HDL Function and Atherosclerosis: Reactive Dicarbonyls as Promising Targets of Therapy.

Epidemiologic studies detected an inverse relationship between HDL (high-density lipoprotein) cholesterol (HDL-C) levels and atherosclerotic cardiovascular disease (ASCVD), identifying HDL-C as a major risk factor for ASCVD and suggesting atheroprotective functions of HDL. However, the role of HDL-C as a mediator of risk for ASCVD has been called into question by the failure of HDL-C-raising drugs to reduce cardiovascular events in clinical trials. Progress in understanding the heterogeneous nature of HDL particles in terms of their protein, lipid, and small RNA composition has contributed to the realization that HDL-C levels do not necessarily reflect HDL function. The most examined atheroprotective function of HDL is reverse cholesterol transport, whereby HDL removes cholesterol from plaque macrophage foam cells and delivers it to the liver for processing and excretion into bile. Indeed, in several studies, HDL has shown inverse associations between HDL cholesterol efflux capacity and ASCVD in humans. Inflammation plays a key role in the pathogenesis of atherosclerosis and vulnerable plaque formation, and a fundamental function of HDL is suppression of inflammatory signaling in macrophages and other cells. Oxidation is also a critical process to ASCVD in promoting atherogenic oxidative modifications of LDL (low-density lipoprotein) and cellular inflammation. HDL and its proteins including apoAI (apolipoprotein AI) and PON1 (paraoxonase 1) prevent cellular oxidative stress and LDL modifications. Importantly, HDL in humans with ASCVD is oxidatively modified rendering HDL dysfunctional and proinflammatory. Modification of HDL with reactive carbonyl species, such as malondialdehyde and isolevuglandins, dramatically impairs the antiatherogenic functions of HDL. Importantly, treatment of murine models of atherosclerosis with scavengers of reactive dicarbonyls improves HDL function and reduces systemic inflammation, atherosclerosis development, and features of plaque instability. Here, we discuss the HDL antiatherogenic functions in relation to oxidative modifications and the potential of reactive dicarbonyl scavengers as a therapeutic approach for ASCVD.

Scavenging dicarbonyls with 5'-O-pentyl-pyridoxamine increases HDL net cholesterol efflux capacity and attenuates atherosclerosis and insulin resistance.

OBJECTIVE: Oxidative stress contributes to the development of insulin resistance (IR) and atherosclerosis. Peroxidation of lipids produces reactive dicarbonyls such as Isolevuglandins (IsoLG) and malondialdehyde (MDA) that covalently bind plasma/cellular proteins, phospholipids, and DNA leading to altered function and toxicity. We examined whether scavenging reactive dicarbonyls with 5'-O-pentyl-pyridoxamine (PPM) protects against the development of IR and atherosclerosis in Ldlr-/- mice. METHODS: Male or female Ldlr-/- mice were fed a western diet (WD) for 16 weeks and treated with PPM versus vehicle alone. Plaque extent, dicarbonyl-lysyl adducts, efferocytosis, apoptosis, macrophage inflammation, and necrotic area were measured. Plasma MDA-LDL adducts and the in vivo and in vitro effects of PPM on the ability of HDL to reduce macrophage cholesterol were measured. Blood Ly6Chi monocytes and ex vivo 5-ethynyl-2'-deoxyuridine (EdU) incorporation into bone marrow CD11b+ monocytes and CD34+ hematopoietic stem and progenitor cells (HSPC) were also examined. IR was examined by measuring fasting glucose/insulin levels and tolerance to insulin/glucose challenge. RESULTS: PPM reduced the proximal aortic atherosclerosis by 48% and by 46% in female and male Ldlr-/- mice, respectively. PPM also decreased IR and hepatic fat and inflammation in male Ldlr-/- mice. Importantly, PPM decreased plasma MDA-LDL adducts and prevented the accumulation of plaque MDA- and IsoLG-lysyl adducts in Ldlr-/- mice. In addition, PPM increased the net cholesterol efflux capacity of HDL from Ldlr-/- mice and prevented both the in vitro impairment of HDL net cholesterol efflux capacity and apoAI crosslinking by MPO generated hypochlorous acid. Moreover, PPM decreased features of plaque instability including decreased proinflammatory M1-like macrophages, IL-1ß expression, myeloperoxidase, apoptosis, and necrotic core. In contrast, PPM increased M2-like macrophages, Tregs, fibrous cap thickness, and efferocytosis. Furthermore, PPM reduced inflammatory monocytosis as evidenced by decreased blood Ly6Chi monocytes and proliferation of bone marrow monocytes and HSPC from Ldlr-/- mice. CONCLUSIONS: PPM has pleotropic atheroprotective effects in a murine model of familial hypercholesterolemia, supporting the therapeutic potential of reactive dicarbonyl scavenging in the treatment of IR and atherosclerotic cardiovascular disease.

The FoxOs are in the ApoM house.

The prevalence of metabolic syndrome continues to increase globally and heightens the risk for cardiovascular disease (CVD). Insulin resistance is a core pathophysiologic mechanism that causes abnormal carbohydrate metabolism and atherogenic changes in circulating lipoprotein quantity and function. In particular, dysfunctional HDL is postulated to contribute to CVD risk in part via loss of HDL-associated sphingosine-1-phosphate (S1P). In this issue of the JCI, Izquierdo et al. demonstrate that HDL from humans with insulin resistance contained lower levels of S1P. Apolipoprotein M (ApoM), a protein constituent of HDL that binds S1P and controls bioavailability was decreased in insulin-resistant db/db mice. Gain- and loss-of-function mouse models implicated the forkhead box O transcription factors (FoxO1,3,4) in the regulation of both ApoM and HDL-associated S1P. These data have important implications for potential FoxO-based therapies designed to treat lipid and carbohydrate abnormalities associated with human metabolic disease and CVD.

Kidney injury-mediated disruption of intestinal lymphatics involves dicarbonyl-modified lipoproteins.

Kidney disease affects intestinal structure and function. Although intestinal lymphatics are central in absorption and remodeling of dietary and synthesized lipids/lipoproteins, little is known about how kidney injury impacts the intestinal lymphatic network, or lipoproteins transported therein. To study this, we used puromycin aminoglycoside-treated rats and NEP25 transgenic mice to show that proteinuric injury expanded the intestinal lymphatic network, activated lymphatic endothelial cells and increased mesenteric lymph flow. The lymph was found to contain increased levels of cytokines, immune cells, and isolevuglandin (a highly reactive dicarbonyl) and to have a greater output of apolipoprotein AI. Plasma levels of cytokines and isolevuglandin were not changed. However, isolevuglandin was also increased in the ileum of proteinuric animals, and intestinal epithelial cells exposed to myeloperoxidase produced more isolevuglandin. Apolipoprotein AI modified by isolevuglandin directly increased lymphatic vessel contractions, activated lymphatic endothelial cells, and enhanced the secretion of the lymphangiogenic promoter vascular endothelial growth factor-C by macrophages. Inhibition of isolevuglandin synthesis by a carbonyl scavenger reduced intestinal isolevuglandin adduct level and lymphangiogenesis. Thus, our data reveal a novel mediator, isolevuglandin modified apolipoprotein AI, and uncover intestinal lymphatic network structure and activity as a new pathway in the crosstalk between kidney and intestine that may contribute to the adverse impact of kidney disease on other organs.

Macrophage SR-BI modulates autophagy via VPS34 complex and PPARα transcription of Tfeb in atherosclerosis.

Autophagy modulates lipid turnover, cell survival, inflammation, and atherogenesis. Scavenger receptor class B type I (SR-BI) plays a crucial role in lysosome function. Here, we demonstrate that SR-BI regulates autophagy in atherosclerosis. SR-BI deletion attenuated lipid-induced expression of autophagy mediators in macrophages and atherosclerotic aortas. Consequently, SR-BI deletion resulted in 1.8- and 2.5-fold increases in foam cell formation and apoptosis, respectively, and increased oxidized LDL-induced inflammatory cytokine expression. Pharmacological activation of autophagy failed to reduce lipid content or apoptosis in Sr-b1-/- macrophages. SR-BI deletion reduced both basal and inducible levels of transcription factor EB (TFEB), a master regulator of autophagy, causing decreased expression of autophagy genes encoding VPS34 and Beclin-1. Notably, SR-BI regulated Tfeb expression by enhancing PPARα activation. Moreover, intracellular macrophage SR-BI localized to autophagosomes, where it formed cholesterol domains resulting in enhanced association of Barkor and recruitment of the VPS34-Beclin-1 complex. Thus, SR-BI deficiency led to lower VPS34 activity in macrophages and in atherosclerotic aortic tissues. Overexpression of Tfeb or Vps34 rescued the defective autophagy in Sr-b1-/- macrophages. Taken together, our results show that macrophage SR-BI regulates autophagy via Tfeb expression and recruitment of the VPS34-Beclin-1 complex, thus identifying previously unrecognized roles for SR-BI and potentially novel targets for the treatment of atherosclerosis.

Perioperative high density lipoproteins, oxidative stress, and kidney injury after cardiac surgery.

Oxidative stress promotes acute kidney injury (AKI). Higher HDL cholesterol concentrations are associated with less AKI. To test the hypothesis that HDL antioxidant activity is associated with AKI after cardiac surgery, we quantified HDL particle (HDL-P) size and number, paraoxonase-1 (PON-1) activity, and isofuran concentrations in 75 patients who developed AKI and 75 matched control patients. Higher preoperative HDL-P was associated with less AKI (OR: 0.80; 95% CI, 0.71-0.91; P = 0.001), higher PON-1 activity ( P < 0.001), and lower plasma concentrations of isofurans immediately after surgery (P = 0.02). Similarly, higher preoperative small HDL-P was associated with less AKI, higher PON-1 activity, and lower isofuran concentrations. Higher intraoperative particle losses were associated with less AKI (OR: 0.79; 95% CI 0.67-0.93; P = 0.005), and with decreased postoperative isofuran concentrations (P = 0.04) . Additionally, higher preoperative small HDL-P and increased intraoperative small particle loss were associated with improved long-term renal function (P = 0.003, 0.01, respectively). In conclusion, a higher preoperative concentration of HDL-P, particularly small particles, is associated with lower oxidative damage and less AKI. Perioperative changes in HDL-P concentrations are also associated with AKI. Small HDL-P may represent a novel modifiable risk factor for AKI.

Scavenging of reactive dicarbonyls with 2-hydroxybenzylamine reduces atherosclerosis in hypercholesterolemic Ldlr-/- mice.

Lipid peroxidation generates reactive dicarbonyls including isolevuglandins (IsoLGs) and malondialdehyde (MDA) that covalently modify proteins. Humans with familial hypercholesterolemia (FH) have increased lipoprotein dicarbonyl adducts and dysfunctional HDL. We investigate the impact of the dicarbonyl scavenger, 2-hydroxybenzylamine (2-HOBA) on HDL function and atherosclerosis in Ldlr-/- mice, a model of FH. Compared to hypercholesterolemic Ldlr-/- mice treated with vehicle or 4-HOBA, a nonreactive analogue, 2-HOBA decreases atherosclerosis by 60% in en face aortas, without changing plasma cholesterol. Ldlr-/- mice treated with 2-HOBA have reduced MDA-LDL and MDA-HDL levels, and their HDL display increased capacity to reduce macrophage cholesterol. Importantly, 2-HOBA reduces the MDA- and IsoLG-lysyl content in atherosclerotic aortas versus 4-HOBA. Furthermore, 2-HOBA reduces inflammation and plaque apoptotic cells and promotes efferocytosis and features of stable plaques. Dicarbonyl scavenging with 2-HOBA has multiple atheroprotective effects in a murine FH model, supporting its potential as a therapeutic approach for atherosclerotic cardiovascular disease.

Reactive Dicarbonyl Scavenging Effectively Reduces MPO-Mediated Oxidation of HDL and Restores PON1 Activity.

Atheroprotective functions of high-density lipoproteins (HDL) are related to the activity of HDL-associated enzymes such as paraoxonase 1 (PON1). We examined the impact of inhibition of myeloperoxidase (MPO)-mediated HDL oxidation by PON1 on HDL malondialdehyde (MDA) content and HDL function. In the presence of PON1, crosslinking of apoAI in response to MPO-mediated oxidation of HDL was abolished, and MDA-HDL adduct levels were decreased. PON1 prevented the impaired cholesterol efflux capacity of MPO-oxidized HDL from Apoe-/- macrophages. Direct modification of HDL with MDA increased apoAI crosslinking and reduced the cholesterol efflux capacity. MDA modification of HDL reduced its anti-inflammatory function compared to native HDL. MDA-HDL also had impaired ability to increase PON1 activity. Importantly, HDL from subjects with familial hypercholesterolemia (FH-HDL) versus controls had increased MDA-apoAI adducts, and PON1 activity was also impaired in FH. Consistently, FH-HDL induced a pro-inflammatory response in Apoe-/- macrophages and had an impaired ability to promote cholesterol efflux. Interestingly, reactive dicarbonyl scavengers, including 2-hydroxybenzylamine (2-HOBA) and pentyl-pyridoxamine (PPM), effectively abolished MPO-mediated apoAI crosslinking, MDA adduct formation, and improved cholesterol efflux capacity. Treatment of hypercholesterolemic mice with reactive dicarbonyl scavengers reduced MDA-HDL adduct formation and increased HDL cholesterol efflux capacity, supporting the therapeutic potential of reactive carbonyl scavenging for improving HDL function.

Modified sites and functional consequences of 4-oxo-2-nonenal adducts in HDL that are elevated in familial hypercholesterolemia.

The lipid aldehyde 4-oxo-2-nonenal (ONE) is a highly reactive protein crosslinker derived from peroxidation of n-6 polyunsaturated fatty acids and generated together with 4-hydroxynonenal (HNE). Lipid peroxidation product-mediated crosslinking of proteins in high-density lipoprotein (HDL) causes HDL dysfunction and contributes to atherogenesis. Although HNE is relatively well-studied, the role of ONE in atherosclerosis and in modifying HDL is unknown. Here, we found that individuals with familial hypercholesterolemia (FH) had significantly higher ONE-ketoamide (lysine) adducts in HDL (54.6 ± 33.8 pmol/mg) than healthy controls (15.3 ± 5.6 pmol/mg). ONE crosslinked apolipoprotein A-I (apoA-I) on HDL at a concentration of > 3 mol ONE per 10 mol apoA-I (0.3 eq), which was 100-fold lower than HNE, but comparable to the potent protein crosslinker isolevuglandin. ONE-modified HDL partially inhibited HDL's ability to protect against lipopolysaccharide (LPS)-induced tumor necrosis factor α (TNFα) and interleukin-1ß (IL-1ß) gene expression in murine macrophages. At 3 eq, ONE dramatically decreased apoA-I exchange from HDL, from ∼46.5 to ∼18.4% (p < 0.001). Surprisingly, ONE modification of HDL or apoA-I did not alter macrophage cholesterol efflux capacity. LC-MS/MS analysis revealed that Lys-12, Lys-23, Lys-96, and Lys-226 in apoA-I are modified by ONE ketoamide adducts. Compared with other dicarbonyl scavengers, pentylpyridoxamine (PPM) most efficaciously blocked ONE-induced protein crosslinking in HDL and also prevented HDL dysfunction in an in vitro model of inflammation. Our findings show that ONE-HDL adducts cause HDL dysfunction and are elevated in individuals with FH who have severe hypercholesterolemia.

Lipoprotein modulation of proteinuric renal injury.

High-density lipoprotein (HDL) and its main protein, apolipoprotein AI (apoAI), have established benefits in various cells, but whether these cytoprotective effects of HDL pertain to renal cells is unclear. We investigated the in vitro consequences of exposing damaged podocytes to normal apoAI, HDL, and apoAI mimetic (L-4F), and the in vivo effects of L-4F on kidney and atherosclerotic injury in a podocyte-specific injury model of proteinuria. In vitro, primary mouse podocytes were injured by puromycin aminonucleoside (PAN). Cellular viability, migration, production of reactive oxygen species (ROS), apoptosis, and the underlying signaling pathway were assessed. In vivo, we used a proteinuric model, Nphs1-hCD25 transgenic (NEP25+) mice, which express human CD25 on podocytes. Podocyte injury was induced by using immunotoxin (LMB2) and generated a proteinuric atherosclerosis model, NEP25+:apoE-/- mice, was generated by mating apoE-deficient (apoE-/-) mice with NEP25+ mice. Animals received L-4F or control vehicle. Renal function, podocyte injury, and atherosclerosis were assessed. PAN reduced podocyte viability, migration, and increased ROS production, all significantly lessened by apoAI, HDL, and L-4F. L-4F attenuated podocyte apoptosis and diminished PAN-induced inactivation of Janus family protein kinase-2/signal transducers and activators of transcription 3. In NEP25+ mice, L-4F significantly lessened overall proteinuria, and preserved podocyte expression of synaptopodin and cell density. Proteinuric NEP25+:apoE-/- mice had more atherosclerosis than non-proteinuric apoE-/- mice, and these lesions were significantly decreased by L-4F. Normal human apoAI, HDL, and apoAI mimetic protect against podocyte damage. ApoAI mimetic provides in vivo beneficial effects on podocytes that culminate in reduced albuminuria and atherosclerosis. The results suggest supplemental apoAI/apoAI mimetic may be a novel candidate to lessen podocyte damage and its complications.

IL-1 Inhibition and Function of the HDL-Containing Fraction of Plasma in Patients with Stages 3 to 5 CKD.

BACKGROUND AND OBJECTIVES: Systemic inflammation modulates cardiovascular disease risk and functionality of HDL in the setting of CKD. Whether interventions that modify systemic inflammation can improve HDL function in CKD is unknown. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS: We conducted a post hoc analysis of two randomized, clinical trials, IL-1 trap in participants with GFR 15-59 ml/min per 1.73 m2 (study A) and IL-1 receptor antagonist in participants on maintenance hemodialysis (study B), to evaluate if IL-1 blockade had improved the anti-inflammatory activity (IL-6, TNF-α, and Nod-like receptor protein 3), antioxidant function (superoxide production), and net cholesterol efflux capacity of HDL. HDL function was measured using LPS-stimulated THP-1 macrophages or peritoneal macrophages of apoE-deficient mice exposed to the apoB-depleted, HDL-containing fraction obtained from the plasma of the study participants, collected before and after the interventions to block IL-1 effects. Analysis of covariance was used for between group comparisons. RESULTS: The mean age of the participants was 60±13 years, 72% (n=33) were men, and 39% (n=18) were black. There were 32 CKD (16 IL-1 trap and 16 placebo) and 14 maintenance hemodialysis (7 IL-1 receptor antagonist and 7 placebo) participants. Compared with placebo, IL-1 inhibition, in study A and B reduced cellular expression of TNF-α by 15% (P=0.05) and 64% (P=0.02), IL-6 by 38% (P=0.004) and 56% (P=0.08), and Nod-like receptor protein 3 by 16% (P=0.01) and 25% (P=0.02), respectively. The intervention blunted superoxide production in the treated arm compared with placebo, with the values being higher by 17% in the placebo arm in study A (P<0.001) and 12% in the placebo arm in study B (P=0.004). Net cholesterol efflux capacity was not affected by either intervention. CONCLUSIONS: IL-1 blockade improves the anti-inflammatory and antioxidative properties of the HDL-containing fraction of plasma in patients with stages 3-5 CKD, including those on maintenance hemodialysis.

Myeloid-Specific Deletion of Epsins 1 and 2 Reduces Atherosclerosis by Preventing LRP-1 Downregulation.

RATIONALE: Atherosclerosis is, in part, caused by immune and inflammatory cell infiltration into the vascular wall, leading to enhanced inflammation and lipid accumulation in the aortic endothelium. Understanding the molecular mechanisms underlying this disease is critical for the development of new therapies. Our recent studies demonstrate that epsins, a family of ubiquitin-binding endocytic adaptors, are critical regulators of atherogenicity. Given the fundamental contribution lesion macrophages make to fuel atherosclerosis, whether and how myeloid-specific epsins promote atherogenesis is an open and significant question. OBJECTIVE: We will determine the role of myeloid-specific epsins in regulating lesion macrophage function during atherosclerosis. METHODS AND RESULTS: We engineered myeloid cell-specific epsins double knockout mice (LysM-DKO) on an ApoE-/- background. On Western diet, these mice exhibited marked decrease in atherosclerotic lesion formation, diminished immune and inflammatory cell content in aortas, and reduced necrotic core content but increased smooth muscle cell content in aortic root sections. Epsins deficiency hindered foam cell formation and suppressed proinflammatory macrophage phenotype but increased efferocytosis and anti-inflammatory macrophage phenotype in primary macrophages. Mechanistically, we show that epsin loss specifically increased total and surface levels of LRP-1 (LDLR [low-density lipoprotein receptor]-related protein 1), an efferocytosis receptor with antiatherosclerotic properties. We further show that epsin and LRP-1 interact via epsin's ubiquitin-interacting motif domain. ox-LDL (oxidized LDL) treatment increased LRP-1 ubiquitination, subsequent binding to epsin, and its internalization from the cell surface, suggesting that epsins promote the ubiquitin-dependent internalization and downregulation of LRP-1. Crossing ApoE-/-/LysM-DKO mice onto an LRP-1 heterozygous background restored, in part, atherosclerosis, suggesting that epsin-mediated LRP-1 downregulation in macrophages plays a pivotal role in propelling atherogenesis. CONCLUSIONS: Myeloid epsins promote atherogenesis by facilitating proinflammatory macrophage recruitment and inhibiting efferocytosis in part by downregulating LRP-1, implicating that targeting epsins in macrophages may serve as a novel therapeutic strategy to treat atherosclerosis.

Administration of N-Acyl-Phosphatidylethanolamine Expressing Bacteria to Low Density Lipoprotein Receptor-/- Mice Improves Indices of Cardiometabolic Disease.

Obesity increases the risk for cardiometabolic diseases. N-acyl phosphatidylethanolamines (NAPEs) are precursors of N-acylethanolamides, which are endogenous lipid satiety factors. Incorporating engineered bacteria expressing NAPEs into the gut microbiota retards development of diet induced obesity in wild-type mice. Because NAPEs can also exert anti-inflammatory effects, we hypothesized that administering NAPE-expressing bacteria to low-density lipoprotein receptor (Ldlr)-/- mice fed a Western diet would improve various indices of cardiometabolic disease manifested by these mice. NAPE-expressing E. coli Nissle 1917 (pNAPE-EcN), control Nissle 1917 (pEcN), or vehicle (veh) were given via drinking water to Ldlr-/- mice for 12 weeks. Compared to pEcN or veh treatment, pNAPE-EcN significantly reduced body weight and adiposity, hepatic triglycerides, fatty acid synthesis genes, and increased expression of fatty acid oxidation genes. pNAPE-EcN also significantly reduced markers for hepatic inflammation and early signs of fibrotic development. Serum cholesterol was reduced with pNAPE-EcN, but atherosclerotic lesion size showed only a non-significant trend for reduction. However, pNAPE-EcN treatment reduced lesion necrosis by 69% indicating an effect on preventing macrophage inflammatory death. Our results suggest that incorporation of NAPE expressing bacteria into the gut microbiota can potentially serve as an adjuvant therapy to retard development of cardiometabolic disease.

Modification by isolevuglandins, highly reactive γ-ketoaldehydes, deleteriously alters high-density lipoprotein structure and function.

Cardiovascular disease risk depends on high-density lipoprotein (HDL) function, not HDL-cholesterol. Isolevuglandins (IsoLGs) are lipid dicarbonyls that react with lysine residues of proteins and phosphatidylethanolamine. IsoLG adducts are elevated in atherosclerosis. The consequences of IsoLG modification of HDL have not been studied. We hypothesized that IsoLG modification of apoA-I deleteriously alters HDL function. We determined the effect of IsoLG on HDL structure-function and whether pentylpyridoxamine (PPM), a dicarbonyl scavenger, can preserve HDL function. IsoLG adducts in HDL derived from patients with familial hypercholesterolemia (n = 10, 233.4 ± 158.3 ng/mg) were found to be significantly higher than in healthy controls (n = 7, 90.1 ± 33.4 pg/mg protein). Further, HDL exposed to myeloperoxidase had elevated IsoLG-lysine adducts (5.7 ng/mg protein) compared with unexposed HDL (0.5 ng/mg protein). Preincubation with PPM reduced IsoLG-lysine adducts by 67%, whereas its inactive analogue pentylpyridoxine did not. The addition of IsoLG produced apoA-I and apoA-II cross-links beginning at 0.3 molar eq of IsoLG/mol of apoA-I (0.3 eq), whereas succinylaldehyde and 4-hydroxynonenal required 10 and 30 eq. IsoLG increased HDL size, generating a subpopulation of 16-23 nm. 1 eq of IsoLG decreased HDL-mediated [3H]cholesterol efflux from macrophages via ABCA1, which corresponded to a decrease in HDL-apoA-I exchange from 47.4% to only 24.8%. This suggests that IsoLG inhibits apoA-I from disassociating from HDL to interact with ABCA1. The addition of 0.3 eq of IsoLG ablated HDL's ability to inhibit LPS-stimulated cytokine expression by macrophages and increased IL-1ß expression by 3.5-fold. The structural-functional effects were partially rescued with PPM scavenging.

Chronic kidney disease alters lipid trafficking and inflammatory responses in macrophages: effects of liver X receptor agonism.

BACKGROUND: Our aim was to evaluate lipid trafficking and inflammatory response of macrophages exposed to lipoproteins from subjects with moderate to severe chronic kidney disease (CKD), and to investigate the potential benefits of activating cellular cholesterol transporters via liver X receptor (LXR) agonism. METHODS: LDL and HDL were isolated by sequential density gradient ultracentrifugation of plasma from patients with stage 3-4 CKD and individuals without kidney disease (HDLCKD and HDLCont, respectively). Uptake of LDL, cholesterol efflux to HDL, and cellular inflammatory responses were assessed in human THP-1 cells. HDL effects on inflammatory markers (MCP-1, TNF-α, IL-1ß), Toll-like receptors-2 (TLR-2) and - 4 (TLR-4), ATP-binding cassette class A transporter (ABCA1), NF-κB, extracellular signal regulated protein kinases 1/2 (ERK1/2) were assessed by RT-PCR and western blot before and after in vitro treatment with an LXR agonist. RESULTS: There was no difference in macrophage uptake of LDL isolated from CKD versus controls. By contrast, HDCKD was significantly less effective than HDLCont in accepting cholesterol from cholesterol-enriched macrophages (median 20.8% [IQR 16.1-23.7] vs control (26.5% [IQR 19.6-28.5]; p = 0.008). LXR agonist upregulated ABCA1 expression and increased cholesterol efflux to HDL of both normal and CKD subjects, although the latter continued to show lower efflux capacity. HDLCKD increased macrophage cytokine response (TNF-α, MCP-1, IL-1ß, and NF-κB) versus HDLCont. The heightened cytokine response to HDLCKD was further amplified in cells treated with LXR agonist. The LXR-augmentation of inflammation was associated with increased TLR-2 and TLR-4 and ERK1/2. CONCLUSIONS: Moderate to severe impairment in kidney function promotes foam cell formation that reflects impairment in cholesterol acceptor function of HDLCKD. Activation of cellular cholesterol transporters by LXR agonism improves but does not normalize efflux to HDLCKD. However, LXR agonism actually increases the pro-inflammatory effects of HDLCKD through activation of TLRs and ERK1/2 pathways.

SR-BI: A Multifunctional Receptor in Cholesterol Homeostasis and Atherosclerosis.

The HDL receptor scavenger receptor class B type I (SR-BI) plays crucial roles in cholesterol homeostasis, lipoprotein metabolism, and atherosclerosis. Hepatic SR-BI mediates reverse cholesterol transport (RCT) by the uptake of HDL cholesterol for routing to the bile. Through the selective uptake of HDL lipids, hepatic SR-BI modulates HDL composition and preserves HDL's atheroprotective functions of mediating cholesterol efflux and minimizing inflammation and oxidation. Macrophage and endothelial cell SR-BI inhibits the development of atherosclerosis by mediating cholesterol trafficking to minimize atherosclerotic lesion foam cell formation. SR-BI signaling also helps limit inflammation and cell death and mediates efferocytosis of apoptotic cells in atherosclerotic lesions thereby preventing vulnerable plaque formation. SR-BI is emerging as a multifunctional therapeutic target to reduce atherosclerosis development.

Macrophage Apoptosis and Efferocytosis in the Pathogenesis of Atherosclerosis.

Macrophage apoptosis and the ability of macrophages to clean up dead cells, a process called efferocytosis, are crucial determinants of atherosclerosis lesion progression and plaque stability. Environmental stressors initiate endoplasmic reticulum (ER) stress and activate the unfolded protein response (UPR). Unresolved ER stress with activation of the UPR initiates apoptosis. Macrophages are resistant to apoptotic stimuli, because of activity of the PI3K/Akt pathway. Macrophages express 3 Akt isoforms, Akt1, Akt2 and Akt3, which are products of distinct but homologous genes. Akt displays isoform-specific effects on atherogenesis, which vary with different vascular cell types. Loss of macrophage Akt2 promotes the anti-inflammatory M2 phenotype and reduces atherosclerosis. However, Akt isoforms are redundant with regard to apoptosis. c-Jun NH2-terminal kinase (JNK) is a pro-apoptotic effector of the UPR, and the JNK1 isoform opposes anti-apoptotic Akt signaling. Loss of JNK1 in hematopoietic cells protects macrophages from apoptosis and accelerates early atherosclerosis. IκB kinase α (IKKα, a member of the serine/threonine protein kinase family) plays an important role in mTORC2-mediated Akt signaling in macrophages, and IKKα deficiency reduces macrophage survival and suppresses early atherosclerosis. Efferocytosis involves the interaction of receptors, bridging molecules, and apoptotic cell ligands. Scavenger receptor class B type I is a critical mediator of macrophage efferocytosis via the Src/PI3K/Rac1 pathway in atherosclerosis. Agonists that resolve inflammation offer promising therapeutic potential to promote efferocytosis and prevent atherosclerotic clinical events. (Circ J 2016; 80: 2259-2268).

Effect of Drug Therapy on Net Cholesterol Efflux Capacity of High-Density Lipoprotein-Enriched Serum in Rheumatoid Arthritis.

OBJECTIVE: Patients with rheumatoid arthritis (RA) have an increased risk of coronary heart disease (CHD). Some RA therapies may modify this risk, but the underlying mechanisms are unclear. The cholesterol efflux capacity of high-density lipoprotein (HDL) is associated with a reduced CHD risk in non-RA populations; however, inflammation may impair the function of HDL. The aim of this study was to evaluate whether reduced inflammation resulting from treatment with methotrexate (MTX), adalimumab (ADA), or tocilizumab (TCZ) would increase the net cholesterol efflux capacity of HDL in patients with RA. METHODS: A longitudinal multicenter study repository (Treatment Efficacy and Toxicity in Rheumatoid Arthritis Database and Repository) provided clinical information for and serum samples from 70 patients with RA before and 6 months after starting treatment with a new drug (MTX [n = 23], ADA [n = 22], or TCZ [n = 25]). Disease activity was measured using the Disease Activity Score in 28 joints using the erythrocyte sedimentation rate (DAS28-ESR). The net cholesterol efflux capacity was measured in paired serum samples using THP-1 macrophages, and total cellular cholesterol was measured by fluorometric assay. RESULTS: The DAS28-ESR decreased with all treatments (P < 0.001). Net cholesterol efflux capacity was not significantly changed after 6 months of new RA therapy (mean ± SD 36.9 ± 17.3% units at baseline versus 38.0% ± 16.9% units at 6 months [P = 0.58]). However, change in net cholesterol efflux capacity was associated with change in the DAS28-ESR (ρ = -0.25, P = 0.04). In a post hoc analysis of patients with impaired net cholesterol efflux capacity at baseline, treatment with TCZ resulted in significant improvement in net cholesterol efflux capacity (21.9 ± 14.7% units at baseline versus 31.3% ± 12.8% units at 6 months [P < 0.02]), but this was not observed with MTX or ADA. CONCLUSION: Net cholesterol efflux capacity of HDL cholesterol did not change significantly after 6 months of new RA therapy, except in patients with impaired baseline cholesterol efflux capacity who were receiving TCZ. Change in disease activity was associated with change in the net cholesterol efflux capacity.

Net cholesterol efflux capacity of HDL enriched serum and coronary atherosclerosis in rheumatoid arthritis.

BACKGROUND/OBJECTIVES: Cardiovascular (CV) risk is increased in patients with rheumatoid arthritis (RA), but not fully explained by traditional risk factors such as LDL and HDL cholesterol concentrations. The cholesterol efflux capacity of HDL may be a better CV risk predictor than HDL concentrations. We hypothesized that HDL's cholesterol efflux capacity is impaired and inversely associated with coronary atherosclerosis in patients with RA. METHODS: We measured the net cholesterol efflux capacity of apolipoprotein B depleted serum and coronary artery calcium score in 134 patients with RA and 76 control subjects, frequency-matched for age, race and sex. The relationship between net cholesterol efflux capacity and coronary artery calcium score and other clinical variables of interest was assessed in patients with RA. RESULTS: Net cholesterol efflux capacity was similar among RA (median [IQR]: 34% removal [28, 41%]) and control subjects (35% removal [27%, 39%]) (P=0.73). In RA, increasing net cholesterol efflux capacity was not significantly associated with decreased coronary calcium score (OR=0.78 (95% CI 0.51-1.19), P=0.24, adjusted for age, race and sex, Framingham risk score and presence of diabetes). Net cholesterol efflux capacity was not significantly associated with RA disease activity score, C-reactive protein, urinary F2-isoprostanes, or degree of insulin resistance in RA. CONCLUSIONS: Net cholesterol efflux capacity is not significantly altered in patients with relatively well-controlled RA nor is it significantly associated with coronary artery calcium score.

Atherosclerosis following renal injury is ameliorated by pioglitazone and losartan via macrophage phenotype.

OBJECTIVE: Chronic kidney disease (CKD) amplifies atherosclerosis, which involves renin-angiotensin system (RAS) regulation of macrophages. RAS influences peroxisome proliferator-activated receptor-γ (PPARγ), a modulator of atherogenic functions of macrophages, however, little is known about its effects in CKD. We examined the impact of combined therapy with a PPARγ agonist and angiotensin receptor blocker on atherogenesis in a murine uninephrectomy model. METHODS: Apolipoprotein E knockout mice underwent uninephrectomy (UNx) and treatment with pioglitazone (UNx + Pio), losartan (UNx + Los), or both (UNx + Pio/Los) for 10 weeks. Extent and characteristics of atherosclerotic lesions and macrophage phenotypes were assessed; RAW264.7 and primary peritoneal mouse cells were used to examine pioglitazone and losartan effects on macrophage phenotype and inflammatory response. RESULTS: UNx significantly increased atherosclerosis. Pioglitazone and losartan each significantly reduced the atherosclerotic burden by 29.6% and 33.5%, respectively; although the benefit was dramatically augmented by combination treatment which lessened atherosclerosis by 55.7%. Assessment of plaques revealed significantly greater macrophage area in UNx + Pio/Los (80.7 ± 11.4% vs. 50.3 ± 4.2% in UNx + Pio and 57.2 ± 6.5% in UNx + Los) with more apoptotic cells. The expanded macrophage-rich lesions of UNx + Pio/Los had more alternatively activated, Ym-1 and arginine 1-positive M2 phenotypes (Ym-1: 33.6 ± 8.2%, p < 0.05 vs. 12.0 ± 1.1% in UNx; arginase 1: 27.8 ± 0.9%, p < 0.05 vs. 11.8 ± 1.3% in UNx). In vitro, pioglitazone alone and together with losartan was more effective than losartan alone in dampening lipopolysaccharide-induced cytokine production, suppressing M1 phenotypic change while enhancing M2 phenotypic change. CONCLUSION: Combination of pioglitazone and losartan is more effective in reducing renal injury-induced atherosclerosis than either treatment alone. This benefit reflects mitigation in macrophage cytokine production, enhanced apoptosis, and a shift toward an anti-inflammatory phenotype.