Research On The Effects And Mechanisms Of PEDF In Atherosclerosis

BackgroundAs the most common underlying cause of coronary artery disease(CAD),atherosclerosis(AS)is the leading cause of death worldwide.AS is a complex pathological process which leads to artery stenosis and results in cerebrovascular disease and CAD.The rupture of vulnerable AS plaque and thrombogenesis are the main pathological mechanisms of acute vascular event.Early intervention on vulnerable plaque is necessary for reducing the incidence of acute cardiovascular events.The classical studies show that the AS starts from the intimal injury,and the intimal inflammation expands to the adventitia and perivascular adipose tissue(PVAT).The inside-out signaling paradigm has been widely accepted.However,more and more studies reveal that PVAT contributes to the pathogenesis of AS through the outside-in model.The adipokines derived from PVAT can act in the adventitia and intima.There are complex interactions between PVAT and intima.Studies illustrate that increase of angiogenesis is the prerequisite of the vulnerable plaque.The invasion of macrophages,intraplaque hemorrhage,the thin fibrous cap and inflammatory infiltration are closely related to the neovascularization of the adventitial vasa vasorum(VV).During the development of AS,the VV which arises from the vascular adventitia proliferates to adapt to the vascular hypoxia caused by intimal thickening and results in intraplaque hemorrhage.However,the recent evidence shows that the proliferation of VV is earlier than the intima thickening,and the dysfunction PVAT is responsible for the proliferative VV.The VV acts as the pipage for the interaction between PVAT and intima,and transports inflammatory factors and cells to the intima during AS process.PVAT,the special adipose tissue,is also an endocrine organ which secrets amounts of proand anti-inflammatory factors and influences the development of AS.The dysfunction of PVAT caused by obesity can aggravate adventitial inflammatory infiltration and angiogenesis,and accelerate intimal hyperplasia and VV proliferation.Based on these studies,inhibition of adventitia vasa vasorum angiogenesis to weaken the effects of inflammatory cytokines from PVAT on vessels can be the new therapeutic target against atherosclerosis.Our previous research demonstrated that pigment epithelium derived factor(PEDF),a potent endogenous anti-angiogenic factor,could protect against atherosclerosis.Compared with exogenetic angiogenesis inhibitors,PEDF possesses high security which only effects on the abnormal neovascularization without injuring the normal vessels.As a multifunctional factor,PEDF can also inhibit inflammation,resist oxidation and inhibit thrombosis besides the antiangiogenesis effect.The presently known receptors of PEDF include adipose triglyceride lipase/independent phospholipase A2(iPLA2)and 37/67-kDa laminin receptor(LR).Studies shows that LR acts as a mediator of cellular signal transduction,and reacts with many signal pathways.LR is firstly recognized as the 67-kDa binding protein of laminin and distributes in many domains of life.LR is the plasma membrane protein,and participates in many biology processes including cell proliferation,migration and protein synthesis.LR is suggested the real receptor mediated the anti-angiogenetic function.However,the molecular mechanisms of PEDF and LR in anti-antiangiogenesis are still unclear.On the basis of available data,upregulation of PEDF might be an important manner to intervene VV proliferation and stabilize atherosclerosis plaque.The effective agonist of PEDF will be a new therapy for AS.As the representative component of ginseng,Ginsenoside Rb1 exhibits many protective properties,including anti-obesity,anti-oxidative stress,antiangiogenesis and anti-fatigue.Our group proved that Rb1 inhibited HUVECs angiogenesis in vitro through modulating miR-33a and PEDF.However,it remains unclear whether Rbl could inhibit VV proliferation in AS.Although a number of studies have shown that PEDF may be an effective factor in antiangiogenesis of VV and stabilization of plaque,there is no direct evidence at present.Meanwhile,the mechanism of the PEDF anti-angiogenesis effect remains unclear.We will investigate the role of PEDF in PVAT during the development of AS and whether PEDF could inhibit the angiogenesis of VV by modulating PEDF expression in ApoE-/-mice.Dissertation Ⅰ Anti-atherosclerotic Effect of PEDF Secreted by Perivascular Adipose Tissue is Exerted via Inhibiting Adventitia Vasa Vasorum AngiogenesisObjectives1.To investigate the alterations of PEDF expression of pericardial adipose tissue in AS procession.2.To investigate whether PEDF affects the development of AS.3.To investigate whether PEDF in PVAT affects the development of AS through VV.4.To investigate the molecular mechanism of PEDF.Methods1.Explore the change of PEDF expression in CAD patientsA total of 6 patients undergoing thoracotomy(3 CAD patients and 3 without CAD patients)were evaluated.The pericardial adipose tissues were obtained at the beginning of extracorporeal circulation.The serum was collected after preoperative examination for the research.The experiment obtained approval from the ethics board.2.Establish the mouse model of PEDF-knockdown atherosclerosisMale ApoE-/-mice(8 weeks old)were treated with lentivirus PEDF shRNA or lentivirus vector(CTR shRNA).The mice were fed with high-fat diet(HFD,40%fat and 1.25%cholesterol)for 12 weeks.3.Establish the mouse model of transplanted perivascular adipose tissueThe models of transplanted perivascular adipose tissue were build based on the PEDFknockdown mice.The ApoE-/-mice(8 weeks old)were randomly divided into 2 groups:the non-operation group and the operation group.The non-operation group mice were randomly divided into CTR shRNA group(n=15)and PEDF shRNA group(n=15).The operation group mice were randomly divided into the donator group and the receptor group.The donator group mice were randomly divided into D-CTR shRNA and D-PEDF shRNA.The receptor group mice were all treated with PEDF shRNA.Mice in donator groups were anesthetized and separated 10mg VAT.The separated adipose tissues were transplanted to encircle the left carotid artery of the mice in the receptor group.Mice in the receptor group were locally injected with adeno-associated virus(AAV)to over express PEDF(AAV-PEDF)or the AAV vector(AAV-CTR)in the transplanted adipose tissue under ultrasound guidance 2 weeks after the transplant.According to the PEDF expression levels of transplanted adipose tissues,mice in receptor groups were divided into four groups:D-CTR shRNA+AAV-CTR group,D-CTR shRNA+AAV-PEDF group,D-PEDF shRNA+AAV-CTR group,and D-PEDF shRNA+AAV-PEDF group(n=15 per group).All mice were fed with HFD for total 12 weeks.4.Explore the effect of reduced PEDF on inflammationSerum levels of PEDF,tumor necrosis factor-α(TNFα),interleukin-6(IL-6)and interleukin-1β(IL-1β)in CTR shRNA and PEDF shRNA groups were measured by enzyme linked immunosorbent assay(ELISA).The content of inflammatory factors in plaque were detected by IHC.5.Explore the effect of PEDF on lipid levelsThe serum levels of total cholesterol(TC),triacylglycerol(TG),low-density lipoprotein cholesterol(LDL-C)and high-density lipoprotein cholesterol(HDL-C)were measured by biochemical detection.6.Explore the effect of PEDF on atherosclerotic plaque stability(1)HE stainingTissue sections were stained with hematoxylin and eosin to show the morphology of the plaques.(2)Oil O stainingTo evaluate the plaque burden of the aortas and the content of lipid in plaque,the aortas,carotid artery,and aortic root sections were stained with oil-red O solution.(3)Sirius red stainingThe sections of carotid artery and aorta were stained by Sirius red to quantified the collagen content in plaque.(4)Immunofluorescent stainingImmunofluorescent staining was used to analyze macrophages(MOMA2)and smooth muscle cells(αSMA)in plaque.(5)Immunohistochemical(IHC)stainingIHC staining was used to detect the expression levels of PEDF,TNFα,IL-6 and IL-1β in plaque and transplanted PVAT.The density of VV was analyzed by IHC staining with endomucin primary antibody.The levels of PEDF,TNFα,IL-6,IL-1β and CD31 in VAT were analyzed by IHC staining.7.Verify the PEDF impact on AS progress in ApoE-/-/PEDF-/-mice(1)Establish the PEDF-knockout mouse modelMale ApoE-/-/PEDF-/-mice(8 weeks old)were all fed with high-fat diet(HFD)for 12 weeks.Measure the PEDF expression in plaque and VAT.(2)Explore the effect of PEDF on inflammation:Western blot and Immunohistochemistry staining were used to detect the content of inflammatory factors in VAT of each group.(3)Explore the effect of PEDF on serum lipids:TC,TG,LDL-C and HDL-C levels in serum were detected.(4)Explore the effect of PEDF on AS plaque stability1)HE stainingTissue sections were stained with hematoxylin and eosin to show the morphology of the plaques.2)Oil O stainingTo evaluate the plaque burden of the aortas and the content of lipid in plaque,the aortas and aortic root sections were stained with oil-red O solution.3)Sirius red stainingThe sections of aorta were stained by Sirius red to quantified the collagen content in plaque.4)Immunofluorescent stainingImmunofluorescent staining was used to analyze macrophages(MOMA2)and smooth muscle cells(αSMA)in plaque.8.Explore the incubation time of PEDF on Mouse aortic endothelial cells(MAECs)The primary MAECs was purchased from Procell company and cultured with endothelial cell medium(ECM).The MAECs were treated with PEDF recombinant protein for 0,4,8,12,16,20 and 24 h to observe the optimal action time.9.Explore the effect of PEDF on neovascularization of MAECsThe MACEs were treated with PEDF(100 ng/ml)or equal volume PBS(Vehicle)to explore the effect of PEDF on angiogenesis.The MAECs were transfected with LR siRNA or CTR siRNA to study the effect of LR.The MAECs were transfected with Atg5 siRNA or CTR siRNA to study whether autophagy was involved in the anti-angiogenesis of PEDF.The MAECs were divided into four groups:①CTR siRNA + Vehicle group;②CTR siRNA +PEDF group;③LR siRNA+PEDF group;④Atg5 siRNA+PEDF group。Western blot was used to detect the protein levels of VEGFA and VEGFR2 in MAECs.The proliferation of MAECs was measured by 5-ethynyl-2’-deoxyuridine(EdU)method.24-well inserts with 8-μm pores were used for Transwell assay.Cells migration were evaluated by scratch wound healing assay.MAECs were seeded in the 24-well plate coated with Matrigel for analyzing the ability of tube formation.10.Explore the effect of PEDF on autophagy flux of MAECsWestern blot was used to detect the expression of autophagy proteins,including LC3、Atg5、SQSTM1/p62 and Beclin1.The MAECs in four groups were transfected with mRFPGFP-LC3 adenovirus.The number of autophagosomes and autolysosomes were measured under fluorescence microscope.11.Explore the effect of PEDF in adipocytes on MAECsThe mature adipocytes differentiated from 3T3-L1 cells and MAECs were co-cultured to simulate the anatomical location of PVAT and intima.The adipocytes were transfected with PEDF siRNA or CTR siRNA,and co-cultured with MAECs.The PEDF levels in supernatant were detected by ELISA.The MAECs migration were measured by Tranwell assay.12.Statistical analysesAll data were presented as the Mean ± SEM of three independent experiments at least.Statistical analysis was carried out using one-way analysis-of-variance(one-way ANOVA)followed by Turkey’s post hoc test(GraphPad,CA USA).The student’s t-test was used for statistical comparisons between two groups.P<0.05 was considered significant.Results1.The PEDF level was down-regulated in pericardial adipose tissue and serum of CAD patientsThe results showed that protein level of PEDF was significantly decreased in the PC AT of CAD patients.Although there was no statistical difference,mRNA expression of PEDF was also tended to be less in PCAT with CAD.The PEDF levels were reduced in patients with CAD.2.The PEDF level was decreased in PEDF shRNA micePEDF shRNA was injected via tail veins to knock down the PEDF in the whole body.The knock-down efficiency was verified through detect PEDF levels in serum and AS plaque after mice sacrificed.It was illustrated that the PEDF expression was significantly reduced in AS plaque and VAT with PEDF shRNA injection.The PEDF level in serum was also decreased in the PEDF shRNA mice.3.The decrease of PEDF increased the inflammatory factorsThe ELISA analyses illustrated that IL-6 and IL-1β levels in serum were increased in PEDF knock-down mice.4.The decrease of PEDF did not influence the lipid levelsThere were no significant differences in the serum levels of TC,TG,LDL-C and HDL-C between CTR shRNA and PEDF shRNA.5.The decrease of PEDF accelerated AS processionAfter HFD for 12 weeks,the relative en face atherosclerotic area was significant enlarged in PEDF knock-down mice.The sectional lesion size at aortic root was also augmented.It demonstrated the decrease of PEDF accelerated AS procession.The accumulation of macrophages and lipids were increased while the content of SMCs and collagen were decreased in the plaque.These data demonstrated that the plaque vulnerability index was dramatically increased in PEDF knock-down mice.Meanwhile,the results indicated that the expressions of TNFα,IL-6 and IL-1β were significantly increased in the plaque of PEDF knock-down mice.6.The PEDF in PVAT ameliorated the AS plaqueOil O red staining showed that the en face atherosclerosis area of left carotid artery surrounded by PVAT in the operation group was significantly augment compared with the nonoperation group.Oil O red staining showed that the sectional lesion size of carotid artery in operation mice with AAV-PEDF injection were decreased compared with the operation mice with AAV-CTR injection.The accumulation of macrophages and lipid in plaque were decreased and the content of smooth muscle cells and collagen were increased in the operation mice with PEDF overexpression in transplanted PVAT compared with the mice without PEDF overexpression in transplanted PVAT.7.PEDF in PVAT ameliorated the inflammatory state in carotid plaqueTo investigate the mechanisms of PEDF anti-atherosclerosis action,we detected the inflammatory levels in carotid artery plaque.The results of IHC staining showed that the expression of TNFa and IL-6 in plaque were reduced with the overexpression of PEDF in PVAT.8.PEDF in PVAT inhibited the proliferation of VVWe stained the frozen sections of carotid arteries and the surrounded PVAT with endomucin primary antibody to evaluate the angiogenesis of VV.The positive areas of endomucin were smaller in the operation mice with PEDF overexpression in transplanted PVAT compared with the mice without PEDF overexpression in transplanted PVAT.9.The knockout of PEDF accelerated AS processionThe area of plaque of aorta in ApoE-/-/PEDF-/-mice was decreased compared with the ApoE-/-mice.The accumulation of macrophages and lipids were increased while the content of SMCs and collagen were decreased in the plaque of ApoE-/-/PEDF-/-mice.These data demonstrated that the plaque vulnerability index was dramatically increased in ApoE-/-/PEDF/-mice.10.The knockout of PEDF increased the inflammatory factors in VATThe results showed that the expressions of IL-6 and TNFa were significantly increased in VAT of ApoE-/-/PEDF-/-mice compared with ApoE-/-mice.The IL-1β protein level trended to be increased in VAT of ApoE-/-/PEDF-/-mice.11.The knockout of PEDF increased lipid levels in serumThe serum levels of TC,TG and LDL-C were significantly increased in ApoE-/-/PEDF-/mice compared with ApoE-/-mice.The HDL-C levels trended to be decreased in ApoE-/-/PEDF/-mice.12.PEDF inhibited the neovascularization of MAECsVEGFA expression was markedly reduced in MAECs with PEDF treatment.The decrease was depended on incubation time.The ratio of VEGFA to VEGFR2 of MAECs was significantly diminished at 8,12,16,20,24 h of PEDF treatment.To investigate the influence of PEDF on MAECs,the capacity for proliferation,migration and tube formation were evaluated in vitro.The EdU positive ratio was reduced by PEDF treatment.PEDF also inhibited cell migration.Compared with the control group,tubular morphogenesis was also inhibited by PEDF treatment.13.PEDF inhibited the angiogenesis of MAECs through LRThe down-regulation of VEGFA by PEDF was inhibited by LR siRNA transfection.LR also repressed the effect of PEDF on VEGFR2.PEDF reduced the ratio of VEGFA/VEGFR2 of MAECs,and interference of the LR expression could debilitated this regulation.The inhibition effects of PEDF on the proliferation,migration and tube formation were alleviated by LR siRNA transfection.Co-IP results showed that LR combined with RACK1,and inhibition of RACK1 could also reverse the anti-angiogenesis effect of PEDF on MAECs.14.PEDF inhibited the angiogenesis of MAECs by changing the autophagy level through LR pathwayTo investigate the possible molecular mechanism,the Atg5 siRNA and CTR siRNA were transfected into MAECs.We detected the autophagy levels of MAECs with PEDF treatment.The results implicated that PEDF increased the expression of LC Ⅱ and decreased the expression of SQSTM1/p62.This stimulative action was in a time dependent manner.The number of autophagosomes and autolysosomes was increased with PEDF treatment.VEGFA expression was increased in Atg5 siRNA+PEDF group compared with the CTR siRNA+PEDF group.The inhibition effects of PEDF on the proliferation,migration and tube formation were alleviated by Atg5 siRNA transfection,which demonstrated that PEDF inhibited the neovascularization of MAECs through regulating autophagy.The number of autolysosomes was reduced in Atg5 siRNA+PEDF group compared with the CTR siRNA+PEDF group.The accelerative effect of PEDF on autophagy flux was evidently inhibited with LR knockdown.15.PEDF in adipocytes inhibited the migration of MAECsThe PEDF level in the supernatant of PEDF siRNA adipocytes was decreased compared with CTR siRNA adipocytes.The decreased PEDF of adipocytes increased the number of migrated MAECs in the co-culture system.Conclusion1.The PEDF levels in PVAT were reduced in CAD patients.2.PEDF knock-down accelerated AS procession.3.Transplanted PVAT promoted the development of AS.4.PEDF in PVAT inhibited VV proliferation and ameliorated AS procession.5.PEDF inhibited the angiogenesis of MAECs by regulating the autophagy level through LR pathway.Dissertation Ⅱ Ginsenoside Rbl Enhances Plaque Stability and Inhibits Adventitial Vasa Vasorum via the Modulation of miR-33 and PEDFObjectives1.To investigate PEDF as a target gene of mi-33;2.To investigate the effect of Rbl on atherosclerosis.3.To investigate the effect of Rb1 on adventitia vasa vasorum angiogenesis.4.To verify the mechanism of Rbl regulating adventitia vasa vasorum angiogenesis.Methods1.Identification of PEDF as a target gene of miR-33 in HUVECsThe 3’-UTR of SERPINF1 was cloned into the pLightSwitch-3’-UTR vector to construct the wild type plasmid(WT).To confirm the predicted binding site,a construct with mutated complementary to seed region of miR-33a(MUT)were generated.Cells seeded were cotransfected with WT or MUT plasmid,phRL-TK,and miR-33a mimic or control mimic.After 24 h transfection,cells were lysed by lysis buffer and luciferase activity was measured.2.Establish the mouse model of atherosclerosis45 female ApoE-/-mice at 8 week-age with a C57BL/6 background were fed with a highfat diet for 20 weeks.3.Explore the effect of Rbl on PEDF expression and atherosclerotic plaque stability(1)HE stainingTissue sections were stained with hematoxylin and eosin to show the morphology of the plaques.(2)Oil O stainingTo evaluate the plaque burden of the aortas and the content of lipid in plaque,the aortas and aortic root sections were stained with oil-red O solution.(3)Sirius red stainingThe sections of carotid artery and aorta were stained by Sirius red to quantified the collagen content in plaque.(4)Immunohistochemical(IHC)stainingIHC staining was used to detect the expression levels of PEDF,TNFα,IL-6 and IL-lβ in plaque.The positive areas of macrophages(MOMA2)and smooth muscle cells(αSMA)in plaque were also analyzed by IHC staining.4.Verify the specific mechanism of Rbl anti-angiogenesis actionAll ApoE-/-mice were randomly divided into 3 groups(n=15 per group),including Vehicle+LV-vector group,Rb1+LV-vector group,and Rb1+LV-miR-33 group.Mice in Rbl+LV-vector group were treated with lentivirus to overexpress miR-33 and intraperitoneal injection of Rb1(50mg/kg/d for 4 weeks).The mice in Rbl+LV-vector group received empty lentivirus by tail vein injection and Rbl treatment by intraperitoneal injection.The mice in Vehicle+LV-vector group were treated with equal amount of saline and empty lentivirus.RTqPCR was used to detect the miR-33 expression levels in the three groups.VV in adventitia were identified through the perfusion with biotinylated Lycopersicon Esculentum(Tomato)lectin.5.Explore the effect of Rb1 on lipidThe serum levels of TC,TG,LDL-C and HDL-C were measured by biochemical detection.6.Statistical analysesAll data were presented as the Mean ± SEM of three independent experiments at least.Statistical analysis was carried out using one-way analysis-of-variance(one-way ANOVA)followed by Turkey’s post hoc test(GraphPad,CA USA).The student’s t-test was used for statistical comparisons between two groups.P<0.05 was considered significant.Results1.PEDF is a target gene of miR-33Overexpression of miR-33a but not the control mimics substantially repressed the luciferase activity with WT plasmid transfection.In contrast,no effect was found when miR33a-binding sites in the 3’-UTR of PEDF were mutated,which suggested that miR-33a could directly bind to the 3’-UTR of PEDF and regulate its expression.2.Rbl inhibited atherosclerotic plaque growth and enhanced plaque stability in ApoE-/miceThere is a significant reduction of the relative en face atherosclerotic lesion area as well as the cross-sectional lesion size at the aortic root,which demonstrated that the extent of atherosclerosis was significantly decreased in Rbl+LV-vector mice.Furthermore,immunohistochemistry analysis also revealed that Rbl treatment exerted notable effects on upregulating the contents of SMCs and collagen.Conversely,Rbl treatment could also reduce the accumulation of macrophages and lipid.As a consequence,the plaque vulnerability index was dramatically decreased in Rbl treated mice.Meanwhile,the immunohistochemical studies of inflammatory cytokines showed that Rbl treatment significantly reduced the expression levels of IL-1β,IL-6 and TNFa in plaque.All the above results indicated that Rbl treatment could enhance AS plaque stability.3.Rbl inhibited VV proliferation in atherosclerotic plaque of ApoE-/-miceConsidering the anti-angiogenic role of Rbl,average number of vasa vasorum in adventitia surrounding the atherosclerosis plaques in aortic roots were counted to uncover whether Rb1 could inhibit neovascularization in ApoE-/-mice.The results showed that Rbl+LV-vector group had less vasa vasorum,indicating that Rbl could inhibit proliferation of VV in plaques of ApoE-/-mice.4.The beneficial effects of Rb1 on atherosclerosis were attenuated by miR-33 overexpressionIn this study,ApoE-/-mice injected with miR-33 lentivirus were applied to study that miR33 overexpression attenuated the beneficial effects of Rbl on atherosclerosis.Compared with Rbl+LV-vector group,miR-33 overexpression significantly increased the accumulation of macrophages and lipid,decreased the contents of SMCs and collagen.Therefore,the plaque vulnerability index was dramatically increased in miR-33 overexpression mice.Moreover,upregulation of miR-33 could significantly attenuate the anti-inflammation effects of Rbl by decreasing the expression levels of IL-1β,IL-6,and TNFa within AS plaques in ApoE-/-mice.Rbl-mediated VV inhibition was abrogated by overexpression of miR-33.Together,these results demonstrated that miR-33 has profound negative impact on the Rbl-mediated enhancement of plaque stability,reduction of plaque burden,anti-inflammation and inhibition of VV in ApoE-/-mice.5.miR-33 was involved in Rbl-induced PEDF expression in atherosclerotic plaque in ApoE-/-miceRbl treatment led to the induction of PEDF expression in AS plaque.Interestingly,miR33 expression was also significantly downregulated in Rbl treated ApoE-/-mice.Overexpression of miR-33 results in a significant regression of Rbl-mediated PEDF elevation in AS plaque in ApoE-/-mice.6.Rbl had no effect on lipidThere were no significant differences in serum levels of TC,TG,LDL-C and HDL-C among the three groups.Conclusion1.PEDF is a target gene of miR-33.2.Ginsenoside Rbl attenuates plaque growth and enhances plaque stability partially through inhibiting adventitial vasa vasorum proliferation and inflammation in ApoE-/-mice.3.The anti-angiogenic and anti-inflammation effects of Rbl are exerted via the modulation of miR-33 and its target gene PEDF.