Inhibitory Regulation Of Hypoxia-induced Growth By Vasonatrin Peptide In Cardiac Myocytes, Fibroblasts And Vascular Smooth Muscle Cells

Hypoxia is a universal phenomenon of many cardiovascular diseases, it not only occurs during acute myocardial infarction but also exists in many other clinic conditions when the oxygen supply is absolutely or relatively inadequate. Low oxygen stresses are known to modulate cell growth and differentiation by increasing transcription of a number of genes such as growth factor enzyme and stress protein depending on the cellular context. In order to understand the mechanisms of hypoxic diseases in cardiovascular system, it is important to elucidate the effects of hypoxia on cells of heart and blood vessels.The natriuretic peptides(NPs) are a family of vasoactive polypeptides possessing natriuretic .. diuretic and vasodilative actions. Recently, increasing attention has been made to their inhibitory effects on cardiac hypertrophy and vascular remodeling in both basic research and clinical application. Vasonatrin peptide (VNP) is a man-made new member of natriuretic peptide family. Previous studies have demonstrated that VNP was effective in preventing and treating hypoxic pulmonary hypertension and right ventricular hypertrophy, and inhibiting the proliferation of pulmonary artery smooth muscle cells. But it is still not clear whether VNP has direct influences on the growth of cardiac myocytes and fibroblasts independent on its effects on after load. Proliferation of vascular smooth muscle cells is involved not only in the process of pulmonary hypertension but also in many other vascular diseases such as atherosclerosis and restenosis after angioplasty or coronary artery bypass grafting (CABG). However, there is no direct evidence about the influences of VNP on the proliferation of other vascular smooth muscle cells.Three receptors subtypes for natriuretic peptides have been identified, the natriuretic peptide receptor (NPR)-A, NPR-B and NPR-C. The NPR-A and NPR-B are membrane-bound guanylate cyclase coupled to production of 3', 5'-cyclic guanosine-7-monophophate (cGMP). These receptors mediate most of the biological actions of natriuretic peptides. The NPR-C is a membrane-bound receptor that lacks the intracellular guanylate cyclase domains of the biologically active receptors and is therefore uncoupled with cGMP. NPR-C functions as a clearance receptor by binding with and internalizing natriuretic peptides. NPRs are widely and differently distributed in most tissues and can be up- or down-regulated in many pathological processes, which indicates that they have different effects on different tissue and participate in the regulation of their physiological and pathological process.In the present study, we investigated the effects and underlying mechanisms of VNP on hypoxia-induced growth of cultured neonatal rat cardiac myocytes, fibroblasts and human vascular smooth muscle cells in order to provide experimental evidence for the application of VNP in treatment of cardiovascular diseases.Aims:1. To observe the effects of different extent of hypoxia on the growth of cardiac myocytes and influences of VNP on cell hypertrophy and apoptosis induced by hypoxia.2. To observe the influences of VNP on the proliferation of cardiac fibroblasts and collagen synthesis induced by hypoxia, and elucidate its mechanisms in inhibiting cardiac interstitium remodeling.3. To observe the proliferation and apoptosis of vascular smooth muscle cells (VSMCs) both from internal mammary artery (IMA) and saphenous vein (SV) from patients undergoing CABG and to investigate the inhibition of VNP on the proliferation of VSMCs from IMA and SV.Methods:1. In cultured neonatal rat cardiac myocytes, MTT methods, total protein measurement and 3H-leucine incorporation were used to evaluate the cell number and protein synthesisof cardiac myocytes. Cell injury was investigated by lactate dehydrogenase (LDH) activity in culture medium. And cell apoptosis was examined by flow cytometry, TUNEL staining and transmission electron microscopy (TEM). Furthermore, radioimmunoassay was performed to determine the levels of in...