| Objective:Vascular remodeling is not only a compensatory reaction but also one of the major and basic pathological basis which at the mean time the reasons of serious complications and dysfunction of circulatory system.The pathogenesis,prevention and treatment of vascular remodeling have become a research hotspot in both domestic and foreign countries.Although its specific mechanism was still unclear,its common characteristic is the increasing of vascular elasticity or vascular smooth muscle cell contractility and the resistance of peripheral vascular.Vascular phenotypic shifting was involved in all the complicated processes.The research on the vascular phenotypic shifting mechanism had become the effective prevention and treatment hotspot of hypertension.Angiotensin ??Ang ??played an important role in the functional modulation of vascular smooth muscle.It activated many signal paths through combination of the recipient in the cell membrane.The promoting effect of Ang ? took part in the occurrence and development processes of hypertension,atherosclerosis and vascular restenosis.In vascular system,vascular smooth muscles cells was not only the resource of Angiotensin ? but also the target of the modulated function of Ang ?.However,the specific mechanisms of Ang ? affected VSMCs proliferation and migration were still unknown.The family of Bcl-2 associated athanogene is the newly founded protein family that regulated cell apoptosis.Its members can connect with Heat-shock protein through the BAG function part.BAG3 was the member of BAG family which had highly affinity to Hsp70/Hsc70 and had widely cell biological effect through the combination with Hsp70/Hsc70,like suppressed cell apoptosis,promote tumor cell proliferation and migration.The expression level of BAG3 in healthy person tissues was relative low or no expression,however,in some tumor cell like leukemia and solid tumor,the expression of BAG3 was relative high which matins the survived of tumor cells.In some special stress situation like oxidant,high temperature,serum deprivation,heavy metals,HIV-1 infection,low frequency magnetic field explosions,electrophilic reagent stimulation,hemodialysis treatment,pulsed ultrasound,retinal photic injury and transient forebrain ischemia,etc,even in the normal cells?like lymphocytes,epithelial cells,and glial cells?,the expression of BAG3 increased.All the listed researchers suggested that BAG3 can modulate the proliferation,migration and invasion of cells in many levels.Previous studies indicated that the activation of NF-?B pathway can promote the inflammations and oxidative stress and simultaneously it had been proved to be involved in the proliferation and migration of vascular smooth cells.At the meantime,there was already studies confirmed that NF-?B pathway took part in the modulation of expression of BAG3 and be the key part in the modulation.Our previous study had proved that BAG3 took part in the proliferation and migration of vascular smooth cells in the condition of AGE,however,it is still unclear that whether it takes part in the process of Ang ? induced VSMCs proliferation and migration and whether NF-?B pathway takes part in this process.In 1997 year,Kuro ect found that Klotho gene existed in the spontaneous hypertension animal research which was related with aging.Klotho was 50kb long contain 5 exons and4 introns which divided into membrane type and secreted type.Researches showed that the deficiency of Klotho gene would make the mouse showed aging phenotype like growth retardation,atherosclerosis,osteoporosis,emphysema,shortened life,atrophy of skin,infertility,ataxia,etc.Recently,there were already plenty of clinical studies associated with Klotho and its products and showed that Klotho genetic polymorphism was related with CAD.Besides,Klotho is associated with CVD risk factors like chronic kidney failure,diabetes,hyperuricemia,hypertension and dyslipidemia.Hence,our research tends to estimate the possible relationship between Klotho and BAG3 in the Ang ? induced VSMCs proliferation and migration process and figure out the possible mechanism.Furthermore to estimate the possible effect of NF-?B pathway in this process.Clarifying the mentioned questions can provide the new experimental and theoretical basis for the pathogenesis of the atherosclerosis,hypertension and vascular stenosis.And the meantime,it can provide a new specific target to treat the cardiovascular diseases mentioned above.Methods:1.VSMCs,a rat vascular smooth muscle cell line obtained from the China Center for Type Culture Collection,Wuhan,China.Passages 5-9 of the cells were used in this study.VSMCs were grown to 70-80%confluence and then made quiescent by incubation in DMEM without FBS for 24 hours before experiments.2.Cells were treated with various concentrations of Ang ? for 10-6,10-7,10-88 M in cell proliferation assay and migration assay.In order to evaluate the effect of BAG3 on the proliferation and migration of Ang ? induced VMSCs,we used sh-BAG3 to decrease the expression of BAG3 in VSMCs.Then cells pretreated with various concentrations of klotho for 50 to200 ng/ml in cell proliferation assay,200 ng/ml in migration assay,cell morphology and western blotting on quiescent cells with or without 10-7M Ang ? for designated times.3.VSMCs were cultured in 24-pore plates and then counted.4.Cell viability was examined by MTS assay.5.Cell cycle progression was assessed using propidium iodide?PI?staining with fluorescence activated cell sorting?FACS?analysis.6.Wound healing assay and Transwell migration chamber model were performed to examine the migration of VSMCs.7.Cell morphology and actin filaments analysis were determined by rhodamine phalloidin staining and captured using a fluorescence microscope.The area and circularity of VSMCs were calculated.8.Western blot technology was used to detect the expression of contractile markers,such as SM22a and aSMA;the expression of BAG3,p-NF-?B p65,NF-?B p65.The experimental results were analyzed by ANOVA and paired or unpaired t-test for statistical analysis as appropriate.P-value<0.05 was considered as statistical significance.All experiments were performed at least three times.The band intensity was quantified using Image J 1.47 software and relative expression averaged across the three experiments.GraphPad Prism5 and Adobe Photoshop CS6 were used to draw graphs.Resutls:1.Ang ? induced proliferation and migration of VSMCs in a dose-and time-dependent manner.2.Ang ? induced the expression of BAG3 in a dose-and time-dependent manner.When knock down the expression of BAG3 the proliferation and migration effect of Ang ? induced were attenuated which indicated that BAG3 took part in the Ang ? induced VSMCs proliferation and migration.3.NF-?B p65 pathway was activated during the Ang ? induced VMSCs process.We used PDTC block the NF-?B p65 pathway and found that BAG3 expression significantly decreased.Hence,we indicated that Ang ? modulated BAG3 expression through NF-?B p65 pathway.4.Klotho inhibited Ang ?-induced proliferation of VSMCs in a dose-and time-dependent manner.The cell survival decreased distinctively with high dose of klotho?50 ng/ml,100ng/ml,150 ng/ml and 200 ng/ml?.Moreover,a time-dependent decrease in Klotho induced inhibitory effect on Ang ?-induced VSMCs proliferation was also observed at a fixed dose of 200 ng/ml.5.Ang ? increased the percentage of cells into S phase with the concomitant decrease of cells in the G0/G1 phase.However,Klotho significantly reduced the percentage of cells in S phase,indicating that klotho may induce G0/G1 phase in Ang ? induced VSMCs.6.Ang ? markedly increased in the cellular levels of cell cycle regulatory proteins including PCNA,cyclin D1,CDK4,cyclin E and CDK2 in VSMCs.In contrast,Klotho treatment attenuated the expression of PCNA,cyclin D1,CDK4,cyclin E and CDK2 in Ang ? stimulated VSMCs.7.Results of wound healing assay and Transwell migration chamber model showed that the promotive effect of Ang ?-induced migration was conspicuously suppressed by the co-administration with Klotho.8.Klotho inhibited the upregulation of migration-related proteins including ICAM-1,VCAM-1,MCP-1,MMP-2 and MMP-9 induced by Ang ?-induced VSMCs.9.Rhodamine-phalloidin staining revealed that Ang ? stimulated VSMCs exhibited orderless distribution of actin filaments and aggregation around the perinuclear region without clear organized cytoskeleton network.However,Klotho inhibited Ang ?-induced VSMCs morphology and cytoskeleton rearrangement.10.Klotho inhibited the Ang ?-induced reduction of VSMCs BAG3 and marker proteins expression,such as SM22a and PCNA.11.Ang ? treatment markedly increased the phosphorylation of p-NF-?B p65 and BAG3.In addition,Kltoho significantly inhibited Ang ?-induced p65phosphorylation and BAG3 in VSMCs.Conclusions:1.Ang ? induced VSMCs proliferation and migaration while klotho inhibited Ang ?-induced VSMCs proliferation and migration.2.Ang ? induced the proliferation and migration partly by the modulation of BAG3 through the activation of NF-?B p65 pathway.3.Klotho induced G0/G1 phase arrest in Ang ?-induced VSMCs and attenuated the expression of PCNA,cyclin D1,CDK4,cyclin E and CDK2.4.Klotho reduced Ang ?-induced VSMCs migration by suppressing the expression of migration-related proteins,such as MMP2,MMP9,VCAM-1,ICAM-1 and MCP-1.5.Klotho inhibited abnormal proliferation,migration,and phenotypic modulation of Ang ?-induced VSMCs via regulation of NF-?B p65/BAG3 signaling pathway. |
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