Investeşte în oameni!

FONDUL SOCIAL EUROPEANProgramul Operaţional Sectorial Dezvoltarea Resurselor Umane 2007-2013Axa Prioritară: 1. Educaţia şi formarea profesională în sprijinul creşterii economice şi dezvoltării societăţii

Domeniul Major de Intervenţie: 1.5. Programe doctorale şi postdoctorale în sprijinul cercetăriiTitlu proiect: "Sprijinirea tinerilor doctoranzi cu frecvenţă prin acordarea de burse doctorale"

Cod Proiect: POSDRU/6/1.5/S/8

Beneficiar: Universitatea de Medicină şi Farmacie din Craiova

UNIVERSITY OF MEDICINE AND PHARMACY OF CRAIOVA

PhD DissertationAbstract

Role of VEGF in angiogenesis in malignant and premalignant gastric

lesions

PhD Tutor Prof. univ. dr. Mihai Cruce ! ! ! ! ! ! ! !! ! ! ! !! ! ! ! ! ! PhD Student! ! ! Maria-Cristina Soare (Angelescu)

Craiova, 2011

Introduction

Despite the decreasing in incidence and mortality rates observed in the last decades, gastric cancer still represents the fourth cause of cancer and the second cause of cancer-related deaths worldwide. Helicobacter pylori (H. pylori), Gram-negative bacteria classified by World Health Organization as a type I carcinogen, triggers the invasion of gastric tumours by several mechanisms, one of which is the induction of host neo-angiogenesis, by modulation of the expression of various angiogenic factors. Moreover, H. pylori chronic infection stimulates angiogenesis in non-malignant gastric lesions, as gastric ulcerations, chronic gastritis, intestinal metaplasia and dysplasia (IARC, 1994; Ki-datai et al., 2003; Papetti and Herman, 2002). H. pylori infection is often associated with a mild hyper-gastrinemia, which has been proposed to be an important factor in promoting gastric carcinogenesis. Associations between Helicobacter infection, hypergastrinemia and gastric carcino-genesis have been proven, but the exact mechanism of interac-tion between Helicobacter infection and hypergastrinemia has not yet been clarified (Takaishi et al, 2009).

Key words: gastric cancer, angiogenesis, Helicobacter, hypergastrinemia

1

I. LITERATURE REVIEW

Chapter 1, entitled “Gastric carcinogenesis”, describes gastric cancer epidemiology, risk factors involved in gastric carcinogenesis, highlighting the importance of infection with H. pylori and hypergastrinemia, and the importance of using animal models in the study of gastric carcinogenesis.

Chapter 2, entitled “Tumour angiogenesis - particulari-ties in gastro-oesophageal malignant and pre-malignant le-sions”, describes first the process of tumour new blood vessels formation, pro-angiogenic factors, and then analyses the angio-genesis process in gastric and oesophageal malignant and pre-malignant lesions and anti-angiogenic targeted therapy.

II. PERSONAL CONTRIBUTIONS

Chapter 3. Expression analysis of VEGF-A, VEGF-B and EGFR mRNAs in gastro-oesophageal tumours

In this chapter we aimed to quantify the expression of total VEGF-A, several VEGF-A transcript variants, VEGF-B and EGFR genes in human gastro-oesophageal cancers and to analyse the correlations between the expression levels and clinico-pathological features of the tumours.

2

Material and Methods Patients and specimens. Gastro-oesophageal tumour samples were biopsied from 43 patients who had undergone upper gastrointestinal endoscopy and endoscopic ultrasonogra-phy at the Research Centre in Gastroenterology and Hepatol-ogy of Craiova, Romania. All the patients included in the study were infected with H. pylori. Both paired tumour and peritu-mour specimens were biopsied during endoscopy for all the patients. RNA isolation. SV Total RNA Isolation System (Promega) was used for the isolation and purification of total RNA from tumour and peritumur mucosa samples according to manufactures’s instructions. The quality of RNA was assessed by denaturing agarose gel electrophoresis and the concentration and purity were measured spectrophotometrically (Eppendorf Biophotometer). qRT-PCR. Two step quantitative Real-Time PCR was per-formed. In the first step 1µg RNA was reverse transcribed into complementary single stranded DNA (cDNA) using High Ca-pacity cDNA Reverse Transcription Kit (Applied Biosystems) according to manufacturer’s instructions. In the second step, quantitative Real-Time PCR was performed using TaqMan® Gene Expression Master Mix (Applied Biosystems) with spe-cific primers and TaqMan® probes for target genes and for en-dogenous control gene (VEGF-A - Hs00900054_m1, VEGF121 - Hs03929005_m1, VEGF148, VEGF165 and VEGF165b - Hs00900057_m1, VEGF-B - Hs00173634_m1,

3

EGFR - Hs01076092_m1 and GAPDH - Hs99999905_m1). The amplifications were carried out in 20 µl volume, in tripli-cate, on a Mastercycler®ep realplex (Eppendorf). The expres-sion of the target genes was normalised to the GAPDH en-dogenous control gene for both tumour and adjacent mucosa and fold changes were calculated using 2-ΔΔCt method. Statistics. The fold differences between paired samples were considered biologically significant when the mRNA lev-els varied more than 1.8 folds. When variables did not follow normal distributions, non-parametric tests (Wilcoxon matched-pairs signed rank tests, Wilcoxon Signed Rank Test, Mann-Whitney, Kruskal-Wallis tests, Spearman correlation test) were used. Two-tailed p values <0.05 were considered statistically significant.

Results and Discussions

qRT-PCR analysis revealed that VEGF-A, including the studied splice variants, VEGF-B and EGFR mRNAs were ex-pressed in both tumour and peritumour mucosa. The expression pattern for each of the analysed transcript is presented in Figure 1.

4

Fig. 1. Expression pattern for the evaluated transcripts in the paired samples with non-malignant peritumour tissue (n=39)

Total VEGF-A, VEGF121, VEGF148, VEGF165 and VEGF165b mRNA levels were higher in tumour tissue when compared with paired non-invaded peritumour tissue. The dif-ferences in expression levels of total VEGF-A and the studied isoforms between paired samples were statistically significant (p=0.007; p=0.002; and p=0.014 respectively, Wilcoxon matched-pairs signed rank test). VEGF-A protein expression has been found elevated in gastric tumours and it was correlated with microvascular den-sity in the majority of the studies (Zhao et al., 2006; Kolev et

VEGF-A

VEGF121

VEGF148 V

EGF165 V

EGF165b

VEGF-BEGFR

0

20

40

60

80

100

Perc

ent o

f the

cas

es

Higher expression in TIrrelevant differenceHigher expression in PT

5

al., 2007). On the contrary, in a DNA microarray study of VEGF-A expression, Jubb et al. showed that VEGF-A is down-regulated in stomach tumours (Jubb et al., 2004). In our study VEGF-A mRNA expression was higher in tumour tissue in 38.46% of the cases and higher in peritumour tissue in 7.69% of the cases; in the remaining 53.85% of the cases no biologi-cally significant difference was observed. A possible explana-tion for the discrepancies between various studies is the meth-odology used in the study. While most of the studies focused on immunohistochemical expression of VEGF-A, in the present study the expression of VEGF-A was investigated at mRNA level. Furthermore, mRNA levels are not always a true reflec-tion of protein abundance due to post-transcriptional regulation. On the other hand, in peritumour tissue may be an up-regulation of VEGF-A due to H. pylori infection and fold changes between paired samples may not reflect the true VEGF-A up-regulation. Taken into account the very different characteristics and variations in angiogenic effect intensity, it is of major impor-tance to elucidate the mechanisms that lead to alternative splic-ing, but, more importantly, to characterise the presence and level of these variants in pathological conditions. We have found that both VEGF121 and isoforms containing only exon 7 were expressed in both tumour and peritumour tissue and in the majority of the cases the expression was up-regulated in tu-mour tissue or the difference between paired samples was ir-relevant; isoforms expression followed the same expression

6

pattern that was observed for total VEGF-A. Moreover, the ra-tio between the isoforms that contain only exon 7 between exon 5 and exon 8 (VEGF148, VEGF165 and VEGF165b) and the isoform that does not contain exon 6 nor exon 7 (VEGF121) was similar in each sample, either tumour or peritumour tissue. There was no statistically significant difference in the ex-pression of VEGF-B nor EGFR between the paired samples. VEGF-A, VEGF-A studied isoforms, VEGF-B and EGFR mRNA levels were analysed in relation to several patho-logical characteristics: tumour stage, tumour location and his-tological type of tumour. When assessed according to tumour stage, total VEGF-A and its splice variants showed a similar expression pattern. The mRNA levels were similar in T2 and T3 lesions, and tended to be lower in T2 and T3 compared with T4 tumours, and the dif-ference was borderline statistically significant. Although the difference did not reach a statistical significance level, VEGF-A expression might be helpful as a progression indicator in as-sociation with other biological or clinico-pathological parame-ters. No differences in total VEGF-A or it’s splice variants ex-pression between adenocarcinomas and squamous cell carci-nomas were observed. mRNA levels for the analysed tran-scripts tended to be higher in corpus compared with antrum and oesophageal, oesophagogastric junction or cardia tumours. In the majority of the cases, VEGF-B mRNA levels were similar between T2, T3 and T4 tumours. VEGF-B expression pattern differed between squamous cell carcinomas and adeno-

7

carcinomas. VEGF-B expression in distal oesophagus, oeso-phagogastric junction and cardia was similar to that in corpus. Most of the antrum cases showed a lower expression compared with other locations. None of these differences reached a bio-logically nor a statistically significant level. No biologically or statistically significant differences in EGFR mRNA levels were observed between different tumour stages, tumour location or histological type of tumour. Aggregation patterns of the studied genes were assessed by hierarchical clustering, supported by Spearman coefficients (Figure 2).

Fig. 2. Hierarchical cluster (GenexPro 4.4.2.308©)

8

Conclusions 1. VEGF-A expression was significantly higher in tumour compared with peritumour tissue. 2. VEGF-A mRNA levels tended to be higher in tumour invading the serosa (T4) and in tumour located within corpus. 3. VEGF-A investigated isoforms (transcript variant 6, and transcript variants 4, 5, 7) followed the same expression pattern as total VEGF-A in all the cases, in both tumour and peritumour mucosa. 4. VEGF121 and VEGF148, VEGF165, VEGF165b ex-pressed at the same level in each individual sample; this fact suggests that no variant is predominant in gastro-oesophageal tumours or peritumour mucosa. 5. VEGF-B expressed in both tumour and peritumour mucosa in the study group, with no significant difference was noticed between paired samples. 6. EGFR expressed in all the samples, with no tendency of up- or down-regulation. No significant correlation was found between EGFR expression and pathological parameters. 7. VEGF-A, VEGF-B and EGFR were up-regulated in microscopically invaded peritumour mucosa even compared with paired tumour tissue, which suggests that over-expression of these pro-angiogenic molecules and receptor is an early event in gastric carcinogenesis. 8. By using a two-step quantitative PCR, we have ob-tained a cDNA library specific for gastro-oesophageal tumours.

9

9. Protein expression assays of the same cases are neces-sary to elucidate if there is a post-transcriptional or post-translational regulation of the analysed transcripts.

Chapter 4. Helicobacter felis infection and hypergas-trinemia promote angiogenesis in short-term infected mice

We hypothesised that angiogenesis is increased by Helicobacter infection and/or hypergastrinemia. We evaluated microvascular density and VEGF-A expression (both at protein and mRNA levels) in relation to both Helicobacter infection and/or hypergastrinemia by infecting with Helicobacter felis (H. felis) both wild-type and transgenic hypergastrinemic mice for 6 weeks.

Material and Methods Animals. The insulin-gastrin (INS-GAS) mice are trans-genic mice which have a chimeric transgene in which the hu-man gastrin gene is transcribed from the rat insulin promoter (Wang et al., 1993). For infection studies, 12 INS-GAS and 11 FVB/N mice at 4 weeks of age were infected with H. felis (ATCC 49179) by 3 gavages over the course of one week. 12 non-infected INS-GAS and 12 non-infected FVB/N were also included in the study. Mice were killed by exsanguination 6 weeks following infection and whole tissue was collected for histology, protein and RNA studies. The experiments were conducted with UK Home Office Approval.

10

Immunohistochemistry. Standard immunohistochemistry methods were performed. We performed a heat mediated anti-gen retrieval with 10mM citric acid buffer pH 6 for 20 minutes for VEGF-A slides and an enzymatic antigen retrieval with pro-teinase K 20µg/ml at 37˚C for 10 minutes for vW factor stain-ing. Mouse monoclonal to VEGF-A, VG-1 clone (ab1316, Ab-cam, Cambridge, UK) and rabbit polyclonal to von Willebrand factor (ab6994, Abcam, Cambridge, UK) were used as primary antibodies. Western blot. Following protein extraction, Bradford pro-tein assay was performed to determine the concentration of the extracted proteins. Proteins were migrated in a 10%, 1.5 mm thick running gel. Mouse monoclonal to VEGF-A (ab1316, Ab-cam, Cambridge, UK) was used at a concentration of 0.01 mg/ml (dilution 1:100) as primary antibody. Gene expression analysis. After total RNA isolation and purification, VEGF-A expression was assessed by performing a two step qRT-PCR analysis using specific primers for mouse VEGF-A and endogenous control Rn18S. Relative VEGF-A mRNA levels (VEGF-A expression reported to Rn18S) were calculated for each group of animals. Statistics. In order to determine whether the variables fol-lowed a gaussian distribution, Kolmogorov-Smirnov test was performed. In order to assess differences in expression between groups, non-parametric tests (Kruskal-Wallis test) were used. Two-tailed p values <0.05 were considered statistically signifi-cant.

11

Results and Discussions

In corpus, the highest increase in the number of microvessels was observed in H. felis infected hypergastrine-mic INS-GAS mice. For antrum, the results were slightly dif-ferent: the increase in MVD was similar between INS-GAS and FVB/N infected with H. felis (Figures 3, 4, 5).

Fig. 3. Microvessels in corpus mucosa of H. felis infected (a) and non-infected (b) wild type FVB/N mice (original magnification x400).

Fig. 4. Microvessels in corpus mucosa of H. felis infected (a) and non-infected (b) INS-GAS mice (original magnification x400).

12

corpus antrum Fig. 5. MVD in both corpus corpus for each of the groups. Data are presented individually and as mean±SEM. *** = p<0.001, ** = p<0.01, Kruskal-Wallis test

The pattern observed for VEGF-A immunostaining with anti-VEGF-A antibody, VG1 clone, was a cytoplasmic pattern with homogenous distribution. Both corpus and antrum from H. felis infected mice (FVB/N and INS-GAS) stained moderate or strong positive for VEGF-A. VEGF-A expression in corpus of both FVB/N and INS-GAS H. felis infected mice was significantly higher when com-pared with FVB/N non-infected mice - 3.4-fold and 3.16 fold respectively (p<0.01, Kruskal-Wallis test). In antrum, we have observed a 3.61-fold (p<0.01, Kruskal-Wallis test) increase for H. felis infected FVB/N and a 2.2-fold increase (not statisti-cally significant) for transgenic INS-GAS mice infected with H. felis (Figure 6).

FVB INS-GAS FVB INS-GAS 3

4

5

6

7M

VD

H. felis + no H. felis

****

13

corpus antrum! Fig. 6. VEGF-A immunoexpression pattern in antrum. ** = p<0.01, Kruskal-Wallis test

For both corpus and antrum, the percentage of positive cells was higher in H. felis infected INS-GAS mice comparing to FVB/N, but the intensity of immunostaining was higher in FVB/N infected group. In both corpus and antrum, an increase in the intensity of staining for VEGF-A in the glandular compartment of the mu-cosa was observed in H. felis infected mice (both FVB/N and INS-GAS groups) when compared with non-infected mice (Figure 7 and 8). This observation have been also made by Tuccillo et al. in H. pylori infected patients (Tuccillo et al., 2005). We have found a strong positive correlation between VEGF-A immunoexpression and MVD, supported by both lin-ear regression analysis and Spearman coefficient.

FVB INS-GAS FVB INS-GAS 0

1

2

3

4

5

VEG

F-A

imm

unos

tain

ing

scor

e

H. felis + no H. felis

**

14

Fig. 7. VEGF-A immunoexpression in corpus mucosa of H. felis infected (a) and non-infected (b) FVB/N mice (original magnification x400).

Fig. 8. VEGF-A immunoexpression in corpus mucosa of H. felis infected (a - mucosal surface, b - gland base) and non-infected (c - mu-cosal surface, d - gland base) INS-GAS mice (original magnification x400).

In Western blot analysis, anti-VEGF-A antibody detected a 60 kDa band corresponding to the VEGF-A dimer and a 30 kDa band corresponding to the VEGF-A monomer (Figure 9).

15

Fig. 9. Western blot for corpus and antrum VEGF-A protein, with pan-actin as loading control.

VEGF-A protein levels increased 3.1-folds in corpus of H. felis infected FVB/N (p<0.01, Kruskal-Wallis test) and by 2.3-folds in corpus of infected INS-GAS mice (p<0.01, Kruskal-Wallis test) when compared with non-infected FVB/N. The same pattern was observed in antrum, with a 3.8-fold increase in VEGF-A protein levels for H. felis infected FVB/N (p<0.01, Kruskal-Wallis test) and a 2.24-fold increase for H. felis infected INS-GAS. Consistent with VEGF-A protein quantification, mRNA levels tended to increase in H. felis infected FVB/N, H. felis infected INS-GAS and non-infected INS-GAS groups, when compared with non-infected FVB/N mice. In our study VEGF-A expression was up-regulated at both gene and protein levels in H. felis infected corpus of wild type FVB/N and transgenic INS-GAS mice. VEGF-A expres-sion, evaluated by immunohistochemistry, Western blot and

16

qRT-PCR, was enhanced in the stomach of FVB/N compared with INS-GAS mice with H. felis infection for 6 weeks. Fur-thermore, VEGF-A tended to express higher in non-infected hypergastrinemic mice compared with non-infected wild-type mice, which suggest that gastrin per se enhances VEGF-A gene and protein expression. This increase in VEGF-A expression may be the reason for a lower increase in VEGF-A expression in hypergastrinemic infected mice compared with wild-type infected mice observed in our study. In INS-GAS mice, H. felis infection occurs in a stomach with mild hypergastrinemia, in-crease in parietal cell number, enhanced VEGF-A expression and increased MVD, as showed in our study. Thus, the need for new blood vessel formation induced by H. felis infection in hy-pergastrinemic mice is not that prominent as in wild-type mice. Although the increase in VEGF-A expression was more pro-nounced in FVB/N mice with short-term H. felis infection, the MVD was higher in infected INS-GAS mice. These apparently contradictory findings may rely on the continuous actions of gastrin on VEGF-A expression, that started before H. felis in-fection. Moreover hypergastrinemia also leads to an over-expression of other molecules involved in angiogenesis (Takaishi and Wang, 2007).

Conclusions 1. There is a strong positive correlation between VEGF-A immunoexpression and MVD in all groups of mice included in our study.

17

2. Helicobacter infection up-regulates VEGF-A expres-sion and microvessels neo-formation in both corpus and antrum of short term infected animals. 3. Hypergastrinemia up-regulated VEGF-A expression and stimulates angiogenesis in corpus and antrum of transgenic mice that over-express human amidated gastrin. The presence of Helicobacter infection enhances these actions. 4. The presence of H. felis infection in the stomach of both wild-type and hypergastrinemic mice leads to an increase in VEGF-A expression in the glandular compartment of gastric glands. 5. A post-transcriptional regulation of VEGF-A expres-sion by hypergastrinemia and/or Helicobacter infection may be involved. 6. Up-regulation of VEGF-A expression by gastrin or Helicobacter infection involves different mechanisms that need to be elucidated.

Chapter 5. Effects of long-term Helicobacter felis in-fection in modulation of angiogenesis - preliminary data

The last study in this dissertation is represented by a pre-liminary experiment in which we aimed to evaluate angiogene-sis in both short-term and long-term H. felis infected wild type mice. C57BL/6, the most common inbred strain of laboratory mice, have been used in this study. For infection studies, 14

18

C57BL/6 at 4 weeks of age were infected with H. felis (ATCC 49179) by 3 gavages over the course of one week. 5 non-infected C57BL/6 were also included in the study as control.

Results and Discussions

An increase in the number of microvessels was observed in corpus of both short-term and long-term H. felis infected C57BL/6 mice (Figure 5.2). Particularly, MVD increased 1.54-fold (not significant) in corpus of 6 weeks infected mice and 6.05-fold in corpus of 9 months infected C57BL/6 (p<0.001, Kruskal-Wallis test) when compared with non-infected wild-type. Corpus from both short-term and long-term H. felis in-fected C57BL/6 stained moderate or strong positive for VEGF-A. VEGF-A immunoexpression in corpus of long-term in-fected wild type mice had a 4.59-fold increase when compared with non-infected mice (p<0.001, Kruskal-Wallis test). In cor-pus of C57BL/6, 6 weeks post infection, only a 2.02-fold in-crease in VEGF-A immunoexpression was found. We have found a strong positive correlation between VEGF-A immunoexpression and MVD, supported by both lin-ear regression analysis and Spearman coefficient.

19

Conclusions 1. A a strong positive correlation between VEGF-A im-munoexpression and MVD has been found in both 6 weeks and 9 months infected mice. 2. H. felis infection up-regulates VEGF-A expression and microvessels neo-formation in both corpus of both short term and long term infected wild type mice, and the increase in VEGF-A expression and MVD depend on duration of infection.

Final Conclusions

1. VEGF-A mRNA expressed in both tumour and peri-tumour mucosa of H. pylori positive patients diagnosed with gastric cancer. VEGF-A expression is significantly higher in tumour compared with peritumour tissue and tends to be higher in tumour invading the serosa. 2. VEGF-B and EGFR mRNAs express in both tumour and peritumour mucosa in H. pylori positive gastric cancer pa-tients. No significant differences are noted between paired samples nor in relation to pathological parameters for VEGF-B or EGFR expression. 3. Helicobacter infection up-regulates VEGF-A expres-sion and microvessels neo-formation in both corpus and antrum of short term infected animals. 4. Hypergastrinemia tends up-regulated VEGF-A ex-pression and stimulates angiogenesis in corpus and antrum of transgenic mice that over-express human amidated gastrin. The

20

presence of Helicobacter infection enhances the effects of hy-pergastrinemia actions, but not at the same amplitude as H. felis infection alone. 5. VEGF-A is an important pro-angiogenic molecule for gastric angiogenesis. VEGF-A is expressed in gastric mucosa from inflammatory and precancerous lesions to advanced gas-tric cancer.

References Burkitt MD, Varro A, Pritchard DM. Importance of gastrin in the pathogenesis and treatment of gastric tumors. World J Gastroenterol. 2009 Jan 7;15(1):1-16 Caputo R, Tuccillo C, Manzo BA, et al. Helicobacter pylori VacA toxin up-regulates vascular endothelial growth factor expression in MKN 28 gastric cells through an epidermal growth factor receptor-, cyclooxygenase-2-dependent mechanism. Clin Cancer Res. 2003 Jun;9(6):2015-21. Folkman J. Tumor angiogenesis: therapeutic implications. N Engl J Med 1971; 285:1182-6 Fondevila C, Metges JP, Fuster J, et al. p53 and VEGF expression are independent predictors of tumour recurrence and survival following curative resection of gastric cancer. Br J Cancer. 2004 Jan 12;90(1):206-15 International Agency for Research on Cancer Working Group on the Evaluation of Carcinogenic Risks to Humans. Schistosomes, Liver Flukes, and Helicobacter pylori. Lyon. IARC Monogr Eval Carcinog Risks Hum. 1994;61:1-241 Jubb AM, Pham TQ, Hanby AM, et al.. Expression of vascular en-dothelial growth factor, hypoxia inducible factor 1alpha, and carbonic anhy-drase IX in human tumours. J Clin Pathol. 2004 May;57(5):504-12. Kitadai Y, Sasaki A, Ito M, et al. Helicobacter pylori infection in-fluences expression of genes related to angiogenesis and invasion in human gastric carcinoma cells.Biochem Biophys Res Commun. 2003 Nov 28;311(4):809-14 Kolev Y, Uetake H, Iida S, et al. Prognostic significance of VEGF expression in correlation with COX-2, microvessel density, and clinicopa-

21

thological characteristics in human gastric carcinoma. Ann Surg Oncol. 2007 Oct;14(10):2738-47 Mangia A, Chiriatti A, Ranieri G, et al. H pylori status and angio-genesis factors in human gastric carcinoma. World J Gastroenterol. 2006 Sep 14;12(34):5465-72 Nikiteas NI, Tzanakis N, Theodoropoulos G, et al. Vascular endo-thelial growth factor and endoglin (CD-105) in gastric cancer. Gastric Cancer. 2007;10(1):12-7 Papetti M, Herman IM. Mechanisms of normal and tumor-derived angiogenesis. Am J Physiol Cell Physiol. 2002 May;282(5):C947-70. Pritchard DM, Przemeck SM. Review article: How useful are the rodent animal models of gastric adenocarcinoma? Aliment Pharmacol Ther. 2004 Apr 15;19(8):841-59. Review. Silvestre JS, Tamarat R, Ebrahimian TG, et al. Vascular endothelial growth factor-B promotes in vivo angiogenesis. Circ Res. 2003 Jul 25;93(2):114-23 Strowski MZ, Cramer T, Schäfer G, et al. Helicobacter pylori stimulates host vascular endothelial growth factor-A (vegf-A) gene expres-sion via MEK/ERK-dependent activation of Sp1 and Sp3. FASEB J. 2004 Jan;18(1):218-20. Takaishi S, Tu S, Dubeykovskaya ZA, et al. Gastrin is an essential cofactor for helicobacter-associated gastric corpus carcinogenesis in C57BL/6 mice. Am J Pathol. 2009 Jul;175(1):365-75 Takaishi S, Wang TC. Gene expression profiling in a mouse model of Helicobacter-induced gastric cancer. Cancer Sci. 2007 Mar;98(3):284-93. Takaishi S, Tu S, Dubeykovskaya ZA, et al. Gastrin is an essential cofactor for helicobacter-associated gastric corpus carcinogenesis in C57BL/6 mice. Am J Pathol. 2009 Jul;175(1):365-75 Tuccillo C, Cuomo A, Rocco A, et al. Vascular endothelial growth factor and neo-angiogenesis in H. pylori gastritis in humans. J Pathol. 2005 Nov;207(3):277-84. Wang TC, Dangler CA, Chen D, et al. Synergistic interaction be-tween hypergastrinemia and Helicobacter infection in a mouse model of gastric cancer. Gastroenterology. 2000 Jan;118(1):36-47. Zhao HC, Qin R, Chen XX, et al. Microvessel density is a prognos-tic marker of human gastric cancer. World J Gastroenterol. 2006 Dec 21;12(47):7598-603

22

Curriculum Vitae

Family Name: AngelescuMaiden name: SoareFirst Name: Maria CristinaDate and Place of Birth: 11th of June 1984, Craiova, RomaniaCitizenship: Romanian Civil status: Married

Work experience

2009-Present - Teaching Assistant, Department of Cell and Molecular Biol-ogy, Faculty of Medicine, University of Medicine and Pharmacy of Craiova 2009-Present - Resident physician on Medical GeneticsEducation

2008-Present - PhD Student, Department of Cell and Molecular Biology, Faculty of Medicine, University of Medicine and Pharmacy of Craiova 2002-2008 - Faculty of Medicine, University of Medicine and Pharmacy of Craiova. Academinc degree: Medical Doctor.1998-2002 - National College “Fratii Buzesti”, Craiova, Romania.

Specialisations and Trainings

1st of February - 31st of July 2011 - Visiting research student at the Uni-versity of Liverpool, Department of Gastroenterology, Institute of Transla-tional Medicine, The Henry Wellcome Laboratory. 1st-30th of September 2006 - IFMSA-SCORE Scholarship at the Institute of Physiology, 1st Faculty of Medicine, Charles University, Prague, Czech Republic. Research project The properties of muscarinic receptors. 1st-30th of September 2005 - IFMSA-SCORE Scholarship at the Depart-ment of Physiology, Section of Biochemistry and Molecular Biology, Uni-versity of Barcelona, Hospital de Bellvitge, Spain. Research project: Tran-scriptional regulation of glycolytic/gluconeogenic enzymes. 1st-26th of March 2004 - IFMSA-SCORE Scholarship at the Department of Cell and Molecular Biology, Section for Medical Inflammation Research,

23

University of Lund, Sweden. Research project: Effect of increased oxidative burst on T cell populations in the acute phase of pristane induced arthritis in rat.

Articles published in ISI indexed journals in PhD research area

1. Florin Burada, Theo Plantinga, Mihai Ioana, Diana Rosentul, Cristina Angelescu, Leo Joosten, Mihai Netea, Adrian Saftoiu, IRGM gene poly-morphisms and risk of gastric cancer. Journal of Digestive Diseases, 2011 (accepted) . 2. Florin Burada, Cristina Angelescu, Mihai Ioana , Anca-Lelia Riza, Paul Mitrut , Robert Dinu, Emil Moraru, Francisc Mixich, Mihai Cruce,Adrian Saftoiu. Association of Interleukin 1β Gene and Interleukin 1 Receptor An-tagonist Gene Polymorphisms and Gastric Cancer Risk, Advances in Clini-cal and Experimental Medicine Vol 20, 4, 423–429, 2011.3. Mihai Ioana, Cristina Angelescu, Florin Burada, Francisc Mixich, Anca Riza, Theodor Dumitrescu, Dragos Alexandru, Tudorel Ciurea, Mihai Cruce, Adrian Saftoiu (2010). MMR Gene Expression Pattern in Sporadic Colorectal Cancer. Journal of Gastrointestinal and Liver Diseases. Vol.19 No 2, 155-159.

Abstracts published in ISI/International indexed journals in PhD research area

1. F. Burada, C. Angelescu, M. Ioana, F. Mixich, P. Mitrut, S. Popa, E. Buteica, M. Cruce. The IL1-RN +2018T>C polymorphism is associated with increased susceptibility to gastric adenocarcinoma. Abstract of Euro-pean Human Genetics Conference Amsterdam 2011, European Journal of Human Genetics, Volume 19, Supplement 2. Page: 209. P.10.56.,209. 2011. 2. Florin Burada, Cristina Angelescu, Mihai Ioana, Elena Cioboată, Fran-cisc Mixich, Mihai Cruce, Adrian Saftoiu. Cytokine gene polymorphisms and risk of gastric adenocarcinoma. Balkan Journal of Medical Genetics, vol 14, supp, 9th BMHG, 28, 2011. 3. Cristina Angelescu, Florin Burada, Mihai Ioana, Radu Angelescu, Fran-cisc Mixich, Mihai Cruce, Adrian Săftoiu. Expression profile of VEGFA and VEGFB genes in human gastric cancer, Journal of Gastrointestinal and

24

Liver Disease, Abstracts Genetics in Gastrointestinal and Liver Diseases, Cluj Napoca 7-9 April, 2011, Volume 20, Supplement , 26-27, 2011.4. Florin Burada, Cristina Angelescu, Mihai Ioana, Paul Mitrut, Robert Dinu, Emil Moraru, Mihai Cruce, Adrian Saftoiu, Francisc Mixich Genetic. Polymorphism of Interleukin 1β (IL-1B) Gene and Interleukin 1 Receptor Antagonist (IL-1RN) Gene in Gastric Cancer Patients in a Romanian Popu-lation, Journal of Gastrointestinal and Liver Disease, Abstracts Genetics in Gastrointestinal and Liver Diseases, Cluj Napoca 7-9 April, 2011, Volume 20, Supplement , 25-26, 2011.5. F. Burada, C. Soare, M. Ioana, P. Mitrut, R. Angelescu, A. Riza, A. Dobrescu, E. Buteica, M. Cruce, F. Mixich (2010). Interleukin 1B gene (IL-1B) and Interleukin 1 receptor antagonist gene (IL-1RN) polymorphisms and the risk of gastric cancer. European Journal of Human Genetics. Volume 18, Supplement 1. Page: 176

Articles published in CNCSIS (National University Research Council) indexed journals in PhD research fiels

1. Cristina Angelescu, F. Burada, M.Ioana, R.Angelescu, Anca Riza, F.Mixich, Doina Voinescu, M.Cruce, A. Saftoiu. Expression profile of VEGF and EGFR mRNA in esophageal and gastric cancers. Annals of the Romanian Society for Cell Biology. 16, 1, 228-233, 2011.2. F. Burada, Cristina Angelescu, Elena Cioboată, M.Ioana, M.Cruce. The IL-23R R381Q gene polymorphism is not associated with risk of gastric cancer. Annals of the Romanian Society for Cell Biology. 16, 1, 267-271, 2011. 3. F. Burada, Cristina Angelescu, M. Ioana, P. Mitrut, E. Moraru, Anca Riza, Alina Preda, M. Cruce. Ll-10 -1082 A/G polymorphism and risk of the gastric cancer. Annals of the Romanian Society for Cell Biology.. Vol. XV, Issue 1. Page: 93-97.4. E. Moraru, Cristina Angelescu, M. Ioana, N.M. Panduru, F. Burada, Alina Preda, Sanda Marchian, M. Cruce (2010). The correlation between three polymorphisms of TP53 gene and gastric cancer. Annals of the Roma-nian Society for Cell Biology. Issue 1. Page: 276-28193-97

25

5. E. Moraru, Cristina Angelescu, M. Ioana, T. Dumitrescu, F. Burada, Alina Preda, Amelia Barca, M. Cruce. (2009). c-ABL gene expression in gastric cancer. Annals of the Romanian Society for Cell Biology. Vol. XIV, Issue 2, pag. 204-211 6. Alina Preda, F. Burada, Cristina Soare, Amelia Birca , E. Moraru, M. Cruce. (2009). Helicobacter pylori infection and the development of gastric cancer. Annals of the Romanian Society for Cell Biology. Vol. XIV, Issue 2, pag 212-214 7. Fl. Burada, Mihai Ioana, Cristina Soare, Eugenia Roşculescu, Elena Buteică. (2009). Cytokines genes polymorphisms and gastric cancer risk. Modern Medicine. vol. XVI, issue 3, pag. 160-162

Book chapters

Obesity Induced Insulin Resistance and Chronic Viral Hepatitis C. Maria Moţa, Simona Popa. Autor - Chapter 5: Molecular Methods in Hepatitis C Virus Diagnosis and Infection Monitoring., Mihai Ioana, Cristina Soare. Editura Medicală Universitară Craiova, 2009, 197 pages. ISBN: 978-973-106-108-5.

Member of Scientific Societies and Committees:

Member of the European Society Human Genetics (ESHG)Member of the Romanian Society Human Genetics (SRGM)Member of the Romanian Society of Cellular Biology (SRBC)

Participation in programmes and projects as a member of the research team:

1.Seventh Framework Programme - SPIDIA-RNA Program (Standardiza-tion and Improvement of Generic Pre-analytical Tools and Procedures for In-Vitro Diagnostic). Responsible investigator from the Laboratory of Mo-lecular Genetics, UMF Craiova2.Seventh Framework Programme - SPIDIA-DNA Program (Standardiza-tion and Improvement of Generic Pre-analytical Tools and Procedures for

26

In-Vitro Diagnostic). Responsible investigator from the Laboratory of Mo-lecular Genetics, UMF Craiova3.The Genographic Project: Molecular Genetic Analyses of Western/Central/Eastern European populations. Project financed by National Geo-graphic Society and IBM. Project coordinator: Dr. Spencer Wells, PhD. Co-ordinator Balkan populations: Mihai Netea, MD, PhD 4.National Plan of Research, Developement and Inovation - PN II. Program 4: “Partnerships in priority research areas” - type PC. The role of endo-scopic ultrasound and endobronchial ultrasound in comparison with the mo-lecular techniques in the diagnosis and staging of the mediastinal malignant diseases. Project coordinator: Professor Dr. T. Ciurea, PhD, MD 5.National Plan of Research, Developement and Inovation - PN II. Program 4: “Partnerships in priority research areas” - type PC. Comparison between video capsule endoscopy and push-and-pull enteroscopy in patients with small bowel pathology. Project coordinator: Associate professor Dr. Cristin Constantin Vere, PhD, MD

27

28