EXPERIMENTAL AND CLINICAL TRANSPLANTATION

Liver transplant (LT) is the most important therapy for treatment of patients with end-stage liver disease for whom resection has failed because of tumor recurrence or liver insufficiency. Rejection is one of the major difficulties that the patient often faces after LT, despite the use of immunosuppressive agents and advanced methods.¹

Occurrences of acute rejection (AR) after LT are well known. Hessheimer and colleagues² summarized pathological changes of AR after LT in 3 aspects: (1) infiltration of inflammatory cells, mainly mononuclear (including activated and transformed lymphocytes) and various amounts of neutrophils and eosinophils, in the portal area; (2) infiltration of subcutaneous inflammatory cells in the portal vein, terminal hepatic vein, and central vein; and (3) cholangitis and degenerative necrosis of biliary epithelial cells. In an overview, vascular endothelial cells of the portal vein and cholangial epithelial cells were shown to be the main target cells of AR.³

Tambur and colleagues⁴ and Aharinejad and colleagues⁵ found that vascular endothelial growth factor (VEGF) was expressed in human heart allografts and that its expression was correlated with AR and chronic rejection. Berberat and colleagues⁶ found that VEGF had a role in local transport of white blood cells in autografts. Apart from participation in various pathophysiological processes, including embryonic development, repair of traumatized tissue, ischemia, inflammation, and tumor occurrence by promoting angiogenesis, VEGF is a kind of highly specific vascular endothelial cell promoting mitogen.⁷ The VEGF gene is located on chromosome 6p21.3 and consists of 8 exons and 7 introns with alternate splicings in order to form a family of proteins.⁸ Purified VEGF is a protein of approximately 46 kDa.⁹

There are some considerable variations between individuals regarding their VEGF expression. An analysis of the 5′-flanking region of the gene showed the presence of many polymorphisms.^10-12 G/C transition at +405 in the 5′-untranslated region has been implicated in a number of diseases, especially those with angiogenic basis,13,14 while in healthy populations, production of the VEGF protein is known to be associated with +405 G/C polymorphism.¹² An association has been reported in case-control studies between VEGF polymorphisms and diseases such as diabetic retinopathy,¹³ ovarian cancer, and endometriosis.^14,15

Despite the importance of VEGF in liver disorders and transplant, there have been no published studies on the association between VEGF gene polymorphism and LT among Iranian patients. Liver rejection remains a concern, and the possible effects of +405 polymorphism in the promoter region of VEGF on rejection in LT recipients have not yet been explored, to the best of our knowledge.

The aim of this case-control study was to investigate the association between the +405 G/C gene polymorphism and the occurrence of AR in Iranian LT recipients.

Materials and Methods

Study population
This retrospective study, which included data from 2013 to 2015, analyzed 124 LT recipients who were seen at Namazi Hospital in Shiraz, Iran. The patients were divided into 2 groups. The non-AR group consisted of 102 patients without any history of AR during the course of this study. The AR group consisted of 22 patients who experienced AR (rejection group). The non-AR transplant patients were considered as the control group or nonrejection group. The patients were enrolled in the study according to the following inclusion criteria: the recipients had received a LT from a deceased donor, and the recipients were treated with specific immunosuppressive drug regimens. The present study protocol was approved by the Ethics Committee of Shiraz University of Medical Sciences, and written informed consents were obtained from patients or their relatives in accordance with the Helsinki Declaration. The demographic data are shown in Table 1.

Crossmatch and ABO grouping were done for all LT recipients and donors. The condition of AR of each patient was confirmed by an expert pathologist according to the level of liver serum enzymes, bilirubin level, histological features of liver biopsies, and clinical and biochemical responses to high-dose steroids.

Blood collection and DNA extraction
Blood samples (5 mL, treated with EDTA) were collected from each patient. Serum and buffy coat from each sample were separated. Genomic DNA was extracted from the buffy coat, using DNP kit (CinnaGen, Iran) according to the manufacturer’s instructions.

Genotyping of vascular endothelial growth factor polymorphisms
Genotyping of the +405 G/C polymorphism in the 5′-untranslated region of the VEGF gene was evaluated with polymerase chain reaction-restriction fragment-length polymorphism (PCR-RFLP), as described by Vanaja and colleagues.¹⁶ In brief, PCR was carried out in a total volume of 25 μL containing MgCl2 (0.75 mM), PCR buffer (10 mM, pH of 8.3), dNTP (0.2 mM), each primer (4 μM), Taq DNA polymerase (1 U) (all from CinnaGen, Iran), and DNA samples. The PCR products were digested by restriction enzyme, and the amplified products were monitored by agarose gel electrophoresis and ethidium bromide staining. Primers, fragment sizes, and restriction enzymes are summarized in Table 2.

Statistical analyses
Allele and genotype frequencies were calculated in the rejection group and control group by direct gene counting. Statistical evaluation was carried out with SPSS (version 16 for Windows) and Epi Info (CDC, Atlanta, GA, USA) software. The frequencies of the alleles/genotypes in the rejection and control groups were compared by the chi-square test or the Fisher exact test, when appropriate. Odds ratios and 95% confidence intervals (CIs) for relative risks were calculated. P < .05 was considered statistically significant. All reported P values were 2-tailed. Hardy-Weinberg equilibrium of the studied alleles was evaluated with Arlequin software (version 3.1.1).

As shown in Table 1, the rejection group included 22 patients, composed of 13 male patients (59.1%) and 9 female patients (40.9%), with a mean age of 40.23 ± 13.368 and 40.11 ± 15.439 years, respectively. The nonrejection (control) group included 102 patients, composed of 46 male patients (45.1%) and 56 female patients (54.9%), with a mean age of 38.63 ± 17.388 and 34.97 ± 16.865 years, respectively.

However, the significance of this allele and genotype did not survive the Bonferroni adjustment, which suggested the striking of the threshold of P from .05 to .017. Genotypes were not in agreement with Hardy-Weinberg equilibrium in both the rejection and control groups.

The underlying diseases of both groups are shown in Table 3, which illustrates the distribution of +405 G/C genotypes, hepatitis B virus, hepatocellular carcinoma, autoimmune hepatitis, and chronic liver disease. There were no statistically significant relationships between hepatitis B virus, hepatocellular carcinoma, autoimmune hepatitis (P = .240), and chronic liver disease (P = .126) and the risk of rejection.

The +405 G/C genotype frequencies in both the nonrejection (G/G = 8, G/C = 20, C/C = 74) and rejection (G/G = 2, G/C = 5, C/C = 15) groups were described. There was no significant relationship between VEGF +405 G/C genotype and alleles and the risk of rejection. Also, in this study, the frequency of allele G and C was shown in the nonrejection (G = 36, C = 168) and the rejected (G = 9, C = 35) groups (Table 4). Statistical analyses indicated no significant differences with regard to sex and age versus rejection.

Logistic regression analysis was used to check the +405 polymorphism and risk of rejection among LT recipients. No significant association between the +405 G/C polymorphism and risk of rejection, age, underlying disease, and sex were observed (Table 5).

Liver tissue adaptive responses against a series of time-dependent immunologic and nonimmunologic injuries after transplantation determine the long-term outcome of the graft. In both physiological and pathological states, VEGF is a key vasculogenic regulator.¹⁷ Zhang and colleagues found a correlation between VEGF expression and AR following orthotopic LT in a rat model.¹⁸

The human VEGF gene is located on chromosome 6p21.3. In the literature, at least 30 single nucleotide polymorphisms (SNPs) have been identified in this gene, so that some functional polymorphisms were shown to be relevant to patients with liver,⁶ lung,¹⁹ colorectal,²⁰ gastric,²¹ ovarian,²² prostate,²³ bladder,²⁴ and breast cancers.²⁵ The prognostic potential of VEGF polymorphisms has been investigated in numerous studies.^25,26

According to a previous study, +405 genotypes have shown the highest VEGF production in G/G genotype and medium production in G/C genotype under in vitro¹² and in vivo¹³ conditions; however, for the C/C genotype, the lowest production was recorded.¹² In addition, the combination of genotype +405 G/G with other polymorphisms increased the activity of the VEGF promoter.²⁷ Other reports have linked the VEGF +405 C/G genotypes to be related to specific pathological conditions, such as lupus nephritis (GG genotype), collateral formation in coronary artery disease (CC genotype), and heart failure (CC genotype).^28-30 In their study of patients with diabetes, Awata and colleagues¹³ reported that genotype distribution of -634 C/G polymorphism was notably different in those with and without retinopathy. Young and colleagues³¹ reported that patients with early onset of psoriasis have a significantly increased frequency of the +405 C/C genotype and the C allele compared with healthy controls. In Hungarian population studies, the C allele of +405 G/C and the T allele of -460 T/C have been considered to be related to preeclampsia and HELLP syndrome (hemolysis, elevated liver enzymes, and low platelet count).^32,33 In our study, in VEGF +405 C/G, no significant difference was observed in the distribution of alleles, genotypes, and polymorphisms.

There are few published studies on the role of VEGF in autoimmune diseases. This study on the VEGF +405 polymorphism in transplant patients with and without rejection has been performed for the first time in Iran. Because there is no previous work on this topic, it is not possible to compare our results.

In conclusion, VEGF is an important factor in the angiogenesis of transplanted tissues. This study was performed on few patients and controls. According to our search, it is the first report on liver graft rejection and VEGF SNPs; therefore, further studies are required to confirm and extend the present results.

Volume: 20 Issue: 6 June 2022

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