Reversal of Right Ventricular Hypertrophy and Dysfunction by Prostacyclin in a Rat Model of Severe Pulmonary Arterial Hypertension.

Prostacyclin analogs are among the most effective and widely used therapies for pulmonary arterial hypertension (PAH). However, it is unknown whether they also confer protection through right ventricle (RV) myocardio-specific mechanisms. Moreover, the use of prostacyclin analogs in severe models of PAH has not been adequately tested. To further identify underlying responses to prostacyclin, a prostacyclin analogue, treprostinil, was used in a preclinical rat Sugen-chronic hypoxia (SuCH) model of severe PAH that closely resembles the human disease. Male Sprague-Dawley rats were implanted with osmotic pumps containing vehicle or treprostinil, injected concurrently with a bolus of Sugen (SU5416) and exposed to 3-week hypoxia followed by 3-week normoxia. RV function was assessed using pressure-volume loops and hypertrophy by weight assessed. To identify altered mechanisms within the RV, tissue samples were used to perform a custom RNA array analysis, histological staining, and protein and transcript level confirmatory analyses. Treprostinil significantly reduced SuCH-associated RV hypertrophy and decreased the rise in RV systolic pressure, mean pulmonary arterial (mPAP), and right atrial (RAP) pressure. Prostacyclin treatment was associated with improvements in RV stroke work, maximum rate of ventricular pressure change (max dP/dt) and the contractile index, and almost a complete reversal of SuCH-associated increase in RV end-systolic elastance, suggesting the involvement of load-independent improvements in intrinsic RV systolic contractility by prostacyclin treatment. An analysis of the RV tissues showed no changes in cardiac mitochondrial respiration and ATP generation. However, custom RNA array analysis revealed amelioration of SuCH-associated increases in newly identified TBX20 as well as the fibrotic markers collagen1α1 and collagen 3α1 upon treprostinil treatment. Taken together, our data support decreased afterload and load-independent improvements in RV function following prostacyclin administration in severe PAH, and these changes appear to associate with improvements in RV fibrotic responses.

DPP4 Inhibition, NPY1-36, PYY1-36, SDF-1α, and a Hypertensive Genetic Background Conspire to Augment Cell Proliferation and Collagen Production: Effects That Are Abolished by Low Concentrations of 2-Methoxyestradiol.

By reducing their metabolism, dipeptidyl peptidase 4 inhibition (DPP4I) enhances the effects of numerous peptides including neuropeptide Y1-36 (NPY1-36), peptide YY1-36 (PYY1-36), and SDF-1α Studies show that separately NPY1-36, PYY1-36 and SDF-1α stimulate proliferation of, and collagen production by, cardiac fibroblasts (CFs), preglomerular vascular smooth muscle cells (PGVSMCs), and glomerular mesangial cells (GMCs), particularly in cells isolated from genetically hypertensive rats. Whether certain combinations of these factors, in the absence or presence of DPP4I, are more profibrotic than others is unknown. Here we contrasted 24 different combinations of conditions (DPP4I, hypertensive genotype and physiologic levels [3 nM] of NPY1-36, PYY1-36, or SDF-1α) on proliferation of, and [3H]-proline incorporation by, CFs, PGVSMCs, and GMCs. In all three cell types, the various treatment conditions differentially increased proliferation and [3H]-proline incorporation, with a hypertensive genotype + DPP4I + NPY1-36 + SDF-1α being the most efficacious combination. Although the effects of this four-way combination were similar in male versus female CFs, physiologic (1 nM) concentrations of 2-methoxyestradiol (2ME; nonestrogenic metabolite of 17ß-estradiol), abolished the effects of this combination in both male and female CFs. In conclusion, this study demonstrates that CFs, PGVSMCs, and GMCs are differentially activated by various combinations of NPY1-36, PYY1-36, SDF-1α, a hypertensive genetic background and DPP4I. We hypothesize that as these progrowth conditions accumulate, a tipping point would be reached that manifests in the long term as organ fibrosis and that 2ME would obviate any profibrotic effects of DPP4I, even under the most profibrotic conditions (i.e., hypertensive genotype with high NPY1-36 + SDF-1α levels and low 2ME levels). SIGNIFICANCE STATEMENT: This work elucidates combinations of factors that could contribute to long-term profibrotic effects of dipeptidyl peptidase 4 inhibitors and suggests a novel drug combination that could prevent any potential profibrotic effects of dipeptidyl peptidase 4 inhibitors while augmenting the protective effects of this class of antidiabetic agents.

Complex III Inhibition-Induced Pulmonary Hypertension Affects the Mitochondrial Proteomic Landscape.

The mitochondria play a vital role in controlling cell metabolism and regulating crucial cellular outcomes. We previously demonstrated that chronic inhibition of the mitochondrial complex III in rats by Antimycin A (AA) induced sustained pulmonary vasoconstriction. On the metabolic level, AA-induced mitochondrial dysfunction resulted in a glycolytic shift that was reported as the primary contributor to pulmonary hypertension pathogenesis. However, the regulatory proteins driving this metabolic shift with complex III inhibition are yet to be explored. Therefore, to delineate the mechanisms, we followed changes in the rat lung mitochondrial proteome throughout AA treatment. Rats treated with AA for up to 24 days showed a disturbed mitochondrial proteome with significant changes in 28 proteins (p < 0.05). We observed a time-dependent decrease in the expression of key proteins that regulate fatty acid oxidation, the tricarboxylic acid cycle, the electron transport chain, and amino acid metabolism, indicating a correlation with diminished mitochondrial function. We also found a significant dysregulation in proteins that controls the protein import machinery and the clearance and detoxification of oxidatively damaged peptides via proteolysis and mitophagy. This could potentially lead to the onset of mitochondrial toxicity due to misfolded protein stress. We propose that chronic inhibition of mitochondrial complex III attenuates mitochondrial function by disruption of the global mitochondrial metabolism. This potentially aggravates cellular proliferation by initiating a glycolytic switch and thereby leads to pulmonary hypertension.

2-Methoxyestradiol Attenuates Angiotensin II-Induced Hypertension, Cardiovascular Remodeling, and Renal Injury.

Estradiol may antagonize the adverse cardiovascular effects of angiotensin II (Ang II). We investigated the effects of 2-methoxyestradiol (2-ME), a nonestrogenic estradiol metabolite, on Ang II-induced cardiovascular and renal injury in male rats. First, we determined the effects of 2-ME on Ang II-induced acute changes in blood pressure, renal hemodynamics, and excretory function. Next, we investigated the effects of 2-ME and 2-hydroxyestardiol (2-HE) on hypertension and cardiovascular and renal injury induced by chronic infusion of Ang II. Furthermore, the effects of 2-ME on blood pressure and cardiovascular remodeling in the constricted aorta (CA) rat model and on isoproterenol-induced (ISO) cardiac hypertrophy and fibrosis were examined. 2-ME had no effects on Ang II-induced acute changes in blood pressure, renal hemodynamics, or glomerular filtration rate. Both 2-ME and 2-HE reduced hypertension, cardiac hypertrophy, proteinuria, and mesangial expansion induced by chronic Ang II infusions. In CA rats, 2-ME attenuated cardiac hypertrophy and fibrosis and reduced elevated blood pressure above the constriction. Notably, 2-ME reduced both pressure-dependent (above constriction) and pressure-independent (below constriction) vascular remodeling. 2-ME had no effects on ISO-induced renin release yet reduced ISO-induced cardiac hypertrophy and fibrosis. This study shows that 2-ME protects against cardiovascular and renal injury due to chronic activation of the renin-angiotensin system. This study reports for the first time that in vivo 2-ME reduces trophic (pressure-independent) effects of Ang II and related cardiac and vascular remodeling.

Estradiol Metabolism: Crossroads in Pulmonary Arterial Hypertension.

Pulmonary arterial hypertension (PAH) is a debilitating and progressive disease that predominantly develops in women. Over the past 15 years, cumulating evidence has pointed toward dysregulated metabolism of sex hormones in animal models and patients with PAH. 17ß-estradiol (E2) is metabolized at positions C2, C4, and C16, which leads to the formation of metabolites with different biological/estrogenic activity. Since the first report that 2-methoxyestradiol, a major non-estrogenic metabolite of E2, attenuates the development and progression of experimental pulmonary hypertension (PH), it has become increasingly clear that E2, E2 precursors, and E2 metabolites exhibit both protective and detrimental effects in PH. Furthermore, both experimental and clinical data suggest that E2 has divergent effects in the pulmonary vasculature versus right ventricle (estrogen paradox in PAH). The estrogen paradox is of significant clinical relevance for understanding the development, progression, and prognosis of PAH. This review updates experimental and clinical findings and provides insights into: (1) the potential impacts that pathways of estradiol metabolism (EMet) may have in PAH; (2) the beneficial and adverse effects of estrogens and their precursors/metabolites in experimental PH and human PAH; (3) the co-morbidities and pathological conditions that may alter EMet and influence the development/progression of PAH; (4) the relevance of the intracrinology of sex hormones to vascular remodeling in PAH; and (5) the advantages/disadvantages of different approaches to modulate EMet in PAH. Finally, we propose the three-tier-estrogen effects in PAH concept, which may offer reconciliation of the opposing effects of E2 in PAH and may provide a better understanding of the complex mechanisms by which EMet affects the pulmonary circulation-right ventricular interaction in PAH.

Recurrent inhibition of mitochondrial complex III induces chronic pulmonary vasoconstriction and glycolytic switch in the rat lung.

BACKGROUND: Pulmonary arterial hypertension (PAH) is a fatal disease; however, the mechanisms directly involved in triggering and the progression of PAH are not clear. Based on previous studies that demonstrated a possible role of mitochondrial dysfunction in the pathogenesis of PAH, we investigated the effects of chronic inhibition of mitochondrial function in vivo in healthy rodents. METHODS: Right ventricle systolic pressure (RVSP) was measured in female rats at baseline and up to 24 days after inhibition of mitochondrial respiratory Complex III, induced by Antimycin A (AA, 0.35 mg/kg, given three times starting at baseline and then days 3 and 6 as a bolus injection into the right atrial chamber). RESULTS: Rodents exposed to AA demonstrated sustained increases in RVSP from days 6 through 24. AA-exposed rodents also possessed a progressive increase in RV end-diastolic pressure but not RV hypertrophy, which may be attributed to either early stages of PAH development or to reduced RV contractility due to inhibition of myocardial respiration. Protein nitration levels in plasma were positively correlated with PAH development in AA-treated rats. This finding was strongly supported by results obtained from PAH humans where plasma protein nitration levels were correlated with markers of PAH severity in female but not male PAH patients. Based on previously reported associations between increased nitric oxide production levels with female gender, we speculate that in females with PAH mitochondrial dysfunction may represent a more deleterious form, in part, due to an increased nitrosative stress development. Indeed, the histological analysis of AA treated rats revealed a strong perivascular edema, a marker of pulmonary endothelial damage. Finally, AA treatment was accompanied by a severe metabolic shift toward glycolysis, a hallmark of PAH pathology. CONCLUSIONS: Chronic mitochondrial dysfunction induces the combination of vascular damage and metabolic reprogramming that may be responsible for PAH development. This mechanism may be especially important in females, perhaps due to an increased NO production and nitrosative stress development.

Captopril Attenuates Cardiovascular and Renal Disease in a Rat Model of Heart Failure With Preserved Ejection Fraction.

Heart failure with preserved ejection fraction (HFpEF), a prevalent form of heart failure, is frequently accompanied by the metabolic syndrome and kidney disease. Because current treatment options of HFpEF are limited, evaluation of therapies in experimental models of HFpEF with the metabolic syndrome and kidney disease is needed. In this study, we evaluated the effects of captopril, furosemide, and their combination in aged, obese ZSF1 rats, an animal model of HFpEF with the metabolic syndrome and chronic kidney disease as comorbidities. Captopril (100 mg/kg), furosemide (50 mg/kg), or their combination was administered orally to obese ZSF1 rats aged 20 to 44 weeks. Untreated ZSF1 rats served as controls. After 24 weeks of treatment, captopril significantly lowered systemic blood pressure and attenuated HFpEF as evidenced by significantly reduced left ventricular end diastolic pressures (10.5 ± 1.4 vs. 4.9 ± 1.3 mm Hg in Control vs. Captopril, respectively) and significantly lower left ventricular relaxation time constants (28.1 ± 2.9 vs. 18.3 ± 3.1 ms in Control vs. Captopril, respectively). The captopril-induced improvement in left ventricular function was associated with reduced cardiac hypertrophy, ischemia, necrosis, and vasculitis. Captopril also increased renal blood flow and glomerular filtration rate, reduced renal vascular resistance and proteinuria, and improved renal histology (ie, reduced renal hypertrophy, glomerulosclerosis, and tubular atrophy/dilation). Furosemide alone provided little benefit; moreover, furosemide did not augment the therapeutic benefits of captopril. This study suggests that chronic administration of captopril, but not furosemide, could be beneficial in patients with HFpEF, particularly in those with comorbidities such as obesity, diabetes, and dyslipidemias.

SIRT3-AMP-Activated Protein Kinase Activation by Nitrite and Metformin Improves Hyperglycemia and Normalizes Pulmonary Hypertension Associated With Heart Failure With Preserved Ejection Fraction.

BACKGROUND: Pulmonary hypertension associated with heart failure with preserved ejection fraction (PH-HFpEF) is an increasingly recognized clinical complication of metabolic syndrome. No adequate animal model of PH-HFpEF is available, and no effective therapies have been identified to date. A recent study suggested that dietary nitrate improves insulin resistance in endothelial nitric oxide synthase null mice, and multiple studies have reported that both nitrate and its active metabolite, nitrite, have therapeutic activity in preclinical models of pulmonary hypertension. METHODS AND RESULTS: To evaluate the efficacy and mechanism of nitrite in metabolic syndrome associated with PH-HFpEF, we developed a 2-hit PH-HFpEF model in rats with multiple features of metabolic syndrome attributable to double-leptin receptor defect (obese ZSF1) with the combined treatment of vascular endothelial growth factor receptor blocker SU5416. Chronic oral nitrite treatment improved hyperglycemia in obese ZSF1 rats by a process that requires skeletal muscle SIRT3-AMPK-GLUT4 signaling. The glucose-lowering effect of nitrite was abolished in SIRT3-deficient human skeletal muscle cells, and in SIRT3 knockout mice fed a high-fat diet, as well. Skeletal muscle biopsies from humans with metabolic syndrome after 12 weeks of oral sodium nitrite and nitrate treatment (IND#115926) displayed increased activation of SIRT3 and AMP-activated protein kinase. Finally, early treatments with nitrite and metformin at the time of SU5416 injection reduced pulmonary pressures and vascular remodeling in the PH-HFpEF model with robust activation of skeletal muscle SIRT3 and AMP-activated protein kinase. CONCLUSIONS: These studies validate a rodent model of metabolic syndrome and PH-HFpEF, suggesting a potential role of nitrite and metformin as a preventative treatment for this disease.

Nitrite Prevents Right Ventricular Failure and Remodeling Induced by Pulmonary Artery Banding.

BACKGROUND: Nitrite has been shown to reduce right ventricle (RV) remodeling in experimental pulmonary hypertension. However, whether this effect is due to a reduction in RV afterload (ie, reduction in pulmonary artery pressure) or a direct effect on the RV itself remains unanswered. We hypothesize that nitrite has direct effects on RV remodeling and studied its effects in mice with pulmonary artery banding (PAB). METHODS AND RESULTS: PAB decreased exercise tolerance and reduced RV systolic and diastolic function. Nitrite treatment attenuated the decrease in RV systolic function and improved the RV diastolic function. Nitrite-treated mice with PAB had similar exercise tolerance compared with a control group. PAB induced RV hypertrophy and fibrosis which were associated with increased expression of phospho-Akt. Interestingly, nitrite treatment attenuated PAB-induced RV hypertrophy and reduced the expression of phospho-Akt in RV tissue from mice with PAB. In neonatal rat cardiac fibroblast, nitrite also attenuated hypoxia-induced increase in expression of phospho-Akt. CONCLUSION: Our study indicates that nitrite treatment has direct beneficial effects on RV and improves function and attenuates remodeling in RV exposed to chronic pressure overload. These beneficial effects, at least in part, could be due to the inhibition of the phospho-Akt (p-Akt) pathway activation.

Dual A1/A2B Receptor Blockade Improves Cardiac and Renal Outcomes in a Rat Model of Heart Failure with Preserved Ejection Fraction.

Heart failure with preserved ejection fraction (HFpEF) is prevalent and often accompanied by metabolic syndrome. Current treatment options are limited. Here, we test the hypothesis that combined A1/A2B adenosine receptor blockade is beneficial in obese ZSF1 rats, an animal model of HFpEF with metabolic syndrome. The combined A1/A2B receptor antagonist 3-[4-(2,6-dioxo-1,3-dipropyl-7H-purin-8-yl)-1-bicyclo[2.2.2]octanyl]propanoic acid (BG9928) was administered orally (10 mg/kg/day) to obese ZSF1 rats (n = 10) for 24 weeks (from 20 to 44 weeks of age). Untreated ZSF1 rats (n = 9) served as controls. After 24 weeks of administration, BG9928 significantly lowered plasma triglycerides (in mg/dl: control group, 4351 ± 550; BG9928 group, 2900 ± 551) without adversely affecting plasma cholesterol or activating renin release. BG9928 significantly decreased 24-hour urinary glucose excretion (in mg/kg/day: control group, 823 ± 179; BG9928 group, 196 ± 80) and improved oral glucose tolerance, polydipsia, and polyuria. BG9928 significantly augmented left ventricular diastolic function in association with a reduction in cardiac vasculitis and cardiac necrosis. BG9928 significantly reduced 24-hour urinary protein excretion (in mg/kg/day: control group, 1702 ± 263; BG9928 group, 1076 ± 238), and this was associated with a reduction in focal segmental glomerulosclerosis, tubular atrophy, tubular dilation, and deposition of proteinaceous material in the tubules. These findings show that, in a model of HFpEF with metabolic syndrome, A1/A2B receptor inhibition improves hyperlipidemia, exerts antidiabetic actions, reduces HFpEF, improves cardiac histopathology, and affords renal protection. We conclude that chronic administration of combined A1/A2B receptor antagonists could be beneficial in patients with HFpEF, in particular those with comorbidities such as obesity, diabetes, and dyslipidemias.

8-Aminopurines in the Cardiovascular and Renal Systems and Beyond.

Screening of compounds comprising 8-substituted guanine revealed that 8-aminoguanosine and 8-aminoguanine cause diuresis/natriuresis/glucosuria, yet decrease potassium excretion. Subsequent investigations demonstrated that 8-aminoguanosine's effects are mediated by its metabolite 8-aminoguanine. The mechanism by which 8-aminoguanine causes diuresis/natriuresis/glucosuria involves inhibition of PNPase (purine nucleoside phosphorylase), which increases renal interstitial inosine levels. Additional evidence suggests that inosine, via indirect or direct adenosine A2B receptor activation, increases renal medullary blood flow which enhances renal excretory function. Likely, 8-aminoguanine has pleiotropic actions that also alter renal excretory function. Indeed, the antikaliuretic effects of 8-aminoguanine are independent of PNPase inhibition. 8-Aminoguanine is an endogenous molecule; nitrosative stress leads to production of biomolecules containing 8-nitroguanine moieties. Degradation of these biomolecules releases 8-nitroguanosine and 8-nitro-2'-deoxyguanosine which are converted to 8-aminoguanine. Also, guanosine and guanine per se may contribute to 8-aminoguanine formation. 8-Aminoinosine, 8-aminohypoxanthine, and 8-aminoxanthine likewise induce diuresis/natriuresis/glucosuria, yet do not reduce potassium excretion. Thus, there are several pharmacologically active 8-aminopurines with nuanced effects on renal excretory function. Chronic treatment with 8-aminoguanine attenuates hypertension in deoxycorticosterone/salt rats, prevents strokes, and increases lifespan in Dahl salt-sensitive rats on a high salt diet and attenuates the metabolic syndrome in rats; 8-aminoguanosine retards progression of pulmonary hypertension in rats and anemia and organ damage in sickle cell mice. 8-Aminoguanine reverses age-associated lower urinary tract dysfunction and retinal degeneration. 8-Aminopurines represent a new class of agents (and potentially endogenous factors) that have beneficial effects on the cardiovascular system and kidneys and may turn back the clock in age-associated diseases.

Endogenous adenosine contributes to renal sympathetic neurotransmission via postjunctional A1 receptor-mediated coincident signaling.

Adenosine A(1) receptor antagonists have diuretic/natriuretic activity and may be useful for treating sodium-retaining diseases, many of which are associated with increased renal sympathetic tone. Therefore, it is important to determine whether A(1) receptor antagonists alter renal sympathetic neurotransmission. In isolated, perfused rat kidneys, renal vasoconstriction induced by renal sympathetic nerve simulation was attenuated by 1) 1,3-dipropyl-8-p-sulfophenylxanthine (xanthine analog that is a nonselective adenosine receptor antagonist, but is cell membrane impermeable and thus does not block intracellular phosphodiesterases), 2) xanthine amine congener (xanthine analog that is a selective A(1) receptor antagonist), 3) 1,3-dipropyl-8-cyclopentylxanthine (xanthine analog that is a highly selective A(1) receptor antagonist), and 4) FK453 (nonxanthine analog that is a highly selective A(1) receptor antagonist). In contrast, FR113452 (enantiomer of FK453 that does not block A(1) receptors), MRS-1754 (selective A(2B) receptor antagonist), and VUF-5574 (selective A(3) receptor antagonist) did not alter responses to renal sympathetic nerve stimulation, and ZM-241385 (selective A(2A) receptor antagonist) enhanced responses. Antagonism of A(1) receptors did not alter renal spillover of norepinephrine. 2-Chloro-N(6)-cyclopentyladenosine (highly selective A(1) receptor agonist) increased renal vasoconstriction induced by exogenous norepinephrine, an effect that was blocked by 1,3-dipropyl-8-cyclopentylxanthine, U73122 (phospholipase C inhibitor), GF109203X (protein kinase C inhibitor), PP1 (c-src inhibitor), wortmannin (phosphatidylinositol 3-kinase inhibitor), and OSU-03012 (3-phosphoinositide-dependent protein kinase-1 inhibitor). These results indicate that adenosine formed during renal sympathetic nerve stimulation enhances the postjunctional effects of released norepinephrine via coincident signaling and contributes to renal sympathetic neurotransmission. Likely, the coincident signaling pathway is: phospholipase C → protein kinase C → c-src → phosphatidylinositol 3-kinase → 3-phosphoinositide-dependent protein kinase-1.

Dimethyl Sulfoxide Induces Hemolysis and Pulmonary Hypertension.

Vascular and lung injury are well established complications associated with hemolytic disorders, and hemolysis associated pulmonary hypertension (PH) has emerged as the most serious complication of sickle cell disease. The causal relationship between intravascular hemolysis and the development of PH is still under investigation. Previously we have shown that repetitive administration of hemolyzed autologous blood causes PH in rats. Dimethyl sulfoxide (DMSO), a widely used solvent and anti-inflammatory agent, induces hemolysis in vivo. We hypothesized that repetitive administration of DMSO would induce PH in rats. We also examined hemolysis-induced release of adenosine deaminase (ADA) and arginase from red blood cells, which may amplify hemolysis-mediated vascular injury. Acute administration of DMSO (1.5ml/30 min into the right atrium) induced intravascular hemolysis and pulmonary vasoconstriction. DMSO-induced increase in right ventricular peak systolic pressure (RVPSP) was associated with increased release of ADA. Notably, the acute increase in RVPSP was attenuated by administration of an adenosine A2A receptor agonist or by pretreatment of animals with ADA inhibitor erythro-9-(2-hydroxy-3-nonyl) adenine (EHNA). Repetitive administration of DMSO for 10 days produced anemia, hemoglobinuria, hemoglobinemia, splenomegaly, and development of PH. Histopathological analysis revealed pulmonary vascular remodeling. The presented data describe a new model of hemolysis induced PH, suggesting that hemolysis is mechanistically related to pulmonary hypertension, and pointing to a potential pathogenic role that adenosine deaminase and accelerated adenosine metabolism may play in hemolysis associated pulmonary hypertension.

2-Methoxyestradiol Attenuates the Development and Retards the Progression of Hypoxia-And Alpha-Naphthylthiourea-Induced Pulmonary Hypertension.

Pulmonary arterial hypertension (PH), a progressive, incurable, and deadly disease, predominantly develops in women. Growing body of evidence suggest that dysregulated estradiol (E2) metabolism influences the development of PH and that some of the biological effects of E2 are mediated by its major non-estrogenic metabolite, 2-metyhoxyestradiol (2ME). The objective of this study was to examine effects of 2ME in chronic hypoxia (CH)-induced PH and alpha-naphthylthiourea (ANTU)-induced acute lung injury and PH. In addition, we investigated the effects of exposure to different levels of CH on development of PH. Chronic exposure to 15% or 10% oxygen produced similar increases in right ventricle peak systolic pressure (RVPSP) and pulmonary vascular remodeling, but oxygen concentration-dependent increase in hematocrit. Notably, right ventricle (RV) hypertrophy correlated with level of hypoxia and hematocrit, rather than with magnitude of RVPSP. The latter suggests that, in addition to increased afterload, hypoxia (via increased hematocrit) significantly contributes to RV hypertrophy in CH model of PH. In CH-PH rats, preventive and curative 2ME treatments reduced both elevated RVPSP and pulmonary vascular remodeling. Curative treatment with 2ME was more effective in reducing hematocrit and right ventricular hypertrophy, as compared to preventive treatment. Single ANTU injection produced lung injury, i.e., increased lungs weight and induced pleural effusion. Treatment with 2ME significantly reduced pleural effusion and, more importantly, eliminated acute mortality induced by ANTU (33% vs 0%, ANTU vs. ANTU+2ME group). Chronic treatment with ANTU induced PH and RV hypertrophy and increased lungs weight. 2-ME significantly attenuated severity of disease (i.e., reduced RVPSP, RV hypertrophy and pulmonary vascular injury). This study demonstrates that 2ME has beneficial effects in chronic hypoxia- and acute lung injury-induced PH and provides preclinical justification for clinical evaluation of 2ME in pulmonary hypertension.

Long-Term Dipeptidyl Peptidase 4 Inhibition Worsens Hypertension and Renal and Cardiac Abnormalities in Obese Spontaneously Hypertensive Heart Failure Rats.

Background The long-term effects of dipeptidyl peptidase 4 (DPP4) inhibitors on blood pressure and cardiovascular and renal health remain controversial. Herein, we investigated the extended (>182 days) effects of DPP4 inhibition in a model of spontaneous hypertension, heart failure, diabetes mellitus, obesity and hyperlipidemia. Methods and Results Adult obese spontaneously hypertensive heart failure rats (SHHF) were implanted with radio transmitters for measurement of arterial blood pressures. Two weeks later, SHHF were randomized to receive either a DPP4 inhibitor (sitagliptin, 80 mg/kg per day in drinking water) or placebo. At the end of the radiotelemetry measurements, renal and cardiac function and histology, as well as other relevant biochemical parameters, were assessed. For the first 25 days, mean arterial blood pressures were similar in sitagliptin-treated versus control SHHF; afterwards, mean arterial blood pressures increased more in sitagliptin-treated SHHF (P<0.000001). The time-averaged mean arterial blood pressures from day 26 through 182 were 7.2 mm Hg higher in sitagliptin-treated SHHF. Similar changes were observed for systolic (8.6 mm Hg) and diastolic (6.1 mm Hg) blood pressures, and sitagliptin augmented hypertension throughout the light-dark cycle. Long-term sitagliptin treatment also increased kidney weights, renal vascular resistances, the excretion of kidney injury molecule-1 (indicates injury to proximal tubules), renal interstitial fibrosis, glomerulosclerosis, renal vascular hypertrophy, left ventricular dysfunction, right ventricular degeneration, and the ratios of collagen IV/collagen III and collagen IV/laminin in the right ventricle. Conclusions These findings indicate that, in some genetic backgrounds, long-term DPP4 inhibitor treatment is harmful and identify an animal model to study mechanisms of, and test ways to prevent, DPP4 inhibitor-induced pathological conditions.

Relaxin Inhibits Ventricular Arrhythmia and Asystole in Rats With Pulmonary Arterial Hypertension.

Pulmonary arterial hypertension (PAH) leads to right ventricular cardiomyopathy and cardiac dysfunctions where in the clinical setting, cardiac arrest is the likely cause of death, in ~70% of PAH patients. We investigated the cardiac phenotype of PAH hearts and tested the hypothesis that the insulin-like hormone, Relaxin could prevent maladaptive cardiac remodeling and protect against cardiac dysfunctions in a PAH animal model. PAH was induced in rats with sugen (20 mg/kg), hypoxia then normoxia (3-weeks/each); relaxin (RLX = 0, 30 or 400 µg/kg/day, n ≥ 6/group) was delivered subcutaneously (6-weeks) with implanted osmotic mini-pumps. Right ventricle (RV) hemodynamics and Doppler-flow measurements were followed by cardiac isolation, optical mapping, and arrhythmia phenotype. Sugen-hypoxia (SuHx) treated rats developed PAH characterized by higher RV systolic pressures (50 ± 19 vs. 22 ± 5 mmHg), hypertrophy, reduced stroke volume, ventricular fibrillation (VF) (n = 6/11) and bradycardia/arrest (n = 5/11); both cardiac phenotypes were suppressed with dithiothreitol (DTT = 1 mM) (n = 0/2/group) or RLX (low or high dose, n = 0/6/group). PAH hearts developed increased fibrosis that was reversed by RLX-HD, but not RLX-LD. Relaxin decreased Nrf2 and glutathione transferases but not glutathione-reductase. High-dose RLX improved pulmonary arterial compliance (measured by Doppler flow), suppressed VF even after burst-pacing, n = 2/6). Relaxin suppressed VF and asystole through electrical remodeling and by reversing thiol oxidative stress. For the first time, we showed two cardiac phenotypes in PAH animals and their prevention by RLX. Relaxin may modulate maladaptive cardiac remodeling in PAH and protect against arrhythmia and cardiac arrest.

Estrogens and development of pulmonary hypertension: interaction of estradiol metabolism and pulmonary vascular disease.

Severe pulmonary arterial hypertension (PAH) is characterized by clustered proliferation of endothelial cells (ECs) in the lumina of small size pulmonary arteries resulting in concentric obliteration of the lumina and formation of complex vascular structures known as plexiform lesions. This debilitating disease occurs more frequently in women, yet both animal studies in classical models of PAH and limited clinical data suggest protective effects of estrogens: the estrogen paradox in pulmonary hypertension. Little is known about the role of estrogens in PAH, but one line of evidence strongly suggests that the vascular protective effects of 17ß-estradiol (estradiol; E2) are mediated largely by its downstream metabolites. Estradiol is metabolized to 2-hydroxyestradiol (2HE) by CYP1A1/CYP1B1, and 2HE is converted to 2-methoxyestradiol (2ME) by catechol-O-methyl transferase. 2ME is extensively metabolized to 2-methoxyestrone, a metabolite that lacks biologic activity, but which may be converted back to 2ME. 2ME has no estrogenic activity, and its effects are mediated by estrogen receptors­independent mechanism(s). Notably, in systemic and pulmonary vascular ECs, smooth muscle cells, and fibroblasts, 2ME exerts stronger antimitotic effects than E2 itself. E2 and 2ME, despite having similar effects on other cardiovascular cells, have opposing effects on ECs; that is, in ECs, E2 is promitogenic, proangiogenic, and antiapoptotic, whereas 2ME is antimitogenic, antiangiogenic, and proapoptotic. This may have significant ramifications in severe PAH that involves uncontrolled proliferation of monoclonal apoptosis-resistant ECs. Based on its cellular effects, 2ME should be expected to attenuate the progression of disease and provide protection in severe PAH. In contrast, E2, due to its mitogenic, angiogenic, and antiapoptotic effects (otherwise desirable in normal quiescent ECs), may even adversely affect endothelial remodeling in PAH, and this may be even more significant if the E2's effects on injured endothelium are not opposed by 2ME (eg, in the event of reduced E2 conversion to 2ME due to hypoxia, inflammation, drugs, environmental factors, or genetic polymorphism of metabolizing enzymes). This review focuses on the effects of estrogens and their metabolites on pulmonary vascular pathobiology and the development of experimental PAH and offers potential explanation for the estrogen paradox in PAH. Furthermore, we propose that unbalanced estradiol metabolism may lead to the development of PAH. Recent animal data and studies in patients with PAH support this concept.

Synergistic therapeutic effects of 2-methoxyestradiol with either sildenafil or bosentan on amelioration of monocrotaline-induced pulmonary hypertension and vascular remodeling.

2-Methoxyestradiol (2ME) is a major nonestrogenic metabolite of estradiol. Our previous studies suggest that 2ME, in several models of cardiac and/or vascular injury, strongly inhibits cardiac and vascular remodeling. Furthermore, our most recent study shows that in male rats, 2ME attenuates the development and retards the progression of monocrotaline (MCT)-induced pulmonary arterial hypertension (PAH), and in female rats, 2ME eliminates the exacerbation of PAH and the increased mortality due to ovariectomy. The current standard of care of patients with PAH includes treatment with an endothelin receptor antagonist (eg, bosentan) or a phosphodiesterase5 inhibitor (eg, sildenafil). Moreover, combination therapy is often prescribed. Therefore, in the present study, we compared the efficacy of 2ME (10 µg · kg(-1) · h(-1), 2ME-10) to the effects of bosentan (200 mg/kg; BOS), sildenafil (50 mg/kg; SIL), and their respective combinations with 2ME-10 (2ME + BOS and 2ME + SIL groups, respectively). Treatments were initiated 12 days after administration of MCT (60 mg/kg). Twenty-eight days after MCT administration, right ventricular peak systolic pressure was measured and morphometric analysis was conducted. 2ME exhibited beneficial effects in pulmonary hypertensive animals and had efficacy comparable to that of BOS and SIL. Importantly, combination treatments had favorable effects on survival, vascular remodeling, and inflammatory response, and the 2ME + SIL combination was significantly more efficacious than any other treatment. These results indicate that 2ME is effective in experimental PAH and suggests that 2ME may provide additional therapeutic benefit over existing drugs used for the treatment of pulmonary hypertension.