componentele matricei celulare.
Pacienţii cu AR au alterată capacitatea antioxidantă a organismului. În acest studiu ne-am propus să evidenţiem şi să cuantificăm
efectele antioxidante ale rutinului şi al acizilor polinesaturaţi (PUFA) omega-3 într-un model de şobolan cu artrită indusă cu adjuvant(AIA). Cinci grupuri de şobolani au fost comparate: grupul de control fără artrită şi patru grupuri cu AIA tratate prin gavaj timp de 21
de zile, după 7 zile de la inducerea AIA, cu vehicol (ser fiziologic), cu un agent antiinflamator non-steroidian (indometacin), cu rutin
sau cu omega-3 (PUFA). Scorul artritic a fost determinat la şobolanii cu AIA ca un indicator clinic al afectării articulare. După 28 dezile a fost măsurat nivelul seric al malondialdehidei (MDA), proteinelor carbonilate (CP), grupărilor sulfhidril (SH), capacităţii dedonor de hidrogen (HD) şi glutationului (GSH). Rezultatele obţinute ne arată o creştere a stresului oxidativ evidenţiat prin creşterea
producţiei de radicali liberi, MDA şi PC, şi o scădere a capacităţii antioxidante prin măsurarea HD, SH şi GSH la şobolanii cu AIA.
Nivelurile MDA şi al CP au scăzut semnificativ, iar nivelurile HD, SH şi GSH au crescut semnificativ după administrarea orală de rutin.
Omega-3 (PUFA) a crescut nivelurile MDA, CP, HD, SH şi a scăzut nivelul GSH. Acest studiu sugerează că tratamentul cu rutin şi
omega-3 (PUFA) poate fi benefic în atenuarea stresului oxidativ asociat AR.
Cuvinte cheie: stres oxidativ, artrită indusă de adjuvant, rutin, şobolani
2013.23.1 (77)  Fiziologia - Physiology

17

QUERCETIN, LYCIUM BARBARUM AND CHITOSANREVERSE THE EFFECTS OF HYPOBARIC HYPOXIAAND EXERT CARDIOPROTECTIVE EFFECTS IN
RATS
ANCA DUMITROVICI1†, IRINA C. CHIŞ2†, ADRIANA MUREŞAN2, ADRIANA MARTON3, REMUS
MOLDOVAN2, DELIA VLAD4, GABRIEL BORZA5, POMPEI BOLFA5
1
Oncologic Institute “I. Chiricuta”, Cluj-Napoca, Romania
2
Department of Physiology, University of Medicine and Pharmacy “Iuliu Hatieganu”, Cluj-Napoca, Romania
3
Nephrology Clinic, Emergency County Hospital, Cluj-Napoca, Romania
4
Section of Cardiology, General Hospital Nikaia Pireus, Grecia
5
Pathology Department, University of Agricultural Sciences and Veterinary Medicine, Cluj-Napoca, Romania†

Equal contributors.

ABSTRACT

Chronic and acute exposure to hypobaric hypoxia induces changes of the pro-oxidant/antioxidant balance in the myocardium andthroughout the body. It has been shown that natural antioxidants supplementation (Quercetin, Lycium barbarum and Chitosan) is
effective in preventing the hypoxic stress. Therefore, the purpose of this study was to observe, highlight and quantify the cardioprotective effects of these natural antioxidants’ administration. The study was conducted on male, Wistar rats, exposed to acute andchronic hypobaric hypoxia (HH) (5500 m, 23h/day) for different periods of time (1 or 14 days) or kept in normoxia for 14 days. Someof the rats were administered natural antioxidants one day before the first HH exposure and 14 days before and prior to every hypoxic
exposure for the next 14 days. At the end of the study the animals were euthanized and the hearts were harvested and stored forfurther analysis. Myocardial sections were obtained and examined histopathologically to determine the cardiomyocyte viability andmorphometry. The results show a negative effect of chronic hypobaric hypoxia on the cardiomyocyte viability and cardio-protective
effects of natural antioxidants’ administration. This study suggests that treatment with Quercetin, Lycium barbarum or Chitosan alone
substantially restored the myocardial architecture and acted like cardioprotectants.
Key words: oxidative stress, hypobaric hypoxia, Lycium barbarum, Quercetin, Chitosan.

INTRODUCTION
Illness related to acute and chronic high-altitude exposure is
primarily caused by hypobaric hypoxia (HH), which is considereda physiological oxidative stress that affects the myocardial tissueand determines several physiological alterations (13, 25). The
severity and duration of the symptoms vary, depending on thealtitude and rate of ascent, sometimes persisting after returning
to lower altitudes (19). The development of these symptoms has
been linked to hypoxia-induced oxidative and nitrosative stress(7). Exposure to high-altitude constitutes an oxidative stress,
the reactive oxygen species (ROS) sources being alterationsof: mitochondrial respiratory chain (11), cellular membrane andcytosol, endothelial cells and lipid and protein perturbations.
ROS generation may be one of the mechanisms involved in thedeath of cardiomyocytes after hypobaric hypoxia exposure (21).
Quercetin is an important flavanone which is known to bea multi-functional agent, due to its anti-inflammatory and anticancerous activities, as well as a potent antioxidant because ofits directly scavenging ROS and free radicals (9, 16, 17, 20).
The fruits of Lycium barbarum (LBG) have a wide antioxidant spectrum and have been used for a very long time in
Easter-Asian medicine, especially in China (29). Recent researchshowed that LBG fruit extract has many beneficial effects, including anti-ageing, anti-cancerous, cytoprotective or immunostimulating properties. Eyesight improvement, blood pressure control,cholesterol level lowering have also been reported. Last but notleast, this extract seems to have protective effects against theoxidative stress of different causes (18, 29).
Chitosan is a α-(1-4)-D-glucosamine polymer, a naturalpolysaccharide present in shellfish, clams, krill, oysters, fungi,etc (10). It has been shown to possess antilipidemic, antiulcerogenic, anti-ageing, membrane-stabilizing and antioxidant
properties (3, 15, 26, 28).
Because hypoxic stress due to either acute or chronic hypobaric hypoxia is one of the mechanisms underlying the initiationof cardiac damage, the aim of this study was to evaluate whether
Quercetin, LBG extract or Chitosan supplementation is effective

Received February 10th 2013. Accepted March 10th 2013. Address for correspondence: Irina Camelia Chis, Lecturer, MD, PhD, Department of Physiology, 1-3,
Clinicilor Street, “Iuliu Hatieganu” University of Medicine and Pharmacy, RO 400023, Cluj-Napoca, Romania; Phone: +40-744-707080; Fax 0040-264-597257;
E-mail: irinnaus@yahoo.com

18

Fiziologia - Physiology  2013.23.1 (77)in preventing cardiac damage.
MATERIALS AND METHODSHistopathological exam
Cardiomyocytes’ diameters in control rats, HH-exposed
rats ant treated HH-exposed rats are illustrated in Figure 1. andTable I. Exposure to acute hypobaric hypoxia does not modify the
cardiomyocytes size but exposure to chronic hypobaric hypoxia
determines a statistically significant decrease in cardiomyocyte
size after 14 days of exposure in all groups, compared to thecontrol group (5th Group). However, the reduction of cardiomyocytes’ size was less severe (cardioprotective effect) in thetreated groups (6th, 7th and 8th group) compared to the 9thGroup (unprotected ctrl group).
Table I. Cardiomyocyte diameter in the 9 experimental groups (Mean±SD, µm)
1st
2nd 3rd 4th
5th 6th
7th
8th
9th
Group Group Group Group Group Group Group Group Group14.1±0.5**

14.4±0.6*

14.4±0.5*

15.5±0.5*

15.5±0.415.8±0.4

16.6±0.7

15.9±0.8

2013.23.1 (77)  Fiziologia - Physiology

RESULTS15.7±0.9

Male Wistar rats (180±10 g) were used for this experiment.
Rats were fed standard laboratory diet and had free access towater. The experimental protocol has been approved by the
Ethics Committee of the “Iuliu Hatieganu” University of Medicine
and Pharmacy from Cluj-Napoca and conforms to the Guide
for the care and use of laboratory animals NIH publication No.
85-23, revised 1996.
The animals were randomly subdivided into nine experimental groups (n=10): 1st Group- unprotected acute hypoxic
hypobaric (HH) rats (control group); 2nd Group- treated acute
HH rats, rats treated with quercetin; 3rd Group- treated acute HHrats, rats treated with LBG extract; 4th Group- treated acute HHrats, rats treated with Chitosan; 5th Group- unprotected normoxia
rats, rats not exposed to HH (control group); 6th Group- treatedchronic HH rats, rats treated with LBG extract; 7th Group- treatedchronic HH rats, rats treated with Quercetin; 8th Group- treatedchronic HH rats, rats treated with chitosan and 9th Group- unprotected chronic HH rats (control group).
The animals were exposed to a simulated altitude of 5500
m in a barochamber (in the Physiology Department of the UMF
˝Iuliu Haţieganu˝ Cluj-Napoca), where temperature and humidity
were maintained at 28ºC and 55-60%, respectively, for one day(acute HH) and for 14 consecutive days (chronic HH). The rats
were taken out of the hypoxic chamber once after every 24h
exposure for 1 hour, for receiving food and water.
Some of the rats received Quercetin (30 mg/kg/day, dissolvedin saline solution) or Lycium barbarum extract (LBG, 30 mg/kg/
day, dissolved in saline solution) via an intragastric tube (0.6 ml/rat) for one day or 14 consecutive days prior to HH exposure
and prior to every HH exposure respectively. Other groups were
given intra-peritoneal injections of Chitosan (0,30-0,35 microg/
animal/day, dissolved in saline solution) for a period of one day
or 14 consecutive days prior to HH exposure and prior to every
HH exposure respectively. The control groups were treated withsaline solution (0.6 ml/rat) via an intragastric tube (ctrl groups).
All rats were employed the next day after the last hypoxic
exposure and sacrificed by decapitation (with sodium pentobarbital, 60 mg/rat ip). Hearts were rapidly excised, washed in cold
saline, than left and right ventricular walls and the septum weredissected, weighed and used for analysis.
For the histopathological examination the myocardial tissues
were fixed in 10% neutral buffered formalin and, after proper
fixation, were dehydrated in graded series of alcohol, cleared in
Xilene and embedded in paraffin wax. Multiple longitudinal andtransversal sections from each block were prepared at 5 µm, andstained with haematoxylin and eosin (H&E). The haematoxylin andeosin staining (H&E) was used, because it allows a good observation of different cell types. For measuring the cardiomyocyte
diameter (morphological study) we used the technique of Aielloet al. (1). Tissue analysis was performed using an Olympus system for image acquisition and analysis, respectively an Olympus

BX51 microscope equipped with Olympus Cell B software. 50
cardiomyocytes were measured from each section, from at least
10 different regions, with 200x or 400x magnification. Statisticalanalysis and all morphological data was performed using ShapiroWilk normality test, followed by the two-sample t-test, using R’software (R Development Core Team, 2010).

*p<0.05; **p<0.01 as compared to the 5th Group (ctrl group)

Fig.1. Quantification of cardiomyocytes diameters in the 9 experimental
Wistar rats groups (Mean ± SD) *p<0.05; **p<0.01 compared to the 5th Group(ctrl group).

To further validate the effects of hypobaric hypoxia on
myocardial tissue histopathologic examination was conducted.
Figures 2-3 present the histopathological analysis of thesections of cardiac tissues stained H&E from the rats exposed
to normoxia, acute hypobaric hypoxia, chronic hypobaric
hypoxia and treated acute or chronic hypobaric hypoxia.
Increased wall thickness, abnormal myocardial architecture
and increased interstitial space were observed in rat hearts
exposed to chronic hypobaric hypoxia (9th Group) comparedto the 5th Group (ctrl group). The rats treated with Quercetin,
LBG extract and Chitosan respectively (6th Group, 7th Groupand 8th Group) and exposed to chronic HH presented a19

restored towards normal myocardial architecture. No obvious
differences of wall thickness, myocardial architecture andinterstitial spaces were observed normoxia group (5th Group)
and acute hypobaric hypoxia group (1st Group).

Fig.2. Cardiac changes of rats exposed to normoxia, acute hypobaric
hypoxia, chronic hypobaric hypoxia and treated acute or chronic hypobaric
hypoxia. (A-J). Representative examples of histopathological analysis in thetransverse sections from the myocardium stained with haematoxylin and eosin (H&E). The images were magnified 200 or 400 times. 1st Group (A), 2ndGroup (B), 3th Group (C) and 4th Group(D): no histological alterations wereobserved; 5th Group (E): no histological changes regarding cardiomyocytes orcapillaries; 6th Group (F), 7th group (G) and 8th group (H): restored towards
normal: mild decrease of cardiomyocytes diameter and increased number ofcapillaries; 9th Group (J): characteristic changes due to hypobaric hypoxia:
moderately decreased diameters of cardiomyocytes and increased capillarydensity (arrows) compared to the 5th Group (ctrl group).

Fig.4. Cardiac and cardiomyopathie changes of rats exposed to normoxia,
acute hypobaric hypoxia, chronic hypobaric hypoxia and treated acute or
chronic hypobaric hypoxia. (A-J) Representative examples of histopathological analysis in the sections of cardiac tissue stained with haematoxylin andeosin (H&E). The images were magnified 200 or 400 times.

As for the non-specific myocardial lesions, we observedin this study that all the rats exposed to hypobaric hypoxia presented in the myocardium (Figure 4) small regions of degenerated cardyomyocytes surrounded by different degrees of moreor less mononuclear inflammatory cells (lympho-histiocytes)- lesions of chronic progressive cardiomyopathy in different
stages. Myocardial lesions were aggravated after chronic hypobaric hypoxia exposure. The rats treated with Quercetin, LBG
extract and Chitosan respectively (6th Group, 7th Group and 8thGroup) and exposed to chronic hypobaric hypoxia presented a
reduction of the myocardial lesions and a minimal mononuclear
cell infiltration.
DISCUSSIONS

Fig.3. Cardiac changes of rats exposed to normoxia, acute hypobaric
hypoxia, chronic hypobaric hypoxia and treated acute or chronic hypobaric
hypoxia. (A-J). Representative examples of histopathological analysis in thelongitudinal sections from the myocardium stained with haematoxylin andeosin (H&E). 1st Group (A), 2nd Group (B), 3th Group (C) and 4th Group (D):no histological alterations from normal cytoarchitecture were observed; 5thGroup (E): no histological changes regarding cardiomyocytes or capillaries;
6th Group (F), 7th Group (G) and 8th Group (H): mild decrease of cardiomyocytes diameter and increased number of capillaries; 9th Group (J): characteristic changes due to hypobaric hypoxia: moderate decreases of cardiomyocytes’ morphometric values (diameter, area and perimeter) and increased
capillary density (arrows) compared to the 5th Group (ctrl group).

20

Coronary artery disease (CAD) is the most common
cardiovascular disease and a leading cause of cardiac death.
Although numerous treatments are available, CAD treatment isstill unsatisfying and limited because of its various side-effects
and because of the lack of a preventive approach. This is why
looking for alternative non-drug therapies for myocardial ischemia
is essential. Long- term high altitude hypoxia has long beenrecognized to increase the resistance of the heart to ischemiahypoxia injury. It has been shown that chronic intermittent hypoxia(CIH) is associated with increased production of reactive oxygen
species that contributes to the adaptive mechanism underlying
Fiziologia - Physiology  2013.23.1 (77)the improved myocardial ischemic tolerance. Multiple mechanisms or pathways have been suggested to contribute to thecardioprotective effects of CIH, such as: increased coronary
flow and myocardial capillary angiogenesis, activation of ATPsensitive potassium channels, activation of protein kinase C,
activation of MnSOD, etc (8).
Recent research suggests that ROS generated the adaptation period a CIH are important components of a signaling
pathway leading to this form of cardioprotection (8). Startingfrom this hypothesis we tried to show in our study that antioxidant
supplementation might have a beneficial effect in reducing theoxidative stress resulted from hypobaric hypoxia exposure, as
well as a cardioprotective effect.
In our study we observed transverse and longitudinal
myocardium stained sections, by histochemical analysis, whichshowed progressive increase of capillary densities associatedwith significant reductions in fiber area and diffusion distances in
the rats exposed to chronic hypobaric hypoxia. Our results are inagreement with previous research (21, 24), and we interpreted
the results as an adaptive response of rat myocardium to amore efficient O2 delivery to the mitochondria of cardiac muscle
cells. Higher capillarization parameters and fiber morphometry
reductions indicate that there is a delay of about 14 days after
the hypoxic stimulus ceases before complete angiogenesis isreached and fiber morphometry changes.
Quercetin is a flavonoid which is frequently used in altitude
illness. Its cardioprotective effects on ischemic lesions in rats
were shown by Annapurna et al. (4) as being due to a reductionof the oxidative stress and an increase of antioxidant enzymes.
We observed in this study that rats exposed to chronic hypobaric
hypoxia and treated with Quercetin presented a restored myocardial structure: cardiomyocytes’ diameters did not decrease toomuch and there was a decrease in capillary densities comparedto the control rats. We interpreted this as a result of Quercetin’s
antioxidant effect, which decreased the oxidative stress inducedby chronic hypobaric hypoxia exposure, thus restoring the architecture of the myocardium.
Hypoxia and oxidative stress were shown to be potential
inducers of cardiac hypertrophy (12). In the present study,
abnormal myocardial architecture and increased interstitialspace were observed after chronic hypobaric hypoxia exposure.
Therefore, we speculate that a longer duration of chronic hypoxia
could induce abnormal myocardial architecture and even leadto cardiac hypertrophy.
Different biological activities of L. barbarum have been demonstrated, including anti-aging, anti-cancerous, immunostimulatory or cytoprotective effects, and many studies suggested thatthe protective effect of L. barbarum mainly depends on its antioxidative action (14, 18, 23). In the present work we observedthat rats exposed to chronic hypobaric hypoxia and treated with
L. barbarum presented myocardial restoration in a way similar to
Quercetin treated rats, clearly showing that myocardial restoration is due to its antioxidant effects.
Recent studies (2, 22) showed that Chitosan plays a role in
the recovery of post-ischemic myocardial lesions by decreasing
2013.23.1 (77)  Fiziologia - Physiology

ROS, attracting chemokines and maintaining a normal level ofantioxidant enzymes, thus proving its cardioprotective effects.
Our study clearly shows that Chitosan administration in rats
exposed to chronic hypobaric hypoxia restored the myocardialarchitecture.
In rats, the terms of progressive or degenerative cardiomyopathy are used to describe a series of degenerative myocardial
lesions. Morphological modifications start around the age of
3-4 months, mainly in the apex, underneath the fibrous rings,
papillary muscles and the free wall of the left ventricle. Sometimes, the modifications can be observed around the intramuralcoronary arteries. The initial lesions are focal accumulations ofmononuclear inflammatory cells, oedema, and degeneratingmuscle fibers. In the more advanced stages, the lesions caneither remain isolated or confluate to larger areas of fibrosis or
cardiomyocyte necrosis (5, 27). The etiology of this cardiomyopathy is not very well known, but it has been suggested that the
myocardial degeneration areas might be due to ischemia, as aresult of a cardiovascular disease (6).
We observed that the rats exposed to acute and chronic
hypobaric hypoxia presented progressive chronic cardiomyopathy lesions, in different evolving stages. Myocardial lesions were
aggravated by the chronic hypobaric hypoxia exposure. The rats
treated with Quercetin, LBG extract and Chitosan respectively,
and exposed to hypobaric hypoxia presented a decrease of the
myocardial lesions and minimal mononuclear cell infiltration.
CONCLUSION
These results demonstrate the beneficial effects of Quercetin, L. barbarum and Chitosan supplementation in natural andenriched foods. These substances presented a cardioprotective
effect induced by hypobaric hypoxia. Our results demonstrate a
cardioprotective role of Quercetin, Chitosan and L. barbarum inmediating the increased tolerance to ischemia observed in thechronically hypoxic heart.
REFERENCES
1. Aiello EA, Villa-Abrille MC, Escudero EM, Portiansky EL, Pérez
NG, de Hurtado MC, Cingolani HE. 2004. Myocardial hypertrophy ofnormotensive Wistar-Kyoto rats. Am J Physiol Heart Circ Physiol, 2004;
286(4):H1229-35.
2. Anandan R, Ganesan B, Obulesu T, Mathew S, Kumar RS, Lakshmanan PT, Zynudheen AA. Dietary chitosan supplementation attenuates isoprenaline-induced oxidative stress in rat myocardium. Int J Biol
Macromol, 2012; 51(5):783-7.
3. Anandan R, Ganesan B, Obulesu T, Mathew S, Asha KK, Lakshmanan PT, Zynudheen AA. Antiaging effect of dietary chitosan supplementation on glutathione-dependent antioxidant system in young and aged
rats. Cell Stress and Chaperones, 2013; 18: 121-125.
4. Annapurna A, Reddy CS, Akondi RB, Rao SR. Cardioprotective
actions of two bioflavonoids, quercetin and rutin, in experimental
myocardial infarction in both normal and streptozotocin-induced type I
diabetic rats. J Pharm Pharmacol, 2009;61 (10): 1365-74.
5. Arceo RJ, Bishop SR, Elwell MR, Kerns WD, Mesfin GM, Ruben Z,
Sandusky GE, Van Vleet JF. Standard nomenclature of spontaneous21

pathological findings in the heart and vasculature of the laboratory rat.
Initial Proposal Society of Toxicologic Pathology, in Guides for Toxicological Pathology STP/ARP/AFIP: Washington, DC. 1990.
6. Ayers KM, Jones SR. The cardiovascular system, in Bernischke K,
Garner FM, Jones TC. (Eds), Pathology of Laboratory Animals. New
York: Springer-Verlag, 1978; Vol. 1, 1-69.
7. Bakonyi T, Radak Z. High altitude and free radicals. J Sports Sci
Med, 2004; 3: 64-69.
8. Balkova P, Hlavackova M, Milerova M, Neckar J, Kolar F, Novak F,
Novakova O. N-acetylcysteine treatment prevents the up-regulationof MnSOD in chronically hypoxic rat hearts. Physiol Res. 2011, 60:
467-474.
9. Boots AW, Haenen GR, Bast A. Health effects of quercetin: fromantioxidant to nutraceutical. Eur J Pharmacol, 2008; 585: 325-37.
10. Cardenas G, Orlando P, Edelio T. Synthesis and applications ofchitosan mercaptanes as heavy retention agent. Int J Biol Macromol.
2001; 28: 167-174.
11. Chandel NS, Maltepe E, Goldwasser E, Mathieu CE, Simon MC,
Schumacker PT. Mitochondrial reactive oxygen species trigger hypoxia
induced transcription. Proc Natl Acad Sci. USA, 1998; 95: 11715-11720.
12. Chen LM, Kuo WW, Yang JJ, Wang SG, Yeh YL, Tsai FJ, Ho YJ et
al. Eccentric cardiac hypertrophy was induced by long-term intermittent
hypoxia in rats. Exp Physiol, 2007; 92: 409-416.
13. Chis I, Ungureanu MI, Simedrea R, Maier M, Muresan A, Marton
A, Decea N. Short and long term hypobaric hypoxia induces oxidative
stress in rats: the protective effects of N-Acetylcysteine. Fiziologia(Physiology). 2008; 18, 3 (59): 20-22.
14. Deng HB, Cui DP, Jiang JM, Feng YC, Cai NS, Li DD. Inhibiting
effects of Achyranthes bidentata polysaccharide and Lycium barbarum
polysaccharide on non-enzyme glycation in D-galactose induced mouseaging model. Biomed Environ Sci, 2003;16: 267-275.
15. Filipovic-Grcic J, Skalko-Basnet N, Jalsenjak I. Mucoadhesive
chitosan-coated liposomes: characteristics and stability. J Microencaps.
2001; 18: 3-12.
16. Hanasaki Y, Ogawa S, Fukui S. The correlation between active
oxygens scavenging and antioxidative effects of flavonoids. Free Radic
Biol Med, 1994;16: 845-50.
17. Galati G, O’Brien PJ. Potential toxicity of flavonoids and otherdietary phenolics: significance for their chemopreventive and anticancer

properties. Free Radic Biol Med. 2004; 37: 287-303.
18. Gan L, Hua ZS, Liang YX, Bi XH. Immunomodulation and antitumor
activity by a polysaccharide-protein complex from Lycium barbarum. Int
Immunopharmacol, 2004; 4:563-569.
19. Imray C, Wright A, Subudhi A, Roach R. Acute mountain sickness:
pathophysiology, prevention, and treatment. Prog Cardiovasc Dis.
2010; 52: 467-484.
20. Jeong SM, Kang MJ, Choi HN, Kim JH, Kim JI. Quercetin ameliorates hyperglycemia and dyslipidemia and improves antioxidant status in
type 2 diabetic db/db mice. Nutrition Research and Practice (Nutr Res
Pract). 2012; 6(3): 201-207.
21. Lee Sd, Kuo WW, Wu CH, Lin YM, Lin JA, Lu MC, Yang AL, Liu JY
et al. Effects of short- and long-term hypobaric hypoxia on Bcl2 family
in rat heart. Intern J of cardiology. 2006; 108: 376-384.
22. Liu Z, Wang H, Wang Y, Lin Q, Yao A, Cao F, Li D, Zhou J, Duan
C, Du Z, Wang Y, Wang C. The influence of chitosan hydrogel on stem
cell engraftment, survival and homing in the ischemic myocardial microenvironment. Biomaterials, 2012; 33 (11): 3093-106.
23. Luo Q, Cai Y, Yan J, Sun M, Corke H. Hypoglycemic and hypolipidemic effects and antioxidant activity of fruit extracts from Lycium
barbarum. Life Sci, 2004; 76: 137-149.
24. Panisello P, Torrella JR, Pagés T, Viscor G. Capillary supply andfiber morphometry in rat myocardium after intermittent exposure tohypobaric hypoxia. High Alt Med Biol. 2007; 8(4): 322-30.
25. Romero RM, Canuelo A, Siles E, Oliver FJ, Lara ME. Nitric oxide
modulates hypoxia-inducible factor-1 and poly(ADP-ribose) polymerase-1 cross talk in response to hypobaric hypoxia. J Appl Physiol.
2012; 112: 816-823.
26. Santhosh S, Sini TK, Anandan R, Mathew PT. Effect of chitosan
supplementation on antitubercular drugs-induced hepatotoxicity in rats,
2006; 219: 53-59.
27. Tucker J. Mary. Diseases of the Wistar Rat. Taylor & Francis Limited,
1 Gunpowder Square, London EC4A 3DA, 1997; 97-101.
28. Xie W, Xu P, Liu Q. Antioxidant activity of water-soluble chitosan
derivatives. Bioorg Med Chem Lett. 2001; 11: 1699-1701.
29. Yu MS, Ho YS, So KF, Yuen WH, Chang RCC. Cytoprotective
effects of Lycium barbarum against reducing stress on endoplasmic
reticulum. International Journal of Molecular Medicine, 2006; 17:1157-1161.

QUERCETINUL, LYCIUM BARBARUM ŞI CHITOSANUL CONTRACAREAZĂ EFECTELE HIPOXIEI HIPOBARE ŞI EXERCITĂ UN RĂSPUNSCARDIOPROTECTOR ÎNTR-UN MODEL DE ŞOBOLAN
REZUMAT

Expunerea la hipoxie hipobară acută şi cronică induce modificarea balanţei oxidanţi/antioxidanţi la nivelul miocardului şi a întreguluiorganism. Cunoscând eficienţa suplimentării cu antioxidanţi naturali (Quercetin, Lycium barbarum şi Chitosan) în prevenirea mai multor
tipuri de leziuni induse de stresul oxidativ în acest studiu ne-am propus să evidenţiem şi să cuantificăm efectele cardioprotectoare aleadministrării unor antioxidanţi naturali. Şobolani masculi, rasa Wistar, adulţi au fost expuşi la hipoxie hipobară (HH) (5500 m, 23h/zi)
pentru diferite perioade de timp (1 şi 14 zile) sau menţinute în normoxie (14 zile). O parte din şobolanii luaţi în studiu au fost trataţi
cu antioxidanţi naturali înaintea expunerii la HH timp de o zi şi respectiv 14 zile şi apoi înaintea fiecărei expuneri la HH. La sfârşitul
studiului toţi şobolanii au fost sacrificaţi şi inimile au fost prelevate şi conservate pentru analize. S-au efectuat secţiuni de miocard
care au fost examinate histopatologic în vederea stabilirii morfologiei cardiomiocitelor (studiul morfometric). Rezultatele obţinuteindică efectul negativ al hipoxiei hipobare cronice asupra arhitecturii miocardice şi efectele cardioprotectoare ale ale antioxidanţilor
naturali administraţi. Rezultatele acestui studiu sugerează că tratamentul cu Quercetin, Lycium barbarum sau Chitosan restaurează
substanţial arhitectura miocardului şi are efecte cardioprotectoare.
Cuvinte cheie: stres oxidativ, hipoxie hipobară, Lycium barbarum, Quercetin, Chitosan.

22

Fiziologia - Physiology  2013.23.1 (77)THE IMPORTANCE OF MYO-INOSITOL IN THE
PHYSIOPATHOLOGY OF HEPATIC ENCEPHALOPATHYSCHEAU CRISTIAN1, LAZĂR MIHAI2, BUNEA MAGDA1, CIORNEI CĂTĂLINA1, PAPACOCEARALUCA1, ION DANIELA ADRIANA2, BĂDĂRĂU ANCA IOANA2
1
Department of Physiology I, “Carol Davila” University of Medicine and Pharmacy, Bucharest
2
Department of Physiopathology, “Carol Davila” University of Medicine and Pharmacy, Bucharest

ABSTRACTHepatic encephalopathy is a clinical entity which is determined by the disturbance in the astrocitary-neuronal metabolic cycle, causedby excessive plasmatic ammonia in the context of liver failure to eliminate this by-product. Myo-inositol decreases were observed in thiscases, as it seems to act as a buffer to compensate the osmotic imbalance. This paper seeks to identify correlations between myo-inositol
levels and osmotic markers values in hepatic encephalopathy, with the intent of clarifying the physiopathological processes involved.
Keywords: Hepatic encephalopathy, physiology, physiopathology, myo-inositol, choline, glutamate, glutamine, MRI, spectroscopy]

INTRODUCTION
Hepatic insufficiency syndrome, indifferent of the determinating cause, has a suite of clinical and paraclinical manifestations,in which ammonia plays one of the leading roles (1), as it is being
accumulated in the plasma due to the incapacity of the liver tometabolize it. Ammonia normally crosses the blood-brain barrier and enters the astrocitary-neuronal metabolic cycle, where
it represents a substrate for glutamate to glutamine conversion.
Its presence here, in amounts higher than usual, overloads thisreaction and leads to increased amounts of glutamine, an osmotically active compound. High osmotic content of the cell producesswelling, and this is the moment when myo-inositol comes into
play, as it is released from the cell to the interstitial space in thetendency to normalize cellular osmolarity (2).
Although these observations have been objectified bynumerous studies dating since the early 2000s, the physiopathological processes are not fully comprehended, and also,
the impact and efficiency of these mechanisms on the underlying
disease and overall evolution of the hepatic encephalopathy are
yet to be established.
Myo-inositol seems to be one of the main osmotically-active
metabolites involved in the regulation of cellular volume in regard
to the osmotic pressure, in the attempt to avoid swelling (3).
Magnetic resonance imaging (MRI) remains the spearhead
in morphological central nervous system evaluation, and due torecent progress in the field, can now assess functional information about specific metabolite dynamics in the brain (4). Magnetic
resonance spectroscopy (MRS) has been successfully used increating a profile spectrum of hepatic encephalopathy, identifying the aforementioned metabolic imbalances as increased
glutamate-glutamine peaks (Glx) and decreased choline (Cho)and myo-inositol (Ins) peaks, respectively. Choline seems tohave an effect similar to myo-inositol in regulating osmotic
disturbances (5).

Magnetic resonance imaging can also quantify the tissularwater amount, defining the grade of edema and thus provide indirect information on the physiopathological processes involved.
The aim of this study is to evaluate the specificity of myoinositol as a marker for minimal hepatic encephalopathy, describe
its variation in relation to the other metabolites involved, andto discuss the dynamics of these metabolites in relation to thetissular water content in specific areas.
PATIENTS AND METHODS
Thirty-two patients were enrolled in our study (18 male and
14 female), aged 26 to 81, averaged 53.3 years. The patients
underwent a neuro-psychological test, followed by an MRI
examination with MRS profiling in the same day.
Twenty-one patients suffering from chronic liver insufficiency
were divided into two groups concordant with the psychologicaltest: one group consisted of patients with minimal hepatic encephalopathy (altered test): 6 male and 4 female; the other groupgathered the patients without any kind of hepatic encephalopathy(normal test). The third lot of patients was healthy controls (6male, 5 female).
The neuro-psychological testing consisted of the PortoSystemic Encephalopathy (PSE) Syndrome Test, a standardized
questionnaire with 5 tests (serial dotting test, line tracing test, twonumber connection tests, digit-symbol test) recommended in theevaluation of minimal hepatic encephalopathy (6).
The MRI examination followed the protocol: T2 weighed
images (axial plane), diffusion-weighed images (par-axial plane),and fluid attenuated inversion-recovery images (coronal plane).
MRS imaging method protocol was the following: single
voxel spectroscopy (SVS), short-TE (30 ms), 8 cm3 voxel
size (2 x 2 x 2 cm), and parietal white matter in the dominanthemisphere (Figure 1). Several calibration tests correlated with

Received 15th of January 2013. Accepted 5th February 2013. Address for correspondence: Scheau Cristian, MD, PhD student, “Carol Davila” University of Medicine andPharmacy, Bucharest, 8 Eroilor Sanitari Boulevard, 5th district, 050474, Romania; telephone: +40723778840, e-mail: cristianscheau@gmail.com

2013.23.1 (77)  Fiziologia - Physiology

23the literature studies confirmed that this particular location was
optimal for detection of the metabolites of interest in this case(see discussions).

myo-inositol (Ins), glutamate-glutamine-gamma amino butyricacid complex (Glx), choline (Cho), N-acetyl aspartate (NAA),
creatine (Cr), and lactate (Lac). Out of these, those relevant
to our study and their inter-relation were summarized in Table
I. Creatine is usually utilized as a reference molecule due toits high stability in various conditions, in different ages andmetabolic states (8).
Table I. Metabolite average values in the study lots. Relevant ratios betweenIns, Glx and Cho.

Fig.1. Normal MRS spectrum acquired in the parietal white matter in thedominant hemisphere. Three dimensional localization system on the righthand side. Assessed metabolites right to left: Lactate (Lac), N-Acetyl-Aspartate (NAA), Glutamate (Glx), Creatine (Cr), Choline (Cho), Myo-inositol (Ins);
secondary and tertiary peaks displayed.

Statistical analysis tests were performed two-sided on thelevel alpha = 0.05; the dependencies between parameters were
tested using Pearson correlation coefficient, and t (student) Test.
RESULTS
Patient distribution was represented in Figure 2. Male tofemale ratio was 1.2 in the control and no hepatic encephalopathy(No HE) lots, and 1.5 in the Minimal HE lot, respectively.

Lots\Parameters M F

Average Ins Ins/CrIns/Glx Cho/Cr Cho/Glx

Control

65126.31±430.56 0.27

0.76

0.37

No HE

6

5

90.44±53.72 0.36 0.20

0.71

0.39

Minimal HE

6463.78±58.95 0.29 0.11

0.66

0.26

The morphological assessment of the general tissular watercontent in the parieto-occipital matter and the cortico-spinal
tracts bilaterally used T2 weighed images. Diffusion weighed
imaging and fluid attenuated inversion recovery sequences were
employed only to clarify the findings in cases of various artifacts.
The results are shown in Table II.
Table II. Tissular water content evaluation through T2 weighed imaging
Lots\Location

Parieto-occipital white matter

Cortico-spinal tracts

Control

336.5 ± 52.15

358.75 ± 49.03

No HE

294 ± 75.81

323.6 ± 53.96

Minimal HE

336.25 ± 22.71

343 ± 15.29

DISCUSSION
Decreased myo-inositol values in the minimal hepatic
encephalopathy lot seemed to correlate with the presence of
this condition (p=0.00505). Also, lower values (though within thenormal range) of myo-inositol were found in lot 2 (patients withchronic liver disease with no HE). This might suggest a tendencyof these patients to evolve towards MHE (Figure 3).

Fig.2. The 3 patients lots distribution by sex (M=male, F=female)

Various studies recommend different areas for MRS measurements in diffuse central nervous system metabolic disease.
Our previous experience in the field of MRS demonstrated thatfor this particular study, the best location of the voxel would bein the parietal white matter. Tests on healthy controls and several
patients suffering of HE showed that this region holds the mostloyal ratio of metabolites measured by MRS compared to theiractual levels in the intracellular and interstitial spaces (7).
The metabolites routinely measured through MRS were
24

Fig. 3. Distribution of myo-inositol values in the 3 lots. Standard deviation overlay

Fiziologia - Physiology  2013.23.1 (77)Nevertheless, we experienced high standard deviation
values in the three lots, which might suggest the need for larger
lots for a better statistical accuracy.
Tissular water content as measured by our method offeredcontroversial data, as it seemed highest in the lot of MHE but
nearly identical in the control group (Figure 4). Lowest values
were found in the No HE lot. In all cases, we delineated higher
values at the level of the corticospinal tracts than those in theparietal white matter. CST values followed the distribution of
those in the parietal region.
In regard to the correlation between the myo-inositol values
and the tissular water content, we failed to obtain statisticalcoherence. Though intuitive that lower values of myo-inositol
measured by MRS suggest its release from the cell in the caseof hyperosmolarity, increased water content in the location of
the measurement was not demonstrated. This might be because
the alteration in water content is very discrete, and though it has
been identified in advanced stages of HE, in MHE the finding
was not confirmed with the methods implied.
Choline variations were coherent with the myo-inositol trend
in the study lots, supporting the idea of these two molecules
having a corroborated effect in osmotic regulation (p<0.001).
Improvement of this study could be achieved by larger
patient lots, and also by repeated MRS measurements to diminish the possibility of patient movement reading errors (image
artifacts).

Fig.4. Tissular water content in the three lots measured at the level of thevoxel placement (P-VOI) and at the level of the cortico-spinal tracts (CST)

CONCLUSION
Our findings confirm the fact that myo-inositol plays animportant role in the cascade of events set in motion by theammonia increase in hepatic encephalopathy. Its decrease correlates with the presence of minimal HE, validating the hypothesisthat it acts as an osmotic buffer.
Choline variations line up with myo-inositol trends in the
study groups proving a correlation of the two in the physiopathologic events of HE.
Higher tissular water content was expected in the caseof MHE compared to the control lots, yet the findings wereinconclusive, suggesting the need of a more sensitive methodfor detection.
ACKNOWLEDGEMENT: This paper is supported by the
Sectoral Operational Programme Human Resources Development (SOP HRD) 2007-2013, financed from the European Social
Fund and by the Romanian Government under the contract
number POSDRU/107/1.5/S/82839
REFERENCES
1. Scheau C, Lazăr M, Bunea M, Ciornei C, Papacocea R, Ion DA,Bădărău AI. Physiology and physiopathology of hepatic encephalopathy.
Fiziologia-Physiology, 2012; 3(75): 23-25.
2. Shawcross D, Jalan R. The pathophysiologic basis of hepatic encephalopathy: central role for ammonia and inflammation. Cell Mol Life
Sci., 2005; 62: 2295-304.
3. Strange K. Regulation of solute and water balance and cell volume inthe central nervous system. J Am Soc Nephrol, 1992; 3: 12-27.
4. Scheau C, Preda EM, Popa GA, Ghergus AE, Capsa RA, Lupescu
IG. Magnetic Resonance Spectroscopy – a non-invasive method in
evaluating focal and diffuse central nervous system disease. J Med
Life, 2012; 5 (4): 423-427.
5. Lee JH, Seo DW, Lee Y, et al. Proton magnetic resonance spectroscopy (1H-MRS) findings for the brain in patients with liver cirrhosis reflect
the hepatic functional reserve. Am J Gastroenterol 1999; 94: 2206-13.
6. Ferenci P, Lockwood AH, Mullen K, et al. Hepatic encephalopathy– definition, nomenclature, diagnosis, and quantification: final reportof the working party at the 11th World Congress of Gastroenterology,
Vienna 1998. Hepatology, 2002; 35: 716-21.
7. Naegele T, et al. MR Imaging and 1H Spectroscopy of Brain Metabolites in Hepatic Encephalopathy: Time-Course of Renormalization afterLiver Transplantation1. Radiology, 2000; 216.3: 683-691.
8. Hennig J, Pﬁster H, Ernst T, Ott D. Absolute quantiﬁcation of metabolites in the human brain with in vivo localized proton spectroscopy.
NMR Biomed. 1992; 5: 193-199.

IMPORTANŢA MIO-INOZITOLULUI IN FIZIOPATOLOGIAENCEFALOPATIEI HEPATICEREZUMAT

Encefalopatia hepatică este o entitate clinică determinată de perturbarea ciclului metabolic astrocitaro-neuronal, produs de valori
plasmatice crescute de amoniu în contextul eşecului ficatului de a elimina acest produs secundar. Scăderi ale valorilor mio-inozitolului
au fost observate în aceste situaţii, întrucât acesta pare să funcţioneze ca un tampon în compensarea dezechilibrelor osmoticeproduse. Această lucrare caută să identifice corelaţii între nivelul mio-inozitolului şi valorile markerilor osmotici în encefalopatia
hepatică, cu intenţia de a clarifica procesele fiziopatologice implicate.
Cuvinte cheie: encefalopatie hepatică, fiziologie, fiziopatologie, mio-inozitol, colină, glutamat, glutamină, IRM, spectroscopie
2013.23.1 (77)  Fiziologia - Physiology

25

PULSE PRESSURE: PROGNOSTIC SIGNIFICANCE
IN THE STRATIFICATION OF CARDIOVASCULARRISKDANA BÂIBÂŢA, GEORGIANA IONESCU, ANGELA DRAGOMIR, SILVIA MANCAȘ
“Victor Babeș” University de Medicine and Farmacy, Timisoara

ABSTRACT

Aim: to evaluate the correlations between the pulse pressure and cardiovascular risk factor: hemodinamic parameters, metabolic
factors and traditional risk factors.
Materials and methods: our survey included 398 consecutive patients with high cardiovascular risk, hospitalized in 2010 in thePreventive Cardiology Clinic of the Institute of Cardiovascular Diseases Timisoara. For every patient, demographic and anamnestic
data as well as common cardiovascular risk factors, metabolic profile and hemodinamic profile (24h-BP Holter survey and 24h-PP)
were registered.
Results: we have obtained highly significant statistical correlations between 24h pulse pressure and hemodinamic parameters:
24h-SBP (p<0.001, r=0.752) and 24h-MBP (p<0.001, r=0.511); significant statistical correlations were found between 24h-PP andmetabolic parameters: LDLc (p=0.038, r=0.156) and glycemia (p=0.001, r=0.162). There were no significant statistical correlations
found between 24h-PP and other common cardiovascular risk factors (smoking, history of coronary artery disease). 24h-PP variability
was determined by linear regression equation by 2 factors: 24h-SBP and 24h-MBP.
Conclusions: the established cut-off value of the 24h-PP validates the fact that people with increased 24h-PP have elevated 24h-SBP,
thus presenting high cardiovascular risk.
We proved the existence of a direct highly significant statistical correlation between 24h-PP and hemodinamic parameters, andbetween 24h-PP and some metabolic factors.
The 24h-PP showed its predicting significance in the assessment of the cardiovascular risk in direct correlation with the SBP and MBP.
Key words: pulse pressure, mean blood pressure , cardiovascular risk factors.

BACKGROUND
The pulse pressure is the pulsatile blood pressure component and is a marker of cardiovascular risk, reflecting large
artery stiffness.
In recent years, seemingly simple direct relationship between cardiovascular risk and systolic and diastolic blood pressure
was complicated by the results of observational studies showingthat the risk is directly proportional to systolic and, for any given
level of systolic blood pressure, the prognosis is inversely proportional to diastolic 1-2 with a strong predictive value of pulsepressure (1). The predictive value of pulse pressure may varydepending on the clinical characteristics of subjects.
In the biggest meta-analasys of the observational surveys
that exists today (61 surveys including almost 1 million subjects
without obvious cardiovascular disease-70% from Europe), both
24h-SBP and 24h-DBP presented independently similar predicting
values concerning mortality due to stroke or coronary disease. In
the case of hypertensive patients presenting cardiovascular riskor any other comorbidities, the 24h-PP was proved to have an
important predicting significance for cardiovascular events (1,2)
Aim - to asses possible correlations between 24h-PP and
the components of global cardiovascular risk: hemodinamic

parameters (24h-SBP, 24h-DBP, 24h-MBP), smoking, metabolic
factors, lipid profile.
MATERIALS AND METHODS
398 consecutive patients with cardiovascular risk, hospitalized in 2010 in the Preventive Cardiology Clinic of the Institute ofCardiovascular Diseases Timisoara were included in this study.
We have used demographic and anamnestic data, full physicalexamination and laboratory examination of the patients. The
inclusion and exclusion criteria are shown in Table I.
Table I. Inclusion and exclusion criteria

Inclusion criteria
•
•
•
•
•

Cardiovascular
asymptomatic (Risk
Score <5%)
Metabolic Syndrome
Dyslipidemia
Diabetus melittus
EF≥40

Exclusion criteria••••

Arrhytmias
Valvulopathies
Congenital
cardiovasculardiseaseEF<40

Received 18th of February 2013. Accepted 15th of March 2013. Address for correspondence: Emilia Dana Bâibâţa, University of Medicine and Pharmacy “Victor Babeş”Timişoara, Eftimie Murgu Square No. 2A, RO-300041, Timisoara, phone: +40745615654; +40737034932, e-mail: danaemilia@yahoo.com

26

Fiziologia - Physiology  2013.23.1 (77)Clinical assessment:
For every patient, we took into consideration:• Anamnestical data
• Family history of cardiovascular disease• The evaluation or common risk factors:
o changeable: hypertension, smoking, TC,
LDL>115mg/dl, HDL<40mg/dl , diabetus
mellitus, BMI
o not changeable: age, male, genetic
susceptibility• For defining the asymptomatc subject, the ESC
criteria were used (1)processed using S.P.S.S. 17.0. Data normality was assessedusing the Shapiro-Wilk test. After that, the data was analysedusing parametric tests like Independent Samples t test for coparing the mean values in two groups, and One Way ANOVA
for comparing the mean values in more than three groups. The
testing of the association between qualitative variables was madeusing Chi square test, and for testing the correlations between
numeric variables, Pearson correlation coefficient was used.
Using a model of variable logistic regression, the 24h binary
dependency was analysed. We included the following independent variables: 24h-SBP, 24h-MBP, glycemia, LDL, HDL, totalcholesterol and tryglicerides.

Assessment of the cardiometabolic profile:
The assessment of the lipid profile and risk categorization
were made according to ESC guidelines recomandations (1).
Indirect measurement of LDL was performed by using theFriedewald equation:

RESULTS
The prevalence of hypertension was 88,5% in the analysedgroup; 27.1% of patients were diagnosed with Diabetes
mellitus type 2 and 10.5% of them, with metabolic syndrome.
Table III. General features

LDLc = total cholesterol – HDL – (triglycerides/5)
NonHDLc was calculated by substracting HDLc from
TC.The metabolic syndrome was defined according to IDF
criteria, if at least 3 of 5 criteria were found (1).
Diabetus mellitus was defined by fasting plasma glucose>126 mg/dl or existing treatment.
Table II. Metabolic syndrome- defining criteria (IDF)

CRITERIA

NCEP-ATP III

IDF

Cardiovascular risk
factors*:

Metabolicprofile **:Primary hypertension

88.5

Diabetes mellitus type 2

27.1

Smokers (active smokers)

9.5

Metabolic syndrome

10.5

Total cholesterol (mg/dl)

194 ± 69.32

Tryglicerides (mg/dl)

151 ± 99.23

LDLc (mg/dl)

100 ± 65.46

Abdominal>88 (F),>102 (M)circumference(cm)>80 (F),>94 (M)*

24H-SBP (mmHg)≥ 130/80≥ 130/80

HDLc (mg/dl)

43 ± 24.53

Glycemia (mg/dl)

> 110

> 100

Tryglerides (mg/dl) >150

>150

Glycemia (mg/dl)

108±36.13

HDL-c (mg/dl)<50(F),<40(M)<50(F),<40(M)* If BMI ≥ 30 kg/m², the measuring of the abdominal circumference is no more
required, according to the IDF

Assessment of the hemodinamic profile:
We assessed the profile of blood pressure according tote outcomes of 24 h recording of the BP, using BTL-08 ABPM.
Measurements were made every 15 minutes during the day (7am- 23 pm) and every 30 minutes during the night (23 pm-7 am),being repeated if more than 30% of them presented artifacts.
During the monitoring period, medication was discontinued. Fromthe protocol of 24 hour BP monitoring, we used the following:
24h-SBP, 24h-DBP, 24h-PP and 24h-MBP (1).
Automatic blood pressure monitoring allowed for defining
hypertension category for the SBP>130mmHg. All the 24hPPvalues above 60 mmHg were considered pathological.
Statistical analysis: All data was stored electronically and*-% of group ; **-mean +/- standard deviation

The correlation between 24h-PP and hemodynamic
parameters: we have obtained a highly significant positive
correlation between 24h-PP and 24h-SBP (p<0.001, r=0.752).

2
4
h
|
S
B
P

24h-PP
Fig.1. Mean 24h-SBP in 24h-PP categories

Fig.1. Mean 24h-SBP in 24h-PP categories

Mean 24h-SBP was significantly higher in the group of subjects with pathological values of 24h-PP. Those with 24h-PP ≥ 60mmHg showeda mean of 24h-SBP=154 ± 22 mmHg. Those with 24h-PP <60 mm Hg had a mean of 24h-SBP=126 ± 14 mmHg (p <0.001) (Figure 1).

2013.23.1 (77)  Fiziologia - Physiology

27

Mean 24h-SBP was significantly higher in the group of subjects with pathological values ​​of 24h-PP. Those with 24h-PP ≥60mmHg showed a mean of 24h-SBP=154 ± 22 mmHg. Thosewith 24h-PP <60 mm Hg had a mean of 24h-SBP=126 ± 14mmHg (p <0.001) (Figure 1).

Correlation between 24h-PP and metabolic parameters: wehave obtained a highly significant statistical correlation between
24h-PP and glycemia (figure no. 3) (p=0.001, r=0.162). The
correlation between 24h-PP and LDLc was also statisticallysignificant (p=0.038, r=0.156) (Table IV).

Fig.2. Correlations between 24h-PP and 24h-SBP
Fig.4. Correlation between 24h-PP and glycemia

We also obtained a highly significant positive correlation
between 24h-PP and 24h-DBP (p<0.001, r=0.246) as well as
between 24h-PP and 24h-MBP (p<0.001, r=0.511) (Table IV).

Fig.5. Correlation between 24h-PP and LDLc

Fig.3. Correlations between 24h-PP and 24h-MBP

There is a statistically significant association between 24hPP, 24h-SBP ( fig.no.3) and 24h-DBP (p=0.003) categories. We
have also obtained a statistically significant association between
24h-PP and 24h-SBP (p<0.001), respectively 24h-DBP (p=0.003)(Table IV)
Table IV. Correlations between 24h-PP and cardio-metabolic risk parameters

Dependent
varianle

Independent Pearson
variablecorrelation
coefficient

Statisticalsignificance24h-SBP24h-PP= pulse
24h-DBPpressure
24h-MBP

r=0.752p<0.001

r=0.246p<0.001

r=0.511p<0.001

glycemia

r=0.162p=0.001

LDLc

r=0.156

p=0.038

28

Tabel V. Mean values of the metabolic parameters analyzed in the 2 groupsof patients

Mean value * 24h-PP≥60mmHg(1) 24h-PP<60mmHg (0)glycemia

112±44105±28

LDLc

53±74

49±59

TC

187±69175±69TG

77±10480±96

nonHDLc

166±65.4

153.5±65.4*mg/dlThe patients with 24h-PP ≥ 60 mm Hg had mean values ofthe metabolic parameters higher compared to those with PPint <60mmHg (Table V). Those with metabolic syndrome had mean
24h-PP of 56.12 ± 17.86 . Only 42.23% of those with metabolic
syndrome had values of 24h-PP ≥ 60mmHg.
We didn’t obtained statistically significant correlations
between: 24h-PP and BMI (p = 0.708), 24h-PP and TC (p =0.118), 24h-PP and TG (p = 0.374), 24h-PP and HDL (p = 0.457)
and 24h-PP and nonHDL (p = 0.133).
The correlations between 24h-PP and BMI (p=0.708), 24hFiziologia - Physiology  2013.23.1 (77)PP and total cholesterol (p=0.118), 24h-PP and tryglicerides(p=0.374), 24h-PP and HDL (p=0.457), 24h-PP and nonHDL(p=0.133) were not statistically significant.
The model of variable logistic regression used for analysing the 24h-PP binary dependency was significant for two
predictors: 24h-MBP and 24h-SBP- p value<0.001.
We have obtained the following logistic regression model
equation:
24h-PP=0.342 x (24h-SBP) – 0.349 x (24h-MBP) – 12.049

The variation of 24h-PP was explained at a rate of 71,9%by the predictor variables: 24h-SBP and 24h-MBP (Rsquare =0.719).
DISCUSSION
The correlation between 24h-PP and the hemodinamic
parameters (24h-SBP, 24h-MBP, 24h-DBP)
In our study, we have obtained statistical significant correlations between 24h-PP and 24h-SBP in the entire group of subjects.
The 24h-PP reflects the tendency in the increasing of 24h-SBP
and in the decreasing of 24h-DBP due to the increased arterial
stiffness (3,4), thus reflecting the stiffness of large arteries.
Thereby, when the 24h-SBP is pharmacologically controlled, thedrecreasing of the 24h-DBP will expose the patient to a 24h-PP> 60 mmHg, thus to risk (5,6).
Therefore, in the assessment of the cardiovascular risk, it’swrong to focus only on one hemodinamic parameter, but we musttake into account both the pulsating part (24h-PP) and 24h-MBP.
Dyer et all, in all four Chicago epidemiological prospective
surveys which included 28360 subjects, men aged between
18-74 and women aged between 40-74, have obtained a statistically significant correlation between 24h-MBP, 24h-PP andcardiovascular risk (7,8).
There is clear evidence that, reflecting the arterial stiffness,the 24h-PP represents an independent and significant cardiovascular risk factor. Madhavan et all reported that pacientswith 24H-PP > 63 mmHg present a high risk for cardiovascularcomplications (9) They have discovered that, in case of severe
drop of 24h-DBP due to antihypertensive medication, there isan increased risk of myocardial infarction. In the asymptomatic
cardiovascular patients, this shows the importance of 24h-PP
and 24h-MBP assessment as variables which are to be considered in order to increase the accuracy of cardiovascular risk
stratification.
The predictive significance of the 24h-PP may vary depending of the clinical features of the subjects. In the biggest
meta-analasys of the observational surveys that exists today(61 surveys including almost 1 million subjects without obvious
cardiovascular disease-70% from Europe (10), both 24h-SBP
and 24h-DBP presented independently similar predicting values concerning mortality due to stroke or coronary disease, at
hypertensive patients with cardiovascular risk factors or any
2013.23.1 (77)  Fiziologia - Physiology

other comorbidities. The 24h-PP was proved to have an important
predicting significance for cardiovascular events (11,12).
Correlation between 24h-PP and metabolic parameters
In our study, we have obtained statistical significant correlations
between the hemodinamic parameters (arterial stiffness-24h-PP)
and LDLc.
The statistical significant correlations between 24h-PP and LDLc
that we have found, suggest the fact that persistent hypertension
combined with persistent lipoprotein molecules such as LDLc
can lead to increased 24h-PP (13) Although Zanchetti et all havefound a statistical significant correlation between 24h-PP and TC(14), at values of TC= 262,4 mg/dl, TG= 181.3 mg/dl, HDLc =141mg/dl, LDLc=141mg/dl, this was not sustained by our study.
This may be due to a mean of TC of only 194 mg/dl in our groupof patients. Also, the mean of TG and HDLc was situated close
to the cardiovascular protection cut-off limit in asymptomaticpatients. We consider that this circumstance has led to lackof statistical significant correlations between 24h 24h-PP and
lipid risk profile. Other studies emphasize the arterial stiffening
condition, related with increased values of TC and LDLc, andanalyzed in terms of 24h-PP behaviour (15,16).
In our group of patients, we have found statistical significant
correlations between the hemodinamic parameters (arterial
stiffness-24h-PP) and glycemia. Miyagi et all, in a multicentercross-sectional study which included 939 hypertenive patients,
obtained a statistical significant correlation between glycemic
index, 24h-PP and the decreased value of HDL (17).
CONCLUSIONS• The established cut-off value for 24h-PP confirm the factthat subjects with increased 24h-PP have increased 24h-SBP• We have demonstrated the existence of a direct highly
significant correlation between 24h-PP and hemodinamic parameters, and between 24h-PP and some of the cardio-metabolic
parameters (LDLc, glycemia)• In hypertensive patients, the 24h-PP cand be considereda factor with prognostic value concerning the cardiovascular risk
stratification, due to its direct connection with BP
REFERENCES
1. European Guidelines on cardiovascular disease prevention in clinical practice (version 2012) The Fifth Joint Task Force of the European
Society of Cardiology and Other Societies on Cardiovascular Disease
Prevention in Clinical Practice (constituted by representatives of ninesocieties and by invited experts) European Heart Journal (2012)33,
1635–1701doi:10.1093/eurheartj/ehs 092
2. Mancia G, et al. The 2007 Guidelines for the Management of Arterial
Hypertension. Europ. Heart J., 2007; 28: 1462-536.
3. Prospective Studies Collaboration. Age-specific relevance of usual
blood pressure to vascular mortality: a meta-analysis of individual
data for one million adults in 61 prospective studies. Lancet, 2002;
360: 1903-1913.
4. Gasowski J, et al. INDANA Project Collaborators. Pulsatile blood

29

pressure component as predictor of mortality in hypertension: a metaanalysis of clinical trial control groups. J Hypertens, 2002; 20:145-151.
5. Safar ME, Blacher J, Jankowski P. Arterial stiffness, pulse pressure,and cardiovascular disease-is it possible to break the vicious circle?
Atherosclerosis, 2011; 218: 263-271.
6. Madhavan S, Ooi WL, Cohen H, Alderman MH. Relation of pulsepressure and blood pressure reduction to the incidence of myocardial
infarction. Hypertension.,1994; 23: 395-401.
7. Safar ME, et al. Pulse pressure not mean pressure determines
cardiovascular risk in older hypertensive patients. Arch Intern Med,
2000;160:1085-1089.
8. Benetos A, Safar M, Rudnichi A, Smulyan H, Richard JL, Ducimetieere
P, Guize L. Pulse pressure: a predictor of long-term cardiovascular mortality in a French male population. Hypertension, 1997; 30:1410-1415.
9. Prospective Studies Collaboration. Age-specific relevance of usual
blood pressure to vascular mortality: a meta-analysis of individual
data for one million adults in 61 prospective studies. Lancet, 2002;
360:1903-1913.
10. Laurent S, et al. On behalf of the European Network for non invasive
investigation of large arteries. Expert consensus document on arterial
stiffness: methodological issues and clinical applications. Eur Heart J,
2006; 27: 2588-2605.
11. Mitchell GF, et al. For the SAVE Investigators (Survival and Ventricular Enlargement). Sphygmomanometrically determined pulse pressure
is a powerful independent predictor of recurrent events after myocardial
infarction in patients with impaired left ventricular function. Circulation,
1997; 96: 4254-60.
12. Verdecchia P, et al. Ambulatory blood pressure: an independent
predictor of prognosis in essential hypertension. Hypertension, 1994;

24: 793-801.
13. Gasowski J, et al. Pulsatile blood pressure component as predictor of mortality in hypertension: a meta-analysis of clinical trial control
groups. J Hypertens, 2002; 20:145-151.
14. Blacher J, Staessen JA, Girerd X, Gasowski J, Thijs L, Liu L, Wang
JG, Fagard RH, Safar ME. Pulse pressure not mean pressure determines cardiovascular risk in older hypertensive patients. Arch Intern
Med 2000; 160:1085-89.
15. Joint National Committee on the Detection, Evaluation, and Treatment of High Blood Pressure. The sixth report of the Joint National
Committee on prevention, detection, evaluation, and treatment of highblood pressure. Arch Intern Med, 1997; 157: 2413-46.
16. Zanchetti A, Crepaldi G, Bond MG, et al. Systolic and pulse bloodpressures (but not diastolic blood pressure and serum cholesterol) areassociated with alterations in carotid intima-media thickness in themoderately hypercholesterolaemic hypertensive patients of the PlaqueHypertension Lipid Lowering Italian Study. Journal of Hypertension,
2001; 19: 79-88.
17. Darne B, Girerd X, Safar M, Cambien F, Guize L. Pulsatile versus
steady component of blood pressure: a cross-sectional analysis anda prospective analysis on cardiovascular mortality. Hypertension.,
1989;13: 392-400.
18. Gaudio G, et al. Correlation between pulse pressure and LDLcholesterol in patients with resistant hypertension. American Journalof Hypertension, 2001; 14: 246A-247A.
19. Miyagi T, et al. Increase in Pulse Pressure Relates to Diabetes
Mellitus and Low HDL Cholesterol, but Not to Hyperlipidemia in Hypertensive Patients Aged 50 Years or Older. Hypertens Res, 2002;
25: 335-341.

PRESIUNEA PULSULUI: SEMNIFICATIA PROGNOSTICA PENTRUSTRATIFICAREA RISCULUI CARDIOVASCULARREZUMAT

Scop: evaluarea corelatiilor dintre presiunea pulsului si factorii de risc cardiovascular: parametrii hemodinamici, factorii metabolici,
factorii de risc traditionali. Materiale si metode: studiul nostru a inclus 398 pacienti cu risc cardiovascular crescut, spitalizati in anul
2010 in Clinica de Cardiologie Preventiva a Institutului de Boli Cardiovasculare Timisoara. Pentru fiecare pacient au fost evaluate
datele anamnestice si demografice, factorii obisnuiti de risc cardiovascular, profilul metabolic si hemodinamic (24h-BP Holter si
24h-PP). Rezultate: am obtinut corelatii statistice inalt semnificative intre presiunea pulsului pe 24h si parametrii hemodinamici:
24h-SBP (p < 0,001, r = 0,752) and 24h-MBP (p < 0,001, r = 0,511); corelatii cu semnificatie statistica au fost evidentiate intre 24hPP si parametrii metabolici: LDLc (p = 0,038, r = 0,156) si glicemie (p = 0,001, r = 0,162). Nu au fost gasite corelatii cu semnificatie
statistica intre 24h-PP si alti factori comuni de risc cardiovascular (fumat, antecedente de boala coronariana). Variabilitatea 24h-PP
a fost determinata prin ecuatia de regresie liniara cu 2 factori: 24h-SBP si 24h-MBP. Concluzii: valoarea cut-off a 24h-PP valideaza
faptul ca pacientii cu 24h-PP crescut au valori crescute si ale 24h-SBP, astfel incat prezinta risc cardiovascular inalt. Am dovedit
existenta unei corelatii inalt semnificative statistic intre 24h-PP si parametrii hemodinamici, precum si intre 24h-PP si anumiti factori
metabolici. 24h-PP este un parametru cu semnificatie predictiva in evaluarea riscului cardiovascular in corelatie directacu SBP si MBP.
Cuvinte cheie: presiunea pulsului, presiune sanguina medie, factori de risc cardiovascular

30

Fiziologia - Physiology  2013.23.1 (77)JUVENILE NASOPHARYNGEAL ANGIOFIBROMA- HISTOLOGICAL AND IMMUNOHISTOCHEMICALLANDMARKS
DANA FLORENTINA GIDEA1,2, GEORGE IOVANESCU1,3, GHIRAN RAMONA MARIA2,4 , MARIA DOMUTA2,5, STAN COTULBEA3,4 , DELIA HORHAT1,3
1
Emergency Hospital Timisoara County
2
MD PhD Student UMF Victor Babes Timisoara
3
UMF Victor Babes Timisoara
4
Municipal Emergency Hospital Timisoara
5
Faculty of Medicine and Pharmacy Oradea

ABSTRACT

Objectives: Juvenile nasopharyngeal angiofibroma is a rare, histologically benign tumor, very aggressive locally, affecting male adolescents or those at puberty (around age 15). In terms of histopathology, it is an angiofibroma, characterized by fibrous swirls andblood lakes, without walls. The fibrous elements initially have only a tumor supporting stroma role, then they become a predominant
structural component, as the effect of estrogen decrease. Patients and methods: This study is a retrospective that includes assessment of tumor resection pieces of 10 patients diagnosed and treated in the ENT Clinic Timisoara between 2002-2012. It showsthe
macroscopic tumoral aspects, the microscopic characteristics and Ac estrogen anti-receptor, Ac progesterone anti-receptor, Ac
antivimentina-Ventana, Ac CD117, Ac muscle specific actin, Ac ki67, CD31, and Ac CD34.Results: All patients were male. The most frequently encountered histological model contains in the stroma immature fusiform fibroblasts and thin-walled blood vessels in different
proportions. The tumor does not have its own capsule. But, around these tumors, usually the non-ulcerative, one can occasionallyobserve a pseudocapsule derived from pharyngeal or nasal mucosa, of atrophic and stretched appearance. The dense fibrousstroma may show areas of hyaline altering or focal myxoid issues. Multinucleated stromal cells may be present, with “ganglion-like”,
intensely eosinophilic cytoplasm.Debate: The ratio between blood vessels and tumor stroma varies based on the tumor thickness.
Ultrastructural studies have shown that stromal cells are myofibroblast, with variable expression of fibroblast and myofibroblast features. Conclusions: Although angiofibroma is a histologically benign tumor, it may have an aggressive behavior through its recurrence
and ability to destroy adjacent bone structures.
Keywords: juvenile nasopharyngeal angiofibroma, benign, microscopical, macroscopical, histological.

INTRODUCTION
Juvenile nasopharyngeal angiofibroma is a rare, histologically benign tumor of the nasopharynx, affecting almost
exclusively male teens or pubers, being diagnosed in boys
between 14-25 years. It is a highly vascularized tumor with hightendency to local and loco-regional invasion, extending into thesubmucosa. For this reason it is a destructive tumor. From thehistological point of view, it’s a mesenchymal, vascular, and highcell tumor,made of fibrous connective tissue and an abundance ofvascular spaces without endothelium (endothelium lined). There
are various theories about the angiofibroma formation, but themost acknowledged theory is the theory of angiogenesis andhistogenesis. This describes the angiofibroma as a pure vasculartumor that proliferates in ahemangiomatous manner and in which
all other components, including fibrous connective tissue arederived from the undifferentiated vasoformative mesenchymal.
The macroscopic appearance indicates the juvenile nasopharyngeal angiofibroma to be a vascular, lobulated, firm, un-encapsulated, reddish blue, sessile or pedunculatedtumor, withnumerous crusts. The tumor surface, if it was not traumatized
during surgery or during hemostasis, is covered by an intense
red mucosa in the case of young subjects, and pale pink in older
patients, or those in which the vascularization was reduced byradiation or hormone therapy (1). In patients who have hada history of bleeding or hemostatic tamponament, the tumor
surface may showulceration or necrosis, with granular tissue
appearing in such areas.
As texture, some tumors can be rubbery, otherscartilaginous andothers soft, edematous, and occasionally brittle. When sectioned,
the tumor may vary in color from pinkish-white to gray-yellow
to red-brown (1). All tumors have a solid appearance, without
cystic degeneration. Tumors that were previously radiated are
pale and of fibrous consistency; tumors of young people andthose not previously treated are soft and richly vascularized (2).
The microscopic appearance shows a tumor composedalmost essentially of network-shaped blood vessels and con-

Received January 4th 2013. Accepted February 20th 2013. Adress for correspondence: Dana Florentina Gidea, MD, PhD Student UMF Victor Babes Timisoara, Emergency
Hospital Timisoara County; Brandusei Street No.9, Timisoara; phone: +40748117540; e-mail: danafv@yahoo.com

2013.23.1 (77)  Fiziologia - Physiology

31nective tissue. Under the microscope, the appearance can varyfrom fully cavernous angioma, developed in a fibrous stroma, tooccasionalmyxomatous fibroma, or dense cellularity.
The most frequently encountered histological patterncontains immature fusiform fibroblasts in the stroma and thinwalled blood vessels in different proportions. In fact, fusiform orstellate fibroblasts can be so numerous, that they may suggest
a fibrosarcoma or angiosarcoma, tumors that are often confusedwith the juvenile nasopharyngeal angiofibroma (3).
In young subjects or untreated tumors, the angiomatous
elements are more abundant, vessels become larger, irregularor sinusoidal. In old tumors or those who have already undergone radiotherapy or intensive androgen therapy,the vascularcomponents are less represented or may disappear altogether,and thereby fibrous elements can predominate.
Lymphocyte holes or plasma cells may be present, especially in ulcerated or injured tumors. Myxomatous changes canbe observed in various proportions, as well as areas of necrosis.
Occasionally hyalinized thrombican be found, especially in old or
previously treated tumors,as well ashyalinized stroma areas (3).
The tumor does not have its own capsule. But, around theseusuallynon-ulcerated tumors, we can occasionally observe thepseudocapsule derived from pharyngeal or nasal mucosa, andit appears atrophic and stretched.
In this study we try to evaluate the histological and immunohistochemical aspects pieces found when examining thetumor resection.

with few cells and partially containing collagen. The fibrous tissue cells are spindle or stellate, with rounded nucleus and small
nucleon,inconsistently noticeable. The mitotic figures are few
and typical in appearance.

Fig.1. Ac Ki67 -Ventana / Roche, monoclonal, clone 30-9; a) lens 20x; b) 40x
objective; c) overview, 20x objective.

Dense fibrous stroma may show areas of altered hyaline or
focal myxoid issues. Collagen fibers are parallel. Vascular spaces
have a dilated or slotappearance; the number, configuration andwall thickness are variable.

PATIENTS AND METHODThis is a retrospective study including the evaluation oftumor resection pieces of 10 patients diagnosed and treated in
the ENT Clinic Timisoara between 2002-2012, and who received
a histological confirmation of the preoperative diagnosis. The
study includes a total of 10 registry blocks. Serial sections of
4-5 μ thick were performed according to the usual histological
technique. For each case the sections made were stained withhematoxylin-eosin forthe histological diagnosis. The macroscopic
aspects were studied retrospectively, as described when collecting the tumor resection pieces, from the pathological diagnosis
report sheets, which confirmed the preoperative diagnosis. The
macroscopic tumoral features are presented, the microscopic
characteristics, as well as the Ac anti-estrogen receptor, progesterone receptor anti-Ac, Ac antivimentina-Ventana, CD117, themuscle specific Ac actin, Ac ki67, Ac CD31, and Ac CD34.

Fig.2. Nasopharyngeal angiofibroma - overview, H.E. stain; a, b) 20x objective.

Fig.3. Nasopharyngeal angiofibroma - detail H.E. staining; a, b) 40x objective.

RESULTS
Macroscopically, it was found that the resected angiofibromas were well delimited, polypoid or lobulated tumors, coveredby smooth and shinymucous membrane. They are like a firm
and gummy mass. On the section surface, they have a fibrous orspongy appearance (due to the presence of numerous vascularspaces) and are gray-colored.
The microscopic aspect diagnosis is that of fibrous stroma,32

Fig.4. Ac antivimentin (Ventana / Roche monoclonal, Clone V9); a) lens20x;b) lens40x.

Fiziologia - Physiology  2013.23.1 (77)They are most often visible as being padded with a single
row of endothelial cells.

Fig.9. Ac CD31 Cell Margue, monoclonal, clone JC 70; a) lens 20x; b) lens 40x
Fig.5. Ac CD31 - overview; a) lens 20x b) lens 40x.

Fig.10. Ac CD34 -a) overview, lens 20x; b) detail, lens 20x.

Fig.6. Ac CD34 – detail, lens 40x.

Fig.11. Ac anti CD34 ; overview, 20x lens b) detail, 40x objective.

Vessels with thicker walls and lacking elastic fibers may bepresent. Few vessels can be surrounded by flat muscle cells.

Fig.12. Ac CD117 (c-kit) - Ventana / Roche, monoclonal, Clone 9.7, a) 20x
objective; b) 40x objective.
Fig.7. Muscle specific Ac actin, monoclonal, clone HHF 35; a) lens 20x; b)lens 40x.

The ratio between the blood vessels and tumor stroma varies
depending on the tumor thickness. Thus, stromal elements are
more numerous in the central area, while blood vessels are muchless numerous. The blood vessels at the edges of the tumor, andare smaller, more uniform in size, thus the fibrosis is minimal.

Fig.8. Ac CD34 - Cell Margue, monoclonal, clone QBE end/10; a) lens 20x;b) lens 40x.

2013.23.1 (77)  Fiziologia - Physiology

The arrangement of the collagen bands is random. Multinucleated stromal cells with intensely eosinophilic, “ganglionlike”cytoplasm may also be present.

Fig.13. Nasopharyngeal angiofibroma -cell morphology; stromal cells withmultiple nuclei: H.E. stain; 40x objective. Tumors showed no receptors for
estrogen or progesterone (Ac anti estrogen receptor, 6F11/Novocastra clone,
Ac anti progesterone clone and 1A6/Novocastra).

33

DISCUSSION
Ultrastructural studies have shown that stromal cells are myofibroblasts, with variable expression of fibroblast and myoblast
features. These cells contain intranuclear dense granules, whichapparently represent RNA-related protein complexes (4, 9, 10).
The angiofibroma has been shown to have receptors for
testosterone (6) but no receptors for estrogen (6, 7). In the
study groups some tumors (little in number) showed positivity
for progesterone. In our study, tumors did not show receptors
for estrogen or progesterone (Ac estrogen anti-receptor, clone
6F11/Novocastna and Ac progesterone anti-receptor, clone
1A6/Novocastra). Preoperative administration of estrogenoften leads to reduced tumor volume and number of bloodvessels, assuming that the estrogen is inhibiting the releaseof gonadotropic hormones (7).
CONCLUSIONS
Although the angiofibroma is a histologically benign tumor, it
may have an aggressive behavior by recurrence and through its
ability to destroy adjacent bone structures. Macroscopically, thejuvenile nasopharyngeal angiofibroma is a vascular, lobulated,
firm, unencapsulated, reddish blue, sessile or pedunculated
tumor. Microscopically, the tumor is composed almost entirelyof network-shaped blood vessels and connective tissue. Ap-

pearance may vary from a complete cavernous angioma,
developed in a fibrous stroma, to occasional or dense cellularity
myxomatous fibroma.
REFERENCES
1. Andrade NA, Andrade JSC, Silva PDM, Oliveira VB, Andrade BB.
Nasopharyngeal angiofibroma: review of the genetic and molecular
aspects. Intl. Arch. Otorhinolaryngol., 2008; 12: 442-9.
2. Schiff M. Juvenile nasopharyngeal angiofibroma: a theory of pathogenesis. Laryngoscope, 1995; 69: 981-1016.
3. Raica M, Iacovliev M, Mederle O, Alexa A, Sarb S. Histologie medicala, Ed. Mirton,Timisoara, 1997.
4. McAvran MH, Sessions DG, Doufaran RF, Davis DO, Ogura JH.
Nasopharyngeal angiofibroma. Anet. Otolaryngol, 1969; 90: 68-78.
5. Neel HB, Whincken JH, Devine KD, Weilard LH. Juvenile angiofibroma. Review of 120 cases. Am J Jung., 1973; 126: 546-556.
6. Lee DA, Rao BR, Meyen JS, Priolean PG, Bauen WC. Hormon
receptor determination in juvenil nasopharyngeal angiofibroma. Cancer,
1980; 46: 547-551.
7. Johns ME, Mac Lead RM, Cantreli RW. Estrogen receptors in nasopharyngeal angiofibroma. Laryngoscope, 1980; 90: 628-634.
8. Johnson S, Klosten JH, Schiff M. The action of hormons on juvenile
nasopharyngeal angiofibroma. Actaotolaryngol., 1966; 61:153-160.
9. Szobola DJ, Kirchnen F. Ultrastructure of NFAF. Cancer, 1966;
19:1949-62.
10. Taxy JB. Juvenile nasopharyngeal angiofibroma. An ultrastructural
study. Cancer, 1977; 39: 1044-54 .

ANGIOFIBROMUL NASOFARINGIAN JUVENIL – CARACERISTICIHISTOLOGICE ŞI IMUNOHISTOCHIMICEREZUMAT

Obiective: Angiofibromul nasofaringian juvenile este o tumoră rară, benign din punct de vedere histologic, dar foarte agresivă la nivel
local, care afectează personale la vârsta pubertăţii sau adolescenţii de sex masculin (în jurul vârstei de 15 ani). Din punct de vedere
histopatologic este un angiofibrom, caracterizat prin vârtejuri fibroase şi efuziuni sanguine, fără limitare clară. Elementele fibroase
au iniţial doar rol de susţinere a stromei tumorale, mai târziu devenind elemente structurale predominante, ca efect al scăderii estrogenilor. Pacienţi şi metode: Acesta este un studiu retrospectiv care include evaluarea a 10 piese de rezecţie chirurgicală obţinutede la pacienţi diagnosticaţi şi trataţi în Clinica ORL Timişoara între 2002-2012. Evaluarea a inclus aspectul macroscopic tumoral,
caracteristicile microscopice şi evidenţierea diferiţilor markeri – receptorul de estrogen, receptorul de progesteron, Vimentina, Ki67,
CD31 şi CD34. Rezultate: Toţi pacienţii investigaţi au fost de sex masculin. Cel mai frecvent model histologic întâlnit a fost acela
cu fibroblaste fusiforme imature prezente la nivel stromal şi vase sanguine cu pereţi subţiri în diferite proporţii. Tumora nu prezintă
capsulă proprie, dar la nivel peri-tumoral, mai ales în cazul tumorile non-ulcerative, se observă ocazional o pseudocapsulă derivată
de la nivelul mucoasei faringiene sau nazale, cu un aspect atrofic. Stroma fibroasă densă poate prezenta arii hialine care alterează
aspectele mixoide focale. Celulele multinucleate stromale sunt prezente, cu citoplasmă intens eozinofilică, ganglion-like. Discuţii:Proporţia dintre vasele sanguine şi stroma tumorală variază în funcţie de dimensiunea tumorii. Studiile ultrastructurale au arătat că
celulele stromale sunt miofibroblaste, cu expresie variabilă a caracteristicilor fibroblastice şi miofibroblastice. Concluzii: Cu toate că
angiofibromul este o tumoră benignă din punct de vedere histologic, poate avea un comportament agresiv datorită recurenţelor şi
capacităţii de a distruge structurile osoase adiacente.
Cuvinte cheie: angiofibrom nasofaringian juvenil, benign, microscopic, macroscopic, histologic

34

Fiziologia - Physiology  2013.23.1 (77)NEW INSIGHT INTO THE PULMONARY VASCULITISMANOLE COJOCARU1, ELENA RUSU1, CAMELIA DOINA VRABIE2, INIMIOARA MIHAELACOJOCARU2
1”Titu Maiorescu” University, Faculty of Medicine, Bucharest
2“Carol Davila” University of Medicine and Pharmacy, Bucharest

ABSTRACT

Pulmonary vasculitides are non-infectious inflammatory disorders that mainly affect the blood vessels of the lung, from the main
pulmonary artery to alveolar capillaries and are usually associated with systemic vasculitis. The presence of pulmonary vasculitis
can be suggested by a clinical presentation that includes diffuse pulmonary hemorrhage, acute glomerulonephritis, chronic refractory
sinusitis or rhinorrhea. Pulmonary vasculitis can occur in isolation without the involvement of other organs. Serologic tests, including
the use of cytoplasmic antineutrophil antibody (ANCA) and perinuclear ANCA, are performed for the differential diagnosis.
Key words: pulmonary vasculitis, autoimmune diseases, pathophysiology, clinical and laboratory findings

INTRODUCTION
Pulmonary vasculitis refers to inflammation and damage toblood vessels in the lungs. Pulmonary vasculitis can be an acutereaction to a respiratory tract infection or a chronic condition
related to an autoimmune disorder (1,2).
Several clinical symptoms and signs are suggestive of
the presence of pulmonary vasculitis. Symptoms can vary, butmany patients exterience shortness of breath, fatigue, and chest
pains. Pulmonary vasculitis is associated with granulomatous,
eozinophilic, lymphoplasmocytic, or neutrophilic inflammatory
diseases and commonly is a manifestation of systemic illnesses
such as primary systemic vasculitides, collagen vasculardiseases, systemic diseases associated with the autoantibodies,and as a side effect of certain drugs. A positive cytoplasmic
ANCA test result is specific enough to make a diagnosis of
ANCA associated granulomatous vasculitis if the clinical features
are typical. Perinuclear ANCA positivity raises the possibility of
Churg-Strauss syndrome or microscopic polyangiitis. Treatment
is generally geared toward eliminating the underlying cause (3).
VASCULITIS ASSOCIATED WITH COLLAGEN VASCULARDISEASE
Pulmonary capillaritis can manifest as a complication of adisease course of various kinds of collagen vascular diseases
such as systemic lupus erythematosus (SLE), rheumatoid arthritis, polymyositis, Sjögren syndrome, scleroderma, and primary
antiphospholipid syndrome. The lungs and pleura are affected
more frequently in SLE (50%–70% of cases) than in other collagen vascular diseases, and lung involvement manifests withdiverse pathologic entities. SLE vasculitis is immune complex
mediated and involves the arterioles and capillaries; necrotizing
arteriolitis (much rarer than capillaritis) results in edema andfibrinoid necrosis in the arterioles within the secondary pulmonary lobule, and acute necrotizing capillaritis (more common
than arteriolitis) chiefly causes diffuse alveolar hemorrhage(DAH). When DAH does occur, it is often seen concomitantlywith other pulmonary manifestations of SLE such as acute lupus
pneumonitis, pulmonary edema, or pleural effusion. DAH occurs
in 4% of hospitalized patients with SLE, representing 22% of thepulmonary complications of SLE. Typically, patients with DAH
present with rapid-onset tachypnea, cough, fever, hypoxia, andhemoptysis while displaying symptoms of generalized SLE
vasculitis such as renal failure, arthritis, or rash. It is known thatthe presence of DAH should exclude scleroderma, rheumatoid
arthritis, and dermatomyositis or polymyositis. However, DAH
has been reported in a small number of patients with mixed
connective tissue disease, and it has occurred predominantly
in association with glomerulonephritis. Similarly, DAH has beenreported in a small number of cases of rheumatoid arthritis. In
SLE, pulmonary hypertension occurs in 0.5%–14% of patients.
The above-mentioned necrotizing vasculitis may be a factor inpulmonary hypertension in SLE patients. Recurrent thromboembolic disease, bland vasculopathy as in primary pulmonary
hypertension, and interstitial pulmonary fibrosis and hypoxia
may also contribute to pulmonary arterial hypertension (4,5).
ETIOLOGY AND PATHOGENESISThe exact mechanisms by which vasculitis occurs in thelungs is not always clear. Vasculitis can be pathologically definedby the presence of cellular inflammation, vessel destruction,and tissue necrosis. Although the inciting antigens or stimuli ofvasculitis are not well understood, an immunologic mechanism
is involved in the pathogenesis of antineutrophil cytoplasmic
antibody (ANCA)-associated vasculitis. In ANCA-associatedvasculitis, an intense infiltration of activated neutrophils results

Received February 2nd 2013. Accepted March 10th 2013. Address for correspondence: Dr. Manole Cojocaru, Titu Maiorescu” University, Faculty of Medicine, Str.
Thomas Masaryk No. 5 Sector 2 Postal Code 020983, Bucharest, Romania, e-mail: mancojocaru@yahoo.com

2013.23.1 (77)  Fiziologia - Physiology

35

in fibrinoid necrosis and dissolution of vessel walls, thus
compromising the vascular lumen. ANCA may be involved in
the pathogenesis not only by activation of neutrophils but also
by enhancement of the adhesion of the activated neutrophils tocytokine-primed endothelial cells. The damage to the integrity ofthe vessels results in the leakage of blood into the alveolar spacefrom the interstitial capillaries and causes stenoses, thrombotic
obstruction, and aneurysms of the corresponding vessels.
Cytoplasmic ANCA is mainly related to antibodies directedagainst proteinase 3 found in azurophilic granules of neutrophils
and monocytes, whereas perinuclear ANCA is associatedwith antibodies directed against a variety of intracellularantigens (most commonly with myeloperoxidase, also foundin azurophilic granules). The production of autoantibodies
such as antiglomerular basement membrane antibody (type2 hypersensitivity reaction), formation of immune complexes(type 3 hypersensitivity reaction), and T cell involvement (type4 hypersensitivity reaction) are likely to link to the pathogenesisof other forms of small-vessel pulmonary vasculitis. This process
may be collectively called immune complex–mediated vasculitis.
Vasculitis in systemic lupus erythematosus (SLE) is typical of thisimmune complex–mediated vascular disease (1-3).
CLINICAL FINDINGS
Most commonly, patients with pulmonary vasculitis present
with various multisystem manifestations. Although combined
manifestations are more common, each of the following symptoms and signs can be seen as an isolated symptom or a sign:diffuse pulmonary hemorrhage, glomerulonephritis, upper
respiratory tract lesions, imaging findings of nodules or cavitarylesions, mononeuritis multiplex, multisystem disease, and palpable purpura. In some instances, laboratory tests and physicalexams may reveal an underlying problem (3).
LABORATORY TESTSANCA tests play an important role in the diagnosis of somepulmonary vasculitides, including ANCA-associated granulomatous vasculitis, CSS, and microscopic polyangiitis, and for
idiopathic pauci-immune rapidly progressive glomerulonephritis.
The use of cytoplasmic ANCA is highly sensitive (90%–95%) for
the detection of active, systemic ANCA-associated granulomatous vasculitis and has a specificity of approximately 90%. In an
adequate clinical setting, a positive cytoplasmic ANCA finding
has sufficient positive predictive value (>90%) so that a biopsy
may be deferred or exempted. Conversely, a positive perinuclear ANCA finding provides no more than suggestive evidenceof CSS, microscopic polyangiitis, or idiopathic pauci-immune
rapidly progressive glomerulonephritis. Perinuclear ANCA canbe found in diseases such as rheumatoid arthritis, Goodpasture
syndrome, infection (hepatitis C, fungal, and mycobacterial),
and inflammatory bowel disease. When cytoplasmic or perinuclear ANCA finding is positive, the addition of proteinase 3 or
myeloperoxidase ANCA enzyme-linked immunosorbent assay
36

test can help increase the specificity in the diagnosis of ANCAassociated vasculitis. The importance of increasing ANCA titers
to predict relapse in patients with vasculitides has also been
evaluated. Current studies suggest that there is insufficientsensitivity and specificity of an isolated increase in the ANCA
titer to accurately predict relapse in ANCA-associated granulomatous vasculitis and other vasculitides. Culture of blood andother affected organs (when applicable) must be obtained to
exclude infection. Routine laboratories (complete blood countwith differential, chemistries, liver tests, blood urea nitrogen,and creatinine) should be obtained; however, the findings are
generally nonspecific. An elevated erythrocyte sedimentation
rate and C-reactive protein are expected in active vasculitis butlack specificity. Urinalysis should be obtained in all patients,
with the microscopic examination performed on a fresh sample(before red cell casts or other diagnostic features degenerate) by personnel skilled in its interpretation. Proteinuria andmicroscopic hematuria are common early findings in WG and
MPA. Anti–glomerular basement membrane antibodies should
be obtained in all patients with pulmonary hemorrhage or apulmonary-renal syndrome and when present are indicative ofGoodpasture’s syndrome. Antinuclear antibodies and rheumatoid factor may be positive in primary vasculitis, although hightiters and disease-specific antibodies, such as dsDNA, SS-A/
SS-B, anti-RNP, and anti-Jo-1, favor a connective tissue disease. IgE should be obtained when CSS is being considered.
Complement (C3 and C4) should be obtained whenever SLE isin the differential. Some fungal, bacterial, and parasitic agentsare capable of causing blood vessel damage in the respiratory
tract and lungs (6-10).
DIAGNOSTIC BIOPSY
Although a confident diagnosis may occasionally be madewithout tissue, diagnostic biopsy remains the bedrock of diagnosis. Diagnostic tissue may infrequently be obtained from easily
accessible sites, such as skin or upper airway lesions. Percutaneous renal biopsy is commonly performed during the evaluationof an acute glomerulonephritis. Although features specific for
vasculitis (e.g., granulomatous inflammation or necrotic vessels)
are rarely found, the finding of a focal, segmental necrotizing glomerulonephritis without immune deposits (pauci-immune) generally reflects a systemic vasculitis. It is important that in addition toconventional histopathology, immunofluorescence (and electron
microscopic when appropriate) studies be performed. The presence of characteristic immunofluorescence patterns, such as IgA
deposition in Henoch-Schönlein purpura, linear IgG depositionin Goodpasture’s syndrome, and irregular immunoglobulin andcomplement deposition in SLE, help provide a specific diagnosis.
When the lung is clinically involved, surgical lung biopsy almost
always provides definitive pathologic evidence. If obtained, theclinician, surgeon, and pathologist must coordinate the management of the sample and collect tissue in saline for culture, frozen
tissue for immunofluorescence, and formalin-fixed tissue forstandard hematoxylin and eosin staining (3, 6-9)
Fiziologia - Physiology  2013.23.1 (77)CONCLUSION
Despite the considerable advances made in the diagnosis and management of the small-vessel vasculitides, themorbidity and mortality of these disorders remains high. A
cytoplasmic ANCA study is both sensitive and specific for
ANCA-associated granulomatous vasculitis in the appropriate
clinical setting. A perinuclear ANCA study is neither sensitive
nor specific, but it is suggestive of the presence of CSS andmicroscopic polyangiitis.
REFERENCES
1. Brown KK. Pulmonary vasculitis. Proc Am Thorac Soc, 2006; 3(1):48-57.
2. Peachell MB, Muller NL. Pulmonary vasculitis. Semin Respir Crit
Care Med, 2004; 25(5): 483-89.
3. Frankel SK, Cosgrove GP, Fischer A, Meehan RT, Brown KK. Updatein the diagnosis and management of pulmonary vasculitis. Chest, 2006;

129(2): 452-465.
4. Kim JS, Lee KS, Koh EM, Kim SY, Chung MP, Han J. Thoracic
involvement of systemic lupus erythematosus: clinical, pathologic, andradiologic findings. J Comput Assist Tomogr, 2000; 24(1): 9-18.
5. Kamen DL, Strange C. Pulmonary manifestations of systemic lupus erythematosus. Clinics in Chest Medicine., 2010; 31(3): 479-88.
6. Newsome BR, Morales JE. Diffuse alveolar hemorrhage. Southern
Medical Journal. 2011;104(4): 269-274.
7. Virdi RPS, Bashir A, Shahzad G, Iqbal J, Mejia JO.Diffuse Alveolar
Hemorrhage: A Rare Life-Threatening Condition in Systemic Lupus
Erythematosus. Case Rep Pulmonol., 2012; doi: 10.1155/2012/836017
8. Schwarz MI, Brown KK. Small vessel vasculitis of the lung. Thorax,
2000; 55: 502-510.
9. Eschun GM, Mink SN, Sharma S. Pulmonary interstitial fibrosis asa presenting manifestation in perinuclear antineutrophilic cytoplasmic
antibody microscopic polyangiitis. Chest, 2003; 123(1): 297-301.
10. Jennette JC, Falk RJ. New insight into the pathogenesis of vasculitis
associated with antineutrophil cytoplasmic autoantibodies. Curr Opin
Rheumatol, 2008; 20(1): 55-60.

ACTUALITĂŢI ÎN VASCULITA PULMONARĂREZUMAT

Vasculitele pulmonare sunt afecţiuni inflamatorii neinfecţioase care afectează în special vasele de sânge ale plămânului, de la artera
pulmonară la capilarele alveolare şi sunt de obicei associate cu vasculită sistemică. Prezenţa vasculitei pulmonare poate să fiesugerată la examenul clinic de hemoragie pulmonară difuză, glomerulonefrită acută, sinuzită cronică recidivantă sau rinoree. Vasculita
pulmonară se poate întâlni izolată, fără implicarea altor organe. Testele serologice cuprind anticorpii anticitoplasma polimorfonuclearului
neutrofil pattern-ul citoplasmatic şi perinuclear, care se utilizează pentru diagnosticul diferenţial.
Cuvinte cheie: vasculita pulmonară, boli autoimune, fiziopatologie, date clinice şi de laborator

2013.23.1 (77)  Fiziologia - Physiology

37

AMIODARONE ASSOCIATED OPTIC NEUROPATHY,IS IT A REAL THING?
N. NICULESCU, M. MUNTEAN, I. ZOLOG
Ophtalmology Clinic Timisoara, “Victor Babes” University of Medicine and Pharmacy Timisoara
ABSTRACTThe purpose of our study was to assess the ophthalmologic impact of amiodarone treatment, especially amiodarone associated optic
neuropathy (AAON). We included in our study 256 clinical cases with ocular changes following amiodarone therapy. All patients
included in our study had a history of complete amiodarone loading dose of 11-12g, with a minimum of 4 months therapy. All patients were on chronic use of amiodarone medication (200-400 mg/daily). The Macaluso et al criteria were used to diagnose AAON.
Amiodarone optic neuropathy is characterized by insidious onset of visual loss, protracted disc edema (for months), and bilateral(usually simultaneous) involvement. We included in our study 256 patients (136 men, 120 women) treated with amiodarone. The
average age of the patients was 45±16 years. The duration of amiodarone medication varied from four months to six years. The ocularcomplication were represented by cornea verticillata, anterior subcapsular lenticular opacities, amiodarone induced neuropathy. Afterinterrupted or discontinued amiodarone therapy 75% of the cases of amiodarone keratopathy were reversible. The most frequentocular complication from amiodarone treatment is cornea verticillata. Despite the fact that amiodarone induced optic neuropathy israre, the screening for diagnosis is important because of clinical impact. We believe that opthalmological surveillance is mandatory
in all patients on long term amiodarone therapy, and those patients should undergo a complete ocular examination every 6 months.
If the diagnosis is suspected we should discontinue amiodarone. The decision regarding the safety of discontinuing the amiodarone
is best left to the cardiologist.
Key words: amiodarone treatment, optic retinopathy

INTRODUCTIONPURPOSE

Amiodarone is a class III antiarrhythmic drug used especially in the treatment of severe arrhythmias, refractory to othermedication, such as malignant ventricular tachycardia and atrial
fibrillation. The widespread use of this drug has increased after
approval, by the American Heart Association, for its use in advanced cardiac life support protocols (1).
Potential adverse effects include corneal microdeposits(70-100%) (2), optic neuropathy/neuritis (≤1%-2%), blue-grayskin discoloration (4%-9%), photosensitivity (25%-75%), hypothyroidism (6-10%), hyperthyroidism (0.9%-2%), pulmonary
toxicity (1%-17%), and hepatotoxicity (elevated enzyme levels,
15%-30%; hepatitis and cirrhosis, <3%). A range of neuropsychiatric adverse effects may also occur. The most common are
tremor and ataxia (3%-35%, depending on dose and duration oftherapy). Peripheral neuropathy is uncommon (0.3% annually)
but may be severe, requiring dose reduction or discontinuationof therapy. Insomnia, memory disturbances, and delirium havebeen reported as well (3-6).
Amiodarone has also been reported to cause optic neuropathy, which may result in permanent visual loss. The clinicalpresentation of amiodarone induced optic neuropathy may besimilar to that of non-arteritic anterior ischaemic optic neuropathy (NAION) (7-10). Casual relationship between amiodarone
and optic neuropathy is subject of intense controversy and thepathophysiology is unknown (11,12).

The purpose of our study was to assess the ophthalmologic impact of amiodarone treatment, especially amiodarone
associated optic neuropathy (AAON).
MATERIAL AND METHODS
We included in our study 256 clinical cases with ocularchanges following amiodarone therapy. All patients included inour study had a history of complete amiodarone loading dose of
11-12g, with a minimum of four months therapy. All patients wereon chronic use of amiodarone medication (200-400 mg/day).
Exclusion criteria included evidence of an alternative systemic
disorder that could explain optic neuropathy and insufficient
clinical information. We recorded for each patient age, sex, amiodarone dose, time of amiodarone therapy, the interval between
initiation of treatment and onset of visual symptoms. All patients
had a complete ocular examination with a particular attention
given to slit-lamp biomicroscopy and fluorescein angiography.
Our study is a retrospective one and we didn’t obtain informed
consent. We used the Macaluso et al (13) criteria to diagnose
AAON. Amiodarone optic neuropathy is characterized by insidious onset of visual loss, protracted disc edema (for months), andbilateral (usually simultaneous) involvement. In contrast, NAION
is characterized by acute unilateral visual loss with resolution ofdisc edema over several weeks.

Received February 22nd 2013. Accepted March 15th 2013.Address for correspondence: Niculescu N, Ophtalmology Clinic, “Victor Babes” University of Medicine andPharmacy Timisoara, 2nd E. Murgu Sq. 300041 Timisoara, RO, phone/fax: +40722620338, e-mail: nico28nov@yahoo.com

38

Fiziologia - Physiology  2013.23.1 (77)RESULTSDISCUSSION

We included in our study 256 patients (136 men, 120
women) treated with amiodarone. The average age of the patients was 45±16 years. The duration of amiodarone medication
varied from four months to six years. The ocular complications
were represented by cornea verticillita (amiodarone keratinopathy-243 patients, 95% - Figure 1), anterior subcapsular lenticularopacities (56 patients, 21.8% - Figure 2), amiodarone induced
neuropathy (4 patients, 1.56% - Figures 3 and 4). From patientswith amiodarone keratinopathy 47 (18.35%) was associatedwith anterior lenticular opacities. We found bilateral asymmetriclesion of cornea vericillita in 170 patients (66.4%). After interrupted or discontinued amiodarone therapy 75% of amiodarone
keratopathy were reversible.

Despite the fact that ultrastructural changes have been
observed in nearly all ocular tissues in patients taking amiodarone, most patients treated with the drug do not develop
visual symptoms. During therapy, intracytoplasmic lamellar
deposits occur in the cornea, lens, retina, and optic nerve.
Although nearly 100% of patients on amiodarone develop a
cornea verticillata (amiodarone keratopathy), this finding only
infrequently produces symptoms of glare, halos, or blurredvision (14,15). The keratopathy is probably dose and durationof treatment-dependant, with usual onset after one or moremonths, and slowly disappearing from the cornea if amiodarone
is discontinued. The consensus in the literature is that it does
not disturb vision. We found in our study that 95% patientsdeveloped cornea vericillata after at least four months oftreatment; in 75% of cases, a resolution after discontinuing
the drug was observed. Of great interest is that amiodarone
is involved in causing an optic neuropathy, findings that are
indistinguishable from those of non-arteritic anterior ischemic
optic neuropathy (NAION), which is frequent in normal people
older then 50 years. The incidence of amiodarone-related optic
neuropathy was estimated by Feiner et al (7) at 1.76% over thepreceding 10 years versus 0.3% in an age-matched population
not using the drug. We used the criteria develop by Macaluso
et al to distinguish between amiodarone induced neuropathy
and non-arteritic anterior ischaemic optic neuropathy. Using
these criteria we found amiodarone associated neuropathy in
4 patients (1.56%), similar to other reports. Johnson et al (16)recently reported a clinical spectrum of amiodarone-induced
optic neuropathy based on an observational case series.
They classified amiodarone-associated optic neuropathy infive categories: insidious onset (43%); acute-onset (28%);
retrobulbar (13%); increased intracranial pressure (8%) anddelayed-progressive onset (8%), although overlap in clinicalpresentation with NAION may occur. The fact that we use
Macaluso ctiteria of AAON can be a limitation of our study, but
it offers a more accurate diagnosis. Amiodarone has a long halflife (21-78 days) therefore the optic disc swelling may persist forweeks to months after the medication is stopped. The diagnosis
is certain only after discontinuing the drug, slow resolution of
optic disc swelling and improvement in visual function.

Fig.1. Cornea verticillata (amiodarone keratopathy); Fig.2. Anterior lenticularopacities

Fig.3. Amiodarone associated optic neuropathy-retinophotography

CONCLUSION

Fig.4. Amiodarone associated optic neuropathy-fluorescein angiography

2013.23.1 (77)  Fiziologia - Physiology

The most frequent ocular complication from amiodarone
treatment is cornea verticillata. Despite the fact that amiodarone
induced optic neuropathy is rare, the screening for diagnosis isimportant because of clinical impact. We believe that opthalmological surveillance is mandatory in all patients on long term
amiodarone therapy, and those patients should undergo acomplete ocular examination every 6 months. If the diagnosis
is suspected we should discontinue amiodarone. The decision
regarding the safety of discontinuing the amiodarone is best left
to the cardiologist.
39

REFERENCES
1. American Heart Association. Guidelines 2000 for cardiopulmonary resuscitation and emergency cardiovascular care. Circulation
2000;102:I86–265
2. Mantyjarvi M, Tuppurainen K, Ikaheimo K. Ocular side effects of
amiodarone. Surv Ophthalmol 1998;42:360–6
3. Zipes DP, Libby PBRO, Braunwald E. Braunwald’s Heart Disease:
A Textbook of Cardiovascular Medicine. 9th ed. Philadelphia, PA: WB
Saunders; 2012
4. Goldschlager N, Epstein AE, Naccarelli G, Olshansky B, Singh B.
Practice Guidelines Subcommittee, North American Society of Pacing
and Electrophysiology.   Practical guidelines for clinicians who treat
patients with amiodarone.  Arch Intern Med. 2000;160(12):1741-174
5. Vorperian VR, Havighurst TC, Miller S, January CT. Adverse
effects of low dose amiodarone: a meta-analysis.   J Am Coll Cardiol. 1997;30(3):791-798
6. Vassallo P, Trohman RG. Prescribing amiodarone: an evidence-based
review of clinical indications. JAMA. 2007 Sep 19;298(11):1312-22.
7. Feiner LA, Younge BR, Kazmier FJ, et al. Optic neuropathy andamiodarone therapy. Mayo Clin Proc 1987;62:702–17
8. Macaluso DC, Shults WT, Fraunfelder FT. Features of amiodarone-

induced optic neuropathy. Am J Ophthalmol 1999;127:610–2
9. Sreih AG, Schoenfeld MH, Marieb MA. Optic neuropathy
following amiodarone therapy. Pacing Clin Electrophysiol
1999;22:1108–10
10. Eryilmaz T, Atilla H, Batioglu F, et al. Amiodarone-related optic
neuropathy. Jpn J Ophthalmol 2000;44:565–8
11. Mantyjarvi M, Tuppurainen K, Ikaheimo K. Ocular side effects of
amiodarone. Surv Ophthalmol 1998;42:360-6.
12. Castello DD, Teitelbaum BA, Tresley DJ. Visual changes
secondary to initiation of amiodarone: A case report and review
involving ocular management in cardiac polypharmacy. Optometry
2002;73:113-21
13. Macaluso DCShults WTFraunfelder FT Features of amiodaroneinduced optic neuropathy. Am J Ophthalmol 1999;127610- 612
14. Pollack PT. Clinical organ toxicity of antiarrhythmic compounds:
Ocular and pulmonary manifestations. Am J Cardiol 1999;84:37R-45.
15. Mantyjarvi M, Tuppurainen K, Ikaheimo K. Ocular side effects of
amiodarone. Surv Ophthalmol 1998;42:360-6.
16. Johnson LN, Krohel GB, Thomas ER. The clinical spectrum of
amiodarone induced optic neuropathy. Invest Ophthalmol Vis Sci
2004;45:E-Abstract 1613

NEUROPATIA ASOCIATA TRATAMENTULUI CU AMIODARONA – ESTE O
REALITATE?
REZUMAT

Scopul studiului nostru a fost de a evalua impactul oftalmologic al tratamentului cu amiodaronă, în special neuropatia optică asociată
cu amiodarona (AAON). Am inclus în studiul nostru 256 de cazuri clinice cu modificări oculare în urma terapiei cu amiodaronă. Toţipacienţii incluşi în studiul nostru aveau un istoric de doză completă de amiodaronă de 11-12g, urmând terapia de cel puţin 4 luni.
Toţi pacienţii utilizau cronic amiodaronă (200-400 mg/zi). Pentru a diagnostica AAON s-au utilizat criteriile enunţate de Macaluso et
al. Neuropatia optică indusă de amiodaronă este caracterizată prin instalare insidioasă a pierderii vedererii, edem de disc prelungit(timp de luni de zile) şi implicare bilaterală (de obicei simultană). Am inclus în studiul nostru 256 de pacienţi (136 de bărbaţi, 120 defemei) trataţi cu amiodaronă. Vârsta medie a pacienţilor a fost de 45±16 ani. Durata administrării medicaţiei a fluctuat între patru
luni şi şase ani. Complicaţiile oftalmologice au fost reprezentate de cornea verticillata, opacităţi lenticulare subcapsulare anterioare,neuropatie indusă de amiodaronă. După întreruperea sau terminarea terapiei cu amiodaronă, 75% dintre cazurile de keratopatie au
fost reversibile. Cea mai frecventă complicaţie oftalmologică în urma tratamentului cu amiodaronă este cornea verticillata. În ciuda
faptului că neuropatia optică indusă de amiodaronă este rară, screeningul pentru diagnostic este important din cauza impactului
clinic. Considerăm că supravegherea oftalmologică este obligatorie la toţi pacienţii care urmează terapie cu amiodaronă pe termenlung; aceşti pacienţi ar trebui să fie supuşi unui examen oftalmologic complet o dată la 6 luni. Dacă se suspectează acest diagnostic,
tratamentul cu amiodaronă trebuie suprimat. Este bine ca decizia cu privire la siguranţa încetării tratamentului cu amiodaronă să
fie luată de cardiolog.
Key words: tratament cu amiodaronă, retinopatie optică

40

Fiziologia - Physiology  2013.23.1 (77)