Role And Molecular Mechanism Of Salusin-? In Hypertension,Vascular Injury And Formation Of Foam Cells

Salusins are identified to be peptides of 28 and 20 amino acids with peripheral hypotensive,bradycardic and mitogenic effects,and to be designated as salusin-a and salusin-? in 2003.They are translated from an alternatively splicedmRNA of TOR2A,a gene encoding a protein of the torsion dystonia family.Salusin-? promoted but salusin-a suppressed the formation of human macrophage foam cells in atherosclerosis.Salusin-? immunoreactivity was found to be dominant in the VSMCs and fibroblasts in coronary atherosclerotic lesions rather than salusin-a.The thesis is divided into three parts,respectively.(1)effects of salusin-? on the proliferation of VSMCs,vascular fibrosis and vascular remodeling in hypertension.(2)effects of salusin-? on the migration of VSMCs and intimal hyperplasia after vascular injury.(3)effects of salusin-? on the foam cell formation and monocyte adhesion in VSMCs.Our results showed that salusin-? promoted VSMC proliferation,fibrosis,migration,foam cell formation and adhesion of monocytes,suggesting that salusin-? may become one of the important targets in the treatment of hypertension,vascular injury,atherosclerosis and other vascular remodeling diseases.Part 1 Salusin-? contributes to vascular remodeling associated with hypertension via promoting vascular smooth muscle cell proliferation and vascular fibrosis1.Background Proliferation of vascular smooth muscle cells(VSMCs)and vascular fibrosis are closely linked with many clinical diseases including hypertension,atherosclerosis and diabetes and their target organ damage.Excess synthesis and accumulation of extracellular matrix(ECM)mainly in the vascular wall responses to chronically elevated blood pressure induces vascular stiffening,which reinforce the development of hypertension,thus reflecting a vicious circle.The vascular lesions are mainly manifested as reduced vascular compliance,increased stiffness and arterial wall thickening in hypertension.The proliferation of VSMCs is a major contributor in the vascular wall thickening.The vascular disease is not only an important pathological change in hypertension,but also fundamental structural components for maintenance and development of high blood pressure.Thus reversing or inhibition of vascular remodeling has become one of the important strategies for the prevention and treatment of hypertension and its target organ damage.However,the role of salusin-? in vascular remodeling of hypertension is not clear.In this study,we determined the effects of salusin-? on VSMCs proliferation and vascular fibrosis and their downstream signal pathway in human VSMCs.2.ObjectiveThe study was to investigate the role and molecular mechanism of salusin-? on the proliferation and fibrosis of VSMCs and to determine the effects of persistently increased expression of salusin-? on the vascular remodeling and blood pressure in rats.3.MethodsHuman aortic VSMCs were cultured in F12K Kaighn's modification medium supplemented with 10%fetal bovine serum(FBS)and 1× Antibiotic-Antimycotic Solution at 37?,humidified atmosphere containing 5%C02.EdU incorporation assay was employed to determine the DNA synthesis of VSMCs in vitro.The distribution of various phases of the cell cycle was used to evaluate the VSMCs proliferation with flowcytometry.The intracellular cAMP and salusin-? levels were determined by an enzyme immunoassay kit.The phosphorylation of ERK1/2,JNK,p38,PKA,histone H3,cAMP response element binding protein(CREB),epidermal growth factor receptor(EGFR)and smad2/3 were determined with Western blot.The mRNA of collagen-?,collagen-?,fibronectin,transforming growth factor-?1(TGF-?1)and connective tissue growth factor(CTGF)were analyzed by real-time quantitative PCR.Rats were randomly divided into control(Ctrl),Vector and Salusin-? groups,which were respectively subjected to injection of PBS,lentivirus expressing GFP or lentivirus-expressing both GFP and salusin-?.Systolic blood pressure(SBP)of tail artery was measured in conscious state by using a noninvasive computerized tail-cuff system.The descending thoracic aorta,main renal artery and the third-order branches of the mesenteric artery were stained with Masson's trichromestaining for assessment of vascular remodeling.4.Results(1)Salusin-? dose-relatedly promotes VSMCs proliferation,reaching its maximal effects at the concentration of 10nmol/L at 48 h after salusin-? treatment.Salusin-P reduced the cell populations in the G0/G1 phases and increased those in the S phase comparedwith vehicle treatment in VSMCs,and increased Edu-positive cells in VSMCs.(2)Salusin-? increased cAMP levels in VSMCs(Fig.2A).It promoted the phosphorylation of PKA,which was abolished by an adenylate cyclase(AC)inhibitor SQ22536.Salusin-P promoted the ERK1/2 phosphorylation,reaching its maximal effect at 30 min and lasting at least 2 h,which was abolished by the pretreatment with SQ22536,Rp-cAMP or AG1478.Moreover,salusin-? promoted the phosphorylation of CREB and EGFR,which were abolished by the pretreatment with SQ22536,Rp-cAMP or AG1478.The effect of salusin-? on proliferation was prevented by SQ22536,PKA inhibitor Rp-cAMP,EGFR tyrosine kinase inhibitor AG1478,ERK inhibitor U0126 or CREB inhibitor KG501.(3)Salusin-? increased the TGF-?1,collagen-I,collagen-? and fibronectin mRNA expressions in VSMCs,which were abolished by anti-salusin-? IgG,and not by anti-salusin-a IgG.(4)ALK5 inhibitor A83-01 prevented the effect of salusin-? on collagen-?,collagen-? and fibronectin mRNA expressions in VSMCs.Salusin-? promoted the phosphorylation of smad2/3 and increased the CTGF mRNA expression,which were abolished by A83-01.(5)Salusin-? levels in aorta,renal artery and mesentery artery were higher in rats with salusin-? overexpression than control rats.The plasma salusin-? level in rats with salusin-? overexpression was increased to 6-8 folds of those in control rats.The media thickness and the ratio of media thickness to lumen diameter were significantly increased in aorta,renal artery and mesentery artery of the rats with salusin-?overexpression.Moreover,the lumen diameter of renal artery and mesentery artery was significantly reduced in rats with salusin-? overexpression.Masson's trichromestaining showed the increased adventitia thickness and collagen content in the adventitia of arteries in the rats with salusin-? overexpression.(6)Salusin-? overexpression promoted the phosphorylation of CREB,EGFR and ERK 1/2,but had no significant effect on the phosphorylation of JNK and p38 in aorta,renal artery and mesentery artery of rats.It increased the PCNA and phosphorylated histone H3 levels(markers of proliferation)in the media of aorta,renal artery and mesentery artery.The salusin-? overexpression increased the collagen-I,collagen-III and fibronectin mRNA expression in these arteries of rats.(7)Salusin-? overexpression caused a severe and long-lasting hypertension accompanied by tachycardia in rats.SBP in conscious rats was significantly increased since the 3rd day after the salusin-? gene transfer,reached its maximum at the end of the 2ndweek and maintained at very high level from this time on.HR in conscious rats was significantly increased since the 2nd week after the salusin-? gene transfer.Hypertension and tachycardia were further confirmed by the measurement of carotid MAP and HR under anaesthesia.5.ConclusionsSalusin-? promotes VSMC proliferation via cAMP-PKA-EGFR-CREB/ERK pathway and vascular fibrosis via TGF-?1-Smad pathway.Increased salusin-?contributes to vascular remodeling and hypertension.Part 2 Salusin-? promotes vascular smooth muscle cell migration and intimal hyperplasia after vascular injury via ROS/NF?B/MMP-9 pathway1.BackgroundThe proliferation and apoptosis of vascular smooth muscle cells(VSMCs)are in a dynamic balance in physiological conditions.The VSMCs will be transformed,and then start the proliferation and migration process in the case of pathological changes,which plays a key role in the pathogenesis of atherosclerosis and restenosis after percutaneous coronary intervention.Media-to-intima migration of vascular smooth muscle cells(VSMCs)is critical to intimal thickening in atherosclerosis and restenosis after coronary angioplasty.The growth factor(PDGF),epidermal growth factor(EGF),fibroblast growth factor(FGF)and vascular endothelial growth factor(VEGF)are the main factors to promote the proliferation and migration of VSMCs.Therefore,it is necessary to find an important target molecule for VSMC proliferation and migration,which is one of the important strategies for the treatment of atherosclerosis and restenosis after percutaneous coronary intervention.However,it is not clear whether salusin-? played a role in the migration of vascular smooth muscle cells and vascular injury.2.ObjectiveThe study was to investigate the effect of salusin-? on the migration of VSMCs and its underlying signal pathway and to determine the effects of down-regulation salusin-? on vascular injury-induced intimal hyperplasia.3.MethodsThe primary culture of VSMCs from the rat aorta was performed with enzymatic digestion.Unilateral carotid artery ligation was performed to induce vascular injury in 8-week-old rats.The arteries were fixed in 4%paraformaldehyde solution,were embedded in paraffin,and then were transversely cut into 5-?m sections.The serial sections were subjected to hematoxylin-eosin(HE).Immunohistochemical staining or dual immunofluorescence was used to determine the expression of salusin-?.Real-time PCR was employed to measure the mRNA expressions of MMP-9,MMP-2 and salusin-?.The protein levels of NOX-2,NOX-4,p65,I?B?,phosphor-I?B?,Lamin B1,GAPDH,MMP-2 and MMP-9 were assessed by Western blot.The expression of salusin-? was also detected with ELISA method.VSMC migration was evaluated by scratch-induced wound healing assay or a Boyden chamber assay.The small interference RNA(siRNA)sequences were transiently transfected into the VSMCs using Lipofectamine.ChIP assay was conducted with a standard protocol and kit to evaluate whether NF?B-p65 binds to the MMP-9 promoter in salusin-?-stimulated VSMCs.Dihydroethidium(DHE)and 2',7'-dichlorodihydrofluorescein diacetate(DCF-DA)were used to evaluate the intracellular superoxide anion and hydrogen peroxide,respectively.4.Results(1)Moderate intimal hyperplasia developed in ligated carotid arteries at 4 weeks ofpost-injury and became much more severe at 8 weeks.Immunofluorescence double staining showed the low levels of salusin-? within normal vascular walls,primarily in the adventitia of arteries.Salusin-? positive staining was obviously increased in the neointima in response to injury and was predominantly to VSMCs in neointima.Similar change was observed by immunohistochemical assay.Salusin-? mRNA level was greatly increased in injured carotid arteries during the 4 to 8 weeks after injury.(2)Both scratch-wound assay and Boyden chamber chemotaxis assay were used to determine the effect of salusin-? on the VSMCs migration in vitro.Salusin-?dose-dependently stimulated the migration of VSMCs including increased migrated distance and number of migrated cells,almost reaching its maximal effects at the concentration of 10 nM.(3)Salusin-? increased MMP-9 expression in VSMCs in a time-related and dose-related manner,but had no significant effect on MMP2 expression.Similarly,salusin-P increased MMP-9 mRNA levels but not MMP-2 levels in VSMCs.Luciferase reporter gene assay showed that salusin-? enhanced the reporter activity of MMP-9 promoter construct.Importantly,salusin-?-induced VSMC migration was prevented by MMP-9 inhibition rather than MMP-2 inhibition.(4)Incubation of VSMCs with salusin-? promoted the phosphorylation and degradation of I?B?.The p65 level in the cytoplasm was relatively high compared with that in the nucleus in the VSMCs without salusin-? treatment After stimulation of salusin-?,the p65 level in the cytoplasm was decreased with concomitant increase in the nucleus in time-dependent manner.(5)Salusin-? significantly increased binding activities of the NF?B motifs in the MMP-9 promoter.Knockdown of NF?B with siRNA suppressed the salusin-p-induced MMP9 expression responses to salusin-?.Bay 11-7082,an inhibitor of NF?B showed similar inhibitory effects to the NF?B knockdown.Importantly,knockdown of NF?B with siRNA or inhibition of NF?B inhibition with Bay 11-7082 prevented the VSMC migration response to salusin-?.(6)Salusin-? increased NAD(P)H oxidase 2(NOX2)expression in a time-dependent manner,but had no significant effect on NOX4 expression.It increased the production of superoxide anions,which was prevented by NOX2-siRNA transfection,but not by NOX4-siRNA transfection.Moreover,knockdown of NOX2 with NOX2-siRNA inhibited salusin-?-induced MMP-9 expression and p65-NF?B nuclear translocation.Treatment with N-acetyl-cysteine(NAC,a ROS scavenger)or apocynin(a NAD(P)H oxidase inhibitor)prevented salusin-(3-induced MMP-9 expression and p65-NF?B nuclear translocation.Chemotaxis assay showed that treatment with NAC or apocynin prevented the salusin-(?-induced VSMC migration.Similarly,VSMC migration response to salusin-? was reduced by NOX2-siRNA,but not by NOX4-siRNA.(7)Ad-salusin-? shRNA reduced neointima formation,neointima area and the ratio of neointima area to medial area in injured carotid arteries of rats.It prevented the injury-induced MMP-9 and NOX2 expression as well as p65-NF?B nuclear translocation.Dihydroethidium(DHE)fluorescent dye showed that the production of superoxide anions in the injured carotid arteries was increased in the rats,which were prevented by Ad-salusin-? shRNA.On the other hand,salusin-? mRNA and protein expressions were increased in injured carotid arteries of rats,which were attenuated by Ad-salusin-? shRNA.5.Conclusions Our data provide a novel view that salusin-? promotes migration of vascular smooth muscle cells(VSMCs)and contributes to the exacerbated intimal hyperplasia of post-injury.NAD(P)H oxidase 2(NOX2)-derived reactive oxygen species(ROS)were important initiating factors for salusin-?-induced p65-NF?B nuclear translocation,which positively regulates the activity and expression of MMP-9 and thus induces VSMC migration.Knockdown of salusin-? attenuates the vascular injury-induced NOX2 expression,ROS production,intimal thickening and intimal hyperplasia.Our findings provide new insights that intervention of salusin-? may be a promising therapeutic strategy for some vascular diseases such as atherosclerosis and stent restenosis.Part 3 Salusin-p induces foam cell formation and monocyte adhesion in human vascular smooth muscle cells via--miR155/NOX2/NF?B pathway1.BackgroundVascular smooth muscle cells(VSMCs)are indispensible components in foam cell formation.The foam cell formation is an essential hallmark in the advanced atherosclerosis lesion and the susceptibility of rupture of atherosclerotic plaque.Most of foam cells have been found to be derived from macrophages,but VSMCs give rise to a considerable number of foam cells as well.On the other hand,leukocyte recruitment from blood stream to the intima of vessels is a crucial event in the pathogenesis of atherosclerosis and a potential candidate for therapeutic approaches of atherosclerosis.However,the roles of salusin-? in the foam formation and monocyte adhesion in human VSMCs are rarely known.2.ObjectiveThe study was to investigate the effect of salusin-? on the foam formation and monocyte adhesion of VSMCs and its underlying signal pathway.3.MethodsHuman aortic VSMCs were cultured in F12K Kaighn's modification medium supplemented with 10%fetal bovine serum(FBS)and 1× Antibiotic-Antimycotic Solution at 37?,humidified atmosphere containing 5%CO2.Oil red O staining was used to detect lipid deposition in vascular smooth muscle cells and intracellular total cholesterol level was determined by enzymatic assay.The small interference RNA(siRNA)sequences were transiently transfected into the VSMCs using Lipofectamine.The protein levels of NOX-2,p65,Lamin B1,GAPDH,ACAT-1 and VACM-1 were assessed by Western blot.Real-time PCR was employed to measure the expressions of ACAT-1,VCAM-land miR155.ChIP assay was conducted with a standard protocol and kit to evaluate whether NF?B-p65 binds to the ACAT-1 and VCAM-1 promoter in salusin-?-stimulated VSMCs.Dihydroethidium(DHE)was used to evaluate the intracellular superoxide anion in VSMCs.4.Results(1)Salusin-? dose-and time-relatedly increased the accumulation of lipid droplets in VSMCs and the adhesion of monocyte to VSMCs,and the maximal effects were observed at the dose of 30 nM of salusin-? for 48 h.(2)Salusin-? upregulated the ACAT-1 and VCAM-1 protein levels in VSMCs in concentration-and time-dependent manner.Furthermore,it increased ACAT-1 and VCAM-1 mRNA levels,which were parallel to those of protein expressions.Luciferase reporter genes assay showed that salusin-? enhanced the promoter activities in the ACAT-1 and VCAM-1 truncated forms containing proximal NF-?B binding sites.Knockdown of ACAT-1 impeded salusin-?-induced accumulation of lipid droplets in VSMCs and increase in total intracellular cholesterol contents,while knockdown of VCAM-1 prevented salusin-?-induced monocyte adhesion in VSMCs.(3)Salusin-? elicited an increase in nuclear p65-NF?B levels and a reduction in cytoplasmic p65-NFB levels in VSMCs in dose-related manner and time-related manner.As expected,salusin-? increased the binding of NF-?B to ACAT-1 and VCAM-1 promoter in VSMCs.The up-regulated ACAT-1 and VCAM-1 expressions in response to salusin-? were abrogated by inhibition of NF?B with Bay11-7082.Pretreatment with Bay11-7082 inhibited the salusin-?-induced foam cell formation and adhesion of monocyte to VSMCs.(4)Incubation of VSMCs with salusin-? dose-and time-relatedly increased NAD(P)H oxidase 2(NOX2)expressions and ROS production.Salusin-?-induced ACAT-1 and VCAM-1 expressions,and p65-NF?B nuclear translocation in VSMCs were prevented by the treatment with a ROS scavenger N-acetyl,cysteine(NAC),a NAD(P)H oxidase inhibitor apocynin or knockdown of NOX2 with siRNA.Importantly,incubation of VSMCs with NAC,apocynin or NOX2 siRNA abolished the salusin-?-induced foam cell formation and adhesion of monocyte to VSMCs.(5)Salusin-? time-relatedly increased miR155 expression in VSMCs.Knockdown of miR155 with siRNA in VSMCs prevented salusin-?-induced foam cell formation,monocyte adhesion to VSMCs,ROS production and NOX2 expression.Furthermore,salusin-?-induced ACAT-1 and VCAM-1 expressions,and p65-NF?B nuclear translocation were diminished by miR-155 siRNA transfection in VSMCs.5.ConclusionsSalusin-? upregulates miR155 in VSMCs,which increases NOX2-derived ROS production and subsequent nucleus translocation of p65-NF?B.The activation of NF?B promotes ACAT1 and VCAM-1 expressions,and then causes foam cell formation from VSMCs and monocyte adhesion to VSMCs.Intervention of salusin-?may be a promising strategy for retarding the vascular lesions in atherosclerosis.