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.

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.

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.

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).

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.

Macrophage SR-BI mediates efferocytosis via Src/PI3K/Rac1 signaling and reduces atherosclerotic lesion necrosis.

Macrophage apoptosis and efferocytosis are key determinants of atherosclerotic plaque inflammation and necrosis. Bone marrow transplantation studies in ApoE- and LDLR-deficient mice revealed that hematopoietic scavenger receptor class B type I (SR-BI) deficiency results in severely defective efferocytosis in mouse atherosclerotic lesions, resulting in a 17-fold higher ratio of free to macrophage-associated dead cells in lesions containing SR-BI(-/-) cells, 5-fold more necrosis, 65.2% less lesional collagen content, nearly 7-fold higher dead cell accumulation, and 2-fold larger lesion area. Hematopoietic SR-BI deletion elicited a maladaptive inflammatory response [higher interleukin (IL)-1ß, IL-6, and TNF-α lower IL-10 and transforming growth factor ß]. Efferocytosis of apoptotic thymocytes was reduced by 64% in SR-BI(-/-) versus WT macrophages, both in vitro and in vivo. In response to apoptotic cells, macrophage SR-BI bound with phosphatidylserine and induced Src phosphorylation and cell membrane recruitment, which led to downstream activation of phosphoinositide 3-kinase (PI3K) and Ras-related C3 botulinum toxin substrate 1 (Rac1) for engulfment and clearance of apoptotic cells, as inhibition of Src decreased PI3K, Rac1-GTP, and efferocytosis in WT cells. Pharmacological inhibition of Rac1 reduced macrophage efferocytosis in a SR-BI-dependent fashion, and activation of Rac1 corrected the defective efferocytosis in SR-BI(-/-) macrophages. Thus, deficiency of macrophage SR-BI promotes defective efferocytosis signaling via the Src/PI3K/Rac1 pathway, resulting in increased plaque size, necrosis, and inflammation.

Isolevuglandin-type lipid aldehydes induce the inflammatory response of macrophages by modifying phosphatidylethanolamines and activating the receptor for advanced glycation endproducts.

AIMS: Increased lipid peroxidation occurs in many conditions associated with inflammation. Because lipid peroxidation produces lipid aldehydes that can induce inflammatory responses through unknown mechanisms, elucidating these mechanisms may lead to development of better treatments for inflammatory diseases. We recently demonstrated that exposure of cultured cells to lipid aldehydes such as isolevuglandins (IsoLG) results in the modification of phosphatidylethanolamine (PE). We therefore sought to determine (i) whether PE modification by isolevuglandins (IsoLG-PE) occurred in vivo, (ii) whether IsoLG-PE stimulated the inflammatory responses of macrophages, and (iii) the identity of receptors mediating the inflammatory effects of IsoLG-PE. RESULTS: IsoLG-PE levels were elevated in plasma of patients with familial hypercholesterolemia and in the livers of mice fed a high-fat diet to induce obesity and hepatosteatosis. IsoLG-PE potently stimulated nuclear factor kappa B (NFκB) activation and expression of inflammatory cytokines in macrophages. The effects of IsoLG-PE were blocked by the soluble form of the receptor for advanced glycation endproducts (sRAGE) and by RAGE antagonists. Furthermore, macrophages derived from the bone marrow of Ager null mice failed to express inflammatory cytokines in response to IsoLG-PE to the same extent as macrophages from wild-type mice. INNOVATION: These studies are the first to identify IsoLG-PE as a mediator of macrophage activation and a specific receptor, RAGE, which mediates its biological effects. CONCLUSION: PE modification by IsoLG forms RAGE ligands that activate macrophages, so that the increased IsoLG-PE generated by high circulating cholesterol levels or high-fat diet may play a role in the inflammation associated with these conditions.

Macrophage apoAI protects against dyslipidemia-induced dermatitis and atherosclerosis without affecting HDL.

Tissue cholesterol accumulation, macrophage infiltration, and inflammation are features of atherosclerosis and some forms of dermatitis. HDL and its main protein, apoAI, are acceptors of excess cholesterol from macrophages; this process inhibits tissue inflammation. Recent epidemiologic and clinical trial evidence questions the role of HDL and its manipulation in cardiovascular disease. We investigated the effect of ectopic macrophage apoAI expression on atherosclerosis and dermatitis induced by the combination of hypercholesterolemia and absence of HDL in mice. Hematopoietic progenitor cells were transduced to express human apoAI and transplanted into lethally irradiated LDL receptor(-/-)/apoAI(-/-) mice, which were then placed on a high-fat diet for 16 weeks. Macrophage apoAI expression reduced aortic CD4(+) T-cell levels (-39.8%), lesion size (-25%), and necrotic core area (-31.6%), without affecting serum HDL or aortic macrophage levels. Macrophage apoAI reduced skin cholesterol by 39.8%, restored skin morphology, and reduced skin CD4(+) T-cell levels. Macrophage apoAI also reduced CD4(+) T-cell levels (-32.9%) in skin-draining lymph nodes but had no effect on other T cells, B cells, dendritic cells, or macrophages compared with control transplanted mice. Thus, macrophage apoAI expression protects against atherosclerosis and dermatitis by reducing cholesterol accumulation and regulating CD4(+) T-cell levels, without affecting serum HDL or tissue macrophage levels.

Dysfunctional high-density lipoproteins in children with chronic kidney disease.

OBJECTIVES: Our aim was to determine if chronic kidney disease (CKD) occurring in childhood impairs the normally vasoprotective functions of high-density lipoproteins (HDLs). MATERIALS AND METHODS: HDLs were isolated from children with end-stage renal disease on dialysis (ESRD), children with moderate CKD and controls with normal kidney function. Macrophage response to HDLs was studied as expression of inflammatory markers (MCP-1, TNF-α, IL-1ß) and chemotaxis. Human umbilical vein endothelial cells were used for expression of adhesion molecules (ICAM-1, VCAM-1, E-selectin) and adhesion. Cellular proliferation, apoptosis, and necrosis of endothelial cells were measured by MTS/PMS reagent-based assay, flow cytometry, and ELISA. Cholesterol efflux was assessed by gas chromatographic measurements of cholesterol in macrophages exposed to HDLs. RESULTS: Compared with HDL(Control), HDL(CKD) and HDL(ESRD) heightened the cytokine response and disrupted macrophage chemotaxis. HDL(Control) reduced endothelial expression of ICAM-1, VCAM-1, E-selectin, whereas HDL(CKD) and HDL(ESRD) were less effective and showed reduced capacity to protect endothelial cells against monocyte adhesion. Compared with a dramatically enhanced endothelial proliferation following injurious stimulus by HDL(Control), neither HDL(CKD) nor HDL(ESRD) caused proliferative effects. HDLs of all three groups were equally protective against apoptosis assessed by flow cytometry and cleaved caspase-3 activity. Compared to HDL(Control), HDL(CKD) and HDL(ESRD) trended toward reduced capacity as cholesterol acceptors. CONCLUSION: CKD in children impairs HDL function. Even in the absence of long-standing and concomitant risk factors, CKD alters specific HDL functions linked to control of inflammation and endothelial responses.

Dysfunctional high-density lipoprotein in patients on chronic hemodialysis.

OBJECTIVES: This study examined the functionality of high-density lipoprotein (HDL) in individuals with end-stage renal disease on dialysis (ESRD-HD). BACKGROUND: The high rate of cardiovascular disease (CVD) in chronic kidney disease is not explained by standard risk factors, especially in patients with ESRD-HD who appear resistant to benefits of statin therapy. HDL is antiatherogenic because it extracts tissue cholesterol and reduces inflammation. METHODS: Cellular cholesterol efflux and inflammatory response were assessed in macrophages exposed to HDL of patients with ESRD-HD or controls. RESULTS: HDL from patients with ESRD-HD was dramatically less effective than normal HDL in accepting cholesterol from macrophages (median 6.9%; interquartile range [IQR]: 1.4% to 10.2%) versus control (median 14.9%; IQR: 9.8% to 17.8%; p < 0.001). The profound efflux impairment was also seen in patients with ESRD-HD and diabetes compared with patients with diabetes without renal disease (median 8.1%; IQR: 3.3% to 12.9%) versus control (median 13.6%; IQR: 11.0% to 15.9%; p = 0.009). In vitro activation of cellular cholesterol transporters increased cholesterol efflux to both normal and uremic HDL. HDL of patients with ESRD-HD had reduced antichemotactic ability and increased macrophage cytokine response (tumor necrosis factor-alpha, interleukin-6, and interleukin-1-beta). HDL of patients with ESRD-HD on statin therapy had reduced inflammatory response while maintaining impaired cholesterol acceptor function. Interestingly, impaired HDL-mediated efflux did not correlate with circulating C-reactive protein levels or cellular inflammatory response. CONCLUSIONS: These findings suggest that abnormal HDL capacity to mediate cholesterol efflux is a key driver of excess CVD in patients on chronic hemodialysis and may explain why statins have limited effect to decrease CV events. The findings also suggest cellular cholesterol transporters as potential therapeutic targets to decrease CV risk in this population.

Macrophage polarization by angiotensin II-type 1 receptor aggravates renal injury-acceleration of atherosclerosis.

OBJECTIVE: Angiotensin II is a major determinant of atherosclerosis. Although macrophages are the most abundant cells in atherosclerotic plaques and express angiotensin II type 1 receptor (AT1), the pathophysiologic role of macrophage AT1 in atherogenesis remains uncertain. We examined the contribution of macrophage AT1 to accelerated atherosclerosis in an angiotensin II-responsive setting induced by uninephrectomy (UNx). METHODS AND RESULTS: AT1(-/-) or AT1(+/+) marrow from apolipoprotein E deficient (apoE(-/-)) mice was transplanted into recipient apoE(-/-) mice with subsequent UNx or sham operation: apoE(-/-)/AT1(+/+)→apoE(-/-)+sham; apoE(-/-)/AT1(+/+) →apoE(-/-)+UNx; apoE(-/-)/AT1(-/-)→apoE(-/-)+sham; apoE(-/-)/AT1(-/-)→apoE(-/-)+UNx. No differences in body weight, blood pressure, lipid profile, and serum creatinine were observed between the 2 UNx groups. ApoE(-/-)/AT1(+/+) →apoE(-/-)+UNx had significantly more atherosclerosis (16907±21473 versus 116071±8180 µm(2), P<0.05). By contrast, loss of macrophage AT1 which reduced local AT1 expression, prevented any effect of UNx on atherosclerosis (77174±9947 versus 75714±11333 µm(2), P=NS). Although UNx did not affect total macrophage content in the atheroma, lesions in apoE(-/-)/AT1(-/-)→apoE(-/-)+UNx had fewer classically activated macrophage phenotype (M1) and more alternatively activated phenotype (M2). Further, UNx did not affect plaque necrosis or apoptosis in apoE(-/-)/AT1(-/-)→apoE(-/-) whereas it significantly increased both (by 2- and 6-fold, respectively) in apoE(-/-)/AT1(+/+) →apoE(-/-) mice. Instead, apoE(-/-)/AT1(-/-)→apoE(-/-) had 5-fold-increase in macrophage-associated apoptotic bodies, indicating enhanced efferocytosis. In vitro studies confirmed blunted susceptibility to apoptosis, especially in M2 macrophages, and a more efficient phagocytic function of AT1(-/-) macrophages versus AT1(+/+). CONCLUSIONS: AT1 receptor of bone marrow-derived macrophages worsens the extent and complexity of renal injury-induced atherosclerosis by shifting the macrophage phenotype to more M1 and less M2 through mechanisms that include increased apoptosis and impaired efferocytosis.

Low-density lipoprotein receptor-related protein 1 prevents early atherosclerosis by limiting lesional apoptosis and inflammatory Ly-6Chigh monocytosis: evidence that the effects are not apolipoprotein E dependent.

BACKGROUND: We previously demonstrated that macrophage low-density lipoprotein receptor (LDLR)-related protein 1 (LRP1) deficiency increases atherosclerosis despite antiatherogenic changes including decreased uptake of remnants and increased secretion of apolipoprotein E (apoE). Thus, our objective was to determine whether the atheroprotective effects of LRP1 require interaction with apoE, one of its ligands with multiple beneficial effects. METHODS AND RESULTS: We examined atherosclerosis development in mice with specific deletion of macrophage LRP1 (apoE(-/-) MΦLRP1(-/-)) and in LDLR(-/-) mice reconstituted with apoE(-/-) MΦLRP1(-/-) bone marrow. The combined absence of apoE and LRP1 promoted atherogenesis more than did macrophage apoE deletion alone in both apoE-producing LDLR(-/-) mice (+88%) and apoE(-/-) mice (+163%). The lesions of both mouse models with apoE(-/-) LRP1(-/-) macrophages had increased macrophage content. In vitro, apoE and LRP1 additively inhibit macrophage apoptosis. Furthermore, there was excessive accumulation of apoptotic cells in lesions of both LDLR(-/-) mice (+110%) and apoE(-/-) MΦLRP1(-/-) mice (+252%). The apoptotic cell accumulation was partially due to decreased efferocytosis as the ratio of free to cell-associated apoptotic nuclei was 3.5-fold higher in lesions of apoE(-/-) MΦLRP1(-/-) versus apoE(-/-) mice. Lesion necrosis was also increased (6 fold) in apoE(-/-) MΦLRP1(-/-) versus apoE(-/-) mice. Compared with apoE(-/-) mice, the spleens of apoE(-/-) MΦLRP1(-/-) mice contained 1.6- and 2.4-fold more total and Ly6-C(high) monocytes. Finally, there were 3.6- and 2.4-fold increases in Ly6-C(high) and CC-chemokine receptor 2-positive cells in lesions of apoE(-/-) MΦLRP1(-/-) versus apoE(-/-) mice, suggesting that accumulation of apoptotic cells enhances lesion development and macrophage content by promoting the recruitment of inflammatory monocytes. CONCLUSION: Low-density lipoprotein receptor protein 1 exerts antiatherogenic effects via pathways independent of apoE involving macrophage apoptosis and monocyte recruitment.

Oral activated charcoal adsorbent (AST-120) ameliorates extent and instability of atherosclerosis accelerated by kidney disease in apolipoprotein E-deficient mice.

BACKGROUND: Accelerated atherosclerosis and increased cardiovascular events are not only more common in chronic kidney disease (CKD) but are more resistant to therapeutic interventions effective in the general population. The oral charcoal adsorbent, AST-120, currently used to delay start of dialysis, reduces circulating and tissue uremic toxins, which may contribute to vasculopathy, including atherosclerosis. We, therefore, investigated whether AST-120 affects CKD-induced atherosclerosis. METHODS: Apolipoprotein E-deficient mice, a model of atherosclerosis, underwent uninephrectomy, subtotal nephrectomy or sham operation at 8 weeks of age and were treated with AST-120 after renal ablation. Atherosclerosis and its characteristics were assessed at 25 weeks of age. RESULTS: Uninephrectomy and subtotal nephrectomised mice had significantly increased acceleration of atherosclerosis. AST-120 treatment dramatically reduced the atherosclerotic burden in mice with kidney damage, while there was no beneficial effect in sham-operated mice. The benefit was independent of blood pressure, serum total cholesterol or creatinine clearance. AST-120 significantly decreased necrotic areas and lessened aortic deposition of the uremic toxin indoxyl sulfate without affecting lesional macrophage or collagen content. Furthermore, AST-120 lessened aortic expression of monocyte chemoattractant protein-1, tumor necrosis factor-α and interleukin-1ß messenger RNA. CONCLUSIONS: AST-120 lessens the extent of atherosclerosis induced by kidney injury and alters lesion characteristics in apolipoprotein E-deficient mice, resulting in plaques with a more stable phenotype with less necrosis and reduced inflammation.