Effects of activity levels on aortic calcification in hyperlipidemic mice as measured by microPETmicroCT.

BACKGROUND AND AIMS: Cardiovascular disease risk is associated with coronary artery calcification and is mitigated by regular exercise. Paradoxically, elite endurance athletes, who have low risk, are likely to have more coronary calcification, raising questions about the optimal level of activity. METHODS: Female hyperlipidemic (Apoe-/-) mice with baseline aortic calcification were subjected to high-speed (18.5 m/min), low-speed (12.5 m/min), or no treadmill exercise for 9 weeks. 18F-NaF microPET/CT images were acquired at weeks 0 and 9, and echocardiography was performed at week 9. RESULTS: In controls, aortic calcium content and density increased significantly. Exercise regimens did not alter the time-dependent increase in content, but the increase in mean density was blunted. Interestingly, the low-speed regimen significantly reduced 18F-NaF uptake, a marker of surface area. Left ventricular (LV) systolic function was lower while LV diameter was greater in the low-speed group compared with controls or the high-speed group. In the low-speed group, vertebral bone density by CT decreased significantly, contrary to expectations. Male hyperlipidemic (Apoe-/-) mice were fed a Western diet and also subjected to low-speed or no exercise followed by imaging at weeks 0 and 9. In males, exercise also did not alter the time-dependent increase in aortic calcification. Exercise did not affect 18F-NaF uptake or bone mineral density, but it blunted the diet-induced LV hypertrophy seen in controls. CONCLUSIONS: These results suggest that, in mice, exercise has differential effects on aortic calcification, cardiac function, and skeletal bone mineral density.

Tm4sf1-marked Endothelial Subpopulation Is Dysregulated in Pulmonary Arterial Hypertension.

The identification and role of endothelial progenitor cells in pulmonary arterial hypertension (PAH) remain controversial. Single-cell omics analysis can shed light on endothelial progenitor cells and their potential contribution to PAH pathobiology. We aim to identify endothelial cells that may have stem/progenitor potential in rat lungs and assess their relevance to PAH. Differential expression, gene set enrichment, cell-cell communication, and trajectory reconstruction analyses were performed on lung endothelial cells from single-cell RNA sequencing of Sugen-hypoxia, monocrotaline, and control rats. Relevance to human PAH was assessed in multiple independent blood and lung transcriptomic data sets. Rat lung endothelial cells were visualized by immunofluorescence in situ, analyzed by flow cytometry, and assessed for tubulogenesis in vitro. A subpopulation of endothelial cells (endothelial arterial type 2 [EA2]) marked by Tm4sf1 (transmembrane 4 L six family member 1), a gene strongly implicated in cancer, harbored a distinct transcriptomic signature enriched for angiogenesis and CXCL12 signaling. Trajectory analysis predicted that EA2 has a less differentiated state compared with other endothelial subpopulations. Analysis of independent data sets revealed that TM4SF1 is downregulated in lungs and endothelial cells from patients and PAH models, is a marker for hematopoietic stem cells, and is upregulated in PAH circulation. TM4SF1+CD31+ rat lung endothelial cells were visualized in distal pulmonary arteries, expressed hematopoietic marker CD45, and formed tubules in coculture with lung fibroblasts. Our study uncovered a novel Tm4sf1-marked subpopulation of rat lung endothelial cells that may have stem/progenitor potential and demonstrated its relevance to PAH. Future studies are warranted to further elucidate the role of EA2 and Tm4sf1 in PAH.

A novel negative pressure isolation device reduces aerosol exposure: A randomized controlled trial.

Background and aim: The COVID-19 pandemic has led to a proliferation of intubation barriers designed to protect healthcare workers from infection. We developed the Suction-Assisted Local Aerosol Containment Chamber (SLACC) and tested it in the operating room. The primary objectives were to determine the ease and safety of airway management with SLACC, and to measure its efficacy of aerosol containment to determine if it significantly reduces exposure to health care workers. Methods: In this randomized clinical trial, adult patients scheduled to undergo elective surgery with general endotracheal anesthesia were screened and informed consent obtained from those willing to participate. Patients were randomized to airway management either with or without the SLACC device. Patients inhaled nebulized saline before and during anesthesia induction to simulate the size and concentration of particles seen with severe symptomatic SARS-CoV-2 infection. Results: 79 patients were enrolled and randomized. Particle number concentration (PNC) at the patients' and healthcare workers' locations were measured and compared between the SLACC vs. control groups during airway management. Ease and success of tracheal intubation were recorded for each patient. All intubations were successful and time to intubation was similar between the two groups. Healthcare workers were exposed to significantly lower particle number concentrations (#/cm3) during airway management when SLACC was utilized vs. control. The particle count outside SLACC was reduced by 97% compared to that inside the device. Conclusions: The SLACC device does not interfere with airway management and significantly reduces healthcare worker exposure to aerosolized particles during airway management.

Bmpr2 mutant mice are an inadequate model for studying iron deficiency in pulmonary hypertension.

As bone morphogenetic protein receptor type II (Bmpr2) mutations are the most common genetic cause of pulmonary arterial hypertension (PAH), and iron deficiency (ID) is associated with worse clinical outcomes in PAH patients, we proposed to use Bmpr2 ± mice to induce a model of ID in pulmonary vascular disease. Our study shows that these transgenic mice are not a good model for this clinical phenomenon.

Transcriptomic Analysis of Right Ventricular Remodeling in Two Rat Models of Pulmonary Hypertension: Identification and Validation of Epithelial-to-Mesenchymal Transition in Human Right Ventricular Failure.

BACKGROUND: Right ventricular (RV) dysfunction is a significant prognostic determinant of morbidity and mortality in pulmonary arterial hypertension (PAH). Despite the importance of RV function in PAH, the underlying molecular mechanisms of RV dysfunction secondary to PAH remain unclear. We aim to identify and compare molecular determinants of RV failure using RNA sequencing of RV tissue from 2 clinically relevant animal models of PAH. METHODS: We performed RNA sequencing on RV from rats treated with monocrotaline or Sugen with hypoxia/normoxia. PAH and RV failure were confirmed by catheterization and echocardiography. We validated the RV transcriptome results using quantitative real-time polymerase chain reaction, immunofluorescence, and Western blot. Immunohistochemistry and immunofluorescence were performed on human RV tissue from control (n=3) and PAH-induced RV failure patients (n=5). RESULTS: We identified similar transcriptomic profiles of RV from monocrotaline- and Sugen with hypoxia-induced RV failure. Pathway analysis showed genes enriched in epithelial-to-mesenchymal transition, inflammation, and metabolism. Histological staining of human RV tissue from patients with RV failure secondary to PAH revealed significant RV fibrosis and endothelial-to-mesenchymal transition, as well as elevated cellular communication network factor 2 (top gene implicated in epithelial-to-mesenchymal transition/endothelial-to-mesenchymal transition) expression in perivascular areas compared with normal RV. CONCLUSIONS: Transcriptomic signature of RV failure in monocrotaline and Sugen with hypoxia models showed similar gene expressions and biological pathways. We provide translational relevance of this transcriptomic signature using RV from patients with PAH to demonstrate evidence of epithelial-to-mesenchymal transition/endothelial-to-mesenchymal transition and protein expression of cellular communication network factor 2 (CTGF [connective tissue growth factor]). Targeting specific molecular mechanisms responsible for RV failure in monocrotaline and Sugen with hypoxia models may identify novel therapeutic strategies for PAH-associated RV failure.

Oral 15-Hydroxyeicosatetraenoic Acid Induces Pulmonary Hypertension in Mice by Triggering T Cell-Dependent Endothelial Cell Apoptosis.

Pulmonary arterial hypertension (PAH) is a fatal disease characterized by increased mean pulmonary arterial pressure. Elevated plasma and lung concentrations of oxidized lipids, including 15-hydroxyeicosatetraenoic acid (15-HETE), have been demonstrated in patients with PAH and animal models. We previously demonstrated that feeding mice with 15-HETE is sufficient to induce pulmonary hypertension, but the mechanisms remain unknown. RNA sequencing data from the mouse lungs on 15-HETE diet revealed significant activation of pathways involved in both antigen processing and presentation and T cell-mediated cytotoxicity. Analysis of human microarray from patients with PAH also identified activation of identical pathways compared with controls. We show that in both 15-HETE-fed mice and patients with PAH, expression of the immunoproteasome subunit 5 is significantly increased, which was concomitant with an increase in the number of CD8/CD69 (cluster of differentiation 8 / cluster of differentiation 69) double-positive cells, as well as pulmonary arterial endothelial cell apoptosis in mice. Human pulmonary arterial endothelial cells cultured with 15-HETE were more prone to apoptosis when exposed to CD8 cells. Cultured intestinal epithelial cells secreted more oxidized lipids in response to 15-HETE, which is consistent with accumulation of circulating oxidized lipids in 15-HETE-fed mice. Administration of an apoA-I (apolipoprotein A-I) mimetic peptide, Tg6F (transgenic 6F), which is known to prevent accumulation of circulating oxidized lipids, not only inhibited pulmonary arterial endothelial cell apoptosis but also prevented and rescued 15-HETE-induced pulmonary hypertension in mice. In conclusion, our results suggest that (1) 15-HETE diet induces pulmonary hypertension by a mechanism that involves oxidized lipid-mediated T cell-dependent pulmonary arterial endothelial cell apoptosis and (2) Tg6F administration may be a novel therapy for treating PAH.

The Role of Estrogen Receptors in Cardiovascular Disease.

Cardiovascular Diseases (CVDs) are the leading cause of death globally. More than 17 million people die worldwide from CVD per year. There is considerable evidence suggesting that estrogen modulates cardiovascular physiology and function in both health and disease, and that it could potentially serve as a cardioprotective agent. The effects of estrogen on cardiovascular function are mediated by nuclear and membrane estrogen receptors (ERs), including estrogen receptor alpha (ERα), estrogen receptor beta (ERß), and G-protein-coupled ER (GPR30 or GPER). Receptor binding in turn confers pleiotropic effects through both genomic and non-genomic signaling to maintain cardiovascular homeostasis. Each ER has been implicated in multiple pre-clinical cardiovascular disease models. This review will discuss current reports on the underlying molecular mechanisms of the ERs in regulating vascular pathology, with a special emphasis on hypertension, pulmonary hypertension, and atherosclerosis, as well as in regulating cardiac pathology, with a particular emphasis on ischemia/reperfusion injury, heart failure with reduced ejection fraction, and heart failure with preserved ejection fraction.

Histological hallmarks and role of Slug/PIP axis in pulmonary hypertension secondary to pulmonary fibrosis.

Pulmonary hypertension secondary to pulmonary fibrosis (PF-PH) is one of the most common causes of PH, and there is no approved therapy. The molecular signature of PF-PH and underlying mechanism of why pulmonary hypertension (PH) develops in PF patients remains understudied and poorly understood. We observed significantly increased vascular wall thickness in both fibrotic and non-fibrotic areas of PF-PH patient lungs compared to PF patients. The increased vascular wall thickness in PF-PH patients is concomitant with a significantly increased expression of the transcription factor Slug within the macrophages and its target prolactin-induced protein (PIP), an extracellular matrix protein that induces pulmonary arterial smooth muscle cell proliferation. We developed a novel translational rat model of combined PF-PH that is reproducible and shares similar histological features (fibrosis, pulmonary vascular remodeling) and molecular features (Slug and PIP upregulation) with human PF-PH. We found Slug inhibition decreases PH severity in our animal model of PF-PH. Our study highlights the role of Slug/PIP axis in PF-PH.

Estrogen rescues heart failure through estrogen receptor Beta activation.

BACKGROUND: Recently, we showed that exogenous treatment with estrogen (E2) rescues pre-existing advanced heart failure (HF) in mice. Since most of the biological actions of E2 are mediated through the classical estrogen receptors alpha (ERα) and/or beta (ERß), and both these receptors are present in the heart, we examined the role of ERα and ERß in the rescue action of E2 against HF. METHODS: Severe HF was induced in male mice by transverse aortic constriction-induced pressure overload. Once the ejection fraction (EF) reached ~ 35%, mice were treated with selective agonists for ERα (PPT, 850 µg/kg/day), ERß (DPN, 850 µg/kg/day), or E2 (30 µg/kg/day) together with an ERß-antagonist (PHTPP, 850 µg/kg/day) for 10 days. RESULTS: EF of HF mice was significantly improved to 45.3 ± 2.1% with diarylpropionitrile (DPN) treatment, but not with PPT (31.1 ± 2.3%). E2 failed to rescue HF in the presence of PHTPP, as there was no significant improvement in the EF at the end of the 10-day treatment (32.5 ± 5.2%). The improvement of heart function in HF mice treated with ERß agonist DPN was also associated with reduced cardiac fibrosis and increased cardiac angiogenesis, while the ERα agonist PPT had no significant effect on either cardiac fibrosis or angiogenesis. Furthermore, DPN improved hemodynamic parameters in HF mice, whereas PPT had no significant effect. CONCLUSIONS: E2 treatment rescues pre-existing severe HF mainly through ERß. Rescue of HF by ERß activation is also associated with stimulation of cardiac angiogenesis, suppression of fibrosis, and restoration of hemodynamic parameters.

The protective role of estrogen and estrogen receptors in cardiovascular disease and the controversial use of estrogen therapy.

Epidemiologic studies have previously suggested that premenopausal females have reduced incidence of cardiovascular disease (CVD) when compared to age-matched males, and the incidence and severity of CVD increases postmenopause. The lower incidence of cardiovascular disease in women during reproductive age is attributed at least in part to estrogen (E2). E2 binds to the traditional E2 receptors (ERs), estrogen receptor alpha (ERα), and estrogen receptor beta (ERß), as well as the more recently identified G-protein-coupled ER (GPR30), and can exert both genomic and non-genomic actions. This review summarizes the protective role of E2 and its receptors in the cardiovascular system and discusses its underlying mechanisms with an emphasis on oxidative stress, fibrosis, angiogenesis, and vascular function. This review also presents the sexual dimorphic role of ERs in modulating E2 action in cardiovascular disease. The controversies surrounding the clinical use of exogenous E2 as a therapeutic agent for cardiovascular disease in women due to the possible risks of thrombotic events, cancers, and arrhythmia are also discussed. Endogenous local E2 biosynthesis from the conversion of testosterone to E2 via aromatase enzyme offers a novel therapeutic paradigm. Targeting specific ERs in the cardiovascular system may result in novel and possibly safer therapeutic options for cardiovascular protection.

Severe pulmonary hypertension in aging female apolipoprotein E-deficient mice is rescued by estrogen replacement therapy.

BACKGROUND: Apolipoprotein E (ApoE) is a multifunctional protein, and its deficiency leads to the development of atherosclerosis in mice. Patients with pulmonary hypertension (PH) have reduced expression of ApoE in lung tissue. ApoE is known to inhibit endothelial and smooth muscle cell proliferation and has anti-inflammatory and anti-platelet aggregation properties. Young ApoE-deficient mice have been shown to develop PH on high fat diet. The combined role of female sex and aging in the development of PH has not been investigated before. Here, we investigated the development of PH in young and middle-aged (MA) female ApoE-deficient mice and explored the role of exogenous estrogen (E2) replacement therapy for the aging females. METHODS: Wild type (WT) and ApoE-deficient female mice (Young and MA) were injected with a single intraperitoneal dose of monocrotaline (MCT, 60 mg/kg). Some ApoE-deficient MA female mice that received MCT were also treated with subcutaneous E2 pellets (0.03 mg/kg/day) from day 21 to 30 after MCT injection. Direct cardiac catheterization was performed terminally to record right ventricular systolic pressure (RVSP). Right ventricular (RV), left ventricular (LV), and interventricular septum (IVS) were dissected and weighed. Lung sections were examined using trichrome and immunofluorescence staining. Western blot analyses of lung and RV lysates were performed. RESULTS: In WT female mice, the severity of PH was similar between young and MA mice as RVSP was not significantly different (RVSP = 38.2 ± 1.2 in young vs. 40.5 ± 8.3 mmHg in MA, p < 0.05). In ApoE-deficient mice, MA females developed significantly severe PH (RVSP = 63 ± 10 mmHg) compared to young females (RVSP; 36 ± 3 mmHg, p < 0.05 vs. MA female). ApoE-deficient MA females also developed more severe RV hypertrophy compared to young females (RV hypertrophy index (RV/[LV + IVS]) = 0.53 ± 0.06 vs. 0.33 ± 0.01, p < 0.05). ApoE-deficient MA female mice manifested increased peripheral pulmonary artery muscularization and pulmonary fibrosis. E2 treatment of MA female ApoE-deficient mice resulted in a significant decrease in RVSP, reversal of pulmonary vascular remodeling, and RV hypertrophy. In MA female ApoE-deficient mice with PH, only the expression of ERß in the lungs, but not in RV, was significantly downregulated, and it was restored by E2 treatment. The expression of ERα was not affected in either lungs or RV by PH. GPR30 was only detected in the RV, and it was not affected by PH in MA female ApoE-deficient mice. CONCLUSIONS: Our results suggest that only aging female ApoE-deficient but not WT mice develop severe PH compared to younger females. Exogenous estrogen therapy rescued PH and RV hypertrophy in aging female ApoE-deficient mice possibly through restoration of lung ERß.

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.

Role of miR206 in genistein-induced rescue of pulmonary hypertension in monocrotaline model.

Pulmonary hypertension (PH) is a progressive lung disease associated with proliferation of smooth muscle cells and constriction of lung microvasculature, leading to increased pulmonary arterial pressure, right ventricular failure, and death. We have previously shown that genistein rescues preexisting established PH by significantly improving lung and heart function. (Matori H, Umar S, Nadadur RD, Sharma S, Partow-Navid R, Afkhami M, Amjedi M, Eghbali M. Hypertension 60: 425-430, 2012). Here, we have examined the role of microRNAs (miRs) in the rescue action of genistein in monocrotaline (MCT)-induced PH in rats. Our miR microarray analysis on the lung samples from control, PH, and genistein-rescue group revealed that miR206, which was robustly upregulated to ∼11-fold by PH, was completely normalized to control levels by genistein treatment. Next, we examined whether knockdown of miR206 could reverse preexisting established PH. PH was induced in male rats by 60 mg/kg of MCT, and rats received three intratracheal doses of either miR206 antagomir (10 mg/kg body wt) or scrambled miR control at days 17, 21, and 26. Knockdown of miR206 resulted in significant improvement in the cardiopulmonary function, as right ventricular pressure was significantly reduced to 38.6 ± 3.61 mmHg from 61.2 ± 5.4 mmHg in PH, and right ventricular hypertrophy index was decreased to 0.35 ± 0.04 from 0.59 ± 0.037 in PH. Knockdown of miR206 reversed PH-induced pulmonary vascular remodeling in vivo and was associated with restoration of PH-induced loss of capillaries in the lungs and induction of vascular endothelial growth factor A expression. In conclusion, miR206 antagomir therapy improves cardiopulmonary function and structure and rescues preexisting severe PH in MCT rat model possibly by stimulating angiogenesis in the lung.

Apolipoprotein A-I mimetic peptide 4F rescues pulmonary hypertension by inducing microRNA-193-3p.

BACKGROUND: Pulmonary arterial hypertension is a chronic lung disease associated with severe pulmonary vascular changes. A pathogenic role of oxidized lipids such as hydroxyeicosatetraenoic and hydroxyoctadecadienoic acids is well established in vascular disease. Apolipoprotein A-I mimetic peptides, including 4F, have been reported to reduce levels of these oxidized lipids and improve vascular disease. However, the role of oxidized lipids in the progression of pulmonary arterial hypertension and the therapeutic action of 4F in pulmonary arterial hypertension are not well established. METHODS AND RESULTS: We studied 2 different rodent models of pulmonary hypertension (PH): a monocrotaline rat model and a hypoxia mouse model. Plasma levels of hydroxyeicosatetraenoic and hydroxyoctadecadienoic acids were significantly elevated in PH. 4F treatment reduced these levels and rescued preexisting PH in both models. MicroRNA analysis revealed that microRNA-193-3p (miR193) was significantly downregulated in the lung tissue and serum from both patients with pulmonary arterial hypertension and rodents with PH. In vivo miR193 overexpression in the lungs rescued preexisting PH and resulted in downregulation of lipoxygenases and insulin-like growth factor-1 receptor. 4F restored PH-induced miR193 expression via transcription factor retinoid X receptor α. CONCLUSIONS: These studies establish the importance of microRNAs as downstream effectors of an apolipoprotein A-I mimetic peptide in the rescue of PH and suggest that treatment with apolipoprotein A-I mimetic peptides or miR193 may have therapeutic value.

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, a clinically safe compound, protects the heart against ischemia-reperfusion injury more efficiently than cyclosporine-A.

BACKGROUND: We have recently shown that postischemic administration of intralipid protects the heart against ischemia-reperfusion injury. Here we compared the cardioprotective effects of intralipid with cyclosporine-A, a potent inhibitor of the mitochondrial permeability transition pore opening. METHODS: In vivo rat hearts or isolated Langendorff-perfused mouse hearts were subjected to ischemia followed by reperfusion with intralipid (0.5%, 1% and 2% ex-vivo, and 20% in vivo), cyclosporine-A (0.2 µM, 0.8 µM, and 1.5 µM ex- vivo and 10 mg/kg in vivo), or vehicle. The hemodynamic function, infarct size, calcium retention capacity, mitochondrial superoxide production, and phosphorylation levels of protein kinase B (Akt)/glycogen synthase kinase-3ß (GSK-3ß) were measured. The values are mean ± SEM. RESULTS: Administration of intralipid at reperfusion significantly reduced myocardial infarct size compared with cyclosporine-A in vivo (infarct size/area at risk)%: 22.9 ± 2.5% vs. 35.2 ± 3.5%; P = 0.030, n = 7/group). Postischemic administration of intralipid at its optimal dose (1%) was more effective than cyclosporine-A (0.8 µM) in protecting the ex vivo heart against ischemia-reperfusion injury, as the rate pressure product at the end of reperfusion was significantly higher (mmHg · beats/min: 12,740 ± 675 [n = 7] vs. 9,203 ± 10,781 [n = 5], P = 0.024), and the infarct size was markedly smaller (17.3 ± 2.9 [n = 7] vs. 29.2 ± 2.7 [n = 5], P = 0.014). Intralipid was as efficient as cyclosporine-A in inhibiting the mitochondrial permeability transition pore opening (calcium retention capacity = 280 ± 8.2 vs. 260.3 ± 2.9 nmol/mg mitochondria protein in cyclosporine-A, P = 0.454, n = 6) and in reducing cardiac mitochondrial superoxide production. Unlike intralipid, which increased phosphorylation of Akt (6-fold) and GSK-3ß (5-fold), cyclosporine-A had no effect on the activation of these prosurvival kinases. CONCLUSIONS: Although intralipid inhibits the opening of the mitochondrial permeability transition pore as efficiently as cyclosporine-A, intralipid is more effective in reducing the infarct size and improving the cardiac functional recovery.

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-ß. 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-ß 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 vulnerability to ischemia/reperfusion injury drastically increases in late pregnancy.

Although the murine late pregnant (LP) heart is speculated to be a better functioning heart during physiological conditions, the susceptibility of LP hearts to I/R injury is still unknown. The aims of this study were to investigate the cardiac vulnerability of LP rodents to ischemia/reperfusion (I/R) injury and to explore its underlying mechanisms. In vivo female rat hearts [non-pregnant (NP) or LP] or ex vivo Langendorff-perfused mouse hearts were subjected to I/R. The infarct size was approximately fourfold larger in LP animals compared with NP both in vivo and ex vivo. The heart functional recovery was extremely poor in LP mice compared with NP (~10% recovery in LP vs. 80% recovery in NP at the end of reperfusion, P < 0.01). Interestingly, the poor functional recovery and the larger infarct size in LP were partially restored one day post-partum and almost fully restored 1 week post-partum to their corresponding NP levels. Mitochondrial respiratory function and the threshold for opening of the mitochondrial permeability transition pore were significantly lower in LP compared with NP when they both were subjected to myocardial I/R injury [Respiratory control ratio = 1.9 ± 0.1 vs. 4.0 ± 0.5 in NP, P < 0.05; calcium retention capacity (CRC) = 167 ± 10 vs. 233 ± 18 nmol/mg protein in NP, P < 0.01]. Cardiac reactive oxygen species (ROS) generation, as well mitochondrial superoxide production, was approximately twofold higher in LP compared with NP following I/R. The phosphorylation levels of Akt, ERK1/2, and STAT3, but not GSK3ß, were significantly reduced in the hearts from LP subjected to I/R. In conclusion, increased mitochondrial ROS generation, decreased CRC as well as impaired activation of Akt/ERK/STAT3 at reperfusion are the possible underlying mechanisms for higher vulnerability of LP hearts to I/R.

Reverse right ventricular structural and extracellular matrix remodeling by estrogen in severe pulmonary hypertension.

Chronic pulmonary hypertension (PH) leads to right-ventricular failure (RVF) characterized by RV remodeling. Ventricular remodeling is emerging as an important process during heart failure and recovery. Remodeling in RVF induced by PH is not fully understood. Recently we discovered that estrogen (E2) therapy can rescue severe preexisting PH. Here, we focused on whether E2 (42.5 µg·kg(-1)·day(-1), 10 days) can reverse adverse RV structural and extracellular matrix (ECM) remodeling induced by PH using monocrotaline (MCT, 60 mg/kg). RV fibrosis was evident in RVF males. Intact females developed less severe RV remodeling compared with males and ovariectomized (OVX) females. Novel ECM-degrading disintegrin-metalloproteinases ADAM15 and ADAM17 transcripts were elevated ∼2-fold in all RVF animals. E2 therapy reversed RV remodeling in all groups. In vitro, E2 directly inhibited ANG II-induced expression of fibrosis markers as well as the metalloproteinases in cultured cardiac fibroblasts. Estrogen receptor-ß agonist diarylpropionitrile (DPN) but not estrogen receptor-α agonist 4,4',4″-(4-propyl-[1H]-pyrazole-1,3,5-triyl)trisphenol (PPT) was as effective as E2 in inhibiting expression of these genes. Expression of ECM-interacting cardiac fetal-gene osteopontin (OPN) also increased ∼9-fold in RVF males. Intact females were partially protected from OPN upregulation (∼2-fold) but OVX females were not. E2 reversed OPN upregulation in all groups. Upregulation of OPN was also reversed in vitro by E2. Plasma OPN was elevated in RVF (∼1.5-fold) and decreased to control levels in the E2 group. RVF resulted in elevated Akt phosphorylation, but not ERK, in the RV, and E2 therapy restored Akt phosphorylation. In conclusion, E2 therapy reverses adverse RV remodeling associated with PH by reversing fibrosis and upregulation of novel ECM enzymes ADAM15, ADAM17, and OPN. These effects are likely mediated through estrogen receptor-ß.