RESUMO
We determined the epigenetic mechanisms regulating mean arterial pressure (MAP) and renal dysfunction in guanylyl cyclase/natriuretic peptide receptor-A (GC-A/NPRA) gene-targeted mice. The Npr1 (encoding NPRA) gene-targeted mice were treated with class 1 specific histone deacetylase inhibitor (HDACi) mocetinostat (MGCD) to determine the epigenetic changes in a sex-specific manner. Adult male and female Npr1 haplotype (1-copy; Npr1+/-), wild-type (2-copy; Npr1+/+), and gene-duplicated heterozygous (3-copy; Npr1++/+) mice were intraperitoneally injected with MGCD (2 mg/kg) for 14 days. BP, renal function, histopathology, and epigenetic changes were measured. One-copy male mice showed significantly increased MAP, renal dysfunction, and fibrosis than 2-copy and 3-copy mice. Furthermore, HDAC1/2, collagen1alpha-2 (Col1α-2), and alpha smooth muscle actin (α-SMA) were significantly increased in 1-copy mice compared with 2-copy controls. The expression of antifibrotic microRNA-133a was attenuated in 1-copy mice but to a greater extent in males than females. NF-κB was localized at significantly lower levels in cytoplasm than in the nucleus with stronger DNA binding activity in 1-copy mice. MGCD significantly lowered BP, improved creatinine clearance, and repaired renal histopathology. The inhibition of class I HDACs led to a sex-dependent distinctive stimulation of acetylated positive histone marks and inhibition of methylated repressive histone marks in Npr1 1-copy mice; however, it epigenetically lowered MAP, repaired renal fibrosis, and proteinuria and suppressed NF-kB differentially in males versus females. Our results suggest a role for epigenetic targets affecting hypertension and renal dysfunction in a sex-specific manner.
Assuntos
Pressão Sanguínea , Epigênese Genética , Receptores do Fator Natriurético Atrial , Animais , Feminino , Masculino , Receptores do Fator Natriurético Atrial/genética , Receptores do Fator Natriurético Atrial/metabolismo , Camundongos , Pressão Sanguínea/efeitos dos fármacos , Rim/metabolismo , Rim/patologia , Inibidores de Histona Desacetilases/farmacologia , Nefropatias/genética , Nefropatias/metabolismo , Nefropatias/patologiaRESUMO
BACKGROUND: Hydrogen sulfide is a critical endogenous signaling molecule that exerts protective effects in the setting of heart failure. Cystathionine γ-lyase (CSE), 1 of 3 hydrogen-sulfide-producing enzyme, is predominantly localized in the vascular endothelium. The interaction between the endothelial CSE-hydrogen sulfide axis and endothelial-mesenchymal transition, an important pathological process contributing to the formation of fibrosis, has yet to be investigated. METHODS: Endothelial-cell-specific CSE knockout and Endothelial cell-CSE overexpressing mice were subjected to transverse aortic constriction to induce heart failure with reduced ejection fraction. Cardiac function, vascular reactivity, and treadmill exercise capacity were measured to determine the severity of heart failure. Histological and gene expression analyses were performed to investigate changes in cardiac fibrosis and the activation of endothelial-mesenchymal transition. RESULTS: Endothelial-cell-specific CSE knockout mice exhibited increased endothelial-mesenchymal transition and reduced nitric oxide bioavailability in the myocardium, which was associated with increased cardiac fibrosis, impaired cardiac and vascular function, and worsened exercise performance. In contrast, genetic overexpression of CSE in endothelial cells led to increased myocardial nitric oxide, decreased endothelial-mesenchymal transition and cardiac fibrosis, preserved cardiac and endothelial function, and improved exercise capacity. CONCLUSIONS: Our data demonstrate that endothelial CSE modulates endothelial-mesenchymal transition and ameliorate the severity of pressure-overload-induced heart failure, in part, through nitric oxide-related mechanisms. These data further suggest that endothelium-derived hydrogen sulfide is a potential therapeutic for the treatment of heart failure with reduced ejection fraction.
Assuntos
Insuficiência Cardíaca , Sulfeto de Hidrogênio , Disfunção Ventricular Esquerda , Camundongos , Animais , Sulfeto de Hidrogênio/metabolismo , Células Endoteliais/metabolismo , Óxido Nítrico/metabolismo , Camundongos Knockout , Endotélio Vascular/metabolismo , FibroseRESUMO
Atrial and brain natriuretic peptides (ANP and BNP) bind to guanylyl cyclase-A/natriuretic peptide receptor-A (GC-A/NPRA), stimulating natriuresis and diuresis and reducing blood pressure (BP), but the role of ANP/NPRA signaling in podocytes (highly specialized epithelial cells covering the outer surfaces of renal glomerular capillaries) remains unclear. This study aimed to determine the effect of conditional deletion of podocyte (PD)-specific Npr1 (encoding NPRA) gene knockout (KO) in male and female mice. Tamoxifen-treated wild-type control (PD Npr1 f/f; WT), heterozygous (PD-Cre-Npr1 f/+; HT), and knockout (PD-Cre-Npr1 f/-; KO) mice were fed a normal-, low-, or high-salt diet for 4 weeks. Podocytes isolated from HT and KO male and female mice showed complete absence of Npr1 mRNA and NPRA protein compared to WT mice. BP, plasma creatinine, plasma sodium, urinary protein, and albumin/creatinine ratio were significantly increased, while plasma total protein, albumin, creatinine clearance, and urinary sodium levels were significantly reduced in the HT and KO male and female mice compared to WT mice. These changes were significantly greater in males than females. On a normal-salt diet, glomerular filtration rate (GFR) was significantly decreased in PD Npr1 HT and KO male and female mice compared with WT mice. Immunofluorescence of podocin and synaptopodin were also significantly reduced in HT and KO mice compared to WT mice. These observations suggest that in podocytes, ANP/NPRA signaling may be crucial in the maintenance and regulation of glomerular filtration and BP and serve as a biomarker of renal function in a sex-dependent manner.
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BACKGROUND: Hydrogen sulfide (H2S) exerts mitochondria-specific actions that include the preservation of oxidative phosphorylation, biogenesis, and ATP synthesis, while inhibiting cell death. 3-MST (3-mercaptopyruvate sulfurtransferase) is a mitochondrial H2S-producing enzyme whose functions in the cardiovascular disease are not fully understood. In the current study, we investigated the effects of global 3-MST deficiency in the setting of pressure overload-induced heart failure. METHODS: Human myocardial samples obtained from patients with heart failure undergoing cardiac surgeries were probed for 3-MST protein expression. 3-MST knockout mice and C57BL/6J wild-type mice were subjected to transverse aortic constriction to induce pressure overload heart failure with reduced ejection fraction. Cardiac structure and function, vascular reactivity, exercise performance, mitochondrial respiration, and ATP synthesis efficiency were assessed. In addition, untargeted metabolomics were utilized to identify key pathways altered by 3-MST deficiency. RESULTS: Myocardial 3-MST was significantly reduced in patients with heart failure compared with nonfailing controls. 3-MST KO mice exhibited increased accumulation of branched-chain amino acids in the myocardium, which was associated with reduced mitochondrial respiration and ATP synthesis, exacerbated cardiac and vascular dysfunction, and worsened exercise performance following transverse aortic constriction. Restoring myocardial branched-chain amino acid catabolism with 3,6-dichlorobenzo1[b]thiophene-2-carboxylic acid (BT2) and administration of a potent H2S donor JK-1 ameliorates the detrimental effects of 3-MST deficiency in heart failure with reduced ejection fraction. CONCLUSIONS: Our data suggest that 3-MST derived mitochondrial H2S may play a regulatory role in branched-chain amino acid catabolism and mediate critical cardiovascular protection in heart failure.
Assuntos
Insuficiência Cardíaca , Sulfeto de Hidrogênio , Disfunção Ventricular Esquerda , Trifosfato de Adenosina/metabolismo , Aminoácidos de Cadeia Ramificada/metabolismo , Animais , Insuficiência Cardíaca/metabolismo , Humanos , Sulfeto de Hidrogênio/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Mitocôndrias/metabolismo , Miocárdio/metabolismo , Disfunção Ventricular Esquerda/metabolismoRESUMO
RATIONALE: Neurogenic hypertension is characterized by an increase in sympathetic activity and often resistance to drug treatments. We previously reported that it is also associated with a reduction of angiotensin-converting enzyme type 2 (ACE2) and an increase in a disintegrin and metalloprotease 17 (ADAM17) activity in experimental hypertension. In addition, while multiple cells within the central nervous system have been involved in the development of neurogenic hypertension, the contribution of ADAM17 has not been investigated. OBJECTIVE: To assess the clinical relevance of this ADAM17-mediated ACE2 shedding in hypertensive patients and further identify the cell types and signaling pathways involved in this process. METHODS AND RESULTS: Using a mass spectrometry-based assay, we identified ACE2 as the main enzyme converting angiotensin II into angiotensin-(1-7) in human cerebrospinal fluid. We also observed an increase in ACE2 activity in the cerebrospinal fluid of hypertensive patients, which was correlated with systolic blood pressure. Moreover, the increased level of tumor necrosis factor-α in those cerebrospinal fluid samples confirmed that ADAM17 was upregulated in the brain of hypertensive patients. To further assess the interaction between brain renin-angiotensin system and ADAM17, we generated mice lacking angiotensin II type 1 receptors specifically on neurons. Our data reveal that despite expression on astrocytes and other cells types in the brain, ADAM17 upregulation during deoxycorticosterone acetate-salt hypertension occurs selectively on neurons, and neuronal angiotensin II type 1 receptors are indispensable to this process. Mechanistically, reactive oxygen species and extracellular signal-regulated kinase were found to mediate ADAM17 activation. CONCLUSIONS: Our data demonstrate that angiotensin II type 1 receptors promote ADAM17-mediated ACE2 shedding in the brain of hypertensive patients, leading to a loss in compensatory activity during neurogenic hypertension.
Assuntos
Proteína ADAM17/fisiologia , Hipertensão/metabolismo , Hipotálamo/metabolismo , Neurônios/metabolismo , Peptidil Dipeptidase A/metabolismo , Receptor Tipo 1 de Angiotensina/fisiologia , Adulto , Angiotensina II/metabolismo , Enzima de Conversão de Angiotensina 2 , Animais , Animais Recém-Nascidos , Células Cultivadas , Feminino , Humanos , Masculino , Camundongos , Camundongos Knockout , Camundongos TransgênicosRESUMO
While restoration of ACE2 activity in the pancreas leads to improvement of glycemia in experimental models of Type 2 diabetes, global deficiency in ACE2 disrupts ß-cell function and impairs glucose tolerance in mice, demonstrating the physiological role of ACE2 in glucose homeostasis. Although the contribution of pancreatic ACE2 to glucose regulation has been demonstrated in genetic models of diabetes and in models with overexpression of the renin-angiotensin system (RAS), it is unclear whether islet ACE2 is involved in glycemic control in common models of human Type 2 diabetes. To determine whether diet-induced diabetes deregulates glucose homeostasis via reduction of ACE2 in the pancreatic islets, wild-type (WT) and ACE2 knockout (KO) male mice were fed a high-fat diet (HFD) for 16 wk. ACE2 KO mice were more susceptible than WT mice to HFD-mediated glycemic dysregulation. Islet ACE2 activity and expression of various genes, including ANG II type 1a receptor (mAT1aR) were then assessed. Surprisingly, we observed no change in islet ACE2 activity and expression despite local RAS overactivity, indicated by an upregulation of mAT1aR expression. Despite a predominant expression in islet α-cells, further investigation highlighted a minor role for ACE2 on glucagon expression. Further, pancreatic ACE2 gene therapy improved glycemia in HFD-fed WT mice, leading to enhanced glucose-stimulated insulin secretion, reduced pancreatic ANG II levels, fibrosis, and ADAM17 activity. Altogether, our study demonstrates that HFD feeding increases RAS activity and mediates glycemic dysregulation likely through loss of ACE2 present outside the islets but independently of changes in islet ACE2.
Assuntos
Dieta Hiperlipídica/efeitos adversos , Transtornos do Metabolismo de Glucose/etiologia , Transtornos do Metabolismo de Glucose/metabolismo , Glucose/metabolismo , Ilhotas Pancreáticas/metabolismo , Peptidil Dipeptidase A/metabolismo , Enzima de Conversão de Angiotensina 2 , Animais , Gorduras na Dieta/efeitos adversos , Insulina/metabolismo , Resistência à Insulina , Secreção de Insulina , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos KnockoutRESUMO
We previously reported that type 2 angiotensin-converting enzyme (ACE2) compensatory activity is impaired by the disintegrin and metalloprotease 17 (ADAM17), and lack of ACE2 is associated with oxidative stress in neurogenic hypertension. To investigate the relationship between ADAM17 and oxidative stress, Neuro2A cells were treated with ANG II (100 nM) 24 h after vehicle or α-lipoic acid (LA, 500 µM). ADAM17 expression was increased by ANG II (120.5 ± 9.1 vs. 100.2 ± 0.8%, P < 0.05) and decreased after LA (69.0 ± 0.3 vs. 120.5 ± 9.1%, P < 0.05). In another set of experiments, LA reduced ADAM17 (92.9 ± 5.3 vs. 100.0 ± 11.2%, P < 0.05) following its overexpression. Moreover, ADAM17 activity was reduced by LA in ADAM17-overexpressing cells [109.5 ± 19.8 vs. 158.0 ± 20.0 fluorescence units (FU)·min(-1)·µg protein(-1), P < 0.05], in which ADAM17 overexpression increased oxidative stress (114.1 ± 2.5 vs. 101.0 ± 1.0%, P < 0.05). Conversely, LA-treated cells attenuated ADAM17 overexpression-induced oxidative stress (76.0 ± 9.1 vs. 114.1 ± 2.5%, P < 0.05). In deoxycorticosterone acetate (DOCA)-salt hypertensive mice, a model in which ADAM17 expression and activity are increased, hypertension was blunted by pretreatment with LA (119.0 ± 2.4 vs. 131.4 ± 2.2 mmHg, P < 0.05). In addition, LA improved dysautonomia and baroreflex sensitivity. Furthermore, LA blunted the increase in NADPH oxidase subunit expression, as well as the increase in ADAM17 and decrease in ACE2 activity in the hypothalamus of DOCA-salt hypertensive mice. Taken together, these data suggest that LA might preserve ACE2 compensatory activity by breaking the feedforward cycle between ADAM17 and oxidative stress, resulting in a reduction of neurogenic hypertension.
Assuntos
Proteínas ADAM/metabolismo , Antioxidantes/farmacologia , Hipertensão/metabolismo , Estresse Oxidativo , Ácido Tióctico/farmacologia , Proteínas ADAM/genética , Proteína ADAM17 , Angiotensina II/farmacologia , Enzima de Conversão de Angiotensina 2 , Animais , Antioxidantes/uso terapêutico , Barorreflexo , Linhagem Celular Tumoral , Hipertensão/tratamento farmacológico , Hipotálamo/citologia , Hipotálamo/efeitos dos fármacos , Hipotálamo/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , NADPH Oxidases/metabolismo , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Peptidil Dipeptidase A/metabolismo , Ácido Tióctico/uso terapêuticoRESUMO
Endoplasmic reticulum (ER) stress was previously reported to contribute to neurogenic hypertension while neuronal angiotensin-converting enzyme type 2 (ACE2) overexpression blunts the disease. To assess which brain regions are important for ACE2 beneficial effects and the contribution of ER stress to neurogenic hypertension, we first used transgenic mice harboring a floxed neuronal hACE2 transgene (SL) and tested the impact of hACE2 knockdown in the subfornical organ (SFO) and paraventricular nucleus (PVN) on deoxycorticosterone acetate (DOCA)-salt hypertension. SL and nontransgenic (NT) mice underwent DOCA-salt or sham treatment while infected with an adenoassociated virus (AAV) encoding Cre recombinase (AAV-Cre) or a control virus (AAV-green fluorescent protein) to the SFO or PVN. DOCA-salt-induced hypertension was reduced in SL mice, with hACE2 overexpression in the brain. This reduction was only partially blunted by knockdown of hACE2 in the SFO or PVN, suggesting that both regions are involved but not essential for ACE2 regulation of blood pressure (BP). DOCA-salt treatment did not increase the protein levels of ER stress and autophagy markers in NT mice, despite a significant increase in BP. In addition, these markers were not affected by hACE2 overexpression in the brain, despite a significant reduction of hypertension in SL mice. To further assess the role of ER stress in neurogenic hypertension, NT mice were infused intracerebroventricularlly with tauroursodeoxycholic acid (TUDCA), an ER stress inhibitor, during DOCA-salt treatment. However, TUDCA infusion failed to blunt the development of hypertension in NT mice. Our data suggest that brain ER stress does not contribute to DOCA-salt hypertension and that ACE2 blunts neurogenic hypertension independently of ER stress.
Assuntos
Encéfalo/enzimologia , Acetato de Desoxicorticosterona , Estresse do Retículo Endoplasmático , Retículo Endoplasmático/enzimologia , Hipertensão/prevenção & controle , Peptidil Dipeptidase A/metabolismo , Cloreto de Sódio na Dieta , Enzima de Conversão de Angiotensina 2 , Animais , Biomarcadores/metabolismo , Pressão Sanguínea , Encéfalo/efeitos dos fármacos , Encéfalo/fisiopatologia , Modelos Animais de Doenças , Retículo Endoplasmático/efeitos dos fármacos , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Técnicas de Silenciamento de Genes , Humanos , Hipertensão/enzimologia , Hipertensão/genética , Hipertensão/fisiopatologia , Infusões Intraventriculares , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Núcleo Hipotalâmico Paraventricular/enzimologia , Núcleo Hipotalâmico Paraventricular/fisiopatologia , Peptidil Dipeptidase A/genética , Órgão Subfornical/enzimologia , Órgão Subfornical/fisiopatologia , Ácido Tauroquenodesoxicólico/administração & dosagem , Fatores de Tempo , Regulação para CimaRESUMO
RATIONALE: Overactivity of the brain renin-angiotensin system is a major contributor to neurogenic hypertension. Although overexpression of angiotensin-converting enzyme type 2 (ACE2) has been shown to be beneficial in reducing hypertension by transforming angiotensin II into angiotensin-(1-7), several groups have reported decreased brain ACE2 expression and activity during the development of hypertension. OBJECTIVE: We hypothesized that ADAM17-mediated ACE2 shedding results in decreased membrane-bound ACE2 in the brain, thus promoting the development of neurogenic hypertension. METHODS AND RESULTS: To test this hypothesis, we used the deoxycorticosterone acetate-salt model of neurogenic hypertension in nontransgenic and syn-hACE2 mice overexpressing ACE2 in neurons. Deoxycorticosterone acetate-salt treatment in nontransgenic mice led to significant increases in blood pressure, hypothalamic angiotensin II levels, inflammation, impaired baroreflex sensitivity, and autonomic dysfunction, as well as decreased hypothalamic ACE2 activity and expression, although these changes were blunted or prevented in syn-hACE2 mice. In addition, reduction of ACE2 expression and activity in the brain paralleled an increase in ACE2 activity in the cerebrospinal fluid of nontransgenic mice after deoxycorticosterone acetate-salt treatment and were accompanied by enhanced ADAM17 expression and activity in the hypothalamus. Chronic knockdown of ADAM17 in the brain blunted the development of hypertension and restored ACE2 activity and baroreflex function. CONCLUSIONS: Our data provide the first evidence that ADAM17-mediated shedding impairs brain ACE2 compensatory activity, thus contributing to the development of neurogenic hypertension.
Assuntos
Pressão Sanguínea , Encéfalo/enzimologia , Hipertensão/enzimologia , Peptidil Dipeptidase A/metabolismo , Sistema Renina-Angiotensina , Proteínas ADAM/genética , Proteínas ADAM/metabolismo , Proteína ADAM17 , Angiotensina II/metabolismo , Bloqueadores do Receptor Tipo 1 de Angiotensina II/farmacologia , Enzima de Conversão de Angiotensina 2 , Animais , Anti-Hipertensivos/farmacologia , Sistema Nervoso Autônomo/metabolismo , Sistema Nervoso Autônomo/fisiopatologia , Barorreflexo , Pressão Sanguínea/efeitos dos fármacos , Encéfalo/efeitos dos fármacos , Encéfalo/fisiopatologia , Acetato de Desoxicorticosterona , Modelos Animais de Doenças , Humanos , Hipertensão/induzido quimicamente , Hipertensão/tratamento farmacológico , Hipertensão/genética , Hipertensão/fisiopatologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Peptidil Dipeptidase A/genética , Interferência de RNA , Sistema Renina-Angiotensina/efeitos dos fármacos , Fatores de TempoRESUMO
Pancreatic angiotensin-converting enzyme 2 (ACE2) has previously been shown to be critical for maintaining glycemia and ß-cell function. Efforts to maintain or increase ACE2 expression in pancreatic ß-cells might therefore have therapeutic potential for treating diabetes. In our study, we investigated the transcriptional role of hepatocyte nuclear factor 1α (HNF1α) and hepatocyte nuclear factor 1ß (HNF1ß) in induction of ACE2 expression in insulin-secreting cells. A deficient allele of HNF1α or HNF1ß causes maturity-onset diabetes of the young (MODY) types 3 and 5, respectively, in humans. We found that ACE2 is primarily transcribed from the proximal part of the ACE2 promoter in the pancreas. In the proximal part of the human ACE2 promoter, we further identified three functional HNF1 binding sites, as they have binding affinity for HNF1α and HNF1ß and are required for induction of promoter activity by HNF1ß in insulinoma cells. These three sites are well-conserved among mammalian species. Both HNF1α and HNF1ß induce expression of ACE2 mRNA and lead to elevated levels of ACE2 protein and ACE2 enzymatic activity in insulinoma cells. Furthermore, HNF1α dose-dependently increases ACE2 expression in primary pancreatic islet cells. We conclude that HNF1α can induce the expression of ACE2 in pancreatic islet cells via evolutionarily conserved HNF1 binding sites in the ACE2 promoter. Potential therapeutics aimed at counteracting functional HNF1α depletion in diabetes and MODY3 will thus have ACE2 induction in pancreatic islets as a likely beneficial effect.
Assuntos
Evolução Molecular , Fator 1-alfa Nuclear de Hepatócito/fisiologia , Ilhotas Pancreáticas/enzimologia , Peptidil Dipeptidase A/metabolismo , Enzima de Conversão de Angiotensina 2 , Animais , Sequência de Bases , Linhagem Celular Tumoral , Primers do DNA , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Peptidil Dipeptidase A/genética , Ratos , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
BACKGROUND AND PURPOSE: Sodium glucose cotransporter 2 inhibitors (SGLT2i) have emerged as a potent therapy for heart failure with preserved ejection fraction (HFpEF). Hydrogen sulphide (H2S), a well-studied cardioprotective agent, could be beneficial in HFpEF. SGLT2i monotherapy and combination therapy involving an SGLT2i and H2S donor in two preclinical models of cardiometabolic HFpEF was investigated. EXPERIMENTAL APPROACH: Nine-week-old C57BL/6N mice received L-NAME and a 60% high fat diet for five weeks. Mice were then randomized to either control, SGLT2i monotherapy or SGLT2i and H2S donor, SG1002, for five additional weeks. Ten-week-old ZSF1 obese rats were randomized to control, SGLT2i or SGLT2i and SG1002 for 8 weeks. SG1002 monotherapy was investigated in additional animals. Cardiac function (echocardiography and haemodynamics), exercise capacity, glucose handling and multiorgan pathology were monitored during experimental protocols. KEY RESULTS: SGLT2i treatment improved E/e' ratio and treadmill exercise in both models. Combination therapy afforded increases in cardiovascular sulphur bioavailability that coincided with improved left end-diastolic function (E/e' ratio), exercise capacity, metabolic state, cardiorenal fibrosis, and hepatic steatosis. Follow-up studies with SG1002 monotherapy revealed improvements in diastolic function, exercise capacity and multiorgan histopathology. CONCLUSIONS AND IMPLICATIONS: SGLT2i monotherapy remediated pathological complications exhibited by two well-established HFpEF models. Adjunctive H2S therapy resulted in further improvements of cardiometabolic perturbations beyond SGLT2i monotherapy. Follow-up SG1002 monotherapy studies inferred an improved phenotype with combination therapy beyond either monotherapy. These data demonstrate the differing effects of SGLT2i and H2S therapy while also revealing the superior efficacy of the combination therapy in cardiometabolic HFpEF.
RESUMO
An overactive renin-angiotensin system (RAS) is known to contribute to type 2 diabetes mellitus (T2DM). Although ACE2 overexpression has been shown to be protective against the overactive RAS, a role for pancreatic ACE2, particularly in the islets of Langerhans, in regulating glycemia in response to elevated angiotensin II (Ang II) levels remains to be elucidated. This study examined the role of endogenous pancreatic ACE2 and the impact of elevated Ang II levels on the enzyme's ability to alleviate hyperglycemia in an Ang II infusion mouse model. Male C57bl/6J mice were infused with Ang II or saline for a period of 14 days. On the 7th day of infusion, either an adenovirus encoding human ACE2 (Ad-hACE2) or a control adenovirus (Ad-eGFP) was injected into the mouse pancreas. After an additional 7-8 days, glycemia and plasma insulin levels as well as RAS components expression and oxidative stress were assessed. Ang II-infused mice exhibited hyperglycemia, hyperinsulinemia, and impaired glucose-stimulated insulin secretion from pancreatic islets compared with control mice. This phenotype was associated with decreased ACE2 expression and activity, increased Ang II type 1 receptor (AT1R) expression, and increased oxidative stress in the mouse pancreas. Ad-hACE2 treatment restored pancreatic ACE2 expression and compensatory activity against Ang II-mediated impaired glycemia, thus improving ß-cell function. Our data suggest that decreased pancreatic ACE2 is a link between overactive RAS and impaired glycemia in T2DM. Moreover, maintenance of a normal endogenous ACE2 compensatory activity in the pancreas appears critical to avoid ß-cell dysfunction, supporting a therapeutic potential for ACE2 in controlling diabetes resulting from an overactive RAS.
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Angiotensina II/farmacologia , Diabetes Mellitus Tipo 2/terapia , Terapia Genética/métodos , Hiperglicemia/terapia , Células Secretoras de Insulina/fisiologia , Peptidil Dipeptidase A/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Adenoviridae/genética , Enzima de Conversão de Angiotensina 2 , Animais , Glicemia/efeitos dos fármacos , Glicemia/metabolismo , Peptídeo C/sangue , Diabetes Mellitus Tipo 2/metabolismo , Humanos , Hiperglicemia/metabolismo , Insulina/sangue , Resistência à Insulina/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Peptidil Dipeptidase A/metabolismo , Peptidil Dipeptidase A/farmacologia , Sistema Renina-Angiotensina/efeitos dos fármacos , Sistema Renina-Angiotensina/fisiologia , Vasoconstritores/farmacologiaRESUMO
Background Recent studies have suggested that cardiac nitrosative stress mediated by pathological overproduction of nitric oxide (NO) via inducible NO synthase (iNOS) contributes to the pathogenesis of heart failure with preserved ejection fraction (HFpEF). Other studies have suggested that endothelial NO synthase (eNOS) dysfunction and attenuated NO bioavailability contribute to HFpEF morbidity and mortality. We sought to further investigate dysregulated NO signaling and to examine the effects of a NO-based dual therapy (sodium nitrite+hydralazine) following the onset of HFpEF using a "2-hit" murine model. Methods and Results Nine-week-old male C57BL/6 N mice (n=15 per group) were treated concurrently with high-fat diet and N(ω)-nitro-L-arginine methyl ester (L-NAME) (0.5 g/L per day) via drinking water for 10 weeks. At week 5, mice were randomized into either vehicle (normal saline) or combination treatment with sodium nitrite (75 mg/L in the drinking water) and hydralazine (2.0 mg/kg IP, BID). Cardiac structure and function were monitored with echocardiography and invasive hemodynamic measurements. Cardiac mitochondrial respiration, aortic vascular function, and exercise performance were also evaluated. Circulating and myocardial nitrite were measured to determine the bioavailability of NO. Circulating markers of oxidative or nitrosative stress as well as systemic inflammation were also determined. Severe HFpEF was evident by significantly elevated E/E', LVEDP, and Tau in mice treated with L-NAME and HFD, which was associated with impaired NO bioavailability, mitochondrial respiration, aortic vascular function, and exercise capacity. Treatment with sodium nitrite and hydralazine restored NO bioavailability, reduced oxidative and nitrosative stress, preserved endothelial function and mitochondrial respiration, limited the fibrotic response, and improved exercise capacity, ultimately attenuating the severity of "two-hit" HFpEF. Conclusions Our data demonstrate that nitrite, a well-established biomarker of NO bioavailability and a physiological source of NO, is significantly reduced in the heart and circulation in the "2-hit" mouse HFpEF model. Furthermore, sodium nitrite+hydralazine combined therapy significantly attenuated the severity of HFpEF in the "2-hit" cardiometabolic HFpEF. These data suggest that supplementing NO-based therapeutics with a potent antioxidant and vasodilator agent may result in synergistic benefits for the treatment of HFpEF.
Assuntos
Água Potável , Insuficiência Cardíaca , Camundongos , Masculino , Animais , Insuficiência Cardíaca/tratamento farmacológico , Nitrito de Sódio , Volume Sistólico/fisiologia , NG-Nitroarginina Metil Éster , Modelos Animais de Doenças , Camundongos Endogâmicos C57BL , Hidralazina/farmacologia , Óxido Nítrico SintaseRESUMO
RATIONALE: Angiotensin converting enzyme type 2 (ACE2) is a new member of the brain renin-angiotensin system, that might be activated by an overactive renin-angiotensin system. OBJECTIVE: To clarify the role of central ACE2 using a new transgenic mouse model with human (h)ACE2 under the control of a synapsin promoter, allowing neuron-targeted expression in the central nervous system. METHODS AND RESULTS: Syn-hACE2 (SA) transgenic mice exhibit high hACE2 protein expression and activity throughout the brain. Baseline hemodynamic parameters (telemetry), autonomic function, and spontaneous baroreflex sensitivity (SBRS) were not significantly different between SA mice and nontransgenic littermates. Brain-targeted ACE2 overexpression attenuated the development of neurogenic hypertension (Ang II infusion: 600 ng/kg per minute for 14 days) and the associated reduction of both SBRS and parasympathetic tone. This prevention of hypertension by ACE2 overexpression was reversed by blockade of the Ang-(1-7) receptor (d-Ala7-Ang-[1-7]; 600 ng/kg per minute). Brain angiotensin II type 2 (AT(2))/AT(1) and Mas/AT(1) receptor ratios were significantly increased in SA mice. They remained higher following Ang II infusion but were dramatically reduced after Ang-(1-7) receptor blockade. ACE2 overexpression resulted in increased NOS and NO levels in the brain, and prevented the Ang II-mediated decrease in NOS expression in regions modulating blood pressure regulation. CONCLUSIONS: ACE2 overexpression attenuates the development of neurogenic hypertension partially by preventing the decrease in both SBRS and parasympathetic tone. These protective effects might be mediated by enhanced NO release in the brain resulting from Mas and AT(2) receptor upregulation. Taken together, our data highlight the compensatory role of central ACE2 and its potential benefits as a therapeutic target for neurogenic hypertension.
Assuntos
Encéfalo/metabolismo , Regulação Enzimológica da Expressão Gênica , Hipertensão/genética , Peptidil Dipeptidase A/genética , Angiotensina II/sangue , Angiotensina II/metabolismo , Angiotensina II/farmacologia , Enzima de Conversão de Angiotensina 2 , Animais , Barorreflexo/fisiologia , Pressão Sanguínea/efeitos dos fármacos , Western Blotting , Encéfalo/enzimologia , Tronco Encefálico/enzimologia , Tronco Encefálico/metabolismo , Feminino , Humanos , Hipertensão/enzimologia , Hipertensão/fisiopatologia , Imuno-Histoquímica , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase/genética , Óxido Nítrico Sintase/metabolismo , Sistema Nervoso Parassimpático/fisiologia , Fragmentos de Peptídeos/sangue , Fragmentos de Peptídeos/metabolismo , Fragmentos de Peptídeos/farmacologia , Peptidil Dipeptidase A/metabolismo , Receptores de Angiotensina/genética , Receptores de Angiotensina/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Angiotensin-converting enzyme 2 (ACE2) is a component of the renin-angiotensin system, and its expression and activity have been shown to be reduced in cardiovascular diseases. Enzymatic activity of ACE2 is commonly measured by hydrolysis of quenched fluorescent substrates in the absence or presence of an ACE2-specific inhibitor, such as the commercially available inhibitor DX600. Whereas recombinant human ACE2 is readily detected in mouse tissues using 1 µM DX600 at pH 7.5, the endogenous ACE2 activity in mouse tissues is barely detectable. We compared human, mouse, and rat ACE2 overexpressed in cell lines for their sensitivity to inhibition by DX600. ACE2 from all three species could be inhibited by DX600, but the half maximal inhibitory concentration (IC(50)) for human ACE2 was much lower (78-fold) than for rodent ACE2. Following optimization of pH, substrate concentration, and antagonist concentration, rat and mouse ACE2 expressed in a cell line could be accurately quantified with 10 µM DX600 (>95% inhibition) but not with 1 µM DX600 (<75% inhibition). Validation that the optimized method robustly quantifies ACE2 in mouse tissues (kidney, brain, heart, and plasma) was performed using wild-type and ACE2 knockout mice. This study provides a reliable method for measuring human, as well as endogenous ACE2 activity in rodents. Our data underscore the importance of validating the effect of DX600 on ACE2 from each particular species at the experimental conditions employed.
Assuntos
Inibidores da Enzima Conversora de Angiotensina/farmacologia , Peptídeos/farmacologia , Peptidil Dipeptidase A/metabolismo , Enzima de Conversão de Angiotensina 2 , Animais , Encéfalo/efeitos dos fármacos , Encéfalo/enzimologia , Linhagem Celular Tumoral , Relação Dose-Resposta a Droga , Humanos , Concentração de Íons de Hidrogênio , Hidrólise , Rim/efeitos dos fármacos , Rim/enzimologia , Cinética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Miocárdio/enzimologia , Peptidil Dipeptidase A/deficiência , Peptidil Dipeptidase A/genética , Ratos , Reprodutibilidade dos Testes , Especificidade da Espécie , Espectrometria de Fluorescência , Especificidade por Substrato , TransfecçãoRESUMO
Overactivity of the renin-angiotensin system (RAS) is involved in the pathogenesis of hypertension, and an overactive brain RAS has been highlighted in several genetic and experimental models. Until now, angiotensin II (Ang II) was thought to be the main effector of this system, and the angiotensin-converting enzyme (ACE)-Ang II-Ang II type 1 receptor axis was the main target for antihypertensive therapies. A new member of the RAS, ACE2 (angiotensin-converting enzyme type 2), has been identified in organs and tissues related to cardiovascular function (e.g. heart, kidney and blood vessels) and appears to be part of a counter-regulatory pathway to buffer the excess of Ang II. We recently identified the ACE2 protein in brain regions involved in the central regulation of blood pressure and showed that it regulates, and is regulated by, other components of the RAS. Here, we present evidence for the involvement of brain ACE2 in the central regulation of blood pressure, autonomic and cardiac function. We show that lack of ACE2 is deleterious for the central regulation of blood pressure and that brain ACE2 gene therapy can restore baroreflex and autonomic functions and prevent the development of hypertension. Additionally, and independently of a reduction in Ang II levels, we will highlight some of the mechanisms responsible for the beneficial effects of central ACE2 in cardiovascular function.
Assuntos
Hipertensão/fisiopatologia , Peptidil Dipeptidase A/fisiologia , Angiotensina II/metabolismo , Enzima de Conversão de Angiotensina 2 , Animais , Barorreflexo/efeitos dos fármacos , Pressão Sanguínea/efeitos dos fármacos , Camundongos , Camundongos Transgênicos , Óxido Nítrico Sintase/fisiologiaRESUMO
We recently reported the presence of angiotensin-converting enzyme (ACE)2 in brain regions controlling cardiovascular function; however, the role of ACE2 in blood pressure regulation remains unclear because of the lack of specific tools to investigate its function. We hypothesized that ACE2 could play a pivotal role in the central regulation of cardiovascular function by regulating other renin-angiotensin system components. To test this hypothesis, we generated an adenovirus expressing the human ACE2 cDNA upstream of an enhanced green fluorescent protein (eGFP) reporter gene (Ad-hACE2-eGFP). In vitro characterization shows that neuronal cells infected with Ad-hACE2-eGFP (10 to 100 multiplicities of infection), but not Ad-eGFP (100 multiplicities of infection), exhibit dose-dependent ACE2 expression and activity. In addition, an active secreted form was detected in the conditioned medium. In vivo, Ad-hACE2-eGFP infection (2x10(6) plaque-forming units intracerebroventricularly) produced time-dependent expression and activity (with a peak at 7 days) in the mouse subfornical organ. More importantly, 7 days after virus infection, the pressor response to angiotensin (Ang) II (200 pmol intracerebroventricularly) was significantly reduced in Ad-hACE2-eGFP-treated mice compared with controls. Furthermore, subfornical organ-targeted ACE2 overexpression dramatically reduced the Ang II-mediated drinking response. Interestingly, ACE2 overexpression was associated with downregulation of the Ang II type 1 receptor expression both in vitro and in vivo. These data suggest that ACE2 overexpression in the subfornical organ impairs Ang II-mediated pressor and drinking responses at least by inhibiting the Ang II type 1 receptor expression. Taken together, our results show that ACE2 plays a pivotal role in the central regulation of blood pressure and volume homeostasis, offering a new target for the treatment of hypertension and other cardiovascular diseases.
Assuntos
Angiotensina II/metabolismo , Barorreflexo , Comportamento de Ingestão de Líquido , Neurônios/metabolismo , Peptidil Dipeptidase A/metabolismo , Receptor Tipo 1 de Angiotensina/metabolismo , Órgão Subfornical/metabolismo , Adenoviridae/efeitos dos fármacos , Angiotensina II/administração & dosagem , Enzima de Conversão de Angiotensina 2 , Animais , Barorreflexo/efeitos dos fármacos , Pressão Sanguínea , Linhagem Celular Tumoral , Meios de Cultura/metabolismo , Regulação para Baixo , Comportamento de Ingestão de Líquido/efeitos dos fármacos , Genes Reporter , Vetores Genéticos , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Frequência Cardíaca , Humanos , Injeções Intraventriculares , Camundongos , Camundongos Endogâmicos C57BL , Neurônios/enzimologia , Peptidil Dipeptidase A/genética , Receptor Tipo 1 de Angiotensina/agonistas , Proteínas Recombinantes de Fusão/metabolismo , Transdução de Sinais , Órgão Subfornical/efeitos dos fármacos , Órgão Subfornical/enzimologia , Fatores de Tempo , Transdução Genética , Regulação para CimaRESUMO
Angiotensin-converting enzyme 2 (ACE2) is a new component of the renin-angiotensin system (RAS). Accumulating evidence shows that ACE2 provides protective effects in peripheral tissues and has great potential for the treatment of RAS-related diseases. The role of ACE2 in the central nervous system is not well established. However, in recent years, much more progress has been made on the studies of this carboxypeptidase in the central regulation of blood pressure and cardiovascular function in general. It has been shown that brain ACE2 interacts with the other components of the RAS (ACE, angiotensin II, and angiotensin II type 1 receptor), protects baroreflex and autonomic function, stimulates nitric oxide release, reduces oxidative stress, and prevents the development of or attenuates hypertension. These data support the critical role of ACE2 in the central regulation of cardiovascular function. This review summarizes recently published data on the central effects of ACE2 in the regulation of cardiovascular function.
Assuntos
Fenômenos Fisiológicos Cardiovasculares , Sistema Cardiovascular/enzimologia , Peptidil Dipeptidase A/fisiologia , Sistema Renina-Angiotensina/fisiologia , Enzima de Conversão de Angiotensina 2 , Sistema Nervoso Autônomo/enzimologia , Sistema Nervoso Autônomo/fisiopatologia , Barorreflexo , Carboxipeptidases/metabolismo , Doenças Cardiovasculares/enzimologia , Doenças Cardiovasculares/fisiopatologia , Humanos , Hipertensão/enzimologia , Hipertensão/fisiopatologia , Óxido Nítrico , Óxido Nítrico Sintase/metabolismo , Estresse Oxidativo , Peptidil Dipeptidase A/metabolismoRESUMO
Background Hydrogen sulfide (H2S) is an important endogenous physiological signaling molecule and exerts protective properties in the cardiovascular system. Cystathionine γ-lyase (CSE), 1 of 3 H2S producing enzyme, is predominantly localized in the vascular endothelium. However, the regulation of CSE in vascular endothelium remains incompletely understood. Methods and Results We generated inducible endothelial cell-specific CSE overexpressed transgenic mice (EC-CSE Tg) and endothelial cell-specific CSE knockout mice (EC-CSE KO), and investigated vascular function in isolated thoracic aorta, treadmill exercise capacity, and myocardial injury following ischemia-reperfusion in these mice. Overexpression of CSE in endothelial cells resulted in increased circulating and myocardial H2S and NO, augmented endothelial-dependent vasorelaxation response in thoracic aorta, improved exercise capacity, and reduced myocardial-reperfusion injury. In contrast, genetic deletion of CSE in endothelial cells led to decreased circulating H2S and cardiac NO production, impaired endothelial dependent vasorelaxation response and reduced exercise capacity. However, myocardial-reperfusion injury was not affected by genetic deletion of endothelial cell CSE. Conclusions CSE-derived H2S production in endothelial cells is critical in maintaining endothelial function, exercise capacity, and protecting against myocardial ischemia/reperfusion injury. Our data suggest that the endothelial NO synthase-NO pathway is likely involved in the beneficial effects of overexpression of CSE in the endothelium.
Assuntos
Cistationina gama-Liase/metabolismo , Células Endoteliais/metabolismo , Tolerância ao Exercício/fisiologia , Sulfeto de Hidrogênio/metabolismo , Traumatismo por Reperfusão Miocárdica/metabolismo , Óxido Nítrico/metabolismo , Animais , Aorta Torácica/metabolismo , Aorta Torácica/fisiopatologia , Camundongos , Camundongos Transgênicos , Miocárdio/metabolismo , Miocárdio/patologia , Óxido Nítrico Sintase/metabolismo , Transdução de SinaisRESUMO
Angiotensin (Ang)-converting enzyme (ACE) 2 cleaves Ang-II into the vasodilator peptide Ang-(1-7), thus acting as a pivotal element in balancing the local effects of these peptides. ACE2 has been identified in various tissues and is supposed to be a modulator of cardiovascular function. Decreases in ACE2 expression and activity have been reported in models of hypertension, heart failure, atherosclerosis, diabetic nephropathy and others. In addition, the expression level and/or activity are affected by other renin-angiotensin system components (e.g., ACE and AT1 receptors). Local inhibition or global deletion of brain ACE2 induces a reduction in baroreflex sensitivity. Moreover, ACE2-null mice have been shown to exhibit either blood pressure or cardiac dysfunction phenotypes. On the other hand, over-expression of ACE2 exerts protective effects in local tissues, including the brain. In this review, we will first summarize the major findings linking ACE2 to cardiovascular function in the periphery then focus on recent discoveries related to ACE2 in the CNS. Finally, we will unveil new tools designed to address the importance of central ACE2 in various diseases, and discuss the potential for this carboxypeptidase as a new target in the treatment of hypertension and other cardiovascular diseases.