Tatiane M. Murça

New cardiovascular and pulmonary therapeutic strategies based on the Angiotensin-converting enzyme 2/angiotensin-(1-7)/mas receptor axis.

Angiotensin (Ang)-(1–7) is now recognized as a biologically active component of the renin-angiotensin system (RAS). The discovery of the angiotensin-converting enzyme homologue ACE2 revealed important metabolic pathways involved in the Ang-(1–7) synthesis. This enzyme can form Ang-(1–7) from Ang II or less efficiently through hydrolysis of Ang I to Ang-(1–9) with subsequent Ang-(1–7) formation. Additionally, it is well established that the G protein-coupled receptor Mas is a functional ligand site for Ang-(1–7). The axis formed by ACE2/Ang-(1–7)/Mas represents an endogenous counter regulatory pathway within the RAS whose actions are opposite to the vasoconstrictor/proliferative arm of the RAS constituted by ACE/Ang II/AT1 receptor. In this review we will discuss recent findings concerning the biological role of the ACE2/Ang-(1–7)/Mas arm in the cardiovascular and pulmonary system. Also, we will highlight the initiatives to develop potential therapeutic strategies based on this axis.

Angiotensin-Converting Enzyme 2 Activation Improves Endothelial Function

Diminished release and function of endothelium-derived nitric oxide coupled with increases in reactive oxygen species production is critical in endothelial dysfunction. Recent evidences have shown that activation of the protective axis of the renin–angiotensin system composed by angiotensin-converting enzyme 2, angiotensin-(1–7), and Mas receptor promotes many beneficial vascular effects. This has led us to postulate that activation of intrinsic angiotensin-converting enzyme 2 would improve endothelial function by decreasing the reactive oxygen species production. In the present study, we tested 1-[[2-(dimetilamino)etil]amino]-4-(hidroximetil)-7-[[(4-metilfenil)sulfonil]oxi]-9H-xantona-9 (XNT), a small molecule angiotensin-converting enzyme 2 activator, on endothelial function to validate this hypothesis. In vivo treatment with XNT (1 mg/kg per day for 4 weeks) improved the endothelial function of spontaneously hypertensive rats and of streptozotocin-induced diabetic rats when evaluated through the vasorelaxant responses to acetylcholine/sodium nitroprusside. Acute in vitro incubation with XNT caused endothelial-dependent vasorelaxation in aortic rings of rats. This vasorelaxation effect was attenuated by the Mas antagonist D-pro7-Ang-(1–7), and it was reduced in Mas knockout mice. These effects were associated with reduction in reactive oxygen species production. In addition, Ang II–induced reactive oxygen species production in human aortic endothelial cells was attenuated by preincubation with XNT. These results showed that chronic XNT administration improves the endothelial function of hypertensive and diabetic rat vessels by attenuation of the oxidative stress. Moreover, XNT elicits an endothelial-dependent vasorelaxation response, which was mediated by Mas. Thus, this study indicated that angiotensin-converting enzyme 2 activation promotes beneficial effects on the endothelial function and it is a potential target for treating cardiovascular disease.

Oral Administration of an Angiotensin-Converting Enzyme 2 Activator Ameliorates Diabetes-Induced Cardiac Dysfunction

We evaluated the hypothesis that activation of endogenous angiotensin-converting enzyme (ACE) 2 would improve cardiac dysfunction induced by diabetes. Ten days after diabetes induction (streptozotocin, 50 mg/kg, i.v.), male Wistar rats were treated with the ACE2 activator 1-[[2-(dimethylamino)ethyl]amino]-4-(hydroxymethyl)-7-[[(4-methylphenyl)sulfonyl]oxy]-9H-xanthen-9-one (XNT, 1 mg/kg/day, gavage) or saline (control) for 30 days. Echocardiography was performed to analyze the cardiac function and kinetic fluorogenic assays were used to determine cardiac ACE and ACE2 activities. Cardiac ACE2, ACE, Mas receptor, AT(1) receptor, AT(2) receptor and collagen types I and III mRNA and ACE2, ACE, Mas, AT(1) receptor, AT(2) receptor, ERK1/2, Akt, AMPK-α and AMPK-β(1) protein were measured by qRT-PCR and western blotting techniques, respectively. Histological sections of hearts were analyzed to evaluate the presence of hypertrophy and fibrosis. Diabetic animals presented hyperglycemia and diastolic dysfunction along with cardiac hypertrophy and fibrosis. XNT treatment prevented further increase in glycemia and improved the cardiac function, as well as the hypertrophy and fibrosis. These effects were associated with increases in cardiac ACE2/ACE ratios (activity: ~26%; mRNA: ~113%; and protein: ~188%) and with a decrease in AT(1) receptor expression. Additionally, XNT inhibited ERK1/2 phosphorylation and prevented changes in AMPK-α and AMPK-β(1) expressions. XNT treatment did not induce any significant change in AT(2) receptor and Akt expression. These results indicate that activation of intrinsic cardiac ACE2 by oral XNT treatment protects the heart against diabetes-induced dysfunction through mechanisms involving ACE, ACE2, ERK1/2, AMPK-α and AMPK-β(1) modulations.

Chronic activation of endogenous angiotensin‐converting enzyme 2 protects diabetic rats from cardiovascular autonomic dysfunction

In this study, we evaluated whether the activation of endogenous angiotensin‐converting enzyme 2 (ACE2) would improve the cardiovascular autonomic dysfunction of diabetic rats. Ten days after induction of type 1 diabetes (streptozotocin, 50 mg kg−1i.v.), the rats were treated orally with 1‐[(2‐dimethylamino)ethylamino]‐4‐(hydroxymethyl)‐7‐[(4‐methylphenyl) sulfonyl oxy]‐9H‐xanthene‐9‐one (XNT), a newly discovered ACE2 activator (1 mg kg−1 day−1), or saline (equivalent volume) for 30 days. Autonomic cardiovascular parameters were evaluated in conscious animals, and an isolated heart preparation was used to analyse cardiac function. Diabetes induced a significant decrease in the baroreflex bradycardia sensitivity, as well as in the chemoreflex chronotropic response and parasympathetic tone. The XNT treatment improved these parameters by ∼76% [0.82 ± 0.09 versus 1.44 ± 0.17 Ratio between changes in pulse interval and changes in mean arterial pressure (ΔPI/ΔmmHg)], ∼85% (−57 ± 9 versus−105 ± 10 beats min‐1) and ∼205% (22 ± 2 versus 66 ± 12 beats min‐1), respectively. Also, XNT administration enhanced the bradycardia induced by the chemoreflex activation by ∼74% in non‐diabetic animals (−98 ± 16 versus−170 ± 9 Δbeats min‐1). No significant changes were observed in the mean arterial pressure, baroreflex tachycardia sensitivity, chemoreflex pressor response and sympathetic tone among any of the groups. Furthermore, chronic XNT treatment ameliorated the cardiac function of diabetic animals. However, the coronary vasoconstriction observed in diabetic rats was unchanged by ACE2 activation. These findings indicate that XNT protects against the autonomic and cardiac dysfunction induced by diabetes. Thus, our results provide evidence for the viability and effectiveness of oral administration of an ACE2 activator for the treatment of the cardiovascular autonomic dysfunction caused by diabetes.

Nucleus of the solitary tract (pro)renin receptor-mediated antihypertensive effect involves nuclear factor-κB-cytokine signaling in the spontaneously hypertensive rat.

The importance of the (pro)renin receptor (PRR) in the function of the central nervous system is increasingly evident because PRR seems to play a role in neuronal control of cardiovascular function. PRR expression is elevated in the nucleus of the solitary tract (NTS) of spontaneously hypertensive rats (SHR). In this study, we tested the hypothesis that altered activity of PRR in the NTS is linked to hypertension. Eight weeks of chronic knockdown of the NTS PRR, using recombinant adeno-associated virus type 2 (AAV2)-PRR-small hairpain RNA (shRNA)–mediated gene transduction, caused a significant increase in mean arterial pressure (MAP) in the SHR (shRNA, 173±5; Control, 151±6 mm Hg) but not in Wistar Kyoto rats (shRNA, 108±7; Control, 106±6 mm Hg). The MAP elevation in the SHR was associated with decreased inflammatory markers tumor necrosis factor-&agr;, interleukin-6, C-C motif ligand 5, and their transcription factor, nuclear factor-&kgr;B. Consistent with the pressor effects of the PRR knockdown, acute bilateral NTS injection of human renin (2 pmol/side) decreased MAP and heart rate (HR) in SHR (&Dgr;MAP, −38±4 mm Hg; &Dgr;heart rate, −40±10 bpm), with negligible responses in Wistar Kyoto rats (&Dgr;MAP, −4±3 mm Hg; &Dgr;heart rate, −12±7 bpm). These effects in SHR were attenuated (80%) by prorenin handle region peptide but were not affected by angiotensin II type 1 or angiotensin II type 2 receptor blockers. Finally, PRR activation in SHR neuronal cultures by prorenin activated nuclear factor-&kgr;B and increased mRNA levels of interleukin-1&bgr; (250-fold), tumor necrosis factor-&agr; (32-fold), interleukin-6 (35-fold), C-C motif ligand 5 (12-fold), and interleukin-10 (7-fold) in a nuclear factor-&kgr;B–dependent but angiotensin II type 1 receptor–independent manner. Therefore, NTS PRR mediates antihypertensive effects via an angiotensin II–independent mechanism in SHR, which involves stimulation of the nuclear factor-&kgr;B–cytokine signaling pathway.

Chronic Knockdown of the Nucleus of the Solitary Tract AT1 Receptors Increases Blood Inflammatory-Endothelial Progenitor Cell Ratio and Exacerbates Hypertension in the Spontaneously Hypertensive Rat

AT1 receptor subtype a (AT1Ra) expression is increased in the nucleus of the solitary tract (NTS) in spontaneously hypertensive rat (SHR) compared with Wistar Kyoto controls. However, the chronic role of AT1Ra in the NTS for cardiovascular control is not well understood. In this study, we investigated the hypothesis that the NTS AT1Ra is involved in the neural regulation of the peripheral inflammatory status and linked with hypertension. Transduction of brain neuronal cultures with recombinant adeno-associated virus type 2 (AAV2)-AT1R-small hairpin RNA (shRNA) resulted in a 72% decrease in AT1Ra mRNA and attenuated angiotensin II–induced increase in extracellular signal–regulated kinase 1/2 phosphorylation and neuronal firing. Specific NTS microinjection of AAV2-AT1R-shRNA vector in the SHR resulted in a ≈30 mm Hg increase in the mean arterial pressure compared with control vector–injected animals (Sc-shRNA: 154±4 mm Hg; AT1R-shRNA: 183±10 mm Hg) and induced a resetting of the baroreflex control of heart rate to higher mean arterial pressure. In addition, AAV2-AT1R-shRNA–treated SHRs exhibited a 74% decrease in circulating endothelial progenitor cells (CD90+, CD4−/CD5−/CD8−) and a 300% increase in the circulating inflammatory cells, including CD4+ +CD8+, CD45+/3+ T lymphocytes, and macrophages (CD68+). As a result, the endothelial progenitor cell/inflammatory cells ratio was decreased by 8- to 15-fold in the AT1R-shRNA–treated SHR. However, identical injection of AAV2-AT1R-shRNA into the NTS of Wistar Kyoto rats had no effect on mean arterial pressure and inflammatory cells. These observations suggest that increased expression of the AT1Ra in SHR NTS may present a counterhypertensive mechanism involving inflammatory/angiogenic cells.AT1 receptor subtype a (AT1R1a) expression is increased in the nucleus of the solitary tract (NTS) in Spontaneously Hypertensive Rat (SHR) compared to Wistar Kyoto (WKY) controls. However, the chronic role of AT1Ra in the NTS for cardiovascular control is not well understood. In this study, we investigated the hypothesis that the NTS AT1Ra is involved in neural regulation of the peripheral inflammatory status, and linked with hypertension. Transduction of brain neuronal cultures with AAV2-AT1R-shRNA resulted in a 72% decrease in AT1Ra mRNA, and attenuated AngII-induced increase in ERK1/2 phosphorylation and neuronal firing. Specific NTS microinjection of AAV2-AT1R-shRNA vector in the SHR resulted in a ~30 mmHg increase in the mean arterial pressure (MAP) compared to control vector injected animals (Sc-shRNA: 154±4; AT1R-shRNA: 183±10 mmHg), and induced a resetting of the baroreflex control of heart rate to higher MAP. In addition, AAV2-AT1R-shRNA-treated SHRs exhibited a 74% decrease in circulating endothelial progenitor cells (EPC, CD90+, CD4−/5−/8−), and a 300% increase in the circulating inflammatory cells (IC) including CD4+/CD8+, CD45+/3+ T lymphocytes, and macrophages (CD68+). As a result, the EPC/IC ratio was decreased by 8~15 fold in the AT1R-shRNA-treated SHR. However, identical injection of AAV2-AT1R-shRNA into the NTS of WKY had no effect on MAP and ICs. These observations suggest that increased expression of the AT1Ra in SHR NTS may present a counter-hypertensive mechanism involving inflammatory/angiogenic cells.

Cardiovascular effects of angiotensin A: a novel peptide of the renin-angiotensin system.

Introduction: Angiotensin (Ang) A was first identified in human plasma and it differs from Ang II in Ala1 instead of Asp1. Here, we hypothesized that the actions of this peptide might explain, at least partially, the limited effects of AT1R antagonists in certain cardiovascular diseases. Materials and methods: The effects of Ang A and Ang II on blood pressure (BP) and heart function were compared. Importantly, participation of AT1R in these effects was evaluated. Furthermore, the effects of these two peptides on ischemia/reperfusion arrhythmias and involvement of calcium in these effects were investigated. Results: Administration of increasing doses of these peptides caused elevations in BP at comparable magnitude. AT1R blockade completely abolished these effects. The actions of these peptides in cardiac function were quite similar although the effects of Ang A were only partially blocked by losartan. Interestingly, Ang II elicited an increase in the duration of ischemia/reperfusion arrhythmias while Ang A had no effect on cardiac rhythm during reperfusion. In accordance, differently to Ang II, Ang A did not induce any significant effect on calcium transient during baseline and ischemic stress conditions. Conclusions: These data suggest that the existence of alternative peptides of the renin–angiotensin system (RAS) might contribute to the limited effects of angiotensin receptor blockers (ARBs) in certain pathophysiological circumstances.

Chronic Knockdown of the Nucleus of the Solitary Tract AT1 Receptors Increases Blood Inflammatory-Endothelial Progenitor Cell Ratio and Exacerbates Hypertension in the Spontaneously Hypertensive RatNovelty and Significance

AT1 receptor subtype a (AT1Ra) expression is increased in the nucleus of the solitary tract (NTS) in spontaneously hypertensive rat (SHR) compared with Wistar Kyoto controls. However, the chronic role of AT1Ra in the NTS for cardiovascular control is not well understood. In this study, we investigated the hypothesis that the NTS AT1Ra is involved in the neural regulation of the peripheral inflammatory status and linked with hypertension. Transduction of brain neuronal cultures with recombinant adeno-associated virus type 2 (AAV2)-AT1R-small hairpin RNA (shRNA) resulted in a 72% decrease in AT1Ra mRNA and attenuated angiotensin II–induced increase in extracellular signal–regulated kinase 1/2 phosphorylation and neuronal firing. Specific NTS microinjection of AAV2-AT1R-shRNA vector in the SHR resulted in a ≈30 mm Hg increase in the mean arterial pressure compared with control vector–injected animals (Sc-shRNA: 154±4 mm Hg; AT1R-shRNA: 183±10 mm Hg) and induced a resetting of the baroreflex control of heart rate to higher mean arterial pressure. In addition, AAV2-AT1R-shRNA–treated SHRs exhibited a 74% decrease in circulating endothelial progenitor cells (CD90+, CD4−/CD5−/CD8−) and a 300% increase in the circulating inflammatory cells, including CD4+ +CD8+, CD45+/3+ T lymphocytes, and macrophages (CD68+). As a result, the endothelial progenitor cell/inflammatory cells ratio was decreased by 8- to 15-fold in the AT1R-shRNA–treated SHR. However, identical injection of AAV2-AT1R-shRNA into the NTS of Wistar Kyoto rats had no effect on mean arterial pressure and inflammatory cells. These observations suggest that increased expression of the AT1Ra in SHR NTS may present a counterhypertensive mechanism involving inflammatory/angiogenic cells.AT1 receptor subtype a (AT1R1a) expression is increased in the nucleus of the solitary tract (NTS) in Spontaneously Hypertensive Rat (SHR) compared to Wistar Kyoto (WKY) controls. However, the chronic role of AT1Ra in the NTS for cardiovascular control is not well understood. In this study, we investigated the hypothesis that the NTS AT1Ra is involved in neural regulation of the peripheral inflammatory status, and linked with hypertension. Transduction of brain neuronal cultures with AAV2-AT1R-shRNA resulted in a 72% decrease in AT1Ra mRNA, and attenuated AngII-induced increase in ERK1/2 phosphorylation and neuronal firing. Specific NTS microinjection of AAV2-AT1R-shRNA vector in the SHR resulted in a ~30 mmHg increase in the mean arterial pressure (MAP) compared to control vector injected animals (Sc-shRNA: 154±4; AT1R-shRNA: 183±10 mmHg), and induced a resetting of the baroreflex control of heart rate to higher MAP. In addition, AAV2-AT1R-shRNA-treated SHRs exhibited a 74% decrease in circulating endothelial progenitor cells (EPC, CD90+, CD4−/5−/8−), and a 300% increase in the circulating inflammatory cells (IC) including CD4+/CD8+, CD45+/3+ T lymphocytes, and macrophages (CD68+). As a result, the EPC/IC ratio was decreased by 8~15 fold in the AT1R-shRNA-treated SHR. However, identical injection of AAV2-AT1R-shRNA into the NTS of WKY had no effect on MAP and ICs. These observations suggest that increased expression of the AT1Ra in SHR NTS may present a counter-hypertensive mechanism involving inflammatory/angiogenic cells.

Chronic Knockdown of the NTS AT1R Increases Blood Inflammatory-Endothelial Progenitor Cells Ratio and Exacerbates Hypertension in the SHR

AT1 receptor subtype a (AT1Ra) expression is increased in the nucleus of the solitary tract (NTS) in spontaneously hypertensive rat (SHR) compared with Wistar Kyoto controls. However, the chronic role of AT1Ra in the NTS for cardiovascular control is not well understood. In this study, we investigated the hypothesis that the NTS AT1Ra is involved in the neural regulation of the peripheral inflammatory status and linked with hypertension. Transduction of brain neuronal cultures with recombinant adeno-associated virus type 2 (AAV2)-AT1R-small hairpin RNA (shRNA) resulted in a 72% decrease in AT1Ra mRNA and attenuated angiotensin II–induced increase in extracellular signal–regulated kinase 1/2 phosphorylation and neuronal firing. Specific NTS microinjection of AAV2-AT1R-shRNA vector in the SHR resulted in a ≈30 mm Hg increase in the mean arterial pressure compared with control vector–injected animals (Sc-shRNA: 154±4 mm Hg; AT1R-shRNA: 183±10 mm Hg) and induced a resetting of the baroreflex control of heart rate to higher mean arterial pressure. In addition, AAV2-AT1R-shRNA–treated SHRs exhibited a 74% decrease in circulating endothelial progenitor cells (CD90+, CD4−/CD5−/CD8−) and a 300% increase in the circulating inflammatory cells, including CD4+ +CD8+, CD45+/3+ T lymphocytes, and macrophages (CD68+). As a result, the endothelial progenitor cell/inflammatory cells ratio was decreased by 8- to 15-fold in the AT1R-shRNA–treated SHR. However, identical injection of AAV2-AT1R-shRNA into the NTS of Wistar Kyoto rats had no effect on mean arterial pressure and inflammatory cells. These observations suggest that increased expression of the AT1Ra in SHR NTS may present a counterhypertensive mechanism involving inflammatory/angiogenic cells.AT1 receptor subtype a (AT1R1a) expression is increased in the nucleus of the solitary tract (NTS) in Spontaneously Hypertensive Rat (SHR) compared to Wistar Kyoto (WKY) controls. However, the chronic role of AT1Ra in the NTS for cardiovascular control is not well understood. In this study, we investigated the hypothesis that the NTS AT1Ra is involved in neural regulation of the peripheral inflammatory status, and linked with hypertension. Transduction of brain neuronal cultures with AAV2-AT1R-shRNA resulted in a 72% decrease in AT1Ra mRNA, and attenuated AngII-induced increase in ERK1/2 phosphorylation and neuronal firing. Specific NTS microinjection of AAV2-AT1R-shRNA vector in the SHR resulted in a ~30 mmHg increase in the mean arterial pressure (MAP) compared to control vector injected animals (Sc-shRNA: 154±4; AT1R-shRNA: 183±10 mmHg), and induced a resetting of the baroreflex control of heart rate to higher MAP. In addition, AAV2-AT1R-shRNA-treated SHRs exhibited a 74% decrease in circulating endothelial progenitor cells (EPC, CD90+, CD4−/5−/8−), and a 300% increase in the circulating inflammatory cells (IC) including CD4+/CD8+, CD45+/3+ T lymphocytes, and macrophages (CD68+). As a result, the EPC/IC ratio was decreased by 8~15 fold in the AT1R-shRNA-treated SHR. However, identical injection of AAV2-AT1R-shRNA into the NTS of WKY had no effect on MAP and ICs. These observations suggest that increased expression of the AT1Ra in SHR NTS may present a counter-hypertensive mechanism involving inflammatory/angiogenic cells.