Abstract Purpose Diabesity, the combination of obesity and type 2 diabetes, is an ever-growing global health burden. Diabesity-associated dysbiosis of the intestinal microbiome has gained attention as a potential driver of disease and, therefore, a possible therapeutic target by means of pro- or prebiotic supplementation. This study tested the effects of a multispecies synbiotic (i.e. a combination of probiotics and prebiotics) on glucose metabolism, gut microbiota, gut permeability, neutrophil function and quality of life in treatment-experienced diabesity patients. Methods A randomized, double-blind, placebo-controlled pilot study with 26 diabesity patients was conducted in which patients received a daily dose of a multispecies probiotic and a prebiotic (or a placebo) for 6 months. Results There were no changes in glucose metabolism or mixed meal tolerance test responses throughout the study. The analysis of secondary outcomes revealed beneficial effects on hip circumference [− 1 (95% CI − 4; 3) vs +3 (− 1; 8) cm, synbiotics vs. placebo, respectively, p = 0.04], serum zonulin [− 0.04 (− 0.2; 0.1) vs +0.3 (− 0.05; 0.6) ng/ml, p = 0.004)] and the physical role item of the SF36 quality of life assessment [+ 5.4 (− 1.7; 12.5) vs − 5.0 (− 10.1; 0.2) points, p = 0.02] after 3 months of intervention, and lipoprotein (a) [− 2.1 (− 5.7; 1.6) vs +3.4 (− 0.9; 7.9) mg/dl, p = 0.02] after 6 months. There were no significant differences in alpha or beta diversity of the microbiome between groups or time points. Conclusions Glucose metabolism as the primary outcome was unchanged during the intervention with a multispecies synbiotic in patients with diabesity. Nevertheless, synbiotics improved some symptoms and biomarkers of type 2 diabetes and aspects of quality of life suggesting a potential role as adjuvant tool in the management of diabesity. Graphic abstract
High-density lipoproteins (HDLs) are complex particles with multiple functions. They are thought to have evolved as part of the body's innate defense system against infection. Recent research suggests that HDL levels influence susceptibility to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and the severity of associated complications, making it a promising target for therapeutic intervention.
Experimental data indicate that stimulation of the nitric oxide-soluble guanylate cyclase(sGC)-cGMP-PKG pathway can increase left ventricular (LV) capacitance via phosphorylation of the myofilamental protein titin. We aimed to test whether acute pharmacological sGC stimulation with BAY 41-8543 would increase LV capacitance via titin phosphorylation in healthy and deoxycorticosteroneacetate (DOCA)-induced hypertensive pigs. Nine healthy Landrace pigs and 7 pigs with DOCA-induced hypertension and LV concentric hypertrophy were acutely instrumented to measure LV end-diastolic pressure-volume relationships (EDPVRs) at baseline and during intravenous infusion of BAY 41-8543 (1 and 3 μg·kg −1 ·min −1 for 30 min, respectively). Separately, in seven healthy and six DOCA pigs, transmural LV biopsies were harvested from the beating heart to measure titin phosphorylation during BAY 41-8543 infusion. LV EDPVRs before and during BAY 41-8543 infusion were superimposable in both healthy and DOCA-treated pigs, whereas mean aortic pressure decreased by 20–30 mmHg in both groups. Myocardial titin phosphorylation was unchanged in healthy pigs, but total and site-specific (Pro-Glu-Val-Lys and N2-Bus domains) titin phosphorylation was increased in DOCA-treated pigs. Bicoronary nitroglycerin infusion in healthy pigs ( n = 5) induced a rightward shift of the LV EDPVR, demonstrating the responsiveness of the pathway in this model. Acute systemic sGC stimulation with the sGC stimulator BAY 41-8543 did not recruit an LV preload reserve in both healthy and hypertrophied LV porcine myocardium, although it increased titin phosphorylation in the latter group. Thus, increased titin phosphorylation is not indicative of increased in vivo LV capacitance. NEW & NOTEWORTHY We demonstrate that acute pharmacological stimulation of soluble guanylate cyclase does not increase left ventricular compliance in normal and hypertrophied porcine hearts. Effects of long-term soluble guanylate cyclase stimulation with oral compounds in disease conditions associated with lowered myocardial cGMP levels, i.e., heart failure with preserved ejection fraction, remain to be investigated.
Hypochlorous acid/hypochlorite, generated by the myeloperoxidase/H(2)O(2)/halide system of activated phagocytes, has been shown to oxidize/modify low density lipoprotein (LDL) in vitro and may be involved in the formation of atherogenic lipoproteins in vivo. Accordingly, hypochlorite-modified (lipo)proteins have been detected in human atherosclerotic lesions where they colocalize with macrophages and endothelial cells. The present study investigates the influence of hypochlorite-modified LDL on endothelial synthesis of nitric oxide (NO) measured as formation of citrulline (coproduct of NO) and cGMP (product of the NO-activated soluble guanylate cyclase) upon cell stimulation with thrombin or ionomycin. Pretreatment of human umbilical vein endothelial cells with hypochlorite-modified LDL led to a time- and concentration-dependent inhibition of agonist-induced citrulline and cGMP synthesis compared with preincubation of cells with native LDL. This inhibition was neither due to a decreased expression of endothelial NO synthase (eNOS) nor to a deficiency of its cofactor tetrahydrobiopterin. Likewise, the uptake of l-arginine, the substrate of eNOS, into the cells was not affected. Hypochlorite-modified LDL caused remarkable changes of intracellular eNOS distribution including translocation from the plasma membrane and disintegration of the Golgi location without altering myristoylation or palmitoylation of the enzyme. In contrast, cyclodextrin known to deplete plasma membrane of cholesterol and to disrupt caveolae induced only a disappearance of eNOS from the plasma membrane that was not associated with decreased agonist-induced citrulline and cGMP formation. The present findings suggest that mislocalization of NOS accounts for the reduced NO formation in human umbilical vein endothelial cells treated with hypochlorite-modified LDL and point to an important role of Golgi-located NOS in these processes. We conclude that inhibition of NO synthesis by hypochlorite-modified LDL may be an important mechanism in the development of endothelial dysfunction and early pathogenesis of atherosclerosis.
CVD remains the leading cause of morbidity and mortality in patients with chronic kidney disease (CKD). CKD profoundly affects HDL composition and functionality, but whether abnormal HDL independently contributes to cardiovascular events in CKD patients remains elusive. In the present study, we assessed whether compositional and functional properties of HDL predict cardiovascular outcome among 526 nondialysis CKD patients who participate in the CARE FOR HOMe study. We measured HDL cholesterol, the content of HDL-associated proinflammatory serum amyloid A (SAA), and activities of the HDL enzymes paraoxonase and lipoprotein-associated phospholipase A2 (Lp-PLA2). In addition, we assessed the antioxidative activity of apoB-depleted serum. During a mean follow-up of 5.1 ± 2.1 years, 153 patients reached the predefined primary endpoint, a composite of atherosclerotic cardiovascular events including cardiovascular mortality and death of any cause. In univariate Cox regression analyses, lower HDL-cholesterol levels, higher HDL-associated SAA content, and lower paraoxonase activity predicted cardiovascular outcome, while Lp-PLA2 activity and antioxidative capacity did not. HDL-cholesterol and HDL-paraoxonase activity lost their association with cardiovascular outcome after adjustment for traditional cardiovascular and renal risk factors, while SAA lost its association after further adjustment for C-reactive protein. In conclusion, our data suggest that neither HDL quantity nor HDL composition or function independently predict cardiovascular outcome among nondialysis CKD patients. CVD remains the leading cause of morbidity and mortality in patients with chronic kidney disease (CKD). CKD profoundly affects HDL composition and functionality, but whether abnormal HDL independently contributes to cardiovascular events in CKD patients remains elusive. In the present study, we assessed whether compositional and functional properties of HDL predict cardiovascular outcome among 526 nondialysis CKD patients who participate in the CARE FOR HOMe study. We measured HDL cholesterol, the content of HDL-associated proinflammatory serum amyloid A (SAA), and activities of the HDL enzymes paraoxonase and lipoprotein-associated phospholipase A2 (Lp-PLA2). In addition, we assessed the antioxidative activity of apoB-depleted serum. During a mean follow-up of 5.1 ± 2.1 years, 153 patients reached the predefined primary endpoint, a composite of atherosclerotic cardiovascular events including cardiovascular mortality and death of any cause. In univariate Cox regression analyses, lower HDL-cholesterol levels, higher HDL-associated SAA content, and lower paraoxonase activity predicted cardiovascular outcome, while Lp-PLA2 activity and antioxidative capacity did not. HDL-cholesterol and HDL-paraoxonase activity lost their association with cardiovascular outcome after adjustment for traditional cardiovascular and renal risk factors, while SAA lost its association after further adjustment for C-reactive protein. In conclusion, our data suggest that neither HDL quantity nor HDL composition or function independently predict cardiovascular outcome among nondialysis CKD patients. There is unequivocal evidence of an inverse association between plasma HDL cholesterol (HDL-C) concentrations and the risk of CVD, a finding that has led to the hypothesis that HDL protects from CVD. In line with the HDL hypothesis, HDL exhibits many potential antiatherogenic properties (1.Toth P.P. Barter P.J. Rosenson R.S. Boden W.E. Chapman M.J. Cuchel M. D'Agostino Sr., R.B. Davidson M.H. Davidson W.S. Heinecke J.W. et al.High-density lipoproteins: a consensus statement from the National Lipid Association.J. Clin. Lipidol. 2013; 7: 484-525Abstract Full Text Full Text PDF PubMed Scopus (245) Google Scholar). HDL can act as an acceptor of cellular cholesterol, which constitutes the first step in a pathway known as reverse cholesterol transport (2.Yancey P.G. Bortnick A.E. Kellner-Weibel G. de la Llera-Moya M. Phillips M.C. Rothblat G.H. Importance of different pathways of cellular cholesterol efflux.Arterioscler. Thromb. Vasc. Biol. 2003; 23: 712-719Crossref PubMed Scopus (438) Google Scholar). In addition, HDL isolated from healthy subjects shows potent antiinflammatory and antioxidant properties (3.Barter P.J. Nicholls S. Rye K.A. Anantharamaiah G.M. Navab M. Fogelman A.M. Antiinflammatory properties of HDL.Circ. Res. 2004; 95: 764-772Crossref PubMed Scopus (1056) Google Scholar). apoA-I is the most important structural protein of HDL with antioxidant properties (4.Kontush A. Chapman M.J. Antiatherogenic function of HDL particle subpopulations: focus on antioxidative activities.Curr. Opin. Lipidol. 2010; 21: 312-318Crossref PubMed Scopus (217) Google Scholar, 5.Panzenböck U. Stocker R. Formation of methionine sulfoxide-containing specific forms of oxidized high-density lipoproteins.Biochim. Biophys. Acta. 2005; 1703: 171-181Crossref PubMed Scopus (65) Google Scholar). Consequently, HDL has the capacity to inhibit the oxidative modification of LDL and thereby reduce the atherogenicity of these lipoproteins (4.Kontush A. Chapman M.J. Antiatherogenic function of HDL particle subpopulations: focus on antioxidative activities.Curr. Opin. Lipidol. 2010; 21: 312-318Crossref PubMed Scopus (217) Google Scholar). HDL-associated paraoxonase shows antiinflammatory (6.Mackness B. Mackness M. The antioxidant properties of high-density lipoproteins in atherosclerosis.Panminerva Med. 2012; 54: 83-90PubMed Google Scholar, 7.Efrat M. Aviram M. Paraoxonase 1 interactions with HDL, antioxidants and macrophages regulate atherogenesis—a protective role for HDL phospholipids.Adv. Exp. Med. Biol. 2010; 660: 153-166Crossref PubMed Scopus (46) Google Scholar) and endothelial protective activities (8.Besler C. Heinrich K. Rohrer L. Doerries C. Riwanto M. Shih D.M. Chroni A. Yonekawa K. Stein S. Schaefer N. et al.Mechanisms underlying adverse effects of HDL on eNOS-activating pathways in patients with coronary artery disease.J. Clin. Invest. 2011; 121: 2693-2708Crossref PubMed Scopus (445) Google Scholar). Chronic kidney disease (CKD) profoundly alters enzyme activities involved in HDL metabolism, affecting particle maturation and remodeling, thereby changing HDL composition and function (9.Birner-Gruenberger R. Schittmayer M. Holzer M. Marsche G. Understanding high-density lipoprotein function in disease: recent advances in proteomics unravel the complexity of its composition and biology.Prog. Lipid Res. 2014; 56: 36-46Crossref PubMed Scopus (80) Google Scholar, 10.Marsche G. Saemann M.D. Heinemann A. Holzer M. Inflammation alters HDL composition and function: implications for HDL-raising therapies.Pharmacol. Ther. 2013; 137: 341-351Crossref PubMed Scopus (87) Google Scholar, 11.Holzer M. Schilcher G. Curcic S. Trieb M. Ljubojevic S. Stojakovic T. Scharnagl H. Kopecky C.M. Rosenkranz A.R. Heinemann A. et al.Dialysis modalities and HDL composition and function.J. Am. Soc. Nephrol. 2015; 26: 2267-2276Crossref PubMed Scopus (62) Google Scholar). These compositional alterations include enrichment of uremic HDL with proinflammatory acute-phase protein serum amyloid A (SAA) and lipoprotein-associated phospholipase A2 (Lp-PLA2) content (12.Holzer M. Birner-Gruenberger R. Stojakovic T. El-Gamal D. Binder V. Wadsack C. Heinemann A. Marsche G. Uremia alters HDL composition and function.J. Am. Soc. Nephrol. 2011; 22: 1631-1641Crossref PubMed Scopus (221) Google Scholar, 13.Weichhart T. Kopecky C. Kubicek M. Haidinger M. Doller D. Katholnig K. Suarna C. Eller P. Tolle M. Gerner C. et al.Serum amyloid A in uremic HDL promotes inflammation.J. Am. Soc. Nephrol. 2012; 23: 934-947Crossref PubMed Scopus (181) Google Scholar), whereas antiinflammatory HDL-paraoxonase content and activity are markedly reduced (11.Holzer M. Schilcher G. Curcic S. Trieb M. Ljubojevic S. Stojakovic T. Scharnagl H. Kopecky C.M. Rosenkranz A.R. Heinemann A. et al.Dialysis modalities and HDL composition and function.J. Am. Soc. Nephrol. 2015; 26: 2267-2276Crossref PubMed Scopus (62) Google Scholar). Of note, despite substantial experimental work on HDL in CKD, the clinical implications of most of these structural and functional alterations of HDL have remained enigmatic up to now. Earlier work on HDL functionality largely focused on its promotion of cholesterol efflux from macrophages (HDL-C efflux capacity). In the general population (14.Rohatgi A. Khera A. Berry J.D. Givens E.G. Ayers C.R. Wedin K.E. Neeland I.J. Yuhanna I.S. Rader D.R. de Lemos J.A. et al.HDL cholesterol efflux capacity and incident cardiovascular events.N. Engl. J. Med. 2014; 371: 2383-2393Crossref PubMed Scopus (970) Google Scholar, 15.Saleheen D. Scott R. Javad S. Zhao W. Rodrigues A. Picataggi A. Lukmanova D. Mucksavage M.L. Luben R. Billheimer J. et al.Association of HDL cholesterol efflux capacity with incident coronary heart disease events: a prospective case-control study.Lancet Diabetes Endocrinol. 2015; 3: 507-513Abstract Full Text Full Text PDF PubMed Scopus (348) Google Scholar) and among individuals at elevated cardiovascular risk (16.Ritsch A. Scharnagl H. Marz W. HDL cholesterol efflux capacity and cardiovascular events.N. Engl. J. Med. 2015; 372: 1870-1871PubMed Google Scholar), HDL cholesterol efflux capacity was inversely associated with incident cardiovascular events, even after adjusting for HDL-C levels. Importantly, these associations were not observed among CKD patients within the CARE FOR HOMe (Cardiovascular and Renal Outcome in CKD 2-4 Patients–The Fourth Homburg evaluation) (17.Bauer L. Kern S. Rogacev K.S. Emrich I.E. Zawada A. Fliser D. Heinemann A. Heine G.H. Marsche G. HDL cholesterol efflux capacity and cardiovascular events in patients with chronic kidney disease.J. Am. Coll. Cardiol. 2017; 69: 246-247Crossref PubMed Scopus (33) Google Scholar) and the German Diabetes Dialysis Study (4D Study) (18.Kopecky C. Ebtehaj S. Genser B. Drechsler C. Krane V. Antlanger M. Kovarik J.J. Kaltenecker C.C. Parvizi M. Wanner C. et al.HDL cholesterol efflux does not predict cardiovascular risk in hemodialysis patients.J. Am. Soc. Nephrol. 2017; 28: 769-775Crossref PubMed Scopus (42) Google Scholar). Given that oxidative stress and a chronic inflammatory state are major nontraditional risk factors among CKD patients (19.Locatelli F. Canaud B. Eckardt K.U. Stenvinkel P. Wanner C. Zoccali C. Oxidative stress in end-stage renal disease: an emerging threat to patient outcome.Nephrol. Dial. Transplant. 2003; 18: 1272-1280Crossref PubMed Scopus (621) Google Scholar, 20.Cachofeiro V. Goicochea M. de Vinuesa S.G. Oubina P. Lahera V. Luno J. Oxidative stress and inflammation, a link between chronic kidney disease and cardiovascular disease.Kidney Int. Suppl. 2008; 111: S4-S9Abstract Full Text Full Text PDF PubMed Scopus (443) Google Scholar, 21.Marsche G. Frank S. Hrzenjak A. Holzer M. Dirnberger S. Wadsack C. Scharnagl H. Stojakovic T. Heinemann A. Oettl K. Plasma-advanced oxidation protein products are potent high-density lipoprotein receptor antagonists in vivo.Circ. Res. 2009; 104: 750-757Crossref PubMed Scopus (87) Google Scholar, 22.Witko-Sarsat V. Friedlander M. Capeillere-Blandin C. Nguyen-Khoa T. Nguyen A.T. Zingraff J. Jungers P. Descamps-Latscha B. Advanced oxidation protein products as a novel marker of oxidative stress in uremia.Kidney Int. 1996; 49: 1304-1313Abstract Full Text PDF PubMed Scopus (1633) Google Scholar), tests of antiinflammatory properties of HDL and antioxidative activity of serum may prove useful. In the present study, we investigated whether metrics of HDL composition and function predict future cardiovascular events in a cohort of nondialysis CKD patients. CARE FOR HOMe is an ongoing cohort study including patients with CKD G2–G4 according to the Modification of Diet in Renal Disease equation [estimated glomerular filtration rate (eGFR) 15–89 ml/min/1.73 m2] at baseline (23.National Kidney Foundation K/DOQI clinical practice guidelines for chronic kidney disease: evaluation, classification, and stratification.Am. J. Kidney Dis. 2002; 39: S1-S266PubMed Google Scholar). CARE FOR HOMe excluded transplant recipients, pregnant women, patients under the age of 18, receivers of systemic immunosuppressive medication, and patients infected with human immunodeficiency virus. Clinically apparent infections [defined as C-reactive protein (CRP) levels > 50 mg/l, and/or requiring systemic antibiotic therapy] or active malignancy at baseline were excluded. Finally, patients with acute kidney injury were excluded. All procedures involving human subjects were approved by the local Ethics Committee and carried out in accordance with the Code of Ethics of the World Medical Association (Declaration of Helsinki). Informed consent was obtained from all patients. At baseline, all patients provided a fasting blood and a spot urine sample. A standardized questionnaire was used to collect data, including medical history, current medication, smoking status, and prevalent diabetes mellitus. Prevalent CVD was defined as any of the following: history of myocardial infarction, coronary artery angioplasty/stenting/bypass surgery, major stroke, carotid endarterectomy/stenting, nontraumatic lower extremity amputation, or lower limb artery angioplasty/stenting/bypass surgery. All prevalent cardiovascular events were confirmed by chart review. Patients with self-reported or physician-reported diabetes mellitus, with a fasting blood glucose level of >126 mg/dl or treatment with hypoglycemic medication, were categorized as diabetic. Patients were defined as active smokers if they smoked at study start or had stopped smoking <1 month before entry into the study. Only 10 women reported estrogen treatment. Annually, we invited all patients to our study center for a follow-up examination, in which we collected information on cardiovascular events by a standardized questionnaire. Patients who became dialysis-dependent during follow-up and patients who refused to attend the regular follow-up visit were contacted by telephone, and laboratory information was provided from their treating primary care physician or their nephrologist. Two independent physicians adjudicated all events; in case of disagreement, a third physician was consulted. The primary atherosclerotic cardiovascular endpoint was defined as the first occurrence of any of the following: myocardial infarction, coronary artery angioplasty/stenting/bypass surgery, major stroke, carotid endarterectomy/stenting, nontraumatic lower extremity amputation, and/or lower limb artery angioplasty/stenting/bypass surgery, or death of any cause. As additional endpoints, we defined all-cause death as well as hospital admission for heart failure. All cardiovascular events were confirmed by chart review. A total of 544 patients were included into CARE FOR HOMe between 2008 and 2015. Serum was available from 526 patients for SAA, antioxidative activity, paraoxonase activity, Lp-PLA2 activity, HDL-C efflux capacity, and HDL-C measurements. The samples analyzed for these variables were drawn at baseline. SAA content was quantified by ELISA (human SAA, BioSource Europe S.A., Belgium) as described (24.Pertl L. Kern S. Weger M. Hausberger S. Trieb M. Gasser-Steiner V. Haas A. Scharnagl H. Heinemann A. Marsche G. High-density lipoprotein function in exudative age-related macular degeneration.PLoS One. 2016; 11: e0154397PubMed Google Scholar, 25.Trieb M. Horvath A. Birner-Gruenberger R. Spindelboeck W. Stadlbauer V. Taschler U. Curcic S. Stauber R.E. Holzer M. Pasterk L. et al.Liver disease alters high-density lipoprotein composition, metabolism and function.Biochim. Biophys. Acta. 2016; 1861: 630-638Crossref PubMed Scopus (54) Google Scholar). ApoB-depleted serum was prepared by the addition of 40 μl of polyethylene glycol (20% in 200 mmol/l glycine buffer) to 100 μl of serum. Samples were incubated for 20 min, and the supernatants were recovered after centrifugation (9,703 g, 20 min, 4°C) as described (26.Khera A.V. Cuchel M. de la Llera-Moya M. Rodrigues A. Burke M.F. Jafri K. French B.C. Phillips J.A. Mucksavage M.L. Wilensky R.L. et al.Cholesterol efflux capacity, high-density lipoprotein function, and atherosclerosis.N. Engl. J. Med. 2011; 364: 127-135Crossref PubMed Scopus (1533) Google Scholar, 27.Potočnjak I. Degoricija V. Trbusic M. Teresak S.D. Radulovic B. Pregartner G. Berghold A. Tiran B. Marsche G. Frank S. Metrics of high-density lipoprotein function and hospital mortality in acute heart failure patients.PLoS One. 2016; 11: e0157507Crossref PubMed Scopus (22) Google Scholar). Subsequently, the HDL-containing supernatants (apoB-depleted serum) were collected and stored at –80°C. The antioxidative activity of apoB-depleted serum was determined as previously described (24.Pertl L. Kern S. Weger M. Hausberger S. Trieb M. Gasser-Steiner V. Haas A. Scharnagl H. Heinemann A. Marsche G. High-density lipoprotein function in exudative age-related macular degeneration.PLoS One. 2016; 11: e0154397PubMed Google Scholar, 27.Potočnjak I. Degoricija V. Trbusic M. Teresak S.D. Radulovic B. Pregartner G. Berghold A. Tiran B. Marsche G. Frank S. Metrics of high-density lipoprotein function and hospital mortality in acute heart failure patients.PLoS One. 2016; 11: e0157507Crossref PubMed Scopus (22) Google Scholar). Briefly, dihydrorhodamine (DHR) was suspended in DMSO to a 50 mmol/l stock, which was diluted in HEPES (20 mmol/l HEPES, 150 mmol/l NaCl, pH 7.4) containing 1 mmol/l 2,2'-azobis-2-methyl-propanimidamide-dihydrochloride to a 50 µmol/l working reagent. One microliter of apoB-depleted serum was placed into 384-well plates, 15 µl of DHR working reagent was added, and the volume was completed to 100 µl with HEPES buffer. The increase in fluorescence due to the oxidation of DHR was measured every 2 min for 1 h at 538 nm. The increase in fluorescence per minute was determined for samples containing only DHR and for samples containing DHR and individual serum samples. Ca2+-dependent arylesterase activity of paraoxonase was determined by a photometric assay using phenylacetate as substrates as described (25.Trieb M. Horvath A. Birner-Gruenberger R. Spindelboeck W. Stadlbauer V. Taschler U. Curcic S. Stauber R.E. Holzer M. Pasterk L. et al.Liver disease alters high-density lipoprotein composition, metabolism and function.Biochim. Biophys. Acta. 2016; 1861: 630-638Crossref PubMed Scopus (54) Google Scholar, 27.Potočnjak I. Degoricija V. Trbusic M. Teresak S.D. Radulovic B. Pregartner G. Berghold A. Tiran B. Marsche G. Frank S. Metrics of high-density lipoprotein function and hospital mortality in acute heart failure patients.PLoS One. 2016; 11: e0157507Crossref PubMed Scopus (22) Google Scholar). ApoB-depleted serum was added to 200 μl of buffer containing 100 mmol/l Tris, 2 mmol/l CaCl2 (pH 8.0), and 1 mmol/l phenylacetate. The rate of hydrolysis of phenylacetate was monitored by the increase of absorbance at 270 nm, and readings were taken every 15 s at room temperature to generate a kinetic plot. The slope from the kinetic chart was used to determine ΔAb270 nm/min. Enzymatic activity was calculated with the Beer–Lambert law from the molar extinction coefficient of 1,310 l·mol−1·cm−1. HDL-associated Lp-PLA2 activity was measured in apoB-depleted serum by using a commercially available photometric assay (Cayman Europe, Talinn, Estonia) using 2-thio platelet activating factor as substrate as described previously (24.Pertl L. Kern S. Weger M. Hausberger S. Trieb M. Gasser-Steiner V. Haas A. Scharnagl H. Heinemann A. Marsche G. High-density lipoprotein function in exudative age-related macular degeneration.PLoS One. 2016; 11: e0154397PubMed Google Scholar). We quantified cholesterol efflux capacity with use of a previously published protocol (26.Khera A.V. Cuchel M. de la Llera-Moya M. Rodrigues A. Burke M.F. Jafri K. French B.C. Phillips J.A. Mucksavage M.L. Wilensky R.L. et al.Cholesterol efflux capacity, high-density lipoprotein function, and atherosclerosis.N. Engl. J. Med. 2011; 364: 127-135Crossref PubMed Scopus (1533) Google Scholar, 27.Potočnjak I. Degoricija V. Trbusic M. Teresak S.D. Radulovic B. Pregartner G. Berghold A. Tiran B. Marsche G. Frank S. Metrics of high-density lipoprotein function and hospital mortality in acute heart failure patients.PLoS One. 2016; 11: e0157507Crossref PubMed Scopus (22) Google Scholar, 28.Marsche G. Zelzer S. Meinitzer A. Kern S. Meissl S. Pregartner G. Weghuber D. Almer G. Mangge H. Adiponectin predicts high-density lipoprotein cholesterol efflux capacity in adults irrespective of body mass index and fat distribution.J. Clin. Endocrinol. Metab. 2017; 102: 4117-4123Crossref PubMed Scopus (29) Google Scholar). J774 cells, a mouse macrophage cell line, were plated and labeled for 24 h with 1 μCi/ml [3H]cholesterol (Perkin Elmer, Boston, MA). J774 cells express very low levels of ATP-binding cassette transporter A1 (ABCA1), an important pathway of cholesterol efflux from macrophages. To upregulate ABCA1, cells were stimulated for 6 h with serum-free DMEM containing 0.3 mmol/l 8-(4-chlorophenylthio)-cyclic AMP (Sigma, Darmstadt, Germany). After labeling, cells were washed, and [3H]cholesterol efflux was determined by incubating cells for 4 h with 2.8% apoB-depleted serum as described (26.Khera A.V. Cuchel M. de la Llera-Moya M. Rodrigues A. Burke M.F. Jafri K. French B.C. Phillips J.A. Mucksavage M.L. Wilensky R.L. et al.Cholesterol efflux capacity, high-density lipoprotein function, and atherosclerosis.N. Engl. J. Med. 2011; 364: 127-135Crossref PubMed Scopus (1533) Google Scholar). The quantity of [3H]cholesterol incorporated into cellular lipids was assessed by means of isopropanol extraction of [3H]cholesterol content of J774 cells not exposed to the patients' serum. All steps were performed in the presence of a concentration of 2 μg/ml of the acyl CoA cholesterol acyltransferase inhibitor Sandoz 58-035 (Sigma, Darmstadt, Germany). All assays were performed twice, and samples were assessed in duplicates. To correct for interassay variation across plates, we included a serum control on each plate, and we normalized values for serum samples from patients to this value in subsequent analyses. Across the entire cohort, duplicate measures of efflux capacity were highly correlated (r = 0.91). Categorical variables are presented as percentage of patients and compared by using the chi2 test. Continuous data are expressed as means ± SD, or median (interquartile ranges), as appropriate, and were compared by using one-way ANOVA test partitioning the between-groups sums of squares into trend components; continuous variables are presented as mean ± SD, or median (interquartile range) in case of skewed distribution. We calculated univariate correlation analyses using Spearman coefficients. For outcome analyses, we first performed Kaplan-Meier analysis with consecutive log-rank testing, after stratifying patients into tertiles by their HDL-C, SAA, antioxidative activity, paraoxonase, and Lp-PLA2 activities. Subsequently, we calculated univariate and multivariate Cox models to assess the association of a) HDL-C, b) logarithmized SAA (logSAA), c) antioxidative activity, d) paraoxonase activity, and e) Lp-PLA2 activity and cardiovascular outcome. All variables were considered both as continuous variables and as categorized variables, after stratification of patients into tertiles. We predefined multivariate Cox models with adjustment for age, gender, body mass index, mean blood pressure, smoking status, diabetes mellitus, eGFR, and log-transferred albuminuria (model 2), with additional adjustment for total cholesterol and HDL-C (for analyses with SAA, antioxidative activity, paraoxonase activity, and Lp-PLA2 activity as exposition variable) or log-transformed SAA, respectively (for analyses with HDL-C as exposition variable; model 3), and with additional adjustment for CRP (model 4). The CARE FOR HOMe consists of 526 study patients stratified by Kidney Disease: Improving Global Outcomes stages. The mean age at study entry was 65.1 ± 12.3 years with a mean eGFR of 46 ± 16 ml/min/1.73 m2 (Table 1). Of those, 39% of all patients had prevalent CVD, and about one-third had diabetes mellitus. With declining renal function, people were older and had more cardiovascular comorbidities (Table 1). Serum samples of study subjects with more advanced CKD stages showed similar HDL-C levels (Fig. 1A), increased levels of the HDL associated acute phase protein SAA (P = 0.016) (Fig. 1B), higher antioxidative activity (P < 0.001) (Fig. 1C), and lower paraoxonase activity (P = 0.011) (Fig. 1D), whereas HDL-associated Lp-PLA2 activity was not altered (Fig. 1E). In addition, we stratified the study participants by the presence of diabetes mellitus (supplemental. Fig. S1). CKD patients with diabetes mellitus showed lower HDL-C levels (P < 0.001), increased SAA (P = 0.045), lower arylesterase activity of paraoxonase (P = 0.013), and HDL associated Lp-PLA2 activity (P = 0.010) whereas antioxidative activity was not different (P = 0.935).TABLE 1Baseline characteristics, stratified by eGFR categoriesCKD G 2 (n = 112)CKD G 3a (n = 178)CKD G 3b (n = 141)CKD G 4 (n = 95)Total cohort (n = 526)p-valuesAge (years)58.8 ± 12.165.1 ± 12.568.3 ± 10.968.1 ± 11.465.1 ± 12.3≤0.001Sex (woman)38 (34%)81 (46%)61 (43%)38 (40%)218 (41%)0.252eGFR (ml/min/1.73 m2)69 ± 752 ± 438 ± 423 ± 446 ± 16≤0.001BMI (kg/m2)30.4 ± 5.430.7 ± 5.530.6 ± 6.029.5 ± 4.730.4 ± 5.50.280Diabetes mellitus (yes)40 (36%)69 (39%)54 (38%)40 (42%)203 (39%)0.827Mean BP (mmHg)108 ± 14110 ± 14105 ± 15107 ± 16108 ± 150.104Systolic BP (mmHg)149 ± 20153 ± 24150 ± 24155 ± 27152 ± 240.396Diastolic BP (mmHg)108 ± 14110 ± 14105 ± 15107 ± 16108 ± 15≤0.001Prevalent CVD (yes)18 (16%)55 (31%)61 (43%)31 (33%)165 (31%)≤0.001Current nicotine (yes)19 (17%)15 (8%)12 (9%)8 (8%)54 (10%)0.074CRP (mg/l)2.35 (1.23–4.30)2.5 (1.05–4.90)2.9 (1.2–6.4)3.3 (1.1–6-1)2.7 (1.1–5.1)0.001Triglycerides (mg/dl)157 ± 124159 ± 116165 ± 104176 ± 125163 ± 1170.210Total cholesterol (mg/dl)195 ± 39192 ± 43189 ± 44190 ± 47192 ± 430.290LDL-C (mg/dl)118 ± 35114 ± 37112 ± 36114 ± 39114 ± 360.303HDL-C (mg/dl)53 ± 1653 ± 1851 ± 1749 ± 1752 ± 170.067Lipid-lowering drugs (yes)47 (42%)100 (56%)87 (62%)52 (55%)286 (54%)0.017Statins (yes)43 (38%)94 (53%)84 (60%)50 (53%)271 (52%)0.009Lipid-lowering therapy other than statins (yes)14 (13%)16 (9%)19 (14%)7 (7%)56 (11%)0.365ApoA-I (mg/dl)166.3 ± 30.6168.1 ± 31.0164.5 ± 31.3162.1 ± 35.5165.7 ± 31.90.222ApoB (mg/dl)98.7 ± 24.6100.3 ± 26.498.0 ± 25.499.0 ± 26.499.1 ± 25.70.814Paraoxonase activity (U/ml)423.43 ± 118.45413.21 ± 104.76388.36 ± 112.51392.87 ± 122.51405.05 ± 113.690.011SAA (μg/ml)25.46 (12.61–52.68)33.13 (18.16–59.36)31.13 (14.44–58.53)30.15 (15.79–71.38)29.95 (14.96–61.75)0.016Antioxidative activity (Inhibition of oxidation in %)63.06 ± 5.2965.34 ± 5.4866.01 ± 5.9566.03 ± 5.8965.16 ± 5.74≤0.001Lp-PLA2 activity (U/ml)59.55 ± 10.3160.02 ± 11.9759.25 ± 13.0460.99 ± 9.9259.89 ± 11.580.561Values are means ± SD or interquartile ranges (in parentheses). BP, blood pressure; LDL-C, LDL cholesterol. Open table in a new tab Values are means ± SD or interquartile ranges (in parentheses). BP, blood pressure; LDL-C, LDL cholesterol. Kidney function weakly correlated with lower CRP (P = 0.031), higher HDL-C (P = 0.030), and higher paraoxonase activity (P = 0.014) (Table 2). Interestingly, kidney function was inversely associated with cholesterol efflux capacity (P = 0.006), suggesting that some metrics of HDL function are not affected and even improved in more advanced CKD stages. Moreover, kidney function was inversely associated with antioxidative activity of apoB-depleted serum (P < 0.001). The systemic inflammation marker CRP significantly correlated with lower HDL-C (P = 0.001), Lp-PLA2 activity (P = 0.001), and cholesterol efflux capacity (P < 0.001). As expected, a robust association between the systemic inflammation markers CRP and the HDL-associated SAA (P < 0.001) was observed. Further details and correlations between markers of HDL functionality are shown in Table 2.TABLE 2Univariate Spearman correlation coefficientseGFRCRPHDL-CParaoxonase activitySAAAntioxidative activityLp-PLA2 activityRhoPRhoPRhoPRhoPRhoPRhoPRhoPCRP−0.0940.031————————————HDL-C0.0950.030−0.193<0.001——————————Paraoxonase activity0.1070.014−0.0670.1230.245<0.001————————SAA−0.0730.0920.575<0.0010.0800.0650.0260.557——————Antioxidative activity−0.233<0.0010.0420.342−0.1240.0040.1280.003−0.0430.321————Lp-PLA2 activity−0.0350.428−0.1390.0010.0750.0850.1360.002−0.1110.0110.0880.045——Cholesterol efflux capacity−0.1200.006−0.1480.0010.497<0.0010.289<0.001−0.0140.7510.0700.1110.153<0.001Boldface type indicates significance (P < 0.05). Open table in a new tab Boldface type indicates significance (P < 0.05). During a mean follow-up of 5.1 ± 2.1 years, 153 patients reached the primary cardiovascular endpoint. After stratifying patients in tertiles for levels of markers of HDL-C quantity and functionality, lower levels of HDL-C (P = 0.015) and paraoxonase activity (P < 0.001) were associated with the occurrence of the primary endpoint in Kaplan-Meier analyses (Fig. 2). The primary endpoint was neither predicted by antioxidative activity (P = 0.467) nor by Lp-PLA2 (P = 0.818) nor by SAA (P = 0.054; Fig. 2). As additional endpoints, we defined all-cause death (which is majorly driven by cardiovascular death), as well as hospital admission for heart failure. We recalculated our analyses with these alternative endpoints, but did not find a substantial difference in the main results (supplemental Figs. S2 and S3). Consistently, in univariate Cox regression analyses with HDL-C quantity and function markers considered as continuous variables, lower HD
Lipid abnormalities may have an effect on clinical outcomes of patients on dialysis. Recent studies have indicated that HDL dysfunction is a hallmark of ESRD. In this study, we compared HDL composition and metrics of HDL functionality in patients undergoing hemodialysis (HD) or peritoneal dialysis (PD) with those in healthy controls. We detected a marked suppression of several metrics of HDL functionality in patients on HD or PD. Compositional analysis revealed that HDL from both dialysis groups shifted toward a more proinflammatory phenotype with profound alterations in the lipid moiety and protein composition. With regard to function, cholesterol efflux and anti-inflammatory and antiapoptotic functions seemed to be more severely suppressed in patients on HD, whereas HDL-associated paraoxonase activity was lowest in patients on PD. Quantification of enzyme activities involved in HDL metabolism suggested that HDL particle maturation and remodeling are altered in patients on HD or PD. In summary, our study provides mechanistic insights into the formation of dysfunctional HDL in patients with ESRD who are on HD or PD.
Endothelial lipase (EL) is a potent modulator of the structural and functional properties of HDL. Impact of EL on cholesterol efflux capacity (CEC) of serum and isolated HDL is not well understood and apparently contradictory data were published. Here, we systematically examined the impact of EL on composition and CEC of serum and isolated HDL, in vitro and in vivo, using EL-overexpressing cells and EL-overexpressing mice. CEC was examined in a validated assay using 3H-cholesterol labelled J774 macrophages. In vitro EL-modification of serum resulted in complex alterations, including enrichment of serum with lipid-free/-poor apoA-I, decreased size of human (but not mouse) HDL and altered HDL lipid composition. EL-modification of serum increased CEC, in line with increased lipid-free/-poor apoA-I formation. In contrast, CEC of isolated HDL was decreased likely through altered lipid composition. In contrast to in vitro results, EL-overexpression in mice markedly decreased HDL-cholesterol and apolipoprotein A-I serum levels associated with a decreased CEC of serum. HDL lipid composition was altered, but HDL particle size and CEC were not affected. Our study highlights the multiple and complex effects of EL on HDL composition and function and may help to clarify the seemingly contradictory data found in published articles.
Cholesterol and other lipids carried by lipoproteins play an indispensable role in fetal development. Recent evidence suggests that maternally derived high-density lipoprotein (HDL) differs from fetal HDL with respect to its proteome, size, and function. Compared to the HDL of adults, fetal HDL is the major carrier of cholesterol and has a unique composition that implies other physiological functions. Fetal HDL is enriched in apolipoprotein E, which binds with high affinity to the low-density lipoprotein receptor. Thus, it appears that a primary function of fetal HDL is the transport of cholesterol to tissues as is accomplished by low-density lipoproteins in adults. The fetal HDL-associated bioactive sphingolipid sphingosine-1-phosphate shows strong vasoprotective effects at the fetoplacental vasculature. Moreover, lipoprotein-associated phospholipase A2 carried by fetal-HDL exerts anti-oxidative and athero-protective functions on the fetoplacental endothelium. Notably, the mass and activity of HDL-associated paraoxonase 1 are about 5-fold lower in the fetus, accompanied by an attenuation of anti-oxidative activity of fetal HDL. Cholesteryl ester transfer protein activity is reduced in fetal circulation despite similar amounts of the enzyme in maternal and fetal serum. This review summarizes the current knowledge on fetal HDL as a potential vasoprotective lipoprotein during fetal development. We also provide an overview of whether and how the protective functionalities of HDL are impaired in pregnancy-related syndromes such as pre-eclampsia or gestational diabetes mellitus.
