Rangarajan D. Nadadur

Genistein, a Soy Phytoestrogen, Reverses Severe Pulmonary Hypertension and Prevents Right Heart Failure in Rats

Pretreatment with a phytoestrogen genistein has been shown to attenuate the development of pulmonary hypertension (PH). Because PH is not always diagnosed early, we examined whether genistein could also reverse preexisting established PH and prevent associated right heart failure (RHF). PH was induced in male rats by 60 mg/kg of monocrotaline. After 21 days, when PH was well established, rats received daily injection of genistein (1 mg/kg per day) for 10 days or were left untreated to develop RHF by day 30. Effects of genistein on human pulmonary artery smooth muscle cell and endothelial cell proliferation and neonatal rat ventricular myocyte hypertrophy were assessed in vitro. Severe PH was evident 21 days after monocrotaline, as peak systolic right ventricular pressure increased to 66.35±1.03 mm Hg and right ventricular ejection fraction reduced to 41.99±1.27%. PH progressed to RHF by day 30 (right ventricular pressure, 72.41±1.87 mm Hg; RV ejection fraction, 29.25±0.88%), and mortality was ≈75% in RHF rats. Genistein therapy resulted in significant improvement in lung and heart function as right ventricular pressure was significantly reduced to 43.34±4.08 mm Hg and right ventricular ejection fraction was fully restored to 65.67±1.08% similar to control. Genistein reversed PH-induced pulmonary vascular remodeling in vivo and inhibited human pulmonary artery smooth muscle cell proliferation by ≈50% in vitro likely through estrogen receptor-&bgr;. Genistein also reversed right ventricular hypertrophy (right ventricular hypertrophy index, 0.35±0.029 versus 0.70±0.080 in RHF), inhibited neonatal rat ventricular myocyte hypertrophy, and restored PH-induced loss of capillaries in the right ventricle. These improvements in cardiopulmonary function and structure resulted in 100% survival by day 30. Genistein restored PH-induced downregulation of estrogen receptor-&bgr; expression in the right ventricle and lung. In conclusion, genistein therapy not only rescues preexisting severe PH but also prevents the progression of severe PH to RHF.

Cardiac structural and hemodynamic changes associated with physiological heart hypertrophy of pregnancy are reversed postpartum.

Pregnancy is associated with ventricular hypertrophy and volume overload. Here we investigated whether late pregnancy is associated with cardiac structural and hemodynamic changes, and if these changes are reversed postpartum. Female mice (C57BL/6) were used in nonpregnant diestrus (NP), late-pregnant (LP), or 7-day postpartum (PP7) stages. Echocardiography and cardiac catheterization were performed to monitor cardiac hemodynamics. Transcript expression of proangiogenic vascular endothelial growth factor, cardiac fetal gene osteopontin, cardiac extracellular matrix-degrading enzymes matrix metalloproteinase-2, and a disintegrin and metalloproteinase-15 and -17 were assessed by RT-PCR. Masson trichrome staining for cardiac fibrosis and endothelial marker CD31 immunostaining for angiogenesis were performed. Heart hypertrophy in LP was fully reversed in PP7 (heart weight: NP = 114 ± 4 mg; LP = 147 ± 2 mg; PP7 = 117 ± 8 mg, P < 0.05 for LP vs. PP7). LP had elevated left ventricular (LV) pressure (119 ± 5 mmHg in LP vs. 92 ± 7 mmHg in NP, P < 0.05) that was restored at PP7 (95 ± 8 mmHg, P < 0.001 vs. LP). LP had increased LV contractility (maximal rate of increase of LV pressure = 6,664 ± 297 mmHg/s in LP vs. 4,294 ± 568 mmHg/s in NP, P < 0.01) that was restored at PP7 (5,313 ± 636 mmHg/s, P < 0.05 vs. LP). LV ejection fraction was reduced in LP (LP = 58 ± 1% vs. NP = 70 ± 4%, P < 0.001) and was already restored at PP1 (77 ± 2%, P < 0.001 vs. LP). Myocardial angiogenesis was significantly increased in LP (capillary density = 1.25 ± 0.02 vs. 0.95 ± 0.01 capillaries/myocyte in NP, P < 0.001) and was fully restored in PP7 (0.98 ± 0.01, P < 0.001 vs. LP). Vascular endothelial growth factor was upregulated in LP (LP = 1.4 ± 0.1 vs. NP = 1 ± 0.1, normalized to NP, P < 0.001) and was restored in PP7 (PP7 = 0.83 ± 0.1, P < 0.001 vs. LP). There was no increase in cardiac fibrosis in LP. Matrix metalloproteinase-2 transcript levels were downregulated in LP (LP = 0.47 ± 0.03 vs. NP = 1 ± 0.01, normalized to NP, P < 0.001) and was restored at PP7 (0.70 ± 0.1, P < 0.001 vs. LP). In conclusion, pregnancy-induced heart hypertrophy is associated with transient cardiac dysfunction, increased cardiac angiogenesis, lack of fibrosis, and decreased expression of remodeling enzymes that are reversed postpartum.

Intralipid Prevents and Rescues Fatal Pulmonary Arterial Hypertension and Right Ventricular Failure in Rats

Pulmonary arterial hypertension (PAH) is characterized by pulmonary vascular remodeling leading to right ventricular (RV) hypertrophy and failure. Intralipid (ILP), a source of parenteral nutrition for patients, contains &ggr;-linolenic acid and soy-derived phytoestrogens that are protective for lungs and heart. We, therefore, investigated the therapeutic potential of ILP in preventing and rescuing monocrotaline-induced PAH and RV dysfunction. PAH was induced in male rats with monocrotaline (60 mg/kg). Rats then received daily ILP (1 mL of 20% ILP per day IP) from day 1 to day 30 for prevention protocol or from day 21 to day 30 for rescue protocol. Other monocrotaline-injected rats were left untreated to develop severe PAH by day 21 or RV failure by approximately day 30. Saline or ILP-treated rats served as controls. Significant increase in RV pressure and decrease in RV ejection fraction in the RV failure group resulted in high mortality. Therapy with ILP resulted in 100% survival and prevented PAH-induced RV failure by preserving RV pressure and RV ejection fraction and preventing RV hypertrophy and lung remodeling. In preexisting severe PAH, ILP attenuated most lung and RV abnormalities. The beneficial effects of ILP in PAH seem to result from the interplay of various factors, among which preservation and/or stimulation of angiogenesis, suppression and/or reversal of inflammation, fibrosis and hypertrophy, in both lung and RV, appear to be major contributors. In conclusion, ILP not only prevents the development of PAH and RV failure but also rescues preexisting severe PAH.

Rescue of Pressure Overload‐Induced Heart Failure by Estrogen Therapy

Background Estrogen pretreatment has been shown to attenuate the development of heart hypertrophy, but it is not known whether estrogen could also rescue heart failure (HF). Furthermore, the heart has all the machinery to locally biosynthesize estrogen via aromatase, but the role of local cardiac estrogen synthesis in HF has not yet been studied. Here we hypothesized that cardiac estrogen is reduced in HF and examined whether exogenous estrogen therapy can rescue HF. Methods and Results HF was induced by transaortic constriction in mice, and once mice reached an ejection fraction (EF) of ≈35%, they were treated with estrogen for 10 days. Cardiac structure and function, angiogenesis, and fibrosis were assessed, and estrogen was measured in plasma and in heart. Cardiac estrogen concentrations (6.18±1.12 pg/160 mg heart in HF versus 17.79±1.28 pg/mL in control) and aromatase transcripts (0.19±0.04, normalized to control, P<0.05) were significantly reduced in HF. Estrogen therapy increased cardiac estrogen 3‐fold and restored aromatase transcripts. Estrogen also rescued HF by restoring ejection fraction to 53.1±1.3% (P<0.001) and improving cardiac hemodynamics both in male and female mice. Estrogen therapy stimulated angiogenesis as capillary density increased from 0.66±0.07 in HF to 2.83±0.14 (P<0.001, normalized to control) and reversed the fibrotic scarring observed in HF (45.5±2.8% in HF versus 5.3±1.0%, P<0.001). Stimulation of angiogenesis by estrogen seems to be one of the key mechanisms, since in the presence of an angiogenesis inhibitor estrogen failed to rescue HF (ejection fraction=29.3±2.1%, P<0.001 versus E2). Conclusions Estrogen rescues pre‐existing HF by restoring cardiac estrogen and aromatase, stimulating angiogenesis, and suppressing fibrosis.

Apolipoprotein-A1 Mimetic Peptide 4F Rescues Severe Pulmonary Hypertension in Rats and Inhibits Human Pulmonary Artery Smooth Muscle Cell Proliferation In Vitro

Pulmonary hypertension (PH) is a characterized by arterial obstruction resulting from proliferation of pulmonary artery smooth muscle and endothelial cells. Genetic deletion of apolipoprotein-A1 increases airway hyperresponsiveness, inflammation, and collagen deposition in the lung. Apolipoprotein-A1 mimetic peptide 4F protects endothelial function, causes vasodilation, decreases inflammation and oxidative stress in lungs, yet its role in treating PH and right ventricular (RV) dysfunction is not known. We hypothesized that 4F may rescue pre-existing severe PH. We also investigate the effects of 4F on human pulmonary artery smooth muscle cell (hPASMC) proliferation in vitro as a possible mechanism of rescue by 4F. Twenty three rats were randomly divided into 4-groups. PH was induced by monocrotaline (MCT, 60mg/kg, s.c.). Severe PH was well-established at day-21 (PH, n=6) that progressed to RV failure (RVF, n=6) by day-30. One MCT-group was treated with 4F (50mg/kg/day, s.c., n=6) from day-21 to 30. Saline-treated rats served as control (CTRL, n=5). Serial echocardiography was performed to monitor cardiopulmonary hemodynamics. Cardiac catheterization was performed terminally to record RV-pressure (RVP). hPASMCs proliferation was assessed by MTT-assay. p<0.05 was considered significant. Values were mean±SE. Rats developed severe PH 21-days after MCT (RVP=67.12±1 vs. 29.8±1 mmHg in CTRL, RV/LV+IVS= 0.65±0.05 vs. 0.23±0.02, RV-ejection fraction (RVEF)= 40±1 vs. 65±1%, all p<0.05 vs. CTRL), which progressed to RVF by day-30 [RVP=74±1; RV/(LV+IVS)=0.68±0.05; RVEF=28.6±1%, p<0.05 for all vs. CTRL]. 4F-therapy from day-21 to 30 resulted in rescue of PH (RVP=47±3 mmHg, RV/LV+IVS= 0.38±0.02, RVEF= 51.7±3%, p<0.05 vs. PH and RVF). 4F also inhibited hPASMC proliferation (∼60% inhibition at 100ng 4F/ml medium, p<0.05). In conclusion, 4F rescues pre-existing severe PH and RV-dysfunction. Inhibition of PASMC proliferation may be one of the key mechanisms in this rescue.

Estrogen Receptor Beta, but Not Alpha, is the Key Player in Restoration of Heart Function of Heart Failure Mice by Estrogen Therapy

Recently we discovered that estrogen(E2) therapy can rescue advanced heart failure(HF) induced by pressure overload in mice. Most of the biological actions of estrogen are mediated thorough estrogen receptors alpha(ERa) and beta(ERb), and both of these receptors are present in the heart. Here we investigated which estrogen receptor(s) are involved in the rescue by estrogen. We used the transaortic constriction(TAC) procedure to induce HF. Once the ejection fraction(EF) reached ∼30%, one group of animals was sacrificed(HF group), and the other three groups received either 17b-estradiol (30 ug/kg/day), selective ERa agonist (PPT, 0.625mg/kg/day), or selective ERb agonist (DPN, 0.625mg/kg/day) for 10 days. Serial echocardiography was performed and LV pressure was measured by direct catheterization before sacrifice. As expected, E2 rescued HF by restoring EF from 33.17±1.12% to 53.05±1.29%. Interestingly, mice treated with ERb agonist had a significant improvement in their EF from 33.17±1.12% to 45.25±2.1%(n=7), whereas the EF of mice treated with ERa agonist did not improve at all(31.09±2.3%, n=6). Similar to EF, only fractional shortening of DPN-treated mice improved from 15.7±0.58% in HF to 21.95±1.65% in DPN vs. 14.72±1.24% in PPT). Next, we examined the mechanical performance of the LV in mice treated with DPN and PPT. DPN treatment improved LVDP from 94.68±5.14 in HF to 119.3±5.5 and RPP from 39749.9±4527.03 in HF to 62794.04±5534.62. PPT treatment had no effect on LV mechanical performance as both LVDP and RPP were not significantly different from their corresponding values in HF. The relaxation and contraction defects of HF mice (dP/dtmax and dP/dtmin) were also partially restored by DPN but not by PPT. Our data strongly support the view that ERb is the main player in rescuing advanced HF by E2.

Molecular Mechanisms Involved in the Rescue of Severe Pulmonary Hypertension by Genistein Therapy

Previously, we showed that genistein, a soy isoflavone, rescued severe pulmonary hypertension(PH). However, the mechanisms involved in the rescue were largely unknown. Here, we investigated possible mechanisms of genistein rescue of PH. We induced PH in rats using a single subcutaneous injection of monocrotaline(MCT, 60 mg/kg). By day 21, rats developed severe PH. At this time point, we started genistein therapy(1 mg/kg/day, subcutaneous) to one group(GEN) until day 30. The other group was left untreated and developed RV failure by day 30(MCT group). The control group(CTRL) received saline. At day 30, cardiac catheterization was performed to assess right ventricular pressure(RVP), animals were sacrificed and lungs and hearts were dissected. Immunohistochemistry, Western Blot, and RT PCR were performed. MCT group developed severe PH(RVP 31±1 mmHg in CTRL, n=5 vs. 72±1 mmHg in MCT, n=7). Genistein attenuated severe PH(44±5 mmHg, n=8). Additionally, the RV hypertrophy index(RV/(LV+IVS)) increased(∼3-fold) in MCT and was restored in GEN. We investigated the role of estrogen receptors α and β(ERα and ERβ) in genistein rescue. ERβ protein levels were significantly downregulated in lungs and RV of MCT(∼2-fold and ∼5-fold). Genistein restored lung and RV ERβ protein levels. ERα protein levels did not change in PH. PH led to significantly decreased capillary density in RV and VEGF protein(∼2.5-fold) in both RV and lung. Genistein restored RV capillary density and significantly improved VEGF levels in RV and lung. Furthermore, we found a significant increase in lung Caspase-3(∼3-fold) and pSTAT3/STAT3(∼3.5-fold) proteins in MCT, which were reversed by Genistein. PH was also associated with significantly reduced lung Caveolin-1(∼6-fold) protein levels that were restored in GEN. In conclusion, genistein rescues severe PH through ERβ mediated protection, preserves cardiopulmonary angiogenesis, and restores Caspase-3, pSTAT3/STAT3 and Caveolin-1 in the lungs.

Stimulation of Cardiac Neoangiogenesis by Estrogen Therapy is One of the Key Mechanisms in Reversing Advanced Heart Failure

Estrogen(E2) has been shown to regulate angiogenesis in different tissues, but it is still not known if E2 could stimulate angiogenesis in the heart. Recently, we showed that E2 rescues advanced heart failure by reversing the myocardial contractile deficiency and restoring ejection fraction from ∼30% to ∼55%. Here we examined whether stimulation of angiogenesis in the heart is a mechanism involved in the E2-induced rescue of HF. Trans-aortic constriction(TAC) procedure was used to induce HF. Once the ejection fraction(EF) reached ∼30%, one group of mice was sacrificed and the other two groups were treated with E2(30 μg/kg/day, n=16), or E2 plus the angiogenesis inhibitor TNP-470(TNP, 30 mg/kg, n=4) for 10 days. Serial echocardiography, real-time PCR and immunocytochemistry were performed. RT-PCR showed that the transcript levels of two markers of angiogenesis, vascular endothelial growth factor(VEGF) and hypoxia-inducible factor-1a(HIF1a), were ∼10 fold downregulated in HF(0.17±0.06 for VEGF and 0.26±0.01 for HiF1a; normalized to CTRL). E2 treatment was not only able to reverse VEGF and HIF1a transcript level downregulation observed in HF, but to even upregulate both transcripts 3 fold higher than in healthy controls(3.24±0.1 for VEGF and 3.16±0.09 for HIF1a). Quantification of capillary density also revealed that E2 therapy not only completely reversed the loss of capillaries in HF, but significantly enhanced capillary density by ∼ 4 fold compared to HF(2.83±0.14 in E2 vs. 0.66±0.07 in HF, normalized to CTRL). Interestingly, E2 failed to rescue HF in the presence of TNP-470 (E2+TNP group) as EF (29.3±2.1%) was not significantly improved after 10 days of therapy. The capillary density of HF mice also did not improve in E2+TNP group(0.53±0.07). These data strongly support the vital role of angiogenesis in the rescue action of E2.