The Mechanisms Of Autophagy And MALAT1 On OxLDL-induced Vascular Cell Proliferation

Background and Objective:According to "China Cardiovascular Report 2015",cardiovascular diseases accounted for the first cause of the total deaths of urban and rural residents.The growing burden of cardiovascular diseases has become a major public health problem.Atherosclerosis(AS)is a common and serious pathological change in arterial disease,which often leads to seriously injury to heart,brain and other vital organs.In the process of AS formation,there are three main steps.Endothelium damage is the first step;and then the second step contains a series of vascular wall reactions which include the increase of endothelial permeability and adhesion,blood coagulation and the number of cytokines changes;Finally the vascular wall structure changes which mainly reflects in endometrial lipid infiltration,migration of vascular smooth muscle cell(VSMC)and macrophage and formation of foam cells.During the process of AS formation,VSMCs are the most active cells.Endothelium injury and vascular smooth muscle cell abnormal proliferation are two important steps in the development of AS,which are essential for repairing of endothelial injury and preventing abnormal proliferation of VSMCs to prevention and treatment of AS.So in the light of the repair of endothelial injury and prevention of abnormal proliferation of VSMCs can promote the repair of vascular injury,which are crucial for the prevention and treatment of the AS.Endothelial Progenitor Cells(EPCs)are considered to be key cell for repairing endothelial cells after injury.Therefore,with two different parts,our study aimed to explore the change and the related mechanism of EPCs and VSMCs proliferation under the AS risk factor-oxidized low density lipoprotein(ox-LDL)environment.1.Effects of autophagy on ox-LDL induced EPCs proliferation.EPCs were first discovered and named in 1997.Subsequent studies have found that EPCs can improve endothelial function and increase angiogenesis.And it has been revealed that hyperlipidemia and hypertension are closely related to the decrease of the number of EPCs.The decrease of EPCs can also be regarded as an independent risk factor for AS progression.In animal models such as rabbit cerebral ischemia,acute renal injury in rats,EPCs-based studies have also found that EPCs can improve ischemia-induced damage.But there is a long distance from the basic transplant research to clinical application.Therefore,it is essential to improve the survival of EPCs in the stress environment.Autophagy is a kind of self-protection behavior based on the body’s material in the stress environment.In 2006,Yan,et al.found that autophagy can reduce apoptosis and maintain leukemic cells viability.In 2011,a study in the journal of Science further revealed that the anti-apoptotic effect of autophagy can be achieved by up-regulating antiapoptotic proteins and clearing pro-apoptotic proteins.At present,autophagy has been studied in the cardiovascular system,which includes endothelial cells,VSMCs and myocardial cells.But autophagy for EPCs related research is still relatively rare.Therefore,our study aims to observe the effect of autophagy on the proliferation of EPCs by ox-LDL.2.Effect and mechanism of MALAT1 on ox-LDL induced VSMCs proliferation.The development of AS is closely linked to the proliferation of VSMCs.VSMCs have potent proliferative activity and can secrete a large number of inflammatory factors and adhesion agents to promote arteriosclerosis progression.In addition,the arterial wall of the vascular smooth muscle cell proliferation or hypertrophy is the main reason for hypertensive vascular wall thickening.The main mechanism of restenosis after coronary intervention is neonatal endometrial hyperplasia,and pathology studies show that the VSMCs accounted for 89% in the total proliferation number of cells.Therefore,it is important to study of VSMCs proliferation of the intrinsic mechanism,which is leading to seek a new target to inhibit the proliferation of VSMCs,and further to prevent and treat AS efficiently.Long non-coding RNA MALAT-1(lnc RNA-MALAT1,MALAT1)is also called nuclera-enriched autosomal transcript2(NEAT2),which is an important member of the long non-coding RNA family.MALAT1 was first discovered in non-small cell lung cancer in 2003.MALAT1 is expressed in a variety of tissues.Previous study found that silencing MALAT1 can regulate retinal vascular endothelial cell proliferation,migration and in vitro angiogenesis to improve diabetic retinopathy.It is also noted that MALAT1 affected the function of endothelial cells,in the hypoxia and high glucose environment respectively.However,the effect of MALAT1 on VSMCs,which closely related to cardiovascular disease,has not been reported.Therefore,we aimed to study the effect of MALAT1 on the proliferation of VSMCs by ox-LDL and explored the possible molecular mechanisms.Methods1.Effects of autophagy on ox-LDL induced EPCs proliferation.1.1 EPCs culture and identification.The spleen of rats was separated and crushed,and the cells were harvested by density gradient centrifugation.The morphology of the cells was observed by microscope and identified by Di I-Ac-LDL and FITC-UEA-I double staining.1.2 Detection of EPCs Cell Proliferation by RTCA.EPCs were inoculated into E-plate8 cell culture plates.EPCs were treated with ox-LDL at different concentrations(0μg / ml,10 μg / ml,30 μg / ml,60 μg / ml,100 μg / ml)to detect cell viability or to detect EPCs proliferative activity after inhibiting autophagy.1.3 Detection of EPCs autophagy levels.After treatment of EPCs with the same concentration of ox-LDL,the total protein was extracted at different time points(0h,6h,12 h,18h,and 24h).Western blot analysis was used to reflect the relative expression of autophagy p62 protein level.2.Effect and mechanism of MALAT1 on ox-LDL induced VSMCs proliferation.2.1 We first cultured human coronary artery smooth muscle cells,treated with different concentrations(0μg/ml,5μg/ml,10μg/ml,20μg/ml,50μg/ml,100μg/ml)ox-LDL,and used CCK8 to detect VSMCs changes in cell proliferation capacity.2.2 Total RNA was extracted from cells treated with ox-LDL at different concentrations and real-time quantitative PCR was used to detect the expression of MALAT1 in different groups.2.3 The transfection efficiency was measured using real-time quantitative PCR.2.4 Changes of cell proliferation were measured by CCK8,and the proliferation of si-malalt1 and si-malalt1 + ox-LDL groups were compared.2.5 The expression of p-AKT/AKT in ox-LDL treatment,si-malalt1 treatment and si-malalt1+ox-LDL treatment were detected by western blot.2.6 After treatment with si-malat1,the effect of AKT agonists(SC-79)for the proliferation of VSMCs were observed by CCK8.2.7 The p-AKT/AKT was detected by western blot after treatment with small interfering RNA(si-stim1).The effect of si-stim1 on cell proliferation was detected by CCK8.2.8 Finally,cells were treated with si-malat1 and the protein was extracted to detect the expression of STIM1.The calcium influx was measured by confocal microscopy combined.Results1.Effects of autophagy on ox-LDL induced EPCs proliferation.1.1 EPCs were successfully isolated and cultured,and the cultured cells were elongated to spindle shape,showing typical EPCs morphology.Di I-Ac-LDL and FITC-UEA-I double-stained positive cells reached 85%.1.2 Ox-LDL inhibited the proliferative activity of EPCs in a concentration-dependent manner.1.3 Ox-LDL inhibited the expression of p62 in concentration-and time-dependent manner.1.4 Inhibition of autophagy by Sh Atg7 and 3-MA further aggravated ox-LDL-inhibited EPCs proliferation.2.Effect and mechanism of MALAT1 on ox-LDL induced VSMCs proliferation.2.1 Ox-LDL promoted the proliferation of VSMCs in a concentration and time dependent manner.2.2 Ox-LDL promoted MALAT1 expression in a concentration-dependent manner.2.3 MALAT1 knockdown reversed the increase of VSMCs proliferation by ox-LDL.2.4 Inhibition of MALAT1 reduced ox-LDL induced AKT phaosphorylation.2.5 AKT activation relieved the inhibition effects on VSMCs proliferation by MALAT1 knockdown.2.6 Inhibition of MALAT1 inhibited STIM1 expression and SOCE.2.7 STIM1 knockdown inhibited AKT phosphorylation and VSMCs proliferation.Conclusions1.Effects of autophagy on ox-LDL induced EPCs proliferation.1.1 Ox-LDL inhibits the proliferation of EPCs while promotes the level of autophagy.1.2 Autophagy alleviates ox-LDL inhibited proliferation.2.Effect and mechanisms of MALAT1 on ox-LDL induced VSMCs proliferation.2.1 ox-LDL increases MALAT1 expression while enhances VSMCs proliferation.2.2 MALAT1 affects ox-LDL induced VSMCs proliferation via AKT phosphorylation.2.3 MALAT1 affects VSMCs proliferation through STIM1 expression SOCE and AKT phosphorylation.In conclusion,lnc-RNA MALAT1 participates in ox-LDL enhanced VSMCs proliferation enhancement,partly through regulating STIM1 expression,SOCE and AKT phosphorylation.