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Brown adipose tissue (BAT) is a metabolically active organ that contributes to the thermogenic response to cold exposure. In addition, other thermogenic cells termed beige adipocytes are generated in white adipose tissue (WAT) by cold exposure. Although activation of brown/beige adipose tissue is associated with mobilization of both glucose and lipids, few studies have focused on the role of glycolytic enzymes in regulating adipose tissue function. We generated mouse models with specific deletion of the glycolytic enzyme phosphoglycerate mutase 1 (PGAM1) from adipose tissue. Deletion of Pgam1 from both BAT and WAT promoted whitening of BAT with beiging of visceral WAT, whereas deletion of Pgam1 from BAT alone led to whitening of BAT without beiging of WAT. Our results demonstrate a potential role of glycolytic enzymes in beiging of visceral WAT and suggest that PGAM1 would be a novel therapeutic target in obesity and diabetes.

Cellular senescence plays a pivotal role in aging and age-related disorders. Recently, it was shown that elimination of senescent cells, so-called "senolysis" has the potential to become a next generation therapy for age-related disorders including cardio-metabolic diseases. However, currently there is no senolytic drug available in clinical settings. The present study was aimed to identify a novel senolytic reagent effective for cardiometabolic diseases in compounds already available in clinical settings. We demonstrate a compound called "Seno-7284" exhibits senolytic effect in murine models of type 2 diabetes, atherosclerosis, progeroid and chronological aged mice. Seno-7284 reduced the accumulation of senescent cells in visceral adipose tissue of diabetic mice as senescence-associated-beta-galactosidase (SA-beta-gal) staining exhibits. This associated with the suppression in systemic glucose intolerance, and adipose tissue inflammation in four weeks after the administration of Seno-7284. Administrating Seno-7284 for two weeks also suppressed the progression of atherosclerosis in ApoE-KO mice. This drug significantly improved the lifespan of Zm-KO mice by administering it from 12 weeks old. Administration of Seno-7284 from 50 week-old for 20 weeks improves physical function in chronological aged mice. Further analysis including single cell RNA-seq suggested that Seno-7284 stimulates endogenous senolytic function of NK cells and CD8+ T cells. Our results indicate Seno-7284 mediates its biological effects by inducing senolysis in murine aging models. Seno-7284 would become promising therapies for age-related disorders.

p53 is a guardian of the genome that protects against carcinogenesis. There is accumulating evidence that p53 is activated with aging. Such activation has been reported to contribute to various age-associated pathologies, but its role in vascular dysfunction is largely unknown. The aim of this study was to investigate whether activation of endothelial p53 has a pathological effect in relation to endothelial function. We established endothelial p53 loss-of-function and gain-of-function models by breeding endothelial-cell specific Cre mice with floxed Trp53 or floxed Mdm2/Mdm4 mice, respectively. Then we induced diabetes by injection of streptozotocin. In the diabetic state, endothelial p53 expression was markedly up-regulated and endothelium-dependent vasodilatation was significantly impaired. Impairment of vasodilatation was significantly ameliorated in endothelial p53 knockout (EC-p53 KO) mice, and deletion of endothelial p53 also significantly enhanced the induction of angiogenesis by ischemia. Conversely, activation of endothelial p53 by deleting Mdm2/Mdm4 reduced both endothelium-dependent vasodilatation and ischemia-induced angiogenesis. Introduction of p53 into human endothelial cells up-regulated the expression of phosphatase and tensin homolog (PTEN), thereby reducing phospho-eNOS levels. Consistent with these results, the beneficial impact of endothelial p53 deletion on endothelial function was attenuated in EC-p53 KO mice with an eNOS-deficient background. These results show that endothelial p53 negatively regulates endothelium-dependent vasodilatation and ischemia-induced angiogenesis, suggesting that inhibition of endothelial p53 could be a novel therapeutic target in patients with metabolic disorders.

Brown adipose tissue (BAT) has a role in maintaining systemic metabolic health in rodents and humans. Here, we show that metabolic stress induces BAT to produce coagulation factors, which then—together with molecules derived from the circulation—promote BAT dysfunction and systemic glucose intolerance. When mice were fed a high-fat diet (HFD), the levels of tissue factor, coagulation Factor VII (FVII), activated coagulation Factor X (FXa), and protease-activated receptor 1 (PAR1) expression increased significantly in BAT. Genetic or pharmacological suppression of coagulation factor-PAR1 signaling in BAT ameliorated its whitening and improved thermogenic response and systemic glucose intolerance in mice with dietary obesity. Conversely, the activation of coagulation factor-PAR1 signaling in BAT caused mitochondrial dysfunction in brown adipocytes and systemic glucose intolerance in mice fed normal chow. These results indicate that BAT produces endogenous coagulation factors that mediate pleiotropic effects via PAR1 signaling under metabolic stress.

Prognosis of severe heart failure remains poor. Urgent new therapies are required. Some heart failure patients do not respond to established multidisciplinary treatment and are classified as “non-responders”. The outcome is especially poor for non-responders, and underlying mechanisms are largely unknown. Mitofusin-1 (Mfn1), a mitochondrial fusion protein, is significantly reduced in non-responding patients. This study aimed to elucidate the role of Mfn1 in the failing heart. Twenty-two idiopathic dilated cardiomyopathy (IDCM) patients who underwent endomyocardial biopsy of intraventricular septum were included. Of the 22 patients, 8 were non-responders (left ventricular (LV) ejection fraction (LVEF) of

Introduction: Recent studies have shown elimination of senescent cells, so-called "senolysis" has the potential to become a novel therapy for aging and age-related disorders. However, there is no senolytic drug available in clinical settings currently. Purpose: The present study was aimed to identify a novel senolytic reagent effective for cardiometabolic disease among compounds already available in clinical settings. Here we identified a compound called "seno-7284" which exhibits senolytic effect in murine models of aging and age-related cardiometabolic disorders. Methods: We generated diet-induced obese(DIO) mice model by imposing high-fat diet, atherosclerotic mice model by imposing western diet to ApoE knockout mice. We administered seno-7284 mixed in the diet (0.03% w/w) to each mouse model for 1, 2 or 4 weeks. We harvested tissue samples for molecular biological analysis after measuring glucose/insulin tolerance. We also administered seno-7284 to Zmpste24 homozygous knockout mice as a progeroid mice model from 12-week-old to measure lifespan and to wild-type mice from 1-year-old for 20 weeks to measure physical function. Results: Seno-7284 reduced accumulation of senescent cells in visceral adipose tissue of DIO mice as senescence-associated beta-galactosidase (SA-beta-gal) staining exhibits (figure(fig) a), resulting in improvement of glucose metabolism (fig b). We also found seno-7284 reduced accumulation of senescent cells in the aorta of atherosclerotic mice model (fig c) and inhibited advancing atherosclerosis (fig d). Furthermore, Seno-7284 improved lifespan of progeroid mice (fig e) and physical function of aged mice (fig f). Further analysis including RNA-seq speculated seno-7284 stimulates endogenous senolytic function of immune cells. Conclusion: Our results indicate that seno-7284 would become a promising senolytic drug that exhibits novel therapeutic machinery for aging and age-related cardiometabolic diseases.

Cellular senescence entails an irreversible growth arrest and a pro-inflammatory secretory phenotype, which contributes to aging-associated disorders such as atherosclerosis and diabetes, however, underlying mechanisms are largely unknown. In this study, we identified a novel protein, senescence-associated glycoprotein (SAGP), as a biomarker of cellular senescence and we also found that elimination of senescent cells targeting SAGP attenuated aging-associated disorders such as atherosclerosis, diabetes and frailty. First, we identified that SAGP as a senescent marker by microarray analysis of senescent human endothelial cells compared with young endothelial cells. The expression of SAGP was significantly increased in the aorta of chronological aging mice and ApoE-knockout mice. Then we measured SAGP expression in the patients registered in our hospital and found that mean SAGP expression was significantly higher in patients with atherosclerotic diseases compared to patients without atherosclerotic diseases. These data suggest that SAGP would become the novel marker of cellular senescence and/or aging-associated disorders. We found SAGP co-localized with lysosome and bound to V-ATPase, proton pump in the acid organelles such as lysosome. The electron microscopy analysis revealed that the dysfunctional lysosomes were accumulated in SAGP knockdown endothelial cell. The genetic deletion of SAGP resulted in the increase of lysosomal pH and the suppression of mitochondrial autophagy, mitophagy. And this associated with the high level of mitochondrial reactive oxygen species (ROS) and promoted premature senescence in human endothelial cells. These data suggest that SAGP was induced by the lysosomal stress in the senescent cells to protects senescent cells by maintaining the lysosomal homeostasis. Recently, it is reported that elimination of senescent cells (senolysis) reversibly improved pathological aging phenotypes and also extended the lifespan. We established senolytic therapy targeting SAGP. We generated SAGP-DTR (diphtheria toxin receptor) transgenic mice, in which we could eliminate the SAGP- positive senescent cells using DT (diphtheria toxin). We found elimination of SAGP positive senescent cells significantly reduced the atherosclerotic plaque burden in the aorta of ApoE-KO mice and improved the glucose metabolism of dietary obese mice, indicating that SAGP could be a useful target for senolytic therapy. For clinical implication, we then developed a cytotoxic vaccine targeting SAGP. Treatment with SAGP vaccine successfully eliminated SAGP positive senescent cells and attenuated atherosclerosis and metabolic dysfunction. Surprisingly, administration of SAGP vaccine to Zmpste24-KO mice, premature aging mice, extended the lifespan. These data indicate that targeting SAGP-positive cells could be a novel strategy for senolytic therapy. Effect of SAGP vaccine Funding Acknowledgement Type of funding source: Public grant(s) – National budget only. Main funding source(s): Grant-in-Aid for Scientific Research by Japan Society for the Promotion of Science (JSPS)

Chronic sterile inflammation in visceral fat has causal roles for systemic metabolic disorders in obesity. Inflamed visceral adipose tissue secretes pro-inflammatory adipokines, and this contributes to tissue remodeling under a metabolically stressed condition. Various kinds of white adipokines are broadly studied, however, roles of brown adipose tissue (BAT) derived adipokines (BATokine) remain to be explored. In this project, we tried to characterize pathogenic role of BATokine in obesity related fibrotic disorders, especially focusing on heart failure with preserved ejection fraction (HFpEF). For this purpose, we analyzed two sets of DNA microarray data, and identified an obesity associated pro-fibrotic protein (OAFP) as a possible pathogenic BATokine. Our biobank studies showed OAFP increased in patients with diastolic dysfunction, and E/e' analyzed with cardiac echo increased in direct proportion to circulating OAFP level in humans. We generated dietary obese mice model, and found OAFP increased both in BAT and circulation. We generated a murine systemic or BAT specific OAFP knockout (KO) models, and found that obesity-induced diastolic dysfunction ameliorated in these models. Cardiac fibrosis was also suppressed by genetic depletion of OAFP. We found OAFP increased in circulation in aged humans and mice, and studies in chronologically aged mice showed this molecule increased in BAT with aging. Our results indicate that OAFP is secreted predominantly from BAT, and mediates pathogenic roles by augmenting cardiac fibrosis in dietary obesity or aging. Suppression of OAFP may become a therapy for HFpEF. Funding Acknowledgement Type of funding source: None

Background/Introduction Non-alcoholic steatohepatitis (NASH), driven by the obesity epidemic, has become the most common form of liver disease. Inflamed visceral adipose tissue secretes pro-inflammatory adipokines that are causal for systemic metabolic disorders. Role of adipokines in NASH, especially those from brown adipose tissues (BATokine) remain unclear. Purpose To show the pathogenic role of BATokine in NASH. Methods To identify and characterize the pathological roles of pro-fibrotic BATokine, we generated a murine obese NASH model by imposing a high fat diet in C57BL6/NCr mice, and murine systemic or BAT specific knockout (KO) models. We also conducted functional in-vitro studies with differentiated brown adipocytes. Results Analyzing two sets of DNA micro array data with bioinformatics, we identified a secreted form pro-fibrotic protein (sPFP) expressed in dysfunctional brown adipose tissues (BAT) in mice. Testing our biobank samples, we found this protein increased in plasma of NASH patients. We generated a murine obese NASH model by imposing a high fat diet in C57BL6/NCr mice for 9–10 months since 4 weeks of age, and found that sPFP is produced predominantly by BAT. In this model, we also found that sPFP increased in plasma. We generated a murine systemic or BAT specific sPFP knockout (KO) models and found that liver fibrosis ameliorated in these models. We also suppressed circulating sPFP with a peptide vaccine targeting this molecule, and found that sPFP vaccination therapy inhibited liver fibrosis. Next, we generated sPFP gain of function (GOF) model by the administration of plasmid encoding sPFP into skeletal muscle. Liver fibrosis augmented in sPFP-GOF model, and these results suggested that sPFP has causal role for the progression of fibrotic response in liver. In vitro studies with differentiated brown adipocytes showed that metabolic stress increased c-Fos in nuclear, and this was causal for an increase in sPFP level. Conclusions Our results suggest that one of the BATokines, sPFP, contributes for the progression of fibrotic responses in obese-NASH model. Inhibition of sPFP may become a therapy for NASH or obesity related fibrotic disorders. Funding Acknowledgement Type of funding source: None

Background Cellular senescence occurs as a result of various genotoxic stresses and it plays a pivotal role in aging and age-related disorders. Recently, it was shown that elimination of senescent cells, so-called “senolysis” has the potential to become a promising novel therapy for age-related disorders in several mice models including cardiovascular diseases. However, there is no senolytic drug available in clinical settings currently. Purpose The present study was aimed to identify a novel senolytic reagent effective for cardiometabolic disease among compounds already available in clinical settings. Here we demonstrate that a compound called “seno-7284” exhibits senolytic effect in murine models of type 2 diabetes, atherosclerosis, progeroid and chronological aging. Methods We generated diet-induced obesity/diabetic mice model by imposing high-fat diet from 4-week-old for two months, atherosclerosis mice model by imposing western diet to ApoE homozygous knockout mice (ApoE-KO mice) from 4-week-old for 3 months. We administered seno-7284 mixed in the diet (0.03% w/w) to each mouse model for 1, 2 or 4 weeks. For the analysis, we carried out some physiological examinations including glucose tolerance test (GTT) and insulin tolerance test (ITT), then harvested tissue samples and took them to molecular biological analysis including real-time PCR, western blotting, RNA-sequence, etc. We also generated Zmpste24 homozygous knockout mice (Zmpste24-KO mice) as a progeroid mice model to measure their lifespan. Seno-7284 was administered to Zmpste24-KO mice from 12-week-old to the end of life. We also administrated seno-7284 to chronological aged mice at 1-year-old for 20 weeks and their physical function was examined with rotarod running test and hand-grip test. Results Seno-7284 reduced the accumulation of senescent cells in visceral adipose tissue of dietary obese mice as senescence-associated beta-galactosidase (SA-beta-gal) staining exhibits (Figure 1a). This effect results in ameliorating insulin tolerance (Figure 1b) and adipose tissue inflammation after 4-week administration of seno-7284. We also found administrating Seno-7284 for two weeks also reduced the accumulation of senescent cells in the atherosclerotic lesion in the aorta of ApoE-KO mice (Figure 1c) and inhibited advancing atherosclerosis (Figure 1d). Surprisingly, seno-7284 significantly improved the lifespan of Zmpste24 KO mice (Figure 1e). Seno-7284 also improved the physical function of chronologically aged mice by administrating it from 1-year-old for 20 weeks (Figure 1f). In-vitro studies didn't exhibit seno-7284 kills senescent cells directly, but further analysis including RNA-seq or metabolomic analysis speculated that seno-7284 stimulates endogenous senolytic function of NK-cells and CD8+ T-cells. Conclusion Our results indicate that seno-7284 would become a promising senolytic drug that exhibits novel therapeutic machinery for aging and age-related cardiometabolic diseases. Figure 1 Funding Acknowledgement Type of funding source: Public grant(s) – National budget only. Main funding source(s): Grant-in-Aid for Scientific Research (KAKENHI) C, Niigata University Tsukada medical research grant

The prevalence of obesity is increasing worldwide. Obese individuals are predisposed to cardio-metabolic disorders. Brown adipose tissue (BAT) is an active metabolic organ abundant with mitochondria, and studies suggest a potential role of BAT in the maintenance of metabolic health in rodents and humans. Metabolic stress causes BAT dysfunction, but the underlying mechanisms are largely unknown. Coagulation factor Xa (FXa) is critically involved in a coagulation cascade, and it is also known to mediate biological effects by the activation of protease-activated receptor (PAR)-signaling. Accumulating evidence shows that PAR1 contributes to tissue remodeling in cardiovascular system. Analyzing deposited microarray data, we found transcripts for coagulation factors including factor VII (F7), factor X (F10), and PAR1 receptor were increased in BAT from obese mice. Here we show a previously unknown role of FXa-PAR signaling in promoting BAT dysfunction and systemic metabolic disorder in a murine dietary obese model. Imposing a high fat diet (HFD) on C57BL/6NCr mice led to a marked increase in tissue factor (TF), coagulation factor VII and FXa in BAT. TF-FVIIa (activated form of FVII)-FXa complex is known to activate PAR1, and we found a significant increase in PAR1 expression in BAT upon metabolic stress. Administration of a FXa inhibitor ameliorated BAT whitening, improved thermogenic response and systemic glucose intolerance upon dietary obesity. Fxa inhibition reduced reactive oxygen species (ROS) level in BAT. In contrast, administration of warfarin did not show any phenotype in BAT. BAT specific TF and PAR1 over-expression model showed significant whitening of this tissue, which was associated with systemic glucose intolerance. We generated BAT specific PAR1 KO mice. BAT-PAR1 KO mice exhibited re-browning of BAT along with reduced ROS level in this tissue. In BAT-PAR1 KO mice, glucose intolerance and thermogenic response under a metabolically stressed condition were ameliorated. In differentiated brown adipocytes, FXa markedly increased mitochondrial ROS and reduced mitochondrial membrane potential. Inhibition of PAR1 ameliorated FXa-induced mitochondrial ROS production and reduction in membrane potential. We also found that plasma FXa level did not increase in obese mice as well as in obese individuals. These results suggest the previously unknown role of coagulation systems in promoting BAT dysfunction, leading to systemic metabolic disorders. Maintenance of BAT homeostasis through the suppression of FXa-PAR1 signaling would become a new therapeutic target for obesity and diabetes. Funding Acknowledgement Type of funding source: None

Background Frailty is a multifactorial physiological syndrome most often associated with age but which has received increasing recognition as a component of chronic illnesses such as heart failure. Patients with heart failure are likely to be frail, irrespective of their age. Adipokine dysregulation, which is associated with frailty, occurs in patients with heart failure. In this study, we tested the hypothesis that adipokines are associated with skeletal muscle and bone mineral density that change lead to frailty in patients with heart failure. Methods Thirty-five patients with heart failure (age, 67 ± 14 years; 25 males; left ventricular ejection fraction, 45 ± 19%) were included. Serum adipokine levels, physical performance, and body composition were measured. Results Adiponectin and leptin were inversely correlated with grip strength. Adiponectin was inversely correlated with bone mineral density. Leptin was positively correlated with fat mass. Adipokines were not correlated with skeletal muscle mass. Conclusions Adipokines were associated with grip strength and bone mineral density in patients with heart failure. Adipokine dysregulation may play a role in the development of frailty in heart failure.

Previous studies have suggested that cellular senescence plays a central role in the progression of pathologic changes in the failing heart. It is well known that the sympathetic nervous system is activated in patients with heart failure, and this change is associated with poor clinical outcomes. Sympathetic activation increases the levels of various catecholamines, such as epinephrine and norepinephrine, but the contribution of these catecholamines to cellular senescence associated with heart failure remains to be determined. We found that catecholamine infusion induced senescence of endothelial cells and bone marrow cells, and promoted cardiac dysfunction in mice. In C57BL/6NCr mice, the continuous infusion of isoproterenol-induced cardiac inflammation and cardiac dysfunction. Expression of p53, a master regulator of cellular senescence, was increased in the cardiac tissue and bone marrow cells of these mice. Suppression of cellular senescence by genetic deletion of p53 in endothelial cells or bone marrow cells led to improvement of isoproterenol-induced cardiac dysfunction. In vitro studies showed that adrenergic signaling increased the expression of p53 and adhesion molecules by endothelial cells and macrophages. Our results indicate that catecholamine-induced senescence of endothelial cells and bone marrow cells plays a pivotal role in the progression of heart failure. Suppression of catecholamine-p53 signaling is crucial for inhibition of remodeling in the failing heart.

Vascular cells have a finite lifespan and eventually enter irreversible growth arrest called cellular senescence. We have previously suggested that vascular cell senescence contributes to the pathogenesis of human atherosclerosis. Amlodipine is a mixture of two enantiomers, one of which (S- enantiomer) has L-type channel blocking activity, while the other (R+ enantiomer) shows ~1000-fold weaker channel blocking activity than S- enantiomer and has other unknown effects. It has been reported that amlodipine inhibits the progression of atherosclerosis in humans, but the molecular mechanism of this beneficial effect remains unknown. Apolipoprotein E-deficient mice on a high-fat diet were treated with amlodipine, its R+ enantiomer or vehicle for eight weeks. Compared with vehicle treatment, both amlodipine and the R+ enantiomer significantly reduced the number of senescent vascular cells and inhibited plaque formation to a similar extent. Expression of the pro-inflammatory molecule interleukin-1β was markedly upregulated in vehicle-treated mice, but was inhibited to a similar extent by treatment with amlodipine or the R+ enantiomer. Likewise, activation of p53 (a critical inducer of senescence) was markedly suppressed by treatment with amlodipine or the R+ enantiomer. These results suggest that amlodipine inhibits vascular cell senescence and protects against atherogenesis at least partly by a mechanism that is independent of calcium channel blockade.

Prasugrel, a novel P2Y12 receptor inhibitor, is administered to patients with acute coronary syndrome (ACS) after percutaneous coronary intervention (PCI), but it can increase the risk of bleeding. The Japanese exhibit weaker responses to clopidogrel than other races because of CYP2C19 polymorphisms; thus, it is unclear whether these patients should continue dual antiplatelet therapy (DAPT) using prasugrel or switch to clopidogrel in the chronic phase. Here we evaluated the clinical outcomes of DAPT guided by CYP2C19 polymorphisms after implantation of second-generation drug-eluting stents (DESs) for ACS management. Patients with ACS receiving PCI via DES from November 2011 to March 2015 were divided into two groups: conventional DAPT with clopidogrel (n = 41) and gene-guided DAPT (n = 24). In the gene-guided DAPT group, all patients with ACS were given DAPT using prasugrel as soon as possible; extensive and intermediate metabolizers receiving PCI for the first time were switched to clopidogrel at least 2 weeks after discharge, and intermediate metabolizers with repeated ACS and poor metabolizers continued on DAPT using prasugrel. Notably, gene-guided DAPT significantly reduced major adverse cardiovascular/cerebrovascular events (MACCEs; 22.0% versus 4.2%, hazard ratio [HR]: 0.15, 95% confidence interval [CI]: 0.01-0.81; P = 0.0247). Hemorrhagic complications were observed in 3.1% of patients receiving conventional DAPT and absent in the gene-guided group. Moreover, multivariate analysis showed that gene-guided DAPT significantly decreased MACCE incidence (HR: 0.15, 95% CI: 0.01-0.81; P = 0.033). Collectively, these data suggest that CYP2C19 polymorphism analysis may improve treatment decisions in patients with ACS receiving DES-PCI.

The mechanisms underlying the lower risk of hemorrhagic complications during treatment with direct oral anticoagulants (DOAC) have not been well clarified. Previous studies have evaluated the effects of DOAC on coagulation factors in the stationary state, but the effects of these agents after

Background Empagliflozin is a renal sodium glucose transporter 2 (SGLT2) inhibitor, thereby mediates its anti-diabetic effect via excretion of glucose into urine. EMPA-REG OUTCOME study, the first big randomized control trial of empagliflozin have shown significant reduction of mortality and hospitalization due to heart failure in diabetic patients. This trial hasn't only had a huge impact to cardiovascular field, but also raised a number of questions about underlying mechanisms. It is also uncertain about the efficacy of empagliflozin in non-diabetic heart failure. In this study, we aimed to elucidate the biological effects and its underling mechanism of empagliflozin in a murine non-diabetic heart failure model. Methods We generated a heart failure murine model due to left ventricular (LV) pressure overload by performing transverse aortic constriction (TAC) operation to C57BL/6NCr mice. Two weeks after TAC operation we started empagliflozin administration mixed with diet at the ratio of 0.03% w/w. LV function was measured with echocardiography after administration of empagliflozin for two weeks (four weeks after TAC operation) and compared to a littermate control (no treatment) group. Then, heart samples were collected and subjected to further studies including metabolomic analysis. In-vitro studies including Seahorse Extracellular Flux Analyzer were also conducted with differentiated C2C12 cells and neonatal rat ventricular myocytes (NRVM). Results We found that empagliflozin treatment (Empa) significantly ameliorated LV systolic dysfunction induced by TAC compared to control group (Con) (figure.A) while heart weight/body weight ratio wasn't reduced. To explore key metabolites that can contribute to improvement of LV function, we conducted metabolomic analysis and found that empagliflozin significantly increased plasma acetylcarnitine level both in sham and TAC groups (figure.B). Previous studies have shown that acetylcarnitine acts as a substrate of acetyl CoA to fuel tricarboxylic acid cycle, and we tested the efficacy of acetylcarnitine for mitochondrial respiration capacity in differentiated C2C12 cells with Seahorse Extracellular Flux Analyzer. This analysis revealed that administration of acetylcarnitine resulted in a significant increase of oxygen consumption reflected by enhancing mitochondrial respiration. Similary, acetylcarnitine also markedly ameliorated impairment of mitochondrial respiration induced by isoproterenol in NRVM. Conclusion Our results indicated that empagliflozin has cardioprotective effect in murine heart failure model by enhancing mitochondrial respiration through the increased production of acetylcarnitine. We provide new evidence that empagliflozin would become a promising therapeutic agent to heart failure without diabetes.

Obese individuals are predisposed to cardio-metabolic disorders. Brown adipose tissue (BAT) is an active metabolic organ abundant with mitochondria, and studies suggest a potential role of BAT in the maintenance of metabolic health in rodents and humans. Metabolic stress causes BAT dysfunction, but the underlying mechanisms are largely unknown. Coagulation factor Xa (FXa) is critically involved in a coagulation cascade, and it is also known to mediate biological effects by the activation of protease-activated receptor (PAR)-signaling. Accumulating evidence shows that PAR1 contributes to tissue remodeling in cardiovascular system. Here we show a previously unknown role of FXa-PAR signaling in promoting BAT dysfunction and systemic metabolic disorder in a murine dietary obese model. Imposing a high fat diet (HFD) on C57BL/6NCr mice led to a marked increase in tissue factor (TF), coagulation factor VII and FXa in BAT. TF-FVIIa (activated form of FVII)-FXa complex is known to activate PAR1, and we found a significant increase in PAR1 expression in BAT upon metabolic stress. Administration of a FXa inhibitor ameliorated BAT whitening, improved thermogenic response and systemic glucose intolerance upon dietary obesity. In contrast, administration of warfarin did not show any phenotype in BAT. BAT specific TF and PAR1 over-expression model showed significant whitening of this tissue, which was associated with systemic glucose intolerance. BAT specific PAR1 KO mice improved glucose intolerance and thermogenic response under a metabolically stressed condition. In differentiated brown adipocytes, FXa markedly increased mitochondrial reactive oxygen species (ROS) and reduced mitochondrial membrane potential. Inhibition of PAR1 ameliorated FXa-induced mitochondrial ROS production and reduction in membrane potential. We also found that plasma FXa level did not increase in obese mice as well as in obese individuals. These results suggest the previously unknown role of coagulation systems in promoting BAT dysfunction, leading to systemic metabolic disorders. Maintenance of BAT homeostasis through the suppression of FXa-PAR1 signaling would become a new therapeutic target for obesity and diabetes.

Cellular senescence is defined as a state of irreversible growth arrest and is accompanied by changes of both cell morphology and gene expression. Although accumulation of senescent vascular endothelial cells impair the vessel homeostasis and promote atherosclerotic diseases, underlying mechanisms are largely unknown. In this study, we identified a novel protein, senescence-associated glycoprotein (SAGP), as a biomarker of cellular senescence and we found modulation of SAGP or elimination of senescent cells targeting SAGP would become a novel therapy for atherosclerotic diseases. We found that SAGP expression was significantly increased in human endothelial cells undergoing replicative senescence compared with young endothelial cells. We also found SAGP expression in aorta was significantly increased both in chronological aging mice or ApoE knockout mice. Furthermore, we measured SAGP expression in patients registered in our hospital and found that mean SAGP expression was significantly higher in patients with atherosclerotic diseases compared to patients without atherosclerotic diseases.These data suggest that SAGP would become a novel cellular senescence and/or atherosclerotic disease marker. Genetic deletion of SAGP resulted in high level of mitochondrial reactive oxygen species (ROS) and promoted premature senescence in human endothelial cells. And this associated with suppression of mitochondrial autophagy, mitophagy. We found SAGP co-localized with lysosome by immunocytochemistry. In addition, the electron microscopy analysis revealed that the dysfunctional lysosomes were accumulated in SAGP knockdown endothelial cell, suggesting that SAGP maintain lysosomal homeostasis. Next, wegenerated ApoE-KO/ SAGP overexpression mice and found that atherosclerotic plaque burden was attenuated in these double-transgenic mice. In contrast, SAGP/ApoE double knockout mice showed progression in atherosclerosis. These data suggest that modulation of SAGPwould become a new therapeutic target for atherosclerotic diseases. SAGP vaccine Recently, it is reported that elimination of senescent cells (senolysis) reversibly improved pathological aging phenotypes and also extended the lifespan. We have taken another approach for atherosclerotic diseases, senolytic therapy targeting SAGP. We generated SAGP-DTR (diphtheria toxin receptor) transgenic mice, in which we could eliminate the SAGP- positive senescent cells using DT (diphtheria toxin). We found elimination of SAGP positive senescent cells significantly reduced the atherosclerotic plaque burden, indicating that SAGP would become a useful target for senolytic therapy. We then developed a cytotoxic vaccine targeting SAGP. Treatment with SAGP vaccine successfully eliminated SAGP positive senescent cells. Administration of SAGP vaccine to ApoE-KO mice significantly reduced atherogenesis. These data indicate that targeting SAGP-positive cells could become a strategy for senolytic therapy.

Background Senescence at cellular level develops with various genotoxic stresses and it plays a pivotal role in aging and age-related disorders. Recently, it was shown that elimination of senescent cells, so called “senolysis” has potential to become a next generation therapy for age-related disorders including cardiovascular diseases, pulmonary emphysema, Alzheimer's diseases, etc. However, currently there is no senolytic agent available in clinical settings. Purpose Present study was aimed to identify a novel senolytic agent effective for cardiometabolic diseases in compounds already available in clinical settings. Here we demonstrate a compound called “seno-7284” exhibits senolytic effect in murine models of type 2 diabetes, atherosclerosis and progeroid. Methods We generated 1) diet-induced obase and diabetic model by imposing a high fat diet for two months, 2) atherosclerosis mice model by imposing western diet to ApoE homozygous knockout mice (ApoE-KO mice) for three months, and 3) Zmpste24 homozygous knockout mice (Zmpste24-KO mice) as a progeroid mice model. We administrated seno-7284 by mixing it into the diet (0.03% w/w). In one, two or four weeks after the administration of seno-7284 to each mice model, we collected tissue samples for further analyses. Results Seno-7284 reduced the accumulation of senescent cells in visceral adipose tissue of dietary obese mice as senescence-associated beta-galactosidase (SA-beta-gal) staining exhibits (Figure a). This effect was associated with the suppression in systemic glucose intolerance (Figure b), and adipose tissue inflammation in four weeks after the administration of seno-7284. Administrating seno-7284 for two weeks also reduced accumulation of senescent cells in atherosclerotic lesion in aorta of ApoE-KO mice (Figure c), and inhibited the progression of atherosclerosis (Figure d). Surprisingly, this drug significantly improved the lifespan of Zmpste24-KO mice by administering it from 12 weeks old. Further analysis including RNA-seq or metabolomic analysis suggested that seno-7284 stimulates endogenous senolytic function of NK cells and CD8+ T cells. Conclusion Our results indicate that seno-7284 mediates its biological effects by inducing senolysis in some murine aging models. Seno-7284 would become a promising therapeutic agent for age-related cardiometabolic diseases.

Obese individuals are predisposed to cardio-metabolic disorders. Brown adipose tissue (BAT) is an active metabolic organ abundant with mitochondria, and studies suggest a potential role of BAT in the maintenance of metabolic health in rodents and humans. Metabolic stress causes BAT dysfunction, but the underlying mechanisms are largely unknown. Coagulation factor Xa (FXa) is critically involved in a coagulation cascade, and it is also known to mediate biological effects by the activation of protease-activated receptor (PAR)-signaling. Accumulating evidence shows that PAR1 contributes to tissue remodeling in cardiovascular system. Here we show a previously unknown role of FXa-PAR signaling in promoting BAT dysfunction and systemic metabolic disorder in a murine dietary obese model. Imposing a high fat diet (HFD) on C57BL/6NCr mice led to a marked increase in tissue factor (TF), coagulation factor VII and FXa in BAT. TF-FVIIa (activated form of FVII)-FXa complex is known to activate PAR1, and we found a significant increase in PAR1 expression in BAT upon metabolic stress. Administration of a FXa inhibitor ameliorated BAT whitening, improved thermogenic response and systemic glucose intolerance upon dietary obesity. In contrast, administration of warfarin did not show any phenotype in BAT. BAT specific TF and PAR1 over-expression model showed significant whitening of this tissue, which was associated with systemic glucose intolerance. BAT specific PAR1 KO mice improved glucose intolerance and thermogenic response under a metabolically stressed condition. In differentiated brown adipocytes, FXa markedly increased mitochondrial reactive oxygen species (ROS) and reduced mitochondrial membrane potential. Inhibition of PAR1 ameliorated FXa-induced mitochondrial ROS production and reduction in membrane potential. We also found that plasma FXa level did not increase in obese mice as well as in obese individuals. These results suggest the previously unknown role of coagulation systems in promoting BAT dysfunction, leading to systemic metabolic disorders. Maintenance of BAT homeostasis through the suppression of FXa-PAR1 signaling would become a new therapeutic target for obesity and diabetes

Although accumulation of senescent vascular endothelial cells impairs the vessel homeostasis and promote atherosclerotic diseases, underlying mechanisms are largely unknown. We found that Senescence associated glycoprotein (SAGP) expression was significantly increased in human endothelial cells undergoing replicative senescence. SAGP expression in aorta was significantly increased in Apo-E knockout mice. Furthermore, mean SAGP expression was significantly higher in the leukocytes of the patients with atherosclerotic diseases.These data suggest that SAGP would become the novel cellular senescence and/or atherosclerotic disease marker. The genetic deletion of SAGP resulted in high level of mitochondrial reactive oxygen species (ROS) and promoted premature senescence in human endothelial cells. And this associated with suppression of mitochondrial autophagy, mitophagy, suggesting that SAGP protect cells from ROS by inducing mitophagy. Thus, wegenerated ApoE-KO / SAGP gain-of-function transgenic mice and found that atherosclerotic plaque burden was attenuated in these transgenic mice. In contrast, in SAGP / ApoE double knockout mice atherosclerosis were impaired. These data suggest that modulation of SAGPwould become a new therapeutic target for atherosclerotic diseases.Recently, it is reported that elimination of senescent cells (senolysis) reversibly improved pathological aging phenotypes. We generated SAGP-DTR (diphtheria toxin receptor) transgenic mice,inducible elimination of SAGP positive senescent cells upon administration of diphtheria toxin. Elimination of SAGP expressing cells significantly reduced the atherosclerotic plaque burden. Furthermore, we developed a cytotoxic vaccine targeting SAGP. Administration of SAGP vaccine to ApoE-KO mice significantly reduced atherogenesis. These data indicate that targeting SAGP-positive cells could be a strategy for senolytic therapy.

Analyzing two sets of DNA micro array data with bioinformatics, we identified a secreted form pro-fibrotic protein (sPFP) expressed in dysfunctional brown adipose tissue (BAT) in mice. Testing our biobank samples, we found this protein increased in plasma of non-alcoholic steatohepatitis (NASH) patients or aged individuals. We generated a murine obese NASH model by imposing a high fat diet in C57BL/6NCr mice for 9-10 months since 4 weeks of age, and found that sPFP is produced predominantly by BAT. In this model, we also found that sPFP increased in plasma. We generated a murine systemic sPFP knockout (KO) model and found that liver fibrosis ameliorated in sPFP-KO model. We also suppressed circulating sPFP with a peptide vaccine targeting this molecule, and found that sPFP vaccination therapy inhibited liver fibrosis. Next, we generated sPFP gain of function (GOF) model by the administration of plasmid encoding sPFP into skeletal muscle. Liver fibrosis augmented in sPFP-GOF model, and these results suggested that sPFP has causal role for the progression of fibrotic response in liver. In the obese NASH model, we found that cardiac fibrosis also developed and it ameliorated in sPFP-KO model, indicating that sPFP may have pathological roles for heart failure with preserved ejection fraction (HFpEF) related with age-related disorders. In addition to an increase in circulating sPFP in aged individuals, we found that sPFP increased in BAT of chronological aged mice model. In vitro studies with differentiated brown adipocytes showed that c-Fos upregulated sPFP in transcript level. Our results suggest that sPFP contributes for the progression of fibrotic responses in obese or aged models. Inhibition of sPFP may become a therapy for NASH or HFpEF.

Accumulation of senescent cells is promoted in various organs as aging, and it also contributes to the progression of age-related disorders. Recent reports have demonstrated the elimination of senescent cells, so-called "senolysis" ameliorated various age-related disorders including cardiovascular diseases. However, there is currently no senolytic drug available in clinical settings. Here, we found a novel senolytic drug (termed “seno-7284”) from those already used in clinical setting and it exhibited senolytic effect in murine models of type 2 diabetes, atherosclerosis and progeroid aging. Conducting senescence-associated beta-galactosidase staining(SA-beta gal), we found that administrating seno-7284 for one week significantly reduced the accumulation of senescent cells in viscerala dipose tissue of diabetic mice induced by fed high-fat diet(Figure). This drug also ameliorated systemic glucose metabolism and adipose tissue inflammation without a reduction of body weight. Further analysis including RNA-seq analysis suggested seno-7284 stimulates the endogenous senolytic function of NK cells and CD8+ T cells via the Cxcl9-Cxcr3 axis. We also found administrating seno-7284 for two weeks also reduced the accumulation of senescent cells and atherosclerotic lesions in the aorta of western-diet-fed ApoE knock out mice. Surprisingly, this drug significantly improved the lifespan of Zmpste24 KO progeroid aging mice. Correctively, our results indicate that seno-7284 mediates its senolytic effect through the recruitment of lymphocytes. Senolytics would become a promising therapy for aging and age-related cardiometabolic disorders.

We previously demonstrated that cellular aging signals upregulated a secreted class 3 semaphorin E (Sema3E) and its receptor plexinD1 in the adipose tissue of a murine model of dietary obesity and that Sema3E was a chemoattractant, mediating its biological effects by inducing infiltration of plexinD1-positive inflammatory macrophages into the visceral white adipose tissue. This study was performed to develop a peptide vaccine for Sema3E and test its therapeutic potential in a murine model of dietary obesity. Two antigenic peptides were selected to generate neutralizing antibodies for a vaccine. These peptides were conjugated to keyhole limpet hemocyanin (KLH), and were administered with Freund’s adjuvant to obese wild-type male mice. The Sema3E antibody titer was analyzed by ELISA, and the biological effects of the peptides were tested in mice with dietary obesity. Among the two candidate peptides, the Sema3E antibody titer was significantly increased by injection of KLH-conjugated HKEGPEYHWS (Sema3E vaccine). Administration of Sema3E vaccine suppressed the infiltration of plexinD1-positive cells, ameliorated chronic inflammation in visceral white adipose tissue, and improved systemic glucose intolerance in mice with dietary obesity, suggesting that Sema3E vaccine has the potential to become a next generation therapy for obesity and diabetes.

Pulmonary arterial hypertension (PAH) is characterized by remodeling and narrowing of the pulmonary arteries, which lead to elevation of right ventricular pressure, heart failure, and death. Proliferation of pulmonary artery smooth muscle cells (PASMCs) is thought to be central to the pathogenesis of PAH, although the underlying mechanisms are still being explored. The protein p53 is involved in cell cycle coordination, DNA repair, apoptosis, and cellular senescence, but its role in pulmonary hypertension (PH) is not fully known. We developed a mouse model of hypoxia-induced pulmonary hypertension (PH) and found significant reduction of p53 expression in the lungs. Our in vitro experiments with metabolomic analyses and the Seahorse XF extracellular flux analyzer indicated that suppression of p53 expression in PASMCs led to upregulation of glycolysis and downregulation of mitochondrial respiration, suggesting a proliferative phenotype resembling that of cancer cells. It was previously shown that systemic genetic depletion of p53 in a murine PH model led to more severe lung manifestations. Lack of information about the role of cell-specific p53 signaling promoted us to investigate it in our mouse PH model with the inducible Cre-loxP system. We generated a mouse model with SMC-specific gain or loss of p53 function by crossing Myh11-Cre/ERT2 mice with floxed Mdm4 mice or floxed Trp53 mice. After these animals were exposed to hypoxia for 4 weeks, we conducted hemodynamic and echocardiographic studies. Surprisingly, the severity of PH was similar in both groups of mice and there were no differences between the genotypes. Our findings in these mice indicate that activation or suppression of p53 signaling in SMCs has a minor role in the pathogenesis of PH and suggest that p53 signaling in other cells (endothelial cells, immune cells, or fibroblasts) may be involved in the progression of this condition.

Summary: Brown adipose tissue (BAT) is a metabolically active organ that contributes to the maintenance of systemic metabolism. The sympathetic nervous system plays important roles in the homeostasis of BAT and promotes its browning and activation. However, the role of other neurotransmitters in BAT homeostasis remains largely unknown. Our metabolomic analyses reveal that gamma-aminobutyric acid (GABA) levels are increased in the interscapular BAT of mice with dietary obesity. We also found a significant increase in GABA-type B receptor subunit 1 (GABA-BR1) in the cell membranes of brown adipocytes of dietary obese mice. When administered to obese mice, GABA induces BAT dysfunction together with systemic metabolic disorder. Conversely, the genetic inactivation or inhibition of GABA-BR1 leads to the re-browning of BAT under conditions of metabolic stress and ameliorated systemic glucose intolerance. These results indicate that the constitutive activation of GABA/GABA-BR1 signaling in obesity promotes BAT dysfunction and systemic metabolic derangement. : Brown adipose tissue (BAT) is a metabolically active organ important for systemic metabolism. Here, Ikegami et al. identify a role for gamma-aminobutyric acid (GABA) in metabolic and BAT dysfunction in obese mice and demonstrate that inhibition of GABA/GABA-BR1-mediated signaling and of mitochondrial calcium overload can restore BAT function in obesity. Keywords: brown adipose tissue, BAT, metabolome, gamma-aminobutyric acid, GABA, obesity

There is accumulating evidence that the peripheral blood is a source of pro-angiogenic mononuclear cells (MNCs). These cells were initially described as endothelial progenitor cells (EPCs) expressing CD34 and vascular endothelial growth factor (VEGF) receptor 2. Pro-angiogenic MNCs are now known to represent a mixed population of cells, including hematopoietic stem cells, mesenchymal stem cells, and EPCs. The therapeutic potential of bone marrow or peripheral blood MNCs has been tested in patients with severe peripheral artery disease, showing that implanted cells promote the production of pro-angiogenic molecules in an autocrine and/or paracrine fashion and contribute to the recovery of blood flow in ischemic limbs. The molecular mechanisms underlying therapeutic angiogenesis are yet to be defined, but a number of studies have indicated that injection of either bone marrow or peripheral blood MNCs improves the clinical outcome in patients with severe limb ischemia and importantly achieves this effect with minor adverse events. In addition to controlling classical risk factors such as diabetes and/or hypertension, recent studies have suggested several combination therapies that can contribute to improving the therapeutic effects of these pro-angiogenic cells. Therapeutic angiogenesis promoted by the injection of autologous peripheral blood MNCs is an essential and practical treatment for patients who have no other options.