Involvement Of Profilin-1 In Asymmetric Dimethylarginine-induced Vascular Smooth Muscle Cell Proliferation

BACKGROUNDEssential hypertension is a cardiac chronic medical condition in which the systemic arterial blood pressure is elevated. It is one of the risk factors for stroke, myocardial infarction, heart failure and arterial aneurysm, and is a leading cause of chronic kidney failure. It can increase risk for cerebral, cardiac, and renal events.Remodeling of large and small arteries contributes to the development and complications of hypertension. Vascular remodeling is the basic pathology of cardiovascular diseases, such as systemic hypertension, pulmonary hypertension, atherosclerosis. Remodeling of large and small arteries in hypertension resulted in the elevation of blood pressure, and induces the complications of hypertension.The 12-16 kDa protein pro filin is ubiquitously expressed in organisms and isolated as a 1:1 complex with G-actin, profilins constitute a large family of proteins, among of them, profilin-1 is essential for cell survival and cell division, it plays a critical role in modulating actin dynamics by promoting both actin-filament and depolymerization, moreover, profilin also has been implicated in the proliferation, migration and apoptosis of cells and signal transduction.The primary aim of the study is to detect the level of profilin-1 mRNA and the expression of profilin-1 protein in the spontaneous hypertensive rats, which were treated with losartan, and observe the changes of profilin-1 in the rat aortic of SHR.METHODTwelve-week-old male spontaneous hypertensive rats (SHRs) and the age-and sex-matched normotensive Wistar Kyoto rats (WKYs) as lean controls were obtained from the SLRC Laboratory Animal Inc.(Shanghai, China). SHRs were randomly divided into three groups (n=8):SHR; L-Losartan, rats were orally administered with losartan (15 mg/kg/day) for 8 weeks; H-Losartan, rats were orally administered with losartan (30 mg/kg/day) for 8 weeks. WKYs (n=8) and SHR were orally administered with saline (1 ml/kg/day) for 8 weeks. The systolic blood pressure (SBP) of rats ware measured by the tail-cuff method with an electro-sphygmomanometer coupled to a computerized recorder.The rats were anesthetized and the thoracic aorta and mesenteric arteries were collected. Slices of these arteries were stained with hematoxylin-eosin staining (HE), media thickness (MT), lumen diameter (LD) and MT/LD were measured with the computer-assisted image analysis system. The level and expression of profilin-1 in thoracic aorta were tested by real time-PCR and Western blot analysis respectively.RESULTSCompared with WKY rats, SBP significantly elevated in SHR rats, accompanied by enhanced profilin-1 mRNA and protein levels. Treatment with losartan significantly reduced the SBP and profilin-1 mRNA expression. Compared with WKY rats, MT, LD and MT/LD of thoracic aorta and mesenteric arteries of SHR group were significantly higher than WKY group.CONCLUSIONThe incidence of vascular remodeling in SHR was associated with increased profilin-1 level. ChapterⅡRole of profilin-1 in asymmetric dimethylarginine-induced vascular smooth muscle cells proliferationBACKGROUNDNitric oxide (NO) is an important mediator of vascular tone and cell function, it causes vasodilation and inhibition of both platelet aggregation and proliferation of vascular smooth muscle cells. NO is synthesized by specific oxidation of the terminal guanidine nitrogen of 1-arginine by the action of the NO synthase (NOS).Asymmetric dimethylarginine (ADMA) is the endogenous competitive inhibitor of NOS and one of the important factors that determined NO bioavailability. It is formed by proteinarginine methyltransferase (PRMT) enzymes and metabolized mainly by dimethylarginine dimethylaminohydrolase (DDAH). ADMA plasma concentrations have been demonstrated in patients with hypertension, hyperlipidaemia, hyperhomocysteinaemia, coronary artery disease, and so on.Actin filaments localize to specific regions and are important for oocyte maturation, fertilization and embryo development. Profilin-1 acts by sequestering actin monomers in a 1:1 complex and inhibiting actin polymerization. Many studies demonstrated that profilin-1 contributes to the migration and proliferation of vascular smooth muscle cells, modulation of cell transcription, as well as endothelial dysfunction.In the present study, we examined the effects of profilin-1 on the proliferation of rat aortic smooth muscle cells (RASMCs).METHODRASMCs were treated with different concentrations of ADMA as indicated for 24 h or 30μM ADMA for different periods of time. Cells were transfected with profilin-1 shRNA to interrupt expression of profilin-1 protein. The level and expression of profilin-1 in RASMCs were tested by real time-PCR and Western blot analysis respectively. Cell proliferation was determined by using the 3-(4,5-dim-ethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and flow cytometry analysis.RESULTADMA induced the expression of profilin-1 (both mRNA and protein) in RASMCs in a concentration-and time-dependent manner. Moreover, ADMA induced proliferation of RASCMs in a concentration-and time-dependent manner as show by the increase in formazan absorbance. Compared with both blank control and negative control, loss of profilin-1 expression attenuates the proliferation of RASMCs. Profilin-1 shRNA successfully knocked down profilin-1 mRNA and protein levels in RASCMs.DISCUSSIONThe proliferation of vascular smooth muscle cells induced by ADMA may be related to activation of the profilin-1. ChapterⅢInvolvement of JAK2/STAT3 in asymmetric dimethylarginine-induced vascular smooth muscle cell proliferationBACKGROUNDJanus kinase (JAK)/signal transducers and activators of transcription (STAT) signaling pathway has been shown to mediate the proliferation of vascular smooth muscle cells. JAKs are a family of non-receptor tyrosine kinases that activate the STAT. They are activated by various cytokines and growth factors, such as platelet-derived growth factor, interleukin-6 (IL-6), epidermal growth factor. AG490 is a member of the tyrosine kinase inhibitors. AG490 inhibits the proliferation of several cell types including leukemia cells and fibroblasts.A large body of data indicates that JAK2/STAT3 play an important role in the regulation of the expression of multiple genes involved in controlling cell proliferation and apoptosis. More recently study revealed that 7-ketocholesterol up-regulated expression of profilin-1 in aortic endothelial cells by activation of JAK2/STAT3 pathway.In the study, we explored whether JAK2/STAT3 pathway are involved in ADMA-induced proliferation of RASMCs.METHODRASMCs were treated with ADMA, pretreatment with AG490 (5×10-5 M) or rapamycin (10-8M) were used. The level and expression of profilin-1 in RASMCs were tested by RT-PCR and Western blot analysis respectively. Cell proliferation was determined by using the MTT assay and flow cytometry analysis.RESULTPretreatment with AG490(5×10-5M) and rapamycin(10-8M) inhibited ADMA-induced the expression of profilin-1 and RASMCs proliferation detected by MTT and flow cytometry. AG490 alone had no effect on proliferation of RASMCs.CONCLUSIONThe activation of JAK2/STAT3 contributes to ADMA-induced profilin-1 increasing of RASMC.