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Histone deacetylase (HDAC) 9 is a negative regulator of adipogenic differentiation, which is required for maintenance of healthy adipose tissues. We reported that HDAC9 expression is upregulated in adipose tissues during obesity, in conjunction with impaired adipogenic differentiation, adipocyte hypertrophy, insulin resistance, and hepatic steatosis, all of which were alleviated by global genetic deletion of Hdac9. Here, we developed a novel transgenic (TG) mouse model to test whether overexpression of Hdac9 is sufficient to induce adipocyte hypertrophy, insulin resistance, and hepatic steatosis in the absence of obesity. HDAC9 TG mice gained less body weight than wild-type (WT) mice when fed a standard laboratory diet for up to 40 weeks, which was attributed to reduced fat mass (primarily inguinal adipose tissue). There was no difference in insulin sensitivity or glucose tolerance in 18-week-old WT and HDAC9 TG mice; however, at 40 weeks of age, HDAC9 TG mice exhibited impaired insulin sensitivity and glucose intolerance. Tissue histology demonstrated adipocyte hypertrophy, along with reduced numbers of mature adipocytes and stromovascular cells, in the HDAC9 TG mouse adipose tissue. Moreover, increased lipids were detected in the livers of aging HDAC9 TG mice, as evaluated by oil red O staining. In conclusion, the experimental aging HDAC9 TG mice developed adipocyte hypertrophy, insulin resistance, and hepatic steatosis, independent of obesity. This novel mouse model may be useful in the investigation of the impact of Hdac9 overexpression associated with metabolic and aging-related diseases.

ABSTRACT The vascular endothelial injury occurs in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections, but the mechanisms are poorly understood. We sought to determine the frequency and type of cytokine elevations and their relationship to endothelial injury induced by plasma from patients with SARS-CoV-2 versus controls. Plasma from eight consecutively enrolled patients hospitalized with acute SARS-CoV-2 infection was compared to controls. Endothelial cell (EC) barrier integrity was evaluated using ECIS (electric cell-substrate impedance sensing) on human lung microvascular EC. Plasma from all SARS-CoV-2 but none from controls decreased transendothelial resistance to a greater degree than that produced by tumor necrosis factor-alpha (TNF-α), the positive control for the assay. Thrombin, angiopoietin 2 (Ang2), and vascular endothelial growth factor (VEGF), complement factor C3a and C5a, and spike protein increased endothelial permeability, but to a lesser extent and a shorter duration when compared to SARS-CoV-2 plasma. Analysis of Ang2, VEGF, and 15 cytokines measured in plasma revealed striking patient-to-patient variability within the SARS-CoV-2 patients. Pretreatment with thrombin inhibitors, single, or combinations of neutralizing antibodies against cytokines, Ca3 and C5a receptor antagonists, or with ACE2 antibody failed to lessen the SARS-CoV-2 plasma-induced EC permeability. The EC barrier destructive effects of plasma from patients with SARS-CoV-2 were susceptible to heat inactivation. Plasma from patients hospitalized with acute SARS-CoV-2 infection uniformly disrupts lung microvascular integrity. No predicted single, or set of, cytokine(s) accounted for the enhanced vascular permeability, although the factor(s) were heat-labile. A still unidentified but potent circulating factor(s) appears to cause the EC disruption in SARS-CoV-2 infected patients. IMPORTANCE Lung vascular endothelial injury in SARS-CoV-2 patients is one of the most important causes of morbidity and mortality and has been linked to more severe complications including acute respiratory distress syndrome (ARDS) and subsequent death due to multiorgan failure. We have demonstrated that in eight consecutive patients with SARS-CoV-2, who were not selected for evidence of endothelial injury, the diluted plasma-induced intense lung microvascular damage, in vitro. Known endothelial barrier-disruptive agents and proposed mediators of increased endothelial permeability in SARS-CoV-2, induced changes in permeability that were smaller in magnitude and shorter in duration than plasma from patients with SARS-CoV-2. The effect on endothelial cell permeability of plasma from patients with SARS-CoV-2 was heat-labile. The main plasma factor that causes the increased endothelial permeability remains to be identified. Our study provides a possible approach for future studies to understand the underlying mechanisms leading to vascular injury in SARS-CoV-2 infections.

Myeloid cells, including monocytes/macrophages, primarily rely on glucose and lipid metabolism to provide the energy and metabolites needed for their functions and survival. AMP-activated protein kinase (AMPK, its gene is PRKA for human, Prka for rodent) is a key metabolic sensor that regulates many metabolic pathways. We studied recruitment and viability of Prkaa1-deficient myeloid cells in mice and the phenotype of these mice in the context of cardio-metabolic diseases. We found that the deficiency of Prkaa1 in myeloid cells downregulated genes for glucose and lipid metabolism, compromised glucose and lipid metabolism of macrophages, and suppressed their recruitment to adipose, liver and arterial vessel walls. The viability of macrophages in the above tissues/organs was also decreased. These cellular alterations resulted in decreases in body weight, insulin resistance, and lipid accumulation in liver of mice fed with a high fat diet, and reduced the size of atherosclerotic lesions of mice fed with a Western diet. Our results indicate that AMPKα1/PRKAA1-regulated metabolism supports monocyte recruitment and macrophage viability, contributing to the development of diet-induced metabolic disorders including diabetes and atherosclerosis.

Increased glycolysis and inflammatory responses have been observed in endothelial cells exposed to disturbed flow. However, the role of endothelial glycolysis in atherosclerosis is unclear. Here the authors unveil a protective role for glycolysis by showing that endothelial deletion of Prkaa1 accelerates atherosclerosis in hyperlipidemic mice through a reduction of glycolytic metabolism.

The molecular mechanisms underlying vascular inflammation are unclear. Here the authors show that pro-inflammatory stimuli lead to endothelial inflammation by increasing adenosine kinase expression, and that its knockdown in endothelial cells inhibits atherosclerosis and cerebral ischemic injury in mice.

Pathological angiogenesis in the retina is a major cause of blindness. Here the authors show that adenosine receptor A2A drives pathological angiogenesis in the oxygen-induced retinopathy mouse model by promoting glycolysis in endothelial cells via the ERK/Akt/HIF-1α pathway, thereby suggesting new therapeutic targets for disease treatment.

As fundamental processes of immune homeostasis, autophagy, and apoptosis must be maintained to mitigate risk of chronic inflammation and autoimmune diseases. Periodontitis is a chronic inflammatory disease characterized by oral microbial dysbiosis, and dysregulation of dendritic cell (DC) and T cell responses. The aim of this study was to elucidate the underlying mechanisms by which the oral microbe Porphyromonas gingivalis (P. gingivalis) manipulates dendritic cell signaling to perturb both autophagy and apoptosis. Using a combination of Western blotting, flow cytometry, qRT-PCR and immunofluorescence analysis, we show a pivotal role for the minor (Mfa1) fimbriae of P. gingivalis in nuclear/cytoplasmic shuttling of Akt and FOXO1 in human monocyte-derived DCs. Mfa1-induced Akt nuclear localization and activation ultimately induced mTOR. Activation of the Akt/mTOR axis downregulated intracellular LC3II, also known as Atg8, required for autophagosome formation and maturation. Use of allosteric panAkt inhibitor MK2206 and mTOR inhibitor rapamycin confirmed the role of Akt/mTOR signaling in autophagy inhibition by P. gingivalis in DCs. Interestingly, this pathway was also linked to induction of the anti-apoptotic protein Bcl2, decreased caspase-3 cleavage and decreased expression of pro-apoptotic proteins Bax and Bim, thus promoting longevity of host DCs. Addition of ABT-199 peptide to disrupt the interaction of antiapoptotic Bcl2 and its proapoptotic partners BAK/BAX restored apoptotic death to P. gingivalis-infected DC cells. In summary, we have identified the underlying mechanism by which P. gingivalis promotes its own survival and that of its host DCs.

Background Obesity compromises cardiometabolic function and is associated with hypertension and chronic kidney disease. Exercise ameliorates these conditions, even without weight loss. Although the mechanisms of exercise's benefits remain unclear, augmented lean body mass is a suspected mechanism. Myostatin is a potent negative regulator of skeletal muscle mass that is upregulated in obesity and downregulated with exercise. The current study tested the hypothesis that deletion of myostatin would increase muscle mass and reduce blood pressure and kidney injury in obesity. Methods and Results Myostatin knockout mice were crossed to db/db mice, and metabolic and cardiovascular functions were examined. Deletion of myostatin increased skeletal muscle mass by ≈50% to 60% without concomitant weight loss or reduction in fat mass. Increased blood pressure in obesity was prevented by the deletion of myostatin, but did not confer additional benefit against salt loading. Kidney injury was evident because of increased albuminuria, which was abolished in obese mice lacking myostatin. Glycosuria, total urine volume, and whole kidney NOX‐4 levels were increased in obesity and prevented by myostatin deletion, arguing that increased muscle mass provides a multipronged defense against renal dysfunction in obese mice. Conclusions These experimental observations suggest that loss of muscle mass is a novel risk factor in obesity‐derived cardiovascular dysfunction. Interventions that increase muscle mass, either through exercise or pharmacologically, may help limit cardiovascular disease in obese individuals.

Abstract The objective of this study is to test the hypothesis that increased muscle mass has positive effects on cardiovascular function. Specifically, we tested the hypothesis that increases in lean body mass caused by deletion of myostatin improves cardiac performance and vascular function. Echocardiography was used to quantify left ventricular function at baseline and after acute administration of propranolol and isoproterenol to assess β‐adrenergic reactivity. Additionally, resistance vessels in several beds were removed, cannulated, pressurized to 60 mmHg and reactivity to vasoactive stimuli was assessed. Hemodynamics were measured using in vivo radiotelemetry. Myostatin deletion results in increased fractional shortening at baseline. Additionally, arterioles in the coronary and muscular microcirculations are more sensitive to endothelial‐dependent dilation while nonmuscular beds or the aorta were unaffected. β‐adrenergic dilation was increased in both coronary and conduit arteries, suggesting a systemic effect of increased muscle mass on vascular function. Overall hemodynamics and physical characteristics (heart weight and size) remained unchanged. Myostatin deletion mimics in part the effects of exercise on cardiovascular function. It significantly increases lean muscle mass and results in muscle‐specific increases in endothelium‐dependent vasodilation. This suggests that increases in muscle mass may serve as a buffer against pathological states that specifically target cardiac function (heart failure), the β‐adrenergic system (age), and nitric oxide bio‐availability (atherosclerosis). Taken together, pharmacological inhibition of the myostatin pathway could prove an excellent mechanism by which the benefits of exercise can be conferred in patients that are unable to exercise.

We have recently identified that the role of secreted protein acidic and rich in cysteine (SPARC) in amelioration of peritoneal ovarian carcinomatosis is mediated, at least in part, through mesothelial cell/lysophosphatidic acid-induced inflammatory response in ovarian cancer cells. The aim of this study was to elucidate the molecular mechanisms of the interactions between tumor cells and the cellular components of the ovarian cancer peritoneal microenvironment, specifically, mesothelial cells and macrophages. We found that SPARC not only significantly reduced macrophage chemoattractant protein-1 production and its macrophage chemotactic effect, but also attenuated the response of ovarian cancer cells to the mitogenic and proinvasive effects of macrophage chemo-attractant protein-1 and decreased macrophage-induced cancer cell invasiveness. Overexpression of SPARC in ovarian cancer cells significantly attenuated macrophage- and mesothelial cell-induced production and activity of interleukin-6, prostanoids (prostaglandins E2 and 8-isoprostanes) as well as matrix metalloproteinases and urokinase plasminogen activator. Moreover, the effects of SPARC overexpression in ovarian cancer cells were mediated, in part, through inhibition of nuclear factor-κB promoter activation. These results indicate, for the first time, that the effects of tumor SPARC as a negative regulator of ovarian cancer are mediated through decreased recruitment of macrophages and downregulation of the associated inflammation.

The interplay between peritoneal mesothelial cells and ovarian cancer cells is critical for the initiation and peritoneal dissemination of, and ascites formation in, ovarian cancer. The production of lysophosphatidic acid (LPA) by both peritoneal mesothelial cells and ovarian cancer cells has been shown to promote metastatic phenotype in ovarian cancer. Herein, we report that exogenous addition or ectopic overexpression of the matricellular protein SPARC (secreted protein acidic and rich in cysteine) significantly attenuated LPA-induced proliferation, chemotaxis, and invasion in both highly metastatic SKOV3 and less metastatic OVCAR3 ovarian cancer cell lines. SPARC appears to modulate these functions, at least in part, through the regulation of LPA receptor levels and the attenuation of extracellular signal-regulated kinase (ERK) 1/2 and protein kinase B/AKT signaling. Moreover, our results show that SPARC not only significantly inhibited both basal and LPA-induced interleukin (IL) 6 production in both cell lines but also attenuated IL-6-induced mitogenic, chemotactic, and proinvasive effects, in part, through significant suppression of ERK1/2 and, to a lesser extent, of signal transducers and activators of transcription 3 signaling pathways. Our results strongly suggest that SPARC exerts a dual inhibitory effect on LPA-induced mesothelial-ovarian cancer cell crosstalk through the regulation of both LPA-induced IL-6 production and function. Taken together, our findings underscore the use of SPARC as a potential therapeutic candidate in peritoneal ovarian carcinomatosis.

Abstract The nucleoside adenosine is a potent regulator of vascular homeostasis, but it remains unclear how expression or function of the adenosine‐metabolizing enzyme adenosine kinase (ADK) and the intracellular adenosine levels influence angiogenesis. We show here that hypoxia lowered the expression of ADK and increased the levels of intracellular adenosine in human endothelial cells. Knockdown (KD) of ADK elevated intracellular adenosine, promoted proliferation, migration, and angiogenic sprouting in human endothelial cells. Additionally, mice deficient in endothelial ADK displayed increased angiogenesis as evidenced by the rapid development of the retinal and hindbrain vasculature, increased healing of skin wounds, and prompt recovery of arterial blood flow in the ischemic hindlimb. Mechanistically, hypomethylation of the promoters of a series of pro‐angiogenic genes, especially for VEGFR2 in ADK KD cells, was demonstrated by the Infinium methylation assay. Methylation‐specific PCR, bisulfite sequencing, and methylated DNA immunoprecipitation further confirmed hypomethylation in the promoter region of VEGFR2 in ADK‐deficient endothelial cells. Accordingly, loss or inactivation of ADK increased VEGFR2 expression and signaling in endothelial cells. Based on these findings, we propose that ADK downregulation‐induced elevation of intracellular adenosine levels in endothelial cells in the setting of hypoxia is one of the crucial intrinsic mechanisms that promote angiogenesis.