Background

HCV infection is a worldwide cause of chronic liver diseases [1]. The long-term progression of HCV infection is highly variable. The hepatic pathology progress from minimal histological alterations up to extensive hepatocellular necrosis, fibrosis, and cirrhosis that could be complicated by hepatocellular carcinoma (HCC) [2]. In Egypt, the results of national survey conducted by the Egyptian ministry of health showed that HCV prevalence in those over 18 years was about 4% [3].

Cirrhosis is the common end-result for chronic liver injury regardless of the etiology. Histologically, it is characterized by diffuse hepatocellular necrosis and diffuse nodular regeneration surrounded by dense fibrotic septa causing pronounced distortion of hepatic vascular architecture causing increased resistance to portal blood flow and hence in portal hypertension [4].

Esophageal and gastric varices are present in about 50% of patients with cirrhosis, and hepatic decompensation is linked to higher risk of variceal formation and bleeding [5, 6]. Variceal hemorrhage occurs at a rate of around 10‑15% per year and depends upon the severity of liver disease, degree of portal hypertension, size and location of varices, and bleeding risk signs, e.g., red wale marks, cherry spot, and varices on varices. Six-week mortality is still high (15‑25%) even with advanced medical and endoscopic treatment [7, 8].

Portal hypertension in cirrhosis occurs due to both the increase in intrahepatic vascular resistance (static element) and the increase in blood flow in the splanchnic circulation (kinetic element) [9].

Endothelial dysfunction, occurring in cirrhosis, leads to impaired vasomotor control, inflammation, fibrosis, and impaired liver regeneration [10, 11]. All of which enhance progression of liver cirrhosis and portal hypertension. It also leads to increased vasodilators especially nitric oxide (NO) which is the most potent vasodilator molecule known [12, 13].

Members of the VEGF family are major regulators of blood vessels growth and function [14]. VEGF also regulates endothelial cell survival and apoptosis [15,16,17]. VEGF is the major factor to endothelial proliferation and neoangiogenesis [18]. Activation of VEGF receptor leads to eNOS activation and release [19]. VEGF is also a potent inducer of vascular permeability and inflammation [20].

Aim of the work

The aim of the work was the assessment of effect of VEGF C(+405)G (rs2010963) single nucleotide polymorphism (SNP) on development and grade of esophageal and gastric varices and risk of bleeding in cirrhotic patients with HCV.

Subjects

The study was conducted on 90 subjects, 30 cirrhotic patients with varices on endoscopy (group A), 30 cirrhotic patients without varices on endoscopy (group B), and 30 healthy controls (group C). All subjects with renal impairment, hepatocellular carcinoma (HCC), or other malignancy, on beta-blockers, non-steroidal anti-inflammatory drugs or anticoagulant, patients with platelet count < 50000 cell/cmm or international normalized ratio > 1.5, and patients with any other source of gastric bleeding (ulcer, portal hypertensive gastropathy) were excluded.

Methods

An informed consent was obtained from all participants before enrollment. Based on 800 cirrhotic patients with HCV being admitted to Tropical Medicine Department, Alexandria Main University Hospital annually and there was only 2 previous studies performed on VEGF C(+405)G (rs2010963) in Egyptian patients with the prevalence of GG genotype was about 6.7% and 5.5% with precision of 5 and α of 5% [21,22,23]. The minimum number needed for our study was calculated to be 86 patients [24]. All subjects were submitted to the following, detailed history taking and thorough clinical examination including abdominal examination, laboratory investigations including complete blood picture (CBC), erythrocyte sedimentation rate (ESR), C reactive protein (CRP), liver functions tests ( serum bilirubin (total, direct), albumin, international normalized ratio (INR), liver enzymes (aspartate transferase (AST), alanine transferase (ALT), alkaline phosphatase (ALP)) renal functions tests (blood urea and creatinine), alfa-fetoprotein, HCV antibodies, or polymerase chain reaction (PCR), hepatitis B surface antigen (HBs Ag), AST to platelet ratio index (APRI) score, ultrasound, gastroduodenoscopy for variceal detection, and grading and genotyping for (VEGF) C(+405)G (rs2010963) using allelic discrimination 5′ nuclease assay. Data were fed to the computer and analyzed using the IBM SPSS software package version 20.0. Comparisons between groups for categorical variables were assessed using Chi-square test (Fisher or Monte Carlo). F test (ANOVA) for normally distributed quantitative variables, to compare between more than two groups, and post hoc test (LSD) for pairwise comparisons. Kruskal-Wallis test for abnormally distributed quantitative variables, to compare between more than two studied groups, and post hoc (Dunn’s multiple comparisons test) for pairwise comparisons. Significance of the obtained results was judged at the 5% level.

Results

The current study involved 90 participants divided into 3 groups: group A, 30 patients with HCV-related liver cirrhosis and esophageal varices; group B, 30 patients with HCV-related liver cirrhosis with no esophageal varices group C, 30 apparently healthy controls.

There was no statistically significant difference between the studied groups as regard age (p = 0.749) and sex (p = 0.725) (Table 1). The results of complete blood count, creatinine, ALT, AST, ALP, bilirubin, INR, albumin, splenic bipolar diameter, and portal vein diameter by ultrasound abdomen and APRI; all are summarized in Table 1.

Table 1 Comparison between the studied groups according to demographic data, complete blood count, creatinine, ALT, AST, ALP, bilirubin, INR, albumin, splenic bipolar diameter, and portal vein diameter by ultrasound abdomen and APRI
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The mean of Child-Pugh score was statistically significantly higher in cirrhotic patients with varices (mean 9.57) than cirrhotic patients with no varices (mean 7.17) (P < 0.001) (Table 2).

Table 2 Comparison between groups A and B according to Child-Pugh classification and score
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Endoscopically, using Paquet’s classification for grading of esophageal varices, the most frequent grade was IV (15 patients (50%)), followed by grade 1 with 6 patients (20%), grade 2 in 5 patients (16.7%), and grade 3 in 4 patients (13.3%) [7]. Gastric varices were present in 11 patients (36.7%). Twenty-two patients (73.3%) had endoscopic risk signs of variceal bleeding (white nipple, red wales, clots overlying a varix, cherry red spot, or varix on varix) [25]. Endoscopic therapy (band ligation) had been accomplished in 20 patients (66.7%).

The genotype distribution of VEGF C(+405)G (rs2010963) SNP in all groups was in accordance with Hardy-Weinberg equilibrium [26] (Table 3).

Table 3 Comparison between the three studied groups according to (VEGF) C(+405)G (rs2010963) single nucleotide polymorphism and whether observed genotype frequencies are consistent with Hardy-Weinberg equilibrium [23]
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The GG genotype was statistically significantly more frequent in cirrhotic patients with esophageal varices than cirrhotic patients without esophageal varices and controls (P < 0.001). There was no statistically significant difference between the studied groups as regard the CG genotype (P = 0.866). The G allele was statistically significantly more frequent in group A than group B and group C (P < 0.001). There was no statistically significant difference between group B and controls as regard both C and G alleles (P = 0.838) (Table 3) (Fig. 1).

Fig. 1
figure 1

Comparison between the three studied groups according to VEGF C(+405)G (RS2010963) SNP in the studied groups (n = 90)

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Cirrhotic patients with GG genotype displayed 14.857-fold increased risk for developing esophageal varices than CC genotype and patients with CG genotype displayed 1.905-fold increased risk for developing esophageal varices than patients with CC genotype, while cirrhotic patients carrying G allele had 4.125-fold increased risk for esophageal varices than patient carrying C allele (Table 4).

Table 4 VEGF C(+405)G (rs2010963) SNP genotype and risk for esophageal varices
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Cirrhotic patients with varices (group A) who had the GG genotype had statistically significantly higher grade of esophageal varices, more frequent gastric varices, more evident risk signs of bleeding, and more frequent variceal bleeding when compared to those who had the CC and CG genotype (Table 5).

Table 5 Relation between (VEGF) C(+405)G (rs2010963) SNP with grade of esophageal varices, risk signs of variceal bleeding, gastric varices, and variceal bleeding in group A
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Cirrhotic patients (group A, B) who had the GG genotype had statistically significantly higher APRI, higher Child-Pugh score, and larger portal vein diameter when compared to those who had the CC and CG genotype (Table 6).

Table 6 Relation between (VEGF) C(+405)G (rs2010963) single nucleotide polymorphism gene with APRI, Child score, and portal vein diameter in group A + B (n = 60)
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Discussion

VEGF is produced by the endothelial cells, the macrophages, the T cells, the platelets, and many other cell types. Many researchers outlined the key role of VEGF in many vascular diseases as important pro-angiogenic factor regulating the normal and pathological angiogenic processes [27,28,29,30,31]. Many studies had demonstrated that VEGF had a pivotal role in pathogenesis and progression of portal hypertension in cirrhosis through potentiation of inflammation and enhancement of portal-systemic collateral vessel formation [32,33,34,35,36,37,38]. Watson et al. studied promotor and 5 untranslated region of VEGF gene and found that the GG genotype of VEGF C(+405)G (rs2010963) SNP was associated with higher serum VEGF production level than the CG and CC genotypes [39].

Our results are in ordinance with the previous study performed by Yang et al. who demonstrated the GG genotype of VEGF C(+405)G (rs2010963) SNP was associated with higher incidence of esophageal varices though the patients included were Taiwanese, and different etiologic factors of cirrhosis were included with HBV being the most prominent factor; the odd ratio for the GG genotype was lower than the current study (3.1 for the GG genotype) and no increased risk of variceal bleeding was demonstrated [40].

The more frequent and larger esophageal varices, the more frequent gastric varices and the more frequent variceal bleeding noticed with the GG genotype in the current study could be attained to the fact that this genotype is associated with higher VEGF production [39]. The higher VEGF production enhances angiogenesis [27] leading to more portal collateral circulation [38, 41, 42] and that VEGF is also associated with unregulated hepatic inflammation leading to more cirrhosis progression and higher portal pressure [36,37,38].

Lower serum level of VEGF in cirrhotic patients was shown by Assay et al. who attained it to endothelial dysfunction caused by cirrhosis [36]. Huang et al. hypothesized that the higher levels of serum VEGF in early stages of cirrhosis were due to homeostatic compensatory mechanisms [41]. Desideri et al., Akiyoshi et al., and Robinson et al. noticed that serum VEGF levels decreases as cirrhosis progresses in accordance with the Child–Pugh classification and is influenced by complex factors including etiology of liver disease, platelets dysfunction, and different VEGF isoforms [43,44,45]. All those studied could explain the lower urinary levels of VEGF in cirrhotic patients with esophageal varices than those without demonstrated by Mohamed et al. Low VEGF level could be attained to higher degree endothelial dysfunction and lower platelets count in cirrhotic patients with varices than those without [46]. Those studies also points out to the value of VEGF gene polymorphism over serum level to predict variceal occurrence in cirrhotic patients.

Applying those finding, we could conclude that the GG allele is associated with a better VEGF response to incentive stimuli causing higher VEGF production and effect of VEGF causing enhanced growth of compensatory collateral circulation which facilitates esophageal and gastric varices development and progression and hence higher risk of variceal bleeding. VEGF C(+405)G (rs2010963) SNP is a better marker to predict esophageal varices occurrence over the VEGF serum level which is affected by many factors including the stage and etiology of cirrhosis. The current study highlights the importance of individualized medical care based on genomic sequencing which would hopefully be more widespread and less expensive in the near future.

Limitations

This is a single center experience; the patients were only Caucasians and single SNP was targeted.

Conclusion

VEGF C(+405)G (rs2010963) SNP plays a major role in development of esophageal varices, gastric varices, and risk of variceal bleeding. It also plays a key role in portal hypertension pathogensis and progression.