SCAP Regulates The Activation Of NLRP3 Inflammasome In Vascular Smooth Muscle Cells And Its Role On Atherogenesis

Objective:Atherosclerosis（As）is a chronic inflammatory disease,usually accompanied with hyperlipidemia.Cardiovascular diseases are one of the main causes for resident’s death.There are several theories about the mechanism of As,including:Smooth muscle monoclonal theory,Thromobogenic theory,Lipid deposition theory,Chronic inflammatory response,and Injury response theory.However,they do not work alone,each theory has direct or indirect interaction with each other to promote the occurrence and development of As.During the last 30 years,although the clinical and basic studies about As was significantly increased and many important achievements has been made,it is of great physiological and clinical significance to further study and elucidate the pathogenesis of AS.Sterol regulatory element binding protein（SREBP）cleavage-activating protein（SCAP）is a cholesterol sensor and plays an important role in the maintenance of cholesterol homeostasis.The high expression of SREBP2 in endothelial cells can promote the occurrence and development of As,however,there are few studies on SCAP in vascular smooth muscle cells（VSMCs）and its mechanism in AS.Recent studies have shown that there is a“crosstalk”between SCAP and inflammatory signaling pathways.However,it remains unclear whether SCAP overexpression in the vascular smooth muscle cells（VSMCs）promotes vascular media inflammation and accelerates the initiation and progression of As.Therefore,this study mainly investigated the role and related mechanism of sterol-resistant SCAP（D443N mutation）overexpression in VSMCs in local vascular inflammation and As,so as to provide relevant targets for the prevention and treatment of As.Methods:（1）Firstly,we have established a transgenic knock-in mouse model of As with an activating D443N mutation at the sterol-sensing domain of SCAP(SCAP^D443N)by microinjection.Secondly,we extracted mouse tail DNA by heat lysis to verify the genotype of mice.Finally,to verify whether SCAP^D443N mice were successfully established,we further used q RT-PCR,immunohistochemistry,western blot and immunofluorescence to detect the expression of SCAP in the aortas of mice.（2）In 10 SCAP^D443N/ApoE^-/-mice and 10 ApoE^-/-mice,the animals were sacrificed after 24 weeks of high-fat diet.Serum,aorta,heart and other tissues were collected.Serum lipid levels in each group were detected by Elisa.Lipid accumulation in atherosclerotic plaques was detected by HE staining and oil red O staining.The collagen fiber content in plaque were detected by masson staining and sirius red staining.The m RNA levels of aortic inflammatory factors,chemokines and adhesion molecules were detected by q RT-PCR.The expression of TNF-α,IL-1β,IL-6,IL-18 and CD68 in atherosclerotic plaques were detected by immunohistochemistry.Immunofluorescence was used to detect the expression of SCAP and NLRP3 in the plaque of SCAP^D443N/ApoE^-/-mice and ApoE^-/-mice.Then,SCAP overexpressed lentivirus was transfected into VSMCs to construct SCAP overexpressed VSMCs.After transfection,the expression of NLRP3,procaspase-1,Cleaved caspase-1,IL-1β,and IL-18 in VSMCs were detected by western blot.The contents of IL-1βand IL-18 in SCAP overexpression of VSMCs supernatant culture medium were detected by Elisa.The interaction between NLRP3 and SCAP in VSMCs was detected by co-immunoprecipitation.The co-localization of NLRP3 or SCAP on the Golgi was detected by immunofluorescence.Furthermore,in order to observe whether lycorine could inhibit the activation of NLRP3inflammasome in VSMCs,SCAP overexpression of VSMCs was preincubated with SCAP inhibitor lycorine.Western blot was used to detect the expression of NLRP3,procaspase-1,Cleaved caspase-1,IL-1β,and IL-18 in VSMCs after lycorine pretrement.Meanwhile,the levels of IL-1βand IL-18 in SCAP overexpression of VSMCs supernatant culture medium were detected by Elisa.Immunofluorescence was used to detect the effect of lycorine on co-localization of NLRP3 or SCAP on the Golgi.（3）SCAP^D443N mice and wild-type(SCAP^+/+)mice were used as animal models.After 12 or 24 weeks of high-fat diet,respectively,the aortic valve of the heart was taken for oil red O staining and immunohistochemical staining.According to the results of oil red O staining,the effects of different periods of high-fat diet on lipid accumulation and As formation were analyzed.Lipid levels in each group were detected by Elisa.The m RNA expressions of inflammatory factors,chemokines and adhesion molecules in the aorta of SCAP^+/+and SCAP^D443N mice were detected by q RT-PCR.The expression of NLRP3 in the aorta of SCAP^+/+and SCAP^D443N mice was detected by western blot.Immunohistochemistry was used to detect the expression of vascular cell adhesion protein 1（VCAM-1）and intercellular adhesion molecule1（ICAM-1）in the endothelial cells of the aortic valve of SCAP^D443N mice.After exposed to SCAP overexpression VSMCs conditioned media（SCAP CM）,the apoptosis of endothelial was assessed by flow cytometry analysis.The expression of VCAM-1 and ICAM-1 in endothelial cells was detected by immunofluorescence,q RT-PCR and western blot.Finally,after lycorine preincubation,the SCAP overexpression of VSMCs supernatant was collected for incubation of endothelial cells,and then the apoptosis of endothelial cells was detected by flow cytometry.The expression of VCAM-1 and ICAM-1 in endothelial cells were detected by western blot and immunofluorescence staining.Results:（1）The expression of SCAP in aorta,liver and kidney were detected by q RT-PCR.The results showed that SCAP m RNA expression in the aorta of SCAP^D443N mice was significantly increased.In addition,we also found that the m RNA expression of downstream signaling molecules of SCAP,such as HMGCo AR and LDLr,was also significantly increased in the aorta of SCAP^D443Nmouse.Then,immunohistochemistry and western blot analysis showed a significant increase in SCAP expression in the aorta,but no significant difference in renal and liver.Finally,the co-localization of SCAP andα-SMA was detected by immunofluorescence,and the results showed that the expression of SCAP was significant increased in the aortic VSMCs.（2）The data showed that there were significant increases in the plaques and necrotic core area within plaques of SCAP^D443N/ApoE^-/-mice.HE and oil red O staining showed that compared with ApoE^-/-mice,the atherosclerotic plaque area of SCAP^D443N/ApoE^-/-mice significantly increased.Masson’s trichrome and sirius red staining showed that the collagen-positive area was significantly increased in SCAP^D443N/ApoE^-/-mice,compared with the ApoE^-/-mice.However,there was no significant difference in serum levels of TC,TG,LDL-C and HDL-C between SCAP^D443N/ApoE^-/-mice and ApoE^-/-mice.Notably,the m RNA expression of pro-inflammatory cytokines（IL-1β,IL-6,IL-18 and TNF-α）and chemokines（MCP-1,MIP-1α,MIP-1βand CX3CL1）in the aorta of SCAP^D443N/ApoE^-/-mice was also significantly increased compared to that of ApoE^-/-mice.Immunohistochemical results also showed that increased expression of inflammatory cytokines（TNF-α,IL-1β,IL-6 and IL-18）in the aorta of SCAP^D443N/ApoE^-/-mice.In addition,SCAP also significantly increased macrophage infiltration in atherosclerotic lesions of SCAP^D443N/ApoE^-/-mice.Finally,the co-localization of NLRP3 with SCAP was significantly increased in the atherosclerotic lesions of SCAP^D443N/ApoE^-/-mice.Next,SCAP overexpression in VSMCs significantly increased the protein expression of inflammasome components and inflammatory cytokines,including NLRP3,Procaspase-1,cleaved caspase-1,IL-1β,and IL-18.The levels of proinflammatory cytokines（IL-1βand IL-18）were also remarkably upregulated in the SCAP overexpression cell culture supernatant.Moreover,NLRP3 was also co-expressed with SCAP in VSMCs,and the correlation between them was markedly enhanced after SCAP overexpression as demonstrated by co-immunoprecipitation analysis.In addition,SCAP overexpression significantly increased the co-localization of SCAP and NLRP3 with Golgi in VSMCs.However,the expression levels of NLRP3,caspase-1 activation and the production of IL-1βand IL-18,and the co-localization of SCAP or NLRP3 with the Golgi in VSMCs were significantly inhibited in the presence of lycorine.（3）After 12 or 24 weeks of high-fat diet,oil red O staining showed that the lipid accumulation in the aortic root of SCAP^D443Nmice were significantly increased at either 12 or 24 weeks of high-fat diet.However,TC,TG,LDL,and HDL levels in serum did not show significant changes between SCAP^D443Nmice and control mice after 12 or 24 weeks of high-fat diet.In addition,SCAP overexpression significantly increased the m RNA expression of proinflammatory cytokines（IL-1β,IL-6,IL-18,and TNF-α）,chemokine cytokines（MCP-1,MIP-1α,MIP-1β,and CX3CL1）,and adhesion molecules（ICAM-1 and VCAM-1）in the aorta,compared with the control mice.Furthermore,SCAP enhanced the protein expression of NLRP3 in the aortas of SCAP^D443Nmice.These results indicated that lipid deposition,local inflammation,and NLRP3 protein were increased in the aortas of SCAP^D443N mice.Next,VCAM-1 and ICAM-1 were increased significantly in the aorta of SCAP^D443N mice after 24 weeks of high-fat diet.Flow cytometry analysis demonstrated that SCAP overexpression increased the apoptosis of endothelial cells after exposure to SCAP CM,compared with the SCAP^+/+VSMC-conditioned media（WT CM）group.Immunofluorescence,q RT-PCR and western blot also showed that the expression of ICAM-1 and VCAM-1 in endothelial cells was significantly increased in the SCAP CM group,compared with the WT CM group.Finally,flow cytometry analysis showed that lycorine abrogated the SCAP CM-induced apoptosis of endothelial cells.This inhibitor also decreased the expression of ICAM-1and VCAM-1 in endothelial cells.Conclusion:This study elucidated that increased SCAP（D443N）overexpression in VSMCs enhanced local vascular inflammation and As in mice.In the setting of hypercholesterolemia,SCAP induce inflammation in VSMCs is sufficient to promote endothelial dysfunction in the vessel wall,which has been implicated in atherosclerotic lesion initiation and progression.These result not only showed that initiation of plaques in the aorta of mice requires inflammation in media VSMCs,but also shed new potential therapeutic target for As.