Protective Effects Of Atorvastatin On The Contractile Dysfunction Of Aorta

Part one: Atorvastatin improves aortic contractile dysfunction by regulating Ca2+ Signaling and the mechanismBackground: Atorvastatin(ATORV)is widely used for the treatment of hypercholesterolemia and secondary prevention of cardiovascular and cerebrovascular diseases,and its vascular protective function is independent of its lipid-lowering effect.In recent years,studies on vascular function have focused on vascular endothelial cells and vasodilation.However,in many instances,dysfunction of smooth muscle cells also plays an important role in the Aortic systolic dysfunction.The proliferation and migration of vascular smooth muscle cells(VSMCs)in the formation of atherosclerotic plaques,vascular development and the repair of lesions(eg,the stent restenosis and other pathological conditions)have been found to play a vital role in these pathological and abnormal physiological processes.Phenotypic transformed cells have stronger ability of secretion,synthesis,proliferation and migration.Statins inhibit the phenotypic transformation of VSMCs,but the underlying mechanism is not yet fully elucidated.Ca2+ signal is involved in the contraction of smooth muscle cells.Ca2+ signal is involved in the contraction of smooth muscle cells.The change of intracellular Ca2+ concentration is closely related to the phenotypic transformation of VSMCs.There is a difference in Ca2+ signaling between contractile phenotypes and synthetic cells.The Ca2+ transporters expressed by contractile VSMCs include voltage-gated L-type calcium channels and SERCA pumps and store operated calcium channel(SOCC).They maintain low levels of intracellular Ca2+ and dynamic changes of Ca2+ in space and time.However,systolic VSMCs dependent voltage-gated Ca2+ decreased significantly.Calcium imbalance was closely related to the phenotypic transformation of VSMCs.It is involved in the formation of atherosclerotic plaques,hypertension,diabetes mellitus.But the role of atorvastatinin the protection of aorta from contractile function,and if the calcium signaling pathways and the phenotypic switching of VSMCs are involved is still unclear.Objective: ATORV can inhibit the transformation of VSMCs from the contractile phenotype to the synthetic phenotype.The effect of its pleiotropic mechanism is not clear.This study was to investigate the effect of atorvastatin on the systolic dysfunction of cultured thoracic aorta and explore the regulation of Ca2+ signaling and the phenotypic transformation mechanism of VSMCs.Method: 1.Animal grouping: Male Sprague Dawley rats,SPF grade,weighing 220-250 g.In fresh group,cultured group,cultured + ATORV group.2.Preparation and cultured thoracic aortic vascular rings: rats were sacrificed with CO2 gas,and the thoracic aorta was immediately taken.The thoracic aorta was dissected with a surgical scissors and placed in serum-free Petri dishes with 95% O2 in the mixture.After incubation with 95% O2/5% CO2 incubator for 24 hours without FBS,the cultured thoracic aorta was placed in an already saturated Krebs solution containing 95% O2 and 5% CO2 until the vascular experiment was performed.3.Changes of isometric froce were determined on rat thoracic aortas in myograph: The vasoconstrictor response was induced by Phenylephrine(Phenylephrine,Phe)and high concentration of potassium chloride(60 mm m M KCl)in serum-free cultured of aorta,and the changes in the basic function of blood vessels were observed,and the intervention mechanism of atorvastatin was observed.L type calcium channel blocker nifedipine was used to block L type calcium channel,and the role of L type calcium channel in aortic vasoconstriction was observed.4.The protein expression of anti-?1A receptor,anti-inositol tr-isphosphate(IP3)receptor,anti-protein kinase C(PKC?),anti-matrix sympathotroph 1(STIM1),Orail,anti-High-voltage activated dihydropyridine-sensitive(L-type calcium channel,Cav1.2)channels and contractile phenotypic proteins such as anti-alpha smooth musc-le actin(alpha-SMA),anti-myosin(SM-MHC)were detected by western blot.5.Statistical analysis: All experimental data were analyzed by SPSS22.0 and sigmaplot software was used for the mapping.All data were compared by analysis of variance and expressed as x±s.Results: 1.Muscle tonegram technique is used to detect the response of serum-free cultured thoracic aortic rings for high concentrations of potassium chloride(60 m M KCl)and vasoactive substances such as Phe for 24 h.Compared with the fresh group,cultured aorta group's contractile tension induced by KCl and Phe were decreased significantly(P<0.05).The 20?M of atorvastatin increased the contraction tension of cultured aorta.and the same results were also obtained with Nifeidipine + Phe and calcium-free KH solution + Ca Cl2.2.Compared with fresh aortic group,the aortic group was partially ruptured,and the smooth muscle cells were arranged disorderly.After the acute treatment of ATORV,the elastic layer was partially repaired,and the smooth muscle cells were arranged relatively neatly.3.The proten expression levels of ?-A1 adrenergic receptor,IP3 receptor,PKC?,STIM1,Orail,Cav1.2,and ?-SMA and SM-MHC in cultured aorta group were decreased(P<0.05).In the Cultrued+ATORV group,there was no statistical difference in the expression level of Orail protein,and the expression level of the other target proteins increased,compared with the cultured aorta.Therefore,ATORV could improve the contractions in aorta by regulating Ca2+ sensitivity,releasing calcium from the endoplasmic reticulum,and opening the L-type calcium channel,SOC signaling pathway may play a minor role;At the same time,in the culture of aorta,VSMCs were transferred from synthetic to contractile type.Atorvastatin inhibited the phenotypic transformation of VSMCs to protected blood vessels.The possible mechanism was through the alpha 1-adrenoceptor/PKC Delta /Cav1.2 or alpha 1-adrenoceptor/IP3/STIM1/Orai1 pathway.Conclusion: 1.The contraction dysfunction of cultured aorta may be related to the phenotypic transformation of aortic VSMCs.2.ATORV protects aortic contraction by inhibiting the phenotypic transformation of VSMCs and calcium signaling pathway,which may provide a new pharmacological theoretical basis for statin as a secondary prevention and treatment of cardiovascular and cerebrovascular diseases.Part two: Atorvastatin improves phenotypic transformation and regulates calcium signaling pathways on hyperostatic pressure aortic smooth muscle cellsBackground: Hypertension is one of the independent risk factors for cardiovascular and cerebrovascular diseases.The basic pathological changes are vascular remodeling,which manifests as proliferation,hypertrophy,and migration of VSMCs,which results in the enlargement of the arterial wall and the reduction of the diameter of the media,resulting in an increase in blood flow resistance.Phenotypic transformation of VSMCs throughout the development of hypertensive lesions;L-type calcium channels mediate SMC involvement in vasoconstriction disorders of hypertension,and appearance of smooth muscle cell contractile protein markers(including alpha-actin,SM22?,calponin,and SM-MHC)inhibition.In patients with essential hypertension or animal experiments,atorvastatin was found to inhibit restenosis after balloon dilatation in rabbit aorta,proliferation and migration of VSMCs,inhibit the thickening of the intima and narrowing of the lumen,and improve the reconstructed blood vessels.Therefore,based on the phenotypic transformation of SMC cells,we further investigated the effects of ATORV on the changes of the contractile phenotypic markers of VSMCs induced by high hydrostatic pressure on the basis of the phenotypes of the previous chapters,and explored the mechanisms for the prevention of vascular remodeling.Objective: To explore the phenotype transformation and mechanism of aortic VSMCs under ATORV intervention with high hydrostatic pressure.Method: 1.Primary aortic VSMCs of SD rats were cultured and passaged using tissue adherence method,and VSMCs were identified by immunofluorescent staining.2.The expression of phenotype-associated protein ?-SMA and OPN,IP3 receptor,PKC?,STIM1,Orail and Cav1.2 in VSMCs after treatment with atmospheric pressure and 180 mm Hg pressure in aorta VSMCs and ATORV intervention were detected by western blot.Results: 1.Compared with the atmospheric pressure group,the ?-SMA protein levels in VSMCs cultured under high-hydrostatic pressure was decreased significantly,and OPN protein levels was increased.,Atorvastatin can regulate the imbalance of ?-SMA and OPN protein levels.2.The protein expression levels of Cav1.2,IP3 R,PKC? in VSMCs cultured with high hydrostatic pressure were increased significantly.Atorvastatin treatment could down-regulated the protein levels.But there was no significant difference in the expression of Stim1 and Orai1 between the atmospheric pressure group and high hydrostatic pressure group.Conclusion: 1.The transformation of VSMCs from the contractile phenotype to the synthetic phenotype could be induced by high hydrostatic pressure(180mm Hg).2.In VSMCs cultured with high hydrostatic pressure(180mm Hg),the increased calcium influx was mediated by Cav1.2/IP3R/PKC? pathway,but not,SOC;ATORV may improve phenotypic transformation of VSMCs induced by high pressure through regulating the Ca2+ sensitivity,endoplasmic reticulum calcium release,L-type calcium channel opening,lower effect on SOC was observed and needs to be further confirmed.