Stevan P. Tofovic

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.

Effects of Long-term Caffeine Consumption on Renal Function in Spontaneously Hypertensive Heart Failure Prone Rats

Our previous studies supported the hypothesis that prolonged administration of caffeine to animals with high-renin hypertension causes progressive deterioration of renal function. However, thus far this hypothesis has been tested with only a few animal models of hypertension. The aim of this study was to test this hypothesis further by investigating the effects of long-term caffeine consumption on renal function in adult spontaneously hypertensive heart failure (SHHF/Mcc-fa(cp)) rats, another model of high-renin hypertension. Lean, male, 9-month-old SHHF/Mcc-fa(cp) rats were randomized to receive either normal drinking water (control group) or drinking water containing 0.1% caffeine (caffeine group) for 20 weeks. No changes in body weight, food and fluid intake, urine volume, and sodium and potassium excretion were found in conscious SHHF/Mcc-fa(cp) rats after 10 or 20 weeks of caffeine treatment. However, caffeine treatment accelerated the time-related decline in renal function and augmented urinary protein excretion. Ten weeks into the protocol, creatinine clearance was 3.6+/-0.4 and 5.7+/-0.9 L/kg/day in the caffeine group and control group, respectively (p<0.02), whereas 20 weeks into the study, creatinine clearance was similarly diminished in both groups. Proteinuria was greater in the caffeine group compared with the control group at both 10 (928+/-131 vs. 439+/-21 mg/kg/day, respectively; p<0.02) and 20 weeks (1,202+/-196 vs. 603+/-30 mg/kg/day, respectively; p<0.01) into the protocol. After 20 weeks, all animals were anesthetized and instrumented. Caffeine treatment for 20 weeks had no effects on blood pressure, heart rate, or vascular resistance in four examined vascular beds (abdominal aorta and renal, carotid, and mesenteric arteries). No changes in renal hemodynamics and electrolyte excretion were found, whereas significantly lower glomerular filtration rate (GFR; inulin clearance) and creatinine clearance (p<0.05) were observed in caffeine-treated animals. These data support our hypothesis that prolonged consumption of caffeine has adverse effects on renal function, in high-renin hypertension.

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 17β-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 receptors-independent 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 E2s 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.

Estradiol metabolites attenuate monocrotaline-induced pulmonary hypertension in rats.

Pulmonary arterial hypertension (PH) is a deadly disease characterized by pulmonary arterial vasoconstriction and hypertension, pulmonary vasculature remodeling, and right ventricular hypertrophy. Our previous in vivo studies, performed in several models of cardiac, vascular, and/or renal injury, suggest that the metabolites of 17β-estradiol may inhibit vascular and cardiac remodeling. The goal of this study was to determine whether 2-methoxyestradiol (2ME), major non-estrogenic estradiol metabolite, prevents the development and/or retards the progression of monocrotaline (MCT)-induced PH. First, a total of 27 male Sprague Dawley rats were injected with distillated water (Cont, n = 6) or monocrotaline (MCT; 60 mg/kg, i.p.; n = 21). Subsets of MCT animals (n = 7 per group) received 2ME or its metabolic precursor 2-hydroxyestradiol (2HE; 10 μg/kg/h via osmotic minipumps) for 21 days. Next, an additional set (n = 24) of control and MCT rats was monitored for 28 days, before right ventricular peak systolic pressure (RVPSP) was measured. Some pulmonary hypertensive animals (n = 8) were treated with 2ME (10 μg/kg/h) beginning from day 14 after MCT administration. MCT caused pulmonary hypertension (ie, increased right ventricle/left ventricle + septum [RV/LV+S] ratio and wall thickness of small-sized pulmonary arteries, and elevated RVPSP) and produced high and late (days 22 to 27) mortality. Pulmonary hypertension was associated with strong proliferative response (PCNA staining) and marked inflammation (ED1 + cells) in lungs. Both metabolites significantly attenuated the RV/LV+S ratio and pulmonary arteries media hypertrophy and reduced proliferative and inflammatory responses in the lungs. Furthermore, in diseased animals, 2ME (given from day 14 to 28) significantly decreased RVPSP, RV/LV+S ratio and wall thickness, and reduced mortality by 80% (mortality rate: 62.5% vs. 12.5%, MCT vs. MCT+2ME day 14 to 28). This study provides the first evidence that 2ME, a major non-estrogenic, non-carcinogenic metabolite of estradiol, prevents the development and retards the progression of monocrotaline-induced pulmonary hypertension. Further evaluation of 2ME for management of pulmonary arterial hypertension is warranted.

Renal function and structure in diabetic, hypertensive, obese ZDFxSHHF-hybrid rats.

The obese ZDFxSHHF-fa/facp model was developed by crossing lean female Zucker Diabetic Fatty (ZDF +/fa) and lean male Spontaneously Hypertensive Heart Failure (SHHF/Mcc-facp, +/fa) rats. The purpose of the present study was to determine renal function and morphology, hemodynamics, and metabolic status in ZDFxSHHF rats. Two sets of experiments were conducted. First, we evaluated heart and kidney function and metabolic status in aged (46 weeks old) male obese ZDFxSHHF and age matched obese SHHF rats, lean Spontaneously Hypertensive (SHR) and lean normotensive Wistar Kyoto (WKY) rats. In the second set of experiments, renal function and structure as well as metabolic and lipid status were determined in lean (LN) and obese (OB) adult (29-weeks of age) ZDFxSHHF rats. At 46 weeks of age ZDFxSHHF rats are hypertensive expressing marked cardiac hypertrophy associated with diastolic dysfunction and preserved contractile function. Fasted hyperglycemia and hyperinsulinemia are accompanied by moderate hypercholesterolemia and hypertriglyceridemia. Obese aged ZDFxSHHF have marked renal hypertrophy, a 3–8 fold decrease in creatinine clearance (compared with SHHF, SHR and WKY), a high percent of segmental + global glomerulosclerosis (59.8% ± 10.8), and severe tubulointerstitial and vascular changes. Obese ZDFxSHHF rats die at an early age (∼12 months) from end-stage renal failure. Studies conducted in 29-week animals showed that, although both LN and OB 29-week old animals are hypertensive, OB animals have more severely compromised renal function and structure as compared with lean littermates (kidney weight: 2.56 ± 0.16 vs. 1.61 ± 0.12 g; creatinine clearance: 0.42 ± 0.04 vs. 1.24 ± 0.13 L/g kid/day; renal vascular resistance 12.39 ± 1.4 vs. 6.14 ± 0.42 mmHg/mL/min/g kid; protein excretion: 556 ± 16 vs. 159 ± 9 mg/day/g kid, p < 0.05, OB vs. LN, respectively). Obesity is also associated with hyperglycemia (424 ± 37 vs. 115 ± 11 mg/dL), hyperinsulinemia (117.2 ± 8.8 vs. 42.3 ± 3.5 μU/mL), hypertriglyceridemia (5200 ± 702 vs. 194 ± 23 mg/dL), hypercholesterolemia (632 ± 39 vs. 109 ± 4 mg/dL), and presence of segmental + global glomerulosclerosis (20.1 ± 3.2% vs. 0.1 ± 0.1%) with prominent tubular and interstitial changes (p < 0.05, OB vs. LN, respectively). In summary, the present study indicates that the crossing of rat strains of nephropathy produces hybrids that carry a high risk for severe renal dysfunction. The ZDFxSHHF rats express insulin resistance, hypertension, dislipidemia and obesity and develop severe renal dysfunction. In addition, the hybrids do not develop some of the complications (hydronephrosis or congestive heart failure) common for the parental strains that may compromise studies of renal function and structure. Therefore, the ZDFxSHHF rat may be a useful model fore valuating risk factors and pharmacological interventions in chronic renal failure.

Gender, sex hormones and pulmonary hypertension

Most subtypes of pulmonary arterial hypertension (PAH) are characterized by a greater susceptibility to disease among females, although females with PAH appear to live longer after diagnosis. While this “estrogen paradox” of enhanced female survival despite increased female susceptibility remains a mystery, recent progress has begun to shed light upon the interplay of sex hormones, the pathogenesis of pulmonary hypertension, and the right ventricular response to stress. For example, emerging data in humans and experimental models suggest that estrogens or differential sex hormone metabolism may modify disease risk among susceptible subjects, and that estrogens may interact with additional local factors such as serotonin to enhance the potentially damaging chronic effects of estrogens on the pulmonary vasculature. Regardless, it remains unclear why not all estrogenic compounds behave equally, nor why estrogens appear to be protective in certain settings but detrimental in others. The contribution of androgens and other compounds, such as dehydroepiandrosterone, to pathogenesis and possibly treatment must be considered as well. In this review, we will discuss the recent understandings on how estrogens, estrogen metabolism, dehydroepiandrosterone, and additional susceptibility factors may all contribute to the pathogenesis or potentially to the treatment of pulmonary hypertension, by evaluating current human, cell-based, and experimental model data.

Estradiol metabolites attenuate renal and cardiovascular injury induced by chronic nitric oxide synthase inhibition

Our previous studies in rodent models of nephropathy demonstrate that 2-hydroxyestradiol (2HE), an estradiol metabolite with little estrogenic activity, exerts renoprotective effects. In vivo, 2HE is readily converted to 2-methoxyestradiol (2ME), a major estradiol metabolite with no estrogenic activity. The goal of this study was to determine whether 2ME has renal and cardiovascular protective effects in vivo. First, the acute (90 minutes) and chronic (14 days) effects of 2ME (10 μg/kg/h) on blood pressure and renal function were examined in normotensive and spontaneously hypertensive rats (SHR). Second, a rat model of cardiovascular and renal injury induced by chronic nitric oxide synthase inhibition (Nω-nitro-L-arginine; 40 mg/kg/d; LNNA group) was used to examine the protective effects of estradiol metabolites. Subsets of LNNA-treated rats were administered either 2HE or 2ME (10 μg/kg/h via osmotic minipump; LNNA+2ME and LNNA+2HE groups, respectively. 2-Methoxyestradiol had no acute or chronic effects on blood pressure or renal function in normotensive animals or on hypertension in SHR. Prolonged, 5-week NOS inhibition induced severe cardiovascular and renal disease and high mortality (75%, LNNA group). 2ME, but not 2HE, significantly decreased elevated blood pressure and attenuated the reduction in GFR. 2HE delayed the onset of proteinuria, whereas no proteinuria was detected in the 2-ME group. 2HE and 2ME reduced mortality rate by 66% and 83%, respectively (P < 0.001). In the kidney, 2HE and 2ME abolished LNNA-induced interstitial and glomerular inflammation, attenuated glomerular collagen IV synthesis, and inhibited glomerular and tubular cell proliferation. In the heart, 2HE and 2ME markedly reduced vascular and interstitial inflammation and reduced collagen synthesis and vascular/interstitial cell proliferation. This study provides the first evidence that, in a model of severe cardiovascular and renal injury, 2-methoxyestradiol (a major nonestrogenic estradiol metabolite) exerts renal and cardiovascular protective effects and reduces mortality.

2-methoxyestradiol attenuates bleomycin-induced pulmonary hypertension and fibrosis in estrogen-deficient rats.

Pulmonary hypertension (PH) is a common and life-threatening complication of pulmonary fibrosis. Estradiol (E2) is protective in experimental PH, and its non-estrogenic metabolite 2-methoxyestradiol (2ME) prevents the development and retards the progression of monocrotaline-induced PH in male and female rats. However, the effects of E2 and 2ME on pulmonary fibrosis and associated PH have not been examined. Therefore, we compared the growth inhibitory effects of E2 and 2ME in human lung fibroblasts (hLFs) and pulmonary vascular smooth muscle cells (hPASMCs), and we investigated the effects of estrogen deficiency and 2ME on bleomycin-induced pulmonary fibrosis and PH. Intact and ovariectomized (OVX) female Sprague-Dawley rats were administered intratracheally either saline or bleomycin (15IU/kg), and a subset of OVX bleomycin-treated rats received 2ME (10microg/kg/h) for 21days. Estradiol had only limited inhibitory effects on growth in hPASMCs and no effect in hLFs, whereas 2ME exhibited strong and concentration-dependent (1-10microM) antimitogenic effects in both cell types. Bleomycin caused lung injury/PH (significantly increased lung and right ventricle (RV) weights, RV peak systolic pressure (RVPSP), and RV/left ventricle + septum ratio (RV/LV + S); caused medial hypertrophy and adventitial widening of pulmonary arteries; induced marked focal/diffuse fibrosis with diffuse infiltration of inflammatory (ED1+) cells; and resulted in 30% mortality). OVX exacerbated the disease and increased mortality (to 75%); whereas 2ME tended to reduce mortality (55.5%) and in surviving animals reduced RVPSP and RV/LV + S ratio, and attenuated vascular remodeling, pulmonary inflammation and fibrosis. This study suggests that 2ME may have protective effects in bleomycin-induced PH and fibrosis. Further investigation of 2ME in pulmonary fibrosis and PH is warranted.

2-Hydroxyestradiol Attenuates Renal Disease in Chronic Puromycin Aminonucleoside Nephropathy

It has been previously shown that 2-hydroxyestradiol (2-OHE) attenuates the development of renal disease in genetic nephropathy associated with obesity and the metabolic syndrome. The purpose of this study was to test the hypothesis that 2-OHE, irrespective of its effects on metabolic status and/or obesity, exerts direct renoprotective effects in vivo. First, the effects of increasing doses of 2-OHE on mesangial cell growth, proliferation, and collagen synthesis in isolated rat glomerular mesangial cells were evaluated in vitro. Second, the effects of 12-wk administration of 2-OHE (10 micro g/h per kg) on renal function and structure in chronic puromycin aminonucleoside (PAN)-induced nephropathy in rats were evaluated in vivo. 2-OHE concentration-dependently (0.001 to 1 micro mol/L; P < 0.001) inhibited serum (2.5%)-induced cell growth ((3)H-thymidine incorporation), collagen synthesis ((3)H-proline incorporation), and cell proliferation (cell number). Importantly, the inhibitory effects of 2-OHE (0.1 micro mol/L) were not blocked by ICI182780 (50 micro mol/L), an estrogen receptor antagonist. In vivo, chronic administration of PAN (75 mg/kg + 5 x 20 mg/kg) over 12 wk induced severe chronic renal disease. Chronic treatment with 2-OHE significantly (P < 0.05) attenuated PAN-induced decrease in glomerular filtration, reduced proteinuria, and the elevated BP, and it had no effect on PAN-induced increase in plasma cholesterol and triglycerides levels. 2-OHE had no effects on plasma testosterone levels in male nephropathic animals. Immunohistochemical staining for collagen IV and proliferating cell nuclear antigen (PCNA) in glomeruli and transforming growth factor-beta (TGF-beta) in renal tubular cells were significantly higher in PAN nephropatic rats versus control animals with intact kidneys. PAN also markedly increased glomerular and interstitial macrophage infiltration (ED1(+) cells). 2-OHE had no effects on renal tubular cell TGF-beta, but it significantly reduced glomerular PCNA and collagen IV and glomerular and interstitial macrophage infiltration. In summary, this study provides the first evidence that 2-OHE exerts direct renoprotective effects in vivo. These effects are mediated by estrogen receptor-independent mechanisms and are due, at least in part, to the inhibition of some of the key proliferative mechanisms involved in glomerular remodeling and sclerosis.

Bosentan inhibits oxidative and nitrosative stress and rescues occlusive pulmonaryhypertension

Pulmonary arterial hypertension (PH) is a fatal disease marked by excessive pulmonary vascular cell proliferation. Patients with idiopathic PH express endothelin-1 (ET-1) at high levels in their lungs. As the activation of both types of ET-1 receptor (ETA and ETB) leads to increased generation of superoxide and hydrogen peroxide, this may contribute to the severe oxidative stress found in PH patients. As a number of pathways may induce oxidative stress, the particular role of ET-1 remains unclear. The aim of this study was to determine whether inhibition of ET-1 signaling could reduce pulmonary oxidative stress and attenuate the progression of disease in rats with occlusive-angioproliferative PH induced by a single dose of SU5416 (200 mg/kg) and subsequent exposure to hypoxia for 21 days. Using this regimen, animals developed severe PH as evidenced by a progressive increase in right-ventricle (RV) peak systolic pressure (RVPSP), severe RV hypertrophy, and pulmonary endothelial and smooth muscle cell proliferation, resulting in plexiform vasculopathy. PH rats also had increased oxidative stress, correlating with endothelial nitric oxide synthase uncoupling and NADPH oxidase activation, leading to enhanced protein nitration and increases in markers of vascular remodeling. Treatment with the combined ET receptor antagonist bosentan (250 mg/kg/day; day 10 to 21) prevented further increase in RVPSP and RV hypertrophy, decreased ETA/ETB protein levels, reduced oxidative stress and protein nitration, and resulted in marked attenuation of pulmonary vascular cell proliferation. We conclude that inhibition of ET-1 signaling significantly attenuates the oxidative and nitrosative stress associated with PH and prevents its progression.