The results showed that Kindlin-2 could significantly promote the expression of integrin1 and3 in VSMC, and could eliminate the promotion of Kindlin-2 by inhibiting the FAK and AKT pathways (Fig 3B)

The results showed that Kindlin-2 could significantly promote the expression of integrin1 and3 in VSMC, and could eliminate the promotion of Kindlin-2 by inhibiting the FAK and AKT pathways (Fig 3B). Open in a separate window Fig 3 The effect of Kindlin-2 on integrin1 and3 expression.(A) The expression of Kindlin-2 in VSMC was detected by immunofluorescence. of extracellular matrix of a pathophysiological process [1]. After arterial injury, the blood vessels will respond, which will last for weeks or even months. During this process, intimal hyperplasia will occur. This process mainly involves 3 factors: vascular easy muscle cells, endothelial cells and extracellular matrix [2]. Among them, the proliferation and migration of vascular easy muscle cells are the main pathological features of intimal hyperplasia. Vascular easy muscle cell proliferation is usually evident in 24h after arterial injury and last for at least 2 weeks [3]. At the same time, VSMC, stimulated by TGF-b, PDGF and other factors, secreted a large number of extracellular matrix and accumulated, forming vascular lumen stenosis and occlusion. In clinic, in addition to external injury, cutaneous artery angioplasty, stent implantation, vascular bypass grafting and autologous vein grafting can cause intimal hyperplasia, leading to surgical failure. At present, the specific mechanism of vascular hyperplasia is still under exploration. However, the proliferation and migration of vascular easy muscle cells is one AN3199 of the important causes of vascular hyperplasia [4]. Many factors, such as Wnt, AKT and FAK, contribute to the migration and proliferation of vascular easy muscle cells. In previous studies, researchers have found that vascular proliferation is also associated with integrin activity. Previous studies have shown that classical FAK-PI3K-AKt signaling pathway can activate Integrin activity [5]. Integrin can mediate the connection between extracellular matrix and intracellular signaling pathways. In the process of vascular hyperplasia, integrin will further enhance this process when vascular easy muscle migrates and proliferates [6,7]. Kindlin-2, also known as FERMT2, is usually a scaffolding protein that enhances integrin activation. In particular, Kindlin-2 can enhance integrin mediated cell adhesion and migration [8]. In previous studies, kindlin-2 promoted the invasion and migration of different tumor cells through AKT and NF-kb pathways in various cell lines [9C11]. In particular, kindlin-2 also could promote the migration of gastric cancer cells by promoting phosphorylation of integrin beta 1 and beta 3 in gastric cancer cell lines [12]. However, although the effect of kindlin-2 is usually coincident with the mechanism of promoting vascular hyperplasia, the role of kindlin-2 in vascular hyperplasia has not been studied. Therefore, our study aims to explore the role of kindlin-2 in vascular hyperplasia by using rat vascular easy muscle cells and rat vascular injury and hyperplasia animal model. Materials and methods Antibody In this experiment, we used a variety of antibodies in Western and immunofluorescence, including: anti-kindlin-2(SIGMA), anti-FAK, anti-AKT (Proteintech Group, Wuhan, China), ati-P-AKT, anti-P-FAK(CST), anti-ITGF1, anti-ITGF3(Millipore), anti-P- ITGF1(Bioworld), anti-P- ITGF3(NOVUS), anti-Brdu(CST), HRP labeled Goat anti mouse antibody, HRP labeled MDNCF Goat anti rabbit antibody and HRP labelled rabbit against rat antibody(Boster Biological Technologyco, AN3199 Wuhan, China). Isolation of rat aortic easy muscle cells The rats were AN3199 killed, then opened chest and chest skin. The aorta vessels of rats were removed and placed in a pre cooled DMEM medium. The vascular medium membrane was separated under a microscope. The separated middle membrane was cut and broken by surgical scissors and cultured in DMEN with 20% fetal bovine serum. Identification of rat aortic easy muscle cells When the degree of fusion of vascular easy muscle cells reached about 90%, anti–SMA antibody was used to identify whether the isolated primary cells were vascular easy muscle cells by immunofluorescence staining. Cell culture The VSMC cells were cultured using DMEM medium supplemented with 20% of fetal bovine serum, 1% sodium pyruvate (100 mM), 1% non-essential amino acids (10 mM) and 1% penicillin/streptomycin solution (Invitrogen, Carlsbad, CA). Cells were maintained at 37C at 5% CO2 atmosphere. The cells were passaged every 5 days. Immunofluorescence For immunofluorescence, cells were plated on chamber slides, fixed either with methanol at ?20C for 5?min or with 4% paraformaldehyde at 37C for 15?min depending on the antibodies used. To examine the protein levels at each mitotic stage, cells were synchronized by double-thymidine block and release to fresh media for various times. A staging system was used to identify AN3199 the different phases.