Basic And Clinical Studies On The Role Of Angiotensin-(1-7) In The Treatment And Evaluation Of Diabetic Cardiomyopathy

BackgroundDiabetic cardiomyopathy (DCM), characterized by left ventricular (LV) remodeling and dysfunction, is associated with substantial risk of heart failure and increased mortality. Hyperglycaemia-induced oxidative stress and inflammation has been implicated in the pathogenesis of DCM. Cardiac fibrosis, myocardial hypertrophy and myocyte apoptosis, are considered to be the three major pathological features of the LV remodeling in DCM. Prevention and alleviation of LV remodeling is the keystone in the management of DCM.A wealth of evidence indicates that the renin-angiotensin system (RAS) plays an important role in the pathogenesis of DCM, and angiotensin-converting enzyme (ACE), angiotensin Ⅱ (Ang-Ⅱ), angiotensin-converting enzyme2(ACE2) and angiotensin-(1-7)[Ang-(1-7)] are recognized as important components of RAS. Previous studies have demonstrated that ACE inhibitors and Ang-Ⅱ type1receptor (AT1R) antagonists are efficacious in improving LV remodeling and function in DCM. Recently, Zhong J et al. reported that Ang-Ⅱ-induced myocardial fibrosis, hypertrophy and diastolic dysfunction were exacerbated by ACE2deficiency but substantially attenuated by recombinant human ACE2, which was associated with reduced plasma and myocardial Ang-Ⅱ and increased plasma Ang-(1-7) levels. More recently, we found that that local ACE2overexpression, via downregulating Ang-Ⅱ and upregulating Ang-(1-7) expression in the myocardium, remarkably inhibited interstitial collagen accumulation and improved LV remodeling and function in a rat model of DCM. These studies suggest that Ang-(1-7) may provide a promising molecule in the treatment of DCM.Ang-(1-7), a heptapeptide converted from Ang-Ⅱ by ACE2, binds to a distinct plasma membrane G protein-coupled receptor, the Mas receptor (MasR), and exerts vasodilative, anti-proliferative and anti-inflammatory effects. Our recent study found that ACE2overexpression downregulated AT1R protein expression in vivo and in vitro, suggesting that the effects of Ang-(1-7) may involve receptors other than MasR. Thus, several important issues raised by recent studies need to be clarified. First, what is the dose-effect relationship between Ang-(1-7) and DCM? Second, is Ang-(1-7) superior to ACE inhibition in the treatment of DCM? Third, is the combination of Ang-(1-7) and ACE inhibition better than Ang-(1-7) alone in alleviating DCM? Forth, what are the roles of AT1R, Ang-Ⅱ type2receptor (AT2R) and MasR in mediating the therapeutic effects of Ang-(1-7) in DCM? To address these issues, we hypothesize that chronic infusion of Ang-(1-7) may dose-dependently ameliorates LV remodeling and function in a rat model of DCM, and the combination of Ang-(1-7) and ACE inhibition may be superior to the solo therapy. A series of in vitro and in vivo experiements were designed and performed to validate this hypothesis.ObjectiveThe study was designed to test the hypothesis that chronic infusion of angiotensin-(1-7)[Ang-(1-7)] may dose-dependently ameliorate left ventricular remodeling and function in a rat model of diabetic cardiomyopathy (DCM) and the combination of Ang-(1-7) and angiotensin-converting enzyme inhibition may be superior to the solo therapy.MethodsAnimal ModelOne hundred twenty-six male Wistar rats were divided randomly into model group (n=112) and control group (n=14). Diabetes was induced in the model group by a single intraperitoneal injection of streptozotocin. At the end of week 12after injection, all surviving rats in the model group were randomly divided into8groups for treatment:mock group, perindopril group, low-dose Ang-(1-7) group, moderate-dose Ang-(1-7) group, high-dose Ang-(1-7) group, high-dose Ang-(1-7)+perindopril group, high-dose Ang-(1-7)+A779group and high-dose Ang-(1-7)+PD123319group.Blood Pressure MeasurementPeripheral arterial systolic and diastolic blood pressure was measured by the tail-cuff method.Echocardiographic and Hemodynamic MeasurementEchocardiographic and hemodynamic measurement was performed after treatment.Blood ChemistrySerum levels of total cholesterol, triglycerides and blood glucose were analyzed by use of the Bayer1650blood chemistry analyzer.Histological AnalysisWe used4-μm paraffin-embedded tissue sections for hematoxylin and eosin (H&E) and Masson trichrome staining to assess tissue architecture and interstitial and perivascular fibrosis.Transmission Electron Microscopy (TEM)After hearts were excised, fresh LV tissue was quickly cut into1-mm cubes and underwent standard block preparation for TEM.Real-Time RT-PCRThe mRNA levels of genes were determined using standard methods and their relative levels were quantified by the2ΔΔCT method, with β-actin as the endogenous reference gene.Isolation and Culture of Neonatal Rat Cardiac Fibroblasts and MyocytesNeonatal rat cardiac fibroblasts and myocytes were isolated and cultured using standard methods.3H-Proline Incorporation AssayCollagen synthesis of cultured cardiac fibroblasts was measured by 3H-proline incorporation using standard methods.ZymographyThe activity of matrix metalloproteinase (MMP)-2and MMP-9in cardiac fibroblasts was evaluated by zymography.ELISAELISA was used to measure protein levels of soluble collagen Ⅰ and Ⅲ and transforming growth factor-beta1(TGF-β1) in the medium of cardiac fibroblasts, Ang-(1-7) in rat myocardium, and Ang-Ⅱ in vivo and in vitro.Assessment of Cardiomyocyte HypertrophyThe cross-sectional area of myocytes in cardiac sections was measured by staining with Alexa Fluor(?)488-conjugated wheat germ agglutinin and the surface area of cultured cardiomyocytes was determined by immunostaining with a rabbit polyclonal antibody against myosin heavy chain.Detection and Quantitation of ApoptosisApoptotic cells in tissue sections were quantified by terminal deoxynucleotidyltransferase-mediated dUTP nick-end labeling (TUNEL). Apoptosis of cultured cardiomyocytes was evaluated by double immunofluorescence for myosin heavy chain and TUNEL.Dihydroethidium Fluorescence and Lucigenin-Enhanced ChemiluminescenceThe oxidative fluorescent dye dihydroethidium was used to measure superoxide (O2-) levels in myocardial frozen sections and cultured cardiac fibroblasts and myocytes. NADPH oxidase activity in myocardial tissues and cardiac fibroblasts and myocytes was quantified by lucigenin-enhanced chemiluminescence.Western Blot, Immunohistochemistry, and ImmunocytochemistryWestern blot, immunohistochemistry, and immunocytochemistry were performed using standard methods.Statistical AnalysisSPSS v11.5(SPSS Inc., Chicago, IL) was used for statistical analysis. Continuous data were expressed as mean±SEM and compared by one-way ANOVA, followed by Tukey-Kramer post hoc test and independent samples t test. Survival rates were assessed by Kaplan-Meier analysis and log-rank tests. A P value<0.05was considered statistically significant.ResultsAng-(1-7) treatment dose-dependently ameliorated left ventricular remodeling and dysfunction in DCM rats by attenuating myocardial fibrosis, myocardial hypertrophy and myocyte apoptosis. The molecular mechanisms involved reduced inflammatory cytokine expression, oxidative stress and collagen synthesis, inhibited phosphorylation of extracellular signal-regulated kinase1/2and p38mitogen-activated protein kinase and expression of transforming growth factor-beta1as well as a complex interaction of Mas receptor, angiotensin Ⅱ type2receptor and type1receptor. Furthermore, combination of Ang-(1-7) with perindopril provided additional cardioprotection relative to the solo therapy.Ang-(1-7) Has no Effects on Serum Lipid and Blood Glucose LevelsOne week after streptozotocin injection, fasting blood glucose levels were markedly elevated in the model group and remained high until the end of the experiment. Similarly, serum total cholesterol and triglyceride levels were higher in the mock group than in the control group. At the end of week16, serum total cholesterol and triglyceride and blood glucose levels did not differ between the mock group and the7treatment groups. No apparent side effects were observed in any treatment group. As depicted in Kaplan-Meier survival curves, survival rates did not differ according to treatment.Ang-(1-7) Prevents LV DysfunctionTwelve weeks after streptozotocin injection, rats in the mock group showed decreased LV ejection fraction, fractional shortening, ratio of early to late LV filling velocity, ratio of early to late diastolic peak annular velocity, maximal LV systolic pressure, and maximal rates of pressure rise and fall, as well as increased LV end diastolic diameter and pressure as compared with the control group. These measurements were dose-dependently improved by4-week treatment with Ang-(1-7), and the salutary effects were enhanced by the combination treatment with Ang-(1-7) and perindopril and largely offset by the co-administration of Ang-(1-7) and A779or PD123319. However, Ang-(1-7) had no significant effects on blood pressure and heart rate in the DCM rats.Ang-(1-7) Attenuates Myocardial HypertrophyIntraventricular septal thickness (IVSth), LV posterior wall thickness (LVPWth), heart size and the ratio of heart weight to body weight were significantly increased in the mock group relative to the control group. Treatment with Ang-(1-7) dose-dependently reduced IVSth, LVPWth, heart size and the ratio of heart weight to body weight compared with the mock group. Notably, combined treatment with Ang-(1-7) and perindopril further reduced IVSth, LVPWth, heart size and the ratio of heart weight to body weight relative to the solo therapy groups. However, the effects of Ang-(1-7) on myocardial hypertrophy were largely reversed by co-administration of PD123319or A779.Cardiomyocyte cross-sectional areas were significantly larger in the mock group than the control group, indicative of the presence of myocardial hypertrophy in DCM rats. In contrast, Ang-(1-7)-treated groups showed a dose-dependent decrease in the cardiomyocyte cross-sectional areas as compared with the mock group, and such an effect was enhanced by co-administration of perindopril and reversed by PD123319or A779. Similarly, the mRNA expression of brain natriuretic peptide and β-myosin heavy chain as markers of cardiac hypertrophy was dose-dependently reduced by Ang-(1-7) treatment, which was also enhanced by co-administration of perindopril, completely reversed by PD123319, and partially blocked by A779. Thus, Ang-(1-7) protects diabetic hearts from development of cardiac hypertrophy via both AT2R and MasR.Ang-(1-7) Suppresses Myocardial Fibrosis In the mock group, the collagen volume fraction (CVF) and the ratio of perivascular collagen area to luminal area (PVCA/LA) increased by2.9-and3.1-fold, respectively, as compared with the control group, and these values were significantly reduced by the Ang-(1-7) treatment at the end of week16. Moreover, CVF and PVCA/LA were dramatically decreased with the combined Ang-(1-7) and perindopril treatment relative to the solo therapy with either Ang-(1-7) or perindopril. However, the beneficial effects of Ang-(1-7) on CVF and PVCA/LA were reversed completely by A779and partially by PD123319.In comparison with the mock group, Ang-(1-7) treatment dose-dependently reduced mRNA expression of fibrosis-associated genes, including fibronectin-1, collagen Ⅰ-α1, collagen Ⅲ-α1, and TGF-β1, as well as the ratio of collagen Ⅰ-α1to collagen Ⅲ-α1. Similar effects were observed in the perindopril group. Again, these Ang-(1-7)-induced effects were completely reversed by A779and partially blocked by PD123319. Immunohistochemistry revealed that the contents of collagen Ⅰ and Ⅲ and the ratio of collagen Ⅰ to Ⅲ were significantly increased in the mock group relative to the control group. Compared with the mock group, Ang-(1-7) treatment reduced the content of collagen I and the ratio of collagen Ⅰ to Ⅲ but not the content of collagen Ⅲ. The effect of Ang-(1-7) on collagen Ⅰ expression was reversed completely by A779and partially by PD123319.To explore the mechanisms underlying the effects of Ang-(1-7) on myocardial fibrosis, LV sections were stained for the expression of S100A4as a marker of fibroblasts and expression of a-smooth muscle actin (a-SMA) as a marker of fibroblast transformation with vascular positive staining being excluded. Interstitial S100A4-positive area, α-SMA-positive area and the ratio of α-SMA to S100A4were remarkably increased in the mock group relative to the control group, whereas treatment with Ang-(1-7) or perindopril reduced these values versus the mock group. In contrast, the effects of Ang-(1-7) on fibroblasts were completely reversed by A779and partially blocked by PD123319. In addition, Ang-(1-7) treatment inhibited activation of extracellular signal-regulated kinase1/2(ERK1/2) and p38mitogen-activated protein kinase (p38-MAPK) and TGF-β1protein expression as compared with the mock group. The inhibitory effects of Ang-(1-7) on ERK1/2and p38-MAPK activation and TGF-β1expression were completely reversed by A779and partially blocked by PD123319.Ang-(1-7) Improves LV Morphological and Ultrastructural AbnormalitiesH&E staining of the rat myocardia revealed fiber disarray and fragmentation, and diffused fibrosis, which were substantially alleviated by treatment with Ang-(1-7) or perindopril, especially with their combination.TEM revealed clear sarcomeres and Z lines and apparently normal-sized mitochondria, with normal numbers, in the LV myocytes of the control group but significant swelling and disruption of mitochondria, as well as myofibril disarray, in the LV myocardium of the mock group. However, these abnormalities of the LV ultrastructure were markedly improved by Ang-(1-7) or perindopril treatment. Thus, Ang-(1-7) and perindopril could synergistically restore and maintain LV myocardial integrity. However, the effects of Ang-(1-7) on LV morphological and ultrastructural abnormalities were blocked in part by co-administration of A779or PD123319.Ang-(1-7) Inhibits Cardiac ApoptosisRats in the mock group showed prominent cardiac apoptosis, as indicated by significantly increased proportion of TUNEL-positive cells, abnormal morphology of myocyte nuclei by TEM, increased mRNA and protein expression of Bax and the expression ratio of Bax to Bcl-2and significantly decreased mRNA and protein expression of Bcl-2, all of which were ameliorated by treatment with Ang-(1-7) at a dose more than400ng·kg·1·min-1or with perindopril. Of note, Ang-(1-7) at800ng·kg-1·min-1improved features of cardiomyocyte nuclei. The effects of Ang-(1-7) on cardiac apoptosis were completely reversed by PD123319and partially blocked by A779.Ang-(1-7) Ameliorates Myocardial Oxidative Stress and Inflammation There was an increased superoxide production and NADPH oxidase activation in the myocardia of the mock group versus the control group. Ang-(1-7) treatment dose-dependently attenuated, whereas the combined Ang-(1-7) and perindopril normalized, superoxide production and NADPH oxidase activation. These effects, however, were offset by co-administration of A779or PD123319.Immunohistochemical staining revealed that the protein expression of IL-1β, IL-6and MCP-1was increased in the mock group as compared with the control group. Ang-(1-7) treatment dose-dependently attenuated whereas combined treatment of Ang-(1-7) and perindopril normalized the expression of IL-1β, IL-6and MCP-1. These inhibitory effects were reversed by co-administration with A779or PD123319.Ang-(1-7) Reduces Collagen Synthesis of Cardiac FibroblastsHigh glucose stimulation significantly increased types Ⅰ and Ⅲ collagen content in the cultured media of cardiac fibroblasts which was inhibited by Ang-(1-7) treatment time-and dose-dependently and so was the ratio of collagen Ⅰ to Ⅲ. These inhibitory effects of Ang-(1-7) were enhanced by combined treatment with perindopril and completely reversed by A779.We then studied the ability of Ang-(1-7) to modulate collagen synthesis and degradation by cardiac fibroblasts. Collagen synthesis in cardiac fibroblasts subjected to high glucose with or without Ang-(1-7) was evaluated by3H-proline incorporation, and MMP-2and MMP-9activities were measured by gelatin zymography. High glucose induced a2.2-fold increase in collagen synthesis, which was significantly reduced by10-5M Ang-(1-7) over72hr and was further decreased by combined Ang-(1-7) with perindopril treatment. High glucose increased MMP-9activity but reduced MMP-2activity. Interestingly, Ang-(1-7) or perindopril treatment increased MMP-2activity but had no effects on MMP-9activity. The effects of Ang-(1-7) on collagen synthesis and MMP-2activity were completely blocked by A779.Ang-(1-7) Inhibits Proliferation, Differentiation and Oxidative Stress of Cardiac FibroblastsHigh glucose stimulation significantly augmented fibroblast proliferation as determined by immunofluorescence staining for Ki67. In addition, cardiac fibroblast differentiation to myofibroblasts was increased by high glucose, as determined by a-SMA-positive cells as a percentage of total S100A4-positive cells. Ang-(1-7) significantly decreased fibroblast proliferation and differentiation over72-hr culture versus high-glucose treatment alone, and such an effect of Ang-(1-7) was completely reversed by A779and partially blocked by PD123319. Moreover, combined treatment with Ang-(1-7) and perindopril significantly inhibited fibroblast proliferation and differentiation relative to Ang-(1-7) or perindopril alone.We then performed dihydroethidium staining and lucigenin-enhanced chemiluminescence assay in cultured cardiac fibroblasts. Superoxide production and NADPH oxidase activation was increased by high-glucose stimulation that was attenuated by Ang-(1-7) for72hr. Combined incubation with Ang-(1-7) and perindopril normalized superoxide production and NADPH oxidase activation in cardiac fibroblasts. In addition, the ability of Ang-(1-7) to attenuate oxidative stress of cardiac fibroblasts induced by high glucose was partially blunted by co-administration of A779or PD123319.Ang-(1-7) Inhibits ERK1/2and p38-MAPK Phosphorylation and TGF-β1Expression in Cardiac FibroblastsThe ratio of phosphorylated to total protein expression of ERK1/2and p38-MAPK in cardiac fibroblasts was markedly decreased after Ang-(1-7) treatment relative to the high glucose treatment, suggesting that the MAPK signaling pathway is involved in therapeutic mechanisms of Ang-(1-7) in DCM. Likewise, the protein expression level of TGF-β1was decreased in cardiac fibroblasts incubated with Ang-(1-7) and high glucose as compared with high glucose alone.The effects of Ang-(1-7) on ERK1/2, p38-MAPK and TGF-β1in cardiac fibroblasts incubated with high glucose were inhibited by co-administration of A779or PD123319, indicating that Ang-(1-7) inhibited ERK1/2and p38-MAPK activation and TGF-β1expression in cardiac fibroblasts by binding to both MasR and AT2R.Fibroblast-Myocyte Interaction Increases Collagen and TGF-β1ProductionThe protein expression levels of collagen Ⅰ and Ⅲ and TGF-β1were significantly higher in the fibroblast+non-treated myocyte group than in the fibroblast group. In contrast, the protein expression levels of collagen Ⅰ and Ⅲ and TGF-β1were lower in the fibroblast+Ang-(1-7)-treated myocyte group than in the fibroblast+non-treated myocyte group.The protein expression levels of collagen Ⅰ and Ⅲ and TGF-β1were substantially higher in the fibroblast+non-treated myocyte media group than in the fibroblast group. In contrast, the protein levels of collagen Ⅰ and Ⅲ and TGF-β1were lower in the fibroblast+Ang-(1-7)-treated myocyte media group than in the fibroblast+non-treated myocyte media group.The inhibitory effects of Ang-(1-7) on collagen and TGF-β1production induced by fibroblast-myocyte co-culture were partially blocked by co-administration of A779or PD123319.Ang-(1-7) Prevents Cardiomyocyte Hypertrophy, Apoptosis and Oxidative Stress via MasR and AT2RIn the in vitro experiment, high glucose treatment significantly increased cardiomyocyte size as compared with the normal glucose or osmotic control groups. Ang-(1-7) treatment normalized cardiomyocyte size in comparison with high-glucose alone treatment, which was abrogated by the addition of A779and in particular PD123319. Neither A779nor PD123319alone altered the size of high-glucose-incubated cardiomyocytes.Cardiomyocyte apoptosis was increased by high glucose treatment relative to the normal glucose or osmotic control groups (P<0.05). Ang-(1-7) partially inhibited cardiomyocyte apoptosis induced by high glucose (P>0.05), and the effect was abrogated by co-treatment with either A779or PD123319. We then performed dihydroethidium staining and lucigenin-enhanced chemiluminescence assay in cultured cardiac myocytes. Superoxide production and NADPH oxidase activation was greater in the high-glucose group than in the normal-glucose group (P<0.01). Ang-(1-7) treatment with or without administration of perindopril normalized high glucose-induced superoxide production and NADPH oxidase activation in cardiac myocytes. In addition, the ability of Ang-(1-7) to attenuate oxidative stress of cardiac myocytes after high glucose stimulation was partially blocked by co-administration of A779or PD123319.Ang-(1-7) Downregulates AT1R and Upregulates AT2R in vivo and in vitroThe Ang-(1-7) level in the rat myocardium was significantly increased after4-week treatment with Ang-(1-7) but not with perindopril, although their combination exerted greater effect on Ang-(1-7) level than Ang-(1-7) treatment alone, possibly because of the effect of perindopril in inhibiting the degradation of Ang-(1-7) into inactive Ang-(1-5).The in vivo experiment revealed that AT1R expression was significantly increased while AT2R expression was decreased in the mock group versus the control group. These effects were largely attenuated by Ang-(1-7) treatment at a large dose. In the in vitro study, high glucose induced higher AT1R expression and lower AT2R expression than the normal glucose group in both cardiac fibroblasts and myocytes, which was reversed by Ang-(1-7) treatment. Nevertheless, Ang-(1-7) did not affect MasR expression or Ang-Ⅱ levels in the rat hearts or in cardiac fibroblasts and myocytes cultured in high-glucose medium.The effect of Ang-(1-7) on AT1R expression in rat hearts and cultured cardiac fibroblasts was totally inhibited with co-administration of A779or PD123319, and that in cardiac myocytes was reversed by A779but not by PD123319. The effect of Ang-(1-7) on AT2R expression in rat hearts was completely reversed by PD123319and partially blocked by A779, and that in cardiac fibroblasts was completely blocked by PD123319but not altered by A779, and that in cardiac myocytes was totally inhibited by A779or PD123319.ConclusionChronic Ang-(1-7) treatment ameliorated LV remodeling and dysfunction in DCM via multiple mechanisms involving attenuated inflammation, oxidative stress and collagen synthesis, inhibited ERK1/2and p38-MAPK signaling and TGF-(31expression as well as complex interactions of MasR, AT2R and AT1R. Furthermore, combination of Ang-(1-7) with perindopril provided additional cardioprotection. Thus, Ang-(1-7) administration may provide a novel and promising approach to the treatment of DCM. BackgroundThe renin-angiotensin system (RAS), which is activated in diabetes mellitus (DM), contributes to the development of left ventricular (LV) remodeling and dysfunction, but this association has not been established for right ventricles. Inhibition of a hyperactive RAS by angiotensin-converting enzyme (ACE) inhibitors or angiotensin Ⅱ type1(AT1) receptor blockers protects against LV remodeling, left heart failure, and mortality; however, we lack convincing evidence for use of these therapies in right ventricular (RV) failure. In preliminary studies, we demonstrated that overexpression of ACE2, a zinc metalloproteinase and a new member of the RAS, attenuated LV remodeling and dysfunction induced by type1DM or acute myocardial infarction in rats, which was accompanied by downregulated angiotensin Ⅱ (Ang-Ⅱ) and upregulated angiotensin-(1-7)[Ang-(1-7)] levels. In addition, Johnson and colleagues found that recombinant human ACE2diminished RV hypertrophy and improved RV systolic and diastolic function in association with a marker of intercellular communication in a mouse RV load-stress model of early heart failure.Ang-(1-7) is formed from Ang-Ⅰ and-Ⅱ by several endopeptidases and carboxypeptidases, including ACE and ACE2, and has vasoprotection, atheroprotection and cardioprotection properties. Several studies have shown that endogenous or exogenous Ang-(1-7) leads to enhanced LV functional performance in diabetic and other animal models. Which receptor of Ang-(1-7) is activated to mediate its effects is much speculated. With the discovery of the G-protein-coupled receptor Mas, which is highly expressed in several tissues, including the heart, kidney, and the vasculature, increasing studies have shown that the effects of Ang-(1-7) are mediated through the Mas receptor.Although the literature supports a beneficial role for Ang-(1-7) in LV remodeling and function, the effects of Ang-(1-7) specifically in the setting of RV dysfunction have not been evaluated. Importantly, the response of right ventricles to DM should not be extrapolated from left heart experiments. The function, structure, and embryology of right and left ventricles are unique. The right ventricle is smaller, crescent shaped, and thin-walled and has a much lower afterload than the left ventricle; these differences are augmented by a differing embryologic origin of the right ventricle. Thus, the right ventricle may not respond like the left ventricle to DM and pharmacological therapies.ObjectiveWe aimed to investigate whether LV changes in cardiac fibrosis, hypertrophy, apoptosis and function in DM are paralleled by similar RV alterations in an experimental rat model of type1DM. We also aimed to determine the effect of chronic Ang-(1-7) treatment on alterations in the right ventricle. To elucidate the possible mechanisms, we further investigated the effect of Ang-(1-7) on ACE and ACE2expression and activities as well as protein expression of AT1, AT2and Mas receptors in right ventricles of DM rats.MethodsAnimal ModelMale Wistar rats (approximately200g) were purchased from Vital River Laboratory Animal Technology Co.(Beijing, China). After12weeks of DM induced by a single intraperitoneal injection of streptozotocin (Sigma, St. Louis, MO), rats were treated with saline, Ang-(1-7)(Auspep, Parkville, Australia), perindopril (Servier, Melbourne, Australia), Ang-(1-7) plus perindopril, Ang-(1-7) plus Mas receptor antagonist A779(Auspep, Parkville, Australia), or Ang-(1-7) plus AT2receptor antagonist PD123319(Pfizer, Groton, CT) for4weeks. All procedures were performed in accordance with the Principles of Laboratory Animal Care formulated by the National Society for Medical Research and the Guide for the Care and Use of Laboratory Animals by the National Academy of Sciences and published by the US National Institutes of Health (NIH Publication No.86-23, revised1996).Blood Pressure and RV Pressure MeasurementPeripheral arterial blood pressure was measured by the tail-cuff method and RV pressure by invasive RV catheterization.Blood AnalysisAfter rats fasted overnight, blood samples were collected from jugular veins. The serum lipid profile and fasting blood glucose (FBG) were analyzed by use of the Bayer1650blood chemistry analyzer (Bayer, Tarrytown, NY).Histopathology and ImmunohistochemistryMasson trichrome staining was performed to assess overall tissue architecture and interstitial and perivascular fibrosis. Collagen Ⅰ and Ⅲ content was identified with appropriate antibodies.Assessment of Cardiomyocyte Hypertrophy and ApoptosisThe cross-sectional area of myocytes in cardiac sections was measured by staining with Alexa Fluor(?)488-conjugated wheat germ agglutinin. Apoptotic cells in tissue sections were quantified by terminal deoxynucleotidyltransferase-mediated dUTP nick-end labeling (TUNEL).Real-Time RT-PCRThe mRNA levels of fibronectin-1, transforming growth factor-beta1(TGF-β1), brain natriuretic peptide (BNP), β-myosin heavy chain (β-MHC), Bax, and Bcl-2were quantified.Western Blot AnalysisThe protein levels of TGF-β1, total and phosphorylated Smad3, Smad7, Bax, Bcl-2, ACE, ACE2, AT1receptor, AT2receptor, and Ang-(1-7) Mas receptor were assayed.ACE and ACE2ActivitiesACE and ACE2activities were determined with assays based on internally quenched fluorescent substrates.Dihydroethidium Fluorescence and Lucigenin-Enhanced ChemiluminescenceThe oxidative fluorescent dye dihydroethidium was used to measure superoxide (O2-) levels in myocardial frozen sections. NADPH oxidase activity in myocardial sections was quantified by lucigenin-enhanced chemiluminescence.Enzyme-linked Immunosorbent Assay (ELISA)ELISA was performed to measure the levels of Ang-Ⅱ, TGF-β1, and soluble collagen Ⅰ and Ⅲ proteins.Co-culture of Fibroblasts and MyocytesRV fibroblasts were co-cultured with Ang-(1-7)-treated or nontreated RV myocytes or the conditioned media of these cells, and the levels of collagen Ⅰ and Ⅲ and TGF-β1proteins in the media were determined by ELISA.Statistical AnalysisAll continuous values are expressed as mean±SEM. Data were analyzed by use of SPSS v11.5(SPSS Inc., Chicago, IL). A P<0.05was considered statistically significant.ResultsSerum Lipid Profile and FBG ConcentrationsOne week after streptozotocin injection, FBG was markedly elevated in diabetic rats and remained elevated until the end of the experiment. Simultaneously, serum total cholesterol and triglyceride levels were maintained at higher levels than for controls. At the end of week16, total cholesterol and triglyceride levels or FBG did not differ among the8groups except as compared with the control group (all P>0.05).Hemodynamic MeasurementsDiabetic rats showed significant systolic and diastolic dysfunction, as indicated by decreased RV peak pressure and maximal rate of pressure increase as well as increased RV end-diastolic pressure and relaxation time constant. Four-week Ang-(1-7) treatment prevented RV dysfunction in part, and the effects were suppressed by co-administration of A779or PD123319. However, Ang-(1-7) did not significantly change arterial blood pressure or heart rates of diabetic rats.Pathology Characteristics of Diabetic RatsDM induced LV remodeling, as indicated by cardiac fibrosis as well as hypertrophy and apoptosis of individual cardiomyocytes. In contrast, RV remodeling in diabetic rats was indicated by fibrosis of the RV free wall in the absence of hypertrophy and apoptosis.Ang-(1-7) Suppresses RV Fibrosis Induced by DMAng-(1-7) treatment significantly reduced the collagen volume fraction (CVF) and ratio of perivascular collagen area to luminal area (PVCA/LA) in right ventricles of diabetic rats. The effect of Ang-(1-7) on DM-induced RV fibrosis was completely reversed by A779and partially by PD123319. With Ang-(1-7) treatment, collagen Ⅰ and Ⅲ content and ratio of collagen Ⅰ to Ⅲ were reduced. The effect of Ang-(1-7) on collagen Ⅰ and Ⅲ levels and collagen Ⅰ-to-Ⅲ ratio was enhanced by perindopril co-administration, completely blocked by A779, and partially inhibited by PD123319.Treatment with Ang-(1-7) alone or with perindopril dose-dependently reduced the DM-induced mRNA levels of fibronectin-1and TGF-β1, and the effect of Ang-(1-7) was inhibited by A779or PD123319. In addition, Ang-(1-7) dose-dependently inhibited TGF-β1protein expression and Smad3activation, as well as increased Smad7level in diabetic rats. The effect of Ang-(1-7) on TGF-β1and Smad7level and Smad3activation was inhibited by A779or PD123319.Ang-(1-7) Ameliorated RV Oxidative Stress of Diabetic RatsSuperoxide generation and NADPH oxidase activation mediated by high glucose may be pivotal mechanisms of high-glucose-mediated injury in the cardiovascular system, such as mitochondrial damage. DM exacerbated RV and LV oxidative stress in rats, as indicated by increased superoxide production and...