Vascular Endothelial Growth Factor 165 And Neuropilin-1 MRNA Expression In Esophageal Squamous Cell Carcinoma And The Association With Angiogenesis

Backgrouand and Objective Esophageal carcinoma is a malignancy that exhibits a wide diversity in geographical incidence and mortality. Among them 90 % are squamous cell carcinomas. In China, there are around 150 thousand patients dead of this disease annually. Therefore, treatment is very important for esophageal cancer.Tumor angiogenesis is the proliferation of a network of blood vessels that penetrates into cancerous growths, supplys nutrients and oxygen and removes waste products. Solid tumors depend on angiogenesis for growth and metastasis in a hostile environment. Several angiogenic and anti-angiogenic factors released by tumor play crucial roles in regulating endothelial cell (EC) proliferation, migration, apoptosis or survival, cell-cell and cell-matrix adhesion , this is considered as an essential mechanism during tumor-induced angiogenesis.Vascular endothelial growth factor (VEGF) is the key angiogenic factor in tumor-induced angiogenesis. High levels of VEGF are expressed in various types of tumors. VEGF exists in five different isomers that are produced by alternative splicing from a single gene containing eight exons. Human VEGF isomers consist of monomers of 121, 145, 165, 189 and 206 amino acids. They may induce the proliferation and movement of ECs, remodeling of the extracellular matrix, the formation of capillary tubules, and vascular leakage. VEGF121 and VEGF₁65 are the most abundant in the five known VEGF isomers. The structural difference between these two monomers is the presence of exon 7 in VEGF₁65 of 44 amino acids but lackin VEGFni. The VEGF receptor (VEGFR) family is comprised of VEGFRl/Flt-1, VEGFR2/KDR and VEGFR3/FU-4. Recently, neuropilin-1 (NRP-1) has been found to be a receptor for VEGF and is expressed on the surface of ECs and tumor cells. NRP-1 is able to bindVEGF,65 but not VEGF121. Interestingly, VEGFi65 binds to NRP-1 via the VEGF exon 7-encoded domain that is lacking in VEGF121.Neuropilin-1 (NRP-1) is cell surface glycoproteins and plays a critical role as a receptor in the development of the neuronal and vascular systems. It binds semaphorins and members of the VEGF family and regulates axonal guidance, vasculogenesis and angiogenesis, respectively. NRP-1 is essential for normal development of the nervous and cardiovascular systems. Transgenic overexpression of NRP-1 results in an excess of capillaries and blood vessels and a malformed heart. NRP-l also contributes to tumor angiogenesis. Induction of NRP-1 expression in tumor tissues in vivo results in larger and more vascular tumors. NRP-1 is a receptor for VEGF 165 and acts as a coreceptor to enhance VEGF 165 function through tyrosine kinase VEGF receptor 2 (VEGFR-2). When NRP-1 is coexpressed on VEGFR-2+ ECs, the binding to VEGF 165 and chemotactic activity by VEGF 165 for these cells are upregulated in comparison with those of ECs expressing VEGFR-2 alone. Astrocytic tumor, breast and prostate carcinomas are shown to express high levels of NRP-1. However, expression of NRP-1 in esophageal cancer is not well understood.In this study, we investigated the expression of NRP-1 mRNA, VEGF 165 mRNA and micro vessel density (MVD) value in carcinoma and adjacent normal tissues of the esophageal squamous cancer and attempted to clarify their clinical significance. In addition, we studied whether there was any correlation among them.Materials and Methods Fresh surgical specimens were obtained from 42 patients with primary esophageal squamous cell carcinomas and adjacent normal esophageal tissue. All samples had been proved by means of pathological diagnosis. Reverse transcription-PCR method was used to examine the expression of neuropilin-1 mRNA and VEGF165 mRNA. They were calculated after a correction for the expression of P -actin mRNA. Immunohistochemistry was also used to examine the expression of CD 105. We calculated the number of micro vessel by anti-CD 105monoclonal antibody to achieve MVD in esophageal carcinoma tissue and correspondingly adjacent normal esophageal tissue. Comparisons between esophageal carcinoma tissue and adjacent normal esophageal tissue were performed using paired Mest for dependent samples. Comparisons between groups were performed using Student's Mest and the correlation between two continuous variables was determined using the Pearson rank correlation ( Y) test. P value < 0.05 was considered significant. Results1. Expression of NRP-lmRNA is higher in esophageal carcinoma tissue than adjacent normal esophageal tissue in 37 (89.00 %) of 42 case. The average value is 0.55+0.19 and 0.44+0.19 respectively (P < 0.01). The cases with tumor diameter ^ 3 cm show significantly higher expression of NRP-1 mRNA than those with the diameter < 3cm (P = 0.009). The cases with adventitia infiltration and the cases with lymph node metastasis show significantly higher expression of NRP-1 mRNA than those without adventitia infiltration (P = 0.010) and lymph node metastasis (P = 0.011). Expression of NRP-1 mRNA has no correlation with histological changes (P = 0.325) and sex (P = 0.432).2. In 41 (97.62 %)of 42 cases, expression of VEGFies mRNA is higher in esophageal carcinoma tissue than adjacent normal esophageal tissue, the average value is 0.64+0.18 and 0.49 ±0.18 respectively (PO.01). The cases with tumor diameter ^ 3cm show significantly higher expression of VEGF165 mRNA than those with the diameter < 3cm (P = 0.025). The cases with adventitia infiltration and lymph node metastasis show significantly higher expression of VEGF165 mRNA than those without adventitia infiltration (P = 0.014) and lymph node metastasis (P = 0.011). Expression of VEGF165 mRNA has no correlation with histological changes (P = 0.331) and sex (P = 0.514).3. In 37 (89.00%) of 42 cases, MVD values are greater in esophageal carcinoma tissue than adjacent normal esophageal tissue. The average value is 25.10+10.68 and 16.79 + 8.36 respectively (PO.01). The cases with tumor diameter ^ 3cm show significantly higher MVD values than those with the diameter < 3cm (P = 0.047). The cases with poorer histological classification show significantly higher MVDvalues than those with better histological classification (P = 0.041). The cases with adventitia infiltration and lymph node metastasis show significantly higher MVD values than those without adventitia infiltration (P = 0.039) and lymph node metastasis (P = 0.041). There is no correlation between MVD values and sex (P = 0.286).4. There is positive correlation between the expression of NRP-1 mRNA and the expression of VEGFi65 mRNA ( y = 0.939, P< 0.01).5. There is positive correlation between the expression of NRP-1 mRNA and MVD values ( Y = 0.797, P <0.01).6. There is positive correlation between the expression of VEGF165 mRNA and MVD values (y =0.771, P<0.01).Conclusions1. The expression of NRP-1 mRNA, VEGF165 mRNA and MVD values are significantly higher in esophageal squamous cell carcinomas compared to adjacent normal esophageal tissue.2. The expression of NRP-1 mRNA and VEGF165 mRNA have correlation with the tumor diameter, invasion and metastasis of esophageal squamous cell carcinoma, but no correlation with histological changes and sex.3. MVD values have correlation with the tumor diameter, histological changes, invasion and metastasis of esophageal squamous cell carcinoma, but no correlation with sex.4. The expression of NRP-1 mRNA has positive correlation with the expression of VEGF165 mRNA and MVD values, and the expression of VEGFi6s mRNA has positive correlation with MVD values.5. Overexpression of NRP-1 mRNA and VEGF165 mRNA are involved in tumor angiogenesis.