The Biological Role Of NIX On Vascular Remodeling In Hypertension

Objectives To investigateexpression, cellular distribution and the role in vascular smooth muscle cell(VSMC) in order to find a key molecular in the vascular remodeling induced hypertension, which can provide an evidence for effective controlling and reversing vascular remodeling.Methods 1 The expression and cellular distribution of NIX in vascular remodeling induced hypertension. 1) Abdominal aortic constriction(AAC) were be used to build rat vascular remodeling model. Wistar rats were divided into sham and AAC groups. Each group had 8 rats. The rats were fed about 8 weeks. Carotid stents were used to detect using mean arterial pressure. HE staining was used to detect pathomorphological changes of vascular tissue. Masson staining was applied to detecte vasular fibrosis. Western bloting was used to detece the expression of fibronectin and collagen. All of these were used to verify whether the model was established successfully. In order to make sure the expression and cellular distibution of NIX in the rat model of vascular remodeling induce by hypertension, western blot and immunohistochemical were applied. 2) VSMC fibrosis model was established by different concentrations of NE indicating. VSMCs were divided into 0 mol/L, 10-6 mol/L, 10-5 mol/L, 10-4 mol/L groups, and treated with different concentration of NE intervention for 24 hours. MTT and flow cytometry were used to detect the prolifieration of VSMCs. Western blot was used to detecte the expression of collagen and fibronactin. All of these were used to ensure whether the model was established successfully. Then the expression of NIX was deteced by western blot to test the changes of NIX in VSMCs. 2 The biological effects of NIX in vascular smooth muscle cells induced by NE. 1) The pc DNA3-NIX high expression plasmid was used to transfect VSMCs. The cells were divided into C, pc DNA3, pc DNA3-NIX, NE, NE+pc DNA3, NE+pc DNA3-NIX groups. 24 hours after transfection, the cells was be intervened by NE(10-5mol/L) for 24 hours. Western blot was adopted to detect the effect of transfection of NIX. VSMCs proliferation was examined by MTT and flow cytometry, and the expression changes of collagen and fibronectin were deteced by Western blot. All of these were used to confirm the effect of NIX overexpression on fibrosis of VSMCs induced by NE. 2) The si RNA-NIX was applied to transfect VSMCs. The cells were divided into C, NC, si RNA, NE, NE+NC, NE+si RNA. 24 hours after tranfection, the cells was be intervened by NE(10-5mol/L) for 24 hours Western blot was adopted to detect the effect of transfection of NIX. VSMC proliferation was examined by MTT and flow cytometry, and the expression changes of collagen and fibronectin were deteced by Western blot. In order to confirm the effect of NIX lower expression on fibrosis ofVSMCs induced by NE.Results 1 The expression and cellular distribution of NIX in vascular remodeling induced hypertension. 1) Compared with sham group, mean arterial blood pressurein of AAC group sharply increased(P<0.05). HE showed that tunica media thickness, artery vessel area and thickness/inner diameter in the AAC group also increased(P<0.05). Masson staining showed that the collagen accumulation in AAC group rats were increased. Western bloting also proved that the expression of collagen and fibronectin of AAC group were higher. All of these told us, we had established the model of vasular remodeling induced by hypertension sucessfully. Meaningwhile, the expression of NIX in AAC group was higher than sham group. Immunohistochemistry showed that NIX was up-regulated in vascular smooth muscle cells. 2) The result of MTT and flow cytometry showed the proliferation of VSMCs sharply intensified with the increase of NE concentration(P<0.05). Western bloting showed that the expression of collagen and fibronectin increased when the concentration of NE increasing. All of these told us, we had established the fibrosis model of VSMC sucessfully. At the same time, we also found that when the concentration of NE increased, the expression of NIX was also increased. 2 The biological effects of NIX induced by NE in vascular smooth muscle cells. 1) Western blot results indicated that high NIX expression plasmid was successfully transferred. MTT and flow cytometry showed overexpression of NIX could promote the proliferation of VSMC, and also could promote the proliferation of VSMCs induced by NE(P<0.05). The results of Western blot showed that overexpression of NIX could promote the deposition of collagen and fibronectin, and intensify the increasement of collagen and fibronectin which induced by NE. 2) Western blot results indicated that si RNA-NIX was successfully transferred. MTT and flow cytometry showed inhibition of NIX could inhibit the proliferation of VSMCs, and also could inhibit the proliferation of VSMCs induced by NE(P<0.05). Western blot results showed that inhibition of NIX expression can inhibit the expression of collagen and fibronectin, and also can inhibitory effect on the increasement of collagen and fibronectin induced by NE.Conclusion The increasing expression of NIX is closely related with vascular fibrosis in the vascular remodeling induced by hypertensive. In addition, NIX played an important role in regulation of vascular fibrosis in VSMCs during vascular remodeling.