| Paper One An Experimental Study on the Suppressive Effects of Tumor Necrosis Factorαon the Rate-limiting Subunit of Collagen Synthase(P4Hα1) and the Underlying Molecular Mechanisms1.IntriductionMany pathologic changes in the cardiovascular system can be triggered by inflammation,which contributes to the disease process.Metabolic imbalance in the extracellular matrix(ECM) in the myocardium and arterial wall represents one of the key structural changes that mark the development and progression of most cardiovascular diseases.ECM components,especially collagen—the main constituent of the fibrous cap in atheroma—determines plaque stability and vulnerability to rupture.Furthermore,dysregulated ECM metabolism in the aortic wall,such as inadequate collagen degradation or elastin disruption,leads to aortic aneurysm and rupture.ECM is the structural framework of all tissues including the arterial wall,in which fibrillar proteins(collagen and elastin) and adhesive proteins(eg, laminin and fibronectin) form the structural backbone of the tissue.In the arterial wall,various cells including endothelial cells,smooth muscle cells,and fibroblasts,contribute to ECM metabolism.Collagen is one of the most metabolically active ECM components,with at least 39 subtypes;typesâ… andâ…¢are the ones most commonly found in the arterial wall.The collagen molecule consists of 3 identical polypeptide chains,calledαchains.This molecule has at least 1 triple-helical collagenous domain with repeating (Gly-X-Y) n sequences,ie,a glycine residue at every third amino acid and, frequently,proline and 4-hydroxyproline in the X and Y positions.Collagen biosynthesis involves a number of posttranslational modifications of procollagens and proteolytic conversion to collagens.The intracellular modifications require 5 specific enzymes,including 3 collagen hydroxylases and 2 collagen glycosyltransferases.Prolyl-4-hydroxylase(P4H) is one of the key intracellular enzymes required for the synthesis of all known types of collagens.It catalyzes the formation of hydroxyproline from proline residues located in repeating X-Pro-Gly triplets in the procollagens during posttranslational processing.It is essential for folding the procollagen polypeptide chains into stable triple helical molecules.Inhibition of P4H produces unstable collagen associated with collagen decrease.P4H is composed ofαandβsubunits in whichαsubunit is rate-limiting and essential for collagen maturation and secretion.Cytokines,including TNF-α,transforming growth factor(TGF)-α,and various interleukins,are dysregulated in inflammation and may participate in ECM metabolism by increasing ECM degradation through activation of matrix metalloproteinases(MMPs) and inhibition of collagen synthesis.TNF-α,which is released by activated macrophages,is one of the most potent cytokines involved in cardiovascular pathogenesis1 and actively regulates ECM metabolism.2.Objectives(1) To investigate whether TNF-αexerts an inhibitive effect on P4Hα1 in vitro;(2) To elucidate the underlying molecular mechanisms of TNF-α-mediated inhibition of P4Hα1 in vitro and explore a novel therapeutic target of plaque stabilization.3.Methods3.1 Plasmid ConstructionTo define promoter regions that are responsive to TNF-α-mediated P4Hα1 suppression,we generated P4Hα1 promoter-pGL3 constructs with serial deletions of the P4Hα1 promoter.The pGL3-basic vector(Cat#:E1751, Promega,Madison,Wi) was used for the plasmid construction.We produced plasmids containing P4Hα1 promoter regions from -580,-480,-417,-320,-271, -184,-145,-97,-32,and -18 to +76bp between the multiple cloning sites Kpnl and Hindâ…¢for the purpose of directional cloning.3.2 Cell Transfection and StimulationFor dose-dependent effects,HASMCs were treated with 0,1,10,and 100 ng/mL human recombinant TNF-α(Cat#T6674,Sigma-Aldrich) for 8 hours before cells were harvested for measurements of target gene mRNA levels. For the time-course study,we treated cells for 4,8,24,and 48 hours,1ug recombinant P4Hα-pGL3 plasmids were transfected to HASMCs with Lipofectamine 2000.we used NonO,hnRNP-K,BUB3,and ILF2 specific siRNAs(100 nmol/L) to inhibit the expression of these proteins in cells。3.3 RT-PCRP4Hα1,β-actin and luciferase mRNA levles were measured by RT-PCR after RNA extraction and reverse transcription.3.4 EMSAWe carried out electrophoretic mobility shift assay(EMSA) to ascertain whether the TaRE in the P4Hα1 promoter was binding with any transcription factors.For the antibody-based supershift assay,antibodies against transcription factors were added to a mixture of biotin-labeled oligonucleotide probes and nuclear proteins.3.5 Chromatin Immunoprecipitation Assay The chromatin immunoprecipitation(ChIP) assay was performed with the histone ChIP assay.We also used anti-transcription factor antibodies instead of anti-H3 histone antibody for the immunoprecipitation process.3.6 Western BlotProteins were extracted form HASMC,separated with 10%SDS-PAGE, and transferred to nitrocellulose membranes.After incubation with the primary and secondary antibodies and exposure,protein expression was determined.4.Results4.1.TNF-α-mediated Suppression of P4Hα1 ExpressionWe treated HASMCs with TNF-α100 ng/mL for 4,8,24,and 48 hours.At the end of each of these time points,cells were harvested and P4Hα1 mRNA levels were measured.We found that P4Hα1 mRNA levels were significantly decreased up to 8 hours after treatment.In examining dose-response effects, we observed a linear reduction in P4Hα1 mRNA levels in HASMCs treated with 1 to 100 ng/mL TNF-αfor 8 hours in culture.4.2.Identification of TNF-αRegulatory Element(TaRE) in the P4Hα1 5'-Flanking SequenceWe transfected HASMCs with several pGL3 reporter constructs containing the progressively deleted 5'-flanking regions of the P4Hα1 gene and measured mRNA levels of the luciferase gene after treatment with TNF-α.Compared with no treatment,TNF-αdramatically reduced promoter activity in all P4Hα1 promoter vectors except when the region of -32bp to +18bp was deleted in the promoter region(Figure 2),which abolished more than 80%of the TNF-α-induced inhibition.4.3.Direct Binding of NonO and hnRNP-K to the P4Hα1 PromoterThe density of the supershift band with the anti-NonO antibody was significantly increased when nuclear proteins extracted from TNF-α-treated HASMCs were used in the EMSA.Only TNF-α-treated HASMCs showed a NonO-TaRE interaction in the ChIP assay.NonO silencing abolished more than 60%of TNF-α-mediated P4Hα1 suppression.4.4.Involvement of the ASK1-JNK PathwayJNK inhibitor recovered almost 100%of the TNF-α-mediated P4Hα1 suppression,after treatment with the ASK1 inhibitor,TNF-α-mediated P4Hα1 suppression recovered to a degree similar to that of JNK inhibitor.4.5.Role of DJ-1 in TNFα-MKK4-JNK1-NonO-mediated P4Hα1 SuppressionAfter DJ-1 expression was silenced by DJ-1-specific siRNA in HASMCS,we observed a 50%recovery of P4Hα1 expression in HASMCs treated with TNFα. Using the ChIP assay with the anti-NonO antibody for immunoprecipitation,we showed that TNF-αinduced NonO-TaRE binding was abolished when the expression of DJ-1 was suppressed by gene specific siRNA.The ChIP assay (with anti-oxidized DJ-1 antibody) showed that the binding of DJ-1 to TaRE was abrogated by NonO siRNA.4.6.Role of histone acetylation in TNFα-MKK4-JNK1-NonO-mediated P4Hα1 suppressionIn contrast,although TNFαor JNK1 induced H4 lysine 12(H4L12) acetylation,the treatments induced deacetylation at H3 lysine.HATI abolished the TNFα-induced H4L12 acetylation,and the HDAC inhibitor abrogated TNFα-induced H3L9 deacetylation.These results demonstrated that silencing NonO by gene specific siRNA had no effect on TNFα-induced H4L12 acetylation.However,NonO suppression abolished TNFα-induced H3L9 deacetylation.5.Conclusions(1) Through direct binding to TaRE in the TNF-αpromoter,TNFαactivates the ASK1-MKK4-JNK1-NonO pathway,oxidizes DJ-1 and phosphorylates NonO,thereby suppressing P4Hα1 transcription.(2) These results revealed the key molecules involving inflammation-related collegen degradation and provided novel therapeutic targets for reducing collegen degradation in the fibrous cap of atherosclerotic plaques and in the aortic wall,thereby stabilizing vulnerable plaques and inhibiting the formation of aortic aneurysm. Paper Two An Experimental Study on the Expression Level and Regulatory Mechanisms of Arginaseâ…¡in Atherosclerotic Plaques1.IntroductionIn recent years,atherosclerosis(AS)-induced cardiovascular disease has become the number one killer in many countries of the world.Many studies have demonstrated that vascular endothelial dysfunction is the earliest detectable abnormalities in the long time course of AS and brachial arterial endothelial dysfunction measure by high frequency ultrasound has been used as a surrogate endpoint for acute cardiovascular events in multiple clinical trials.Nitric oxide(NO) produced by endothelial nitric oxide synthase(eNOS) is the most important factor for regulating endothelial function.NO directly dilates blood vessels and inhibits adhesion and aggregation of platelets, proliferation and migration of smooth muscle cells and expression of adhesion molecules by endothelial cells,thereby exerting an anti-atherosclerosis effect. Previous studies have confirmed that the reduction in NO synthesis may lead to endothelial dysfunction,accelerate AS lesions and induce cardiovascular events.Accumulating evidence now exists that NO plays a key role in the development and progression of AS.At present,three types of NO synthase have been reported in the literature.The first type of NO synthase was found in the brain and thus named neuronal nitric oxide synthase(nNOS),although it was detected later in many other cells including vascular endothelial cells.The second type was discovered in macrophages and named inducible nitric oxide synthase(iNOS). The third type was found in the aortic endothelial cells and therefore named eNOS.Among the three types,eNOS is the most important in mediating vascular function,eNOS can be activated by some signals from the endothelial cells and the configuration changes,especially phosphorylation,of eNOS may affect the activity of this particular enzyme.L-arginine is a semi-essential amino acid engaging many pathological processes,which can be catalyzed into L-arginine,L-ornithine or polyamines and serve as substrate of NO synthase.Converted by eNOS in endothelial cells,L-arginine becomes L-citrulline and NO,and hence is the precursor of NO.Because of the key role of NO in vascular protection and the important function of L-arginine in the synthesis of NO,some authors have stepped forward to investigate the function of L-arginine for vessel protection and they are able to show that L-arginine can activate eNOS-NO pathway and treatment with L-arginine can even reduce the incidence of AS-related diseases in animals.Arginase is a manganese metalloprotease which can catalyze L-arginine into L-ornithine and urea.Arginase has two isozymes-arginineâ… and arginineâ…¡, with 60%homologous sequence.Arginaseâ… expresses mainly in the liver and undertakes the most activities of arginase in the body.Arginaseâ…¡expresses in most tissues,in particular,kidney and prostate.Recent studies have reported that vascular tissues including the aortic,pulmonary,carotid and coronary arteries are rich in both arginaseâ… and arginaseâ…¡.Since arginase is capable of catalyzing arginine into L-ornithine and urea, it may compete with eNOS in catalyzing L-arginine and impede the conversion of L-arginine to NO.Metabolic Studies of L-arginine in macrophages found that there was more conversion of L-arginine to urea than to NO.The production of NO was increased after inhibition of arginase both in vitro and in vivo.On the other hand,overexpression of arginaseâ… and arginaseâ…¡reduced the expression levels of L-arginine and NO at the same time.Arginaseâ…¡has been found to be more closely related to AS than arginaseâ… as RNA interference of arginineâ…¡attenuated arginine upregulation by Ox-LDL and enhanced NO expression in human aortic endothelial cells.In the early aortic atherosclerotic lesions in Apo-/- mice,the activity of arginaseâ…¡was increased while the level of NO decreased.These studies,however,were limited to early atherosclerotic lesions and it remains unclear whether the expression level of arginaseâ…¡continues to increase and that of eNOS continues to decrease with increased plaque burden.Therefore,it is still an open question whether arginaseâ…¡can serve as a sensitive biomarker and an effective therapeutic target in the late stage of atherosclerosis with a big plaque burden.Besides,as the transcription process of arginaseâ…¡is unknown,whether arginase can be inhibited at the levels of promoter or transcription factors constitutes another open question.2.Objectives:(1) To establish a carotid atherosclerotic model with a large plaque burden in ApoE-/- mice with placement of a stenotic carotid collar and to measure the expression levels of arginaseâ…¡and eNOS in these plaques;(2) To detect the positive region of arginaseâ…¡promoter and the transcription factors binding with this promoter and thereby clarify arginiaseâ…¡transcription process.3.Methods3.1.Plasmid ConstructionpGL3-basic vector was used to define positive promoter regions of arginaseâ…¡.We generated arginaseâ…¡promoter-pGL3 constructs with serial deletions of the arginaseâ…¡promoter.After the PCR amplification and plasmid construction,we produced plasmids containing arginaseâ…¡promoter regions between the multiple cloning sites Xhoâ… and Hindâ…¢for the purpose of directional cloning. 3.2.Animal Model80 male apoE-/- mouse were fed with an atherogenic chow and underwent right carotid collar placement to induce atherosclerotic lesions with a big plaque burden.The left carotid artery without collar placement was used as a internal control.3.3.Cell CultureHuman Hela cells and endothelial cells were cultured for experimental studies.3.4.Immunohistochemical AnalysisExpression levels of eNOS and arginaseâ…¡proteins in atherosclerotic lesions were measured using appropriate primary antibodies.3.5 Real-time RT-PCRThe gene expression levels of luciferase were quantitatively analyzed using RT-PCR.4.Results4.1.Histological Measurement4.1.1.Hematoxylin and Eosin StainingIncreased intima and media area and significant plaques were detected in the right carotid atherosclerotic lesions.No plaques were found in the left carotid artery.4.1.2.Masson StainingCollagen expression was abundant in the right common carotid artery of ApoE-/- mice.4.1.3.Picrosirius Red StainingThe relative content of typeâ… ,â…¡andâ…¢collagen fibers was increased significantly in atherosclerotic plaques of the right carotid artery4.1.4.Oil Red O StainingThe relative content of lipids was significantly increased in atherosclerotic plaques of the right carotid artery of ApoE-/- mice. 4.2.Expression of eNOS in PlaquesImmunohistochemical staining revealed that the expression level of eNOS proteins was significantly lower in the right carotid artery than that in the left carotid artery.4.3.Expression of Arginaseâ…¡in PlaquesImmunohistochemical staining showed that the expression level of arginaseâ…¡proteins was significantly higher in the right carotid artery than that in the left carotid artery.4.4.Promoter Region Responsible for Arginaseâ…¡Transcription ActivitypGL3-basic vectors combined with arginaseâ…¡promoter regions were transfected into HELA cells for 24h and the expression of luciferase was detected by RT-PCR.The result showed that the region of-704bp to-644bp was responsible for most activities of arginaseâ…¡promoter.4.5.Identification of Transcription Factors Biding with Arginaseâ…¡PromoterWe designed biotin-labeled probe containing the -704bp to -644bp region of arginaseâ…¡promoter.Proteins binding with the probe were pulled-down using immunoprecipitation and stained with Coomassie blue after gel electrophoresis.A clear band was cut for mass spectrometry and proteins PARP1,PSPC1 and SFPQ binding with the positive region of arginaseâ…¡promoter were finally indentified as the transcription factors.5.Conclusions(1) The expression level of arginaseâ…¡was increased whereas that of eNOS decreased in atherosclerotic lesions with a big plaque burden.Transcription factors PARP1,PSPC1and SFPQ bind with the positive region of arginaseâ…¡promoter and mediate the transcription process of arginaseâ…¡.(3) These results suggest that arginaseâ…¡can serve as a biomarker and a therapeutic target in atherosclerosis.By effectively inhibiting the synthesis of arginaseâ…¡,the activity of eNOS can be enhanced and endothelial function improved.Thus,our finding has important theoretical significance as well as application potentials.â… Paper Three An Experimental Study on the Therapeutic Effects of ACE2 Gene Transfection on Early Atheroscleriotic Lesions and the Underlying Molecular Mechanisms1.IntriductionAccumulating evidence indicates that the renin-angiotensin system(RAS) plays an important role in the pathogenesis of atherosclerosis.Angll increases mRNA and protein expression of monocyte chemoattractant protein-1(MCP-1) and lectin-like oxidized low-density lipoprotein receptor-1(LOX-1) and enhances the uptake of low-density lipoprotein(LDL) through LOX-1 by endothelial cells,macrophages and smooth muscle cells.MCP-1 is the major chemotactic factor contributing to monocyte adhesion to endothelial cells,one of the earliest events in the pathogenesis of atherosclerosis.Likewise,LOX-1 plays an essential role in endothelial injury and dysfunction.Therefore, inhibition of ACE by ACE inhibitors or AT1 receptor blockers is effective against atherosclerosis.Angiotensin-converting enzyme 2(ACE2),a zinc-bearing metalloproteinase,is the first angiotensin-converting enzyme(ACE) homologue identified in 2000.The human ACE2 protein consists of 805 amino acids.The expression of ACE2 is highly tissue specific and is found to be distributed mainly in heart,kidney and didymus tissue.Recent studies have demonstrated that in physiological and pathological conditions,ACE2 competes with ACE by converting vasoconstrictive angiotensinâ…¡(Angâ…¡) into vasodilative Ang-(1-7).ACE2 appears to play a protective role in cardiovascular system.Both ACE and ACE2 mRNA and protein expression is increased in rats with acute myocardial infarction.Many studies have found ACE2 protein expressed in atherosclerotic plaques in New Zealand white rabbits,mainly in endothelial cells and foam cells.Cardiac function was shown to decline in ACE2 gene knock-out mice,but overexpression of ACE2 by use of an adenovirus(Ad) vector significantly inhibited the development of myocardial fibrosis.These studies suggest that ACE2 may protect myocardium and vessels by counteracting ACE and relieving the harmful effects of RAS and hence is a potential therapeutic target in cardiovascular diseases.2.Objectives:(1)To attest the therapeutic effects of ACE2 overexpression on early atherosclerotic lesions;(2) To elucidate the molecular mechanisms of ACE2-mediated therapeutic effects on atherosclerotic lesions.3.Methods:3.1.Preparation of ACE2 Ad VectorThe murine ACE2 cDNA was amplified by RT-PCR from RNA of mouse kidney.Recombinant adenoviruses(Ad) carrying the murine ACE2(AdACE2) or a control transgene EGFP(Ad-EGFP) were prepared as previously described with the AdMax system.3.2.Animal Model and Gene TransferOne hundred of New Zealand white rabbits were fed an atherogenic chow and underwent balloon induced arterial endothelial injury after anesthesia using a previously described method.Rabbits were randomly divided to 5 groups(n=20 in each group) and gene therapy was initiated at the end of week 4.Rabbits in group Ad-ACE2 or Ad-EGFP or Ad-ACE2+A779 or A779 or control received a suspension of Ad-ACE2(2.5×109pfu) or Ad-EGFP (2.5×109pfu) or Ad-ACE2+A779 or A779 or no injection,respectively.A779 is an antagonist of Ang-(1-7) receptor and was administered at a dose of 200 ng kg-1 min-1 for 28 days.Rabbits were maintained on the high-cholesterol diet for additional 4 weeks.The rabbits were anesthetized and their aortas were collected for pathological and biochemical analysis.3.3.Cell Culture and Gene TransferThe HUVECs were incubated with Ang-â…¡for 24 hours before they were divided into 10 groups that received Ad-ACE2,Ad-EGFP,Ad-ACE2+A779 (1μmol/l),A779,Ad-ACE2+ERK inhibitor,ERK inhibitor,Ad-ACE2+Akt inhibitor,Akt inhibitor,Ang-(1-7) and no treatment(control group),respectively. Ad-ACE2(1×106 pfu) or Ad-EGFP(1×106 pfu) was transfected into cells per well in a 6-well plate and cells were harvested 24,48 and 72 hours after gene transfection for western blot analysis.Cells in the Ang-(1-7) group were collected 4,8,16 and 24 hours after treatment with Ang-(1-7) at a dose of 1×106 mol.3.4.Measurement of ACE2 and ACE ActivityThe enzymatic activities of ACE2 for converting Ang-â…¡to Ang-(1-7) and those of ACE for converting Ang-â… to Ang-â…¡were evaluated by surface-enhanced laser desorption/ionization time of flight mass spectrometry.3.5.Serum Lipid MeasurementBlood samples were collected at baseline and at the end of week 4 and week 8,respectively,and serum concentrations of total cholesterol(TC) and triglyceride(TG) were determined by enzymatic assays.3.6.Immunohistochemical AnalysisMacrophages,LOX-1,MCP-1,Ang-(1-7),ERK and p38 in atherosclerotic lesions were identified using appropriate primary antibodies.3.7.Real-time RT-PCR The gene expression levels of ACE2 were quantitatively analyzed using RT-PCR.3.8.Western Blot AnalysisThe protein expression of ACE2,ACE,Ang-â…¡,AT1R,MCP-1,LOX-1, Ang-(1-7),ERK,p38,PI3K,AKT and ROS were assayed by Western blot both in Vivo and in Vitro,respectively.4.Results4.1.ACE2 Expression and Activity after Gene TransferACE2 expression was high in atherosclerotic lesions of Ad-ACE2 and Ad-ACE2+A779 groups but low in lesions of Ad-EGFP,A779 and control groups.Similarly,western blot analysis showed that ACE2.protein expression in human endothelial cells was significantly increased 24 hours after Ad-ACE2 transfection in comparison with Ad-EGFP and control groups.4.2.Effects of ACE2 Gene Transfer on Atherosclerotic LesionsMarked intimal thickening was present in the Ad-EGFP,control and A779 groups.However,the intima area and the ratio of intimal area/media area(I/M area) were significantly lower in the Ad-ACE2 group than in the Ad-EGFP, control or A779 groups.Conversely,with the administration of A779 in the Ad-ACE2+A779 group,the intimal area and the ratio of I/M area were significantly increased compared with those in the Ad-ACE2 group.4.3.Effects of ACE2 Gene Transfer on MCP-1 and LOX-1 ExpressionImmunohistochemistry was used to examine the expression of MCP-1 and LOX-1.The abdominal aorta in Ad-EGFP,control and A779 groups were heavily stained,as compared with those in the Ad-ACE2 group.Western blot analysis in cultured endothelial cells demonstrated a lower expression of MCP-1 in the Ad-ACE2 group than that in the Ad-EGFP and the control groups 48 hours after ACE2 transfection.Similarly,LOX-1 was significantly suppressed by ACE2 gene transfer but this effect was not observed until 72 hours after gene transfer. 4.4.Effects of ACE2 Transfection on Ang-(1-7) ExpressionThe Ang-(1-7) protein expression level was higher in the Ad-ACE2 and Ad-ACE2+A779 groups than that in the Ad-EGFP,control and A779 groups in the aortic lesions.Ang-(1-7) protein expression level in the Ad-ACE2 group was significantly increased in comparison with that in the Ad-EGFP and control groups in HECs.4.5.Effects of ACE2 Transfection on ACE ExpressionThe ACE protein expression determined by immuohistochemistry were significantly lower in the Ad-ACE2,Ad-ACE2+A779 group than that in the Ad-EGFP,A779 and control groups.Western blot analysis indicated that the ACE protein expression level was significantly reduced 24 hours after gene transfection in the Ad-ACE2 group compared with that in the Ad-EGFP and control groups.4.6.Effects of ACE2 Transfection on AT1R ExpressionAT1R protein expression level detected by western blot were significantly lower in the Ad-ACE2,Ad-ACE2+A779 group than that in the Ad-EGFP,A779 and control groups.Reduction of the AT1R protein expression level was also observed 48 hours after Ad-ACE2 transfection and 16 hours after Ang-(1-7) treatment in HECs.4.7.Effects of ACE2 on ERK-p38-ACE ExpressionERK and p38 protein expression level was significantly lower in the Ad-ACE2,Ad-ACE2+A779 group than that in the Ad-EGFP,A779 and control groups according to immunohistochemical analysis.The ERK and p38 protein expression level was significantly reduced in the Ad-ACE2 group 24 hours after gene transfection in comparison with that in the control and Ad-EGFP groups.4.8.Effects of ACE2 on PI3K-Akt-MCP1/LOX1 ExpressionThe PI3K and Akt protein expression in atherosclerotic lesions were detected by western blot.The results indicated that protein expression levels of PI3K and Akt were significantly higher in the Ad-ACE2 and Ad-ACE2+A779 group than that in the Ad-EGFP,A779 and control groups.The PI3K and Akt protein expression level in the Ad-ACE2 group 24 hours after gene transfection was significantly higher than that in the control and Ad-EGFP groups.4.9.Effects of AdACE2 on ROS Expression in vitroThe ROS protein expression level was significantly reduced in the Ad-ACE2 group in comparison with that in the control and Ad-EGFP groups and this effect was not enhanced by increasing gene transfection time to 48 and 72 hours5.Conclusions(1) ACE2 overexpression significantly attenuated early atherosclerotic lesions via cross-talk among multiple signaling pathways including Ang-â…¡-AT1R-ERK-p38-ACE,Ang-â…¡-AT1R-ROS-MCP-1/LOX-1, Ang-â…¡-AT1R-PI3K-Akt-LOX-1/MCP-1,Ang-(1-7)-ERK-p38-ACE, Ang-(1-7)-PI3K-Akt-LOX-1/MCP-1 and Ang-(1-7)-ROS-MCP-1/LOX-1 pathways.(2) Through selective enhancement or inhibition of the cross-talk among Ang-â…¡å’ŒAng-(1-7) signaling pathways,early AS lesions may be suppressed.Thus, our results provide multiple novel targets for the treatment of early AS lesions.
|
PDF Full Text Request