Purpose: Vascular endothelial growth factor (VEGF) is one of the most important growth factors for metastatic tumors. To clarify the role of VEGF-A and C in patients with peptic ulcer disease (PUD) or gastric cancer (GC), we evaluated the expression levels of these two molecules. We also analyzed the effect of Helicobacter pylori infection on VEGF-A and C expression levels

Abbasi, A.

Ajami, A.

Ajami, M.

Farazmandfar, T.

Hafezi, N.

Hosseini, V.

Mohammadian, R.

Sankian, M.

Taghizadeh, S.

Tehrani, M.

English

PubMed

Golestan University of Medical Sciences Repository

Original ArticleExpression Levels of Vascular Endothelial Growth Factors A and C in Patients with Peptic Ulcers and Gastric CancerShirin Taghizadeh1, Mojtaba Sankian1, Abolghasem Ajami2,3, Mohsen Tehrani3, Nasim Hafezi3, Rajeeh Mohammadian3, Touraj Farazmandfar4, Vahid Hosseini5, Ali Abbasi6, and Maryam Ajami71Department of Immunology, Faculty of Medicine, Mashhad University of Medical Sciences, Mashhad, 2Molecular and Cell Biology Research Center, Mazandaran University of Medical Sciences, Sari, 3Department of Immunology, Faculty of Medicine, Mazandaran University of Medical Sciences, Sari, 4Faculty of Advanced Medical Science Technology, Golestan University of Medical Sciences, Gorgan, 5Inflammatory Diseases of Upper GI Tract Research Center, Mazandaran University of Medical Sciences, Sari, 6Department of Pathology, Islamic Azad University, Sari Branch, Sari, 7 Department of Immunology, Faculty of Medicine, Tehran University of Medical Sciences, Tehran, IranPurpose: Vascular endothelial growth factor (VEGF) is one of the most important growth factors for metastatic tumors. To clarify the role of VEGF-A and C in patients with peptic ulcer disease (PUD) or gastric cancer (GC), we evaluated the expression levels of these two mol-ecules. We also analyzed the effect of Helicobacter pylori infection on VEGF-A and C expression levels.Materials and Methods: Patients with dyspepsia who needed diagnostic endoscopy were selected and divided into three groups: non-ulcer dyspepsia (NUD), PUD, and GC, according to their endoscopic and histopathological results. Fifty-two patients with NUD, 50 with PUD, and 38 with GC were enrolled in this study. H. pylori infection was diagnosed by the rapid urease test. After RNA extraction and synthesis of cDNA, the expression levels of VEGF-A and C were determined by quantitative reverse transcriptase polymerase chain reac-tion.Results: The VEGF-C expression level in the PUD and GC groups was significantly higher than that in the NUD group. Moreover, the VEGF-A expression level in the PUD and GC groups was higher than in the NUD group, although the differences were not statistically significant. Significant positive correlations were also observed between the expression levels of these two molecules in the PUD and GC groups. In addition, the expression levels of these two molecules were higher in H. pylori positive patients with PUD or GC than in H. pylori negative patients of the same groups; however, these differences did not reach statistical significance.Conclusions: Up-regulation of VEGF-C expression during gastric mucosal inflammation may play a role in the development of peptic ul-cers or GC.Key Words: Vascular endothelial growth factor-A; Vascular endothelial growth factor-C; Stomach neoplasms; Peptic ulcer; Helicobacter pyloriJ Gastric Cancer 2014;14(3):196-203  http://dx.doi.org/10.5230/jgc.2014.14.3.196Correspondence to: Abolghasem AjamiDepartment of Immunology, Faculty of Medicine, Mazandaran University of Medical Sciences, Sari 48175-1665, IranTel: +98-151-3543081, Fax: +98-151-3543248E-mail: ajami36@gmail.comReceived August 19, 2014Revised September 9, 2014Accepted September 21, 2014Copyrights © 2014 by The Korean Gastric Cancer Association www.jgc-online.org This is an open-access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0) which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original work is properly cited.IntroductionDyspeptic disorders such as gastroesophageal reflux, gastritis, peptic ulcer disease (PUD), and gastric cancer (GC) are major medical conditions.1 PUD is usually associated with a reduced health-related quality of life, whereas GC is the fourth most com-mon cancer and the second leading cause of cancer-related deaths worldwide.2,3 Host factors such as genetics and nutrition, and en-vironmental factors such as Helicobacter pylori infection may be involved in the development of these conditions.1,4H. pylori infection has been shown to be a major risk factor for the development of PUD and GC.5,6 However, despite several investigations, it is still not completely understood why the major-VEGF-A and C Expression in Patients with PUD or GC197ity of infected people (80%~90%) carry and spread the bacterium while they are asymptomatic, or why only a small percentage of infected people develop peptic ulcers, whereas others develop GC.6Host immune responses against H. pylori can result in chronic inflammation in the gastric mucosa, which in turn leads to the de-velopment of pathological conditions including PUD and GC.5,6Vascular endothelial growth factors (VEGFs) are glycopro-teins secreted by tumor cells that are the most important factors in angiogenesis and tumor metastasis.7 The VEGF family includes VEGF-A to F and placental growth factor.7,8 Studies have shown that VEGF-A and B play a key role in blood vessel growth, whereas VEGF-C and D are important for the growth of lymphat-ic vessels.9,10 The role of VEGFs, particularly VEGF-A, C, and D in promoting angiogenesis and metastasis of many cancers includ-ing GC, has been previously discussed.11,12 Moreover, inflammatory cytokines such as interleukin (IL)-1, IL-6, and tumor necrosis factor-alpha (TNF-α) are generally responsible for the epigenetic alteration of gastric epithelial cells.13 These cytokines induce the mediators of angiogenesis, including VEGF and IL-8, which pro-mote angiogenesis in cancer. These mediators also promote angio-genesis during chronic inflammation such as cardiovascular disease, rheumatoid arthritis, diabetic retinopathy, delayed-type hypersensi-tivity, and asthma.14 It has been shown that VEGF-A expression is up-regulated in response to H. pylori infection.15 Indeed, H. pylori activates the c-Jun N-terminal Kinases (JNK) signaling pathway, which leads to transactivation of the VEGF-A promoter. VEGFs promote angiogenesis, which is a pathophysiological mechanism that can result in inflammatory and ulcerative epithelial lesions and malignant tumor growth and metastasis.15To understand the role of VEGFs in the pathogenesis of H. pylori-related gastric abnormalities, the mRNA expression levels of VEGF-A and C were determined in patients with peptic ulcers or GC, and compared with those with non-ulcer dyspepsia (NUD).Materials and Methods1. Patients and samplingPatients with dyspepsia who underwent esophagogastroduo-denoscopy at Imam Hospital or Tooba Outpatient Clinic (Mazan-daran University of Medical Sciences, Sari, Iran) were enrolled in the study. All samples were collected between January 2012 and December 2013. The study was approved by the ethics committee of Mazandaran University of Medical Sciences. Clinical history, demographic data, and written informed consent forms were ob-tained from all study subjects. None of the subjects had a history of chronic inflammatory or autoimmune disorders or treatment with H. pylori eradication therapy, nor did they receive any non-steroidal anti-inflammatory drugs for 2 weeks prior to enrollment. Among patients with GC, none had undergone surgery, radiotherapy, or chemotherapy, or received any other medical intervention before sample donation.Based on the endoscopic and histopathological assessments, the patient samples were divided into three groups: NUD, PUD, and GC. The histological grade of the gastric tumors was determined based on the state of differentiation. PUD was defined as a circum-scribed mucosal break (＞5 mm in diameter, with apparent depth) in the stomach or duodenum, covered with exudates. H. pylori infection was diagnosed by histopathological examination (includ-ing Giemsa staining) and a positive result on the rapid urease test performed on at least one additional biopsy sample. Patients were considered H. pylori positive if the results of one or both diagnostic methods were positive, and H. pylori negative if the results of both methods were negative. Patients in NUD group were then divided into two groups: H. pylori positive and H. pylori negative. Tissue samples were obtained from all patients during endoscopy and pre-served in RNALater (Qiagen, Phoenix, AZ, USA).2. RNA isolation and cDNA synthesisEach tissue specimen was homogenized using mortar and pestle at room temperature. Total RNA was extracted from the dissected tissues using commercial RNA extraction kits (RNeasy Minikit; Qiagen), according to the manufacturer’s instructions. The quantity and quality of the extracted RNA were assessed using a nanodrop spectrophotometer (Thermo Fisher Scientific Inc., Waltham, MA, USA) and agarose gel electrophoresis, respectively. RNA (1 μg) was reverse-transcribed into complementary DNA (cDNA) using the RevertAidTM First-Strand cDNA Synthesis Kit (Fermentas, Pitts-burg, PA, USA) primed with random hexamers as per the manu-facturer’s instructions.3. Quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR)VEGF-A, VEGF-C and hypoxanthine-guanine phospho-ribosyl transferase (HGPRT, for normalization), sequences were obtained from the GenBank (Table 1). Primers for amplification of VEGF-A, VEGF-C, and HGPRT were designed using the Bea-con designer 7 software and synthesized by TIBmol (Germany) (Table 1).qRT-PCR was performed using 96 well plates (Bio-Rad Labo-ratories Inc., Hercules, CA, USA) in a volume of 20 μl containing 

Expression levels of vascular endothelial growth factors A and C in patients with peptic ulcers and gastric cancer

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Expression levels of vascular endothelial growth factors A and C in patients with peptic ulcers and gastric cancer

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