WISP1 Promotes Proliferation And Migration Of Human Vascular Smooth Muscle Cells Via Akt Signaling Pathway

BackgroundAtherosclerosis (AS) is a kind of common and frequently-occurring diseases threatening human health, such as cerebrovascular, coronary and peripheral arterial Occlusive Disease. The disease, with high morbidity, disability and mortality, has become the major disease burden in most Western countries and our country. Early pathology of atherosclerosis is described as a kind of end stage degenerative disease, it causes the extensiveness of the artery lumennarrowing, its process and its complexity is determined by various factors involved in chronic inflammatory processes. Vascular smooth muscle cells and endothelial cells, macrophages and the formation of atherosclerotic plaques are closely linked. The current treatment of arteriostenosis, caused by atherosclerosis, includes balloon angioplasty, Stenting and bypass surgery. But any means of treatment will lead to an inevitable complications-Restenosis after vascular surgery. Balloon angioplasty and endovascular stenting implantation, considered to be safe and effective treatment for peripheral arterial Occlusive Disease, has been widely used in recent years. The technology above can improve vascular open rate in short-term. However restenosis seriously effect long-term effectiveness, according to report, femoral popliteal artery stenting implantation potency rate only for 59%-81% after one year, up to 5-10% of patients appeared has acute bracket within thrombosis. Thus, high rate of restenosis after vascular surgery became "Achilles’ heel" of vascular intervention, remains one of important vascular surgical needed solution.In essence, restenosis after vascular injury is composed of a variety of cytokines and growth factorsare mediated by a local repair (wound healing) response. Restenosis is generally considered to be a process that multi-factors and multiple mechanisms are involved, and its core mechanism is vascular smooth muscle cells (VSMC) proliferation and migration. The proliferation and migration of vascular smooth muscle cells in vascular remodeling diseases (VRD) play vital roles in this pathological process. The study on the proliferation and migration of VSMC, cell phenotypic changes of restenosis may clarify the mechanisms and shed light on providing useful ideas for treatment. Our previous study (Liu H, Dong W, Lin Z, Lu J, Wan H, Zhou Z, Liu Z. CCN4 regulates vascular smooth muscle cell migration and proliferation. Mol Cells.2013 Aug;36(2):112-8.) found that CCN4 can enhance the bonding of VSMC cells in vitro, and promote VSMC migration and proliferation. In addition, we also found that after CCN4 treatment, VSMC phenotypic changes in gene expression, some synthetic marker genes (such as elastin and osteopontin, etc) significantly increased, and convergent marker genes (such as Alpha-actin, etc) significantly declined. However, so far, the extracellular matrix (ECM) protein—the regulation of CCN4 on VSMC phenotype, proliferation and migration have not been reported. Relationship between WISP1 and restenosis in vascular diseases remains to be further studied.Recently, it is found that the signaling pathway consisting PI3K and its downstream protein kinase B (PKB or Akt) are closely related with the occurrence and development of restenosis after vascular surgery. The pathway regulates vascular smooth muscle cell proliferation and survival, abnormal activity of which can lead to VSMC proliferation, migration, and the degradation of the extracellular matrix. PI3K-Akt pathway works mainly through activation of Akt and many downstream molecules. Several studies has reported, in vascular injury model, activation of Akt phosphorylation lead to activation or inhibition of its downstream target, such as GSK-3, MMP9, cyclinDl, which regulates the function of vascular smooth muscle cell proliferation, differentiation, and migration, and eventually lead to restenosis.WISP1, the molecular in our study, is secreted by fibroblast cells, smooth muscle cells and other cells. WISP1, also known as WISP1, belonging to the CCN family member (other family members are CYR61, CTGF, NOV, CCN5 and CCN6), is a multifunctional extracellular matrix proteins. WISP1, is a downstream of wnt pathway, regulates migration, proliferation, immunomodulation and tumor formation in vivo. Studies consider it may be a new target for treatment of a range of diseases (such as trauma, neurodegeneration, skeletal muscle diseases, cardiovascular disease, pulmonaryfibrosis, tumor growth and metastasis). Studies have shown that WISP1 can be activate Akt/PKB anti-apoptotic signaling pathways:DNA damage induced apoptosis in P53 activation to mitochondrial release of cytochrome c, raising anti-apoptosis BCL-XL and WISP1 by activating Akt molecular inhibited both activities to inhibit apoptosis.For neurodegeneration, WISP1 protects neurons by activating Akt signal in oxygen-glucose deprivation model. WISP1 activates Akt signaling to regulates fibroblastsis in synovial membrane fibrosis.ObjectiveBase on above information, we hypothesize that in the process of restenosis after stenting, Akt signaling pathways participates in regulating proliferation and migration of VSMC induced by WISP1, contributing to occurrence of restenosis. The aim of this study is to investigate whether and how WISP1 stimulates proliferation and migration of human thoracic aortic smooth cells (HAVSMC). We attempt to further explore regulatory mechanisms of WISP1 on vascular smooth muscle cell proliferation and migration, to provide scientific basis and rationale for finding a new solution of restenosis, and provide new drug targets for drug balloon design.In short, the purpose of this study:(1) detection of WISP1 expression in vascular smooth muscle cells proliferation, (2) detection of WISP1 protein and the relationship between cell proliferation and migration of vascular smooth muscle, and (3) explore the signaling pathway regulating vascular smooth muscle cell proliferation and migration induced by WISP1.Methods(1) Observation change of WISP1 protein expression level in HAVSMC proliferation induced by different concentration FBS:in vitro, using different concentrations of FBS (0%FBS, and 2%FBS, and 10%FBS) culture the proliferation of thoracic aortic smooth cells (HAVSMC) for 48 hours, EdU Incorporation Assay to detect cell proliferation and Western blot to detect WISP1 expression, statistical analysis.(2) Construction and validation of WISP1 over-expression in HAVSMC by ad-virus:using adenovirus transfection technology, cells were transfected by adenovirus containing WISP1 gene or empty virus (MOI=10) respectively, transfection for 24 hours and 48 hours in observed by fluorescent microscope, Western blot to confirm WISP1 expression after transfection for 24 hours.(3) EdU Incorporation Assay to measure cell proliferation in two groups, statistic differences of EdU mark positive cell number ratio between the groups.(4) WISP1-induced cell migration measured by Scratch-induced wound healing assay:after transfection for 24 hours, observe and record cell migration in different time:0 hours,6 hours,12 hours,24 hours,48 hours under microscope, statistics analysis.(5) WISP1-induced cell migration measured by Transwell Migration Assay: after transfection for 24 hours, collect and add cells to the upper chamber (cell number about for 5x104 cell), fixe cells after 24 hours migration, dyeing, microscope record results, Statistic differences of migrated cell number between the two groups.(6) Observation of signaling pathway activation in WISP1-induced HAVSMC proliferation and migration:after transfection for 24 hours, Western blot to detect Akt, p-Akt expression. statistical analysis of p-Akt/Akt ratio differences.(7) Validation WISP1 induce HA VSMC proliferation and the migration via Akt signal:after transfection for 24 hours, treated with Akt inhibitors (concentration=5μm) or not, using the same methods to measure and observe cells proliferation and migration, Western blot to detect expression of downstream of Akt p-GSK3/GSK3, MMP9, cyclinDl.Results(1) Transfection after 24 hours and 48 hours, green fluorescent are observed in two group under fluorescent microscope, Western blot results shows, compare to empty virus (EV), WISP1 protein level significantly increased in WISP1 over-expression group (P<0.05);(2) EdU Incorporation Assay revealed that FBS treatment increased proliferation of human VSMC in a dose-dependent manner and quantification results showed a 2.5-fold and 3.0-fold increase in proliferating cells after cell treated with 2% FBS and 10% FBS, respectively. In addition, we found that WISP1 protein level was enhanced by 3.4-fold in 2% FBS-treated human VSMC and 4.1-fold in 10% FBS-treated human VSMC, respectively;(3) EdU Incorporation Assay revealed that EdU mark positive cell number ratio were increased after cells were transfected with WISP1. Quantification analysis confirmed that WISP1 overexpression significantly increased proliferating cells by 2.98-fold.(4) Scratch-induced wound healing assay showed that WISP1 overexpression increased wound closure rate in a time-dependent manner (6,12,24 and 48 hours after injury) by comparison with EV. WISP1 overexpression significantly increased wound closure rate compared with EV 6 hours (4.56±1.14% vs. 11.23±2.25%, p<0.05),12 hours (9.89±2.42% vs.23.21±3.14%, p<0.05),24 hours (15.67±2.24% vs.46.32±6.33%, p<0.01), and 48 hours (25.25±5.51% vs. 97.54±13.12%, p<0.01) after injury.(5) Similarlly, Transwell Migration Assay also showed that migrated cell numbers were significantly increased in AD-WISP1-infected cells by 2.25-fold compared with EV cells at 24 hours;(6) Western blot results show transfection after 24 hours, compared with EV, WISP1 markedly stimulated phosphorylation of Akt by 3.2-fold 24 hours after transfection (P<0.05);(7) Transfection after 24 hours, with or without inhibitors of Akt (concentration=5μm) treatment after transfection of cells (4 groups). AZD5363 treatment significantly decreased EdU-positive proliferating cells induced by WISP1 overexpression, but had no effect on EV cells. Additionally, AZD5363 treatment caused an apparent inhibition of wound closure rates in Ad-WISP1-infected cells from 12 hours (23.12±3.21% vs.14.50±2.13%, p<0.05) to 48 hours (97.21±10.08% vs.57.81±7.75%, p<0.01) after injury (Fig.4C). Consistently, Transwell Migration Assay also revealed that migrated cell numbers at 24 hours were also significantly decreased in Ad-WISP1-transfected cells after treatment with Akt inhibitor, AZD5363 (P<0.05). Western blot test results indicate WISP1 overexpression markedly stimulated phosphorylation of GSK3α/β by 2.99-fold 24 hours after transfection. CyclinDl and MMP9 protein levels were also increased in human VSMC tranfected with Ad-WISP1 compared to EV tranfected-cells. Pretreatment of AZD5363 inhibited the phosphorylation of GSK3a/p, and reduced CyclinD1 and MMP9 protein levels in Ad-WISP1-transfected cells (P<0.05).Conclusions(1) FBS stimuluses vascular smooth muscle cell (HAVSMC) proliferation by a dose-dependent manner, in proliferation of process, WISP1 expression has positive correlation with cell proliferation level;(2) WISP1 over-expression promotes vascular smooth muscle of proliferation and the migration;(3) WISP1 over-expression promotes vascular smooth muscle of proliferation and the migration process by activating Akt;(4) block the Akt signaling prevents the WISP1-induced proliferation and migration of vascular smooth muscle, and down-regulated levels of Akt downstream:p-GSK3/GSK3, expression of MMP9, cyclinD1. Collectively, we demonstrated that Akt signaling pathway mediates WISP1-induced migration and proliferation of human VSMC, suggesting that WISP1 may act as a novel potential therapeutic target for vascular restenosis.