Integrin Linked Kinase Regulates Vascular Smooth Muscle Cell Phenotypic Modulation

Part Ⅰ:the expression of integrin liked kinase in rat models of carotid balloon injuryObjective Percutaneous coronary intervention (PCI) is the most effective method for treatment of coronary artery disease, however, the occurrence of restenosis (RS) after PCI has restricted the clinical efficacy of PCI. The increased proliferation and migration of vascular smooth muscle cells (VSMCs) are key events in the development of artery restenosis following PCI, and the VSMC from contractile phenotype to synthetic phenotype transformation is the initial stage before the proliferation and migration. Recent studies have found that ILK was required for the maintenance of the contractile SMC phenotype. The purpose of this work is to study the expression of ILK in the course of the carotid artery injury in rats.Methods The carotid artery balloon injury model in rats remains an important method to study the molecular and cellular mechanisms involved in vascular smooth muscle dedifferentiation and vascular restenosis. Then the experiments were performed as follows:(1) ILK levels in serum were measured by ELISA. (2) Real-time PCR was used to evaluate ILK and PCNA mRNA expressions in injured carotid artery. (3) Western blot was used to evaluate the SMC markers and ILK protein expressions. (4) The localization and expression of ILK and SM a-actin were determined with immunofluorescence staining.Results After carotid balloon injury in rats, the new intima was significantly increased, the extracellular matrix increased, and the intima continued to increase, resulting in a marked narrowing of the lumen. (1) ELISA analysis showed that serum ILK levels were significantly decreased at 30.8%(P<0.05) in 7 days after operation compared with pre-injury, and the levels of ILK in 14 days were further reduce compared with 7 days after surgery. (2) Real-time PCR analysis revealed that the mRNA levels of PCNA were increased in 7 days after surgery, and the levels of PCNA were further raised in 14 days (P all<0.05). However, the ILK mRNA expression was markedly reduced by 36% and 48% in 7 and 14 day, respectively(P all<0.05). (3) Western Blot analysis revealed that Osteoptin proteins were markedly increased by 34% and 42% in 7 and 14 days after surgery, respectively(P all<0.05). However, the protein levels of Calponin and SM a-actin were dramatically reduced by 22%(vs 7 days after surgery)、56%(vs 14 days after surgery) and 26%(vs 7 days after surgery)、32%(vs 14 days after surgery) (P all< 0.05). (4) Western Blot analysis revealed that PCNA proteins were markedly increased by 130% and 270% in 7 and 14 days after surgery, respectively(P all< 0.05). However, the protein levels of ILK were dramatically reduced by 50% and 88% in 7 and 14 days after surgery (P all<0.05). and the 14 day levels of ILK were further reduced then 7 days (P<0.05). (5) Immunofluorescence staining showed that ILK was mainly co-localized in cytoplasm with SM-a-actin positive cells in healthy carotid artery. Immunofluorescence also revealed that ILK was markedly down-regulated at the 14 days after balloon injury.Conclusion These results demonstrate ILK was mainly localized in VSMC cells in healthy carotid artery. After balloon injury of carotid artery in rats, the proliferation of VSMC was significantly increased, following the increased SMC synthetic marker and the decreased SMC differentiation marker gene expressions, while the serum ILK levels and the expression of ILK mRNA and protein were significantly decreased synchronously.Part II signaling mechanisms that integrin linked kinase regulate smooth muscle cell differentiationObjective:the VSMC from contractile phenotype to synthetic phenotype transformation is the initial stage before the proliferation and migration. The growth factor PDGF-BB is a major regulator of SMC phenotype. A critical step in the activation of SMC-specific gene expression is serum response factor (SRF) binding to CArG elements, and several mechanisms regulate this interaction. Myocardin is a specific coactivator of SRF that regulates the expression of SMC differentiation marker genes in an SRF-dependent manner. ETS like protein 1 (Elk-1) is another major SRF cofactor, which binding to the same region of SRF and compete for SRF binding with Myocardin. Phosphorylated Elk-1 inhibited SMC differentiation marker gene by displacing Myocardin from the SMC-specific promoters. Our results demonstrated ILK was mainly localized in VSMC cells in healthy carotid artery, while the serum ILK levels and the expression of ILK mRNA and protein were significantly decreased after carotid injury. Recent studies have found that ILK was required for the maintenance of the contractile SMC phenotype. We hypothesis ILK modulates of vascular smooth muscle cell phenotype by Myocardin.Methods:The experiments were performed as follows:Primary VSMC culture and treatment. Construction and transfection of adenovirus vector and plasmid vector. ILK small interfering RNA (SiRNA) plasmid construction and cell transfection. F-actin cell cytoskeleton staining. ILK kinase activity assay. Immunoprecipitation assay (Co-IP). Luciferase reporter gene analysis. Chromatin immunoprecipitation assay (Ch-IP). Western blot analysis, and so on.Results:1. The effects and mechanisms of ILK in early PDGF-BB induced VSMC phenotype transformation.VSMC were incubated with PDGF-BB (25ng/mL) for 1 hours and found:(1) The ILK kinase activity was found to be significantly inhibited by PDGF-BB. (2) Western blot analysis showed that PDGF-BB intervention significantly promoted Elk-1 phosphorylation, but had no effect on ILK and Myocardin protein expression. (3) Co-IP found that Elk-1 competed for SRF binding with Myocardin, which resulted in the decrease of SRP and Myocardin binding, and the combination of Elk-1 and SRP increased. (4) ChIP experiments found that Myocardin and SM a-actin gene promoter binding decreased, while Elk-1 and SM a-actin promoter binding increased.2. The effects and mechanisms of ILK in long-term PDGF-BB induced VSMC phenotype transformation.2.1 Effect of PDGF-BB on the regulation of smooth muscle cell contractile genes and ILK and Myocardin protein expression.After incubation with PDGF-BB (25ng/ml)for 48h, the SM a-actin, SM 22a and Calponin protein expression were significantly inhibited (compared with the control group, P<0.05), while the expression of Osteoptin protein was significantly increased(P>0.05). At the same time, the expression of Myocardin and ILK protein was down regulated, compared with the control group (P<0.05), and the expression of SRP and Elk-1 was not affected compared with the control group (P> 0.05).2.2 ILK is required for maintenance of the VSMC differentiated phenotype.We next investigated the potential effect of ILK on VSMC phenotype transition by SiRNA knockdown. ILK protein level was inhibited by 68%(P< 0.05). The protein level of Myocardin was down-regulated in parallel (P<0.05). Similarly, the protein and mRNA levels of SM a-actin, SM 22a and calponin were markedly reduced by siRNA treatment as compared with scramble siRNA treatment. ILK knockdown by siRNA also markedly blunted the response of differentiation markers to TGF-β. As shown by fluorescence staining, ILK siRNA silencing disintegrated actin fibers into short and disorganized fibers in parallel with polygonal shaped VSMC. These data further support ILK as being required for maintenance of the VSMC differentiated phenotype.2.3 ILK overexpression retains the characteristics of the contractile phenotype in PDGF-BB challenged VSMC.Western blot analysis revealed ILK protein level higher by 70% in VSMC with adenovirus (Ad)-ILK than with Ad-GFP infection. The protein level of Myocardin was up-regulated in parallel (P<0.05). Ad-ILK overexpression significantly circumvented PDGF-BB induced suppression of VSMC marker gene expression at both the mRNA and protein levels. Furthermore, Ad-ILK infected VSMC adopted a spindle-like shape and reorganization of the actin network from randomly oriented filaments to thicker and well-oriented fibers. Thus, ILK overexpression could restore PDGF-BB induced VSMC dedifferentiation and render differentiation in cells.2.4 The mechanisms that ILK inhibiting PDGF-BB signaling induced in VSMC dedifferentiationThe promoter activity was markedly suppressed by PDGF-BB, as supported by the results of transient transfection of SM a-actin-luc or calponin-luc reporter into VSMC by Luciferase reporter gene analysis. Conversely, overexpression of ILK reversed PDGF-BB modulated transcription of VSMC specific genes. Furthermore, Ch-IP assay revealed that ILK circumvented PDGF-BB suppressed SRF binding to both SM a-actin and calponin gene promoter regions.Conclusions:1. Early PDGF-BB treatment induced ILK kinase activity decreased, resulting in the enhancement of Elk-1 phosphorylation, and the combination of SRF and Myocardin, which resulted in the decrease of 22a and SM promoter binding of Myocardin, which resulted in down-regulation of SMC gene expression.2. The expression of Myocardin and ILK protein were down regulated by PDGF-BB long-term treatment. ILK is required for maintenance of the VSMC differentiated phenotype. ILK overexpression retains the characteristics of the contractile phenotype in PDGF-BB challenged VSMC. ILK rescues PDGF-BB promoted VSMC dedifferentiation by negatively regulating PDGF-BB induced Myocardin down-regulation, SRF recruitment to VSMC marker gene promoters and, subsequently, marker gene transcription.Part Ⅲ Digoxin inhibits PDGF-BB induced VSMC proliferation and migration through increasing ILK signaling and attenuates neointima formation after carotid injuryObjective:The increased proliferation and migration of vascular smooth muscle cells (VSMC) are key events in the development of artery restenosis after percutaneous coronary intervention. Digoxin has been previously shown to treat heart failure and even inhibit the proliferation of cancer cells through multiple pathways. However, the potential role of digoxin in regulation of VSMC proliferation and migration and treatment of cardiovascular diseases, such as restenosis, remains unexplored.Methods:In vitro, the changes of VSMC proliferation were determined by Methyl thiazolyl tetrazolium (MTT) assay. Migration assay was performed using the Transwell chamber. Real-time quantitative Polymerase Chain Reaction was performed to quantify mRNA levels of the smooth muscle cell phenotype markers such as SM-a-actin, calponin and SM 22a under PDGF-BB treatment in the absence or presence of digoxin. And the flow cytometric analysis (FCA) and Western blot assay were used. In vivo, we evaluated the effect of digoxin on neointima formation using the rat arterial balloon-injury model.Results:In the present study, we showed that digoxin-induced growth inhibition is associated with the down-regulation of CDK activation and the restoration of p27kipl levels in PDGF-stimulated VSMC. In addition, digoxin restored the PDGF-BB induced inhibition of integrin linked kinase(ILK) expression and prevented the PDGF-BB-induced activation of glycogen synthase kinase(GSK) 3β. Furthermore, digoxin was also found to inhibit adhesion molecule and extracellular matrix relative protein expression. Finally, we found that digoxin significantly inhibited neointima formation, accompanied with reduction of cell proliferation after vascular injury in rats. These effects of digoxin are mediated, in part, through increasing ILK/Akt and blockade GSK 3β signaling cascade in PDGF-BB stimulated VSMC.Conclusions:Digoxin has an inhibitory effect on PDGF-BB-stimulated proliferation, migration, and phenotypic modulation of VSMC, and prevents neointimal formation in rats. These effects of digoxin are mediated, in part, through increasing ILK/Akt and blockade GSK 30 signaling cascade in PDGF-BB stimulated VSMC.