The Relations Between α 7 NAChR In Vascular Endothelium And Angiogenesis

Background Angiogenesis is a crucial mechanism required for a number of physiological and pathological events. In physiological conditions, angiogenesis is a highly regulated phenomenon. It normally occurs during embryonic development, wound healing and menstruation cycle. Unregulated angiogenesis is shown in pathological conditions, such as cancer, diabetic retinopathy, rheumatoid arthritis. The α 7nAChR, a ionotropic receptor formed by five homologous subunits, is involved in the form of nervous pathway and the regulation of physiological functions, and play an important role in the central and peripheral nervous system. In non-excitative cells-endothelial cells, the receptor is involved in the secretion and releasion of bioactive substances, the signal transduction between endothelial cells and other cells, and angiogenesis. Therefore, these results suggest an endogenous cholinergic pathway for angiogenesis. In the first part of the study, we investigated the expression of α 7nAChR (mRNA and protein), the regulation of ligand in HUVECs and endothelial cell of the rat model of myocardial infarction, the cellular function, and angiogenesis in vitro and in vivo. So that we investigate the pharmalogical characteristics on HUVECs and the regulation on angiogenesis when the α 7nAChR may function as a receptor of local "hormone". In the second part of the study,, we screened 25 new derivatives of thalidomide. The goal of our study was to develop new therapeutic angiogenesis drug and new anti-tumor drug which can inhibit the angiogenesis.Methods RT-PCR and immunohistochemistry were used to examine the expression of α 7nAChR (mRNA and protein) and the ligand regulation in HUVECs and endothelial cell of the rat model of myocardial infarction. The cellular function was examined using MTT, fluorescence confocal microscopy, and angiogenesis assay in vitro. The capillary density in the rat model of myocardial infarction and transplant tumor of S180 mice were detected using immunohistochemistry. The body weight and tumor weight of the transplant tumor of S180 mice were measured. 25 new derivatives of thalidomide were screened using MTT in HUVECs. Furtherly, those new derivatives which could inhibit proliferation of ECs were screened using MTT in HepG2Cs. Then, the new derivatives which could inhibit proliferation of ECs but not HepG2Cs were investigated in the transplant tumor model of S180 mice. The body weight and tumor weight were measured. The capillary density in the transplant tumor of S180 mice were investigated using immunohistochemistry. Results The results showed that α7nAChR agonists, nicotine and choline, increased theexpressions of α 7nAChR mRNA and protein, enhanced the intracellular Ca2+ concentration, and promoted proliferation and tube formation of ECs. Reverse effects were observed using mecamylamine and α -BTX, a 7nAChR antagonist. In the rat model of MI, α 7nAChR agonist increased the expressions of α 7nAChR mRNA and protein, and enhanced the capillary density in ischemic tissues, whereas antagonist mecamylamine and α -BTX reduced the expression of a 7nAChR (mRNA and protein) and capillary density. Furthermore, the results revealed that α 7nAChR agonists promoted the transplant tumor growth by stimulating angiogenesis. Reverse effects were observed using mecamylamine and α -BTX. W-37 and W-42, the two new derivative of thalidomide, can inhibit the transplant tumor growth of S180 mice via inhibiting angiogenesis. Conclusion The expression of α 7nAChR mRNA in non-excitative cells is differ from in excitative cells. There may be multiform of α 7nAChR in gene expression and cellular function: ① The structure of α 7nAChR in non-excitative cells is differ from in excitative cells, therefore, the function is different. ②The structure of α 7nAChR is different in the same tissue, therefore, the function is diverse. ③The structure of α 7nAChR in non-excitative cells and excitative cells are identical, but the expression and the function are distinct in different position of the tissue. The α7nAChR of endothelial cells may be a new therapeutical target for angiogenesis.