| Background: Cardiovascular disease is charactered by its high morbidity,high mortality,high morbidity,and endangers people’s health seriously.Vascular homeostasis is the foundation of cardiovascular disease.Vascular endothelial cells are an important component of vascular homeostasis.Vascular endothelial cells are an important barrier between circulating blood flow and the vessel wall.Vascular endothelial cells play an important physiological function under physiological conditions,such as synthesis and release of vasoactive mediators,facilitating the exchange of substances,etc.Shear stress(SS)is an important power acting on the surface of endotelial cells(ECs)and has been paid more and more attention recently.Different levels of shear stress have different effects on the expression of endothelial nitric oxide synthase(e NOS).High SS can improve the expression of e NOS,increase the release of nitric oxide(NO)and play a protective role in atherosclerosis(AS).However,low shear stress is a detrimental cellular stress to ECs.Our study included the following two aspects.PartⅠ Effect of shear stress on expression of endothelial nitric oxide synthase in human aortic endothelial cells under oxidized low-density lipoproteinBackground: Oxidized low density lipoprotein(ox LDL)will lead to the AS by carrying oxygen free radicals,cytotoxicity,regulating endothelial cells secretion and accelerating endothelial dysfunction.NO is secreted by vascular endothelial cells.What’s more,NO plays an important role in regulating vascular tone,lowering lipid levels,and inhibiting the expressions of adhesion molecules,platelet aggregation as well as vascular smooth muscle cell(VSMC)proliferation.NO is synthesized from the amino acid L-arginine by the e NOS.Reduced NO output is a characteristic feature of dysfunction of endothelial cells.However,it’s remain unclear wheater ox-LDL has effect on shear stress-induced e NOS expression in endothelial cells.Objective: To investigate the effect of shear stress on expression of endothelial nitric oxide synthase in human aortic endothelial cells under oxidized low-density lipoproteinMethods: Culturaled HAECs were divided into control group and experimental group.Different SS(statics sub-group: 10 dyne/cm2,low SS sub-group: 4.0 dyne/cm2,and high SS sub-group: 15.0 dyne/cm2,respectively)was given to HAECs using the precision infusion pump.Solely SS was given to HAECs in control group.SS and ox-LDL were given to HAECs in experiment group.The expression of e NOS was assessed using real-time PCR and western blot analysis.Concentration of NO was measured using nitrate reductase method.Results: In control and experimental group,the expression of e NOS and concentration of NO were significant lower in low SS than those in non shear stress.Concentration of NO in 3 sub-groups in experiment group vs.similar sub-groups in control group were:(1.09±0.33)μmol/L vs.(2.27±0.90)μmol/L,(0.20±0.08)μmol/L vs.(0.71±0.36)μmol/L,and(3.03±0.71)μmol/L vs.(4.93±1.16)μmol/L.The e NOS m RNA in in 3 sub-groups in experimental group vs.similar sub-groups in control group were:(0.65±0.20)vs.(1.11±0.32),(0.22±0.13)vs.(0.49±0.22),and(1.05±0.22)vs.(1.90±0.56).The expression of e NOS/β-actin in 3 sub-groups in experimental group vs.similar sub-groups in control sub-groups were:(0.50±0.19)vs.(1.10±0.26),(0.18±0.52)vs.(0.62±0.19),and(1.07±0.20)vs.(1.70±0.38).In conclusion,the concentration of NO and the expression in 3 sub-groups in experimental groups were significantly lower comparred with same sub-groups in control group(P < 0.05).In addition,the declined ratio of concentration of NO,e NOS m RNA and e NOS/β-actin in high SS sub-group were significant lower than those in low SS sub-group(P < 0.05).Conclusion: High shear stress may be weaken the restrain effect of ox-LDL on the expression of endothelial nitric oxide synthase in human aortic endothelial cells.Low shear shear stress could aggravate the dysfunction of endothelial cell under oxidized low-density lipoprotein Part Ⅱ The molecular mechanism of mi R-21 regulates endothelial cell autophagy under low shear stressBackground: Autophagy is an evolutionarily conserved process involved in removing misfolded or aggregated proteins,clearing damaged organelles,and eliminating intracellular pathogens.Previous studies domenstrated that low shear stress have an effect on the autophage in endothelial cells.However,the molecular mechanism of this process remains unclear.Micro RNAs are small,non-coding RNAs of 18-25 nucleotides and function as posttranscriptional regulators of gene expression.Micro RNA molecules play a pivotal role in virtually all cellular functions,including apoptosis,proliferation,migration and differentiation.Previous studies domenstrated that mi RNAs play an important role in the process of shear stress mediated endothelial cells autophagy.However,the mechaisms of this process remain unclear.MiR-21 is one of the most widely studied micro RNAs.High-throughput sequencing revealed that in endothelial cells,the expression of many micro RNAs especially mi R-21 will change to some extent when cells were stimulated by shear stress.Therefore,we speculated that low shear stress influences autopgage in endothelial cells through mi R-21.Human umbilical vein endothelial cells(HUVECs)were involved in our study and to explore the effect of mi R-21 on the autophage in endothelial cells.Objective: To explore the impact of shear stress on autophagy development in endothelial cells and role of mi R-21 in this process.Methods: Low SS was given to HUVECs using the precision infusion pump.Next,mi R-2 1 mimics and inhibitor were transfected into cultured HUVECs.The effect of mi R-21 on the autophage was detected by q-PCR,Western Blotting,Immunofluorescence Staining and Transmission electron microscopy(TEM.).Results: Western Blotting domenstrated that the expression of P62 was decreased from 1.43±0.75 to 0.25±0.05,the expression of LC3Ⅱ/LC3Ⅰwas increased from 1.64±0.22 to 4.8±0.80(P<0.05)after low shear stress was acted on HUVECs.In conclusion,low shear stress significantly increases the expression of LC3Ⅱ/LC3Ⅰand decrease the expression of P62.The expression level of LC3 II / LC3 I in endothelial cells was decreased from 4.80±0.80 to 0.25±0.05 and the expression of P62 was increased from 0.25±0.05 to 0.40±0.05 after infecting mi R-21 mimics.However,after infecting mi R-21 inhibitor,The expression level of LC3 II / LC3 I in endothelial cells was incrased from 4.60±0.44 to 7.00±1.32(P<0.05)and the expression of P62 was decreased from 0.54±0.04 to 0.33±0.42(P<0.05).To sum up,the expression level of LC3 II / LC3 I in endothelial cells was decreased and the expression of P62 was increased in mi R-21 mimics group.Conversely,after transfected mi R-21 inhibitor into endothelial cells,the expression level of P62 decreased and LC3Ⅱ/LC3Ⅰincreased(P <0.05).Immunofluorescence revealed that compared with control group,LC3 fluorescent sports increased from 448.79±118.47 to 1569.16±617.17(P<0.05)in low shear stress group.the LC3 fluorescent sports in endothelial cells were reduced from 1569.16±617.17 to 381.33±108.27(P<0.05)after infecting mi R-21 mimics.However,after transfected with mi R-21 inhibitor into endothelial cells,the LC3 fluorescent sports in endothelial cells were increased from 3245.76±1047.90 to 16092.77±2215.37(P<0.05)(P <0.05).In summary,LC3 fluorescent sports increased in mi R-21 inhibitor group and decreased in mi R-21 mimics group(P <0.05).TEM showed that low shear stress could increase the number of autophagic cells in endothelial cells.The number of autophagic body decreased after transfecting of mi R-21 mimics into endothelial cells.After transfected mi R-21 inhibor into endothelial cells,the number of autophagic body increased in HUVECs(P <0.05).Conclusions1.Low shear stress regulates the levels of mi R-21;2.Low shear stress regulated endothelial cell autophagy through mi R-21. |
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