| Objective: Spontaneous subarachnoid hemorrhage(SAH,subarachnoid hemorrhage)is a neurological critically illness with high incidence,disability rate and mortality rate,aneurysmal subarachnoid hemorrhage occupies the main position.Previous studies have suggested that the cerebral vasospasm(CVS,cerebral vasospasm)is one of the serious complications after subarachnoid hemorrhage,and bears the primary responsibility for the high disability rate and mortality rate.It is also the main cause of delayed ischemic neurological dysfunction(DIND,delayed ischemic neurological deficit)after subarachnoid hemorrhage.However,recently,the dysfunction of cerebral vascular autoregulation was regarded as a critical contributor to the delayed cerebral ischemia,brain swelling and vasogenic edema.As the basic structural and functional component of cerebral vascular neural network,vascular smooth muscle cells(VSMC)is essentially responsible for stabilization of vascular tone and regulation of blood flow.Peroxisome proliferator-activated receptor β/δ(PPARβ/δ)has been proved implicated in the modulation of vascular cells proliferation and vascular homeostasis.In this study,we explore the effect of PPARβ/δ on VSMC phenotype transformation and cerebral vascular remodeling by establishing the model of VSMC hemoglobin stimulation and experimental subarachnoid hemorrhage in rats.Further explore the underlying mechanism,and shed new light on treatment of delayed cerebral ischemia.Methods:1.The primary cerebral vascular smooth muscle cells of circle of Willis arteries and basilar artery of SD rat were cultured and identified.VSMCs less than 6 passages were used in the following experiments.VSMCs were exposed to hemoglobin at different concentrations ranged from 1μM to 20μM for 24 hours before following assays.Phenotypic markers of vascular smooth muscle cell transformation: α-SMA,SM-MHC,OPN,Smemb were detected by Western Blot or double immunofluorescence.2.VSMCs were pre-incubated with 1μM GW0742 for 24 hours or were transfected with Ad-PPARβ/δ for 48 hours,and then all cells were stimulated with 10μM hemoglobin for additional 24 hours.Phenotypic markers of vascular smooth muscle cell transformation: α-SMA,SM-MHC,OPN,Smemb and PPARβ/δ expression were detected by Western Blot or double immunofluorescence.3.VSMCs were transfected with 50 n M of si-PPARβ/δ or NC-si RNA for 48 hours,and then all cells were stimulated with 10μM hemoglobin for additional 24 hours.Phenotypic markers of vascular smooth muscle cell transformation: α-SMA,Smemb,and PPARβ/δ expression were detected by Western Blot.4.VSMCs were pre-incubated with 1μM LY294002 30 minutes before incubation with Ad-PPARβ/δ or GW0742,and all groups were incubated with 10μM hemoglobin for another 24 hours.Western Blot was used to detect phenotypic markers of vascular smooth muscle cell transformation: α-SMA,SM-MHC,OPN,Smemb,and Myocardin,p-Akt expression.Double immunofluorescence was used to detect SRF semi quantitative and nuclear translocation.5.The endovascular perforation model of SAH was implemented in SD rats,and intracerebroventricular injection of Ad-PPARβ/δ or Ad-GFP.Neurological impairments were blindly evaluated using an 18-point score system known as the modified Garcia scale and another 4-point score system known as the beam balance test.The basilar arteries and circle of Willis arteries were used to extract total protein.Western Blot was used to detect phenotypic markers of vascular smooth muscle cell transformation: α-SMA,Smemb,and PPARβ/δ expression.Double immunofluorescence was used to detect semi quantitative of α-SMA and Smemb,and measure the inner diameter and the thickness of basilar artery.Results:1.The α-SMA decrease and Smemb increase was detected within cultured VSMC after hemoglobin treatment.With the increased dose of hemoglobin concentration up to 10μM,the α-SMA and Smemb expression exhibited maximal changes.However,excessive dose of hemoglobin resulted in the declined expression of both proteins.Ten μM(optimal stimulating concentration)was selected to perform subsequent experiments.The expression of α-SMA and SM-MHC in Hb group was significantly suppressed as compared to the control group.In contrast,it enhanced the expression of Smemb and OPN proteins.Likewise,after hemoglobin treatment decreased immunoreactivity of α-SMA and increased immunoreactivity of Smemb was observed within cultured VSMC.2.The expression of α-SMA and SM-MHC in Hb group was significantly decreased as compared to the control group.In contrast,it enhanced the expression of Smemb and OPN proteins,and accompanied by a decrease in PPARβ/δ.However,this change was reversed by GW0742 or Ad-PPARβ/δ treatment.Moreover,GW0742 or Ad-PPARβ/δ significantly enhanced the expression of α-SMA and SM-MHC,while suppressed the OPN and Smemb expression even at the presence of hemoglobin.Double immunofluorescence confirmed that GW0742 increased the α-SMA expression,and decreased the Smemb expression.3.The level of PPARβ/δ in cerebral VSMC was strongly suppressed by transfection of si-PPARβ/δ,whereas NC-si RNA showed no significant effects.After exposure to hemoglobin,however,no significant difference on α-SMA and Smemb expression was induced by PPARβ/δ decline.4.Immunoblotting results indicated no significant difference of Akt activation before and after hemoglobin treatment.However,both GW0742 and Ad-PPARβ/δ treatment induced significant increase in p-Akt level.Then LY294002 was used to inhibit the PI3K/AKT activity,and the effects of Ad-PPARβ/δ and GW0742 on VSMC phenotypic switch were abolished,as indicated by inhibited α-SMA and SM-MHC expression,as well as elevated Smemb and OPN level.After hemoglobin incubation,the Myocardin expression within VSMC was dramatically suppressed.Ad-PPARβ/δ and GW0742 promoted Myocardin expression in the presence of hemoglobin,which was blocked by LY294002.On the other hand,SRF nucleus translocation was impeded by hemoglobin treatment,and overexpression of PPARβ/δ by Ad-PPARβ/δ attenuated the effects of hemoglobin on SRF nuclear translocation.However,LY294002 prevented SRF nuclear translocation in spite of Ad-PPARβ/δ treatment.5.The Smemb expression increased as early as 6 hours after SAH then peaked after 72 hours.Therefore,subsequent studies were performed at 72 hours post-SAH.Immunoblot analysis revealed that PPARβ/δ expression increased with Ad-PPARβ/δ administration,however,α-SMA expression decreased within isolated basilar artery and circle of Willis arteries at 72 hours after SAH,accompanied by Smemb overexpression.Afterwards,the expression of α-SMA was significantly enhanced by Ad-PPARβ/δ treatment,while Smemb expression was inhibited.Consistently,Ad-PPARβ/δ increased intensity of α-SMA and decreased intensity of Smemb was observed after SAH respectively.Furthermore,the thickness of vessel wall and lumen stenosis in basilar artery following SAH was significantly attenuated by Ad-PPARβ/δ treatment.SAH animals exhibited severe behavior deficits compared with sham animals.However,the Ad-PPARβ/δ+SAH group had a significant improvement in neurological function compared with the SAH group both in modified Garcia scale and beam balance test.Conclusion:1.Regulation of PPARβ/δ expression in cultured cerebral VSMC effectively alters its phenotypic switch after hemoglobin incubation.2.PPARβ/δ suppresses cerebral VSMC phenotypic switch partially through activating PI3K/AKT pathway.3.PPARβ/δ-induced PI3K/AKT activation contributes to Myocardin expression and SRF nuclear translocation.4.PPARβ/δ regulates VSMC phenotypic switch and pathologic vascular remodeling in SAH rats,and alleviated neurological impairment. |
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