Targeting the Anti-Inflammatory Effects of High-Density Lipoprotein

Targeting the Anti-Inflammatory Effects of High-Density Lipoprotein Benjamin J. Ansell, MD The effects of systemic inflammation can impair the anti-inflammatory functions of high-density lipoprotein (HDL) particles. In patients with atherosclerosis and/or inflammatory conditions, HDLs can be modified such that they paradoxically increase the recruitment and activation of macrophages, upregulate the expression of endothelial cell adhesion molecules, and participate in the oxidation of low-density lipoproteins (LDLs). Statins, apolipoprotein A-I mimetic peptides, and therapeutic lifestyle changes appear to mitigate these proinflammatory features of HDLs. In the future, characterizing and targeting functional aspects of HDLs may prove to offer therapeutic advantages over current treatment strategies. © 2007 Elsevier Inc. All rights reserved. (Am J Cardiol 2007;100[suppl]:3N–9N)

In addition to its well-documented role in facilitating reverse cholesterol transport, high-density lipoprotein (HDL) serves several other important antiatherogenic functions, including limitation of the oxidation of phospholipids within low-density lipoprotein (LDL).1 These oxidized lipids stimulate arterial wall production of monocyte chemoattractant protein (MCP)–1, which increases recruitment of mononuclear cells, and the expression of cellular adhesion molecules that promote leukocyte interaction with endothelial cells and entry into atheroma.2,3 By limiting LDL oxidation, HDL thus plays a key antiinflammatory role in slowing atherogenesis. The ability of HDL to inhibit LDL oxidation has been known for ⬎1 decade.4 In the setting of systemic inflammation, however, the atheroprotective character of HDL can markedly diminish, even to the point where it becomes proinflammatory.1,5 Its ability to promote reverse cholesterol transport can become impaired. Inflammation can paradoxically modify HDL such that it promotes oxidation of LDL, production of MCP-1, and expression of cellular adhesion molecules.6,7 These proinflammatory features can resolve along with the course of the associated systemic inflammation, or they may be modified by several different evolving therapeutic approaches. High-Density Lipoprotein as an Anti-Inflammatory Particle When LDL enters the subendothelial space, it can become modified in a way that promotes atherogenesis. Some of the phospholipids that form the surface layer of lipoproteins Atherosclerosis Research Unit, Division of Cardiology, Department of Medicine, David Geffen School of Medicine at University of California, Los Angeles, Los Angeles, California, USA. Statement of author disclosure: Please see the Author Disclosures section at the end of this article. Address for reprints: Benjamin J. Ansell, MD, 100 UCLA Medical Plaza, Suite 525, Los Angeles, California 90095. E-mail address: [email protected]. 0002-9149/07/$ – see front matter © 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.amjcard.2007.08.006

contain arachidonic acid, which can become oxidized by products of the 12-lipoxygenase pathway and a number of other pathways.8 These oxidized phospholipids stimulate endothelial cell production of MCP-1, leading to recruitment of circulating monocytes from the arterial lumen.9 Modified LDL can be taken up by the scavenger receptors of these activated macrophages, leading to foam cell formation.10 Other cytokines such as tumor necrosis factor–␣ and interleukin-1 are produced by macrophages within atheroma in response to modified LDL and activate production of vascular cell adhesion molecule (VCAM)–1 and intercellular adhesion molecule (ICAM)–1 on the surface of endothelial cells that bind more mononuclear cells.11 Together, modified LDL, MCP, and these cellular adhesion molecules form a potentially escalating cascade of inflammatory cell entry and LDL oxidation that are atherogenic. HDL regulates these processes via several key mechanisms. Under normal circumstances, HDL inhibits the oxidation of LDL through a combination of antioxidant enzymes and apolipoprotein (apo) A-I.12 HDL also normally attenuates cytokine-induced adhesion molecule expression on endothelial cells.3 HDL can also promote efflux of cholesterol out of vascular macrophages and the arterial wall itself, limiting the formation of inflammatory foam cells.13 HDL therefore is capable of multiple anti-inflammatory effects. High-Density Lipoprotein as a Proinflammatory Particle In recent years, it has become apparent that, in the presence of systemic inflammation, HDL’s protective effects are often adversely impacted. Infection, diabetes mellitus, coronary artery disease (CAD), smoking, hemodialysis, and rheumatologic conditions can paradoxically create a “chronic acute-phase response” that has been described by Gabay and Kushner14 and Navab and colleagues.5 This inflammatory milieu can lead to a proinflammatory phenotype of HDL that increases LDL oxidation, enhances vascular inwww.AJConline.org

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flammation, and is less, if at all, effective in reverse cholesterol transport.6 The oxidative products generated within the subendothelial space generate a number of potential oxidants through 12-lipoxygenase and other pathways.8,15 In the process, the essential fatty acids lineolic acid and arachidonic acid can become oxidized and form the pro-oxidant HPODE and HPETE, which themselves can then go on to oxidize phospholipids and create proinflammatory molecules.15 These include conversion of the relatively benign phospholipids in lipoproteins such as 1-palmitoyl-2-arachidonyl-phosphatidylcholine (PAPC) to proinflammatory phospholipids such as oxovaleroyl (POVPC), glutaroyl (PGPC), and epoxyisoprostane (PEIPC).15 These proinflammatory molecules lead to perpetuation of the inflammatory cycle and further oxidation of LDL and also HDL particles.

Measuring the Anti-Inflammatory/Proinflammatory Effects of High-Density Lipoprotein Several evolving methods of identifying these processes have been described, including the monocyte chemotaxis assay (MCA) and a cell-free assay (CFA). The MCA measures HDL’s ability to alter LDL-induced chemotaxis in a human artery wall coculture, whereas the CFA evaluates HDL’s ability to alter oxidized phospholipid-induced fluorescence in a cell-free system.9,16 The CFA is a model of LDL oxidation, whereas MCA reflects cellular inflammation as measured by MCP-1 expression and recruitment of monocytes. The results of the MCA can be indexed to 1.0, the level of monocyte chemotaxis that is produced by LDL alone. The addition of anti-inflammatory HDL should lead to a decrease in the monocyte chemotaxis induced by LDL, ie, an inflammatory index of ⬍1.0. An increase in MCA to ⬎1.0 indicates the effect of proinflammatory HDL. The CFA can similarly be normalized to 1.0, reflecting the fluorescent activity without HDL present.17

Clinical Evidence of Proinflammatory High-Density Lipoprotein Using the CFA and MCA, Ansell and colleagues17 examined the characteristics of HDL sampled from patients who developed CAD despite very high HDL cholesterol levels (ie, HDL ⱖ84 mg/dL). The patients were naive to lipidlowering medications, did not smoke, and did not have diabetes. The results were compared with those from healthy age- and sex-matched controls. Although HDL cholesterol levels were slightly higher in the controls, the LDL cholesterol and triglyceride levels were similar in both groups.17 In contrast, the difference in the anti-inflammatory potential of HDL was marked: those individuals who had CAD despite supernormal levels of HDL cholesterol had uniformly proinflammatory MCA results (inflammatory in-

dex ⫽ 1.28 ⫾ 0.29).17 In contrast, the HDL levels of healthy controls inhibited most of the monocyte chemotaxis that was induced by LDL (inflammatory index ⫽ 0.35 ⫾ 0.11), allowing for a stark distinction on the basis of the functional properties of HDL.17 The results of the CFA were qualitatively similar (inflammatory index ⫽ 1.37 ⫾ 0.19 for the patients, 0.66 ⫾ 0.21 for the controls).17 To determine whether this proinflammatory phenotype is present in patients with CAD and more typical HDL cholesterol levels, Ansell and colleagues enrolled 26 adults with stable CAD or coronary risk equivalents that were naive to hyperlipidemic medications.17 The study also recruited ageand sex-matched controls, with the 2 groups of subjects well matched for LDL cholesterol and high-sensitivity C-reactive protein levels, with the controls having slightly higher HDL cholesterol and lower triglyceride levels.17 The patients with CAD or CAD risk equivalents were treated with simvastatin 40 mg daily for 6 weeks. After the patients were treated with simvastatin, mean LDL cholesterol fell by 38%, while mean HDL cholesterol increased by 8%.17 As seen in Figure 1A, the controls exhibited anti-inflammatory HDL in the MCA assay (inflammatory index ⫽ 0.38 ⫾ 0.14).17 In contrast, in almost all cases, the patients with CAD or CAD risk equivalents had proinflammatory HDL (inflammatory index ⫽ 1.38 ⫾ 0.91).17 After simvastatin treatment, there was a significantly reduced level of monocyte chemotaxis among these patients (inflammatory index ⫽ 1.08 ⫾ 0.71; p ⫽ 0.002), but on average they still showed proinflammatory HDL. The results of the CFA (Figure 1B) were qualitatively similar. The healthy controls had anti-inflammatory HDL (inflammatory index ⫽ 0.53 ⫾ 0.15), while most of those with CAD risk equivalents had proinflammatory HDL (inflammatory index ⫽ 1.19 ⫾ 0.19).17 After 6 weeks of simvastatin, the HDL phenotype returned to mildly antiinflammatory (inflammatory index ⫽ 0.91 ⫾ 0.28), but not nearly to the level of the controls.17 More than by their lipid levels, the patients with CAD and the healthy controls were very distinguishable on the basis of their HDL anti-inflammatory/proinflammatory properties as measured by these assays.17 In addition, some of the benefits of 3-hydroxy-3methylglutaryl coenzyme A reductase inhibitor (statin) therapy in high-risk patients may in fact reflect improved antiinflammatory capacity of their HDL particles.

The Effect of Glycemia on High-Density Lipoprotein Anti-Inflammatory/Proinflammatory Properties The CAD/CAD risk equivalent cohort above included some patients with type 2 diabetes, who on average exhibited proinflammatory HDL. Gowri and coworkers18 reported that HDL isolated from subjects with poorly controlled diabetes was ineffective at promoting cholesterol efflux from macrophages. There is mounting evidence that glycated protein targets within blood vessel cells contribute to

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Figure 1. Comparison of monocyte chemotaxis assay (MCA) (A) and cell-free assay (CFA) (B) determination of high-density lipoprotein (HDL) inflammatory index in patients before and after a 6-week course of simvastatin 40 mg daily compared with healthy matched controls. The assay activities are normalized to a value of 1.0, the results in the absence of test HDL. (Reprinted from Circulation.17)

atherogenesis. Furthermore, glycation of HDL itself may adversely impact its normal protective functions. Hedrick and colleagues19 assessed the impact of incubating glucose with the key HDL-associated antioxidant enzyme paraoxonase on the enzyme’s activity. After 1 week, a significant 65% decrease in paraoxonase activity was evident when the enzyme was mixed in glucose solution. From these data, it can be concluded that nonenzymatic glycation clearly impairs the antioxidant function of HDL. In addition, incubation of HDL with glucose also led to inability to slow monocyte chemotaxis.19 Another study by Ferretti and colleagues20 showed similar results in patients with both high and low paraoxonase levels. HDL that was isolated from these individuals and then incubated with 0, 50, or 100 mmol/L glucose for 0, 48, or 72 hours, respectively. A dose-dependent, time-dependent increase in TBARS production was observed in accordance with the concentration of glucose, evident to a greater extent in individuals with low baseline levels of paraoxonase.20 The model that has been proposed by Ferretti and colleagues suggests that glycation of both LDL and HDL have potential synergy in contributing to the development of dysfunctional HDL and increased atherogenesis. As seen in Figure 2, HDL can become glycated, leading to altered reverse cholesterol transport and greater susceptibility to oxidation, while it is less able to protect LDL from oxidation.21 LDL itself can also become glycated, increasing its potential to become oxidized as well. Oxidized, glycated LDL undergoes enhanced phagocytosis by the scavenger receptors in foam cell formation, ultimately accelerating atherosclerotic plaque formation.21

The Effects of Nonvascular Systemic Inflammation In addition to patients with CAD, CAD risk equivalents, and diabetes, those affected by other forms of systemic inflammation also exhibit a proinflammatory HDL phenotype. For

example, surgery is associated with a transient conversion from anti-inflammatory to proinflammatory HDL that resolves in convalescence.22 The same is true in a mouse model of influenza infection.23 Myocardial infarction (MI) has been associated with flu epidemics,24 suggesting that there may be similar effects in humans. It is also now well recognized that patients with certain rheumatologic conditions are at significantly increased risk for CAD. For example, systemic lupus erythematosus carries a 50-fold increased risk for MI,25 whereas the diagnosis of rheumatoid arthritis portends a 2- to 3-fold increase in the risk for CAD that is not explained by traditional risk factors.26 McMahon and colleagues27 compared cohorts of patients with lupus and active rheumatoid arthritis with age- and sex-matched controls and found that 44.7% of the lupus patients and 20.1% of the rheumatoid arthritis patients showed proinflammatory HDL by the CFA, compared with 4.1% of controls (p ⫽ 0.003 and p ⫽ 0.006, respectively). This suggests a significant potential interaction between chronic inflammation, HDL, and the increased vascular risk in these patients. Recognition of this relationship prompted consideration of potential treatment strategies to target the inflammatory changes in HDL of rheumatologic patients. In the Statins’ Anti-Rheumatic Activity (SARA) trial, 30 patients with active rheumatoid arthritis despite the use of multiple disease-modifying agents were randomized to treatment with either atorvastatin 80 mg or matching placebo for 12 weeks.28 There was no difference in rheumatic disease activity or tolerability associated with either therapy. Compared with baseline, the cell-free measurement of inflammation decreased by 14.8% in HDL from atorvastatin subjects compared with a 7.1% increase in those receiving placebo (p ⫽ 0.026). Both treatments were associated with no significant change in HDL cholesterol levels.28 In addition to statin therapy, some specific lifestyle interventions appear to enhance HDL anti-inflammatory function. Roberts and colleagues29 studied a cohort of patients

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Figure 2. Interaction of glycation and oxidation of both low-density lipoprotein (LDL) and high-density lipoprotein (HDL) in formation of atherosclerotic plaque. Gly-LDL ⫽ glycated LDL; Gly-HDL ⫽ glycated HDL; Gly-ox-LDL ⫽ oxidized glycated LDL; Gly-ox-HDL ⫽ oxidized glycated HDL. (Reproduced with permission from Atherosclerosis.21)

who were overweight with metabolic syndrome and offered them a 21-day intensive lifestyle intervention with (1) high fiber grains, (2) increased vegetables and fruits, (3) reduction in saturated fat, (4) increase in plant/fish protein sources, and (5) a daily supervised walk of up to 1 hour. The subjects showed modest reductions in body mass index from 33.1 to 32.1, LDL cholesterol levels from 126 to 94 mg/dL, and HDL cholesterol levels from 44 to 39 mg/dL (as can occur with low-fat diets). Despite a reduction in HDL cholesterol concentration, the characteristics of the HDL particles improved substantially. As measured by the MCA, the inflammatory index fell from 1.14 to 0.94 following the lifestyle changes.29 Levels of inflammatory lipid hydroperoxides fell and the antioxidant enzyme platelet-activating factor–acetylhydrolase increased after the intervention. Nicholls and colleagues30 reported that an even more focal dietary change—altering fat composition—was also associated with improved measures of HDL anti-inflammatory function. Using a crossover design, subjects were fed a meal primarily including polyunsaturated fat or one largely consisting of saturated fat. The characteristics of the subjects’ HDL were assessed while fasting and at 3 and 6 hours

after the high-fat meals. As shown in Figure 3, ICAM-1 expression and VCAM-1 expression both varied according to time and the type of fat ingested. The polyunsaturated fat meal was associated with a significant reduction in both ICAM-1 and VCAM-1 expression.30 The effect on both adhesion cell molecule levels was evident as early as 3 hours after the meal and was maintained at 6 hours. In contrast, the saturated fat diet resulted in a proinflammatory HDL by 6 hours as evident in levels of both cellular adhesion molecules.30 In addition to the rationale for use of a low saturated fat diet as a means to reduce LDL cholesterol levels, this dietary intervention may also be justified by beneficial changes in the nature of the resultant HDL particles. Assessing Other Aspects of High-Density Lipoprotein Function Other assays measuring different aspects of HDL function including aspects of reverse cholesterol transport (eg, macrophage cholesterol efflux, fecal sterol excretion, and HDL tracer kinetic studies) have also been reported.31 There is an inverse correlation between monocyte chemotaxis and pro-

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Figure 3. The effects of a predominantly saturated fat (gray bars) versus polyunsaturated fat (white bars) meal on intercellular adhesion molecule–1 (A) and vascular cellular adhesion molecule–1 (B) expression at fasting state (black bars), and 3 hours and 6 hours after ingestion. (Reprinted with permission from J Am Coll Cardiol.30)

motion of cholesterol efflux, suggesting that HDL’s reverse cholesterol transport function is highly conserved along with its anti-inflammatory capacity.5 The same close linkage is evident between HDL’s ability to inhibit monocyte chemotaxis and the ability to slow LDL oxidation.17 Although these different functions of HDL may seem disparate, they probably reflect common chemical characteristics of apoA-I, phospholipids, and/or antioxidant enzymes within HDL. Moreover, impaired reverse cholesterol transport leads to increased levels of proinflammatory, oxidized lipoprotein, and lipid particles.32,33

Structural Determinants of High-Density Lipoprotein Function Efforts are underway to correlate chemical and structural changes within HDL with modification of its functionality. Among these, it appears that enzymatic and structural alterations in HDL that are associated with the acute-phase response are particularly important. During this systemic inflammation, serum amyloid A and apo-J are incorporated into the HDL particles while levels of apoA-1, apoA-2, paraoxonase, and lecithin:cholesterol acyltransferase are reduced.34 The replacement of these more beneficial subcomponents of the surface of HDL leads with acute-phase reactants correlated with the conversion to a more proinflammatory HDL. The phospholipid composition of the HDL is critically important in determining the functionality of HDL. Comparing the effects of HDL that was reconstituted with 1 of 3 different types of phospholipid 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC), PAPC, or 1-palmitoyl-2-linoleoylphosphatidylcholine (PLPC), Baker and colleagues33 reported that expression of endothelial cell VCAM-1 was inhibited by 16%, 70%, and 95% respectively. Therefore, both enzyme and phospholipid content can affect the functionality of HDL. The leukocyte product myeloperoxidase can oxidatively modify apoA-I, leading to nitrotyrosination and chloroty-

rosination at specific amino acid sites, and thus leading to a proinflammatory phenotype.35 So there appear to be multiple mechanisms whereby HDL can acquire proinflammatory characteristics.

Modifying Anti-inflammatory Properties of High-Density Lipoprotein Several therapeutic interventions appear to be likely to reduce the potential for HDL to become proinflammatory and enhance its anti-inflammatory potential, including use of statins, emphasizing polyunsaturated fats in lieu of saturated fatty acids in the diet, and intensive lifestyle changes. Some emerging therapeutic strategies, such as analogues of apoA-I, delipidation of HDL, and other developmental HDL-based compounds may be effective in enhancing HDL’s anti-inflammatory effects as well. Of the HDL-based compounds that are currently in development, one that has already demonstrated an ability to render HDL more anti-inflammatory is an orally active 18-amino acid peptide called D-4F. When administered to animal and human subjects, D-4F stimulates the formation of small HDL-like particles with a pre-␤ migration pattern that contain cholesterol, apoA-I, and paraoxonase activity.36 In animal studies, these particles are capable of removing lipid oxidation products and are able to promote cholesterol efflux and inhibit inflammation within the vasculature.36 In a study of cynomolgus monkeys fed a chow diet, HDL was proinflammatory, in that it increased monocyte chemotaxis relative to the effects of LDL alone, with an inflammatory index of 1.2.2 In contrast, HDL that was isolated from these same animals just 2 hours after oral administration of D-4F was clearly anti-inflammatory, with an inflammatory index of just 0.5 (p ⬍0.01).2 In association with this change in the MCA, levels of lipid hydroperoxides decreased for both HDL and LDL after treatment with D-4F, suggesting improvement in the proinflammatory characteristics of both particles.2 In addition, HDL that was isolated from this group of monkeys had relatively limited ability to

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enhance HDL-mediated cholesterol efflux before treatment, but this also substantially improved just 2 hours after administration of the D-4F.2

Conclusion Systemic inflammation can modify HDL such that it becomes ineffective or frankly proinflammatory. Most patients with CAD and likely other inflammatory diseases have proinflammatory HDL. Strategies with proven cardiovascular benefits such as statins and lifestyle changes can partially reverse this phenotype. In addition, the ability of apoA-I and apo and mimetic peptides to reduce levels of oxidized lipids and improve reverse cholesterol transport may also have therapeutic potential. Conversely, conditions and treatments that increase proinflammatory HDL appear likely to increase cardiovascular risk.

Author Disclosures The author who contributed to this article has disclosed the following industry relationships. Benjamin J. Ansell, MD, has received honoraria from Pfizer Inc, AstraZeneca Pharmaceuticals LP, Merck & Co., Inc., and Takeda Pharmaceuticals; serves as a consultant to Pfizer Inc, AstraZeneca Pharmaceuticals LP, Merck & Co Inc., and Reliant Pharmaceuticals; and holds equity interest in Bruin Pharma. 1. Ansell BJ, Fonarow GC, Fogelman AM. High-density lipoprotein: is it always atheroprotective? Curr Atheroscler Rep 2006;8:405– 411. 2. Navab M, Anantharamaiah GM, Reddy ST, Van Lenten BJ, Ansell BJ, Fonarow GC, Vahabzadeh K, Hama S, Hough G, Kamranpour N, et al. The oxidation hypothesis of atherogenesis: the role of oxidized phospholipids and HDL. J Lipid Res 2004;45:993–1007. 3. Cockerill GW, Rye K-A, Gamble JR, Vadas MA, Barter PJ. Highdensity lipoproteins inhibit cytokine-induced expression of endothelial cell adhesion molecules. Arterioscler Thromb Vasc Biol 1995;15: 1987–1994. 4. Watson A, Berliner J, Hama S, La Du BN, Faull K, Fogelman A, Navab M. Protective effect of high density lipoprotein associated paraoxonase: inhibition of the biological activity of minimally oxidized low density lipoprotein. J Clin Invest 1995;96:2882–2891. 5. Navab M, Anantharamaiah GM, Reddy ST, Van Lenten BJ, Ansell BJ, Hama S, Hough G, Bachini E, Grijalva VR, Wagner A, Shaposhnik Z, Fogelman AM. The double jeopardy of HDL. Ann Med 2005;37:1– 6. 6. Ansell BJ, Watson KE, Fogelman AM, Navab M, Fonarow GC. High-density lipoprotein function: recent advances. J Am Coll Cardiol 2005;46:1792–1798. 7. Fogelman AM. When good cholesterol goes bad. Nat Med 2004;10: 902–903. 8. Fogelman AM. When pouring water on the fire makes it burn brighter. Cell Metab 2005;2:6 – 8. 9. Navab M, Imes SS, Hama SY, Hough GP, Ross LA, Bork RW, Valente AJ, Berliner JA, Drinkwater DC, Laks H, et al. Monocyte transmigration induced by modification of low density lipoprotein in cocultures of human aortic wall cells is due to induction of monocyte chemotactic protein 1 synthesis and is abolished by high density lipoprotein. J Clin Invest 1991;88:2039 –2046.

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