| BackgroundDiabetes mellitus is the most common metabolic disease in the world. The marco-and micro-vessel disorders, which could result in vascular homeostasis imbalance and vascular dysfunction, are the main causes of morbidity and mortality in the diabetic patients. Cellular senescence is a relative steady state in which the cell permanently and irreversibly loses the ability of proliferation. Endothelial cell senescence has been related to cardiovascular disease and involved in the formation and development of atherosclerosis, vascular remodeling, inflammation and thrombosis. Recent studies have demonstrated that many factors could accelerate endothelial cell senescence, such as hyperglycemia, advanced glycation end products, hydrogen peroxide, oxidized low denesity lipid, radiation, and so on. Endothelial cell senescence is also involved in the development of diabetic vascular disease.Rho-associated kinase (ROCK) is a kind of serine/threonine kinases with two isoforms, ROCK1and ROCK2. ROCK was primarily found to be involved in RhoA mediated formation of stress fibers and focal adhesion. Recent studies showed that ROCK was involved in the regulation of endothelial cell dysfunction, blood vessel elasticity, inflammation, oxidative stress and played important roles in many cardiovascular diseases, such as atherosclerosis, pulmonary hypertension, heart failure et al.Many studies have shown that high glucose could activate ROCK in endothelial cells, which is related to endothelial cell dysfunction cultured in high glucose medium. Studies in vitro found that inhibition of ROCK improved endothelial nitric oxide synthase (eNOS) mRNA stability and enhanced its activity, thereby promoting synthesis and release of nitric oxide (NO). NO level is closely related to endothelial cell senescence. Increase the level of NO in endothelial cell could decrease endothelial cell senescence, while eNOS inhibitors, asymmetric dimethyl-L-arginine and L-NAME, could promote endothelial cell senescence. Animal experiment and clinical study have shown that inhibition of ROCK activity can reduce diabetes mellitus-induced microvascular injury. These studies suggest that inhibition of ROCK may decrease high glucose-induced endothelial cell senescence. Objectives1To investigate the effects of ROCK inhibitor Y27632on high glucose-induced endothelial cell senescence.2To investigate the effects of ROCK knock-down on high glucose-induced endothelial cell senescence and compare the differences between ROCK1+/-and ROCK2+/-endothelial cells.3To explore the mechanism involved in the above-mentioned effects.Methods1Murine heart endothelial cells (MHECs) were isolated by enzymatic method and purified twice with anti-CD31antibody and anti-CD102antibody conjugated magnetic beads separately. Morphological characteristics were observed with microscopy and cells were identified by immunological staining with anti-vWF antibody.2MHECs were cultured in22mmol/L glucose for72hours to induce cellular senescence. ROCK inhibitor Y27632(10umol/L) was used in one group. The ratio of senescent cells was identified by senescence-associated β-galactosidase (SA-βG) staining and flow cytometry was used to analyse the cell cycle.Telo TAGGG Telomerase PCR ELISAPLUS kit was used to detect telomerase activity in endothelial cells and the expression of ROCK1, ROCK2, p-MBS(Thr853), MBS, p21was explored by Western blot. Nitric oxide assay kit was used to detect NO level in culture medium.3Wild type(WT) MHECs, ROCK1+/-MHECs and ROCK2+A MHECs were obtained and cellular senescence was induced with22mmol/L glucose for72hours. PI3K inhibitor LY294002(20umol/L) was used to explore the possible mechanisms involved. The ratio of senescent cells was identified by senescence-associated β-galactosidase (SA-βG) staining and the telomerase activity in endothelial cells was investigated. The expression of eNOS mRNA was detected by Real-Time PCR and the expression of ROCK1, ROCK2, p-MBS(Thr853), MBS, p-Akt(Ser473), Akt, p-eNOS(Ser1177), eNOS was explored by Western blot. Nitric oxide assay kit was used to detect NO level in cell culture medium.Results1MHECs were harvested and they exhibited typical cobblestone endothelial morphology. Immunological staining showed more than95%cells were vWF positive and believed to be endothelial cells.2Treatment of high glucose could significantly increase the ratio of senescent cells, enhance ROCK activity, increase the ratio of cells in Go/G1stage and the expression of p21, inhibit telemorase activity of the cells and NO level in culture medium (all p<0.05). However, ROCK inhibitor Y27632could greatly inhibit ROCK activity and effectively decrease the ratio of senescent cells (both p<0.05). Also, Y27632could decrease the ratio of cells in Go/G1stage and the expression of p21and reverse the decrease of telomerase activity and NO level in cells cultured in high glucose medium (all p<0.05).3Compared to the WT MHECs, ROCK activities were significantly lower in ROCK1+/-MHECs and ROCK2+/-MHECs. Knock-down of ROCK1or ROCK2could effectively decrease the ratio of senescent cells, increase the expression of eNOS mRNA, promote the phosphorylation and activation of eNOS, increase the telomerase activity and NO level in culture medium (all p<0.05). There were no differences in the aboved-mentioned indices between ROCK1+/-MHECs and ROCK2+/-MHECs (all p>0.05). However, LY294002could significantly reverse the above-mentioned effects of knock-down of ROCK1and ROCK2(all p<0.05).Conclusion1MHECs were successfully obtained with high purity from murine hearts by purifying with antibodies conjugated magnetic beads.2Activation of ROCK was involved in the endothelial cell senescence induced by high glucose.3Both ROCK inhibitor Y27632and knock-down of ROCK could effectively inhibit high glucose-induced endothelial cell senescence.4Similar effects on high glucose-induced endothelial cell senescence were observed in ROCK1+/-MHECs and ROCK2+/-MHECs.5PI3K/Akt/eNOS pathway was invloved in the role of ROCK in high glucose-induced endothelial cell senescence. |
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