Meliana Riwanto

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.

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.

Characterization of Levels and Cellular Transfer of Circulating Lipoprotein-Bound MicroRNAs

Objective—MicroRNAs are important intracellular regulators of gene expression, but also circulate in the blood being protected by extracellular vesicles, proteins, or high-density lipoprotein (HDL). Here, we evaluate the regulation and potential function of HDL- and low-density lipoprotein–bound miRs isolated from healthy subjects and patients with coronary artery disease. Approach and Results—HDL-bound miRs with known effects in the cardiovascular system were analyzed in HDL isolated from healthy subjects (n=10), patients with stable coronary artery disease (n=10), and patients with an acute coronary syndrome (n=10). In HDL from healthy subjects, miR-223 was detected at concentrations >10 000 copies/µg HDL, and miR-126 and miR-92a at about 3000 copies/µg HDL. Concentrations of most miRs were substantially higher in HDL as compared with low-density lipoprotein. However, HDL-bound miR-223 contributed to only 8% of the total circulating miRs. The signatures of miRs varied only slightly in HDL derived from patients with coronary artery disease. We did not observe a significant uptake of HDL-bound miRs into endothelial cells, smooth muscle cells, or peripheral blood mononuclear cells. However, patient-derived HDL transiently reduced miR expression particularly when incubated with smooth muscle and peripheral blood mononuclear cells. Conclusions—Circulating miRs are detected in HDL and to a lesser extent in low-density lipoprotein, and the miR-signatures are only slightly altered in patients with coronary artery disease. Lipoprotein-bound miRs were not efficiently delivered to endothelial, smooth muscle, and peripheral blood mononuclear cells suggesting that the lipoprotein-associated pool of miRs is not regulating the function of the studied cells in vitro.

Myeloperoxidase, paraoxonase-1, and HDL form a functional ternary complex

Myeloperoxidase (MPO) and paraoxonase 1 (PON1) are high-density lipoprotein-associated (HDL-associated) proteins mechanistically linked to inflammation, oxidant stress, and atherosclerosis. MPO is a source of ROS during inflammation and can oxidize apolipoprotein A1 (APOA1) of HDL, impairing its atheroprotective functions. In contrast, PON1 fosters systemic antioxidant effects and promotes some of the atheroprotective properties attributed to HDL. Here, we demonstrate that MPO, PON1, and HDL bind to one another, forming a ternary complex, wherein PON1 partially inhibits MPO activity, while MPO inactivates PON1. MPO oxidizes PON1 on tyrosine 71 (Tyr71), a modified residue found in human atheroma that is critical for HDL binding and PON1 function. Acute inflammation model studies with transgenic and knockout mice for either PON1 or MPO confirmed that MPO and PON1 reciprocally modulate each others function in vivo. Further structure and function studies identified critical contact sites between APOA1 within HDL, PON1, and MPO, and proteomics studies of HDL recovered from acute coronary syndrome (ACS) subjects revealed enhanced chlorotyrosine content, site-specific PON1 methionine oxidation, and reduced PON1 activity. HDL thus serves as a scaffold upon which MPO and PON1 interact during inflammation, whereupon PON1 binding partially inhibits MPO activity, and MPO promotes site-specific oxidative modification and impairment of PON1 and APOA1 function.

High density lipoproteins and endothelial functions: mechanistic insights and alterations in cardiovascular disease

Prospective population studies in the primary prevention setting have shown that reduced plasma levels of HDL cholesterol are associated with an increased risk of coronary disease and myocardial infarction. Experimental and translational studies have further revealed several potential anti-atherogenic effects of HDL, including protective effects on endothelial cell functions. HDL has been suggested to protect endothelial cell functions by prevention of oxidation of LDL and its adverse endothelial effects. Moreover, HDL from healthy subjects can directly stimulate endothelial cell production of nitric oxide and anti-inflammatory, anti-apoptotic, and anti-thrombotic effects as well as endothelial repair processes. However, several recent clinical trials using HDL cholesterol-raising agents, such as torcetrapib, dalcetrapib, and niacin, did not demonstrate a significant reduction of cardiovascular events in patients with coronary disease. Of note, growing evidence suggests that the vascular effects of HDL can be highly heterogeneous and vasoprotective properties of HDL are altered in patients with coronary disease. Characterization of underlying mechanisms and understanding of the clinical relevance of this “HDL dysfunction” is currently an active field of cardiovascular research. Notably, in some recent studies no clear association of higher HDL cholesterol levels with a reduced risk of cardiovascular events was observed in patients with already established coronary disease. A greater understanding of mechanisms of action of HDL and its altered vascular effects is therefore critical within the context of HDL-targeted therapies. In this review, we will address different effects of HDL on endothelial cell functions potentially relevant to atherosclerotic vascular disease and explore molecular mechanisms leading to “dysfunctional HDL”.

Exercise Training in Patients with Chronic Heart Failure Promotes Restoration of High-Density Lipoprotein Functional Properties

Rationale: High-density lipoprotein (HDL) exerts endothelial-protective effects via stimulation of endothelial cell (EC) nitric oxide (NO) production. This function is impaired in patients with cardiovascular disease. Protective effects of exercise training (ET) on endothelial function have been demonstrated. Objective: This study was performed to evaluate the impact of ET on HDL-mediated protective effects and the respective molecular pathways in patients with chronic heart failure (CHF). Methods and Results: HDL was isolated from 16 healthy controls (HDLhealthy) and 16 patients with CHF-NYHA-III (HDLNYHA-IIIb) before and after ET, as well as from 8 patients with CHF-NYHA-II (HDLNYHA-II). ECs were incubated with HDL, and phosphorylation of eNOS-Ser1177, eNOS-Thr495, PKC-&bgr;II-Ser660, and p70S6K-Ser411 was evaluated. HDL-bound malondialdehyde and HDL-induced NO production by EC were quantified. Endothelial function was assessed by flow-mediated dilatation. The proteome of HDL particles was profiled by shotgun LC-MS/MS. Incubation of EC with HDLNYHA-IIIb triggered a lower stimulation of phosphorylation at eNOS-Ser1177 and a higher phosphorylation at eNOS-Thr495 when compared with HDLhealthy. This was associated with lower NO production of EC. In addition, an elevated activation of p70S6K, PKC-&bgr;II by HDLNYHA-IIIb, and a higher amount of malondialdehyde bound to HDLNYHA-IIIb compared with HDLhealthy was measured. In healthy individuals, ET had no effect on HDL function, whereas ET of CHF-NYHA-IIIb significantly improved HDL function. A correlation between changes in HDL-induced NO production and flow-mediated dilatation improvement by ET was evident. Conclusions: These results demonstrate that HDL function is impaired in CHF and that ET improved the HDL-mediated vascular effects. This may be one mechanism how ET exerts beneficial effects in CHF.

Exercise Training in Patients with Chronic Heart Failure Promotes Restoration of HDL Functional Properties

Rationale: High-density lipoprotein (HDL) exerts endothelial-protective effects via stimulation of endothelial cell (EC) nitric oxide (NO) production. This function is impaired in patients with cardiovascular disease. Protective effects of exercise training (ET) on endothelial function have been demonstrated. Objective: This study was performed to evaluate the impact of ET on HDL-mediated protective effects and the respective molecular pathways in patients with chronic heart failure (CHF). Methods and Results: HDL was isolated from 16 healthy controls (HDLhealthy) and 16 patients with CHF-NYHA-III (HDLNYHA-IIIb) before and after ET, as well as from 8 patients with CHF-NYHA-II (HDLNYHA-II). ECs were incubated with HDL, and phosphorylation of eNOS-Ser1177, eNOS-Thr495, PKC-&bgr;II-Ser660, and p70S6K-Ser411 was evaluated. HDL-bound malondialdehyde and HDL-induced NO production by EC were quantified. Endothelial function was assessed by flow-mediated dilatation. The proteome of HDL particles was profiled by shotgun LC-MS/MS. Incubation of EC with HDLNYHA-IIIb triggered a lower stimulation of phosphorylation at eNOS-Ser1177 and a higher phosphorylation at eNOS-Thr495 when compared with HDLhealthy. This was associated with lower NO production of EC. In addition, an elevated activation of p70S6K, PKC-&bgr;II by HDLNYHA-IIIb, and a higher amount of malondialdehyde bound to HDLNYHA-IIIb compared with HDLhealthy was measured. In healthy individuals, ET had no effect on HDL function, whereas ET of CHF-NYHA-IIIb significantly improved HDL function. A correlation between changes in HDL-induced NO production and flow-mediated dilatation improvement by ET was evident. Conclusions: These results demonstrate that HDL function is impaired in CHF and that ET improved the HDL-mediated vascular effects. This may be one mechanism how ET exerts beneficial effects in CHF.

Peripheral blood monocyte Sirt1 expression is reduced in patients with coronary artery disease.

Background Inflammation plays a key role in atherosclerosis. Sirt1 regulates transcription factors involved in inflammatory processes and blunts atherosclerosis in mice. However, its role in humans remains to be defined. This study was therefore designed to investigate the role of Sirt1 in the development of atherosclerosis. Methods and Results 48 male subjects admitted for cardiac catheterization were subdivided into healthy subjects, patients with stable coronary artery disease (CAD), and with acute coronary syndromes (ACS). Monocytes were isolated and Sirt1 mRNA levels were determined. Sirt1 gene expression was higher in healthy subjects as compared to patients with CAD or ACS (P<0.05), respectively. Interestingly, HDL levels correlated positively with Sirt1 expression. Thus, HDL from the three groups was isolated and incubated with THP-1 monocytes to determine the effects of HDL on Sirt1 protein in controlled experimental conditions. HDL from healthy subjects stimulated Sirt1 expression in THP-1 monocytes to a higher degree than HDL from CAD and ACS patients (P<0.05). Paraoxonase-1 (PON-1), a HDL-associated enzyme, showed a reduced activity in HDL isolated from CAD and ACS patients as compared to the controls (P<0.001). Conclusions Monocytic Sirt1 expression is reduced in patients with stable CAD and ACS. Experiments on THP-1 monocytes suggest that this effect is HDL-dependent and is mediated by a reduced activity of HDL-associated enzyme PON1.

The impact of partial and complete loss-of-function mutations in endothelial lipase on high-density lipoprotein levels and functionality in humans

Background—Endothelial lipase is a phospholipase with activity against high-density lipoprotein. Although a small number of mutations in LIPG have been described, the role of LIPG in protection against atherosclerosis is unclear. Methods and Results—We identified 8 loss-of-function (LOF) mutations in LIPG in individuals with high-density lipoprotein cholesterol. Functional analysis confirmed that most rare mutations abolish lipase activity in vitro, indicating complete LOF, whereas 2 more common mutations N396S and R476W reduce activity by ≈50%, indicating partial LOF and implying ≈50% and ≈75% remaining endothelial lipase function in heterozygous complete LOF and partial LOF mutation carriers, respectively. complete LOF mutation carriers had significantly higher plasma high-density lipoprotein cholesterol levels compared with partial LOF mutation carriers. Apolipoprotein B-depleted serum from complete LOF carriers showed significantly enhanced cholesterol efflux acceptor capacity, whereas only trends were observed in partial LOF carriers. Carriers of LIPG mutations exhibited trends toward reduced coronary artery disease in 4 independent cohorts (meta-analysis odds ratio, 0.7; P=0.04). Conclusions—Our data suggest that the impact of LIPG mutations is directly related to their effect on endothelial lipase function and support that antagonism of endothelial lipase function improves cardioprotection.

Plasmalogens of high-density lipoproteins (HDL) are associated with coronary artery disease and anti-apoptotic activity of HDL

OBJECTIVE Low high-density lipoprotein (HDL) cholesterol and loss of atheroprotective functions of HDL are associated with coronary artery disease (CAD). Here, we investigated the associations of HDL phospholipids with acute and stable CAD as well as with the anti-apoptotic activity of HDL. METHODS 49 species of phosphatidylcholines (PCs), lysophosphatidylcholines and sphingomyelins (SMs) as well as three species of sphingosine-1-phosphate (S1P) were quantified by liquid chromatography - mass spectrometry in HDL isolated from 22 healthy subjects as well as 23 and 22 patients with stable CAD and acute coronary syndrome (ACS), respectively. Native HDL and artificially reconstituted HDL (rHDL) were tested for their capacity to inhibit apoptosis of endothelial cells (ECs) induced by serum deprivation. RESULTS HDL of CAD or ACS patients differed from HDL of healthy controls by the content in nine of the 52 quantified phospholipid species as well as reduced anti-apoptotic activity. The capacity of HDL to inhibit EC apoptosis correlated significantly with five of eleven odd-chain PCs (= plasmalogens), two S1Ps, SM42:2, PC34:2, and PC32:0. An orthogonal partial least square - discriminant analysis revealed independent associations of stable CAD with HDL-associated PC34:2, PC33:3 and PC35:2 as well as anti-apoptotic activity of HDL and of ACS with HDL-associated PC33:3, PC35:2, SM42:1, PC34:2 and PC36:2. rHDL reconstituted with apoA-I, PC34:1, and PC35:2 inhibited apoptosis of ECs more effectively than rHDL containing only apoA-I and PC34:1. CONCLUSIONS The inverse association of HDL-plasmalogen levels with both stable and acute CAD may reflect direct anti-apoptotic effects of plasmologens on ECs.