Effect Of G Protein-coupled Receptor Kinase 4 On Proliferation Of Vascular Smooth Muscle Cells In Atherosclerosis

Background:There is a variety of mechanisms for the generation and development of atherosclerosis(AS), among them, The disorder of lipid metabolism is an independent risk factor of the occurrence and development of AS, leading to vascular endothelial cell injury, chronic inflammation, prompting monocyte/macrophages, proliferation and migration of vascular smooth muscle cell(VSMC), as well as releasing a variety of inflammatory factor and so on. Oxidized low density lipoprotein(ox-LDL) is the main factor involved in the pathogenesis of AS. VSMC is the important part of vessel wall, the abnormal proliferation of VSMC can lead to the intimal thickening and formation of fiber lipid plaque, which contribute to the development of AS. As the most important factor in the AS, ox-LDL can induce VSMC proliferation through various pathways, blocking the pathway can inhibit the proliferation of VSMC and slow down the further development of AS.G protein coupled receptor kinases(GRK), belonging to the family of serine/threonine kinases, exert a variety of biological functions via regulating membrane protein receptor. GRKs are composed of seven isoforms divided into three families. GRK1 and GRK7 are expressed exclusively in retina. GRK2, GRK3, GRK5 and GRK6 are widely distributed in mammalian tissues, GRK4 is predominantly found in testis and to lesser extent in Purkinje cells and kidney. Our previous study reported GRK4 was expressed in the vascular smooth muscle and involved in the regulation of vascular smooth muscle function. GRK4 are associated with angiotensin II receptors and dopamine receptors, contributing to the regulation of blood pressure. However, whether GRK4 are involved in the proliferation of VSMC are still unclear. Therefore, in the present study, we determined if GRK4 is associated with the AS via affecting the proliferation of VSMC induced by Ox-LDL, and explore the mechanism involved.Method:1. Mice thoracic aortic vascular smooth muscle tissue of, rat carotid artery of after balloon injury were isolated; Rat thoracic aortic smooth muscle cell line(A10 cells) was used in the experiments.2. The protein expression of GRK4 in smooth muscle tissue of ApoE knockout mice(ApoE- /- mice) and control mice(C57BL / 6J mice) were observed using western blot analysis; the protein expression of GRK4 expression in smooth muscle tissue of rat carotid artery from SD rats with or without carotid artery balloon injury.3. The effect of GRK4 on ox-LDL-induced A10 cells proliferation and the effect of GRK4 siRNA on ox-LDL-induced A10 cells proliferation were observed using CCK8 method.4. The protein expression of GRK4 in A10 cells with ox-LDL treatment using Western blot.5. The signaling pathways involving in the effect of GRK4 on ox-LDL-induced VSMC proliferation were explored using western blot method.Results:1. The formation of plaques in ApoE-/- mice were induced by high-fat diet; rat vascular intimal thickening and vascular smooth muscle cell proliferation were caused by carotid artery balloon injury.2. The GRK4 protein expression significantly increased in the VSMC from ApoE-/- mice compared with control mice on high-fat diet; the GRK4 protein expression also increased in the carotid artery from rats with carotid artery balloon injury relative to the control rats.3. ox-LDL promoted the proliferation of A10 cells, and the interference of GRK4 expression in A10 cells significantly inhibit the proliferation of A10 cells induced by ox-LDL, and this effect was concentration-dependent within a certain range.4. c-Myc inhibitors, 10074-G5 inhibited the increased expression of GRK4 induced by ox-LDL in A10 cells. MAPK/ERK inhibitors, PD98059 inhibited the proliferation of A10 cells induced by ox-LDL. The interference expression of GRK4 in A10 cells significantly inhibited the MAPK/ERK1/2 signaling pathway.Conclusion:The expression of GRK4 on the model of AS is significantly increased, GRK4 is involved in the formation of the AS and associated with the proliferation of VSMC. Oxidized LDL enhances GRK4 expression and further improves the proliferation of smooth muscle cells, which may be related to the MAPK/ERK signaling pathway.