The Study Of The Molecular Mechanisms Of HSP60and Vascular Smooth Muscle Cell Migration And Proliferation

Objective: Atherosclerosis (AS) is a common diseases and frequently-occurringdisease, which severely threaten human health and often leads to fatal cardiovascularand cerebrovascular diseases, such as cerebral infarction, myocardial infarction.Atherosclerosis early pathology was described as a end stage degenerative disease, itwill lead to the extensiveness of the arterial lumen of the stenosis, the process of theiroccurrence and their complexity, by a variety of factors involved in chronicinflammatory processes. In atherosclerotic plaques biological characteristics,endothelial cells, vascular smooth muscle cells and inflammatory are significantassociation.Phenotypic transformation of vascular smooth muscle cells (VSMCs)，proliferation,migration is a key factor in atherosclerosis incidence.Vascular smooth muscle cells aredifferent from other cells, When it is subjected to outsiders injury, the cells will reduceits specificity the telescopic protein expression, and thus can increase cell proliferationand migration, at the same time, the matrix metalloproteinase synthesis will be acorresponding increase, this process can promote the repair of vascular injury, If thisrepair process is abnormal, it will lead to some diseases, such as hypertension,atherosclerosis and vascular stenosis and other vascular diseases. Therefore, thechange of phenotype of vascular smooth muscle cells of vascular smooth muscle cellsplays a very important role in the formation of vascular disease. Smooth muscle cellstate is divided into "synthesis" and "expansion", which show the shift between thesetwo states. When the cells exhibited a "synthetic" state, the "expansion" state of thegene in the inhibition state, and the proliferation and migration of some genes related tothe high expression. This kind of "synthetic" state of the cells can secrete the relevantmatrix metalloproteinase and elastin, which eventually led to the repair and remodeling of damaged blood vessels. In atherosclerotic plaque formation, plaque rupture,associated with smooth muscle cell proliferation.Heat shock protein (HSP) is a biological subject to outside adverse stimuli reactionof the stress protein, exists in prokaryotes and eukaryotes organisms, it may be theimmune system as exogenous molecules to induce autoimmune reactions. The role ofthis protein is involved in cell proliferation, cell cycle and apoptosis. So far, it has beenfound over twenty kinds of HSP. Many of them are named in molecular weight, such asHSP10, HSP20, of HSP27, HSP40, HSP60, of HSP70etc. Some studies have shownthat the HSP and atherosclerosis closely related to the development process occurs.Such as HSP27can regulate actin protein activity, anti-apoptotic, anti-oxidative stress,anti-inflammatory effect. In atherosclerosis plaques, HSP27gene expression can bedetected. Since HSP27and HSP20in the structure are high homology, HSP20in thesmooth muscle cells can also be highly expressed, and play a regulatory role on the cellmigration, muscle contraction, and then participate in the formation of atherosclerosisartery. In addition, in patients with coronary atherosclerosis, the serum is detected theexpression of HSP70. Some studies have shown that the proliferation of smooth musclecells in rabbit atherosclerotic model with HSP70gene expression was positivelycorrelated. With the thickening of the intima, the HSP70gene expression levels weresignificantly increased.The current research data of epidemiology, pathology and animal models showsthat HSP-60is closely related to the development of the AS. Studies have shown thatendogenous of HSP60can be detected within the cells in the human atherosclerotic area,and the extent of disease and the amount of the expression of HSP60in a positivecorrelation. Also some research found that HSP60as a pro-inflammatory factor, showdifferent expression levels in patients with heart disease and different degrees ofcoronary artery disease in patients with serum. And higher expression of HSP60serumlevel was positively correlated with the severity of the patient’s condition. Serum HSP60expression level is higher, the possibility of occurrence of cardiovascular disease isgreater.The existing literature suggests HSP-60induced AS may occur mainly for tworeasons.(1) biological vessel wall to produce a large number of endogenous HSP-60subject to a variety of external stimuli, as a self-antigen can stimulate the localimmunepathogen infection host response, damage vascular endothelial system topromote the AS occurred development.(2) pathogen infection, for exogenous antigen and pathogens antibody generated complex deposition in the vessel wall, and thusactivate complement body, regulating T-cell function, inducing the production ofcytokines and adhesion molecules in vascular wall, to promote the progress of AS.HSP60as the antigen is recognized by the cell surface receptor of immune cell,such as TLR like receptors (TLR-2or TLR-4). A number of studies have reported thatTLR-like receptor is a kind of small molecule HSP receptor. The receptor can activateNF-κB pathway, and then release proinflammatory cytokine. And the formation ofTLR-4gene mutation and polymorphism and atherosclerosis are closely related.Although many studies have shown that HSP60with AS are closely related,however, research on the molecular mechanisms of HSP-60and AS formation are rarelyreported. This study will focus on the role of proliferation and migration and the relatedsignal transduction pathway by HSP60in rat aortic vascular smooth muscle cells (A7r5),in order to futher explore the molecular mechanisms of HSP-60and AS formation,study for the pathogenesis of AS and explore new areas..Methods:(1) In cultured vascular smooth cells (A7r5) under the conditions of42℃for1hour, and then after37℃for24h after extraction of cytosolic protein, theexpression of HSP60was detected by Western blot, and cultured at37℃and normalA7r5cells extracted proteins were compared, thermal stimulation on the expression ofendogenous HSP60.(2) Boyden Chamber method to observe the effect of HSP60oncell migration. By different concentration of HSP60(1ng/ml,5ng/ml,10ng/ml,20ng/ml,50ng/ml) were used to stimulate VSMC5h and24h, to observe whether it can inducecell migration, and determine the best cell migration induced by concentration of10μmol/L respectively;,20μ mol/L U0126(p-ERK inhibitor) pretreatment of cells for1hours, then the cells stimulated with10ng/ml HSP6024h, observation of cell migrationis suppressed; the TLR4antibody a closed cell surface receptor for1hours, then10ng/ml stimulated with/ml HSP6024h, observation of cell migration is suppressed.(3)Effect of HSP60on the scratch test cell migration. By using10ng/ml HSP60stimulationof VSMC for24h,48h, the cell migration was observed in the microscope.(4) TheMTT method to observe the changes of cell proliferation. By different concentrations ofHSP60(1ng/ml,5ng/ml,10ng/ml,20ng/ml,50ng/ml) stimulation of A7r5cells for24h,To observe the effect on the regulation of cell proliferation, to determine the optimumconcentration of inducing cell proliferation. The cells were prereated with10μ mol/L,20μ mol/L U0126for1hours, and then cell proliferation was observated. A closed cellsurface receptor antibodies in1hours, then the cells stimulated with10ng/ml HSP60for 24h, cell proliferation was detected.(5) By using10ng/ml HSP60stimulation of VSMCfor24h, the changes of cell cycle was detected by flow cytometry.(6) By differentconcentrations of HSP60(1ng/ml,10ng/ml,20ng/ml,50ng/ml,100ng/ml) in differenttime points (10min,30min,45min,1H,2h,4h) respectively after VSMC stimulation,the expression of p-ERK induced by HSP60was detected by Western blot, to observethe cell proliferation and migration.(7) The cell proliferation and migration induced byHSP60through the cell surface receptor TLR2or TLR4mediated were detected byReal Time PCR method.(8) A7r5were pretreated with TLR4antibody for1hours, andthen by10ng/ml HSP60treated for24hours, the expression of p-ERK was detected byWestern blot.Results:(1) after42℃cells cultured for1hours, extraction of protein and normalcultured at37℃for intracellular proteins, endogenous HSP60expression increasedsignificantly.(2) the Boyden Chamber results showed that: different concentrations ofHSP60(1ng/ml,5ng/ml,10ng/ml,20ng/ml,50ng/ml) after VSMC stimulation, caninduce smooth muscle cell migration, and cell migration quantity was positivelycorrelated with concentration. The largest number of cellmigration with theconcentration of10ng/ml HSP60(P<0.05), so the concentration of10ng/ml isdetermined as the optimal induction concentration. With10μ mol/L and20μ mol/LU0126pretreatment of cells for1hours, the number of migrated cells reduced, showedthat HSP60induced VSMC migration is related with ERK signaling pathway. Aclosed cell surface receptor with TLR4antibody for1hours, cell migration was alsodecreased, showed that HSP60induced VSMC migration and TLR4receptor related.(3)the scratch results showed that cells in10ng/ml HSP-60for24h, and48h stimulation,migrated cells are obvious.(4) the MTT method to observe the cell proliferation resultsshowed: by using different concentrations of HSP-60(1ng/ml,5ng/ml,10ng/ml,20ng/ml,50ng/ml) after stimulating, the cells proliferation in1ng/ml and5ng/ml HSP60did not change significantly.However, with10ng/ml or20ng/ml HSP60stimulation, cellproliferation was increased significantly (P <0.05), in which10ng/ml HSP60cellproliferation was more obvious. With10μ mol/L or20μ mol/L U0126pretreatment ofcells for1hours, cell proliferation decreased significantly (P <0.05). Similar the TLR4antibody a closed cell surface receptor for1hours, cell proliferation quantity alsodecreased significantly (P <0.05).(5) Clls stimulated with10ng/ml HSP60, significantlyincreased the cell cycle was detected by flow cytometry in the S stage.(6) of differentconcentrations (1ng/ml,10ng/ml, HSP6020ng/ml,50ng/ml,100ng/ml) after VSMC stimulation, the expression of p-ERK was increased by Western blot detection, andp-ERK protein expression was increased obviously at10ng/ml concentration of HSP60.The different time points (10min,30min,1H,2h,4h) after VSMC stimulation, p-ERKexpression was also increased, the expression of p-ERK was increased obviously at10min.(7) of different concentrations of HSP60(1ng/ml,10ng/ml,20ng/ml,50ng/ml,100ng/ml) after VSMC stimulation, the expression of TLR-2and TLR-4receptors byReal Time PCR method in the mRNA level showed that HSP60could promote theexpression of TLR-4receptor, but not TLR-2receptor. In the10ng/ml HSP60stimulation, the highest expression of TLR-4.(8) With TLR4blocking antibody, thep-ERK expression decreased obviously by Western blot.Conclusions:(1) HSP60is able to promote the expression of endogenous HSP60in42℃.(2) HSP60can induce the proliferation and migration of vascular smoothmuscle cells, and are positively with concentration, time. The cell proliferation andmigration is more obvious in the10ng/ml HSP60.(3) HSP-60promote the proliferationand migration of vascular smooth muscle cells is related to MAPK signal pathway byincreasing ERK phosphorylation.(4) Proliferation and migration of vascular smoothmuscle cells by the regulation of HSP60are interacted with cell surface receptorsTLR-4, ERK phosphorylation to achieve intracellular.