Role And Mechanism Of Kindlin-2on Vascular Smooth Muscle Cells Proliferation, Migration,Adhesion And Intimal Hyperplasia

Objective1. To observe the effects of Kindlin-2RNA interference(RNAi) on rat vascular smooth muscle cells(VSMCs) proliferation, migration and adhesion, and explore the possible molecular mechanisms and the associated signal transduction pathways.2. To investigate the effects of Kindlin-2RNAi on intimal hyperplasia in the balloon injured rat carotid arteries and the possible mechnisms.Methods1. Based on rat Kindlin-2gene sequence and RNAi sequence design principles, three RNAi target sequences were designed and the most effective target sequence was chosen. Single-stranded DNA oligo contained RNAi sequence was synthesized and then annealed to produce double-stranded pairs. Double-stranded pairs was directly connected into the digested lentiviral vectors by its restriction sites. The ligation product was transfered into the bacteria competent cells. The positive recombinant was identified by PCR technique and gene sequencing. Finally, the lentiviral vectors were packed using293T cells. The harvested lentivirus was concentrated and the titer was detected.2. Primary rat VSMCs were cultured by tissue adherent and the cells were identified using a-actin antibody. The VSMCs used for all experiments were between the3rd and6th passages. Rat VSMCs were infected with Kindlin-2siRNA lentiviral vectors, multiplicity of infection was100. The expression of Kindlin-2and β1integrin in VSMCs was detected by immunofluorescence. VSMCs proliferation induced through recombinant Wnt3a protein was assessed by CCK-8and BrdU technique. The expression level of Kindlin-2, c-myc and cyclinDl mRNA in VSMCs was detected by real-time quantitative PCR. The relationship between Kindlin-2and β-catenin was investigated by immunoprecipitation. The protein expression of Kindlin-2, β-catenin, phosphor-β-catenin (Ser675), GSK-3β and phosphor-GSK-3β(Ser9) in VSMCs was detected by western blot.3. Primary VSMCs were cultured, then infected with Kindlin-2siRNA lentiviral vectors. To understand the ability of VSMCs migration by Transwell experiment and wound healing assay. And to explore the ability of VSMCs adhesion to extracelluar matrix by cell-extracelluar matrix adhesion assay. The Kindlin-2and β1-integrin mRNA and protein expression levels in VSMCs were detected by real-time quantitative PCR and western blot. Total β1-integrin and active β1-integrin expression on the surface of VSMCs was evaluated by flow cytometry.4. Healthy male SD rats were randomly divided into three groups:sham operation group, negative control group and Kindlin-2group. The balloon injured rat carotid arteries were infected with Kindlin-2siRNA lentivirus. Carotid arteries were collected at1and4week after operation. The expression level of Kindlin-2mRNA in the rat carotid arteries was detected by real-time quantitative PCR technique. The degree of intimal hyperplasia4weeks after balloon injury was assessed by HE staining. The immunofluorescence and histochemistry were used to detect the protein expression of Kindlin-2, a-actin, PCNA and β1-integrin in the carotid arteries. The synthesis of collagen fibers in the carotid arteries was investigated by Masson staining.Results1. The recombinant positive clones were consisitent with the expected results by PCR technique. The sequence map was legible and amplified sequence was fully consistent with designed Kindlin-2RNAi sequence. Lentivirus packed by293T cells concentrated and the titer was close to1x109TU/ml. The lentiviral titer achieved test requirements.2. Kindlin-2siRNA lentivirus can effectively infect VSMCs and change the subcellular distribution of Kindlin-2and β1integrin in VSMCs. Kindlin-2RNAi can significantly inhibit Wnt3a-induced VSMC proliferation(P<0.05). Compared with the control group and the negative control group, the mRNA expression levels of Kindlin-2, c-myc and cyclinDl in the Kindlin-2RNAi group and Kindlin-2RNAi+Wnt3a group were significantly decreased(P<0.05). The interaction between kindlin2and β-catenin was identified by immunoprecipitation in VSMC. And Kindlin-2RNAi can significantly reduce the protein expression levels of Kindlin-2, β-catenin, phosphor-β-catenin (Ser675), GSK-3β and phosphor-GSK-3β(Ser9) in VSMC(P<0.05).3. Kindlin-2RNAi significantly reduced the Kindlin-2mRNA and protein expression levels of VSMCsCs(P<0.05). And significantly inhibited VSMCs migration and adhesion(P<0.05). However, there was no significant effect on the β1-integrin mRNA and protein expression(P>0.05). But Kindlin-2RNA interference was able to influence β1-integrin activation on the surface of VSMCs(P<0.05).4. Kindlin-2RNAi can significantly reduce the Kindlin-2mRNA expression level in the rat carotid arteries at1week(P<0.05). And significantly inhibited intimal hyperplasia of carotid arteries4weeks after balloon injury (P<0.05). Compared with the negative control group, the protein expression levels of Kindlin-2, α-actin, PCNA and β1-integrin in the carotid arteries of the Kindlin-2group4weeks after operation were significantly decreased. The synthesis of collagen fibers in the carotid arteries intima and media was also reduced.Conclusions1. Synthetic Kindlin-2RNAi sequences can build an effective lentiviral vector.2. Kindlin-2RNAi can inhibit VSMC proliferation via the Wnt/β-catenin signaling pathway.3. Kindin-2can regulate VSMCs migration and adhesion through the activity of β1-integrin.4. Kindlin-2RNAi may inhibit VSMCs proliferation, migration and cell-extracellular matrix adhesion to alleviate intimal hyperplasia.