Association between VEGF-634G>C Gene Polymorphism with

Degree of Neutrophil and Lymphocyte Infiltration in Helicobacter

pylori Gastritis Patients

Zaimah Z. Tala

, Gontar Alamsyah Siregar

and Ginanda Putra Siregar

Department of Nutrition, Faculty of Medicine, Universitas Sumatera Utara, Medan, Indonesia

Division of Gastroentero-Hepatology, Department of Internal Medicine, Universitas Sumatera Utara, Haji Adam Malik

General Hospital, Medan, Indonesia

Division of Urology, Department of Surgery, Faculty of Medicine, Universitas Sumatera Utara, Dr Mansyur 5, Medan,

Indonesia

Keywords: Helicobacter pylori, VEGF, Polymorphism, Neutrophil, Lymphocyte.

Abstract: Previous study showed that VEGF-634G>C polymorphism was associated with VEGF expression. VEGF

can enhance inflammatory processes, resulting in more severe inflammation. This study held to analyze

association between VEGF -634G>C polymorphism and the degree of gastric neutrophil and lymphocyte

infiltration in H. pylori gastritis patients. The aim of this study is to investigate association between VEGF -

634G>C polymorphism and the degree of gastric neutrophil and lymphocyte infiltration in H. pylori

gastritis patients.This cross-sectional study included patients with H. pylori gastritis at Haji Adam Malik

General Hospital, Permata Bunda General Hospital, and Universitas Sumatera Utara Hospital, Medan,

Indonesia. Detection of H. pylori infection was made using positive results of 14C-UBT, and/ or rapid

urease test. gaster. The degree of neutrophil and lymphocyte infiltrations were evaluated from biopsies of

the mucosa gaster, referring to visual analogue scale of the updated Sydney System. Real time polymerase

chain reaction (RT-PCR) was used to examine VEGF -634G>C gene polymorphism. Data were analyzed

using SPSS version 22. There was significant association between VEGF-634 G>C polymorphism and

degree of neutrophil infiltration. Patients with the G allele were at risk of 2.07 times for moderate + severe

degree of neutrophil infiltration compared to C allele (p = 0.008). There was no significant association

between VEGF -634 G>C polymorphism and degree of lymphocyte infiltration (p>0.05). G allele of VEGF-

634 G>C polymorphism was associated with moderate + severe neutrophil infiltration.

1 INTRODUCTION

Helicobacter pylori (H. pylori) is the main etiology

of chronic gastritis that plays a role in activation of

host angiogenesis. In all angiogenic factors, vascular

endothelial growth factor (VEGF) is the most potent

neoangiogenesis stimulus (Siregar, 2017). H. pylori

upregulates VEGF expression in gastric epithelial

cells through several mechanisms such as NF-ĸβ,

cyclooxygenase-2 (COX-2), and epidermal growth

factor receptor (EGFR) signaling (Kang, 2014).

VEGF levels can be influenced by inflammation,

hypoxia, oncogenes, tumor suppressor gene, and

genetic factors, one of which variations of VEGF

gene (Lee, 2015; Logsdon, 2014). VEGF gene

located in 6p21.3 chromosome which is consist of 8

exons separated by 7 introns as known very

polymorphic (has 140 variants) (Eng, 2012; Eng,

2013). Several single nucleotide polymorphisms

(SNPs) on the VEGF gene are thought to affect their

expressions. A previous study by Oh et al found that

the GG genotype of VEGF-634G>C polymorphism

was associated with higher VEGF serum levels (Oh,

2013). VEGF can enhance inflammatory processes,

resulting in more severe inflammation, so that VEGF

is not the only mediator of angiogenesis but also acts

as inflammatory mediator (Shaik-Dasthagirisaheb,

2013). This study held to analyze association

between VEGF -634G>C polymorphism and the

degree of gastric neutrophil and lymphocyte

infiltration in H. pylori gastritis patients.

Tala, Z., Siregar, G. and Siregar, G.

Association between VEGF-634G>C Gene Polymorphism with Degree of Neutrophil and Lymphocyte Inﬁltration in Helicobacter pylori Gastritis Patients.

DOI: 10.5220/0010069904150419

In Proceedings of the International Conference of Science, Technology, Engineering, Environmental and Ramiﬁcation Researches (ICOSTEERR 2018) - Research in Industry 4.0, pages

415-419

ISBN: 978-989-758-449-7

415

2 METHODS

2.1 Patients Selection

This cross-sectional study was perfomed on 80

consecutive H. pylori gastritis patients who were

admitted to the Endoscopy Unit at Haji Adam Malik

General Hospital, Permata Bunda General Hospital,

and Universitas Sumatera Utara Hospital, Medan,

Indonesia between April and June 2018. Inclusion

criteria include gastritis patients diagnosed based on

histopathological examination, positive results of

C-UBT and/ or rapid urease test, at least 18 years

old, and willing to take part in the study. Exclusion

criteria are as follows: history of H. pylori

eradication treatment in the last 6 months or

currently on antibiotics therapy commonly used in

H. pylori eradication; history of proton pump

inhibitor, H2 receptor antagonist, NSAID or steroid

1 month prior to the study; patients with systemic

disease, and malignancy. This study was approved

by the local ethics committee of Universitas

Sumatera Utara.

A routine endoscopy was conducted in order to

evaluate gastric mucosa for the presence of edema,

erythema (spotted, patchy, linear), exudate, bleeding,

and erosion; as well as to take a tissue sample for the

rapid urease test and histopathology. Additionally,

tissue biopsy was performed within the greater and

lesser curvature of the distal antrum, the lesser

curvature at incisura angularis, the anterior and

posterior wall of the proximal corpus. An additional

biopsy was also carried out in suspicious regions that

were not included in the preliminary biopsy.

2.2 Helicobacter pylori Detection

A positive result of

C-UBT and / or rapid urease

test will confirm the diagnosis of H. pylori infection.

Participants were asked to fast for at least 6 h, prior

C-UBT examination, usually overnight. Patients

swallowed 37 kBq (1 μCi) of encapsulated

urea/citric acid composition in 25 ml water.

Heliprobe Breath Cards (Noster system) 10 min after

administration of

C urea were used. The next step

is to exhale into the breath cards until its color

indicator changed from orange to yellow. The breath

samples were measured using the Heliprobe analyzer

(Noster system), and the activity was counted for

250s. Results were articulated as counts per minute

(cpm) and counts < 25 cpm were defined as

Heliprobe 0 = not infected, counts between 25 cpm

and 50 cpm as Heliprobe 1 = equivocal and counts >

25 cpm as Heliprobe 2 = infected (Ghanaei, 2011).

The rapid urease test (Pronto Dry®, France) was

also another primary tool for diagnosis of H. pylori

infection. When the color from amber to pink-red at

room temperature within 24 hours, it would confirm

a positive result. A negative result would divulge a

yellow colour on the indicator (Rojborwonwitaya,

2005).

2.3 Degree of Neutrophil and

Lymphocyte Infiltration

The degree of neutrophil and lymphocyte

infiltrations were evaluated from biopsies of the

mucosa gaster. Biopsy specimens were fixed in 10%

formalin and embedded in paraffin. The samples

were stained using Hematoxylin-Eosin and were

evaluated by the Pathologist of anatomic pathology

of the medical faculty of the Universitas Sumatra

Utara referring to visual analogue scale of the

updated Sydney System. The degree of neutrophil

and lymphocyte infiltration were scored 0 to 3, i.e.,

normal (0), mild (1), moderate (2), and severe (3)

(Rugge, 2011).

2.4 VEGF -634 G>C Polymorphism

Genomic DNA was extracted and purified from

peripheral blood smear using High Pure PCR

Template Preparation Kit (Roche Applied Science),

and stored until processed for genotyping. Analysis

of the VEGF SNP -634G>C was performed using

real time polymerase chain reaction (RT-PCR). The

PCR primers used for the –634G>C polymorphisms

were 5’-CGACGGCTTGGGGAGATTGC-3’

(forward) and 5'-GGGCGGTGTCTGTCTGTCTG-

3’ (reverse). The PCR cycle conditions consisted of

an initial denaturation step at 94 °C for 5 min,

followed by 35 cycles of 30 s at 94 °C, 30 s at 62 °C,

30 s at 72 °C, and a final elongation at 72 °C for 10

min.

2.5 Statistical Methods

Data analysis was performed through univariate,

bivariate (Chi-Square test) analyses using SPSS 22

version (SPSS Inc., Chicago). A value of p < 0.05

with 95% confidence interval was considered

statistically significant.

ICOSTEERR 2018 - International Conference of Science, Technology, Engineering, Environmental and Ramiﬁcation Researches

416

3 RESULTS

3.1 Baseline and Clinical

Characteristics of Subjects

The characterized by male (61.3%) and mean age

50.8, and most of the subject were Bataknese

(53.8%). There were 43 polymorphism VEGF-

634G>C GC genotype patient (53.8%), followed 22

patient (27.5%) GG genotype and 15 patient (18.8)

CC genotype (Table 1).

Table 1. Baseline and clinical characteristics of subjects

Characteristics n=80

Gender

Male

Female

49 (61.3%)

31 (38.8%)

e, mean + SD

ears

)

50.8 + 12.2

Ethnicity

Batak

Javanese

Aceh

Malay

43 (53.8%)

17 (21.3%)

11 (13.8%)

9 (11.3%)

Occupation

Entrepreneur

Housewife

Employee

Civil servants

University students

14 (17.5%)

31 (38.8%)

29 (36.3%)

4 (5%)

2 (2.5%)

Education

Elementary school

Middle school

High school

University

6 (7.5%)

10 (12.5%)

53 (66.3%)

11 (13.8%)

VEGF-634 G>C

polymorphism

GG genotype

GC genotype

enot

22 (27.5%)

43 (53.8%)

(

18.8%

)

3.2 Degree of Neutrophil and

Lymphocyte Infiltration

Lymphocyte infiltration degree showed normal +

mild degrees (55%) and moderate + severe degrees

(45%). Neutrophil infiltration degree showed normal

+ mild degrees (71.2%) and moderate-severe

degrees (28.8%) (Table 2).

Table 2. Degree of neutrophil and lymphocyte

Degree of

Neutrophil

Degree of

lymphocyte

Total

Moderat

e +

Severe

Normal

+ Mild

Moderate

+ Severe

Normal

+ Mild

(28.8%)

(71.2%)

36 (45%) 44

(55%)

(100%)

3.3 Association between VEGF -634

G>C Polymorphism and Degree of

Neutrophil and Lymphocyte

Infiltration

There was a significant association between VEGF-

634 G>C polymorphism and degree of neutrophil.

Patients with the G allele were at risk of 2.07 times

for moderate + severe degree of neutrophil

infiltration compared to C allele (p = 0.008) (Table

3). There was no significant association between

VEGF -634 G>C polymorphism and degree of

lymphocyte infiltration (p>0.05) (Table 4).

Table 3. Association between VEGF -634 G>C polymorphism and degree of neutrophil

VEGF-634G>C

Polymorphism

Degree of neutrophil Total

p PR (95% CI)

Moderate + Severe

Normal +

Mil

Genotype

10 (45.5%)

12 (27.9%)

(

6.7%

)

12 (54.5%)

31 (72.1%)

(

93.3%

)

22 (100%)

43 (100%)

(

100%

)

0.051

0.150

6.82 (0.97-47.83)

4.19 (0.59-29.5)

(

ref.

)

GG+GC

Allele

22 (33.8%)

1 (6.7%)

32 (36.8%)

14 (19.2%)

43 (66.2%)

14 (93.3%)

55 (63.2%)

59 (80.8%)

65 (100%)

15 (100%)

87 (100%)

73 (100%)

0.058

0.008*

5.08 (0.74-34.76)

1 (ref.)

2.07 (1.17-3.63)

1 (ref.)

*p<0.05

Association between VEGF-634G>C Gene Polymorphism with Degree of Neutrophil and Lymphocyte Inﬁltration in Helicobacter pylori

Gastritis Patients

417

Table 4. Association between VEGF -634 G>C polymorphism and degree of lymphocyte

VEGF-634G>C

Polymorphism

ree of l

hoc

te Total

p PR (95% CI)

Moderate + Severe

Normal +

Mil

Genotype

11 (50%)

21 (48.8%)

(

26.7%

)

11 (50%)

22 (51.2%)

(

73.3%

)

22 (100%)

43 (100%)

(

100%

)

0.156

0.135

1.88 (0.73-4.79)

1.83 (0.75-4.47)

(

ref.

)

Genotype

GG+GC

32 (49.2%)

4 (26.7%)

33 (50.8%)

11 (73.3%)

65 (100%)

15 (100%)

0.113

1.85 (0.77-4.43)

1 (ref.)

Allele

43 (49.4%)

29 (39.7%)

44 (50.6%)

44 (60.3%)

87 (100%)

73 (100%)

0.219

1.24 (0.87-1.77)

1 (ref.)

4 DISCUSSION

Inflammatory process are characterized by

neutrophil and lymphocyte activity. Neutrophil

activity in gastritis indicates an active course of the

disease, whereas lymphocyte activity indicates a

chronic course of disease. When mucosal damage

occurs due to both infection and other stimulating

factors, IL-8 inflammatory mediator expenditure

acts as a chemotactic agent for neutrophils. IL-8

will activate neutrophils to release the lysosomal

enzyme and induce neutrophil adherence in

endothelial cells. This adherence is followed by

neutrophil migration from the capillaries to the

lamina propria and arise between the epithelial

cells. These neutrophils can be found in gastric

mucosa during acute phase gastritis and H. pylori-

associated gastritis. This theory is in accordance

with the results of the research presented by

Atayan et al that there is a significant correlation

between H. pylori infection with chronic gastritis

severity with neutrophil and lymphocyte

infiltration (r= 0.309, 0.226, respectively) (Atayan,

2017). The higher density of neutrophils, the

higher density of bacterial infections (Dhakwa,

2012; Tuccillo, 2005). H. pylori infection in the

gastric mucosa induces the production of IL-1β,

IL-6, IL-8, and TNF-α cytokines. IL-1 or TNF-α

alone, as well as TNF-α synergize with IFN-γ

induce IL-8 production in gastric cells (Caputo,

2003; Tuccillo, 2005).

Induction of pathological angiogenesis is that

inflammation precedes and is accompanied by the

formation of neovessels as evidenced by increased

vascular permeability and the recruitment of

inflammatory cells. However, VEGF itself will not

only promote angiogenesis, but also has the

potential to induce inflammatory response. VEGF

can influence the inflammatory process in several

ways. VEGF increases vascularization at the site of

inflammation causing the reaction to be more

severe. Furthermore, VEGF can promote the

recruitment of inflammatory cells (Sinnathamby,

2015; Angelo, 2007).

Certain SNP on the VEGF gene is thought to

affect its expression. Allele variation may lead to

overexpression of the transcription factor that will

bind to the promoter site, which serves as the

initial RNA polymerase binding site that will

initiate transcription (Corvalan, 2012). Results of

this study showed that there was a significant

association between VEGF-634 G>C

polymorphism and degree of neutrophil. Patients

with the G allele were at risk of 2.07 times for

moderate + severe degree of neutrophil infiltration

compared to C allele. Patients with GG genotype

were more likely to have moderate+severe degree

of lymphocyte infiltration than patients with CC

genotypes, however not statistically significant

(p>0.05). The association between VEGF-634

G>C polymorphism with degree of neutrophil

infiltration may be due to elevated levels of VEGF

as a result of the G allele that plays a role in

increasing neutrophil cell recruitment in gastric

mucosa. Oh et al showed that patients with GG

genotype of VEGF-634G>C polymorphism had

significantly higher VEGF serum level than

patients with CC genotype (Oh, 2013).

It was concluded that the G allele of VEGF-634

G>C polymorphism was associated with moderate

+ severe neutrophil infiltration. The limitations of

our study include the small sampel size which

might make our study underpowered. Further

studies are necessary to examine other SNPs that

may affect the degree of neutrophil and

lymphocyte infiltration.

ICOSTEERR 2018 - International Conference of Science, Technology, Engineering, Environmental and Ramiﬁcation Researches

418

5 CONCLUSION

G allele of VEGF-634 G>C polymorphism was

associated with moderate + severe neutrophil

infiltration

ACKNOWLEDGEMENT

The authors would like to thank for the funding

support by Research Institute, Universitas

Sumatera Utara (Contract number :

59/UN5.2.3.1/PPM-KP-TALENTAUSU/2018)

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Association between VEGF-634G>C Gene Polymorphism with Degree of Neutrophil and Lymphocyte Inﬁltration in Helicobacter pylori

Gastritis Patients

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