Lucia Rohrer

Mechanisms underlying adverse effects of HDL on eNOS-activating pathways in patients with coronary artery disease

Therapies that raise levels of HDL, which is thought to exert atheroprotective effects via effects on endothelium, are being examined for the treatment or prevention of coronary artery disease (CAD). However, the endothelial effects of HDL are highly heterogeneous, and the impact of HDL of patients with CAD on the activation of endothelial eNOS and eNOS-dependent pathways is unknown. Here we have demonstrated that, in contrast to HDL from healthy subjects, HDL from patients with stable CAD or an acute coronary syndrome (HDLCAD) does not have endothelial antiinflammatory effects and does not stimulate endothelial repair because it fails to induce endothelial NO production. Mechanistically, this was because HDLCAD activated endothelial lectin-like oxidized LDL receptor 1 (LOX-1), triggering endothelial PKCβII activation, which in turn inhibited eNOS-activating pathways and eNOS-dependent NO production. We then identified reduced HDL-associated paraoxonase 1 (PON1) activity as one molecular mechanism leading to the generation of HDL with endothelial PKCβII-activating properties, at least in part due to increased formation of malondialdehyde in HDL. Taken together, our data indicate that in patients with CAD, HDL gains endothelial LOX-1- and thereby PKCβII-activating properties due to reduced HDL-associated PON1 activity, and that this leads to inhibition of eNOS-activation and the subsequent loss of the endothelial antiinflammatory and endothelial repair-stimulating effects of HDL.

Endothelial-Vasoprotective Effects of High-Density Lipoprotein Are Impaired in Patients With Type 2 Diabetes Mellitus but Are Improved After Extended-Release Niacin Therapy

Background— High-density lipoprotein (HDL)–raising therapies are currently under intense evaluation, but the effects of HDL may be highly heterogeneous. We therefore compared the endothelial effects of HDL from healthy subjects and from patients with type 2 diabetes mellitus and low HDL (meeting the criteria for metabolic syndrome), who are frequently considered for HDL-raising therapies. Moreover, in diabetic patients, we examined the impact of extended-release (ER) niacin therapy on the endothelial effects of HDL. Methods and Results— HDL was isolated from healthy subjects (n=10) and patients with type 2 diabetes (n=33) by sequential ultracentrifugation. Effects of HDL on endothelial nitric oxide and superoxide production were characterized by electron spin resonance spectroscopy analysis. Effects of HDL on endothelium-dependent vasodilation and early endothelial progenitor cell–mediated endothelial repair were examined. Patients with diabetes were randomized to a 3-month therapy with ER niacin (1500 mg/d) or placebo, and endothelial effects of HDL were characterized. HDL from healthy subjects stimulated endothelial nitric oxide production, reduced endothelial oxidant stress, and improved endothelium-dependent vasodilation and early endothelial progenitor cell–mediated endothelial repair. In contrast, these beneficial endothelial effects of HDL were not observed in HDL from diabetic patients, which suggests markedly impaired endothelial-protective properties of HDL. ER niacin therapy improved the capacity of HDL to stimulate endothelial nitric oxide, to reduce superoxide production, and to promote endothelial progenitor cell–mediated endothelial repair. Further measurements suggested increased lipid oxidation of HDL in diabetic patients, and a reduction after ER niacin therapy. Conclusions— HDL from patients with type 2 diabetes mellitus and metabolic syndrome has substantially impaired endothelial-protective effects compared with HDL from healthy subjects. ER niacin therapy not only increases HDL plasma levels but markedly improves endothelial-protective functions of HDL in these patients, which is potentially more important. Clinical Trial Registration— clinicaltrials.gov. Identifier: NCT00346970.

Altered Activation of Endothelial Anti- and Proapoptotic Pathways by High-Density Lipoprotein from Patients with Coronary Artery Disease Role of High-Density Lipoprotein–Proteome Remodeling

Background— Endothelial dysfunction and injury are thought to play an important role in the progression of coronary artery disease (CAD). High-density lipoprotein from healthy subjects (HDLHealthy) has been proposed to exert endothelial antiapoptotic effects that may represent an important antiatherogenic property of the lipoprotein. The present study therefore aimed to compare effects of HDLCAD and HDLHealthy on the activation of endothelial anti- and proapoptotic pathways and to determine which changes of the lipoprotein are relevant for these processes. Methods and Results— HDL was isolated from patients with stable CAD (HDLsCAD), an acute coronary syndrome (HDLACS), and healthy subjects. HDLHealthy induced expression of the endothelial antiapoptotic Bcl-2 protein Bcl-xL and reduced endothelial cell apoptosis in vitro and in apolipoprotein E–deficient mice in vivo. In contrast, HDLsCAD and HDLACS did not inhibit endothelial apoptosis, failed to activate endothelial Bcl-xL, and stimulated endothelial proapoptotic pathways, in particular, p38-mitogen-activated protein kinase–mediated activation of the proapoptotic Bcl-2 protein tBid. Endothelial antiapoptotic effects of HDLHealthy were observed after inhibition of endothelial nitric oxide synthase and after delipidation, but not completely mimicked by apolipoprotein A-I or reconstituted HDL, suggesting an important role of the HDL proteome. HDL proteomics analyses and subsequent validations and functional characterizations suggested a reduced clusterin and increased apolipoprotein C-III content of HDLsCAD and HDLACS as mechanisms leading to altered effects on endothelial apoptosis. Conclusions— The present study demonstrates for the first time that HDLCAD does not activate endothelial antiapoptotic pathways, but rather stimulates potential endothelial proapoptotic pathways. HDL-proteome remodeling plays an important role for these altered functional properties of HDL. These findings provide novel insights into mechanisms leading to altered vascular effects of HDL in coronary disease.

High density lipoproteins in the intersection of diabetes mellitus, inflammation and cardiovascular disease.

Purpose of review Low HDL-cholesterol, diabetes mellitus and elevated C-reactive protein as well as various inflammatory diseases are risk factors for coronary heart disease. Both diabetes mellitus and inflammation decrease HDL-cholesterol. We summarize recent findings on the mechanisms underlying low HDL-cholesterol in diabetes and inflammation, as well as on novel functions of HDL that may protect not only from atherosclerosis but also from diabetes mellitus and inflammation-induced organ damage. Recent findings Elevated levels of non-esterified fatty acids and disturbed insulin action contribute to low HDL-cholesterol in diabetes mellitus by modifying lipolysis, apolipoprotein A-I production, as well as the activities of adenosine triphosphate-binding cassette transporter A1 and lipid transfer. Inflammation causes low HDL-cholesterol by increasing the activities of endothelial lipase and soluble phospholipase A2 and by replacing apolipoprotein A-I in HDL with serum amyloid A. HDL and lysosphingolipids therein have been identified as activators of the protein kinase Akt, which in turn is a regulator of apoptosis in beta-cells, endothelial cells, and smooth muscle cells, as well as a regulator of nitric oxide production and adhesion molecule expression in endothelial cells. Summary The protective properties of HDL in cytokine production, lipid oxidation, cholesterol efflux and reverse cholesterol transport make HDL a protective agent in inflammation-induced organ damage including diabetes mellitus. However, inflammation and diabetes cause a decrease in HDL-cholesterol concentrations and impair HDL function, placing HDL into the centre of a vicious cycle that may escalate into diabetes mellitus, inflammation-induced organ damage and atherosclerosis.

Abnormal High-Density Lipoprotein Induces Endothelial Dysfunction via Activation of Toll-like Receptor-2

Endothelial injury and dysfunction (ED) represent a link between cardiovascular risk factors promoting hypertension and atherosclerosis, the leading cause of death in Western populations. High-density lipoprotein (HDL) is considered antiatherogenic and known to prevent ED. Using HDL from children and adults with chronic kidney dysfunction (HDL(CKD)), a population with high cardiovascular risk, we have demonstrated that HDL(CKD) in contrast to HDL(Healthy) promoted endothelial superoxide production, substantially reduced nitric oxide (NO) bioavailability, and subsequently increased arterial blood pressure (ABP). We have identified symmetric dimethylarginine (SDMA) in HDL(CKD) that causes transformation from physiological HDL into an abnormal lipoprotein inducing ED. Furthermore, we report that HDL(CKD) reduced endothelial NO availability via toll-like receptor-2 (TLR-2), leading to impaired endothelial repair, increased proinflammatory activation, and ABP. These data demonstrate how SDMA can modify the HDL particle to mimic a damage-associated molecular pattern that activates TLR-2 via a TLR-1- or TLR-6-coreceptor-independent pathway, linking abnormal HDL to innate immunity, ED, and hypertension.

Low- and High-Density Lipoproteins Modulate Function, Apoptosis, and Proliferation of Primary Human and Murine Pancreatic β-Cells

A low high-density lipoprotein (HDL) plasma concentration and the abundance of small dense low-density lipoproteins (LDL) are risk factors for developing type 2 diabetes. We therefore investigated whether HDL and LDL play a role in the regulation of pancreatic islet cell apoptosis, proliferation, and secretory function. Isolated mouse and human islets were exposed to plasma lipoproteins of healthy human donors. In murine and human beta-cells, LDL decreased both proliferation and maximal glucose-stimulated insulin secretion. The comparative analysis of beta-cells from wild-type and LDL receptor-deficient mice revealed that the inhibitory effect of LDL on insulin secretion but not proliferation requires the LDL receptor. HDL was found to modulate the survival of both human and murine islets by decreasing basal as well as IL-1beta and glucose-induced apoptosis. IL-1beta-induced beta-cell apoptosis was also inhibited in the presence of either the delipidated protein or the deproteinated lipid moieties of HDL, apolipoprotein A1 (the main protein component of HDL), or sphingosine-1-phosphate (a bioactive sphingolipid mostly carried by HDL). In murine beta-cells, the protective effect of HDL against IL-1beta-induced apoptosis was also observed in the absence of the HDL receptor scavenger receptor class B type 1. Our data show that both LDL and HDL affect function or survival of beta-cells and raise the question whether dyslipidemia contributes to beta-cell failure and hence the manifestation and progression of type 2 diabetes mellitus.

SIRT1 decreases Lox-1-mediated foam cell formation in atherogenesis.

Aims Endothelial activation, macrophage infiltration, and foam cell formation are pivotal steps in atherogenesis. Our aim in this study was to analyse the role of SIRT1, a class III deacetylase with important metabolic functions, in plaque macrophages and atherogenesis. Methods and results Using partial SIRT1 deletion in atherosclerotic mice, we demonstrate that SIRT1 protects against atherosclerosis by reducing macrophage foam cell formation. Peritoneal macrophages from heterozygous SIRT1 mice accumulate more oxidized low-density lipoprotein (oxLDL), thereby promoting foam cell formation. Bone marrow-restricted SIRT1 deletion confirmed that SIRT1 function in macrophages is sufficient to decrease atherogenesis. Moreover, we show that SIRT1 reduces the uptake of oxLDL by diminishing the expression of lectin-like oxLDL receptor-1 (Lox-1) via suppression of the NF-κB signalling pathway. Conclusion Our findings demonstrate protective effects of SIRT1 in atherogenesis and suggest pharmacological SIRT1 activation as a novel anti-atherosclerotic strategy by reducing macrophage foam cell formation.

Current understanding of the metabolism and biological actions of HDL.

Purpose of reviewA low concentration of HDL-cholesterol is an important risk factor for coronary heart disease. The purpose of this review is to summarize the novel functions of HDL that may protect not only from atherosclerosis, but also from inflammation-induced organ damage. Recent findingsHDL interacts with several cellular receptors and lipid transporters. The interactions of HDL or apolipoprotein A-I with the scavenger receptor BI or adenosine triphosphate binding cassette transporters A1, G1 and G4 induce cholesterol efflux. Apolipoproteins and enzymes carried by HDL exert antioxidative functions. Some oxidative modifications of apolipoprotein A-I, for example of tyrosine residues, may however interfere with anti-atherosclerotic activities. The interactions of HDL and lysosphingolipids therein with scavenger receptor BI and sphingolipid receptors, respectively, elicit signals activating the protein kinase Akt, which in turn is a regulator of apoptosis in beta, endothelial and smooth muscle cells as well as a regulator of nitric oxide production and adhesion molecule expression in endothelial cells. Other signal transduction cascades are also elicited by HDL, some of which induce cholesterol efflux or activate mitogen-activated protein kinases. SummaryProperties with respect to cytokine production, lipid oxidation, cholesterol efflux and reverse cholesterol transport make HDL a protective agent and thus an interesting therapeutic target in atherosclerosis and inflammation-induced organ damage. Abbreviations ABC: adenosine triphosphate binding cassette transporter; ATP: adenosine triphosphate; CETP: cholesteryl ester transfer protein; EGD: endothelial gene differentiation receptor; LXR: liver X receptor; NO: nitric oxide; PLTP: phospholipid transfer protein; RXR: retinoid X receptor; S1P: sphingosine-1-phosphate; SR-BI: scavenger receptor BI.

The SPTLC3 Subunit of Serine Palmitoyltransferase Generates Short Chain Sphingoid Bases

The enzyme serine palmitoyltransferase (SPT) catalyzes the rate-limiting step in the de novo synthesis of sphingolipids. Previously the mammalian SPT was described as a heterodimer composed of two subunits, SPTLC1 and SPTLC2. Recently we identified a novel third SPT subunit (SPTLC3). SPTLC3 shows about 68% identity to SPTLC2 and also includes a pyridoxal phosphate consensus motif. Here we report that the overexpression of SPTLC3 in HEK293 cells leads to the formation of two new sphingoid base metabolites, namely C16-sphinganine and C16-sphingosine. SPTLC3-expressing cells have higher in vitro SPT activities with lauryl- and myristoyl-CoA than SPTLC2-expressing cells, and SPTLC3 mRNA expression levels correlate closely with the C16-sphinganine synthesis rates in various human and murine cell lines. Approximately 15% of the total sphingolipids in human plasma contain a C16 backbone and are found in the high density and low density but not the very low density lipoprotein fraction. In conclusion, we show that the SPTLC3 subunit generates C16-sphingoid bases and that sphingolipids with a C16 backbone constitute a significant proportion of human plasma sphingolipids.

Transendothelial lipoprotein transport and regulation of endothelial permeability and integrity by lipoproteins

Purpose of review Previously, the endothelium was considered as a passive exchange barrier of lipoproteins between plasma and extravascular tissues. This dogma is challenged by recent findings on a dual relationship between lipoproteins and endothelial permeability. Recent findings LDL and HDL as well as apolipoprotein A-I pass the intact endothelium through transcytosis by processes, which involve caveolin-1, the LDL-receptor, ATP-binding cassette transporters A1 and G1 or scavenger receptor BI. Moreover, HDL help the endothelium to maintain structural integrity and hence selective permeability for biomolecules by keeping interendothelial junctions closed, by inhibiting endothelial cell apoptosis and by stimulating endothelial proliferation, migration and tube formation as well as the recruitment and differentiation of endothelial progenitor cells in damaged parts of the endothelium. Both apolipoprotein A-I and sphingosin-1-phosphate mediate many of the protective effects of HDL on the endothelium by interacting with endothelial scavenger receptor BI and sphingosin-1-phosphate receptors, respectively, and by activating intracellular signalling cascades, including the small G protein Rac, src-kinase, phosphoinositol 3 kinase, protein kinase B (Akt) and mitogen-activated protein kinases. Summary The endothelium actively controls the trafficking of lipoproteins between intravascular and extravascular compartments. In addition, lipoproteins affect the integrity and permeability of the endothelium.