HDAC1/2 inhibitor therapy improves multiple organ systems in aged mice.

Aging increases the risk of age-related diseases, imposing substantial healthcare and personal costs. Targeting fundamental aging mechanisms pharmacologically can promote healthy aging and reduce this disease susceptibility. In this work, we employed transcriptome-based drug screening to identify compounds emulating transcriptional signatures of long-lived genetic interventions. We discovered compound 60 (Cmpd60), a selective histone deacetylase 1 and 2 (HDAC1/2) inhibitor, mimicking diverse longevity interventions. In extensive molecular, phenotypic, and bioinformatic assessments using various cell and aged mouse models, we found Cmpd60 treatment to improve age-related phenotypes in multiple organs. Cmpd60 reduces renal epithelial-mesenchymal transition and fibrosis in kidney, diminishes dementia-related gene expression in brain, and enhances cardiac contractility and relaxation for the heart. In sum, our two-week HDAC1/2 inhibitor treatment in aged mice establishes a multi-tissue, healthy aging intervention in mammals, holding promise for therapeutic translation to promote healthy aging in humans.

MicroRNA-216a is essential for cardiac angiogenesis.

While it is experimentally supported that impaired myocardial vascularization contributes to a mismatch between myocardial oxygen demand and supply, a mechanistic basis for disruption of coordinated tissue growth and angiogenesis in heart failure remains poorly understood. Silencing strategies that impair microRNA biogenesis have firmly implicated microRNAs in the regulation of angiogenesis, and individual microRNAs prove to be crucial in developmental or tumor angiogenesis. A high-throughput functional screening for the analysis of a whole-genome microRNA silencing library with regard to their phenotypic effect on endothelial cell proliferation as a key parameter, revealed several anti- and pro-proliferative microRNAs. Among those was miR-216a, a pro-angiogenic microRNA which is enriched in cardiac microvascular endothelial cells and reduced in expression under cardiac stress conditions. miR-216a null mice display dramatic cardiac phenotypes related to impaired myocardial vascularization and unbalanced autophagy and inflammation, supporting a model where microRNA regulation of microvascularization impacts the cardiac response to stress.

Empagliflozin and Dapagliflozin Reduce ROS Generation and Restore NO Bioavailability in Tumor Necrosis Factor α-Stimulated Human Coronary Arterial Endothelial Cells.

BACKGROUND/AIMS: Heart failure is characterized by chronic low-grade vascular inflammation, which in itself can lead to endothelial dysfunction. Clinical trials showed reductions in heart failure-related hospitalizations of type 2 diabetic patients using sodium glucose co-transporter 2 inhibitors (SGLT2i's). Whether and how SGLT2i's directly affect the endothelium under inflammatory conditions is not completely understood. The aim of the study was to investigate whether the SGLT2i Empagliflozin (EMPA) and Dapagliflozin (DAPA) reduce tumor necrosis factor α (TNFα) induced endothelial inflammation in vitro. METHODS: Human coronary arterial endothelial cells (HCAECs) and human umbilical vein endothelial cells (HUVECs) were (pre-)incubated with 1 µM EMPA or DAPA and subsequently exposed to 10 ng/ml TNFα. ROS and NO were measured using live cell imaging. Target proteins were either determined by infrared western blotting or fluorescence activated cell sorting (FACS). The connection between Cav-1 and eNOS was determined by co-immunoprecipitation. RESULTS: Nitric oxide (NO) bioavailability was reduced by TNFα and both EMPA and DAPA restored NO levels in TNFα-stimulated HCAECs. Intracellular ROS was increased by TNFα, and this increase was completely abolished by EMPA and DAPA in HCAECs by means of live cell imaging. eNOS signaling was significantly disturbed after 24 h when cells were exposed to TNFα for 24h, yet the presence of both SGLT2is did not prevent this disruption. TNFα-induced enhanced permeability at t=24h was unaffected in HUVECs by EMPA. Similarly, adhesion molecule expression (VCAM-1 and ICAM-1) was elevated after 4h TNFα (1.5-5.5 fold increase of VCAM-1 and 4-12 fold increase of ICAM-1) but were unaffected by EMPA and DAPA in both cell types. Although we detected expression of SGLT2 protein levels, the fact that we could not silence this expression by means of siRNA and the mRNA levels of SGLT2 were not detectable in HCAECs, suggests aspecificity or our SGLT2 antibody and absence of SGLT2 in our cells. CONCLUSION: These data suggest that EMPA and DAPA rather restore NO bioavailability by inhibiting ROS generation than by affecting eNOS expression or signaling, barrier function and adhesion molecules expression in TNFα-induced endothelial cells. Furthermore, the observed effects cannot be ascribed to the inhibition of SGLT2 in endothelial cells.

Cardiac Microvascular Endothelial Enhancement of Cardiomyocyte Function Is Impaired by Inflammation and Restored by Empagliflozin.

The positive findings of the EMPA-REG OUTCOME trial (Randomized, Placebo-Controlled Cardiovascular Outcome Trial of Empagliflozin) on heart failure (HF) outcome in patients with type 2 diabetes mellitus suggest a direct effect of empagliflozin on the heart. These patients frequently have HF with preserved ejection fraction (HFpEF), in which a metabolic risk-related pro-inflammatory state induces cardiac microvascular endothelial cell (CMEC) dysfunction with subsequent cardiomyocyte (CM) contractility impairment. This study showed that CMECs confer a direct positive effect on contraction and relaxation of CMs, an effect that requires nitric oxide, is diminished after CMEC stimulation with tumor necrosis factor-α, and is restored by empagliflozin. Our findings on the effect of empagliflozin on CMEC-mediated preservation of CM function suggests that empagliflozin can be used to treat the cardiac mechanical implications of microvascular dysfunction in HFpEF.

FGF23 impairs peripheral microvascular function in renal failure.

Cardiovascular diseases account for ~50% of mortality in patients with chronic kidney disease (CKD). Fibroblast growth factor 23 (FGF23) is independently associated with endothelial dysfunction and cardiovascular mortality. We hypothesized that CKD impairs microvascular endothelial function and that this can be attributed to FGF23. Mice were subjected to partial nephrectomy (5/6Nx) or sham surgery. To evaluate the functional role of FGF23, non-CKD mice received FGF23 injections and CKD mice received FGF23-blocking antibodies after 5/6Nx surgery. To examine microvascular function, myocardial perfusion in vivo and vascular function of gracilis resistance arteries ex vivo were assessed in mice. 5/6Nx surgery blunted ex vivo vasodilator responses to acetylcholine, whereas responses to sodium nitroprusside or endothelin were normal. In vivo FGF23 injections in non-CKD mice mimicked this endothelial defect, and FGF23 antibodies in 5/6Nx mice prevented endothelial dysfunction. Stimulation of microvascular endothelial cells with FGF23 in vitro did not induce ERK phosphorylation. Increased plasma asymmetric dimethylarginine concentrations were increased by FGF23 and strongly correlated with endothelial dysfunction. Increased FGF23 concentration did not mimic impaired endothelial function in the myocardium of 5/6Nx mice. In conclusion, impaired peripheral endothelium-dependent vasodilatation in 5/6Nx mice is mediated by FGF23 and can be prevented by blocking FGF23. These data corroborate FGF23 as an important target to combat cardiovascular disease in CKD. NEW & NOTEWORTHY In the present study, we provide the first evidence that fibroblast growth factor 23 (FGF23) is a cause of peripheral endothelial dysfunction in a model of early chronic kidney disease (CKD) and that endothelial dysfunction in CKD can be prevented by blockade of FGF23. This pathological effect on endothelial cells was induced by long-term exposure of physiological levels of FGF23. Mechanistically, increased plasma asymmetric dimethylarginine concentrations were strongly associated with this endothelial dysfunction in CKD and were increased by FGF23.

Normal and high eNOS levels are detrimental in both mild and severe cardiac pressure-overload.

Nitric oxide (NO) produced by endothelial NO synthase (eNOS) exerts beneficial effects in a variety of cardiovascular disease states. Studies on the benefit of eNOS activity in pressure-overload cardiac hypertrophy and dysfunction produced by aortic stenosis are equivocal, which may be due to different expression levels of eNOS or different severities of pressure-overload. Consequently, we investigated the effects of eNOS-expression level on cardiac hypertrophy and dysfunction produced by mild or severe pressure-overload. To unravel the impact of eNOS on pressure-overload cardiac dysfunction we subjected eNOS deficient, wildtype and eNOS overexpressing transgenic (eNOS-Tg) mice to 8weeks of mild or severe transverse aortic constriction (TAC) and studied cardiac geometry and function at the whole organ and tissue level. In both mild and severe TAC, lack of eNOS ameliorated, whereas eNOS overexpression aggravated, TAC-induced cardiac remodeling and dysfunction. Moreover, the detrimental effects of eNOS in severe TAC were associated with aggravation of TAC-induced NOS-dependent oxidative stress and by further elevation of eNOS monomer levels, consistent with enhanced eNOS uncoupling. In the presence of TAC, scavenging of reactive oxygen species with N-acetylcysteine reduced eNOS S-glutathionylation, eNOS monomer and NOS-dependent superoxide levels in eNOS-Tg mice to wildtype levels. Accordingly, N-acetylcysteine improved cardiac function in eNOS-Tg but not in wildtype mice with TAC. In conclusion, independent of the severity of TAC, eNOS aggravates cardiac remodeling and dysfunction, which appears due to TAC-induced eNOS uncoupling and superoxide production.

Tackling endothelial dysfunction by modulating NOS uncoupling: new insights into its pathogenesis and therapeutic possibilities.

Endothelial nitric oxide synthase (eNOS) serves as a critical enzyme in maintaining vascular pressure by producing nitric oxide (NO); hence, it has a crucial role in the regulation of endothelial function. The bioavailability of eNOS-derived NO is crucial for this function and might be affected at multiple levels. Uncoupling of eNOS, with subsequently less NO and more superoxide generation, is one of the major underlying causes of endothelial dysfunction found in atherosclerosis, diabetes, hypertension, cigarette smoking, hyperhomocysteinemia, and ischemia/reperfusion injury. Therefore, modulating eNOS uncoupling by stabilizing eNOS activity, enhancing its substrate, cofactors, and transcription, and reversing uncoupled eNOS are attractive therapeutic approaches to improve endothelial function. This review provides an extensive overview of the important role of eNOS uncoupling in the pathogenesis of endothelial dysfunction and the potential therapeutic interventions to modulate eNOS for tackling endothelial dysfunction.