The Effect And Mechanism Of WISP1 On Atherosclerotic Plaque Formation And Stability

BackgroundAtherosclerosis is one of common cardiovascular diseases.Its occurrence and development are complicated processes,including injury and dysfunction of endothelial cells,lipid deposition of macrophages,and proliferation and migration of vascular smooth muscle cells(VSMCs).The formation of foam cells is the characteristic of early atherosclerosis.Oxidized lowdensity lipoprotein(ox-LDL)is uptaken by macrophage with the help of scavenger receptor(SR)and is deposited in the cell to form foam cells.Ox-LDL,lysophosphatidyl choline and oxidized fatty acid induce the expression of the scavenger receptors,such as CD36,SR-A and Lectin-like oxidized low density lipoprotein receptor-1(LOX-1).CD36 belongs to scavenger receptor family B,and can be found in many cells such as monocytes/macrophages,adipocytes,endothelial cells and VSMCs.Ox-LDL,apoptotic cell and advanced glycation end products(AGEs)is able to interact with CD36 and participated in the development of atherosclerosis together.In addition,it is reported that SR-A which is a kind of cell surface glycoproteins belonging to SR family,can accelerate atherosclerosis.WNT signal pathway is a conservative signal pathway,which is involved in embryogenesis development and cell differentiation.The relationship between WNT pathway and cardiovascular diseases has attracted much attention recent years.There are studies found that WNT signal pathway involved in the adhesion between endothelial cells and monocytes,regulating on the function of VSMCs,the promotion in vascular calcification and played the important role in adipogenesis.Wnt-1 induced signal pathway protein 1(WISP1)belongs to the CCN family of extracellular matrix proteins.It is a classical downstream target gene of WNT signaling pathway,which is expressed in many organs,mainly participating in several important physiological activities such as embryogenesis,cell migration and proliferation,inflammatory reaction and tissue repairing process.Some studies have found that WISP1 plays an important role in the process of inflammation,and it is closely related to the severity of osteoarthritis.In addition,the expression of WISP1 is significantly elevated in neurons under the condition of oxidative stress and fracture repair.Some studies have shown that WISP1 can accelerate the migration and proliferation of VSMCs,resulting in the thickening of vascular intima.Other studies have shown that obesity leads to the up regulation of WISP1,and obesity is closely related to atherosclerosis,but whether WISP1 plays a role in atherosclerosis remains unclear.This study explored the relationship between WISP1 and atherosclerosis,the role of WISP1 in the formation and development of atherosclerotic plaque,and the specific function and molecular mechanism of WISP1.Our study investigated the possible mechanism of WISP1 in the formation of atherosclerosis via vitro and vivo experiments.Objectives1.To explore the effect of WISP1 on the formation of atherosclerosis;2.To explore the effect and relative mechanism of WISP1 on lipid deposition of macrophages;3.To explore the effect and relative mechanism of ox-LDL on WISP1 in macrophages.MethodsFor the purpose of investigating the relationship and mechanism between WISP1 and the of AS plaques,our experiments can be divided into two parts:vivo and vitro experiments.In vivo experiments,ApoE-/-mice were divided into 5 groups:control group,HFD group,NC group,IvWISP1 group and shWISP1 group.Except the control group,all groups were fed with high fat.The mice in NC group,IvWISP1 group and shWISP1 group were injected with lentivirus transfection via the caudal vein respectively.After lentivirus intervention,all mice were fed for 12 weeks before euthanasia and gathering of heart and aorta.The aortic sinus was stained with oil red O,Immunohistochemical staining of WISP1 and MOMA2 and immunofluorescence staining of CD36.In vitro experiments,primary peritoneal macrophages were used for oil-red O staining and DIL-ox-LDL uptake experiments,and RAW264.7 cell lines were used for other experiments.NC group,shWISP1 group and IvWISP1 group were treated with null lentivirus(MOI100,5'-GTTCTCCGAACGTGTCACGT-3'),WISP1-shRNA lentivirus(MOI 100,5'-CCACTA GAG GAA ACG ACTA-3')and WISP1 overexpression lentivirus(MOI 120)respectively.The proteins from mouse aorta and RAW264.7 macrophages were extracted and used to detected the expression levels of WISP 1,CD36,SR-A,PPAR?,?catenin and Wnt5a.Result1.General situation of miceCompared with control group,the body weight,TC and LDL-C levels in HFD group increased obviously,while the level of H-LDL in HFD group was lower than control group.However,there was no significant difference in body weight and blood lipid levels among HFD group,NC group,IvWISP1 group and shWISP1 group,indicating that WISP1 had no significant effect on body weight and blood lipid level of mice.2.Effect of WISP1 lentivirus transfection on lipid deposition in atherosclerotic aortic plaqueThe results of oil-red O staining of mouse whole aortas showed that the plaque area of HFD group was significantly higher than that of control group;The plaque area of IvWISP1 group was smaller than NC group;While the plaque area of shWISP1 group was significantly larger than that of NC group.The results of oil-red O staining of cross-sections from aortic sinuses were also consistent with those of whole aortas oil-red O staining mentioned above.In conclusion,overexpression of WISP1 can alleviate lipid deposition in atherosclerotic mice,while inhibition of WISP 1 can increase atherosclerotic lesions.3.Effect of WISP1 lentivirus transfection on macrophages in mouse aortic root plaqueThe results of MOMA2 immumohistochemistry of frozen section of mouse aortic root showed that the number of macrophages in HFD group was more than control group;The number of macrophages in plaque of IvWISP1 group was significantly lower than that of NC group,while the number of macrophages in shWISP1 group was significantly higher than that in NC group.In conclusion,overexpression of WISP1 can reduce the recruitment of macrophages in plaque,while inhibition of WISP1 can increase the recruitment of macrophages.4.The expression of CD36 and SR-A was significantly affected by WISP1 lentivirus transfectionThe result of CD36 immunofluorescence staining of frozen section of mouse aortic root showed that the content of CD36 in plaque of HFD group was higher than control group.Western blot of aortic tissue results showed that the protein contents of CD36 and SR-A in HFD group were higher than control group;The expression of CD36 and SR-A proteins in IvWISP1 group was significantly lower than that in NC group;On the contrary,the expression of CD36 and SR-A proteins in shWISP1 group was significantly higher than that in NC group.5.High fat diet increases WISP1 expression and its mechanismWestern blot of aortic tissue results showed that the content of Wnt5a,?-catenin and WISP1 in increased after high fat diet.We can conclude that high fat diet can increase the expression of WISP 1 by activating Wnt5a/?-catenin pathway.6.Ox-LDL can stimulate the production of ROS and activate Wnt5a/?-catenin signaling pathway to promote the expression of WISP1 in macrophagesAfter the macrophages were intervened with ox-LDL,the detection of ROS level indicated that ox-LDL could stimulate the production of ROS,and the expression of Wnt5a,?-catenin and WISP1 was significantly boosted at the meantime.While all of the activation could be inhibited with the ROS inhibitor NAC.7.WISP1 regulates lipid deposition in macrophages through PPAR?/CD36 signaling pathwayMacrophage oil-red O staining and DIL-ox-LDL uptake experiments showed the lipid deposition in the cytoplasm of IvWISP1 group was significantly less than that of NC group,while the lipid deposition in shWISP1 group was significantly more than that of NC group.Western blot results showed that PPARy and CD36 in IvWISP1 group were significantly lower than those in NC group.The level of PPAR? and CD36 in shWISP1 group was significantly higher than that in NC group.This increase,together with its downstream lipid deposition in macrophages,could be inhibited by T0070907,an inhibitor of PPARy signaling pathway.This suggests that overexpression of WISP 1 can inhibit lipid deposition by inhibiting PPARy/CD36 signaling pathway,and interference with WISP1 can promote lipid deposition by activating PPARy/CD36 signaling pathway.Conclusion1.The expression of WISP1 is increased in atherosclerotic plaque.2.WISP1 alleviates lipid deposition in atherosclerotic plaque.3.WISP1 can downregulate macrophages in atherosclerotic plaque.4.Ox-LDL can upregulate the expression of WISP1 by promoting ROS production and activating Wnt5a/?-catenin signaling pathway in macrophages.5.WISP1 can alleviate lipid deposition through inhibiting PPARy/CD36 signaling pathway in macrophages.BackgroundAtherosclerosis is a chronic progressive disease of large and medium-sized arteries,and it is also the first killer threatening human life in the world.Coronary atherosclerosis finally could lead to acute ischemic disease,which is also the main cause of unstable angina pectoris,myocardial infarction and sudden death.The main pathophysiological mechanisms include endothelial injury,lipid deposition,infiltration of inflammatory cells,proliferation and migration of smooth muscle cells(VSMC),which result in the formation of atherosclerotic plaque.With the development and deterioration of AS,plaque rupture can cause acute cardiovascular events.Rupture of atherosclerotic plaque and subsequent thromboembolic occlusion are the causes of acute coronary syndrome and stroke.Atherosclerotic plaques that are prone to rupture are characterized by plaque rupture,which consists of a thin fibrous cap covered with a large number of macrophages and lymphocytes,with a small amount of smooth muscle cells(SMCs)in the fibrous cap.Reversely,the fibrous cap of stable plaque is thick and rich in SMCs and collagen.Therefore,the stability of atherosclerotic plaque is the key to atherosclerosis,and the stable plaque is of great clinical value in the treatment of atherosclerosis.Proliferation and migration of VSMCs are keys to the stability of AS plaque,and integrin plays an important role in mediating the adhesion and migration of VSMCs.Integrin,as a receptor of extracellular matrix(ECM)proteins,not only mediates the interaction between cells and ECM,cells and cells,but also integrates extracellular signal transduction into cells.Focal adhesion kinase(FAK),as a key protein connecting integrin and its downstream signaling molecules in the process of integrin signaling,is the intersection of multiple signaling pathways in cells.FAK can catalyze the activation of a series of signal molecules such as PI3K with the help of multiple tyrosine phosphorylation sites in its molecule,and then trigger a cascade phosphorylation reaction to transmit the signal from integrin downstream.This signal pathway is important to cell's contraction,deformation and movement.On the other hand,the binding of ligands to integrins results in phosphorylation of FAK,which can activate MEK/ERK signaling pathway.MEK/ERK participates in a variety of cell functions,including cell migration and proliferation;Inhibiting this pathway can prevent the migration and proliferation of many types of cells.As another protein family closely related to cell proliferation and migration,the activation of MAPK/ERK signal can promote its migration and proliferation in smooth muscle cells.Mitogen activated protein kinases(MAPKs)belong to serine/threonine protein kinases family and are one of the extracellular signals.There are three members in the MAPK family:extracellular signal regulated protein kinase(ERK)which has a thru-glu-tyr motif,p38 which has a thra-ala-tyr motif and Jun N-terminal kinase(JNK)which has a thri-pro-tyr motif.MAPKs play an important role in cell migration,proliferation,differentiation,inflammation,tumorigenesis and stress response.WNT-inducible signaling pathway protein-1(WISP1)belongs to the CCN family of extracellular matrix proteins and has been identified as one of the downstream target genes of Wnt signaling pathway.WISP 1 is involved in mechanisms such as apoptosis,autophagy,migration,proliferation,angiogenesis,immunity and tumorigenesis.It has been found that WISP1 plays an important role in proliferation and migration.For example,Wnt2 could promotes the migration of VSMCs and thickens the intima with the help of WISP 1.In addition,WISP1 can accelerate wound healing by regulating the migration of skin fibroblasts and the expression of ECM.However,the mechanism of WISP1 in promoting the migration of smooth muscle cells is not complete,and the research of WISP1 on the stability of atherosclerotic plaque has not been reported at home and abroad.We aimed to study the relationship between WISP1 and atherosclerotic plaque stability and its relative mechanism.Our study investigated the possible mechanism of WISP1 and atherosclerotic plaque stability by in vitro and in vivo experiments.Objectives1.To study the stability of atherosclerotic plaque by transfecting WISP1 with lentivirus.2.To explore the effect of WISP1 on proliferation and migration of VSMCs.3.To study the effect of WISP 1 on integrin ?5?1 and FAK/MEK/ERK pathway.4.To investigate the effect of WISP1 on MAPK signaling pathway.MethodsFor the purpose of investigating the relationship and mechanism between WISP1 and the of AS plaques,our experiments can be divide into two parts:vivo and vitro experiments.In vivo experiments,ApoE-/-mice were divided into 3 groups:NC group(null lentivirus group),shWISP1 group(shRNA-WISP1 group)andIvWISP1 group(lentivirus WISP group).After 8 weeks high fat diet,the mice were locally incubated with null lentivirus(NC group),WISP1?shRNA(shWISP1 group)or lentivirus WISP1(IvWISP1 group)with the dose of 2X107 TU/mouse.After lentivirus intervention,all mice were fed for 4 weeks before euthanasia and gathering carotid artery.A thickness of 5?m of frozen section of carotid artery was conducted after OCT embedding.After the carotid artery was made into frozen sections,hematoxylin eosin(HE)staining and oil red O staining were used to observe the lipid content in carotid plaque,Masson staining and Sirius red staining were used to observe the content of collagen fibers in the plaque.In vitro experiments,the NC group,shWISP1 group and IvWISP1 group of mouse vascular smooth muscle cells(MOVAS)were treated with null lentivirus,WISP1-shRNA lentivirus and WISP1 overexpression lentivirus,respectively.After 72 hours of intervention,the stable transgenicstrains were screened by puromycin.The migration and proliferation ability of vascular smooth muscle cells was observed by transwell?wound healing and CCK8 experiment.Mouse carotid artery protein and mouse smooth muscle cell(MOVAS)protein were collected and prepared for detection of the expression levels of WISP1,P-FAK,FAK,P-MEK,MEK,PERK,ERK,P-JNK,JNK,P-P38 and P38.Result1.General situation of miceThere was no obvious diversity in body weight and serum TC,LDL-C and HDL-C levels among NC group,IvWISP1 group and shWISP1 group,indicating that local infiltration of WISP1 had no significant effect on body weight and blood lipid levels of ApoE-/-mice.2.The effect of WISP1 lentivirus transfection on macrophages and lipid in carotid artery of miceThe number of macrophages in Ivwispl group was significantly lower than that in NC group,according to the results of MOMA-2 immunofluorescence in frozen sections of mouse carotid artery,while the number of macrophages in shWISP1 group was significantly higher than that in NC group.The frozen sections of carotid artery in mice were stained with oil-red O to observe the lipid content in carotid plaque.The results showed that the lipid content in plaque of IvWISP1 group was significantly less than that of NC group,while the lipid content of carotid plaque in shWISP1 group elevated than NC group.In conclusion,WISP1 can significantly inhibit lipid deposition and macrophage aggregation,in carotid artery,while inhibiting WISP1 can promote lipid deposition and macrophage aggregation.3.The effect of WISP1 lentivirus transfection on smooth muscle and collagen expression cells in mouse carotid arteryThe result of immunofluorescence of ?-SMA in frozen sections of mouse carotid artery showed that the number of smooth muscle cells in IvWISP1 group was significantly higher than that in NC group,while the number of smooth muscle cells in shWISP1 group was significantly lower than that in NC group.Masson staining and Sirius red staining were used to observe the content of collagen fibers in carotid plaque.The results showed that the content of collagen in IvWISP1 group was significantly higher than that in NC group,while the content of collagen in carotid plaque of shWISP1 group was significantly less than that of NC group.In conclusion,WISP1 can increase the content of collagen and number of smooth muscle cell in carotid plaque of mice,while downregulation of WISP1 can reduce the content of collagen and number of smooth muscle cell in carotid plaque of mice.4.The effect of WISP1 lentivirus transfection on the stability of carotid plaque in miceThrough the assessment of plaque vulnerability index,we could come to the determination that the vulnerability index of IvWISP1 group was significantly lower than that of NC group,while the vulnerability index of shWISP1 group was significantly higher than that of NC group,which concluded that WISP1 can stabilize the carotid atherosclerotic plaque,while interference with WISP1 can reduce the stability of the plaque.5.The effect of WISP1 on migration and proliferation of VSMCs.The migration and proliferation ability of vascular smooth muscle cells was observed by transwell?wound healing and CCK8 experiment.The results showed that the number of VSMC migrated in IvWISP1 group was significantly higher than that in NC group,while the number of SMCs migrated in shWISP1 group was significantly lower.Besides,the IvWISP1 showed an increase in cell proliferation rate compared with the NC group,however,the shWISP1 group showed a marked decrease in that compared with the NC group,suggesting that WISP1 promotes the proliferation of VSMCs.In conclusion,WISP1 can significantly promote the migration and proliferation of VSMCs,while interference with WISP1 can inhibit the migration and proliferation.6.The effect of WISP1 on MAPK signaling pathwayBy means of western blot used to detect the expression of P-P3 8?P3 8?P-JNK?JNK?P-ERK and ERK,it is found that the phosphorylation level of ERK in IvWISP1 group was significantly higher than that in NC group,while that in shWISP1 group was significantly lower than that in NC group.However,there was no significant difference in P38 and JNK phosphorylation among IvWISP1?NC and shWISP1 groups.In conclusion,WISP1 can significantly promote ERK phosphorylation,interference with WISP1 can significantly inhibit ERK phosphorylation,but WISP1 has no significant effect on P38 and JNK phosphorylation.7.WISP1 regulates the phosphorylation of FAK and its downstream MEK?ERK pathway through integrin ?5?1?and then affects smooth muscle cell migration and proliferationThe expressions of P-FAK?FAK?P-MEK?MEK?P-ERK and ERK were detected with the help of western blot,the results showed that the phosphorylation levels of FAK?MEK and ERK in IvWISP1 group were significantly higher than those in NC group,while the phosphorylation levels of FAK?MEK and ERK in shWISP1 group were significantly lower than those in NC group.For the purpose of further investigating the effects of WISP1 on FAK?MEK?ERK signaling pathway and proliferation and migration of VSMCs,we used Ab?5?1 and FAK inhibitor Y15 to down regulate integrin ?5?1 and the phosphorylation levels of FAK.The results showed that the phosphorylation levels of FAK?MEK and ERK in IvWISP1 group were significantly increased,and the ability of migration and proliferation in VSMCs was also elevated in comparison with NC group.Yet this increased phosphorylation and the ability of migration and proliferation on smooth muscle were inhibited by Aba5?1 and Y15.In conclusion,WISP1 can promote the phosphorylation of FAK and its downstream MEK?ERK pathways through integrin ?5?1,thus promoting the migration and proliferation of VSMCs.Conclusion1.WISP1 can increase the stability of atherosclerotic plaque.2.WISP1 can accelerate the migration and proliferation of vascular smooth muscle cells.3.WISP1 can promote the migration and proliferation of vascular smooth muscle cellsThrough integrin ?5?1 and FAK pathway and their downstream MEK?ERK pathway.