Endothelial specific prolyl hydroxylase domain-containing protein 2 deficiency attenuates aging-related obesity and exercise intolerance.

Obesity and exercise intolerance greatly reduce the life quality of older people. Prolyl hydroxylase domain-containing protein 2 (PHD2) is an important enzyme in modulating hypoxia-inducible factor-alpha (HIF) protein. Using vascular endothelial cell-specific PHD2 gene knockout (PHD2 ECKO) mice, we investigated the role of endothelial PHD2 in aging-related obesity and exercise capacity. Briefly, PHD2 ECKO mice were obtained by crossing PHD2-floxed mice with VE-Cadherin (Cdh5)-Cre transgenic mice. The effect of PHD2 ECKO on obesity and exercise capacity in PHD2 ECKO mice and control PHD2f/f mice were determined in young mice (6 to 7 months) and aged mice (16-18 months). We found that aged PHD2 ECKO mice, but not young mice, exhibited a lean phenotype, characterized by lower fat mass, and its ratio to lean weight, body weight, or tibial length, while their food uptake was not reduced compared with controls. Moreover, as compared with aged control mice, aged PHD2 ECKO mice exhibited increased oxygen consumption at rest and during exercise, and the maximum rate of oxygen consumption (VO2 max) during exercise. Furthermore, as compared with corresponding control mice, both young and aged PHD2 ECKO mice demonstrated improved glucose tolerance and lower insulin resistance. Together, these data demonstrate that inhibition of vascular endothelial PHD2 signaling significantly attenuates aging-related obesity, exercise intolerance, and glucose intolerance.

p53 Acetylation Exerts Critical Roles in Pressure Overload-Induced Coronary Microvascular Dysfunction and Heart Failure in Mice.

BACKGROUND: Coronary microvascular dysfunction (CMD) has been shown to contribute to cardiac hypertrophy and heart failure (HF) with preserved ejection fraction. At this point, there are no proven treatments for CMD. METHODS: We have shown that histone acetylation may play a critical role in the regulation of CMD. By using a mouse model that replaces lysine with arginine at residues K98, K117, K161, and K162R of p53 (p534KR), preventing acetylation at these sites, we test the hypothesis that acetylation-deficient p534KR could improve CMD and prevent the progression of hypertensive cardiac hypertrophy and HF. Wild-type and p534KR mice were subjected to pressure overload by transverse aortic constriction to induce cardiac hypertrophy and HF. RESULTS: Echocardiography measurements revealed improved cardiac function together with a reduction of apoptosis and fibrosis in p534KR mice. Importantly, myocardial capillary density and coronary flow reserve were significantly improved in p534KR mice. Moreover, p534KR upregulated the expression of cardiac glycolytic enzymes and Gluts (glucose transporters), as well as the level of fructose-2,6-biphosphate; increased PFK-1 (phosphofructokinase 1) activity; and attenuated cardiac hypertrophy. These changes were accompanied by increased expression of HIF-1α (hypoxia-inducible factor-1α) and proangiogenic growth factors. Additionally, the levels of SERCA-2 were significantly upregulated in sham p534KR mice, as well as in p534KR mice after transverse aortic constriction. In vitro, p534KR significantly improved endothelial cell glycolytic function and mitochondrial respiration and enhanced endothelial cell proliferation and angiogenesis. Similarly, acetylation-deficient p534KR significantly improved coronary flow reserve and rescued cardiac dysfunction in SIRT3 (sirtuin 3) knockout mice. CONCLUSIONS: Our data reveal the importance of p53 acetylation in coronary microvascular function, cardiac function, and remodeling and may provide a promising approach to improve hypertension-induced CMD and to prevent the transition of cardiac hypertrophy to HF.

TIGAR Deficiency Blunts Angiotensin-II-Induced Cardiac Hypertrophy in Mice.

Hypertension is the key contributor to pathological cardiac hypertrophy. Growing evidence indicates that glucose metabolism plays an essential role in cardiac hypertrophy. TP53-induced glycolysis and apoptosis regulator (TIGAR) has been shown to regulate glucose metabolism in pressure overload-induced cardiac remodeling. In the present study, we investigated the role of TIGAR in cardiac remodeling during Angiotensin II (Ang-II)-induced hypertension. Wild-type (WT) and TIGAR knockout (KO) mice were infused with Angiotensin-II (Ang-II, 1 µg/kg/min) via mini-pump for four weeks. The blood pressure was similar between the WT and TIGAR KO mice. The Ang-II infusion resulted in a similar reduction of systolic function in both groups, as evidenced by the comparable decrease in LV ejection fraction and fractional shortening. The Ang-II infusion also increased the isovolumic relaxation time and myocardial performance index to the same extent in WT and TIGAR KO mice, suggesting the development of similar diastolic dysfunction. However, the knockout of TIGAR significantly attenuated hypertension-induced cardiac hypertrophy. This was associated with higher levels of fructose 2,6-bisphosphate, PFK-1, and Glut-4 in the TIGAR KO mice. Our present study suggests that TIGAR is involved in the control of glucose metabolism and glucose transporters by Ang-II and that knockout of TIGAR attenuates the development of maladaptive cardiac hypertrophy.

The Therapeutic Potential of Targeting Ferroptosis in the Treatment of Mitochondrial Cardiomyopathies and Heart Failure.

ABSTRACT: Ferroptosis is a form of iron-regulated cell death implicated in a wide array of diseases, including heart failure, hypertension, and numerous cardiomyopathies. In addition, mitochondrial dysfunction has been associated with several of these same disease states. However, the role of the mitochondrion in ferroptotic cell death remains debated. As a major regulator of cellular iron levels, the mitochondria may very well play a crucial role in the mechanisms behind ferroptosis, but at this point, this has not been adequately defined. Emerging evidence from our laboratory and others indicates a critical role of mitochondrial Sirtuin 3, a deacetylase linked with longevity and protection against numerous conditions, in the prevention of cardiovascular diseases. Here, we provide a brief overview of the potential roles of Sirtuin 3 in mitochondrial iron homeostasis and its contribution to the mitochondrial cardiomyopathy of Friedreich's ataxia and diabetic cardiomyopathy. We also discuss the current knowledge of the involvement of ferroptosis and the mitochondria in these and other cardiovascular disease states, including doxorubicin-induced cardiomyopathy, and provide insight into areas requiring further investigation.

Inhibition of NK1.1 signaling attenuates pressure overload-induced heart failure, and consequent pulmonary inflammation and remodeling.

Background: Inflammation contributes to heart failure (HF) development, the progression from left ventricular failure to pulmonary remodeling, and the consequent right ventricular hypertrophy and failure. NK1.1 plays a critical role in Natural killer (NK) and NK T (NKT) cells, but the role of NK1.1 in HF development and progression is unknown. Methods: We studied the effects of NK1.1 inhibition on transverse aortic constriction (TAC)-induced cardiopulmonary inflammation, HF development, and HF progression in immunocompetent male mice of C57BL/6J background. Results: We found that NK1.1+ cell-derived interferon gamma+ (IFN-γ+) was significantly increased in pulmonary tissues after HF. In addition, anti-NK1.1 antibodies simultaneously abolished both NK1.1+ cells, including the NK1.1+NK and NK1.1+NKT cells in peripheral blood, spleen, and lung tissues, but had no effect on cardiopulmonary structure and function under control conditions. However, systemic inhibition of NK1.1 signaling by anti-NK1.1 antibodies significantly rescued mice from TAC-induced left ventricular inflammation, fibrosis, and failure. Inhibition of NK1.1 signaling also significantly attenuated TAC-induced pulmonary leukocyte infiltration, fibrosis, vessel remodeling, and consequent right ventricular hypertrophy. Moreover, inhibition of NK1.1 signaling significantly reduced TAC-induced pulmonary macrophage and dendritic cell infiltration and activation. Conclusions: Our data suggest that inhibition of NK1.1 signaling is effective in attenuating systolic overload-induced cardiac fibrosis, dysfunction, and consequent pulmonary remodeling in immunocompetent mice through modulating the cardiopulmonary inflammatory response.

SIRT3 Deficiency Enhances Ferroptosis and Promotes Cardiac Fibrosis via p53 Acetylation.

Cardiac fibrosis plays an essential role in the development of diastolic dysfunction and contributes to heart failure with preserved ejection fraction (HFpEF). Our previous studies suggested Sirtuin 3 (SIRT3) as a potential target for cardiac fibrosis and heart failure. In the present study, we explored the role of SIRT3 in cardiac ferroptosis and its contribution to cardiac fibrosis. Our data showed that knockout of SIRT3 resulted in a significant increase in ferroptosis, with increased levels of 4-hydroxynonenal (4-HNE) and downregulation of glutathione peroxidase 4 (GPX-4) in the mouse hearts. Overexpression of SIRT3 significantly blunted ferroptosis in response to erastin, a known ferroptosis inducer, in H9c2 myofibroblasts. Knockout of SIRT3 resulted in a significant increase in p53 acetylation. Inhibition of p53 acetylation by C646 significantly alleviated ferroptosis in H9c2 myofibroblasts. To further explore the involvement of p53 acetylation in SIRT3-mediated ferroptosis, we crossed acetylated p53 mutant (p534KR) mice, which cannot activate ferroptosis, with SIRT3KO mice. SIRT3KO/p534KR mice exhibited a significant reduction in ferroptosis and less cardiac fibrosis compared to SIRT3KO mice. Furthermore, cardiomyocyte-specific knockout of SIRT3 (SIRT3-cKO) in mice resulted in a significant increase in ferroptosis and cardiac fibrosis. Treatment of SIRT3-cKO mice with the ferroptosis inhibitor ferrostatin-1 (Fer-1) led to a significant reduction in ferroptosis and cardiac fibrosis. We concluded that SIRT3-mediated cardiac fibrosis was partly through a mechanism involving p53 acetylation-induced ferroptosis in myofibroblasts.

High Glucose Activates Prolyl Hydroxylases and Disrupts HIF-α Signaling via the P53/TIGAR Pathway in Cardiomyocyte.

The induction of hypoxia tolerance has emerged as a novel therapeutic strategy for the treatment of ischemic diseases. The disruption of hypoxic signaling by hyperglycemia has been shown to contribute to diabetic cardiomyopathy. In this study, we explored the potential molecular mechanisms by which high glucose (HG) impairs hypoxia-inducible factor-α (HIF-α) signaling in cardiomyocytes. The exposure of H9c2 cell lines to HG resulted in time- and concentration-dependent decreases in HIF-1α and HIF-2α expression together with an increase in prolyl hydroxylase-1,2 (PHD1 and PHD2) expression, the main regulators of HIF-α destabilization in the heart. The exposure of H9c2 cells to normal glucose (5.5 mM) and high glucose (15, 30, and 45 mM) led to dose-dependent increases in p53 and TIGAR and a decrease in SIRT3 expression. The pretreatment of H9c2 with p53 siRNA to knockdown p53 attenuated PHD1 and PHD2 expression, thus significantly enhancing HIF-1α and HIF-2α expression in H9c2 cells under HG conditions. Interestingly, pretreatment with p53 siRNA altered H9c2 cell metabolism by reducing oxygen consumption rate and increasing glycolysis. Similarly, pretreatment with TIGAR siRNA blunted HG-induced PHD1 and PHD2 expression. This was accompanied by an increase in HIF-1α and HIF-2α expression with a reduction in oxygen consumption rate in H9c2 cells. Furthermore, pretreatment with adenovirus-SIRT3 (Ad-SIRT3) significantly reduced the HG-induced expression of p53 and PHDs and increased HIF-1α levels in H9c2 cells. Ad-SIRT3 treatment also regulated PHDs-HIF-1α levels in the hearts of diabetic db/db mice. Our study revealed a novel role of the HG-induced disruption of PHDs-HIF-α signaling via upregulating p53 and TIGAR expression. Therefore, the p53/TIGAR signaling pathway may be a novel target for diabetic cardiomyopathy.

IL-12α deficiency attenuates pressure overload-induced cardiac inflammation, hypertrophy, dysfunction, and heart failure progression.

IL-12α plays an important role in modulating inflammatory response, fibroblast proliferation and angiogenesis through modulating macrophage polarization or T cell function, but its effect on cardiorespiratory fitness is not clear. Here, we studied the effect of IL-12α on cardiac inflammation, hypertrophy, dysfunction, and lung remodeling in IL-12α gene knockout (KO) mice in response to chronic systolic pressure overload produced by transverse aortic constriction (TAC). Our results showed that IL-12α KO significantly ameliorated TAC-induced left ventricular (LV) failure, as evidenced by a smaller decrease of LV ejection fraction. IL-12α KO also exhibited significantly attenuated TAC-induced increase of LV weight, left atrial weight, lung weight, right ventricular weight, and the ratios of them in comparison to body weight or tibial length. In addition, IL-12α KO showed significantly attenuated TAC-induced LV leukocyte infiltration, fibrosis, cardiomyocyte hypertrophy, and lung inflammation and remodeling (such as lung fibrosis and vessel muscularization). Moreover, IL-12α KO displayed significantly attenuated TAC-induced activation of CD4+ T cells and CD8+ T cells in the lung. Furthermore, IL-12α KO showed significantly suppressed accumulation and activation of pulmonary macrophages and dendritic cells. Taken together, these findings indicate that inhibition of IL-12α is effective in attenuating systolic overload-induced cardiac inflammation, heart failure development, promoting transition from LV failure to lung remodeling and right ventricular hypertrophy.

P53 Acetylation Exerts Critical Roles In Pressure Overload Induced Coronary Microvascular Dysfunction and Heart Failure.

Coronary microvascular dysfunction (CMD) has been shown to contribute to cardiac hypertrophy and heart failure with preserved ejection fraction. At this point, there are no proven treatments for CMD. We have shown that histone acetylation may play a critical role in the regulation of CMD. By using a mouse model that replaces lysine with arginine at residues K98/117/161/162R of p53 (p534KR), preventing acetylation at these sites, we test the hypothesis that acetylation-deficient p534KR could improve coronary microvascular dysfunction and prevent the progression of hypertensive cardiac hypertrophy and heart failure. Wild-type (WT) and p534KR mice were subjected to pressure overload (PO) by transverse aortic constriction to induce cardiac hypertrophy and heart failure (HF). Echocardiography measurements revealed improved cardiac function together with reduction of apoptosis and fibrosis in p534KR mice. Importantly, myocardial capillary density and coronary flow reserve (CFR) were significantly improved in p534KR mice. Moreover, p534KR upregulated the expression of cardiac glycolytic enzymes and glucose transporters, as well as the level of fructose-2,6-biphosphate; increased PFK-1 activity; and attenuated cardiac hypertrophy. These changes were accompanied by increased expression of HIF-1α and proangiogenic growth factors. Additionally, the levels of SERCA-2 were significantly upregulated in sham p534KR mice as well as in p534KR mice after TAC. In vitro, p534KR significantly improved endothelial cell (EC) glycolytic function and mitochondrial respiration, and enhanced EC proliferation and angiogenesis. Similarly, acetylation-deficient p534KR significantly improved CFR and rescued cardiac dysfunction in SIRT3 KO mice. Our data reveal the importance of p53 acetylation in coronary microvascular function, cardiac function, and remodeling, and may provide a promising approach to improve hypertension-induced coronary microvascular dysfunction (CMD) and to prevent the transition of cardiac hypertrophy to heart failure.

MTM-HCC at Previous Liver Resection as a Predictor of Overall Survival in Salvage Liver Transplantation.

OBJECTIVES: Salvage liver transplantation (sLT) is considered an effective method to treat hepatocellular carcinoma (HCC) recurrence. This multicenter research aimed to identify the prognostic factors associated with recurrence-free survival (RFS) and overall survival (OS) after sLT. MATERIAL AND METHODS: A retrospective analysis of 114 patients who had undergone sLT for recurrent HCC between February 2012 and September 2020 was performed. The baseline and clinicopathological data of the patients were collected. RESULTS: The 1-, 3-, and 5-year RFS rates after sLT were 88.9%, 75.2%, and 69.2%, respectively, and the OS rates were 96.4%, 78.3%, and 70.8%. A time from liver resection (LR) to recurrence < 1 year, disease beyond the Milan criteria at sLT and macrotrabecular massive (MTM)-HCC were identified as risk factors for RFS and were further identified as independent risk factors. A time from LR to recurrence < 1 year, disease beyond the Milan criteria at sLT and MTM-HCC were also risk factors for OS and were further identified as independent risk factors. CONCLUSIONS: Compared with primary liver transplantation (pLT), more prognostic factors are available from patients who had undergone LR. We suggest that in cases of HCC recurrence within 1 year after LR, disease beyond the Milan criteria at sLT and MTM-HCC patients, sLT should be used with caution.

Knockout of TIGAR enhances myocardial phosphofructokinase activity and preserves diastolic function in heart failure.

Hypertension is an important risk factor in the pathogenesis of diastolic dysfunction. Growing evidence indicates that glucose metabolism plays an essential role in diastolic dysfunction. TP53-induced glycolysis and apoptosis regulator (TIGAR) has been shown to regulate glucose metabolism and heart failure (HF). In the present study, we investigated the role of TIGAR in diastolic function and cardiac fibrosis during pressure overload (PO)-induced HF. WT mice subjected to transverse aortic constriction (TAC), a commonly used method to induce diastolic dysfunction, exhibited diastolic dysfunction as evidenced by increased E/A ratio and E/E' ratio when compared to its sham controls. This was accompanied by increased cardiac interstitial fibrosis. In contrast, the knockout of TIGAR attenuated PO-induced diastolic dysfunction and interstitial fibrosis. Mechanistically, the levels of glucose transporter Glut-1, Glut-4, and key glycolytic enzyme phosphofructokinase 1 (PFK-1) were significantly elevated in TIGAR KO subjected to TAC as compared to that of WT mice. Knockout of TIGAR significantly increased fructose 2,6-bisphosphate levels and phosphofructokinase activity in mouse hearts. In addition, PO resulted in a significant increase in perivascular fibrosis and endothelial activation in the WT mice, but not in the TIGAR KO mice. Our present study suggests a necessary role of TIGAR-mediated glucose metabolism in PO-induced cardiac fibrosis and diastolic dysfunction.

TIGAR deficiency sensitizes angiotensin-II-induced renal fibrosis and glomerular injury.

Angiotensin II (Ang-II) is one of the major contributors to the progression of renal fibrosis, inflammation, glomerular injury, and chronic kidney disease. Emerging evidence suggests that renal glycolysis plays an important role in renal fibrosis and injury. TP53-induced glycolysis and apoptosis regulator (TIGAR) has been shown to regulate glycolysis. In the present study, we investigated the role of TIGAR in renal glycolysis, fibrosis, and glomerular injury during Ang-II-induced hypertension. Wild-type (WT) and TIGAR knockout (KO) mice were infused with Ang-II (1 µg/kg/min) via mini-pumps for 4 weeks. The mean arterial pressure was similar between the WT and TIGAR KO mice, associated with a comparable increase in plasma creatinine level. Ang-II infusion resulted in a significant increase in renal interstitial fibrosis and more mesangial expansion and collapsed glomerular structure in the TIGAR KO mice. These were associated with elevated expression of hypoxia-inducible factor-1 alpha, glycolytic enzymes, and transforming growth factor beta 1 in the TIGAR KO mice after Ang-II infusion when compared to that of the WT mice. The coupled-enzyme method revealed that PFK-1 activity was similarly increased in WT and TIGAR KO mice after Ang-II infusion. Our present study suggests that TIGAR is involved in Ang-II-induced renal fibrosis and glomerular injury, although it has little effect on blood pressure and renal function. Knockout of TIGAR sensitizes Ang-II-induced renal fibrosis and injury. This study provides new insights into the role of TIGAR in renal metabolism and pathological remodeling during Ang-II-induced hypertension.

Emerging Role of Pericytes and Their Secretome in the Heart.

Pericytes, as mural cells covering microvascular capillaries, play an essential role in vascular remodeling and maintaining vascular functions and blood flow. Pericytes are crucial participants in the physiological and pathological processes of cardiovascular disease. They actively interact with endothelial cells, vascular smooth muscle cells (VSMCs), fibroblasts, and other cells via the mechanisms involved in the secretome. The secretome of pericytes, along with diverse molecules including proinflammatory cytokines, angiogenic growth factors, and the extracellular matrix (ECM), has great impacts on the formation, stabilization, and remodeling of vasculature, as well as on regenerative processes. Emerging evidence also indicates that pericytes work as mesenchymal cells or progenitor cells in cardiovascular regeneration. Their capacity for differentiation also contributes to vascular remodeling in different ways. Previous studies primarily focused on the roles of pericytes in organs such as the brain, retina, lung, and kidney; very few studies have focused on pericytes in the heart. In this review, following a brief introduction of the origin and fundamental characteristics of pericytes, we focus on pericyte functions and mechanisms with respect to heart disease, ending with the promising use of cardiac pericytes in the treatment of ischemic heart failure.

Regulatory role of TIGAR on endothelial metabolism and angiogenesis.

Endothelial glycolytic metabolism plays an important role in the process of angiogenesis. TP53-induced glycolysis and apoptosis regulator (TIGAR) is a significant mediator of cellular energy homeostasis. However, the role of TIGAR in endothelial metabolism, angiogenesis, and coronary flow reserve (CFR) has not been studied. The present study investigated whether knockout (KO) of TIGAR improves endothelial glycolytic function and angiogenesis. In vitro, aortic endothelial cells (ECs) from TIGAR KO mice exhibited increased expression of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase isoform-3 (PFKFB3) and increased glycolytic function. These were accompanied by increased mitochondrial basal/maximal respiration and ATP production. Furthermore, knockout of TIGAR in ECs enhanced endothelial proliferation, migration, and tube formation. Knockout of TIGAR also significantly increased aortic sprouting ex vivo. In vivo, knockout of TIGAR increased the expression of proangiogenic factor, angiopoietin-1 (Ang-1) in mouse hearts. Knockout of TIGAR also significantly increased coronary capillary density with enhanced CFR in these hearts. Furthermore, TIGAR KO mice subjected to pressure overload (PO), a common model to study angiogenesis and cardiac hypertrophy, exhibited elevated expression of Ang-1, VEGF, and PFKFB3 than that of the wild-type (WT) mice. WT mice subjected to PO exhibited a significant reduction of coronary capillary density and impaired CFR, but TIGAR KO mice did not. In addition, knockout of TIGAR blunted TAC-induced cardiac hypertrophy and dysfunction seen in the WT mice. In conclusion, knockout of TIGAR improves endothelial angiogenetic capabilities by enhancing the endothelial glycolytic function, mitochondrial respiration, and proangiogenic signaling, which leads to increased coronary capillary density and vascular function and protects against chronic stress.

Sirtuin 3 Alleviates Diabetic Cardiomyopathy by Regulating TIGAR and Cardiomyocyte Metabolism.

Background Impairment of glycolytic metabolism is suggested to contribute to diabetic cardiomyopathy. In this study, we explored the roles of SIRT3 (Sirtuin 3) on cardiomyocyte glucose metabolism and cardiac function. Methods and Results Exposure of H9c2 cardiomyocyte cell lines to high glucose (HG) (30 mmol/L) resulted in a gradual decrease in SIRT3 and 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase isoform 3 (PFKFB3) expression together with increases in p53 acetylation and TP53-induced glycolysis and apoptosis regulator (TIGAR) expression. Glycolysis was significantly reduced in the cardiomyocyte exposed to HG. Transfection with adenovirus-SIRT3 significantly increased PFKFB3 expression and reduced HG-induced p53 acetylation and TIGAR expression. Overexpression of SIRT3 rescued impaired glycolysis and attenuated HG-induced reactive oxygen species formation and apoptosis. Knockdown of TIGAR in cardiomyocytes by using siRNA significantly increased PFKFB3 expression and glycolysis under hyperglycemic conditions. This was accompanied by a significant suppression of HG-induced reactive oxygen species formation and apoptosis. In vivo, overexpression of SIRT3 by an intravenous jugular vein injection of adenovirus-SIRT3 resulted in a significant reduction of p53 acetylation and TIGAR expression together with upregulation of PFKFB3 expression in the heart of diabetic db/db mice at day 14. Overexpression of SIRT3 further reduced reactive oxygen species formation and blunted microvascular rarefaction in the diabetic db/db mouse hearts. Overexpression of SIRT3 significantly blunted cardiac fibrosis and hypertrophy and improved cardiac function at day 14. Conclusions Our study demonstrated that SIRT3 attenuated diabetic cardiomyopathy via regulating p53 acetylation and TIGAR expression. Therefore, SIRT3 may be a novel target for abnormal energy metabolism in diabetes mellitus.

Direct Interaction of Daxx and Androgen Receptor Is Required for Their Regulatory Activity in Cholesterol Biosynthesis.

INTRODUCTION: Homeostasis of cholesterol is crucial for cellular function, and dysregulated cholesterol biosynthesis is a metabolic event that can lead to hepatic and cardiovascular abnormalities. OBJECTIVE: The aim of this study was to investigate the effects and mechanisms of domain-associated protein (Daxx) and androgen receptor (AR) on intracellular cholesterol synthesis. METHODS: HepG2 cells were transfected with pCDNA3.1(+)/Daxx plasmid or treated with testosterone propionate to observe the effects of Daxx and AR on intracellular cholesterol levels. Co-immunoprecipitation experiments were performed to identify the interaction between Daxx and AR and to explore the regulatory effects of this interaction on cholesterol synthesis. RESULTS: Our experiments showed that AR promoted cholesterol synthesis and accumulation by activating sterol-regulatory element-binding protein isoform 2. AR-induced cholesterol synthesis was inhibited by Daxx; however, the expression of AR was not affected. Further studies demonstrated the existence of direct binding between Daxx and AR and this interaction was required to suppress AR activity. CONCLUSIONS: The Daxx-mediated antagonism of AR depicts a more complete picture as to how Daxx regulates intracellular cholesterol level and provides a new target for treatment of atherosclerosis.

A Sex-Specific Role of Endothelial Sirtuin 3 on Blood Pressure and Diastolic Dysfunction in Female Mice.

BACKGROUND: Heart failure with preserved ejection fraction (HFpEF) is characterized by a diastolic dysfunction and is highly prevalent in aged women. Our study showed that ablation of endothelial Sirtuin 3 (SIRT3) led to diastolic dysfunction in male mice. However, the sex-specific role of endothelial SIRT3 deficiency on blood pressure and diastolic function in female mice remains to be investigated. METHODS AND RESULTS: In this study, we demonstrate that the ablation of endothelial SIRT3 in females elevated blood pressure as compared with control female mice. Diastolic function measurement also showed that the isovolumic relaxation time (IVRT) and myocardial performance index (MPI) were significantly increased, whereas the E' velocity/A' velocity (E'/A') ratio was reduced in the endothelial-specific SIRT3 knockout (SIRT3 ECKO) female mice. To further investigate the regulatory role of endothelial SIRT3 on blood pressure and diastolic dysfunction in metabolic stress, SIRT3 ECKO female mice were fed a normal diet and high-fat diet (HFD) for 20 weeks. The knockout of endothelial SIRT3 resulted in an increased blood pressure in female mice fed with an HFD. Intriguingly, SIRT3 ECKO female mice + HFD exhibited impaired coronary flow reserve (CFR) and more severe diastolic dysfunction as evidenced by an elevated IVRT as compared with control female mice + HFD. In addition, female SIRT3 ECKO mice had higher blood pressure and diastolic dysfunction as compared to male SIRT3 ECKO mice. Moreover, female SIRT3 ECKO mice + HFD had an impaired CFR and diastolic dysfunction as compared to male SIRT3 ECKO mice + HFD. CONCLUSIONS: These results implicate a sex-specific role of endothelial SIRT3 in regulating blood pressure and diastolic function in mice. Deficiency of endothelial SIRT3 may be responsible for a diastolic dysfunction in aged female.

SIRT3 Deficiency Sensitizes Angiotensin-II-Induced Renal Fibrosis.

BACKGROUND: Sirtuin 3 (SIRT3) has a crucial role in the cardiovascular diseases. Our previous study revealed that SIRT3 knockout (SIRT3KO) promoted cardiac pericyte-fibroblast transition. In this study, we investigated the involvement of pericyte and iron in angiotensin II (Ang-II)-mediated renal fibrosis in the SIRT3KO mice. METHODS AND RESULTS: NG2-DsRed mice and NG2-DsRed-SIRT3 knockout (SIRT3KO) mice were infused with saline or Ang-II (1000 ng/kg/min) for 4 weeks. Renal fibrosis, iron content and reactive oxygen species (ROS) were measured. Masson's trichrome staining showed that SIRT3KO enhanced Ang-II-induced renal fibrosis. Immunostaining showed that Ang-II treatment increased the number of NG2-DsRed+ cells in the kidney, and SIRT3KO further enhanced NG2-DsRed+ cells. Moreover, SIRT3KO promoted pericyte differentiation into fibroblasts as evidenced by co-staining NG2-DsRed/FSP-1. Furthermore, DsRed/FSP-1+ and DsRed/transforming growth factor-ß1 (TGF-ß1)+ fibroblasts were elevated by SIRT3KO after Ang-II infusion. Ang-II-induced collagen I and TGF-ß1 expression was also enhanced in the SIRT3KO mice. SIRT3KO significantly exacerbated Ang-II-induced iron accumulation. This was accompanied by an increase in acetyl-p53, HO-1 and FPN expression. Further, SIRT3KO sensitized Ang-II-induced upregulation of p47phox and gp91phox together with increased ROS formation in the kidney. CONCLUSION: Our study suggests that SIRT3 deficiency sensitized Ang-II-induced renal fibrosis by the mechanisms involved in promoting differentiation of pericytes into fibroblasts, exacerbating iron overload and accelerating NADPH oxidase-derived ROS formation.

Histone Acetyltransferase p300 Inhibitor Improves Coronary Flow Reserve in SIRT3 (Sirtuin 3) Knockout Mice.

Background Coronary microvascular dysfunction is common in patients of myocardial infarction with non-obstructive coronary artery disease. Coronary flow reserve (CFR) reflects coronary microvascular function and is a powerful independent index of coronary microvascular dysfunction and heart failure. Our previous studies showed that knockout of SIRT3 (Sirtuin 3) decreased CFR and caused a diastolic dysfunction. Few studies focus on the treatment of impaired CFR and heart failure. In the present study, we explored the role of C646, a histone acetyltransferase p300 inhibitor, in regulating CFR and cardiac remodeling in SIRT3 knockout (SIRT3KO) mice. Methods and Results After treating with C646 for 14 days, CFR, pulse-wave velocity, and cardiac function were measured in SIRT3KO mice. SIRT3KO mice treated with C646 showed a significant improvement of CFR, pulse-wave velocity, ejection fraction, and fractional shortening. Treatment with C646 reversed pre-existing cardiac fibrosis, hypertrophy, and capillary rarefaction in SIRT3KO mice. Mechanistically, knockout of Sirtuin 3 resulted in significant increases in p300 expression and H3K56 acetylation. Treatment with C646 significantly reduced levels of p300 and H3K56 acetylation in SIRT3KO mice. Furthermore, treatment with C646 increased endothelial nitric oxide synthase expression and reduced arginase II expression and activity. The expression of NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells) and VCAM-1 (vascular cell adhesion molecule 1) was also significantly suppressed by C646 treatment in SIRT3KO mice. Conclusions C646 treatment attenuated p300 and H3K56 acetylation and improved arterial stiffness and CFR via improvement of endothelial cell (EC) dysfunction and suppression of NF-κB.

Endothelial prolyl hydroxylase 2 is necessary for angiotensin II-mediated renal fibrosis and injury.

Angiotensin II (ANG II) is the key contributor to renal fibrosis and injury. The present study investigated the role of endothelium prolyl hydroxylase 2 (PHD2) in ANG II-mediated renal fibrosis and injury. In vitro, endothelial cells (ECs) were isolated from PHD2f/f control [wild-type (WT)] mice or PHD2 EC knockout (PHD2ECKO) mice. In vivo, WT and PHD2ECKO mice were infused with ANG II (1,000 ng·kg-1·min-1) for 28 days. Renal fibrosis, reactive oxygen species (ROS), and iron contents were measured. Knockout of PHD2 resulted in a significant increase in the expression of hypoxia-inducible factor (HIF)-1α and HIF-2α in ECs. Intriguingly, knockout of PHD2 significantly reduced expression of the ANG II type 1 receptor (AT1R) in ECs. WT mice infused with ANG II caused increases in renal fibrosis, ROS formation, and iron contents. ANG II treatment led to a downregulation of PHD1 expression and upregulation of HIF-1α and HIF-2α in the renal cortex and medulla. Knockout of PHD2 in EC blunted ANG II-induced downregulation of PHD1 expression. Furthermore, knockout of PHD2 in ECs attenuated ANG II-induced expression of HIF-1α, HIF-2α, transforming growth factor-ß1, p47phox, gp91phox, heme oxygenase-1, and ferroportin. This was accompanied by a significant suppression of renal fibrosis, ROS formation, and iron accumulation. In summary, knockout of endothelial PHD2 suppressed the expression of AT1R in ECs and blunted ANG II-induced downregulation of PHD1 and upregulation of HIF-α in the kidney. Our study, for the first time, demonstrates a necessary role of endothelial PHD2 in ANG II-mediated renal fibrosis and injury.