Molecular studies of the vascular endothelial growth factor (Vegf-A) and VEGF-receptor (Flk-1) genes of grass carp

Abstract

VEGF-A is a key regulator of angiogenesis and upregulated expression of the VEGF-A gene by hypoxia is due to both transcriptional activation by HIF-1 and stabilization of the normally labile VEGF mRNA. In vertebrates, several different isoforms of the VEGF-A protein - VEGF-A121; VEGF-A145; VEGF-A165; VEGF-A189; and VEGF-A206 - are generated via alternative splicing of the VEGF-A gene, which differ in biological properties (Ferrara, 2004). Apart from its adaptive role in hypoxia, VEGF is an important angiogenic factor in various estrogen target tissues such as the placenta (Pepe and Albrecht, 1999), pituitary (Ochoa et al., 2000), testis (Rudolfsson et al., 2004), ovary and uterus (Charnock-Jones et al., 2002), and it has been demonstrated that estrogens may increase VEGF expression via the binding of estrogen receptors (ERs) a and b to the estrogen-responsive elements (EREs) of the VEGF-A gene (Hyder et al., 2000; Mueller et al., 2000). In this project, I have isolated and characterized the genomic gene and several full-length cDNAs encoding the VEGF-A (gcVEGF-A) protein, and a partial cDNA encoding the VEGF receptor-2 (gcVEGF-R2) protein of the hypoxia-tolerant grass carp. In mammals, VEGF-A gene expression is responsive to hypoxia whereas response of the VEGF-R2 gene to hypoxia is still controversial. Thus, the expression profiles of the gcVEGF-A gene and gcVEGF-R2 gene under normoxic and hypoxic conditions at the mRNA level in different tissues of grass carp were examined by Northern hybridization in this study. Additionally, in an attempt to gain a better insight into the functional role(s) of gcVEGF-A, the cellular expression pattern of the gcVEGF-A protein in gill, kidney and liver tissues of normoxic grass carp was also examined by Western blotting and immunohistochemistry. Two genomic clones from grass carp, RK3000 (6385 bp in size) and RK3001 (1734 bp in size), were isolated using a combination of molecular biological techniques. Computer analysis showed that RK3000 contains 8 exons and 7 introns and that the coding sequence (CDS) shares high amino acid sequence identity with the VEGF-A protein of zebrafish (93%), quail (84%), chicken (84%) and human (80%). On the other hand, RK3001 was found to contain 4 exons and 4 introns, and the CDS shares high nucleotide sequence identity with the VEGF-R2 of human (69.6%), mouse (67.2%) and zebrafish (65.9%). Based on computer analysis, Clone RK3000 is believed to represent the grass carp VEGF-A genomic gene while Clone RK3001 represents a partial sequence of the grass carp VEGF-R2 gene. Using full-length RT-PCR, four different splice variants of the grass carp VEGF-A gene (gcVEGF-A) were isolated which encode for gcVEGF-A121, gcVEGF-A165, gcVEGF-A189 and gcVEGF-A206. Western blot analysis showed that heart, kidney and muscle under normoxic conditions express all four of these gcVEGF-A isoforms, while brain, eye and gill express only the gcVEGF-A121 and gcVEGF-A165 isoforms, and liver only expresses gcVEGF-A121. Whereas gcVEGF-A121 showed high expression levels in all tissues examined, gcVEGF-A189 and gcVEGF-A206 only showed low levels of expression. Moreover, upregulated expression of selected gcVEGF-A isoforms in gill, liver and kidney was also observed under hypoxia by RT-PCR; where liver expresses only the gcVEGF-A121 isoform while gill and kidney express both gcVEGF-A121 and gcVEGF-A165. Collectively, Northern blot and Western blot analyses provided evidence that gcVEGF-A expression is regulated at both the transcriptional and translational levels. Upregulated expression of gcVEGF-A in hypoxic liver probably occurs in pancreatic cells and not in heptocytes. As revealed by Western blot analysis, only gcVEGF-A121 is expressed in liver which is a diffusible isoform that may be translocated to various target organs to exert its effect. In kidney, only distal tubules, proximal tubules and endothelium of renal arterioles showed gcVEGF-A expression by immunohistochemistry; no signal was observed in glomerulus. In gill, both primary and secondary lamellae showed strong immunoreactivity, which was not detectable in chloride cells and endothelial cells of the afferent and efferent gill capillaries. These observations suggested that the effect of gcVEGF-A on blood vessel remodelling is likely to take place in the primary or secondary lamellae.

Date of Award	15 Jul 2005
Original language	English
Awarding Institution	City University of Hong Kong
Supervisor	Yuen Chong Richard KONG (Supervisor)