Vaibhav B. Patel

Loss of Apelin Exacerbates Myocardial Infarction Adverse Remodeling and Ischemia-reperfusion Injury: Therapeutic Potential of Synthetic Apelin Analogues

Background Coronary artery disease leading to myocardial ischemia is the most common cause of heart failure. Apelin (APLN), the endogenous peptide ligand of the APJ receptor, has emerged as a novel regulator of the cardiovascular system. Methods and Results Here we show a critical role of APLN in myocardial infarction (MI) and ischemia‐reperfusion (IR) injury in patients and animal models. Myocardial APLN levels were reduced in patients with ischemic heart failure. Loss of APLN increased MI‐related mortality, infarct size, and inflammation with drastic reductions in prosurvival pathways resulting in greater systolic dysfunction and heart failure. APLN deficiency decreased vascular sprouting, impaired sprouting of human endothelial progenitor cells, and compromised in vivo myocardial angiogenesis. Lack of APLN enhanced susceptibility to ischemic injury and compromised functional recovery following ex vivo and in vivo IR injury. We designed and synthesized two novel APLN analogues resistant to angiotensin converting enzyme 2 cleavage and identified one analogue, which mimicked the function of APLN, to be markedly protective against ex vivo and in vivo myocardial IR injury linked to greater activation of survival pathways and promotion of angiogenesis. Conclusions APLN is a critical regulator of the myocardial response to infarction and ischemia and pharmacologically targeting this pathway is feasible and represents a new class of potential therapeutic agents.

Loss of Angiotensin-Converting Enzyme-2 Exacerbates Diabetic Cardiovascular Complications and Leads to Systolic and Vascular Dysfunction A Critical Role of the Angiotensin II/AT1 Receptor Axis

Rationale: Diabetic cardiovascular complications are reaching epidemic proportions. Angiotensin-converting enzyme-2 (ACE2) is a negative regulator of the renin-angiotensin system. We hypothesize that loss of ACE2 exacerbates cardiovascular complications induced by diabetes. Objective: To define the role of ACE2 in diabetic cardiovascular complications. Methods and Results: We used the well-validated Akita mice, a model of human diabetes, and generated double-mutant mice using the ACE2 knockout (KO) mice (Akita/ACE2−/y). Diabetic state was associated with increased ACE2 in Akita mice, whereas additional loss of ACE2 in these mice leads to increased plasma and tissue angiotensin II levels, resulting in systolic dysfunction on a background of impaired diastolic function. Downregulation of SERCA2 and lipotoxicity were equivalent in Akita and Akita/ACE2KO hearts and are likely mediators of the diastolic dysfunction. However, greater activation of protein kinase C and loss of Akt and endothelial nitric oxide synthase phosphorylation occurred in the Akita/ACE2KO hearts. Systolic dysfunction in Akita/ACE2KO mice was linked to enhanced activation of NADPH oxidase and metalloproteinases, resulting in greater oxidative stress and degradation of the extracellular matrix. Impaired flow-mediated dilation in vivo correlated with increased vascular oxidative stress in Akita/ACE2KO mice. Treatment with the AT1 receptor blocker, irbesartan rescued the systolic dysfunction, normalized altered signaling pathways, flow-mediated dilation, and the increased oxidative stress in the cardiovascular system. Conclusions: Loss of ACE2 disrupts the balance of the renin-angiotensin system in a diabetic state and leads to an angiotensin II/AT1 receptor-dependent systolic dysfunction and impaired vascular function. Our study demonstrates that ACE2 serves as a protective mechanism against diabetes-induced cardiovascular complications.

Angiotensin 1–7 Ameliorates Diabetic Cardiomyopathy and Diastolic Dysfunction in db/db Mice by Reducing Lipotoxicity and Inflammation

Background— The angiotensin-converting enzyme 2 and angiotensin-(1–7) (Ang 1–7)/MasR (Mas receptor) axis are emerging as a key pathway that can modulate the development of diabetic cardiomyopathy. We studied the effects of Ang 1–7 on diabetic cardiomyopathy in db/db diabetic mice to elucidate the therapeutic effects and mechanism of action. Methods and Results— Ang 1–7 was administered to 5-month-old male db/db mice for 28 days via implanted micro-osmotic pumps. Ang 1–7 treatment ameliorated myocardial hypertrophy and fibrosis with normalization of diastolic dysfunction assessed by pressure–volume loop analysis and echocardiography. The functional improvement by Ang 1–7 was accompanied by a reduction in myocardial lipid accumulation and systemic fat mass and inflammation and increased insulin-stimulated myocardial glucose oxidation. Increased myocardial protein kinase C levels and loss of phosphorylation of extracellular signal-regulated kinase 1/2 were prevented by Ang 1–7. Furthermore, Ang 1–7 treatment decreased cardiac triacylglycerol and ceramide levels in db/db mice, concomitantly with an increase in myocardial adipose triglyceride lipase expression. Changes in adipose triglyceride lipase expression correlated with increased SIRT1 (silent mating type information regulation 2 homolog 1) levels and deacetylation of FOXO1 (forkhead box O1). Conclusions— We identified a novel beneficial effect of Ang 1–7 on diabetic cardiomyopathy that involved a reduction in cardiac hypertrophy and lipotoxicity, adipose inflammation, and an upregulation of adipose triglyceride lipase. Ang 1–7 completely rescued the diastolic dysfunction in the db/db model. Ang 1–7 represents a promising therapy for diabetic cardiomyopathy associated with type 2 diabetes mellitus.

Agonist-Induced Hypertrophy and Diastolic Dysfunction Are Associated With Selective Reduction in Glucose Oxidation A Metabolic Contribution to Heart Failure With Normal Ejection Fraction

Background—Activation of the renin-angiotensin and sympathetic nervous systems may alter the cardiac energy substrate preference, thereby contributing to the progression of heart failure with normal ejection fraction. We assessed the qualitative and quantitative effects of angiotensin II (Ang II) and the &agr;-adrenergic agonist, phenylephrine (PE), on cardiac energy metabolism in experimental models of hypertrophy and diastolic dysfunction and the role of the Ang II type 1 receptor. Methods and Results—Ang II (1.5 mg·kg−1·day−1) or PE (40 mg·kg−1·day−1) was administered to 9-week-old male C57/BL6 wild-type mice for 14 days via implanted microosmotic pumps. Echocardiography showed concentric hypertrophy and diastolic dysfunction, with preserved systolic function in Ang II- and PE-treated mice. Ang II induced marked reduction in cardiac glucose oxidation and lactate oxidation, with no change in glycolysis and fatty acid &bgr;-oxidation. Tricarboxylic acid acetyl coenzyme A production and ATP production were reduced in response to Ang II. Cardiac pyruvate dehydrogenase kinase 4 expression was upregulated by Ang II and PE, resulting in a reduction in the pyruvate dehydrogenase activity, the rate-limiting step for carbohydrate oxidation. Pyruvate dehydrogenase kinase 4 upregulation correlated with the activation of the cyclin/cyclin-dependent kinase-retinoblastoma protein-E2F pathway in response to Ang II. Ang II type 1 receptor blockade normalized the activation of the cyclin/cyclin-dependent kinase-retinoblastoma protein-E2F pathway and prevented the reduction in glucose oxidation but increased fatty acid oxidation. Conclusions—Ang II- and PE-induced hypertrophy and diastolic dysfunction is associated with reduced glucose oxidation because of the cyclin/cyclin-dependent kinase-retinoblastoma protein-E2F–induced upregulation of pyruvate dehydrogenase kinase 4, and targeting these pathways may provide novel therapy for heart failure with normal ejection fraction.

Angiotensin II induced proteolytic cleavage of myocardial ACE2 is mediated by TACE/ADAM-17: a positive feedback mechanism in the RAS.

Angiotensin converting enzyme (ACE) 2 is a key negative regulator of the renin-angiotensin system where it metabolizes angiotensin (Ang) II into Ang 1-7. We hypothesize that Ang II suppresses ACE2 by increasing TNF-α converting enzyme (TACE) activity and ACE2 cleavage. Ang II infusion (1.5 mg/kg/day) in wild-type mice for 2 weeks resulted in substantial decrease in myocardial ACE2 protein levels and activity with corresponding increase in plasma ACE2 activity, prevented by AT1R blockade. Ang II resulted in AT1R-mediated increase in myocardial TACE expression and activity, and membrane translocation of TACE. Ang II treatment in Huh7 cells exhibited AT1R-dependent metalloproteinase mediated shedding of ACE2 while transfection with siTACE prevented shedding of ACE2; cardiomyocyte-specific deletion of TACE also prevented shedding of ACE2. Reactive oxygen species played a key role since p47(phox)KO mice were resistant to Ang II-induced TACE phosphorylation and activation with preservation of myocardial ACE2 which dampened Ang II-induced cardiac dysfunction and hypertrophy. In conclusion, Ang II induces ACE2 shedding by promoting TACE activity as a positive feedback mechanism whereby Ang II facilitates the loss of its negative regulator, ACE2. In HF, elevated plasma ACE2 activity likely represents loss of the protective effects of ACE2 in the heart.

Cardioprotective Effects Mediated by Angiotensin II Type 1 Receptor Blockade and Enhancing Angiotensin 1-7 in Experimental Heart Failure in Angiotensin-Converting Enzyme 2–Null Mice

Loss of angiotensin (Ang)-converting enzyme 2 (ACE2) and inability to metabolize Ang II to Ang 1-7 perpetuate the actions of Ang II after biomechanical stress and exacerbate early adverse myocardial remodeling. Ang receptor blockers are known to antagonize the effect of Ang II by blocking Ang II type 1 receptor (AT1R) and also by upregulating the ACE2 expression. We directly compare the benefits of AT1R blockade versus enhancing Ang 1-7 action in pressure-overload–induced heart failure in ACE2 knockout mice. AT1R blockade and Ang 1-7 both resulted in marked recovery of systolic dysfunction in pressure-overloaded ACE2-null mice. Similarly, both therapies attenuated the increase in NADPH oxidase activation by downregulating the expression of Nox2 and p47phox subunits and also by limiting the p47phox phosphorylation. Biomechanical stress-induced increase in protein kinase C-&agr; expression and phosphorylation of extracellular signal–regulated kinase 1/2, signal transducer and activator of transcription 3, Akt, and glycogen synthase kinase 3&bgr; were normalized by irbesartan and Ang 1-7. Ang receptor blocker and Ang 1-7 effectively reduced matrix metalloproteinase 2 activation and matrix metalloproteinase 9 levels. Ang II–mediated cellular effects in cultured adult cardiomyocytes and cardiofibrolasts isolated from pressure-overloaded ACE2-null hearts were inhibited to similar degree by AT1R blockade and stimulation with Ang 1-7. Thus, treatment with the AT1R blocker irbesartan and Ang 1-7 prevented the cardiac hypertrophy and improved cardiac remodeling in pressure-overloaded ACE2-null mice by suppressing NADPH oxidase and normalizing pathological signaling pathways.

Angiotensin 1-7 mediates renoprotection against diabetic nephropathy by reducing oxidative stress, inflammation, and lipotoxicity

The renin-angiotensin system, especially angiotensin II (ANG II), plays a key role in the development and progression of diabetic nephropathy. ANG 1-7 has counteracting effects on ANG II and is known to exert beneficial effects on diabetic nephropathy. We studied the mechanism of ANG 1-7-induced beneficial effects on diabetic nephropathy in db/db mice. We administered ANG 1-7 (0.5 mg·kg(-1)·day(-1)) or saline to 5-mo-old db/db mice for 28 days via implanted micro-osmotic pumps. ANG 1-7 treatment reduced kidney weight and ameliorated mesangial expansion and increased urinary albumin excretion, characteristic features of diabetic nephropathy, in db/db mice. ANG 1-7 decreased renal fibrosis in db/db mice, which correlated with dephosphorylation of the signal transducer and activator of transcription 3 (STAT3) pathway. ANG 1-7 treatment also suppressed the production of reactive oxygen species via attenuation of NADPH oxidase activity and reduced inflammation in perirenal adipose tissue. Furthermore, ANG 1-7 treatment decreased lipid accumulation in db/db kidneys, accompanied by increased expressions of renal adipose triglyceride lipase (ATGL). Alterations in ATGL expression correlated with increased SIRT1 expression and deacetylation of FOXO1. The upregulation of angiotensin-converting enzyme 2 levels in diabetic nephropathy was normalized by ANG 1-7. ANG 1-7 treatment exerts renoprotective effects on diabetic nephropathy, associated with reduction of oxidative stress, inflammation, fibrosis, and lipotoxicity. ANG 1-7 can represent a promising therapy for diabetic nephropathy.

Differential role of TIMP2 and TIMP3 in cardiac hypertrophy, fibrosis, and diastolic dysfunction

AIMS Tissue inhibitor of metalloproteinases (TIMPs) can mediate myocardial remodelling, hypertrophy, and fibrosis in heart disease. We investigated the impact of TIMP2 vs. TIMP3 deficiency in angiotensin II (Ang II)-induced myocardial remodelling and cardiac dysfunction. METHODS AND RESULTS TIMP2(-/-), TIMP3(-/-), and wild-type (WT) mice received Ang II/saline (Alzet pump) for 2 weeks. Ang II infusion resulted in enhanced myocardial hypertrophy and lack of fibrosis in TIMP2(-/-), and conversely, excess fibrosis without hypertrophy in TIMP3(-/-) mice. Echocardiographic imaging revealed preserved ejection fraction in all groups; however, exacerbated left ventricular (LV) diastolic dysfunction was detected in Ang II-infused TIMP2(-/-) and TIMP3(-/-) mice, despite the suppressed Ang II-induced hypertension in TIMP3(-/-) mice. Enhanced hypertrophy in TIMP2(-/-) mice impaired active relaxation, while excess fibrosis in TIMP3(-/-) mice increased LV passive stiffness. Adult WT cardiomyocytes, only when co-cultured with cardiac fibroblasts, exhibited Ang II-induced hypertrophy which was suppressed in TIMP3(-/-) cardiomyocytes. In vitro studies on adult cardiofibroblasts (quiescent and cyclically stretched), and in vivo analyses, revealed that the increased fibrosis in TIMP3(-/-)-Ang II hearts is due to post-translational stabilization and deposition of collagen by matricellular proteins [osteopontin and Secreted Protein Acidic and Rich in Cysteine (SPARC)], which correlated with increased inflammation, rather than increased de novo synthesis. Reduced cross-linking enzymes, LOX and PLOD1, could underlie suppressed collagen deposition in TIMP2(-/-)-Ang II hearts. CONCLUSION TIMP2 and TIMP3 play fundamental and differential roles in mediating pathological remodelling, independent from their MMP-inhibitory function. TIMP2(-/-) and TIMP3(-/-) mice provide a unique opportunity to study myocardial hypertrophy and fibrosis independently, and their impact on cardiac dysfunction.

Loss of p47phox Subunit Enhances Susceptibility to Biomechanical Stress and Heart Failure Because of Dysregulation of Cortactin and Actin Filaments

Rationale: The classic phagocyte nicotinamide adenine dinucleotide phosphate oxidase (gp91phox or Nox2) is expressed in the heart. Nox2 activation requires membrane translocation of the p47phox subunit and is linked to heart failure. We hypothesized that loss of p47phox subunit will result in decreased reactive oxygen species production and resistance to heart failure. Objective: To define the role of p47phox in pressure overload–induced biomechanical stress. Methods and Results: Eight-week-old male p47phox null (p47phox knockout [KO]), Nox2 null (Nox2KO), and wild-type mice were subjected to transverse aortic constriction–induced pressure overload. Contrary to our hypothesis, p47phoxKO mice showed markedly worsened systolic dysfunction in response to pressure overload at 5 and 9 weeks after transverse aortic constriction compared with wild-type–transverse aortic constriction mice. We found that biomechanical stress upregulated N-cadherin and &bgr;-catenin in p47phoxKO hearts but disrupted the actin filament cytoskeleton and reduced phosphorylation of focal adhesion kinase. p47phox interacts with cytosolic cortactin by coimmunoprecipitation and double immunofluorescence staining in murine and human hearts and translocated to the membrane on biomechanical stress where cortactin interacted with N-cadherin, resulting in adaptive cytoskeletal remodeling. However, p47phoxKO hearts showed impaired interaction of cortactin with N-cadherin, resulting in loss of biomechanical stress–induced actin polymerization and cytoskeletal remodeling. In contrast, Nox2 does not interact with cortactin, and Nox2-deficient hearts were protected from pressure overload–induced adverse myocardial and intracellular cytoskeletal remodeling. Conclusions: We showed a novel role of p47phox subunit beyond and independent of nicotinamide adenine dinucleotide phosphate oxidase activity as a regulator of cortactin and adaptive cytoskeletal remodeling, leading to a paradoxically enhanced susceptibility to biomechanical stress and heart failure.

Loss of p47phox Subunit Enhances Susceptibility to Biomechanical Stress and Heart Failure due to Dysregulation of Cortactin and Actin Filaments

Rationale: The classic phagocyte nicotinamide adenine dinucleotide phosphate oxidase (gp91phox or Nox2) is expressed in the heart. Nox2 activation requires membrane translocation of the p47phox subunit and is linked to heart failure. We hypothesized that loss of p47phox subunit will result in decreased reactive oxygen species production and resistance to heart failure. Objective: To define the role of p47phox in pressure overload–induced biomechanical stress. Methods and Results: Eight-week-old male p47phox null (p47phox knockout [KO]), Nox2 null (Nox2KO), and wild-type mice were subjected to transverse aortic constriction–induced pressure overload. Contrary to our hypothesis, p47phoxKO mice showed markedly worsened systolic dysfunction in response to pressure overload at 5 and 9 weeks after transverse aortic constriction compared with wild-type–transverse aortic constriction mice. We found that biomechanical stress upregulated N-cadherin and &bgr;-catenin in p47phoxKO hearts but disrupted the actin filament cytoskeleton and reduced phosphorylation of focal adhesion kinase. p47phox interacts with cytosolic cortactin by coimmunoprecipitation and double immunofluorescence staining in murine and human hearts and translocated to the membrane on biomechanical stress where cortactin interacted with N-cadherin, resulting in adaptive cytoskeletal remodeling. However, p47phoxKO hearts showed impaired interaction of cortactin with N-cadherin, resulting in loss of biomechanical stress–induced actin polymerization and cytoskeletal remodeling. In contrast, Nox2 does not interact with cortactin, and Nox2-deficient hearts were protected from pressure overload–induced adverse myocardial and intracellular cytoskeletal remodeling. Conclusions: We showed a novel role of p47phox subunit beyond and independent of nicotinamide adenine dinucleotide phosphate oxidase activity as a regulator of cortactin and adaptive cytoskeletal remodeling, leading to a paradoxically enhanced susceptibility to biomechanical stress and heart failure.