Endoscopic Disease Activity and Biologic Therapy Are Independent Predictors of Suboptimal Bowel Preparation in Patients with Inflammatory Bowel Disease Undergoing Colonoscopy.

BACKGROUND AND AIMS: Optimal bowel preparation (BP) is critical for endoscopic assessment of inflammation and dysplasia in patients with inflammatory bowel disease (IBD). Comorbidities and patient-related factors have been associated with suboptimal BP (SOBP) in the general population. We sought to identify disease-specific characteristics that may impact the quality of BP in patients with IBD. METHODS: We conducted a retrospective analysis of adult IBD patients who underwent outpatient colonoscopies between January 2014 and September 2020 at a large academic medical center. Quality of BP was documented using the Boston Bowel Preparation Scale (BBPS) or the Aronchick scale and dichotomized into "suboptimal" (BBPS 0-5 or Aronchick "fair," "poor," unsatisfactory") and "optimal" (BBPS 6-9 or Aronchick "excellent," "good"). IBD-specific and other factors associated with SOBP were evaluated using logistic regression analyses. RESULTS: Among a total of 395 IBD patients [54% males, mean age 40 years, 63% with Crohn's disease (CD), 35% with ulcerative colitis (UC)], 24.8% had SOBP. On multivariable analysis, moderate-to-severe endoscopic disease vs mild or inactive disease was associated with a higher odds of SOBP [adjusted OR 2.7(95% CI 1.52-4.94)], whereas baseline biologic use was associated with a lower odds of SOBP [aOR 0.24(0.09-0.65)] among the overall IBD cohort. Additionally, age > 65 years [aOR 2.99(1.19-7.54)] and single-dose vs split-dose BP [aOR 2.37(1.43-3.95)] were predictors of SOBP. In the subgroup analysis by IBD type, moderate-to-severe endoscopic disease predicted SOBP among both CD and UC cohorts. CONCLUSION: Endoscopic disease activity was predictive of SOBP, and biologic therapy was protective against SOBP among IBD patients.

Renin angiotensin aldosterone system in pulmonary fibrosis: Pathogenesis to therapeutic possibilities.

Pulmonary fibrosis is a devastating lung disease with multifactorial etiology characterized by alveolar injury, fibroblast proliferation and excessive deposition of extracellular matrix proteins, which progressively results in respiratory failure and death. Accumulating evidence from experimental and clinical studies supports a central role of the renin angiotensin aldosterone system (RAAS) in the pathogenesis and progression of idiopathic pulmonary fibrosis. Angiotensin II (Ang II), a key vasoactive peptide of the RAAS mediates pro-inflammatory and pro-fibrotic effects on the lungs, adversely affecting organ function. Recent years have witnessed seminal discoveries in the field of RAAS. Identification of new enzymes, peptides and receptors has led to the development of several novel concepts. Of particular interest is the establishment of a protective axis of the RAAS comprising of Angiotensin converting enzyme 2 (ACE2), Angiotensin-(1-7) [Ang-(1-7)], and the Mas receptor (the ACE2/Ang-(1-7)/Mas axis), and the discovery of a functional role for the Angiotensin type 2 (AT2) receptor. Herein, we will review our current understanding of the role of RAAS in lung fibrogenesis, provide evidence on the anti-fibrotic actions of the newly recognized RAAS components (the ACE2/Ang-(1-7)/Mas axis and AT2 receptor), discuss potential strategies and translational efforts to convert this new knowledge into effective therapeutics for PF.

Hypertension-Linked Pathophysiological Alterations in the Gut.

RATIONALE: Sympathetic nervous system control of inflammation plays a central role in hypertension. The gut receives significant sympathetic innervation, is densely populated with a diverse microbial ecosystem, and contains immune cells that greatly impact overall inflammatory homeostasis. Despite this uniqueness, little is known about the involvement of the gut in hypertension. OBJECTIVE: Test the hypothesis that increased sympathetic drive to the gut is associated with increased gut wall permeability, increased inflammatory status, and microbial dysbiosis and that these gut pathological changes are linked to hypertension. METHODS AND RESULTS: Gut epithelial integrity and wall pathology were examined in spontaneously hypertensive rat and chronic angiotensin II infusion rat models. The increase in blood pressure in spontaneously hypertensive rat was associated with gut pathology that included increased intestinal permeability and decreased tight junction proteins. These changes in gut pathology in hypertension were associated with alterations in microbial communities relevant in blood pressure control. We also observed enhanced gut-neuronal communication in hypertension originating from paraventricular nucleus of the hypothalamus and presenting as increased sympathetic drive to the gut. Finally, angiotensin-converting enzyme inhibition (captopril) normalized blood pressure and was associated with reversal of gut pathology. CONCLUSIONS: A dysfunctional sympathetic-gut communication is associated with gut pathology, dysbiosis, and inflammation and plays a key role in hypertension. Thus, targeting of gut microbiota by innovative probiotics, antibiotics, and fecal transplant, in combination with the current pharmacotherapy, may be a novel strategy for hypertension treatment.

Myeloid-derived Suppressor Cells Are Necessary for Development of Pulmonary Hypertension.

Pulmonary hypertension (PH) complicates the care of patients with chronic lung disease, such as idiopathic pulmonary fibrosis (IPF), resulting in a significant increase in morbidity and mortality. Disease pathogenesis is orchestrated by unidentified myeloid-derived cells. We used murine models of PH and pulmonary fibrosis to study the role of circulating myeloid cells in disease pathogenesis and prevention. We administered clodronate liposomes to bleomycin-treated wild-type mice to induce pulmonary fibrosis and PH with a resulting increase in circulating bone marrow-derived cells. We discovered that a population of C-X-C motif chemokine receptor (CXCR) 2+ myeloid-derived suppressor cells (MDSCs), granulocytic subset (G-MDSC), is associated with severe PH in mice. Pulmonary pressures worsened despite improvement in bleomycin-induced pulmonary fibrosis. PH was attenuated by CXCR2 inhibition, with antagonist SB 225002, through decreasing G-MDSC recruitment to the lung. Molecular and cellular analysis of clinical patient samples confirmed a role for elevated MDSCs in IPF and IPF with PH. These data show that MDSCs play a key role in PH pathogenesis and that G-MDSC trafficking to the lung, through chemokine receptor CXCR2, increases development of PH in multiple murine models. Furthermore, we demonstrate pathology similar to the preclinical models in IPF with lung and blood samples from patients with PH, suggesting a potential role for CXCR2 inhibitor use in this patient population. These findings are significant, as there are currently no approved disease-specific therapies for patients with PH complicating IPF.

Involvement of bone marrow cells and neuroinflammation in hypertension.

RATIONALE: Microglial activation in autonomic brain regions is a hallmark of neuroinflammation in neurogenic hypertension. Despite evidence that an impaired sympathetic nerve activity supplying the bone marrow (BM) increases inflammatory cells and decreases angiogenic cells, little is known about the reciprocal impact of BM-derived inflammatory cells on neuroinflammation in hypertension. OBJECTIVE: To test the hypothesis that proinflammatory BM cells from hypertensive animals contribute to neuroinflammation and hypertension via a brain-BM interaction. METHODS AND RESULTS: After BM ablation in spontaneously hypertensive rats, and reconstitution with normotensive Wistar Kyoto rat BM, the resultant chimeric spontaneously hypertensive rats displayed significant reduction in mean arterial pressure associated with attenuation of both central and peripheral inflammation. In contrast, an elevated mean arterial pressure along with increased central and peripheral inflammation was observed in chimeric Wistar-Kyoto rats reconstituted with spontaneously hypertensive rat BM. Oral treatment with minocycline, an inhibitor of microglial activation, attenuated hypertension in both the spontaneously hypertensive rats and the chronic angiotensin II-infused rats. This was accompanied by decreased sympathetic drive and inflammation. Furthermore, in chronic angiotensin II-infused rats, minocycline prevented extravasation of BM-derived cells to the hypothalamic paraventricular nucleus, presumably via a mechanism of decreased C-C chemokine ligand 2 levels in the cerebrospinal fluid. CONCLUSIONS: The BM contributes to hypertension by increasing peripheral inflammatory cells and their extravasation into the brain. Minocycline is an effective therapy to modify neurogenic components of hypertension. These observations support the hypothesis that BM-derived cells are involved in neuroinflammation, and targeting them may be an innovative strategy for neurogenic resistant hypertension therapy.

High-fat diet-induced glucose dysregulation is independent of changes in islet ACE2 in mice.

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.

Diminazene protects corpus cavernosum against hypercholesterolemia-induced injury.

INTRODUCTION: Angiotensin-converting enzyme 2 (ACE2) is a key enzyme of the renin angiotensin system, which breaks down angiotensin II and forms angiotensin-(1-7). In erectile tissues, it has been documented that angiotensin II contributes to the development of erectile dysfunction (ED), while treatment with angiotensin-(1-7) improves penile erection. However, the expression and function of ACE2 in erectile tissues have never been investigated. AIM: Here, we examined the expression of ACE2 in erectile tissues and its actions against hypercholesterolemia-induced corpus cavernosum (CC) injury. METHODS: Hypercholesterolemic apolipoprotein E knockout (ApoE(-/-) ) mice, a well-known model of ED, were treated with diminazene aceturate (DIZE), an ACE2 activator compound, or vehicle for 3 weeks. Reactive oxygen species (ROS), collagen content, and protein expression of ACE2, neuronal nitric oxide synthase (nNOS), endothelial nitric oxide synthase (eNOS), nicotinamide adenine dinucleotide phosphate-oxidase (NADPH) subunits were evaluated in the penis of DIZE-treated and untreated ApoE(-/-) mice. Functional studies were performed in CC strips. MAIN OUTCOME MEASURES: ACE2 expression and its role in modulating nitric oxide (NO)/ROS production and fibrosis within the CC of hypercholesterolemic mice were the main outcome measures. RESULTS: ACE2 was expressed in smooth muscle and endothelial cells of mouse CC. Interestingly, ACE2 was downregulated in penis of hypercholesterolemic mice with ED, suggesting a protective role of ACE2 on the CC homeostasis. In accordance with that, pharmacological ACE2 activation by DIZE treatment reduced ROS production and NADPH oxidase expression, and elevated nNOS and eNOS expression and NO bioavailability in the penis of ApoE(-/-) mice. Additionally, DIZE decreased collagen content within the CC. These beneficial actions of DIZE on the CC were not accompanied by improvements in atherosclerotic plaque size or serum lipid profile. CONCLUSION: ACE2 is expressed in erectile tissue and its reduction is associated with hypercholesterolemia-induced ED. Additionally, treatment with DIZE improved hypercholesterolemia-induced CC injury, suggesting ACE2 as a potential target for treating ED. .

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.

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.

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.

Moving AACP Curriculum Quality Survey Results from Good to Great.

Although the American Association of Colleges of Pharmacy (AACP) Curriculum Quality Surveys (CQS) are required for programs to distribute and utilize as part of accreditation standards, programs face challenges in survey administration and timing, interpreting data and results, and following up on action plans. Because the CQS surveys are standardized, they can allow for greater comparison among institutions, yet interpretation of the items can vary considerably. Programs have flexibility in determining samples for administration and timing of administration (ie, number of years), but some participants (such as preceptors) can suffer from survey overload if multiple institutions administer in the same year. Determining thresholds for action and providing feedback to stakeholders on improvements made based on data triangulations can be daunting. These are a few of the elements that programs must consider when determining their own approach to the AACP CQS. Thus, the purpose of this Commentary is to describe good practices for using the AACP CQS, discuss challenges associated with the surveys, and recommend how to move the utilization of the surveys from good to great.

Moderate cardiac-selective overexpression of angiotensin II type 2 receptor protects cardiac functions from ischaemic injury.

We hypothesized that moderate cardiac-selective overexpression of the angiotensin II type 2 receptor (AT2R) would protect the myocardium from ischaemic injury after a myocardial infarction (MI) induced by coronary artery ligation. For in vitro studies, adenoviral vector expressing genomic DNA of AT2R and enhanced green fluorescence protein (EGFP) was used to overexpress AT2R in rat neonatal cardiac myocytes. Expression of AT2R, measured by real-time PCR and immunostaining, demonstrated efficient transduction of AT2R in a dose-dependent pattern. The AT2R constitutively induced apoptosis in rat neonatal cardiac myocytes in dose-dependent patterns. For in vivo studies, 4 × 10(10) vector genomes (vg) of recombinant adeno-associated virus serotype 9 (rAAV9)-chicken ß actin promoter-AT2R was injected into the left ventricle of 5-day-old Sprague-Dawley rats. At 6 weeks of age, hearts were harvested and expression of AT2R determined by real-time PCR and Western blotting. Expression was increased onefold over control hearts, and no apoptosis was detected. Two subsequent in vivo studies were performed. In a prevention study, 4 × 10(10) vg of rAAV9-CBA-AT2R was injected into the left ventricle of 5-day-old Sprague-Dawley rats and MI was induced at 6 weeks of age. For a post-treatment study, 4 × 10(10) vg of rAAV9-CBA-AT2R was administrated to the peri-infarcted myocardium area immediately after MI in 6-week-old animals. For both in vivo studies, cardiac functions were assessed using echocardiography and haemodynamic measurements 4 weeks after coronary artery ligation. In the in vivo studies, the rats subjected to MI showed significant decreases in fractional shortening and rate of change of left ventricular pressure, with increased left ventricular end-diastolic pressure and ventricular hypertrophy. For the prevention study, the moderate cardiac-selective overexpression of AT2R attenuated these MI-induced impairments and also caused a decrease in ventricular wall thinning. In the post-treatment study, the overexpression of AT2R partly reversed the MI-induced cardiac dysfunction. Myocardial infarction also induced the upregulation of angiotensin II type 1 receptor, angiotensin-converting enzyme and collagen I mRNA expression, all of which were attenuated by the overexpression of AT2R. It is concluded that moderate cardiac-selective overexpression of AT2R protects heart function from ischaemic injury, which may be mediated, at least in part, through modulation of components of the cardiac renin-angiotensin system and collagen levels in the myocardium.

Unraveling the Differentially Articulated Axes of the Century-Old Renin-Angiotensin-Aldosterone System: Potential Therapeutic Implications.

Among numerous choices in cardiovascular therapies used for the management of hypertension and heart failure, drugs affecting the renin-angiotensin-aldosterone system (RAAS) hold substantial therapeutic roles. Therapies aimed at modifying the RAAS and its overactivation are employed for the management of various insidious disorders. In the pharmacologic perspective, RAAS is one of the frequently manipulated systems for the management of hypertension, heart failure, myocardial infarction, and renal disease. The RAAS pharmacologic interventions principally include the ACE inhibitors, the angiotensin II-AT1 receptor blockers, the mineralocorticoid receptor antagonists, and the direct renin inhibitors. In addition, therapeutic implication of ACE2/angiotensin (1-7)/Mas receptor activation using various ligands is being explored owing to their anti-inflammatory, anti-fibrotic, vasodilatory, and cardiovascular defensive roles. Moreover, being considered as the counter-regulatory arm of AT1 receptor, the potential role of AT2 receptor activation using selective AT2 receptor agonist is currently investigated for its efficacy in pulmonary complications. As an important regulator of fluid volume, blood pressure, and cardiovascular-renal function, the RAAS has been documented as a diversified intricate system with several therapeutic possibilities coupled with their fundamental structural and functional modulatory roles in cardiovascular, renal, and other systems. The RAAS possesses a number of regulatory, deregulatory, and counter-regulatory axes of physiopathologic importance in health and disease. The counter-regulatory arms of the RAAS might play an essential role in mitigating cardiovascular, renal, and pulmonary pathologies. In light of this background, we sought to explore the classical and counter-regulatory axes/arms of the RAAS and their imperative roles in physiologic functions and disease pathogenesis.

Angiotensin-converting enzyme 2 activation protects against hypertension-induced cardiac fibrosis involving extracellular signal-regulated kinases.

Our previous studies have indicated that chronic treatment with 1-[(2-dimethylamino) ethylamino]-4-(hydroxymethyl)-7-[(4-methylphenyl) sulfonyl oxy]-9H-xanthene-9-one (XNT), an angiotensin-converting enzyme 2 (ACE2) activator, reverses hypertension-induced cardiac and renal fibrosis in spontaneously hypertensive rats (SHRs). Furthermore, XNT prevented pulmonary vascular remodelling and right ventricular hypertrophy and fibrosis in a rat model of monocrotaline-induced pulmonary hypertension. The aim of this study was to determine the mechanisms underlying the protective effects of XNT against cardiac fibrosis. Hydroxyproline assay was used to measure cardiac collagen content in control and XNT-treated (200 ng kg(-1) min(-1) for 28 days) SHRs. Cardiac ACE2 activity and protein levels were determined using the fluorogenic peptide assay and Western blot analysis, respectively. Extracellular signal-regulated kinases (ERKs; p44 and p42) and angiotensin II type 1 (AT(1)) receptor levels were quantified by Western blotting. Cardiac ACE2 protein levels were ∼15% lower in SHRs compared with Wistar-Kyoto control animals (ACE2/glyceraldehyde 3-phosphate dehydrogenase ratio: Wistar-Kyoto, 1.00 ± 0.02 versus SHR, 0.87 ± 0.01). However, treatment of SHRs with XNT completely restored the decreased cardiac ACE2 levels. Also, chronic infusion of XNT significantly increased cardiac ACE2 activity in SHRs. This increase in ACE2 activity was associated with decreased cardiac collagen content. Furthermore, the antifibrotic effect of XNT correlated with increased cardiac angiotensin-(1-7) immunostaining, though no change in cardiac AT(1) protein levels was observed. The beneficial effects of XNT were also accompanied by a reduction in ERK phosphorylation (phospho-ERK/total ERK ratio: Wistar-Kyoto, 1.00 ± 0.04; control SHR, 1.46 ± 0.25; treated SHR, 0.86 ± 0.02). Our observations demonstrate that XNT activates cardiac ACE2 and inhibits fibrosis. These effects are associated with increases in angiotensin-(1-7) and inhibition of cardiac ERK signalling.

Lentivirus-mediated overexpression of angiotensin-(1-7) attenuated ischaemia-induced cardiac pathophysiology.

Myocardial infarction (MI) results in cell death, development of interstitial fibrosis, ventricular wall thinning and ultimately, heart failure. Angiotensin-(1-7) [Ang-(1-7)] has been shown to provide cardioprotective effects. We hypothesize that lentivirus-mediated overexpression of Ang-(1-7) would protect the myocardium from ischaemic injury. A single bolus of 3.5 × 10(8) transducing units of lenti-Ang-(1-7) was injected into the left ventricle of 5-day-old male Sprague-Dawley rats. At 6 weeks of age, MI was induced by ligation of the left anterior descending coronary artery. Four weeks after the MI, echocardiography and haemodynamic parameters were measured to assess cardiac function. Postmyocardial infarction, rats showed significant decreases in fractional shortening and dP/dt (rate of rise of left ventricular pressure), increases in left ventricular end-diastolic pressure, and ventricular hypertrophy. Also, considerable upregulation of cardiac angiotensin-converting enzyme (ACE) mRNA was observed in these rats. Lentivirus-mediated cardiac overexpression of Ang-(1-7) not only prevented all these MI-induced impairments but also resulted in decreased myocardial wall thinning and an increased cardiac gene expression of ACE2 and bradykinin B2 receptor (BKR2). Furthermore, in vitro experiments using rat neonatal cardiac myocytes demonstrated protective effects of Ang-(1-7) against hypoxia-induced cell death. This beneficial effect was associated with decreased expression of inflammatory cytokines (tumour necrosis factor-α and interleukin-6) and increased gene expression of ACE2, BKR2 and interleukin-10. Our findings indicate that overexpression of Ang-(1-7) improves cardiac function and attenuates left ventricular remodelling post-MI. The protective effects of Ang-(1-7) appear to be mediated, at least in part, through modulation of the cardiac renin-angiotensin system and cytokine production.

The angiotensin-converting enzyme 2/angiogenesis-(1-7)/Mas axis confers cardiopulmonary protection against lung fibrosis and pulmonary hypertension.

RATIONALE: An activated vasoconstrictive, proliferative, and fibrotic axis of the renin angiotensin system (angiotensin-converting enzyme [ACE]/angiotensin [Ang]II/AngII type 1 receptor) has been implicated in the pathophysiology of pulmonary fibrosis (PF) and pulmonary hypertension (PH). The recent discovery of a counterregulatory axis of the renin angiotensin system composed of ACE2/Ang-(1-7)/Mas has led us to examine the role of this vasoprotective axis on such disorders. OBJECTIVES: We hypothesized that Ang-(1-7) treatment would exert protective effects against PF and PH. METHODS: Lentiviral packaged Ang-(1-7) fusion gene or ACE2 cDNA was intratracheally administered into the lungs of male Sprague Dawley rats. Two weeks after gene transfer, animals received bleomycin (2.5 mg/kg). In a subsequent study, animals were administered monocrotaline (MCT, 50 mg/kg). MEASUREMENTS AND MAIN RESULTS: In the PF study, bleomycin administration resulted in a significant increase in right ventricular systolic pressure, which was associated with the development of right ventricular hypertrophy. The lungs of these animals also exhibited excessive collagen deposition, decreased expression of ACE and ACE2, increased mRNA levels for transforming growth factor ß and other proinflammatory cytokines, and increased protein levels of the AT1R. Overexpression of Ang-(1-7) significantly prevented all the above-mentioned pathophysiological conditions. Similar protective effects were also obtained with ACE2 overexpression. In the PH study, rats injected with MCT developed elevated right ventricular systolic pressure, right ventricular hypertrophy, right ventricular fibrosis, and pulmonary vascular remodeling, all of which were attenuated by Ang-(1-7) overexpression. Blockade of the Mas receptor abolished the beneficial effects of Ang-(1-7) against MCT-induced PH. CONCLUSIONS: Our observations demonstrate a cardiopulmonary protective role for the ACE2/Ang-(1-7)/Mas axis in the treatment of lung disorders.

Selective tropism of the recombinant adeno-associated virus 9 serotype for rat cardiac tissue.

BACKGROUND: Cardiac gene transfer may serve as a novel therapeutic approach for heart disease. Numerous serotypes of recombinant adeno-associated virus (rAAV) have been identified with variable tropisms to cardiac tissue. METHODS: Both in vitro and in vivo experiments were undertaken to compare cardiac tropisms of rAAV-2, 5, 7, 8 and 9. For the in vitro studies, 10(7) vector genome (vg) of rAAV-2, 5, 7, 8 or 9 were used to transduce both rat neonatal cardiac myocytes (RNCM) and fibroblasts (RNCF). For the in vivo studies, 4 x 10(10) vg of rAAV-2, 5, 7, 8 or 9, and 4 x 10(11) vg of rAAV8 or 9 were administered in 5-day-old rats via a relatively non-invasive intracardiac injection. One and two months post-administration, green fluorescent protein (GFP) expression in tissues was visualized and GFP mRNA was quantified by the real-time polymerase chain reaction. RESULTS: At 3 days post-viral transduction, rAAV9 and rAAV2 produced the highest transducing efficiency in RNCM. Only rAAV2 elicited any transduction in the RNCF. The results obtained in vivo indicated that the order for transduction efficiency in the heart was: rAAV9 > rAAV8 > rAAV7 > rAAV2 = rAAV5. The transduction efficiency order in the liver was: rAAV2 > rAAV5 > rAAV7 > rAAV8 > rAAV9. Injection of a higher dose (4 x 10(11) vg) of rAAV9 provided more widespread and highly cardiac-selective GFP expression in the heart than rAAV8. Zero to minimal expression of GFP was found in the lung and kidney for both doses of all rAAV serotypes utilized. CONCLUSIONS: Collectively, the results obtained in the present study suggest that rAAV9 provides the most selective and stable transduction efficiency in cardiac tissue, and this expression was primarily exhibited in cardiac myocytes.

Evidence for angiotensin-converting enzyme 2 as a therapeutic target for the prevention of pulmonary hypertension.

RATIONALE: It has been proposed that an activated renin angiotensin system (RAS) causes an imbalance between the vasoconstrictive and vasodilator mechanisms involving the pulmonary circulation leading to the development of pulmonary hypertension (PH). Recent studies have indicated that angiotensin-converting enzyme 2 (ACE2), a member of the vasoprotective axis of the RAS, plays a regulatory role in lung pathophysiology, including pulmonary fibrosis and acute lung disease. Based on these observations, we propose the hypothesis that activation of endogenous ACE2 can shift the balance from the vasoconstrictive, proliferative axis (ACE-Ang II-AT1R) to the vasoprotective axis [ACE2-Ang-(1-7)-Mas] of the RAS, resulting in the prevention of PH. OBJECTIVES: We have taken advantage of a recently discovered synthetic activator of ACE2, XNT (1-[(2-dimethylamino) ethylamino]-4-(hydroxymethyl)-7-[(4-methylphenyl) sulfonyl oxy]-9H-xanthene-9-one), to study its effects on monocrotaline-induced PH in rats to support this hypothesis. METHODS: The cardiopulmonary effects of XNT were evaluated in monocrotaline-induced PH rat model. MEASUREMENTS AND MAIN RESULTS: A single subcutaneous treatment of monocrotaline in rats resulted in elevated right ventricular systolic pressure, right ventricular hypertrophy, increased pulmonary vessel wall thickness, and interstitial fibrosis. These changes were associated with increases in the mRNA levels of renin, ACE, angiotensinogen, AT1 receptors, and proinflammatory cytokines. All these features of PH were prevented in these monocrotaline-treated rats by chronic treatment with XNT. In addition, XNT caused an increase in the antiinflammatory cytokine, IL-10. CONCLUSIONS: These observations provide conceptual support that activation of ACE2 by a small molecule can be a therapeutically relevant approach for treating and controlling PH.