FIZIOLOGIA (PHYSIOLOGY) 2007; vol.17, supplement
Abstracts of the conference: Endothelial Cell and Tumor Angiogenesis

1. ENDOTHELIAL CELL: FUNCTION AND DYSFUNCTION
Maya Simionescu
Institute of Cellular Biology and Pathology “Nicolae Simionescu”, Bucharest
ABSTRACTA cell with a simple structure and complex functions. A squamous type of epithelium considered
initially a gratuitous cellophane-like sheet, the endothelial cells (EC) have earned in time, the respectof biologists and pathologists. Biologists discovered that the EC are endowed with all common
cellular organelles, coated pits and vesicles, a high number of vesicles (caveolae), transendothelial
channels, specific Weibel-Palade bodies, and differentiated microdomains of the plasmalemma. They
are social cells establishing homotypic and heterotypic intercellular junctions. ECs monitor
transcytosis of plasma molecules, function in endocytosis, synthesis and secretion of matrix
components, guard the vascular tone, administer hemostasis, immunity, angiogenesis and have otherfunctions that insure the body homeostasis. Furthermore, the EC possess an innate heterogeneity,
expressed by differences in their structure and function according to the tissues in which they reside.
Aggressive factors induce EC dysfunction. Pathologists discovered that the heterogeneity of EC
concurs to a blood vessel-specific pathology: i.e. atherosclerotic plaques develop in arteries,
thrombosis in veins and vascular leakage occurs in venules. To insults EC respond gradually,
initially by modulation of constitutive functions, which is followed by EC dysfunction, and only
ultimately by injury and apoptosis. As example, hyperlipemia with or without hyperglycemia leads toenhanced transcytosis of plasma LDL, increases EC biosynthetic activity expressed by augmented
synthesis of matrix components, MCP-1 and new cell adhesion molecules which function in
recruitment and diapedesis into the intima of inflammatory circulating cells. Ultimately, lipid accrualleads to the formation of EC-derived foam cells and eventually to EC death.
Endothelial Cells: a therapeutic target and therapeutic tool. Modern approaches for the treatmentof cardiovascular disease (CVD) include specific targeting of EC altered mechanisms and molecules
such as MCP-1, chemokine receptor antagonists, drugs designed to act on specific enzymes involvedin the intracellular signaling cascade or inhibitors of vascular specific NAD(P)H oxidase. As tools,
endothelial progenitor cells derived from bone marrow or vessel wall stem cells are a novel option
for replacement of damaged EC (re-endothelialization) as well as for neovascularization of ischemic
tissues.
Instead of conclusion: the wealth of data on the vital role of EC in health and diseases generated acomplex discipline that theoretically includes: endotheliology, the investigation of normal EC
functions; endotheliopathy, the search for the altered mechanisms in vascular diseases andendotheliotherapy, the quest for novel drugs targeted specifically to dysfunctional EC, a promisingvenue for the reversal of CVD.
Acknowledgements. I am grateful to my mentors, Prof. N. Simionescu and Prof. G.E. Palade and to
all my collaborators who inspired or contributed to this work. I express my thanks to Institutions that
granted this work: NIH-USA, Romanian Academy, Ministry of Education and Research andEuropean Community.
2. THE ROLE OF MAST CELLS IN TUMOR ANGIOGENESIS
Domenico Ribatti
Department of Human Anatomy and Histology, University of Bari Medical School, Bari, Italy
ABSTRACTThe current wisdom is that tumors are endowed with an angiogenic capability and that their growth,invasion and metastasis are angiogenesis-dependent. It is now well documented that neoplastic cells
are influenced by the their microenvironment and vice versa. The specific organ microenvironment

determines the extent of cancer cell proliferation, angiogenesis, invasion and survival. Tumor cells
are surrounded by an infiltrate of inflammatory cells, namely lymphocytes, neutrophils,
macrophages and mast cells (MC), which communicate via a complex network of intercellularsignaling pathways, mediated by surface adhesion molecules, cytokines and their receptors.
In my presentation, I will summarize:i)the MC mediators involved in angiogenesis;ii)
the experimental evidence concerning the role played by MC in angiogenesis;iii)
the list of solid and haematological tumors in which a close relationship between
angiogenesis, tumor progression and MC has been demonstrated;the circumstances in which MC are a critical source of angiogenic factors in vivo, and in such cases,
the signals that regulate their production and secretion that need to be determined as a prelude to theelaboration of new therapeutic strategies associated with MC presence and activation.
3. CLINICAL SIGNIFICANCE OF VASCULAR ENDOTHELIAL GROWTH FACTOR(VEGF) IMMUNOHISTOCHEMICAL EXPRESSION IN SOLID TUMORS
Marius Raica, Anca Maria Cîmpean„Victor Babeş” University of Medicine and Pharmacy, Department of Histology, Timişoara,
Romania
ABSTRACT
Angiogenesis is the process of blood vessels formation from preexisting vessels and has been
demonstrated to play a crucial role in growth and differentiation. As Folkman pointed out three
decades ago, solid tumors without neovascularization do not grow over 2-3 mm in diameter. The
angiogenic phenotype of a malignant tumor is mainly but not exclusively, characterized by the abilityof tumor cells to secrete growth factors. From them, vascular endothelial growth factor (VEGF)
seems to be the most powerful in terms of proliferation and migration of endothelial cells.
The immunohistochemical demonstration of VEGF in normal tissues and malignant tumors became ausual investigation of the angiogenic switch and angiogenic phenotype. The final reaction product iscytoplasmic with granular pattern. VEGF is secreted by a large variety of normal cells, as others andwe demonstrated in the normal human kidney, stomach, thymus, epidermis, and urothelium. A strong
reaction is constantly noticed in the epithelial cells of the kidney tubules that probably represent thebest external control of the reaction.
Many solid human tumors express VEGF, but the incidence of positive cases depends on the site of
the primary. VEGF scoring includes the intensity of the final reaction product and number of positive
cells. In renal cell carcinoma VEGF expression was found in 75.5% of cases and results does notcorrelate with microvessel density, histopathologic form and Fuhrman’s nuclear grade. In thesecases, tumor cells also express VEGF-1 and –2. In gastric carcinoma a positive reaction was found in
70% of cases; a positive reaction was noticed in the associated intestinal metaplasia, and in mucussecreting glands of the normal gastric antrum. The colorectal carcinoma became a favorite target for
anti-VEGF humanized monoclonal antibody, based on the expression of VEGF by tumor cells inover 50% of cases. In thymoma, the VEGF expression correlates with the pathological form and has
the strongest intensity in thymoma B3. A correlation was found between VEGF expression,
microvessel density and the invasive character of thymoma. Of particular interest is the breast
cancer, which express VEGF in early stages, but just rarely in advanced stage; in this tumor, theinitiation of tumor angiogenesis is induced only by VEGF, and as the tumor progresses, otherangiogenic factors are secreted by both tumor cells and normal cells of the stroma. This could be an
explanation for the reduced number of cases with advanced-stage breast cancer that express VEGF.
Besides its prognostic value, as demonstrated by others for many solid tumors, VEGF expression(together with specific phosphorilated receptors) is a marker of the angiogenic phenotype and atarget for therapy.

4. HUMAN ENDOTHELIAL CELL CULTURE: A USEFUL TOOL FOR ENDOTHELIALRESEARCH
Daciana Carmen Nistor, Ioana-Raluca Siska, Erika Deak, Gabriela Tănasie, Carmen Tatu,
Carmen Bunu, Florina Mirea, V. Paunescu
Department of Physiology and Immunology, University of Medicine and Pharmacy "Victor Babeş"Timişoara
ABSTRACT
Blood vessels consist of endothelial cells (EC) that are in direct contact with the blood, and pericytes,
smooth muscle cells, fibroblasts, basement membrane and extracellular matrix, which are
subendotelially located. Depending on body region, the cellular and non-cellular constituents of thevasculature differ in composition, phenotype and function.
The endothelium is a confluent monolayer of thin, flattened, rhomboid-shaped cells lining the intimal
surface of all blood vessels, including veins. In the adult human, the endothelium is composed ofapproximately 1 to 6 x 1013 cells. The net mass of the endothelium is 1 kg and has a surface area ofapproximately 5,000 m2.
The endothelium plays a key role in cardiovascular homeostasis through its diverse influences onblood vessel structure and function. Regulation of vascular smooth muscle tone, hemostasis andfibrinolysis control, participation to immune surveillance, inflammation, and atheroma formation, as
well as metastasis development are the most important phenomena in which endothelial cell are
involved.
The following human endothelial cell cultures are available:− umbilical vein endothelial cells - represent the most used and studied human endothelial cells,
because they are easily available, are normal cells, and probably physiologically more relevant
than many established cell lines.
− arterial endothelial cells (aortic EC, pulmonary artery EC, coronary artery EC, iliac artery EC).
− venous endothelial cells (saphenous vein EC, iliac vein EC).
− microvascular endothelial cells; microvascular EC have been successfully isolated from many
human tissues (adipose, brain, dermal, endometrial, gastric, heart, intestinal, liver, lung, placenta,
renal, synovial, and tonsil tissue) using paramagnetic dynabeads coated with a specific ligand(such as CD31/PECAM).
This presentation reviews human endothelial cells types available for research, the protocol for
isolation and culture of human umbilical vein endothelial cells, as well as human endothelial cells
derived from hematopoietic stem cells. Finally, aspects concerning endothelial seeding of vasculargrafts, and circulating endothelial cells are detailed.
Key words: human endothelial cells, culture, stem cells, endotheliazation of vascular grafts
5. TUMOR ANGIOGENESIS: REGULATION BY HYPOXIA AND MACROPHAGES
Karl H. Plate
Neurologisches Institut (Edinger Institut), Frankfurt
ABSTRACT
Aims: We have investigated the molecular mechanisms of angiogenesis in stroke, breast cancer,
and two distinct types of brain tumors, namely glioblastoma and hemangioblastoma.
Methods: Northern blotting, Western blotting, Luciferase assays, in situ hybridization andimmunohistochemistry was performed. Transgenic and knock-out mice were analyzed in addition.
Results: In all pathologies examined, we observed upregulation of VEGF and of VEGF receptors –1 and –2 in a cell type scecific fashion. VEGF was predominantly expressed around necrotic areas,suggesting a hypoxia-dependent upregulation of the VEGF gene in vivo. Whereas Angiopoietin-1
and tie2 expression was not significantly altered, we observed a cell-type specific upregulation ofAngiopoietin-2 in vascular cells, suggesting a modulation of tie2 function mainly via Ang-2
expression levels. The expression of VEGF, VEGFR-2 and Ang-2 witin the same tumor
microenvironment (e.g. in perinecrotic regions) suggests a connection between VEGFR-2 and Tie-2

signaling pathways. The hypoxia-inducible transcription factors HIF-1a and HIF-2 a were also upregulated in stroke and tumor specimens, suggesting that hypoxic induction of several genes
underlies the observed vascular phenotypes.
Conclusions: Our results thus suggest a coordinate upregulation of genes (synexpression groups)
mediated by hypoxia or by hypoxic mimicry (e.g. von Hippel Lindau Tumor Suppressor gene lossof function) which mediate the vascular and metabolic responses to hypoxic/ischemic andneoplastic brain injury.

6. PRO-ANGIOGENIC MYELOID PROGENITORS CIRCULATE IN BLOOD AND
TARGET TUMORS
E. Deak1, S. Göttig1, B. Rüster1, E. Seifried1, J. Gille2, R. Henschler1
1Institut für Transfusionsmedizin und Immunhämatologie, Frankfurt ,D2
Zentrum der Dermatologie und Venerologie, Johann Wolfgang Goethe Universität Frankfurt, D
ABSTRACT
Objective: Angiogenesis has been shown to be a prerequisite for tumor growth and metastasis, and anumber of anti-angiogenic molecular therapies have been developed to suppress tumor growth. Proangiogenic bone marrow-derived progenitor cell populations (BM-PCs) which home to tumors havebeen shown to be suitable cellular vectors to efficiently eradicate tumors by suicide gene therapy (DePalma, Nat Med 14:1193, 2003; De Palma, Cancer Cell 8:211, 2005). We therefore asked which typeof HPCs may be mobilized into blood and whether they have the capacity to form pro-angiogenic
cells in solid tumors or to metastases. Methods: The Lewis Lung Carcinoma (LLC) and the highly
metastatic melanoma B16 (B16-F10) cell lines were established in C57/BL6 mice. Transplantation of
CD45.1 and CD45.2 mouse strains and of donor bone marrow cells from beta-actin/GFP C57/BL6
mice was used to follow bone marrow origin of the BM-PCs. Flow cytometric analysis and cellsorting were used to enumerate and to purify BM-PCs, and immunohistochemistry to follow the
intra-tissue localisation of bone marrow-derived cells with proangiogenic phenotypes. Results: We
found incorporation of bone marrow-derived cells into both LLC tumors and B16 metastases in
C57/BL6 mice within a period of ~ 9-20 days. Tissue localisation of migrated BM-PCs in the tumors
was confirmed using immunohistochemistry of CD45 (BM-PCs) and CD31 (endothelial cells)
antigens. Flow-cytometric analysis demonstrated the presence of the pro-angiogenic Tie 2-expressing
monocytes (TEMs, CD45+Tie2+) and endothelial progenitor cells (EPCs), which wereCD45+VEGFR2+. Both populations co-expressed CD11b/Mac1. In their blood, tumor-bearing mice
showed increased numbers of myelopoietic colony-forming cells (CFCs) and lineage marker (lin)Stem Cell Antigen (Sca 1) + progenitor cells.
Flow cytometric cell sorting identified a population of lineage marker (CD4, CD8, CD45R0,
TER119)-negative, CD11b+VEGFR2- cells from bone marrow as a precursor for EPCs inclonogenic assays, which during culture generate CD45+VEGFR2+CD11b+ EPCs. In contrast to thelin- CD11b+ VEGFR2- HPCs, both CD45+VEGFR2+CD11b+ EPCs and CD45+tie2+CD11b+
TEMs isolated from the tumors were found to be non-clonogenic in these progenitor assays.
Conclusion: Myeloid lin- progenitors circulate in the blood of tumor-bearing mice and are a potent
source for tumor-infiltrating pro-angiogenic cells. Tumor-bearing mice show enhanced mobilisationof these myeloid progenitors, resulting in accumulation of pro-angiogenic TEMs and EPCs intumors. BM-PCs generated from lin- progenitors could thus be an attractive cellular therapeutic totarget the tumor vasculature.
7. HETEROGENEITY OF TUMOR BLOOD VESSELS: CLINICAL AND THERAPEUTICASPECTS
Anca Maria Cimpean, Marius Raica”Victor Babes” University of Medicine and Pharmacy, Department of Histology Timisoara, Romania
ABSTRACTTumor vasculature is irregular, abnormal, and essential for tumor growth. Heterogeneity of bloodvessels distribution is well documented in surgical specimens. Each tissue has a specific vascular
network adapted to its function. The heterogeneity of tumor blood vessels is closely linked to thetumor microenvironment. By the time of diagnosis, malignant neoplasm is biologicallyheterogeneous and consists of cells with different properties, morphology, growth rate and ability to
induce expression of specific receptors. Some angiogenic factors are overexpressed at the beginningof the process and other in the late stage of tumor blood vessels development. Also, the distributionof angiogenic factors is not similar for various parts of a tumor. The density of tumor blood vessels is
not the same for the whole tumor; the tumor exhibits intralesional or zonal heterogeneity of MVD.
Tumor vasculature has a chaotic pattern of development. It is composed of vessels with different
size, irregular thickness of the wall that is tortuous and leaky. The wall of the tumor blood vessels
consists of various types of cells: endothelial cells, endothelial progenitors cells, tumor cells. Basal
membrane has different features compared with normal one. Pericyte coverage seems to be an
important step in the vascular stabilization but the origin and structure of perivascular cells is subjectof debate. Also the extracellular matrix shows structural and molecular differences between tumors
Genetic instability of activated endothelial cells could interfere with the efficiency of theantiangiogenic therapy. No model for study of angiogenesis is perfect. None of them characterizeproperly tumor angiogenesis. Linked with tumor angiogenesis heterogeneity the proper therapy
might cover all steps of it. Diversity of tumor vessels formation as multistep process makes us tothink that antivascular therapy could be better than antiangiogenic one.
8. EXPERIMENTAL MODEL FOR IN VITRO STUDY AND QUANTIFICATION OF
NEOANGIOGENESISOlga Soritau1, Ioana Brie1, V. Foris2, V. Lung-Illes2, V. Cernea1,2
1“I.K iricuta” Cancer Institute, Cluj-Napoca, Radiobiology Laboratory
2“Iuliu Hatieganu”University of Medicine and Pharmacy, Cluj-Napoca
ABSTRACTIntroduction: Knowledge of angiogenesis molecular mediators is important for understanding its
control mechanisms and for the therapeutic approach. Anti-angiogenic tumor therapies requirecomplementary reproductible tools and techniques to assess the individual pattern of tumor
neoangiogenesis in order to define the optimal therapeutic approach and to monitor its efficacy foreach patient.
Purpose: 1. In vitro quantification of neoangiogenesis 2. The study of endothelial cells response tovarious growth factors (VEGF, FGF-basic, IGF, BMP2, BMP4) and angiogenic inhibitors (TGFβ1,
heparin, chemotherapeutic drugs).
Material and method: HUVEC cells were seeded in serum-free medium supplemented withendothelial growth factors, on a fibrin gel, at a concentration of 5 x 104 cells/ml. After 24 h anotherfibrin gel was added on the cell monolayer surface. The probes were visualized with an inverted
microscope with X100 and X200 magnitude.
Results: Capillary tube formation was tracked at 12h intervals. An angiogenic response was alreadyobserved within the first 24h, consisting in the formation of tubular structures. In the next timeintervals, the elongation of the existing structures and appearance of new, vasculary-like, tubular
structures was noticed. A neoangiogenesis evaluation score was elaborated based on the following
criteria: cell migration and alignment, formation of capillary tubes and their ramification, formationof polygonal structures and capillary network.
Conclusions: Our method of obtaining the fibrin gel is adequate for the in vitro formation ofcapillary tubes from HUVEC cells and probably for the study of angiogenesis in tumoral fragments.
This experimental model will allow the evaluation of the angiogenic potential of individual tumors aswell as the answer to different antiangiogenic agents.
9. THE EFFECT OF PBLAST49-HVEGFCHORIOALLANTOIC MEMBRANEON

VASCULATURE

OF

CHICK

Anca Maria Cîmpean1, Marius Raica1, Andrei Anghel2, Edward Seclaman2, Florin Olimpiu2,
Pusa Gaje1, Ovidiu Ferician1, Diana Tătucu1
1
Department of Histology
2
Department of Biochemistry“Victor Babes” University of Medicine and Pharmacy Timisoara, Romania
ABSTRACT
Aim. There are few studies with pBLAST49-hVEGF gene in wound healing and treatment of
pulmonary hypertension on animal model. The aim of the present study was to describe the dinamicchanges of morphology and growth pattern of chick CAM blood vessels after applying several dosesof pBLAST49-hVEGF.
Material and methods. Fertilized chicken eggs were prepared following standard protocol. On day
three of incubation 2 ml of albumen were removed and we made an window in the shell egg. We
started to apply 40 μL diluted pBLAST49-hVEGF on day 7 after initial incubation for the next fivedays. We monitorized the macroscopic features of the blood vessels growth and also a microscopicchanges during the procedure. The fresh CAM was examined by microscopy. An alcian blue
saphranin stain was applied to detect mast cells.
Results. The growth of the vessels was rapidly increased after the second application of
pBLAST49-hVEGF. There were significantly differences in the number of vessels day by day. We
observed a dense network of perfused small vessels with narrow lumen, which had a different growth
pattern compared with control egg. The number of mast cells increased after the fourth day oftreatment. Ghost vessels phenomenon was observed in few cases. No bleeding, spot hemorrhages or
inflammatory events were observed.
Conclusions. The pBLAST49-hVEGF stimulates blood vessels formation in a dose dependent
manner and promotes the maturation of functional vascular network. This might be a therapeutic
alternative for patients with myocardial or lower limb ischemia.

10. ENDOTHELIAL CELL PROLIFERATION RATE DURING ANGIOGENESIS IN SOFT
TISSUE TUMOURS
1
Marius Raica, 1Aurora Alexa, 1Anca Maria Cîmpean, 2Virginia Baneth
1„Victor Babes” University of Medicine and Pharmacy Timisoara, Romania, Department ofHistology
2
Arad County Hospital, Pathology Laboratory
ABSTRACTIntroduction. The pathologist finds it difficult to diagnose soft tissue tumours (STT), as they havean unpredictable evolution, and the prognostic tissue factors are less known. The present data doesn’t
include STT on the list of potential targets for antiangiogenic therapy. The results on the angiogenic
phenotype of the STT are controversial, with regard not only to the microvascular density, but also to
the angiogenic factors expression. At present, as there are no references on the proliferative potentialof endothelial cells from the vessels of these tumors, we have set out to focus on that subject in thepresent paper.
Material and methods. 54 patients with soft tissue tumours, of which 43 malignant, and 11 benign,
were examined. The biopsy samples obtained surgically were processed using the traditional
techniques, the sections having been stained, using conventional methods of histopathologicaldiagnosis and grading. The immunohistochemical profile contributed to establishing the final
diagnosis of liposarcoma (n=15), malignant fibrous hystiocytoma (MFH) (n=14), leiomyosarcoma(n=9), rhabdomyosarcoma (n=3), fibrosarcoma (n=2) and benign tumors (n=11). The methods for
PCNA and Ki67 (clone MIB-1) were used for assessing the proliferation rate. The observations
focused on the positive reaction within the nuclei of endothelial and perivascular cells, asemiquantitative type of estimations.
Results. The tumoral blood vessels express Ki67 and PCNA, most endothelial cells being positive at
nuclear level, even though the vessel was very big. The reaction for the proliferation marker in theperivascular cells was more often than not negative, and very rarely positive. However, we noticed aconstant correlation between the positivation of tumoral cells and the positive reaction for the
proliferation marker at the level of endothelial cells. For most intratumoral blood vessels, thepositivation rate for MIB-1 and PCNA was between 48 and 62%. The highest values were obtained
for the vessels of the immature and intermediate type. In tumoral cells the index of postive nuclei
was significantly higher for PCNA then for MIB-1, with no significant differences for endothelial
cells. Perivascular cells are positive only when the endothelial ones are negative, marker of the bloodvessel maturation. The endothelial cells proliferation index is correlated with the differentiation
degree (p<0.0001). Very rarely did we notice a positive reaction in endothelial cells from benign
tumours, with a proliferation index below 2%.
Conclusions. The study of the proliferation rate of endothelial and perivascular cells of STT
highlights the steps of positivation of Ki67 and PCNA. The endothelial cells within tumour area havehigh proliferative rate and the reactions are negative in the vessels from the periphery of the tumour.
Our results suggest that the proliferation index of endothelial cells may represent useful criteria forthe characterisation of the angiogenic phenotype of sarcomas.

11. TUMOR LYMPHANGIOGENESIS. A NEW ROLE OF TUMOR ASSOCIATED
MACROPHAGES
Sebastian F. Schoppmann
Medical University of Vienna, Department of Surgery, Austria
ABSTRACTMetastasis of malignant tumors to regional lymph nodes is one of the early signs of cancer spread inpatients, and it occurs at least as frequently as haematogenous metastasis.[1] However, little is known
about the role of tumor lymphangiogenesis in metastasis and whether this process is important for
spread via the lymphatics. During the last few years significant insight has been provided into themolecular mechanisms underlying the development of lymphatic vessels and the role oflymphangiogenesis in health and disease due to the discovery of the key lymphatic growth factors
VEGF-C and –D and their corresponding receptor VEGFR-3, and, more recently, due to theidentification of several specific molecular markers allowing to distinguish blood from the lymphatic
endothelium.[2-4]Breast lymphangiogenesis, as measured by staining with the lymphatic specific marker Podoplanin,
is increased significantly during the development of cancer and is clearly associated with lymph nodemetastasis and worse clinical outcome. [5]Lymphatic vessels generally arise within the peritumoral stroma, although the lymphangiopoietic
vascular endothelial growth factors VEGF-C and –D are produced by tumor cells.[6] In squamous cell
carcinoma of uterine cervix, a tumor with an active peritumoral inflammatory reaction, we showed aclose association between the amount of peritumoral inflammatory reaction and the peritumoral
lymphangiogenesis and lymphovascular invasion, providing evidence for a close relationshipbetween tumor-associated inflammation and tumor-lymphangiogenesis. Further a subpopulation of
CD14+, VEGFR-3-expressing monocytes recruited to, and activated at the site of tumor plays animportant role in tumor-associated lymphangiogenesis producing large amounts of VEGF-C andinducing local sprouting of pre-existing lymphatic endothelial cells in breast cancer.[7],[8]Understanding this novel mechanism for lymphangiogenesis and this new role of specialized TAMs
may allow to open important ramifications for anti-lymphangiogenic therapies within the complexityof human cancer.
1.
2.

Alitalo, K., T. Tammela, and T.V. Petrova, Lymphangiogenesis in development and humandisease. Nature, 2005. 438(7070): p. 946-53.
Kriehuber, E., et al., Isolation and characterization of dermal lymphatic and bloodendothelial cells reveal stable and functionally specialized cell lineages. J Exp Med, 2001.
194(6): p. 797-808.

3.
4.
5.
6.
7.
8.

Schoppmann, S.F., et al., Telomerase-Immortalized Lymphatic and Blood Vessel Endothelial
Cells are Functionally Stable and Retain Their Lineage Specificity. Microcirculation, 2004.
11(3): p. 261-9.
Jussila, L. and K. Alitalo, Vascular growth factors and lymphangiogenesis. Physiol Rev,
2002. 82(3): p. 673-700.
Schoppmann, S.F., et al., Prognostic value of lymphangiogenesis and lymphovascularinvasion in invasive breast cancer. Ann Surg, 2004. 240(2): p. 306-12.
Jain, R.K. and B.T. Fenton, Intratumoral lymphatic vessels: a case of mistaken identity or
malfunction? J Natl Cancer Inst, 2002. 94(6): p. 417-21.
Schoppmann, S.F., et al., Tumor-associated macrophages express lymphatic endothelial
growth factors and are related to peritumoral lymphangiogenesis. Am J Pathol, 2002. 161(3):p. 947-56.
Schoppmann, S.F., et al., VEGF-C expressing tumor-associated macrophages in lymph node
positive breast cancer: impact on lymphangiogenesis and survival. Surgery, 2006. 139(6): p.
839-46.

12. MOLECULAR FEATURES OF THE LYMPHANGIOGENIC VEGFS
Michael Jeltsch
Molecular Cancer Biology Laboratory, University of Helsinki, Finland
ABSTRACTIn the last 10 years lymphangiogenesis has quickly evolved from an antiquated niche discipline into
a mainstream research topic. The relevance of the lymphatic system for medical intervention,
especially in the prevention of cancer metastasis, has led to the generation of a substantial amount ofscientific data. Nevertheless, many of the interesting questions in lymphatic research remainunanswered. Much effort in clinical research has been spent trying to correlate the newly discovered
lymphatic-specific markers with disease or disease outcome, while most of the experimental work
has been devoted to various mouse models. Recently lymphangigenesis models have been describedfor experimental animals whose lymphatic system had been poorly described before, but it is still
unknown which of the models is appropriate for which questions. At the molecular level, the multistage biosynthesis of the lymphangiogenic growth factors VEGF-C and VEGF-D has precluded aclear view on how the multiple forms of VEGF-C and VEGF-D differ from each other and which of
the forms act as the immediate effectors of lymphangiogenesis in vivo. Despite using VEGF-D inhuman gene therapy trials, its physiological role remains entirely enigmatic, as it seems dispensable,
at least for mice. In addition, fundamental questions of embryonic lymphatic development are still
debated, for example the relative contributions of lymphangiogenesis versus lymphvasculogenesis.
While more and more interactions between the molecular players in lymphangiogenesis are
uncovered, the role of the "classical" VEGF receptors for lymphangiogenesis remains incompletely
understood. Attempts to dissect the roles of the individual receptors for angiogenesis andlymphangiogenesis at a comprehensive level have so far not produced unequivocal results. It will beinteresting to see whether the clinical applications of lymphangiogenesis and antilymphangiogenesis will live up to expectations despite our incomplete understanding of the biology
underneath.
13. ENDOTHELIAL MOLECULAR MARKERS IN THE DIAGNOSISANGIOSARCOMA
CorneliaAmalinei1, Raluca Balan1, Irina-Draga Caruntu1, Maricica Pavaleanu1
1Histology Department, University of Medicine and Paharmacy „Gr. T. Popa” Iasi, Romania
2
IIIrd Clinic of Obstetrics and Gynecology Iasi, Romania

OF

ABSTRACT
Molecular markers, including vascular types, may be useful tools in the management ofmalignancies, like breast cancer, facilitating positive and differential diagnosis, orientatingpostoperative therapy and prognosis. Primary epithelial tumors are dominant among breast
malignant tumors, mesenchymal or mixed tumors presenting a relatively low incidence.
Differential immunohistochemistry, using a panel of endothelial-specific markers versus apanel of epithelial-specific markers, certifies the histopathological diagnosis. The paper
presents clinical, histopathological and immunohistochemical features of a rare breast tumor.
Tumor had gross findings characterized by large size, the largest dimension being about 15 cm,
imprecisely delimited margins, and spongy consistency. Cut surface showed extensive
hemorrhagic areas, alternating with necrotic zones. Routine microscopy showed numerous
interanastomosing vascular channels, of variable caliber, forming blood pools, micropapillae
and solid areas composed of atypical spindle-shaped and polyhedrical cells, exhibiting frequentmitoses. Tumor limits were inflitrative into the glandular and adipose tissue. Final diagnosis
required special stainings (van Gieson, Szekelly) and immunohistochemistry (using antibodies
against Factor VIII, CD34, vimentin, desmin, citokeratin, and protein S100). Corroborating theresults of the complex microscopical investigation, diagnosis of high grade breast
angiosarcoma was established. Differential diagnosis with a carcinoma, imposed by thepresence of solid epithelioid areas, was based on the negativity of epithelial-specific markers.
14.
IMMUNOHISTOCHEMICALEXPRESSIONOF
HIF-1Α
ENCHONDROMAS
R Hertzog1, M Marculescu2, C Damaschin2, B Patranichi1, Z Marculescu1
1
Army Center for Medical Research, Bucharest
2
Central Military Hospital, Bucharest

IN

HUMAN

ABSTRACT
Background: The aim of this study is to evaluate the expression of the transcription factor Hif-1α(hypoxia-inducible factor-1 alpha) in human enchondromas. Hif-1α plays a major role in theadaptative response of cells to hypoxia. In normoxia, Hif-1 is maintained at low and often
undetectable levels in cytoplasmic compartment by continuous proteasome-dependent degradation.
Under hypoxia, Hif-1 traslocates into the nucleus and activates transcription of several genes
including VEGF and EPO, which stimulate angiogenesis and erythropoiesis, respectively. Hypoxic
regions are a common feature of solid tumors and Hif-1 indirectly promotes neoangiogenesis,
essential for tumor growth and progression.
Material/Method: Surgical specimens of femoral enchondromas were obtained from 3 pacients.
Immunohistochemical staining was performed on 4 μm thick sections from formalin-fixed paraffinembedded tissue. After deparaffinization / dehydration, antigen retrival through pressure cooking,Hif-1α expression was detected using a standard streptavidin-biotin immunoperoxidase ABC
technique with DAB as chromogen, Methyl Green conterstaining and mounting. Anti-human Hif-1α
MAb (Chemicon) was used as primary antibody (dilution 1:500).
Results: We found that in all the tumors of patients, there are chondrocytes showing cytoplasmic
localization of Hif-1, whereas nuclear Hif-1 staining was detected in just a few cells.
Conclusions: The presence of some chondrocytes showing nuclear localization of Hif-1 could be
responsible via VEGF for enhancement of angiogenesis in chondromas and increasing the risk fortumor growth and malignancy. Accumulation of Hif-1 in the nuclei of chondrocytes could be used asa marker in pre- and post-operative surgical assessment of enchondromas.
15. SIMULTANOUS ASSESSMENT OF LYMPHATIC VESSEL DENSITY AND MASTCELL DENSITY BY COMBINED IMMUNOHISTOCHEMICAL AND HISTOCHEMICALMETHODS IN LIP TUMOURS
Dragoş Andrei Izvernariu, Anca Maria Cîmpean, Marius Raica
Histology Department, University of Medicine and Pharmacy Timişoara
ABSTRACTIntroduction. Mast cells are ubicuous in the connective tissues, particularly in the loose connective
tissue associated with mucosae and of the perineural sheaths. They were found to associate withlymph vessels in old studies, and they are accumulating peritumorally in several malignant tumours,
where they have been found to secrete growth factors and cytokines, including vascular endothelial
growth factor (VEGF – A). Several studies document the presence of VEGF – A and VEGF – C
positive cells with morphology of mast cells / macrophages around different malignant tumours. A
single study documents the role of mast cells in tumour lymphangiogenesis so far, referring topancreatic cancer.
Aim, materials and methods. To study the correlation between mast cell density and lymphatic
density in lip malignant tumours. Immunohistochemical identification of lymphatic vessels wasmade with podoplanin, whereas mast cells presence was assessed with alcian blue – safranin in 22
cases of lip malignant tumours. Hot spots of lymphatic vessels were identified first at 20x, then thelymphatic microvascular density was counted at 400x in the peritumoral area. Hot spots of mast cells
were identified at 400x, and the ones in close vicinity of lymphatic vessels hot – spots were counted.
Results. Lymphatic vessels were identified accurately by this staining method, although positive
postcapilary venules were identified also (they were excluded in counting by their erythrocytes
content and by the presence of pericytes). Podoplanin expression was also noticed in tumor cells,
Schwann cells, myoepithelial cells, ductal and mucus secretor cells of the minor labial salivary
glands. Mast cells were all alcianophyllic. Statistic data regarding the series of different lip tumors
are listed in the table below.
SeriesStatistic
variable
Squamous
cells Mean
carcinoma
95% CI
99% CI
Basal cells carcinoma Mean
95% CI
99% CI
Adenocarcinoma
Mean
95% CI
99% CI

Mast cells density

Lymphatic vessels density

4.98 ± 2.86
3.32011; 6.49589
2.70711; 7.10889
7.8325 ± 1.59965
5.28105; 10.38395
3.15351; 12.51149
6.66333 ± 3.17997- 1.24865; 14.57632- 11.56744; 18.86415

5.642 ± 2.03723
4.51118; 6.77292
4.07449; 7.20951
3.665 ± 1.98065
0.50587; 6.82413- 2.1284; 9.4584
6.44 ± 2.16709
1.04745; 11.83255- 5.98415; 18.86415

Correlation analysis by simple regression test showed that mast cells density correlates withlymphatic vessels density only in squamous cells carcinoma (p = 1,011), whereas in basal cells
carcinoma and adenocarcinoma there is no correlation between these two variables (p=0,849 and
0,271, respectively).
Conclusions:
1. The combined method shown above identifies accurately lymphatic vessels and mast cells.
2. Hot spots of lymphatic vessels and mast cells were often in the same microscopic field.
3. Podoplanin expression was noticed also in tumour cells, Schwann cells, myoepithelial cells
and both secretory and ductal cells of the minor labial salivary glands.
4. Lymphatic vessels density correlated significantly with mast cells density only in squamous
cells carcinoma, whereas in basal cells and adenocarcinoma no signifficant correlations may
be found.
16. INTRACORONARY INFUSION OF BONE MARROW-DERIVED PROGENITORCELLS IN ACUTE MYOCARDIAL INFARCTION: A RANDOMIZED, DOUBLE-BLIND,
PLACEBO-CONTROLLED MULTICENTER TRIAL (REPAIR - AMI)
Volker Schächinger, MD, Sandra Erbs, MD, Albrecht Elsässer, MD, Werner Haberbosch, MD,
Rainer Hambrecht, MD, Hans Hölschermann, MD, Jiangtao Yu, MD, Roberto Corti, MD, Detlef
Mathey, MD, Christian Hamm, MD, Tim Süselbeck, MD, Birgit Assmus, MD, Torsten Tonn, MD,
Stefanie Dimmeler, PhD, Andreas M. Zeiher, MD
Institut für Transfusionsmedizin und Immunhämatologie, Frankfurt ,DABSTRACT
Background: Experimental studies indicated that mononuclear progenitor cells derived from thebone marrow may contribute to the functional regeneration of freshly infarcted myocardium.
Meanwhile, a number of small clinical pilot trials using intracoronary infusion of bone marrowderived progenitor cells indeed suggested that such a strategy appears to be feasible and safe inpatients with acute myocardial infarction (AMI).
Methods: Therefore, we initiated the Reinfusion of Enriched Progenitor Cells And Infarct
Remodeling in Acute Myocardial Infarction (REPAIR - AMI) trial, which is the first double-blind,randomized, placebo controlled, clinical multicenter trial assessing the effects of intracoronary
progenitor cell therapy after AMI. In this investigators-initiated trial performed in 17 centers inGermany and Switzerland, a total of 204 patients with an AMI have been randomized to receive
intracoronary infusion into the infarct artery of either progenitor cells (BMC group), isolated from 50
ml of bone marrow aspirates by Ficoll density gradient centrifugation, or placebo medium 3-6 days
after AMI. Bone marrow aspirates were shipped via courier to a central cell processing lab andisolated cell suspension or placebo medium was sent back in a blinded fashion and infused into theinfarct related artery by stop-flow technique within 24 hours thereafter. The primary endpoint isabsolute improvement of cardiac pump function (global left ventricular ejection fraction, LVEF),
measured by quantitative left ventricular angiography after 4 months, as assessed by a core lab.
Results: At the time of study therapy, LVEF was similar in the Placebo and the BMC group(47±1.1% vs. 48±1.5% [mean ±SEM], p=0.3). At 4 months, LVEF had significantly increased inboth groups. However, the absolute increase in LVEF as the primary endpoint of the study wassignificantly (p = 0.014) greater in the BMC group (+ 5.5 ± 0.7%) compared to the Placebo group (+3.0 ± 0.7 %). At 4 months follow-up, LVEF was significantly (p=0.021) higher in the BMC group(54 ±1.1%) compared to the Placebo group (50 ±1.5%). Thus, the results of the trial confirm the
hypothesis, that intracoronary infusion of BMC provides a significant benefit on recovery of left
ventricular contractile function in patients with successfully revascularized AMI.
Subgroup analysis revealed that the benefit of intracoronary progenitor cell therapy is mostpronounced in patients with large myocardial infarctions. Moreover, intracoronary infusion of BMC
prevented left ventricular endsystolic volume expansion indicating a beneficial effect on postinfarction remodeling processes.
Finally, BMC administration significantly (p=0.002 versus Placebo) improved blood flow reserve in
the infarct artery documenting profoundly enhanced neovascularization within the infarct region. The
beneficial effects on cardiac remodeling processes were paralleled by a reduction in the combinedclinical endpoint death, myocardial infarction and rehospitalization due to heart failure.
Conclusions: Intracoronary infusion of autologous bone marrow-derived progenitor cells in patientswith reperfused acute myocardial infarction is associated with improved global cardiac pumpfunction, prevents left ventricular endsystolic volume expansion, and profoundly augments bloodflow in the infarct artery within 4 months. Large-scale clinical endpoint trials are warranted to assess
the potential of bone marrow-derived progenitor cell therapy to limit the development of
postinfarction heart failure.
17. ANGIOGENIC GENE THERAPY IN PATIENTS WITH SEVERE CHRONIC LOWER
LIMB ISCHAEMIA
Andrei Anghel1, Bogdan Mut-Vitcu2, Catalin Marian1, Edward Seclaman1, Raluca Iman1,
Adriana-Maria Neghina1, Stefan I. Dragulescu2
1
Department of Biochemistry “Victor Babes” University of Medicine and Pharmacy Timisoara
2
Timisoara Institute of Cardiovascular Medicine, Romania
ABSTRACT
Aim: The present study focuses on the application of a therapeutic strategy in patients with chronic
severe lower limb ischaemia using a plasmid vector encoding the vascular endothelial growth factor(VEGF 165). It has been shown that VEGF promotes neovascularization and blood vessel network
formation and thus might have the ability to improve blood-flow at the level of the affected limbs.

However, little information is available regarding the necessary level of expression of VEGF and its
possible related adverse effects.
Material and methods: We have sub-cloned VEGF 165 isophorm into pCMV-Scriopt expression
vector (Stratagene) under the control of CMV promoter. Three patients with chronic ischaemia of thelower limb, considered as not suitable for surgical revascularization received intramuscular injection
with 0.5 ml saline solution containing 10" copies of VEGF165 plasmid
Results: The clinical evolution has been monitored by angiography and estimated by walking timeon the rolling carpet (Gardner protocol). Two month after therapy all three patients showed complete
relief of rest pain, improvement of ischaemic ulcer lesions and increased walking distance on therolling carpet most probably due to appearance of newly formed collateral vessels.
Conclusions: Given the failure of previous conventional therapy we are tempted to state that overexpression of VEGF at the level of the thight and calf muscles prevented the amputation which
would have been the only alternative left for the three patients
18. CHARACTERISTICS AND POTENTIAL OF MSC FROM CHILDREN FOR SAFE
CLINICAL APPLICATION
H. Dimitriou PhD
Department of Pediatric Hematology-Oncology, University Hospital Of Heraklion, University ofCrete Medical School, Heraklion, Greece
ABSTRACT
Mesenchymal Stromal Cells (MSC) became the focus of cellular therapeutics but little is known
regarding bone marrow (BM) MSC derived from children. Since MSC are constituents of the bonemarrow microenvironment which participates in the regulation of hemopoietic stem/ progenitor cellproliferation and differentiation we examined properties and potential, and if any, differences in BM
MSC coming from various hematological diseases.
BM Mononuclear cells (MNC) from children with idiopathic thrombocytopenic purpura,autoimmune neutropenia of childhood and B cell acute lymphoblastic leukemia (at diagnosis andafter the end of treatment) were isolated and expanded. MSCs were immunophenotypicallycharacterized and their functional characteristics were assessed by CFU-F assay and cell doublingtime calculation. Their ability for tri- lineage differentiation was verified by molecular andhistochemical methods. Apoptosis was evaluated and clonal analysis was performed. MSCs wereisolated from BM of all groups. They acquired the mesenchymal-related markers from the firstpassage with simultaneous decrease of haematopoietic markers. A very low percentage of apoptotic
cells were detected in all passages. The proliferative and clonogenic capacity did not differ amonggroups with the exception of ALL at diagnosis in which they were defective. Histochemical andmolecular analysis of differentiated MSCs yielded characteristics for adipocytes, osteoblasts andchondrocytes. Clonal analysis in a number of BM samples revealed a highly heterogeneous
population of cells within each clone.
For the study of spontaneous MSC apoptosis throughout sequential passaging, BM MNC culturesfrom children with benign hematological disorders and solid tumours without BM involvement were
initiated and grew for 10 passages. The expression of CD105, CD146 and CD95 as well as apoptosis
by 7AAD staining, were evaluated by flow cytometry. In every passage CFU-F assay was performed
and the cell doubling time was calculated. Cell cycle characteristics were assessed by propidium
iodide staining at P2 and P6.
In order to test if CD95 which is highly expressed on MSCs as previously described is functional,
induction of apoptosis was triggered by the addition of an agonistic antibody (anti-Fas CH-11) understandard culture conditions. No apoptosis was induced suggesting no leading role of this molecule in
the apoptotic event of MSC. Additionally, serum deprivation of MSCs for up to 72 hours revealed no
substantial apoptotic effect although the number of cells after trypsinization was slightly decreased inserum deprivation conditions. Furthermore, MSC deprived of serum for 72 hours were sequentially
set in culture under standard culture conditions for 2 additional passages and tested as to theirfunctional and cell cycle characteristics and spontaneous apoptotic events in two experiments. The

number of CFU-F and doubling time was not affected and no apoptosis was observed while cell
cycle pattern was maintained irrespectively of the serum free or serum complemented condition
initially used. This finding could be very useful in the setting of transplantation as long as serum
deprivation for up to 72 hours does not seem to affect in vitro expanded MSC while it favors theiruse in the clinical setting avoiding the possible risk of animal contaminants and immune responses
attributable to serum contamination.
According to the cell cycle analysis, cells at P2 are mostly at GoG1 phase while at P6 there is a slightincrease in the cells that have entered cell cycle.
We also studied the expression profile of oncogenes and tumour suppressor genes (TSG), known toplay an important role in the malignant transformation of cells since recent studies have shown that
MSCs can become targets of neoplastic transformation leading to cells with malignant potential. We
assessed the expression levels of p53, p16, Rb and H-Ras genes in BM-MNCs and compared them tothe respective ones in MSCs as well as their levels in different MSC passages by RT-PCR.
MNCs and MSCs were isolated from BM of children diagnosed with idiopathic thrombocytopenic
purpura autoimmune neutropenia, solid tumours without BM involvement and B cell acutelymphoblastic leukaemia (ALL) at diagnosis. MSCs were cultured for 6 passages. RNA was isolated
from MNCs as well as from MSCs at P2 and P6. GAPDH housekeeping gene was used as internal
control and HUVEC cells as normal control for adherent cells.
In all groups studied, MSCs at P2 expressed higher levels of p53, Rb and H-Ras and lower levels of
p16 in comparison to MNCs from the same patients. However, there were no differences in
expression levels between passages P2 and P6 and between P2 and HUVEC. In ALL samples
though, expression levels were similar between MNCs and MSCs at P2.
These results indicate that despite the differences in oncogene and TSG expression levels between
MNCs and MSCs, the profile of the same genes does not change during cultivation of MSCs up topassage 6. Additional experiments of MSC growth on soft agar showed no colony formation,
confirming the anchorage dependence of cells.
As cord blood (CB) constitutes an easily accessible source of hematopoietic progenitors suitable fortransplantation and recently its feasibility as a source of mesenchymal stromal cells (MSC) isassessed we tried to establish the optimal culture conditions for the expansion of CB MSC, to assess
their functional and immunophenotypic characteristics and to compare them with bone marrow (BM)MSC. Culture conditions were initially based on the technique used for the isolation of BM MSC andsequentially different culture conditions were tried in order to optimize MSC expansion. They
included different cell concentrations, enrichment of the culture medium with FGF-2, different
concentrations of the lot selected FCS, different culture surface area and pretreatment of the culture
plate with FCS.
Isolation of CB MSC was achieved in 25% of the samples cultured under optimal conditions whichwere found to be the higher initial concentration of cells (3x106 cells/cm2), the enrichment of theculture medium with FCS (20% compared to 10% of the BM protocol), the addition of 5ng/ml FGF2 (1ng/ml in the BM protocol), the pretreatment of the culture surface with FCS and the initial
volume of the sample (> 40 ml). MSC were morphologically similar to the ones derived from BM,
but appeared late in culture requiring 34 days to reach confluency and 55 days from P1 to P2.
Immunophenotypic analysis of P1 cells showed no expression of CD34 while CD45 ranged from 017.83% and CD105 ranged from 49-83%. CFU-F colonies were developed in one case.
Conclusion
These findings suggest that CB cannot be regarded so far as a sufficient source of MSC for clinicaluse. MSC from bone marrow of children with haematological disorders -with the exception of ALL
at diagnosis –though, can be isolated in sufficient number and quality and moreover retain theirfunctional characteristics throughout serial passages and are very stable under conditions that usuallycause apoptosis although progression in passages results in a lower number of cells maintaining
quiescence. The constant oncogene and TSG expression levels for a number of serial passages
combined with the similar expression levels between MSCs and HUVEC cells, imply that MSCs
may not be transformed during long term culture and thus they can serve as a potential source forsafe clinical applications.

19. ISOLATION OF BONE MARROW PROGENITOR CELLS ON PERCOLL GRADIENT
Ana-Maria Rosca, Alexandrina Burlacu, Irina Titorencu, Emanuel Dragan, Maya Simionescu
Institute of Cellular Biology and Pathology “N. Simionescu”, Bucharest
ABSTRACT
Purpose: Bone marrow (BM) represents the main source of progenitor cells for the entire lifespan of
adult individuals. Their plasticity is being highly investigated in tissue engineering for restoring thefunction of damaged organs. We checked whether the Percoll gradient separation of adult BM cells
generates subpopulations enriched in c-kit+/ Sca-1+ progenitor cells.
Matherial and methods: BM aspirate isolated from adult mouse tibia and femurs, was layered on a
discontinuous Percoll gradient (1,046 - 1,052 - 1,060 - 1,064 - 1,070 - 1,075 g/ml) and 6 cell subpopulations (I-VI) were separated by centrifugation (1500g, 20 min, 4oC). The progenitor cellmarkers and the potential to generate differentiated cells were checked in each subpopulation throughfluorescence microscopy and flow citometry assays before and after cell cultivation for 2 weeks.
Results: Progenitor cells (c-kit+ and Sca-1+) were identified mainly in fractions I-IV. When cultured
for 2 weeks, the adherent cells within these subpopulations (mesenchymal progenitor cells) showed agreat capacity of proliferation and generated various cell types (fibroblast-like, neuronal-like andendothelial-like cells). Fractions III and IV proved to be enriched in CD 45+ and CD 68+ cells,
demonstrating the presence of white lineage cells in these subpopulations. By culturing, some ofthese cells adhered and accumulated lipids, becoming foam cells, as demonstrated by Oil Red
staining. Fractions V and VI contained mesenchymal enlarged cells which stained positive forsmooth muscle actin.
Conclusion: centrifugation of BM aspirate on Percoll gradient is a simple and inexpensive
technique for isolation of a progenitor cells enriched subpopulation. These cells can be further
used in FACS analysis to obtain the pure population of progenitor cells subpopulations neededin cellular therapy. By culturing, some of these cells adhered and accumulated lipids, becoming
foam cells, as demonstrated by Oil Red staining. Fractions V and VI contained mesenchymal
enlarged cells which stained positive for smooth muscle actin.
In conclusion, centrifugation of BM aspirate on Percoll gradient is a simple and inexpensive
technique for isolation of a subpopulation enriched in progenitor cells. These cells can befurther used in FACS analysis to obtain the pure population of progenitor cells needed incellular therapy
20. 5-AZACYTIDINE SUPPORTS THE MYOGENIC DIFFERENTIATION OF BONE
MARROW STROMAL CELLS
Alexandrina
Burlacu,
Ana-Maria
Rosca,
Horia
Maniu,
Irina
Fleseriu,
Emanuel Dragan, Victor Jinga, Maya Simionescu
Institute of Cellular Biology and Pathology “Nicolae Simionescu” Bucharest, Romania
ABSTRACT
Background: Bone marrow stromal cells (BMSC) are multipotent cells able to give rise to various
cell types, among which the myocytes, which may be highly valuable in cellular therapy of
myocardial infarction.
Purpose: In an attempt to increase the myogenic commitment, we investigated whether the exposureof cultured BMSC to the demethylation agent, 5-azacytidine, could improve or accelerate theirdifferentiation, prior to be utilized in cell-mediated therapy.
Materials and methods: BMSC isolated from the adult rat tibia were exposed in culture to 5
microM 5-azacytidine for 24 hours, one day after seeding; the treatment was repeated at weeklyintervals for four passages and the expression of muscle specific morphology, proteins and genes wasassessed by Western blot, immunocytochemistry and RT-PCR.
Results: Microscopic examination, as well as immunocytochemistry, Western blot, and RT-PCR
experiments revealed that cultured cells lost the native expression of Osteocalcin and Alkaline
phosphatase as a function of time and began to express Connexin 43. As compared to control

(untreated cells), the exposure to 5-azacytidine of BMSC induced a myocyte-resembling phenotype
except the sarcomeric organization, and the expression of muscle specific proteins (Troponin T,
alpha-sarcomeric actin, Desmin and GATA-4) and genes (GATA-4, myoD and desmin).
Conclusions: Taken together, the results show that in vitro treatment with 5-azacytidine promotes thecommitment of BMSC towards cells that express several features of cardiomyocytes; one can safely
assume that his agent could serve to prime bone marrow cells prior to their transplantation to
improve myocardial remodeling after acute injury.
21. PRACTICAL APPLICATION OF STEM CELL TRANSPLANT IN THERAPY OF
MYOCARDIAL INFARCTION
Carmen Bunu1, S Drăgulescu2, B Mut-Vitcu2, Carmen Tatu1, Gabriela Tănasie1, Florina Mirea1
and V Păunescu1
1
Department if Physiology and Immunology, University of Medicine and Pharmacy “Victor Babes”
Timisoara
2
Cardiology Clinic, Institute of Cardiovascular Medicine Timisoara
ABSTRACT
Objectives: The present study tested the safety and feasibility of bone marrow derived
autologous cells transplant, using intra-coronary infusion at the level of infarcted artery, inpatients suffering from recent and acute myocardial infarction. Different studies suggested thatstem cells transplant can have a positive contribution both in regeneration of damaged
myocardium, as well as in neovascularization of ischemic area.
Methods: Our study comprised 11 patients suffering from extended acute or recent myocardial
infarction (1-28 days), which were already committed to successful reperfusion (PCI and stentimplant at the level of the damaged coronary artery) and had a 40% ejection fraction of the left
ventricle. The bone marrow derived AC133+ cells were harvested from the posterior iliac crest,
isolated, washed and resuspended in order to be injected through a catheter. Cells were
gradually administrated by intra-coronary injection at the level of infarcted coronary artery.
After transplant and 6 months evaluation comprised clinical and laboratory examinations,
ECG, 48 hours Holter monitoring, trans-thoracic echocardiography, stress echocardiography,
SPECT – using Technetium 99 sestamibi, left ventricle angiography.
Results: There were no complications occurring during the procedures of intra-coronary
infusion of bone marrow derived cells, and no malign arrhythmias were recorded during 48
hours Holter monitoring. Angina pectoris was not present in any patient and there were nosigns of clinical worsening. Side effects did not appear at 6 months evaluation and neither did
malign ventricular arrhythmias when the 48 hours Holter monitoring was used.
Conclusions: Bone marrow derived autologous cells transplant proved to be a secure andfeasible method. Long term clinical reports will confirm the benefits of this new therapeutic
approach.
22. ANTIANGIOGENIC THERAPY BY M TOR BLOCKADE AGAINST SOLID TUMORGROWTHS AND METASTASIS
C. J. Bruns
Department of Surgery, University of Munich-Großhadern, Germany
ABSTRACT
Angiogenesis, the formation of new blood vessels from the endothelium of the existing vasculature,
is fundamental in tumor growth, progression and metastasis. There is preclinical as well as clinicalevidence that inhibiting angiogenesis will suppress tumor growth and tumor metastasis.
Antiangiogenic therapy in general is a concept that targets the vasculature of tumors rather thantumor cells themselves, which has traditionally been the case by chemo- or radiotherapy. Endothelial
derived growth factor (VEGF) and its receptors have been the focus of antiangiogenic research.

Other molecular targets are matrix metalloproteinases (MMPs), Cyclooxygenase 2 (COX-2) and thetubulin cytoskeleton.
Over the last 30 years numerous pro- and antiangiogenic molecules, their ligands and intracellularsignalling pathways have been identified. Enormous efforts have been undertaken in order to develop
antiangiogenic strategies for clinical cancer treatment. Despite numerous promising results in
preclinical models, several initial clinical trials gave no convincing evidence for efficient antitumoral
therapy by classical antiangiogenic agents as monotherapy. This has led to the development of newantiangiogenic compounds and successful combination of angiogenesis inhibitors with classicalcytotoxic chemotherapy and radiotherapy. Combined with chemotherapy, anti-angiogenesis has
proven its clinical efficiency in patients suffering from advanced colorectal cancer leading to an
improved patient survival time. In 2004 the first antiangiogenic compound Bevacizumab (Avastin)
was therefore approved by the FDA as first line therapy in combination with standard 5-Fluorouracilbased chemotherapy in patients with advanced colorectal cancer.
Conventional immunosuppressive drugs have been used effectively to prevent immunologic rejectionin organ transplantation. However, cancer development and recurrence are ominous risk factors forthese immunocompromised patients. Recently, we showed that the new immunosuppressive drug
rapamycin may have a unique ability to reduce the risk of cancer development, while simultaneously
providing effective immunosuppression. Experimentally, rapamycin inhibited metastatic tumor
growth and angiogenesis in in vivo mouse models. Normal immunosuppressive doses of rapamycin
effectively controlled the growth of established tumors. From a mechanistic perspective, rapamycin
demonstrated antiangiogenic activities linked to a decrease in vascular endothelial growth factor
production and to a markedly inhibited response of vascular endothelial cells to vascular endothelial
growth factor stimulation. In pancreatic cancer, rapamycin therapy alone inhibited tumor growth andmetastasis more than gemcitabine, with remarkable long-term tumor control when the drugs werecombined. Mechanistically, H&E analysis revealed tumor vessel endothelium damage andthrombosis with rapamycin treatment. Furthermore, terminal deoxynucleotidyl transferase-mediatednick end labeling/CD31 double staining of orthotopic tumors demonstrated apoptotic endothelial
cells with rapamycin treatment, which also occurred with human umbilical vein endothelial cells in
vitro. Indeed, it has been shown that the mammalian target of rapamycin (mTOR) inhibitor
rapamycin, when administered to tumor-bearing mice, selectively induced extensive local
microthrombosis of the tumor microvasculature. Importantly, rapamycin administration had nodetectable effect on the peritumoral or normal tissue. Intravital microscopy analysis of tumors
implanted into skinfold chambers revealed that rapamycin led to a specific shutdown of initiallypatent tumor vessels. In human umbilical vein endothelial cells vascular endothelial growth factor(VEGF)–induced tissue
factor expression was strongly enhanced by rapamycin. We further showed by Western blot analysis
that rapamycin interferes with a negative feedback mechanism controlling this pathologic VEGFmediated tissue factor expression. This thrombogenic alteration of the endothelial cells was
confirmed in a one-step coagulation assay. The circumstance that VEGF is up-regulated in mosttumors may explain the remarkable selectivity of tumor vessel thrombosis under rapamycin therapy.
Taken together, the data suggest that rapamycin, besides its known antiangiogenic properties, has astrong tumor-specific, antivascular effect in tumors.
23. IMMUNOHISTOCHEMICAL ASPECTS OF SYNCHRONOUS COLORECTALCARCINOMAS
Simona Gurzu, I. Jung, T. Mezei
University of Medicine and Pharmacy of Tg. Mures, Morphopathology Department
Introduction: The synchronous tumours are neoplasias that appear in the same or different organs ina less than 6 months interval. In the literature they are reported as a rare phenomenon and for theiridentification require detailed investigations.
Material and method: We studied 791 surgical specimens with colorectal carcinomas (2003-2006).
We followed the classic clinico-pathological parameters of single colorectal carcinomas and wecompared them with those of the synchronous tumours of this organ. In the synchronous tumours wefollowed the following immunohistochemical markers: p53 protein, c-erbB2 and bcl-2 oncoproteins,chromogranin A and the markers of angiogenesis (CD31, CD105, VEGF).
Results: We identified 15 cases of synchronous colorectal carcinomas (1.9% of the total number ofcases). The peak incidence was in the 71-80 age group. 66% of cases had been associated with
polyps. 12 were adenocarcinomas with different grades of differentiation, and 3 were mucinous
carcinomas. One of the cases presented an association between a squamous carcinoma arising in theanal mucosa and an adenocarcinoma of the rectum. In 6 cases, one component of the synchronous
tumours presented neuroendocrine differentiation. Expression of the p53 protein was notably higher
in the tumours of the left colon and in 9 cases the expression of this protein was the same between
the tumours. Expression of the c-erbB2 and bcl-2 proteins was demonstrated in 11 and 4 cases,
respectively (in 4, respectively 2 cases both tumours were positive for this marker). In 10 cases
microvascular density was the same in the tumours, although CD31 expression did not correlate withthe expression of CD105, nor VEGF.
24. CORRELATION BETWEEN MICROVASCULAR DENSITY AND DIFFERENTIATIONGRADE IN SOFT TISSUE TUMOURS
Mederle Ovidiu, Izvernariu Dragos Andrei, Raica Marius
Histology Department, University of Medicine and Pharmacy, Timisoara
ABSTRACTIntroduction: Although there are thousands of publications on the subject of tumour angiogenesis,
their great majority refer to carcinomas, and the publications on the subject in soft tissue tumours arevery rare and, until present, with discordant results. According to this perspective, we have made theassessment of microvascular density in soft tissue tumours.
Materials and methods: 54 cases of soft tissue tumours (43 malignant and 11 benign tumours) werestudied according to the following protocol: surgical samples were fixed in buffered formalin,embedded in paraffin, sectioned at 5 μ, then stained for morphological diagnosis (H&E), and for
immunohistochemical detection of microvessels (smooth muscle actin – SMA, CD31 and CD34).
Weidnet method of microvessel counting was used.
Results: All the tumours included in the study have presented rich blood vessel network, themajority (>80%) of the blood vessels from within the tumour area being of immature andintermediate type (positive for the endothelial markers and negative for SMA). This aspect was more
obvious in the case of liposarcoma and malignant fibrous histiocytoma, but with no correlation withthe differentiation degree. The highest values of microvascular density (MVD) were recorded inliposarcoma and malignant fibrous histiocytoma, in straight correlation with the high number of
immature and intermediate blood vessels. We have noticed major differences in the blood vessel
number between benign and malignant tumours, which were statistically significant (p<0.02). We
have also noticed significant differences between the group composed of leiomyosarcomas andfibrosarcomas on one side, and liposarcoma and malignant fibrous histiocytoma on the other side
(p<0.05). The results of microvascular density assessments were concordant for either endothelial
marker used (table). Microvascular density was significantly higher in the malignant tumours than in
the benign ones.
TumourBenign tumours
Fibrosarcoma
Liposarcoma
Malignant fibrous histiocytoma
Leiomyosarcoma

MVD/CD34
10.3
34.6
61.2
58.9
21.5

MVD/CD31
9.4
32.2
58.3
55.7
22.4

Conclusions: Malignant soft tissue tumours have a rich blood vessel network, especially theliposarcoma and malignant fibrous histiocytoma. The majority of the blood vessels from within thetumour area are of immature and intermediate type. MVD does not correlate with the differentiation
degree, but have significantly higher values in malignant tumours then in the benign ones.

25. CD34/AAS TECHNICAL DETAILS OF MALIGNANT TUMORS AT THE LIP AND
PHARYNX LEVELSElena Viorica Bocan, Puşa Gaje, Anca Maria Cîmpean
Histology Department, “Victor Babeş” University of Medicine and Pharmacy, Timisoara, Romania
ABSTRACTThe immunohistochemical methods identify a certain tisular antigen through an antigen-antibody
reaction, revealed with a substance with cromogene role. Histochemical methods provide
information about cellular functions and extracellular tisular components. The aim of this study wasto perform a double immunohistochemical / histochemical technique, i.e. the differentiating clone(CD34) and alcian blue safranine. Using the immunohistochemical technique we have revealed theendothelial cells from the blood vessels and mastocites in the conjunctive tissue were revealed using
the alcian blue safranine method. The immunohistochemical method used the QEBnd 10
differentiating clone.
The principle of the technique consists of the following: sections are deparaphinated, then they are
hydrated with alcohols of descending concentrations (100% - 70%), down to distilled water. The
principle of antigen unmasking is not well known. It is done using a solution of buffer citrate(phosphate – citric acid) with a pH of 6 for 20 minutes at a temperature of 95-99 degrees Celsius.
The blocking of endogene peroxidase, used for eliminating the non-specific coloration of the
background, is performed with oxygenated water 3% for 5 minutes, followed by successive washes
with distilled water and buffer phosphate, pH 7. The application of the primary antibody (30minutes) is followed by the En Vision system, based on dextran polymers. The secondary reactive
includes a dextran skeleton which binds several molecule enzymes, causing the increase of themethod sensibility, the decrease of the non-specific background coloration and work time.
The reaction was revealed using the DAB cromogen. The Lillie hematoxylin countercoloration was
not applied; instead the histochemical coloration blue alcyan saphranine coloration was used toreveal the mastocytes. The histochemical technique consisted of the application of the coloring AAS
with pH 1,42, followed by a bath of distilled water and one of absolute ethylic alchohol, every few
seconds. Sections were clarified in benzene and mounted with Canada balm.
The endothelial cells were identified in dark brown in the normal blood vessels and the cells
involved in tumoral angiogenesis. The ancyanophile mast cells were identified in light blue. The
vascular microdensity is correlated to the mastocitary vascular with respect to the tumoral type andlocalization.
26. CORRELATION OF THE CD34 EXPRESSION AND MAST CELL IN VARIOUS TYPES
OF MALIGNANT TUMORSPuşa Gaje, Viorica Bocan, Anca Maria Cîmpean, Marius Raica
Histology Department, “Victor Babeş” University of Medicine and Pharmacy, Timisoara, Romania
Angiogenesys is an important prognostic factor mediated by the angiogenic factors released by themalignant cells and the immunitary system cells. Among these we can find mast cells involved in thetumoral progression through angiogenetic promotion.
The aim of the study was to develop a correlation between the identification of endothelial cells innormal and tumoral blood vessels and their possible association to mast cells according to the tumor
type and localization.
The study was performed on a series of 17 cases of pharinx carcinoma and 21 cases of lip carcinoma,
through the CD34/AAS double coloration using the En Vision/DAB work system and the QBEnd10
clone. Mast cell coloration was performed with blue alcyan safranine. Sections had a width of 5
microns. The normal morphological coloration used to establish the histopathologic diagnostic was
the hematoxylin-eosin. 16 cases of scuamo-cellular carcinoma and 1 adenocarcinoma were found inpharinx, and 17 cases of scuamo-cellular carcinoma and 4 basocellular carcinoma were found at theouter lip margin.

Conclusions: A correlation between the number of vessels and mastocitary density was observed in
the scuamo-cellular carcinoma of lip and pharinx. Vessels are sinuous, small, with no lumen andwith intensely positive endothelial cells for CD34. These are associated to a large number ofalcialphile mast cells, fully and partially degranulated. The distribution of vessels and mast cells is at
the edge of tumor area. Intratumoral mast cells were not observed. The number of mast cells lowers
with the dimension increase of the blood vessels. In case of adenocarcinoma, mast cells are
numerous, associated to sinuous mast cells, compared to scuamocellular carcinoma laid aroundmalignant-transformed glands. A lower number of mast cells were seen in the basocellular
carcinoma, laid in the vicinity of the tumor. Our findings are consistent with the literature data andour study from the previous year.

27. MAST CELL REACTION IN MALIGNANT LARYNGEAL NEOPLASM
Balica N, Raica M, Cotulbea S, Doroş C, Bocan Viorica
University of Medicine and Pharmacy “Victor Babes”, Timisoara, Romania
ABSTRACTIntroduction: Mast cells are normal connective tissue residents; they are present predominantly
around vessels. Mast cell densities vary from an organ to other, but are constantly well represented
in respiratory tract segments. Mast cell hyperplasia was found in many malignant tumors, but thesignificance of this phenomenon is stilt unknown. In the literature there are few data about mast cell
reaction in malignant laryngeal neoplasm.
Material and methods: We studied archive blocks from 127 cases of laryngeal carcinoma. For
histological diagnosis and grading 2 sections were prepared for Hematoxylin-Eozin staining and for
identifying mast cells we used Alcian Blue Safranin histochemistry at 0.2 pH. Examination has
been performed with Nikon Eclipse 600 microscope, the images were recorded in .jpeg format andthe microscopic images were analyzed with Lucia G program. Microvessel density was calculatedusing the hot spot method.
Results: Most of the cases were squamous cell carcinoma with a great variety of grading (G1(24.4%, 31 cases), G2 (56.69%, 72 cases), G3 (18.11%, 23 cases) and in 1 case we encounteredadenoid cystic carcinoma (0.78%, 1 case)). Invasive squamous cell carcinoma mast cell
microdensity was 2.19, in microinvasive squamous cell carcinoma mast cell microdensity was 4.66,
in cases of malignant laryngeal papillomatosis the mast cell microdensity was 9.33, and in the caseof adenoid cystic carcinoma the mast cell microdensity was 46.66. In carcinoma-associated mastcell hyperplasia, the large majority of mast cells were alcian blue positive.
Conclusions: In early stages the mast cells identified with Alcian Blue Safranin are numerous(microinvasive squamous cell carcinoma mast cell microdensity 4.66) and rare or even absent inlate stages (invasive squamous cell carcinoma mast cell microdensity 2.19). In the case of
malignant laryngeal papillomatosis mast cell microdensity was 9.33, and in case of the cystic
carcinoma the mast cell microdensity was 46.66. Alcianophile mast cells are present in tumor area,and safraninophile mast cells are residents of connective and muscular tissue, at a distance from thetumor.
Key words: carcinoma, larynx, mast cell, histochemistry, Alcian-Blue Safranin
28. EXERCISE TRAINING IMPACT ON ARTERIAL RIGIDITY PARAMETERS IN
HYPERTENSIVE PATIENTSStela Iurciuc, M. Iurciuc, Adina Avram, S. Ursoniu, D. Duda-Seiman, Laura Craciun, C.A.
Sarau, C. Avram, Silvia Mancas
University of Medicine and Pharmacy “Victor Babes”, Timisoara, Romania
ABSTRACT
Objectives: Assessment of an individualized exercise training program efficacy on hemodynamic
parameters, arterial rigidity parameters and cardiovascular risk factors in hypertensive subjects withhigh normal and stage 1 and mild and moderate additional risk blood pressure (BP).

Patients and methods: We studied 229 hypertensive patients (69% men, 31% women, average age
58.6±1016, recently diagnosed with high normal and stage 1 and mild and moderate additional risk
BP, who did not require antihypertensive drugs (ESC/ESH 2003 Guidelines). The patients were
included in an individualized comprehensive rehabilitation program, training level representing 80%
from maximum achieved in exercise testing. Before and at the end of the 3 month period there wereassessed the hemodynamic parameters (automatic ambulatory monitoring of BP) and arterial rigidity
parameters through PVWc-r (carotid-radial pulse wave velocity) measured using the Compliormethod. It was also studied the trend of cardiovascular risk factors.
Results: Pulse pressure (PP) dynamic/analyze measured through BP automatic ambulatory
monitoring/24 hours revealed a significant decrease of PP after the 3 month program (from62.27±8.01 mmHg to 56.18±8.24 mmHg) (p<0.0001). We also noticed a reduction of PWVc-r (from9.95±2.08 m/s to 9.11±1.52 m/s). A significant correlation was found between PP and SBP (systolicblood pressure) values and also between PWVc-r and total serum cholesterol levels (TC) at baseline
and after the 3 month training program.
Conclusions:
1. Exercise training has a favorable impact on pulsated component of BP and consequently onarterial compliance of elastic arteries. Due to exercise training impact on arterial compliance,
it can slow down/postpone the arterial aging.
2. The favorable results following exercise training were also observed in muscular arteries
compartment. Pulse wave velocity measured at distal arteries level can represent a key point
in assessment of exercise training outcomes in hypertensive patients.
3. Exercise training has a positive impact on dynamic dysfunction of vascular compartment(endothelial dysfunction) and also on structural changes of arterial wall (arterial rigidity).
4. BP control according to guideline recommendations can have a favorable impact on arterial
compliance.
29. BENEFIT OF EXERCISE TRAINING ON CARDIOVASCULAR RISK IN STAGE 2
HYPERTENSIVE PATIENTS TREATED WITH ANTIHYPERTENSIVE DRUGS
M. Iurciuc, S. Ursoniu, D. Gaita, Simona Dragan, Laura David, Adina Avram, Stela Iurciuc,
Silvia Mancas, C. Avram
University of Medicine and Pharmacy “Victor Babes”, Timisoara
Cardiovascular Prevention and Rehabilitation Clinic, Timisoara, Romania
Introduction/Background: Cardiovascular risk represents a major clinical priority for hypertensive
patients in choosing the right therapy and life style recommendations. It is well-known the impact of
exercise training on cardiovascular risk factors.
Methods: We studied 120 hypertensive patients classified as stage 2, moderate additional risk,
following antihypertensive therapy for at least 3 months and no more than 1 year, with bloodpressure values bellow the target recommended by the guidelines. Patients were evaluated at baseline
and at the end of the study. Hemodynamic parameters, measured using blood pressure Holter
monitoring/24h, were: pulse pressure (PP) and pulse wave velocity (PWVc-r). Patients followed a 6months rehabilitation program. Cardiovascular risk was assessed using Heart SCORE Chart.
Results:
Parameter
At baseline
After 6 monthsp
SCORE Risk (%)
7,26 ± 4,70
5,84 ± 4,06
0,01314
PP/24h (mmHg)
56,99 ± 8,82
53,67 ± 9,75
0,00613
PWVc-r (m/s)
12,14 ± 2,35
11,00 ± 2,00
< 0,001
Smoking (number)
39 (32,5)26 (21,6)<0,05
Total cholesterol (mg/dl):
194,66 ± 38,41
176,18 ± 29,28<0,0001
BMI (kg/sm)
27,50 ± 4,47
26,38 ± 3,90
0,01
Mean value ± Standard Deviation (%)
Conclusions:
1. Beneficial interventions on cardiovascular risk factors modify absolute risk of fatal
cardiovascular disease to a different extent.

2. After a 6 months period we noticed a significant reduction of absolute risk of fatal
cardiovascular diseases in 10 years.
3. Absolute risk reduction was due to exercise training impact on associated risk factors as
smoking and total cholesterol, even though the final value was still in high additional riskarea.
4. The benefits of exercise training were also noticed on arterial rigidity parameters which, eventhough they are not components of Heart Score chart, have an important significance inassessing hypertensive patient status.
30. SOURCES OF ENDOTHELIAL CELLS FOR STENTS ENDOTHELISATION
Daciana Carmen Nistor, Ioana Siska, Gabriela Tanasie, Florina Mirea, Ramona Cioaca, Diana
Szilagy*, Simona Anghel*, Carmen Bunu, Erika Deak, Georgeta Mihalas, Virgil Paunescu
Departament of Physiology and Immunology, University of Medicine and Pharmacy "Victor
Babes" Timisoara* County Hospital Timisoara
ABSTRACT
Objectives: 1. Culture of endothelial-like cells obtained from CD34+ cells isolated from peripheral
blood and umbilical cord blood. 2. Cultivation of endothelial-like cells obtained from bone marrow
AC133+ cells.
Material and method: Isolation of human haematopoietic stem cells (hHSC) was performed using a
positive selection with immunomagnetic beads directly conjugate with CD34 monoclonal antibodies
and AC133 monoclonal antibodies. We used DYNAL Biotech and Miltenyi Biotech separation
complete kits. hHSC differentiation to endothelial lineage was performed by cytokines addition:
VEGF, SCGF and ECGS. The cells were seeded in multi-well plates coated with fibronectine or
gelatin and were maintained at 37°C in a 5% CO2 humidified atmosphere.
Endothelial character of cells obtained from CD34+ was assessed by identification of the following
markers: AC133, CD34, CD31/PECAM, von Willebrandt factor, VE-cadherin, VEGFR2/KDR/flk1,
eNOS and several adhesion molecules (CD54, CD62E, CD106) by flow cytometry,immunohistochemistry and RT-PCR.
Results: Flow cytometry analysis of cells obtained from peripheral blood showed that all CD34+
cells exhibit AC133 marker. Approximately 10% of CD34+ cells obtain from umbilical cord bloodcultivated in medium supplemented with ECGS expressed morphological characters similar toendothelial cells. Immunohistochemical analysis of endothelial-like cells showed that only 3.25 ±
1.25% of this cells were PECAM/CD31 positive and von Willebrand factor positive.
The flowcytometric analysis of the immunomagnetic separated bone marrow-isolated cells with
AC133 MicroBeads showed that 90±5% of the cells are pure. The AC133+ fraction included 95±4%of CD34+ cells. After 3 weeks, all cells were AC133-; in 2/3 of the experiments 5-37% of cells wereCD34+. Gene expression analysis by RT-PCR of mature endothelial cells specific markers - VEcadherin, VEGFR-2, eNOS - showed that most of the cells expressed these markers.
Discussions: Bone marrow and peripheral blood could represent good sources of endothelial cells forstents endothelisation. VEGF is essential for endothelial cells development.
31. PULMONARY VASCULAR REMODELLING IN COPD
Ovidiu Fira-Mladinescu1, 2, Liliana Vasile3, Cristian Suciu3, Corneluţa Fira-Mladinescu1, Danina
Muntean2, Voicu Tudorache4, Georgeta Mihalas5
1
Research Centre in Preventive Medicine
2
Department of Pathophysiology
3
Department of Histology
4
Department of Pneumology
5
Department of Physiology
Victor Babes University of Medicine and Pharmacy, Timisoara, Romania

ABSTRACT
Pulmonary vascular remodelling in COPD patients represents more than a simple hypertrophy of
media triggered by long-term hypoxic vasoconstriction. The aim of the present study was tocharacterise structural changes of pulmonary vasculature in both smokers and patients with mild
COPD in order to assess the involvement of cigarette smoking in vascular remodelling initiation.
Histological examination of tissue sections dissected from exeresis fragments was followed by themorphometric analysis of muscular pulmonary arteries and a semi-quantitative evaluation of elastin
and collagen intimal deposits based on a visual scale. Our results suggest that pulmonary vascularabnormalities present in both smokers and COPD patients with early disease are intimal thickeningof small muscular arteries (intimal index was 23.11 ± 3.04 in smokers, respectively 25.62 ± 3.06 in
COPD group, v. 17.86 ± 2.96 in control group, p = 0.005) and arteriolar muscularisation. In patientswith mild COPD, the substrate of intimal thickening is determined initially by the local predominantdeposition of elastin and further, by colagen deposition. Moreover, intima thickness was found to besignificantly associated with cigarette consumption expressed as packs-years (r = 0.65, p = 0.04) andwith vascular diameter (r = – 0.59, p = 0.02). Immune labelling with anti-CD34 antibodies showed
the swelling of endothelial cells as a sign of vascular injury. In conclusion, the direct effect of smoke
components on the endothelium may be considered as an important factor responsible for both
pulmonary endothelial dysfunction and vascular remodelling in patients with COPD.