Study On The Mechanism Of Integrin Receptors In Regulating Cardiovascular Disease Progression

BackgroundPrevious studies have shown that obesity is an independent risk factor for acute coronary syndrome in patients with coronary heart disease.However,the mechanism by which obesity mediated adverse events of coronary heart disease is still lack of research.It has been reported that adipose tissue,as a kind of endocrine organ,can secrete a large number of bioactive adipokines,and widely participate in the regulation of inflammation and metabolism.Omentin-1 is a new type of anti-inflammatory adipokine,which is mainly expressed and secreted by visceral adipose tissue.It has been found that it has significant cardiovascular protection function,for example,Omentin-1 can improve arterial stenosis caused by guide wire strain by inhibiting the proliferation and migration of smooth muscle;In the mice model of myocardial infarction,overexpression of Omentin-1 can alleviate myocardial ischemia-reperfusion injury and reduce infarct size.In addition,clinical studies suggest that the expression of Omentin-1 in epicardial adipose tissue of patients with coronary heart disease is negatively correlated with the stability of coronary plaque in the segment surrounding by adipose tissue site.A series of evidences suggest that Omentin-1 may play a crucial role in the occurrence of adverse cardiovascular events in patients with coronary heart disease.Objective1.Build the atherosclerotic mouse model and investigate the effect of Omentin-1 on the stability of atherosclerotic plaque;2.To study the effect of omentin-1 on the function of macrophages-derived foam cells;3.To study the mechanism by which omentin-1 regulates the function of macrophagederived foam cells.Methods1.Animal model construction and plaque phenotype analysis:ApoE-/-mice and Ldlr-/-mice were fed on western diet to construct atherosclerosis model.Minipumps were implanted in mouse model of atherosclerosis to achieve continuous delivering of Omentin1 into mice.The root of mouse aorta was sliced to assess the formation of necrotic cores,collagen content and lipid content in the atherosclerotic plaque.Meanwhile,the expression of inflammatory factors(including TNF-α and IL-1β)in plaque was analyzed by immunohistochemistry and Western blot.Immunofluorescence staining was used to analyze the expression level of endogenous and exogenous Omentin-1,macrophage infiltration level and apoptotic cell content in plaque.2.Detection of Omentin-1 binding to integrin receptor on macrophage membrane:coimmunoprecipitation technology,confocal microscopy and flow cytometry analysis was used to demonstrate the binding of omentin-1 to integrin receptor αvβ3 and αvβ5 in vitro.Additionally,confocal microscopy was used to analysis the co-localization of omentin-1 and integrin receptor αvβ3 and αvβ5 in atherosclerotic lesion of aortic root.3.Regulation of function of macrophage-derived foam cells by omentin-1:mouse macrophage cell line RAW264.7 was cultured in vitro and transformed into foam macrophages by adding oxidized low-density lipoprotein(ox LDL).After that,omentin-1 was added to investigate its effect on apoptosis and inflammatory factors(including TNFa and IL-1β)expression of foam cells.Knock down of integrin subunit av by small interfering RNA,as well as application of integrin antagonist cilengitde,was used to demonstrate the role played by integrin αvβ3 and αvβ5 in transducing omentin-1’s biological effects.4.Regulation of function of macrophage-derived foam cells by omentin-1:mouse macrophage cell line RAW264.7 was cultured in vitro.After adding omentin-1,the activation of downstream signal molecules of integrin receptor FAK,p38 MAPK and ERK was detected by Western blot,and the activation effect of omentin-1 on Akt and AMPK in macrophages was also investigated.The effect of integrin receptors on transducing omentin-1 signaling was demonstrated by using integrin receptor antagonist cilentide andαv siRNA.Results1.Intravenous infusion of Omentin-1 by minipump can elevate the retention of Omentin-1 in mouse atherosclerotic plaque;2.Omentin-1 can significantly improve the stability of atherosclerotic plaque in ApoE-/-mice and Ldlr-/-mice;3.Omentin-1 can significantly reduce the infiltration of macrophages in the plaque of ApoE-/-mice and Ldlr-/-mice,and reduce the number of apoptotic cells and expression of inflammatory factor(including TNF-α and IL-1β)in the plaque.4.Omentin-1 can interact with the integrin receptor αvβ3 and αvβ5 on the membrane of macrophages and regulate apoptosis and inflammatory factor expression of macrophage-derived foam cells.5.Omentin-1 can bind to integrin receptor αvβ3 and αvβ5 and activate downstream signaling molecules FAK,p38 MAPK,ERK,Akt and AMPK.ConclusionsThe administration of adipokine omentin-1 can inhibit the necrotic cores formation and pro-inflammatory cytokines expression within the AS lesion.The mechanisms may include the suppression of apoptosis and pro-inflammatory cytokines expression in the macrophage by binding to the integrin receptors αvβ3 and αvβ5.BackgroundHigh glucose environment can promote endothelial cell apoptosis and eventually lead to microvascular dysfunction in patients with diabetes(DM).Previous studies have reported that the expression of integrin as well as its ligand in diseased vessels of diabetes patients was significantly elevated.Fibronectin is a major ligand of integrin,which is mainly involved in regulating cell structure stability and maintaining cell survival.Some studies have observed that fibronectin was significantly increased in the vasculature of diabetes patients,but the association between fibronectin and diabetic vascular complication is unknown.Integrin αvβ5,which is one of the key receptors of fibronectin,was observed to be upregulated by high glucose environment in patients with DM.And our preliminary research indicated that αvβ5 showed significant spatial co-localization with fibronectin in vitro.The above evidence indicates that fibronectin/αvβ5 axis may play an important role in high glucose-induced vascular disease.However,there is no relevant report at present.Objective1.Animal model of spontaneous hyperglycemia(db/db mice)was used to validate the effect of high glucose on the capillary density and apoptosis of vascular endothelial cells in myocardium.Endothelial cells were extracted from diabetic mice myocardium and subjected to immunoblotting analysis to verify the influence of hyperglycemia on the expression of fibronectin,integrin αv subunit(ITGAV),integrins β5 subunit(ITGB5)and autophagosome marker LC3 Ⅰ/Ⅱ.Selective knockdown of ITGB5 in vascular endothelial cells was performed to validate the role played by integrin in regulating hyperglycemiainduced microvascular rarefaction and endothelial cell apoptosis.2.Investigate the effect of high glucose environment on expression of fibronectin and integrin αvβ5(includingITGAV,ITGB5and αvβ5 heterodimer)in human umbilical vein endothelial cells(HUVECs);3.Determine the role played by fibronectin/αvβ5 axis in regulating high glucoseinduced HUVECs apoptosis and autophagy activation.4.Investigate downstream target of β5 integrin in mediating high glucose-induced cell autophagic apoptosis.Methods1.Abundance of capillaries was detected by CD31(a marker of vascular endothelial cells)immunofluorescent staining in diabetic db/db mice and non-diabetic db/m mice(control group).The proportion of CD31+/TUNEL+cells to all CD31+ cells was analyzed to assess the apoptosis activity in the vascular endothelial cells.Vascular endothelial cells(CD31+)were extracted from mouse myocardium and analyzed by immunoblotting to evaluate the expression of fibronectin,ITGAV,ITGB5 and the autophagosome marker LC3 Ⅰ/Ⅱ.2.Vascular endothelium-targeted adeno-associated virus carrying ITGB5 shRNA was injected in diabetic db/db mice to selectively knock down ITGB5 in vascular endothelial cell.And capillary density and endothelial cell apoptosis in myocardium was assessed by immunofluorescent staining.This experiment was designed to verify the effect of ITGB5 deletion on hyperglycemia-induced endothelium impairment.1.We stimulated HUVECs with high-glucose culture medium,and investigated expression of fibronectin,αv integrin,β5 integrin and αvβ5 heterodimer in HUVECs by using RT-qPCR,immunoblotting analysis,immunofluorescence microscopy.Immunofluorescence double staining was used to determine the spatial co-localization of fibronectin and integrin αvβ5.2.Small intervening RNA(siRNA)was used to knock down the expression of fibronectin,αv subunit and ITGB5respectively in order to validate the effect of each protein on high glucose-induced HUVECs apoptosis.3.Integrin β5,which was proved to be the key regulator of high glucose-induced apoptosis in HUVECs,was knocked down by siRNA in order to validate its influence on autophagy.HUVECs were transfected by adenovirus that encoding mRFP-GFP-LC3 to monitor the high glucose-induced autophagy flux change.4.High throughput transcriptome sequencing was used to determine the change of gene expressing profile after ITGB5knock down.The downstream target of ITGB5was screened by analyzing signaling enrichment pattern.Immunoblotting analysis and immunofluorescence were conducted to verify the result getting from RNA-seq analysis.5.Beclinl(key initiator of autophagy)and FoxO1(master regulator of autophagy)were knocked down by siRNA in order to verify the effect of autophagy activity on high glucose-induced apoptosis.6.The overexpression of FoxO1 in HUVECs was induced by adenovirus transfection.And whether FoxO1-overexpression could reverse the protective effect given by β5 knockdown was validated by apoptosis activity analysis.Results1.Diabetic db/db mice on a regular diet exhibited elevated fasting blood glucose level compared to db/m mice,along with a significant decrease in capillary density and an increase in endothelial cell apoptosis ratio.Examination of vascular endothelial cells isolated from the myocardium revealed that hyperglycemia significantly elevated fibronectin,ITGAV and ITGB5 expression in vascular endothelial cells.Besides,a significant increase in autophagic activity(LC3 Ⅱ level)was observed in vascular endothelial cells.2.Upon selective knockdown of ITGB5 expression in endothelial cells,db/db mice showed a significant increase in capillary density and a significant decrease in the level of endothelial apoptosis.Meanwhile,LC3 Ⅱ expression was significantly reduced in the vascular endothelium,suggesting that autophagy was downregulated in the endothelium after ITGB5 knockdown.1.High glucose environment remarkably stimulated overexpression of fibronectin,integrin αv and integrin β5 in HUVECs.And fibronectin exhibited significant spatial colocalization with αvβ5 in HUVECs.2.High glucose promoted HUVECs apoptosis by inducing fibronectin overexpression.Integrin ITGB5knock down attenuated both high glucose-induced and fibronectin-induced apoptosis.3.Both RNA-seq analysis and functional experiment indicated that FoxO1 was the downstream target of integrin β5,which regulates the activation of autophagy and apoptosis induced by high glucose.Besides,knock down of beclin1(the key regulator of autophagy)reduced high glucose-induced apoptosis,which proved that autophagy activation was associated with apoptosis under high glucose condition.4.Overexpression of FoxO1 ablated bioeffect given by β5 knockdown on HUVECs autophagy and apoptosis,which further demonstrated that FoxO1 was the downstream target of ITGB5in regulating HUVECs autophagic apoptosis.ConclusionsWe found that hyperglycemia stimulated apoptosis of mouse endothelial cells and reduced the density of capillaries in the myocardium.Protein level analysis revealed that the high glucose environment promoted overexpression of αvβ5 and its ligand,fibronectin.Knockdown of ITGB5 alleviated high glucose-induced vascular endothelial cell apoptosis and increased capillary density in the myocardium of diabetic mice.Cell level analysis revealed that ITGB5 deletion regulated high glucose-induced apoptosis in endothelial cells by inhibiting autophagy activity.The transcription factor FoxO1 was the downstream target of ITGB5 in mediating autophagic apoptosis.