Impaired HDL antioxidant and anti-inflammatory functions are linked to increased mortality in acute heart failure patients.

AIMS: Acute heart failure (AHF) is typified by inflammatory and oxidative stress responses, which are associated with unfavorable patient outcomes. Given the anti-inflammatory and antioxidant properties of high-density lipoprotein (HDL), this study sought to examine the relationship between impaired HDL function and mortality in AHF patients. The complex interplay between various HDL-related biomarkers and clinical outcomes remains poorly understood. METHODS: HDL subclass distribution was quantified by nuclear magnetic resonance spectroscopy. Lecithin-cholesterol acyltransferase (LCAT) activity, cholesterol ester transfer protein (CETP) activity, and paraoxonase (PON-1) activity were assessed using fluorometric assays. HDL-cholesterol efflux capacity (CEC) was assessed in a validated assay using [3H]-cholesterol-labeled J774 macrophages. RESULTS: Among the study participants, 74 (23.5 %) out of 315 died within three months after hospitalization due to AHF. These patients exhibited lower activities of the anti-oxidant enzymes PON1 and LCAT, impaired CEC, and lower concentration of small HDL subclasses, which remained significant after accounting for potential confounding factors. Smaller HDL particles, particularly HDL3 and HDL4, exhibited a strong association with CEC, PON1 activity, and LCAT activity. CONCLUSIONS: In patients with AHF, impaired HDL CEC, HDL antioxidant and anti-inflammatory function, and impaired HDL metabolism are associated with increased mortality. Assessment of HDL function and subclass distribution could provide valuable clinical information and help identify patients at high risk.

Sex-Related Differences in the Associations between Adiponectin and Serum Lipoproteins in Healthy Subjects and Patients with Metabolic Syndrome.

The strong associations between the serum levels of adiponectin and the lipoprotein subclasses observed in healthy subjects are much weaker in patients with metabolic syndrome (MS). However, the impact of sex on these associations remained unexplored. Therefore, in the present study, we examined associations between adiponectin and the lipoprotein subclasses, analyzed by nuclear magnetic resonance spectroscopy, separately in healthy females and males, as well as in females and males with MS. We observed negative correlations between adiponectin and VLDL, IDL, and small-dense LDL in healthy males, but neither in healthy females nor in females or males with MS. Additionally, adiponectin was positively correlated with some HDL subclasses in healthy males and females with MS, but not in healthy females or males with MS. Adjusting for age and either body mass index, waist circumference, C-reactive protein, or interleukin-6 weakened the associations between adiponectin and VLDL and IDL but not small-dense LDL. The adjustment weakened the associations between adiponectin and HDL in healthy males but not in females with MS. Based on our results, we conclude that sex and the presence of MS are strong determinants of the associations between adiponectin and serum lipoproteins and that the complex regulatory network comprising adiponectin and other molecular players involved in the regulation of lipoprotein metabolism is primarily operative in healthy males and females with MS.

Homocysteine contributes to atherogenic transformation of the aorta in rabbits in the absence of hypercholesterolemia.

Atherosclerosis, the leading cause of cardiovascular disease, cannot be sufficiently explained by established risk factors, including cholesterol. Elevated plasma homocysteine (Hcy) is an independent risk factor for atherosclerosis and is closely linked to cardiovascular mortality. However, its role in atherosclerosis has not been fully clarified yet. We have previously shown that rabbits fed a diet deficient in B vitamins and choline (VCDD), which are required for Hcy degradation, exhibit an accumulation of macrophages and lipids in the aorta, aortic stiffening and disorganization of aortic collagen in the absence of hypercholesterolemia, and an aggravation of atherosclerosis in its presence. In the current study, plasma Hcy levels were increased by intravenous injections of Hcy into balloon-injured rabbits fed VCDD (VCDD+Hcy) in the absence of hypercholesterolemia. While this treatment did not lead to thickening of aortic wall, intravenous injections of Hcy into rabbits fed VCDD led to massive accumulation of VLDL-triglycerides as well as significant impairment of vascular reactivity of the aorta compared to VCDD alone. In the aorta intravenous Hcy injections into VCDD-fed rabbits led to fragmentation of aortic elastin, accumulation of elastin-specific electron-dense inclusions, collagen disorganization, lipid degradation, and autophagolysosome formation. Furthermore, rabbits from the VCDD+Hcy group exhibited a massive decrease of total protein methylated arginine in blood cells and decreased creatine in blood cells, serum and liver compared to rabbits from the VCDD group. Altogether, we conclude that Hcy contributes to atherogenic transformation of the aorta not only in the presence but also in the absence of hypercholesterolemia.

Serum Levels of Adiponectin Are Strongly Associated with Lipoprotein Subclasses in Healthy Volunteers but Not in Patients with Metabolic Syndrome.

Metabolic syndrome (MS) is a widespread disease in developed countries, accompanied, among others, by decreased adiponectin serum levels and perturbed lipoprotein metabolism. The associations between the serum levels of adiponectin and lipoproteins have been extensively studied in the past under healthy conditions, yet it remains unexplored whether the observed associations also exist in patients with MS. Therefore, in the present study, we analyzed the serum levels of lipoprotein subclasses using nuclear magnetic resonance spectroscopy and examined their associations with the serum levels of adiponectin in patients with MS in comparison with healthy volunteers (HVs). In the HVs, the serum levels of adiponectin were significantly negatively correlated with the serum levels of large buoyant-, very-low-density lipoprotein, and intermediate-density lipoprotein, as well as small dense low-density lipoprotein (LDL) and significantly positively correlated with large buoyant high-density lipoprotein (HDL). In patients with MS, however, adiponectin was only significantly correlated with the serum levels of phospholipids in total HDL and large buoyant LDL. As revealed through logistic regression and orthogonal partial least-squares discriminant analyses, high adiponectin serum levels were associated with low levels of small dense LDL and high levels of large buoyant HDL in the HVs as well as high levels of large buoyant LDL and total HDL in patients with MS. We conclude that the presence of MS weakens or abolishes the strong associations between adiponectin and the lipoprotein parameters observed in HVs and disturbs the complex interplay between adiponectin and lipoprotein metabolism.

Advanced Oxidation Protein Products Are Strongly Associated with the Serum Levels and Lipid Contents of Lipoprotein Subclasses in Healthy Volunteers and Patients with Metabolic Syndrome.

The association between advanced oxidation protein products (AOPPs) and lipoprotein subclasses remains unexplored. Therefore, we performed comprehensive lipoprotein profiling of serum using NMR spectroscopy and examined the associations of lipoprotein subclasses with the serum levels of AOPPs in healthy volunteers (HVs) and patients with metabolic syndrome (MS). The serum levels of AOPPs were significantly positively correlated with the serum levels of very-low-density lipoprotein (VLDL), intermediate-density lipoprotein (IDL), and low-density lipoprotein (LDL); however, they were significantly negatively correlated with high-density lipoprotein (HDL). These lipoproteins (and their subclasses) differed markedly regarding the direction of correlations between their lipid contents and AOPPs. The strength of the correlations and the relative contributions of the subclasses to the correlations were different in the HVs and patients with MS. As revealed by orthogonal partial least squares discriminant analyses, the serum levels of IDL were strong determinants of AOPPs in the HVs, whereas the serum levels of VLDL and the lipid content of LDL were strong determinants in both groups. We conclude that IDL, VLDL, and LDL facilitate, whereas HDL diminishes the bioavailability of serum AOPPs. The presence of MS and the lipid contents of the subclasses affect the relationship between lipoproteins and AOPPs.

Association between Serum Free Fatty Acids and Clinical and Laboratory Parameters in Acute Heart Failure Patients.

Very little is known about the association between individual serum free fatty acids (FFAs) and clinical and laboratory parameters (indicators of heart failure severity) in acute heart failure (AHF) patients. Here, the baseline serum levels of FFAs, 16:0 (palmitic acid), 16:1 (palmitoleic acid), 18:0 (stearic acid), 18:1 (oleic acid), 18:2 (linoleic acid), 18:3 (alpha-linolenic acid or gamma-linolenic acid), 20:4 (arachidonic acid), 20:5 (eicosapentaenoic acid), and 22:6 (docosahexaenoic acid), were determined in 304 AHF patients (94.7% belonged to New York Heart Association functional class IV) using gas chromatography. Spearman correlation coefficients were used to examine the associations between the individual and total (the sum of all FFAs) FFAs and clinical and laboratory parameters. After applying a Bonferroni correction to correct for multiple testing, the total FFAs, as well as the individual FFAs (except FFAs 18:0, 20:5, and 22:6), were found to be significantly positively correlated with serum albumin. Only a few additional associations were found: FFA 16:0 was significantly negatively correlated with systolic pulmonary artery pressure, FFA 18:3 was significantly negatively correlated with C-reactive protein and body mass index, and FFA 20:4 was significantly negatively correlated with blood urea nitrogen. Based on our results, we conclude that in patients with severe AHF, individual and total serum FFAs are slightly associated with established laboratory and clinical parameters, which are indicators of heart failure severity.

Genetic deletion of MMP12 ameliorates cardiometabolic disease by improving insulin sensitivity, systemic inflammation, and atherosclerotic features in mice.

BACKGROUND: Matrix metalloproteinase 12 (MMP12) is a macrophage-secreted protein that is massively upregulated as a pro-inflammatory factor in metabolic and vascular tissues of mice and humans suffering from cardiometabolic diseases (CMDs). However, the molecular mechanisms explaining the contributions of MMP12 to CMDs are still unclear. METHODS: We investigated the impact of MMP12 deficiency on CMDs in a mouse model that mimics human disease by simultaneously developing adipose tissue inflammation, insulin resistance, and atherosclerosis. To this end, we generated and characterized low-density lipoprotein receptor (Ldlr)/Mmp12-double knockout (DKO) mice fed a high-fat sucrose- and cholesterol-enriched diet for 16-20 weeks. RESULTS: DKO mice showed lower cholesterol and plasma glucose concentrations and improved insulin sensitivity compared with LdlrKO mice. Untargeted proteomic analyses of epididymal white adipose tissue revealed that inflammation- and fibrosis-related pathways were downregulated in DKO mice. In addition, genetic deletion of MMP12 led to alterations in immune cell composition and a reduction in plasma monocyte chemoattractant protein-1 in peripheral blood which indicated decreased low-grade systemic inflammation. Aortic en face analyses and staining of aortic valve sections demonstrated reduced atherosclerotic plaque size and collagen content, which was paralleled by an improved relaxation pattern and endothelial function of the aortic rings and more elastic aortic sections in DKO compared to LdlrKO mice. Shotgun proteomics revealed upregulation of anti-inflammatory and atheroprotective markers in the aortas of DKO mice, further supporting our data. In humans, MMP12 serum concentrations were only weakly associated with clinical and laboratory indicators of CMDs. CONCLUSION: We conclude that the genetic deletion of MMP12 ameliorates obesity-induced low-grade inflammation, white adipose tissue dysfunction, biomechanical properties of the aorta, and the development of atherosclerosis. Therefore, therapeutic strategies targeting MMP12 may represent a promising approach to combat CMDs.

Low Valine Serum Levels Predict Increased 1-Year Mortality in Acute Heart Failure Patients.

Considering the relationship between disease severity and the extent of metabolic derangement in heart failure, we hypothesized that the serum levels of metabolites may have prognostic value for 1-year mortality in acute heart failure (AHF). The AHF study was a prospective, observational study enrolling consecutive patients hospitalized due to AHF. Metabolites were measured in serum collected at admission using NMR spectroscopy. Out of 315 AHF patients, 118 (37.5%) died within 1 year after hospitalization for AHF. The serum levels of 8 out of 49 identified metabolites were significantly different between patients who were alive and those who died within 1 year after hospitalization for AHF. Of these, only valine was significantly associated with 1-year mortality (hazard ratio 0.73 per 1 standard deviation increase, 95% confidence interval: 0.59-0.90, p = 0.003) in the multivariable Cox regression analyses. Kaplan-Maier analysis showed significantly higher survival rates in AHF patients with valine levels above the median (>279.2 µmol/L) compared to those with valine levels ≤ 279.2 µmol/L. In a receiver operating characteristics curve analysis, valine was able to discriminate between the two groups with an area under the curve of 0.65 (95% CI 0.59-0.72). We conclude that valine serum levels might be of prognostic value in AHF.

Associations between Endothelial Lipase and Apolipoprotein B-Containing Lipoproteins Differ in Healthy Volunteers and Metabolic Syndrome Patients.

The association between serum levels of endothelial lipase (EL) and the serum levels and composition of apolipoprotein B (apoB)-containing lipoproteins in healthy subjects and patients with metabolic syndrome (MS) remained unexplored. Therefore, in the present study, we determined the serum levels and lipid content of apoB-containing lipoproteins using nuclear magnetic resonance (NMR) spectroscopy and examined their association with EL serum levels in healthy volunteers (HVs) and MS patients. EL was significantly negatively correlated with the serum levels of cholesterol in large very low-density lipoprotein (VLDL) particles, as well as with total-cholesterol-, free-cholesterol-, triglyceride-, and phospholipid-contents of VLDL and intermediate-density lipoprotein particles in MS patients but not in HVs. In contrast, EL serum levels were significantly positively correlated with the serum levels of apoB, triglycerides, and phospholipids in large low-density lipoprotein particles in HVs but not in MS patients. EL serum levels as well as the serum levels and lipid content of the majority of apoB-containing lipoprotein subclasses were markedly different in MS patients compared with HVs. We conclude that EL serum levels are associated with the serum levels and lipid content of apoB-containing lipoproteins and that these associations are markedly affected by MS.

Associations between Endothelial Lipase, High-Density Lipoprotein, and Endothelial Function Differ in Healthy Volunteers and Metabolic Syndrome Patients.

Metabolic syndrome (MS) is characterized by endothelial- and high-density lipoprotein (HDL) dysfunction and increased endothelial lipase (EL) serum levels. We examined the associations between EL serum levels, HDL (serum levels, lipid content, and function), and endothelial function in healthy volunteers (HV) and MS patients. Flow-mediated dilation (FMD), nitroglycerin-mediated dilation (NMD), serum levels of HDL subclasses (measured by nuclear magnetic resonance (NMR) spectroscopy), and EL serum levels differed significantly between HV and MS patients. The serum levels of triglycerides in large HDL particles were significantly positively correlated with FMD and NMD in HV, but not in MS patients. Cholesterol (C) and phospholipid (PL) contents of large HDL particles, calculated as HDL1-C/HDL1-apoA-I and HDL1-PL/HDL1-apoA-I, respectively, were significantly negatively correlated with FMD in HV, but not in MS patients. Cholesterol efflux capacity and arylesterase activity of HDL, as well as EL, were correlated with neither FMD nor NMD. EL was significantly negatively correlated with HDL-PL/HDL-apoA-I in HV, but not in MS patients, and with serum levels of small dense HDL containing apolipoprotein A-II in MS patients, but not in HV. We conclude that MS modulates the association between HDL and endothelial function, as well as between EL and HDL. HDL cholesterol efflux capacity and arylesterase activity, as well as EL serum levels, are not associated with endothelial function in HV or MS patients.

Cholesterol Content of Very-Low-Density Lipoproteins Is Associated with 1-Year Mortality in Acute Heart Failure Patients.

Considering the relationship between the extent of metabolic derangement and the disease severity in heart failure, we hypothesized that the lipid content of very-low-density lipoprotein (VLDL) may have prognostic value for 1 year mortality in acute heart failure (AHF). Baseline serum levels of VLDL cholesterol (VLDL-C), VLDL triglycerides (VLDL-TG), VLDL phospholipids (VLDL-PL), and VLDL apolipoprotein B (VLDL-apoB) were measured using NMR spectroscopy. We calculated the ratios of the respective VLDL lipids and VLDL apoB (VLDL-C/VLDL-apoB, VLDL-TG/VLDL-apoB, and VLDL-PL/VLDL-apoB), as estimators of the cholesterol, triglyceride, and phospholipid content of VLDL particles and tested their association with mortality. Out of 315 AHF patients, 118 (37.5%) patients died within 1 year after hospitalization for AHF. Univariable Cox regression analyses revealed a significant inverse association of VLDL-C/VLDL-apoB (hazard ratio (HR) 0.43, 95% confidence interval (CI) 0.29−0.64, p < 0.001), VLDL-TG/VLDL-apoB (HR 0.79, 95% CI 0.71−0.88, p < 0.001), and VLDL-PL/VLDL-apoB (HR 0.37, 95% CI 0.25−0.56, p < 0.001) with 1 year mortality. Of the tested parameters, only VLDL-C/VLDL-apoB remained significant after adjustment for age and sex, as well as other clinical and laboratory parameters that showed a significant association with 1 year mortality in the univariable analyses. We conclude that cholesterol content of circulating VLDL (VLDL-C/VLDL-apoB) might be of prognostic value in AHF.

Deficiency of B vitamins leads to cholesterol-independent atherogenic transformation of the aorta.

Atherosclerosis, the leading cause of cardiovascular disease responsible for the majority of deaths worldwide, cannot be sufficiently explained by established risk factors, including hypercholesterolemia. Elevated plasma homocysteine is an independent risk factor for atherosclerosis and is strongly linked to cardiovascular mortality. However, the role of homocysteine in atherosclerosis is still insufficiently understood. Previous research in this area has been also hampered by the lack of reproducible in vivo models of atherosclerosis that resemble the human situation. Here, we have developed and applied an automated system for vessel wall injury that leads to more homogenous damage and more pronounced atherosclerotic plaque development, even at low balloon pressure. Our automated system helped to glean vital details of cholesterol-independent changes in the aortic wall of balloon-injured rabbits. We show that deficiency of B vitamins, which are required for homocysteine degradation, leads to atherogenic transformation of the aorta resulting in accumulation of macrophages and lipids, impairment of its biomechanical properties and disorganization of aortic collagen/elastin in the absence of hypercholesterolemia. A combination of B vitamin deficiency and hypercholesterolemia leads to thickening of the aorta, decreased aortic water diffusion, increased LDL-cholesterol and impaired vascular reactivity compared to any single condition. Our findings suggest that deficiency of B vitamins leads to atherogenic transformation of the aorta even in the absence of hypercholesterolemia and aggravates atherosclerosis development in its presence.

HDL-apoA-II Is Strongly Associated with 1-Year Mortality in Acute Heart Failure Patients.

The prognostic value of the subset of high-density lipoprotein (HDL) particles containing apolipoprotein (apo)A-II (HDL-apoA-II) in acute heart failure (AHF) remains unexplored. In this study, baseline serum levels of HDL-apoA-II (total and subfractions 1−4) were measured in 315 AHF patients using NMR spectroscopy. The mean patient age was 74.2 ± 10.5 years, 136 (43.2%) were female, 288 (91.4%) had a history of cardiomyopathy, 298 (94.6%) presented as New York Heart Association class 4, and 118 (37.5%) patients died within 1 year after hospitalization for AHF. Multivariable Cox regression analyses, adjusted for age and sex as well as other clinical and laboratory parameters associated with 1-year mortality in the univariable analyses, revealed a significant inverse association of HDL-apoA-II (hazard ratio (HR) 0.67 per 1 standard deviation (1 SD) increase, 95% confidence interval (CI) 0.47−0.94, p = 0.020), HDL2-apoA-II (HR 0.72 per 1 SD increase, 95% CI 0.54−0.95, p = 0.019), and HDL3-apoA-II (HR 0.59 per 1 SD increase, 95% CI 0.43−0.80, p < 0.001) with 1-year mortality. We conclude that low baseline HDL-apoA-II, HDL2-apoA-II, and HDL3-apoA-II serum levels are associated with increased 1-year mortality in AHF patients and may thus be of prognostic value in AHF.

Nicotinamide for the treatment of heart failure with preserved ejection fraction.

Heart failure with preserved ejection fraction (HFpEF) is a highly prevalent and intractable form of cardiac decompensation commonly associated with diastolic dysfunction. Here, we show that diastolic dysfunction in patients with HFpEF is associated with a cardiac deficit in nicotinamide adenine dinucleotide (NAD+). Elevating NAD+ by oral supplementation of its precursor, nicotinamide, improved diastolic dysfunction induced by aging (in 2-year-old C57BL/6J mice), hypertension (in Dahl salt-sensitive rats), or cardiometabolic syndrome (in ZSF1 obese rats). This effect was mediated partly through alleviated systemic comorbidities and enhanced myocardial bioenergetics. Simultaneously, nicotinamide directly improved cardiomyocyte passive stiffness and calcium-dependent active relaxation through increased deacetylation of titin and the sarcoplasmic reticulum calcium adenosine triphosphatase 2a, respectively. In a long-term human cohort study, high dietary intake of naturally occurring NAD+ precursors was associated with lower blood pressure and reduced risk of cardiac mortality. Collectively, these results suggest NAD+ precursors, and especially nicotinamide, as potential therapeutic agents to treat diastolic dysfunction and HFpEF in humans.

Endothelial Lipase Modulates Paraoxonase 1 Content and Arylesterase Activity of HDL.

Endothelial lipase (EL) is a strong modulator of the high-density lipoprotein (HDL) structure, composition, and function. Here, we examined the impact of EL on HDL paraoxonase 1 (PON1) content and arylesterase (AE) activity in vitro and in vivo. The incubation of HDL with EL-overexpressing HepG2 cells decreased HDL size, PON1 content, and AE activity. The EL modification of HDL did not diminish the capacity of HDL to associate with PON1 when EL-modified HDL was incubated with PON1-overexpressing cells. The overexpression of EL in mice significantly decreased HDL serum levels but unexpectedly increased HDL PON1 content and HDL AE activity. Enzymatically inactive EL had no effect on the PON1 content of HDL in mice. In healthy subjects, EL serum levels were not significantly correlated with HDL levels. However, HDL PON1 content was positively associated with EL serum levels. The EL-induced changes in the HDL-lipid composition were not linked to the HDL PON1 content. We conclude that primarily, the interaction of enzymatically active EL with HDL, rather than EL-induced alterations in HDL size and composition, causes PON1 displacement from HDL in vitro. In vivo, the EL-mediated reduction of HDL serum levels and the consequently increased PON1-to-HDL ratio in serum increase HDL PON1 content and AE activity in mice. In humans, additional mechanisms appear to underlie the association of EL serum levels and HDL PON1 content.

Endothelial Dysfunction Following Enhanced TMEM16A Activity in Human Pulmonary Arteries.

Endothelial dysfunction is one of the hallmarks of different vascular diseases, including pulmonary arterial hypertension (PAH). Ion channelome changes have long been connected to vascular remodeling in PAH, yet only recently has the focus shifted towards Ca2+-activated Cl- channels (CaCC). The most prominent member of the CaCC TMEM16A has been shown to contribute to the pathogenesis of idiopathic PAH (IPAH) in pulmonary arterial smooth muscle cells, however its role in the homeostasis of healthy human pulmonary arterial endothelial cells (PAECs) and in the development of endothelial dysfunction remains underrepresented. Here we report enhanced TMEM16A activity in IPAH PAECs by whole-cell patch-clamp recordings. Using adenoviral-mediated TMEM16A increase in healthy primary human PAECs in vitro and in human pulmonary arteries ex vivo, we demonstrate the functional consequences of the augmented TMEM16A activity: alterations of Ca2+ dynamics and eNOS activity as well as decreased NO production, PAECs proliferation, wound healing, tube formation and acetylcholine-mediated relaxation of human pulmonary arteries. We propose that the ERK1/2 pathway is specifically affected by elevated TMEM16A activity, leading to these pathological changes. With this work we introduce increased TMEM16A activity in the cell membrane of human PAECs for the development of endothelial dysfunction in PAH.

Dipeptidyl peptidase 3 modulates the renin-angiotensin system in mice.

Dipeptidyl peptidase 3 (DPP3) is a zinc-dependent hydrolase involved in degrading oligopeptides with 4-12 amino acid residues. It has been associated with several pathophysiological processes, including blood pressure regulation, pain signaling, and cancer cell defense against oxidative stress. However, the physiological substrates and the cellular pathways that are potentially targeted by DPP3 to mediate these effects remain unknown. Here, we show that global DPP3 deficiency in mice (DPP3-/-) affects the renin-angiotensin system (RAS). LC-MS-based profiling of circulating angiotensin peptides revealed elevated levels of angiotensin II, III, IV, and 1-5 in DPP3-/- mice, whereas blood pressure, renin activity, and aldosterone levels remained unchanged. Activity assays using the purified enzyme confirmed that angiotensin peptides are substrates for DPP3. Aberrant angiotensin signaling was associated with substantially higher water intake and increased renal reactive oxygen species formation in the kidneys of DPP3-/- mice. The metabolic changes and altered angiotensin levels observed in male DPP3-/- mice were either absent or attenuated in female DPP3-/- mice, indicating sex-specific differences. Taken together, our observations suggest that DPP3 regulates the RAS pathway and water homeostasis by degrading circulating angiotensin peptides.

Empagliflozin protects heart from inflammation and energy depletion via AMPK activation.

AIMS: Sodium-glucose co-transporter 2 (SGLT2) were originally developed as kidney-targeting anti-diabetic drugs. However, due to their beneficial cardiac off-target effects (as SGLT2 is not expressed in the heart), these antagonists currently receive intense clinical interest in the context of heart failure (HF) in patients with or without diabetes mellitus (DM). Since the mechanisms by which these beneficial effects are mediated are still unclear yet, inflammation that is present in DM and HF has been proposed as a potential pharmacological intervention strategy. Therefore, we tested the hypothesis that the SGLT2 inhibitor, empagliflozin, displays anti-inflammatory potential along with its glucose-lowering property. METHODS AND RESULTS: Lipopolysaccharide (LPS) was used to induce inflammation in vitro and in vivo. In cardiomyocytes and macrophages empagliflozin attenuated LPS-induced TNFα and iNOS expression. Analysis of intracellular signalling pathways suggested that empagliflozin activates AMP kinase (AMPK) in both cell types with or without LPS-treatment. Moreover, the SGLT2 inhibitor increased the expression of anti-inflammatory M2 marker proteins in LPS-treated macrophages. Additionally, empagliflozin-mediated AMPK activation prevented LPS-induced ATP/ADP depletion. In vivo administration of LPS in mice impaired cardiac contractility and aortic endothelial relaxation in response to acetylcholine, whereby co-administration of empagliflozin preserved cardiovascular function. These findings were accompanied by improved cardiac AMPK phosphorylation and ATP/ADP, reduced cardiac iNOS, plasma TNFα and creatine kinase MB levels. CONCLUSION: Our data identify a novel cardio protective mechanism of SGLT2 inhibitor, empagliflozin, suggesting that AMPK activation-mediated energy repletion and reduced inflammation contribute to the observed cardiovascular benefits of the drug in HF.

Circulating cord blood HDL-S1P complex preserves the integrity of the feto-placental vasculature.

Perinatal and long-term offspring morbidities are strongly dependent on the preservation of placental vascular homeostasis during pregnancy. In adults, the HDL-apoM-S1P complex protects the endothelium and maintains vascular integrity. However, the metabolism and biology of cord blood-derived HDLs (referred to as neonatal HDL, nHDL) strikingly differ from those in adults. Here, we investigate the role of neonatal HDLs in the regulation of placental vascular function. We show that nHDL is a major carrier of sphingosine-1-phosphate (S1P), which is anchored to the particle through apoM (rs = 0.90, p < 0.0001) in the fetal circulation. Furthermore, this complex interacts with S1P receptors on the feto-placental endothelium and activates specifically extracellular signal-regulated protein kinases 1 and 2 (ERK) and phospholipase C (PLC) downstream signaling, promotes endothelial cell proliferation and calcium flux. Notably, the nHDL-S1P complex triggers actin filaments reorganization, leading to an enhancement of placental endothelial barrier function. Additionally, nHDL induces vasorelaxation of isolated placental chorionic arteries. Taken together, these results suggest that circulating nHDL exerts vasoprotective effects on the feto-placental endothelial barrier mainly via S1P signaling.

Endothelial lipase increases eNOS activating capacity of high-density lipoprotein.

Endothelial lipase (EL) changes structural and functional properties of high-density lipoprotein (HDL). HDL is a relevant modulator of endothelial nitric oxide synthase (eNOS) activity, but the effect of EL on HDL induced eNOS-activation has not yet been investigated. Here, we examined the impact of EL-modified HDL (EL-HDL) on eNOS activity, subcellular trafficking, and eNOS- dependent vasorelaxation. EL-HDL and empty virus (EV)-HDL as control were isolated from human serum incubated with EL-overexpressing or EV infected HepG2 cells. EL-HDL exhibited higher capacity to induce eNOS phosphorylation at Ser1177 and eNOS activity in EA.hy 926 cells, as well as eNOS-dependent vasorelaxation of mouse aortic rings compared to control HDL. As revealed by confocal and structured illumination-microscopy EL-HDL-driven induction of eNOS was accompanied by an increased eNOS-GFP targeting to the plasma membrane and a lower eNOS-GFP colocalization with Golgi and mitochondria. Widefield microscopy of filipin stained cells revealed that EL-HDL lowered cellular free cholesterol (FC) and as found by thin-layer chromatography increased cellular cholesterol ester (CE) content. Additionally, cholesterol efflux capacity, acyl-coenzyme A: cholesterol acyltransferase activity, and HDL particle uptake were comparable between EL-HDL and control HDL. In conclusion, EL increases eNOS activating capacity of HDL, a phenomenon accompanied by an enrichment of the plasma membrane eNOS pool, a decreased cell membrane FC and increased cellular CE content.