Complementary Embryonic and Adult Cell Populations Enhance Myocardial Repair in Rat Myocardial Injury Model.

We compared the functional outcome of Isl-1+ cardiac progenitors, CD90+ bone marrow-derived progenitor cells, and the combination of the two in a rat myocardial infarction (MI) model. Isl-1+ cells were isolated from embryonic day 12.5 (E12.5) rat hearts and expanded in vitro. Thy-1+/CD90+ cells were isolated from the bone marrow of adult Sprague-Dawley rats by immunomagnetic cell sorting. Six-week-old female Sprague-Dawley rats underwent permanent left anterior descending (LAD) coronary artery ligation and received intramyocardial injection of either saline, Isl-1+ cells, CD90+ cells, or a combination of Isl-1+ and CD90+ cells, at the time of infarction. Cells were delivered transepicardially to the peri-infarct zone. Left ventricular function was assessed by transthoracic echocardiography at 1- and 4-week post-MI and by Millar catheterization (-dP/dt and +dP/dt) at 4-week post-MI. Fluorescence in situ hybridization (Isl-1+cells) and monochrystalline iron oxide nanoparticles labeling (MION; CD90+ cells) were performed to assess biodistribution of transplanted cells. Only the combination of cells demonstrated a significant improvement of cardiac function as assessed by anterior wall contractility, dP/dt (max), and dP/dt (min), compared to Isl-1+ or CD90+ cell monotherapies. In the combination cell group, viable cells were detected at week 4 when anterior wall motion was completely restored. In conclusion, the combination of Isl-1+ cardiac progenitors and adult bone marrow-derived CD90+ cells shows prolonged and robust myocardial tissue repair and provides support for the use of complementary cell populations to enhance myocardial repair.

Involvement of Neuroinflammation in the Pathogenesis of Monocrotaline-Induced Pulmonary Hypertension.

Pulmonary hypertension (PH) is a devastating disease and its successful treatment remains to be accomplished despite recent advances in pharmacotherapy. It has been proposed that PH be considered as a systemic disease, rather than primarily a disease of the pulmonary vasculature. Consequently, an investigation of the intricate interplay between multiple organs such as brain, vasculature, and lung in PH could lead to the identification of new targets for its therapy. However, little is known about this interplay. This study was undertaken to examine the concept that altered autonomic-pulmonary communication is important in PH pathophysiology. Therefore, we hypothesize that activation of microglial cells in the paraventricular nucleus of hypothalamus and neuroinflammation is associated with increased sympathetic drive and pulmonary pathophysiology contributing to PH. We utilized the monocrotaline rat model for PH and intracerebroventricular administration of minocycline for inhibition of microglial cells activation to investigate this hypothesis. Hemodynamic, echocardiographic, histological, immunohistochemical, and confocal microscopic techniques assessed cardiac and pulmonary function and microglial cells. Monocrotaline treatment caused cardiac and pulmonary pathophysiology associated with PH. There were also increased activated microglial cells and mRNA for proinflammatory cytokines (IL [interleukin]-1ß, IL-6, and TNF [tumor necrosis factor]-α) in the paraventricular nucleus. Furthermore, increased sympathetic drive and plasma norepinephrine were observed in rats with PH. Intracerebroventricular infusion of minocycline inhibited all these parameters and significantly attenuated PH. These observations implicate a dysfunctional autonomic-lung communication in the development and progression of PH providing new therapeutic targets, such as neuroinflammation, for PH therapy.

The Selective Angiotensin II Type 2 Receptor Agonist, Compound 21, Attenuates the Progression of Lung Fibrosis and Pulmonary Hypertension in an Experimental Model of Bleomycin-Induced Lung Injury.

Idiopathic Pulmonary Fibrosis (IPF) is a chronic lung disease characterized by scar formation and respiratory insufficiency, which progressively leads to death. Pulmonary hypertension (PH) is a common complication of IPF that negatively impacts clinical outcomes, and has been classified as Group III PH. Despite scientific advances, the dismal prognosis of IPF and associated PH remains unchanged, necessitating the search for novel therapeutic strategies. Accumulating evidence suggests that stimulation of the angiotensin II type 2 (AT2) receptor confers protection against a host of diseases. In this study, we investigated the therapeutic potential of Compound 21 (C21), a selective AT2 receptor agonist in the bleomycin model of lung injury. A single intra-tracheal administration of bleomycin (2.5 mg/kg) to 8-week old male Sprague Dawley rats resulted in lung fibrosis and PH. Two experimental protocols were followed: C21 was administered (0.03 mg/kg/day, ip) either immediately (prevention protocol, BCP) or after 3 days (treatment protocol, BCT) of bleomycin-instillation. Echocardiography, hemodynamic, and Fulton's index assessments were performed after 2 weeks of bleomycin-instillation. Lung tissue was processed for gene expression, hydroxyproline content (a marker of collagen deposition), and histological analysis. C21 treatment prevented as well as attenuated the progression of lung fibrosis, and accompanying PH. The beneficial effects of C21 were associated with decreased infiltration of macrophages in the lungs, reduced lung inflammation and diminished pulmonary collagen accumulation. Further, C21 treatment also improved pulmonary pressure, reduced muscularization of the pulmonary vessels and normalized cardiac function in both the experimental protocols. However, there were no major differences in any of the outcomes measured from the two experimental protocols. Collectively, our findings indicate that stimulation of the AT2 receptor by C21 attenuates bleomycin-induced lung injury and associated cardiopulmonary pathology, which needs to be further explored as a promising approach for the clinical treatment of IPF and Group III PH.

Coupling corticotropin-releasing-hormone and angiotensin converting enzyme 2 dampens stress responsiveness in male mice.

This study used mice to evaluate whether coupling expression of corticotropin-releasing hormone (CRH) and angiotensin converting enzyme 2 (ACE2) creates central interactions that blunt endocrine and behavioral responses to psychogenic stress. Central administration of diminazene aceturate, an ACE2 activator, had no effect on restraint-induced activation of the hypothalamic-pituitary-adrenal (HPA) axis; however, mice that ubiquitously overexpress ACE2 had reduced plasma corticosterone (CORT) and pituitary expression of POMC mRNA. The Cre-LoxP system was used to restrict ACE2 overexpression to CRH synthesizing cells and probe whether HPA axis suppression was the result of central ACE2 and CRH interactions. Within the paraventricular nucleus of the hypothalamus (PVN), mice with ACE2 overexpression directed to CRH had a ≈2.5 fold increase in ACE2 mRNA, which co-localized with CRH mRNA. Relative to controls, mice overexpressing ACE2 in CRH cells had a decreased CORT response to restraint as well as decreased CRH mRNA in the PVN and CEA and POMC mRNA in the pituitary. Administration of ACTH similarly increased plasma CORT, indicating that the blunted HPA axis activation that accompanies ACE2 overexpression in CRH cells is centrally mediated. Anxiety-like behavior was assessed to determine whether the decreased HPA axis activation was predictive of anxiolysis. Mice with ACE2 overexpression directed to CRH cells displayed decreased anxiety-like behavior in the elevated plus maze and open field when compared to that of controls. Collectively, these results suggest that exogenous ACE2 suppresses CRH synthesis, which alters the central processing of psychogenic stress, thereby blunting HPA axis activation and attenuating anxiety-like behavior.

Beneficial Effects of Angiotensin-(1-7) on CD34+ Cells From Patients With Heart Failure.

The dysfunctional nature of CD34 cells from patients with heart failure (HF) may make them unsuitable for autologous stem-cell therapy. In view of evidence that the vasoprotective axis of the renin-angiotensin system (RAS) improves CD34 cell functions, we hypothesized that CD34 cells from patients with HF will be dysfunctional and that angiotensin-(1-7) [Ang-(1-7)] would improve their function. Peripheral blood was collected from New York Heart Association class II-IV patients with HF (n = 31) and reference subjects (n = 16). CD34 cell numbers from patients with HF were reduced by 47% (P < 0.05) and also displayed 76% reduction in migratory capacity and 56% (P < 0.05) lower production of nitric oxide. These alterations were associated with increases in RAS genes angiotensin-converting enzyme and AT2R (595%, P < 0.05) mRNA levels and 80% and 85% decreases in angiotensin-converting enzyme 2 and Mas mRNA levels, respectively. Treatment with Ang-(1-7) enhanced CD34 cell function through increased migratory potential and nitric oxide production, and reduced reactive oxygen species generation. These data show that HF CD34 cells are dysfunctional, and Ang-(1-7) improves their functions. This suggests that activation of the vasoprotective axis of the RAS may hold therapeutic potential for autologous stem-cell therapy in patients with HF.

Therapeutic potential of adipose stem cell-derived conditioned medium against pulmonary hypertension and lung fibrosis.

BACKGROUND AND PURPOSE: Pulmonary hypertension (PH) and pulmonary fibrosis (PF) are life threatening cardiopulmonary diseases. Existing pharmacological interventions have failed to improve clinical outcomes or reduce disease-associated mortality. Emerging evidence suggests that stem cells offer an effective treatment approach against various pathological conditions. It has been proposed that their beneficial actions may be mediated via secretion of paracrine factors. Herein, we evaluated the therapeutic potential of conditioned media (CM) from adipose stem cells (ASCs) against experimental models of PH and PF. EXPERIMENTAL APPROACH: Monocrotaline (MCT) or bleomycin (Bleo) was injected into male Sprague-Dawley rats to induce PH or PF respectively. A subset of MCT and Bleo animals were treated with ASCs or CM. Echocardiographic and haemodynamic measurements were performed at the end of the study. Lung and heart tissues were harvested for RNA, protein and histological measurements. KEY RESULTS: CM treatment attenuated MCT-induced PH by improving pulmonary blood flow and inhibiting cardiac remodelling. Further, histological studies revealed that right ventricular fibrosis, pulmonary vessel wall thickness and pericyte distribution were significantly decreased by CM administration. Likewise, CM therapy arrested the progression of PF in the Bleo model by reducing collagen deposition. Elevated expression of markers associated with tissue remodelling and inflammation were significantly reduced in both PF and PH lungs. Similar results were obtained with ASCs administration. CONCLUSIONS AND IMPLICATIONS: Our study indicates that CM treatment is as effective as ASCs in treating PH and PF. These beneficial effects of CM may provide an innovative approach to treat cardiopulmonary disorders.

Increasing brain angiotensin converting enzyme 2 activity decreases anxiety-like behavior in male mice by activating central Mas receptors.

Over-activation of the brain renin-angiotensin system (RAS) has been implicated in the etiology of anxiety disorders. Angiotensin converting enzyme 2 (ACE2) inhibits RAS activity by converting angiotensin-II, the effector peptide of RAS, to angiotensin-(1-7), which activates the Mas receptor (MasR). Whether increasing brain ACE2 activity reduces anxiety by stimulating central MasR is unknown. To test the hypothesis that increasing brain ACE2 activity reduces anxiety-like behavior via central MasR stimulation, we generated male mice overexpressing ACE2 (ACE2 KI mice) and wild type littermate controls (WT). ACE2 KI mice explored the open arms of the elevated plus maze (EPM) significantly more than WT, suggesting increasing ACE2 activity is anxiolytic. Central delivery of diminazene aceturate, an ACE2 activator, to C57BL/6 mice also reduced anxiety-like behavior in the EPM, but centrally administering ACE2 KI mice A-779, a MasR antagonist, abolished their anxiolytic phenotype, suggesting that ACE2 reduces anxiety-like behavior by activating central MasR. To identify the brain circuits mediating these effects, we measured Fos, a marker of neuronal activation, subsequent to EPM exposure and found that ACE2 KI mice had decreased Fos in the bed nucleus of stria terminalis but had increased Fos in the basolateral amygdala (BLA). Within the BLA, we determined that ∼62% of GABAergic neurons contained MasR mRNA and expression of MasR mRNA was upregulated by ACE2 overexpression, suggesting that ACE2 may influence GABA neurotransmission within the BLA via MasR activation. Indeed, ACE2 overexpression was associated with increased frequency of spontaneous inhibitory postsynaptic currents (indicative of presynaptic release of GABA) onto BLA pyramidal neurons and central infusion of A-779 eliminated this effect. Collectively, these results suggest that ACE2 may reduce anxiety-like behavior by activating central MasR that facilitate GABA release onto pyramidal neurons within the BLA.

Angiotensin-(1-7) Decreases Cell Growth and Angiogenesis of Human Nasopharyngeal Carcinoma Xenografts.

Angiotensin-(1-7) [Ang-(1-7)] is an endogenous, heptapeptide hormone acting through the Mas receptor (MasR), with antiproliferative and antiangiogenic properties. Recent studies have shown that Ang-(1-7) has an antiproliferative action on lung adenocarcinoma cells and prostate cancer cells. In this study, we report that MasR levels were significantly upregulated in nasopharyngeal carcinoma (NPC) specimens and NPC cell lines. Viral vector-mediated expression of Ang-(1-7) dramatically suppressed NPC cell proliferation and migration in vitro. These effects were completely blocked by the specific Ang-(1-7) receptor antagonist A-779, suggesting that they are mediated by the Ang-(1-7) receptor Mas. In this study, Ang-(1-7) not only caused a significant reduction in the growth of human nasopharyngeal xenografts, but also markedly decreased vessel density, suggesting that the heptapeptide inhibits angiogenesis to reduce tumor size. Mechanistic investigations revealed that Ang-(1-7) inhibited the expression of the proangiogenic factors VEGF and PlGF. Taken together, the data suggest that upregulation of MasR could be used as a diagnostic marker of NPC and Ang-(1-7) may be a novel therapeutic agent for nasopharyngeal cancer therapy because it exerts significant antiangiogenic activity.

Angiotensin-converting enzyme 2 inhibits high-mobility group box 1 and attenuates cardiac dysfunction post-myocardial ischemia.

High-mobility group box 1 (HMGB1) triggers and amplifies inflammation cascade following ischemic injury, and its elevated levels are associated with adverse clinical outcomes in patients with myocardial infarction (MI). Angiotensin-converting enzyme 2 (ACE2), a key member of vasoprotective axis of the renin-angiotensin system (RAS), regulates cardiovascular functions and exerts beneficial effects in cardiovascular disease. However, the association between HMGB1 and ACE2 has not been studied. We hypothesized that overexpression of ACE2 provides cardioprotective effects against MI via inhibiting HMGB1 and inflammation. ACE2 knock-in (KI) mice and littermate wild-type (WT) controls were subjected to either sham or coronary artery ligation surgery to induce MI. Heart function was assessed 4 weeks after surgery using echocardiography and Millar catheterization. Tissues were collected for histology and analysis of the expression of HMGB1, RAS components, and inflammatory cytokines. ACE2 in the heart of the ACE2 KI mice was 58-fold higher than WT controls. ACE2-MI mice exhibited a remarkable preservation of cardiac function and reduction of infarct size in comparison to WT-MI mice. Notably, ACE2 overexpression significantly reduced the MI-induced increase in apoptosis, macrophage infiltration, and HMGB1 and proinflammatory cytokine expression (TNF-α and IL-6). Moreover, in an in vitro study, ACE2 activation prevented the hypoxia-induced cell death and upregulation of HMGB1 in adult cardiomyocytes. This protective effect is correlated with downregulation of HMGB1 and downstream proinflammatory cascades, which could be useful for the development of novel treatment for ischemic heart disease.

ACE2 and Microbiota: Emerging Targets for Cardiopulmonary Disease Therapy.

The health of the cardiovascular and pulmonary systems is inextricably linked to the renin-angiotensin system (RAS). Physiologically speaking, a balance between the vasodeleterious (Angiotensin-converting enzyme [ACE]/Angiotensin II [Ang II]/Ang II type 1 receptor [AT1R]) and vasoprotective (Angiotensin-converting enzyme 2 [ACE2]/Angiotensin-(1-7) [Ang-(1-7)]/Mas receptor [MasR]) components of the RAS is critical for cardiopulmonary homeostasis. Upregulation of the ACE/Ang II/AT1R axis shifts the system toward vasoconstriction, proliferation, hypertrophy, inflammation, and fibrosis, all factors that contribute to the development and progression of cardiopulmonary diseases. Conversely, stimulation of the vasoprotective ACE2/Ang-(1-7)/MasR axis produces a counter-regulatory response that promotes cardiovascular health. Current research is investigating novel strategies to augment actions of the vasoprotective RAS components, particularly ACE2, in order to treat various pathologies. Although multiple approaches to increase the activity of ACE2 have displayed beneficial effects against experimental disease models, the mechanisms behind its protective actions remain incompletely understood. Recent work demonstrating a non-catalytic role for ACE2 in amino acid transport in the gut has led us to speculate that the therapeutic effects of ACE2 can be mediated, in part, by its actions on the gastrointestinal tract and/or gut microbiome. This is consistent with emerging data which suggest that dysbiosis of the gut and lung microbiomes is associated with cardiopulmonary disease. This review highlights new developments in the protective actions of ACE2 against cardiopulmonary disorders, discusses innovative approaches to targeting ACE2 for therapy, and explores an evolving role for gut and lung microbiota in cardiopulmonary health.

Oral delivery of Angiotensin-converting enzyme 2 and Angiotensin-(1-7) bioencapsulated in plant cells attenuates pulmonary hypertension.

Emerging evidences indicate that diminished activity of the vasoprotective axis of the renin-angiotensin system, constituting angiotensin-converting enzyme 2 (ACE2) and its enzymatic product, angiotensin-(1-7) [Ang-(1-7)] contribute to the pathogenesis of pulmonary hypertension (PH). However, long-term repetitive delivery of ACE2 or Ang-(1-7) would require enhanced protein stability and ease of administration to improve patient compliance. Chloroplast expression of therapeutic proteins enables their bioencapsulation within plant cells to protect against gastric enzymatic degradation and facilitates long-term storage at room temperature. Besides, fusion to a transmucosal carrier helps effective systemic absorption from the intestine on oral delivery. We hypothesized that bioencapsulating ACE2 or Ang-(1-7) fused to the cholera nontoxin B subunit would enable development of an oral delivery system that is effective in treating PH. PH was induced in male Sprague Dawley rats by monocrotaline administration. Subset of animals was simultaneously treated with bioencapsulaed ACE2 or Ang-(1-7) (prevention protocol). In a separate set of experiments, drug treatment was initiated after 2 weeks of PH induction (reversal protocol). Oral feeding of rats with bioencapsulated ACE2 or Ang-(1-7) prevented the development of monocrotaline-induced PH and improved associated cardiopulmonary pathophysiology. Furthermore, in the reversal protocol, oral ACE2 or Ang-(1-7) treatment significantly arrested disease progression, along with improvement in right heart function, and decrease in pulmonary vessel wall thickness. In addition, a combination therapy with ACE2 and Ang-(1-7) augmented the beneficial effects against monocrotaline-induced lung injury. Our study provides proof-of-concept for a novel low-cost oral ACE2 or Ang-(1-7) delivery system using transplastomic technology for pulmonary disease therapeutics.

Neuroinflammation in pulmonary hypertension: concept, facts, and relevance.

Pulmonary hypertension (PH) is a progressive lung disease characterized by elevated pressure in the lung vasculature, resulting in right-sided heart failure and premature death. The pathogenesis of PH is complex and multifactorial, involving a dysregulated autonomic nervous system and immune response. Inflammatory mechanisms have been linked to the development and progression of PH; however, these are usually restricted to systemic and/or local lung tissue. Inflammation within the CNS, often referred to as neuroinflammation involves activation of the microglia, the innate immune cells that are found specifically in the brain and spinal cord. Microglial activation results in the release of several cytokines and chemokines that trigger neuroinflammation, and has been implicated in the pathogenesis of several disease conditions such as Alzheimer's, Parkinson's, hypertension, atherosclerosis, and metabolic disorders. In this review, we introduce the concept of neuroinflammation in the context of PH, and discuss possible strategies that could be developed for PH therapy based on this concept.

The angiotensin type 2 receptor agonist Compound 21 elicits cerebroprotection in endothelin-1 induced ischemic stroke.

Evidence indicates that angiotensin II type 2 receptors (AT2R) exert cerebroprotective actions during stroke. A selective non-peptide AT2R agonist, Compound 21 (C21), has been shown to exert beneficial effects in models of cardiac and renal disease, as well as hemorrhagic stroke. Here, we hypothesize that C21 may exert beneficial effects against cerebral damage and neurological deficits produced by ischemic stroke. We determined the effects of central and peripheral administration of C21 on the cerebral damage and neurological deficits in rats elicited by endothelin-1 induced middle cerebral artery occlusion (MCAO), a model of cerebral ischemia. Rats infused centrally (intracerebroventricular) with C21 before endothelin-1 induced MCAO exhibited significant reductions in cerebral infarct size and the neurological deficits produced by cerebral ischemia. Similar cerebroprotection was obtained in rats injected systemically (intraperitoneal) with C21 either before or after endothelin-1 induced MCAO. The protective effects of C21 were reversed by central administration of an AT2R inhibitor, PD123319. While C21 did not alter cerebral blood flow at the doses used here, peripheral post-stroke administration of this agent significantly attenuated the MCAO-induced increases in inducible nitric oxide synthase, chemokine (C-C) motif ligand 2 and C-C chemokine receptor type 2 mRNAs in the cerebral cortex, indicating that the cerebroprotective action is associated with an anti-inflammatory effect. These results strengthen the view that AT2R agonists may have potential therapeutic value in ischemic stroke, and provide the first evidence of cerebroprotection induced by systemic post stroke administration of a selective AT2R agonist.

Diminazene aceturate enhances angiotensin-converting enzyme 2 activity and attenuates ischemia-induced cardiac pathophysiology.

Angiotensin-converting enzyme 2 (ACE2) plays a critical role against myocardial infarction (MI). We hypothesized that activation of intrinsic ACE2 would be protective against ischemia-induced cardiac pathophysiology. Diminazene aceturate (DIZE), a small molecule ACE2 activator, has been used to evaluate this hypothesis. DIZE (15 mg/kg per day, s.c.) was injected 2 days before MI surgery and continued throughout the study period. MI rats showed a 62% decrease in fractional shortening (%; control, 51.1±3.2; DIZE alone, 52.1±3.2; MI, 19.1±3.0), a 55% decrease in contractility (dP/dtmax mm Hg/s; control, 9480±425.3; DIZE alone, 9585±597.4; MI, 4251±657.7), and a 27% increase in ventricular hypertrophy (mg/mm; control, 26.5±1.5; DIZE alone, 26.9±1.4; MI, 33.4±1.1). DIZE attenuated the MI-induced decrease in fractional shortening by 89%, improved dP/dtmax by 92%, and reversed ventricular hypertrophy by 18%. MI also significantly increased ACE and angiotensin type 1 receptor levels but decreased ACE2 activity by 40% (control, 246.2±25.1; DIZE alone, 254.2±20.6; MI, 148.9±29.2; RFU/min), which was reversed by DIZE treatment. Thus, DIZE treatment decreased the infarct area, attenuated LV remodeling post-MI, and restored normal balance of the cardiac renin-angiotensin system. In addition, DIZE treatment increased circulating endothelial progenitor cells, increased engraftment of cardiac progenitor cells, and decreased inflammatory cells in peri-infarct cardiac regions. All of the beneficial effects associated with DIZE treatment were abolished by C-16, an ACE2 inhibitor. Collectively, DIZE and DIZE-like small molecules may represent promising new therapeutic agents for MI.

Diminazene aceturate improves autonomic modulation in pulmonary hypertension.

We have previously demonstrated that diminazene aceturate (DIZE), a putative angiotensin 1-7 converting enzyme activator, protects rats from monocrotaline (MCT)-induced pulmonary hypertension (PH). The present study was conducted to determine if the beneficial effects of DIZE are associated with improvements in autonomic nervous system (ANS) modulation. PH was induced in male rats by a single subcutaneous injection of MCT (50 mg/kg). A subset of MCT rats were treated with DIZE (15 mg/kg/day) for a period of 21 days, after which the ANS modulation was evaluated by spectral and symbolic analysis of heart rate variability (HRV). MCT administration resulted in a significant (P<0.001) increase in the right ventricular systolic pressure (62 ± 14 mmHg) when compared with other experimental groups (Control: 26 ± 6; MCT + DIZE: 31 ± 7 mmHg), while DIZE treatment was able to decrease this pressure. Furthermore MCT-treated rats had significantly reduced total power of HRV than the controls. On the other hand, although not significant, a trend towards increased HRV was observed in the MCT + DIZE group (Control: 108 ± 47; MCT: 12 ± 8.86 and MCT + DIZE: 40 ± 14), suggesting an improvement of the cardiac autonomic modulation. This observation was further confirmed by the low-frequency/high-frequency index of spectral analysis (Control: 0.74 ± 0.62; MCT: 1.45 ± 0.78 and MCT + DIZE: 0.34 ± 0.49) which showed that DIZE treatment was able to recover the ANS imbalance observed in the MCT-induced pulmonary hypertensive rats. Collectively, our results demonstrate that MCT-induced PH is associated with a significant increase in sympathetic modulation and a decrease in HRV, which are markedly improved by DIZE treatment.

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.

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.

Diminazene attenuates pulmonary hypertension and improves angiogenic progenitor cell functions in experimental models.

RATIONALE: Studies have demonstrated that angiotensin-converting enzyme 2 (ACE2) plays a protective role against lung diseases, including pulmonary hypertension (PH). Recently, an antitrypanosomal drug, diminazene aceturate (DIZE), was shown to exert an "off-target" effect of enhancing the enzymatic activity of ACE2 in vitro. OBJECTIVES: To evaluate the pharmacological actions of DIZE in experimental models of PH. METHODS: PH was induced in male Sprague Dawley rats by monocrotaline, hypoxia, or bleomycin challenge. Subsets of animals were simultaneously treated with DIZE. In a separate set of experiments, DIZE was administered after 3 weeks of PH induction to determine whether the drug could reverse PH. MEASUREMENTS AND MAIN RESULTS: DIZE treatment significantly prevented the development of PH in all of the animal models studied. The protective effects were associated with an increase in the vasoprotective axis of the lung renin-angiotensin system, decreased inflammatory cytokines, improved pulmonary vasoreactivity, and enhanced cardiac function. These beneficial effects were abolished by C-16, an ACE2 inhibitor. Initiation of DIZE treatment after the induction of PH arrested disease progression. Endothelial dysfunction represents a hallmark of PH pathophysiology, and growing evidence suggests that bone marrow-derived angiogenic progenitor cells contribute to endothelial homeostasis. We observed that angiogenic progenitor cells derived from the bone marrow of monocrotaline-challenged rats were dysfunctional and were repaired by DIZE treatment. Likewise, angiogenic progenitor cells isolated from patients with PH exhibited diminished migratory capacity toward the key chemoattractant stromal-derived factor 1α, which was corrected by in vitro DIZE treatment. CONCLUSIONS: Our results identify a therapeutic potential of DIZE in PH therapy.

Comparison of the transduction efficiency of tyrosine-mutant adeno-associated virus serotype vectors in kidney.

Gene therapy has a distinct potential to treat kidney diseases. However, the efficient transduction of a significant number of renal cells by viral vectors has been difficult to accomplish. Previous studies indicate that adeno-associated virus (AAV) can transduce renal cells with variable and suboptimal efficiency. Because new and innovative mutants of AAV are now available, we compared their efficacy in transducing rat kidneys. We compared five types of AAV mutants (AAV2 mut-triple, AAV2 sextuple, AAV8 mut447, AAV8 mut733 and AAV9 mut446) carrying a green fluorescence protein (GFP) reporter gene. A pressure microinjection technique was used to inject either 1.5 × 10(11) vector genome (vg) AAV mutants or three dose of AAV2 sextuple into the renal cortex of rats. The microinjection approach has not been used in AAV-mediated renal gene transfer thus far. Slow and sustained microinjection enables continuous administration of the viral vector to the kidney cortex and limits any damage to the kidney, because the tip of a glass micropipette is very small. Three weeks after injection, the kidneys were collected and evaluated for GFP expression. Among the various mutated AAV serotypes studied, only AAV2 sextuple showed robust GFP expression in renal tissue. The AAV2 sextuple serotype appears to be an efficient gene transfer vector to preferentially target renal tubular epithelial cells. A combination of the AAV2 sextuple and the microinjection technique holds the key to the future of therapeutic treatments for kidney diseases.