P-4: Role of A2b Receptors in Development and Its Implications in Angiogenesis-A Study Using Zebra Fish Embryo Model
Objective: Angiogenesis is the physiological process of developing new blood vessels. The growth of blood vessels is essential for organ growth and repair. This normal process is critical to the body for functions such as wound healing and fetus development. Zebra fish are rapidly becoming an important model organism for studying vertebrate development. The advantages of zebra fish are short reproductive cycle, production of numerous transparent, synchronously developing embryos, low cost, and etc. Adenosine A2b receptors were cloned from rat hypothalamus and membrane structure of A2b receptors is typical of G protein–coupled receptors, with seven transmembrane domains connected by three extracellular and three intracellular loops. Recent finding of the presence of A2b receptors in some vascular beds raised the possibility that they participate in the regulation of vascular tone. Physiological functions of A2b receptors consist of Control of Vascular Tone, Cardiac Myocyte Contractility, Cell Growth, and angiogenesis. Materials and Methods: In this study, we Adult zebra fish lay hundreds of fertilized eggs each morning. Embryos are arrayed in assay plates with appropriate medium, and different doses of caffeine are added to the medium in each well. Embryos are allowed to develop and are screened visually for developmental defects. Some embryos were collected for RT-PCR. The amino acid sequence homology, 3D structure of A2b receptor between human, zebra fish and mouse were analyzed by bioinformatics browsers and software. Results: Amino acid sequence analysis shows a 61% homology between A2b receptor of human and zebra fish and 59% homology between A2b receptor of mice, zebra fish. Secondary structure analysis using SOPMA & Hy<font><font><font><font>drop</font></font></font></font>athy plot indicate that A2b is indeed a seven transmembrane helix receptor. 3-D structural analysis using SWISS MODEL gives almost a similar structure for A2b receptor of human and zebrafish. Morphological analysis of Zebra fish embryos after caffeine treatment shows pericardial edema, hemorrhage, abnormalities in heart and brain development, body curvature, developmental delay and complete absence of vasculature. These results suggest that caffeine may inhibit angiogenesis via A2b receptor. Gene expression analysis for VEGF-A in control and caffeine treated embryos shows a decrease in expression of VEGF-A. This shows that caffeine may inhibit angiogenesis via A2b receptor by inhibiting the expression of VEGF-A. Conclusion: Data shows that A2b receptor plays an important role in angiogenesis and morphological development in zebra fish due to control of vasculature and angiogenesis.