| Objective: Smooth muscle 22 alpha(SM22?),an actin-binding protein,is highly expressed in differentiated VSMCs and involves in cytoskeletal rearrangements and phenotype regulation of VSMCs.Our group has previously demonstrated that SM22? deletion aggravates arterial inflammation.The effects of SM22? on VSMCs have been well documented.The indirect effect of SM22? on ECs is unclear.In the present study,the EC functions were detected after treated with VSMC conditioned medium(VSMC-CM)from wild type(WT)or SM22? knockout(SM22?-/-)mice.Methods:1 Cultured VSMCs from WT or SM22?-/-mice were treated with AngII(100 nmol/L)for 24 h,and the interaction between vascular cell adhesion molecule 1(VCAM-1)and smooth muscle ?-actin(SM ?-actin)was detected by immunoprecipitation and double immunofluorescence staining.2 VSMC-CM were collected from WT or SM22?-/-mice after 24 h treatment with AngII(100 nmol/L),then treated the human umbilical vein endothelial cells(HUVECs)with VSMC-CM for 24 h.The expressions of endothelial nitric oxide synthase(eNOS)and monocyte chemoattractant protein-1(MCP-1)were detected using Western blot in HUVECs.The content of nitric oxide in the cultured medium was analyzed by Nitric Oxide Assay Kit.3 After treatment with WT or SM22?-/-VSMC-CM,the chemotactic activity for macrophages was measured by the modified Boyden chamber transmembrane migration assay in HUVECs.Results:1 SM22? deletion promotes AngII-induced membrane-bound VCAM-1 expression in VSMCsIn order to illuminate the relationship between the expressions of F-actin and VCAM-1 on the membrane surface,the F-actin fraction was isolated and precipitated with SM ?-actin antibody,and then detected by Western blot.The results showed that VCAM-1 was almost undetectable from F-actin in the non-stimulated WT VSMCs,while a mild interaction of VCAM-1 with F-actin fraction was found in the non-stimulated SM22?-/-VSMCs.After stimulation with AngII,the VCAM-1 binding to SM ?-actin was significantly increased in SM22?-/-VSMCs compared with that of WT VSMCs.These results were verified by immunofluorescence staining.The abundant actin stress fibers arranged parallelly in bundles in WT VSMCs,whereas SM22? deletion decreased the density of stress fibers,and increased cortical F-actin polymerization.After treatment with AngII,the co-localization of cortical F-actin and VCAM-1 was significantly increased in SM22?-/-VSMCs.The results indicate that SM22? deletion promotes AngII-induced the expression of membrane-bound VCAM-1 in VSMCs.2 SM22?-/-VSMC-CM inhibits the expression of eNOS and releasing of NO in HUVECs.Western blot showed that the expression of eNOS was inhibited in endothelial cells after incubation with SM22?-/-VSMC-CM.AngII significantly enhanced the inhibition of eNOS expression in HUVECs after treated with SM22?-/-VSMC-CM for 24 h compared with WT(P<0.01).The releasing of NO was significantly decreased in HUVECs treated with SM22?-/-VSMC-CM compared with WT(P<0.01),which showed the similar pattern in consistent with eNOS expression in each group.3 SM22?-/-VSMC-CM induces the secretion of MCP-1 in endothelial cellsWestern blot assay showed that the expression of MCP-1 was significantly increased in HUVECs after treatment with SM22?-/-VSMC-CM,and AngII enhanced the induction activity of SM22?-/-VSMCs-CM by compared with WT(P<0.05).Furthermore,HUVECs treated with SM22?-/-VSMC-CM increased the transmembrane migration activity of macrophages,which was positively correlated with the expression of MCP-1.The results indicated that SM22? deletion enhances AngII-induced EC dysfunction.Conclusions:1 SM22? deletion promotes VSMC inflammatory response.2 SM22? deletion induces EC dysfunction through VSMC paracrine pathway. |
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