ABSTRACT

 

Atherosclerosis is the underlying cause of heart attack and stroke. Early observations that cholesterol is a key component of arterial plaques gave rise to the cholesterol hypothesis for the pathogenesis of atherosclerosis. Population studies have demonstrated that elevated levels of LDL cholesterol and apolipoprotein B (apoB) 100, the main structural protein of LDL, are directly associated with risk for atherosclerotic cardiovascular events (ASCVE). Indeed, infiltration and retention of apoB containing lipoproteins in the artery wall is a critical initiating event that sparks an inflammatory response and promotes the development of atherosclerosis. Arterial injury causes endothelial dysfunction promoting modification of apoB containing lipoproteins and infiltration of monocytes into the subendothelial space. Internalization of the apoB containing lipoproteins by macrophages promotes foam cell formation, which is the hallmark of the fatty streak phase of atherosclerosis. Macrophage inflammation results in enhanced oxidative stress and cytokine/chemokine secretion, causing more LDL/remnant oxidation, endothelial cell activation, monocyte recruitment, and foam cell formation. HDL, apoA-I, and endogenous apoE prevent inflammation and oxidative stress and promote cholesterol efflux to reduce lesion formation. Macrophage inflammatory chemoattractants stimulate infiltration and proliferation of smooth muscle cells. Smooth muscle cells produce the extracellular matrix providing a stable fibrous barrier between plaque prothrombotic factors and platelets. Unresolved inflammation results in formation of vulnerable plaques characterized by enhanced macrophage apoptosis and defective efferocytosis of apoptotic cells resulting in necrotic cell death leading to increased smooth muscle cell death, decreased extracellular matrix production, and collagen degradation by macrophage proteases. Rupture of the thinning fibrous cap promotes thrombus formation resulting in clinical ischemic ASCVE. Surprisingly, native LDL is not taken up by macrophages in vitro but has to be modified to promote foam cell formation. Oxidative modification converts LDL into atherogenic particles that initiate inflammatory responses. Uptake and accumulation of oxidatively modified LDL (oxLDL) by macrophages initiates a wide range of bioactivities that may drive development of atherosclerotic lesions. Lowering LDL-cholesterol with statins reduces risk for cardiovascular events, providing ultimate proof of the cholesterol hypothesis. All of the apoB containing lipoproteins are atherogenic, and both triglyceride rich remnant lipoproteins and Lp(a) promote atherothrombosis. Non-HDL cholesterol levels capture all of the apoB containing lipoproteins in one number and are useful in assessing risk in the setting of hypertriglyceridemia. Measures of apoB and LDL-P are superior at predicting risk for ASCVE, when levels of LDL-C and LDL-P are discordant.  Here, we also describe the current landscape of HDL metabolism. Epidemiological studies have consistently shown that HDL-C levels are inversely related to ASCVE. We highlight recent clinical trials aimed at raising HDL-C that failed to reduce CVE and the shifting clinical targets of HDL-C, HDL particle numbers, and HDL function (e.g. cholesterol efflux capacity). Furthermore, we describe many beneficial properties of HDL that antagonize atherosclerosis and how HDL dysfunction may promote cardiometabolic disease.

 

PATHOPHYSIOLOGY OF ATHEROSCLEROSIS

 

Atherosclerosis in Cardiovascular Disease

 

As the underlying cause of heart attack, stroke, and peripheral vascular disease, atherosclerosis is the major cause of death and morbidity in the United States and the industrial world (1). The discovery by Virchow more than 100 years ago that atheroma contained a yellow fatty substance, later identified as cholesterol by Windaus, suggested a role for lipids in the pathogenesis of atherosclerosis (2). Indeed, the goal of this chapter is to focus on the role of lipids and lipoproteins in the pathogenesis of atherosclerosis as well as their critical roles in risk assessment and as targets of therapy. The recognition that atherosclerosis is an inflammatory disease has led to tremendous progress in our understanding of the pathogenesis of atherosclerosis (3). First, we provide brief description of the cellular and molecular events in the key stages of atherosclerosis.

 

Initiation and Fatty Streak Phase of Atherosclerotic Lesions

 

The endothelial lining of arteries responds to mechanical and molecular stimuli to regulate tone, (4)hemostasis, (5)and inflammation (6)throughout the circulation. Endothelial cell dysfunction is an initial step in atherosclerotic lesion formation and is more likely to occur at arterial curves and branches that are subjected to low shear stress and disturbed blood flow (atherosclerosis prone areas) (7,8). These mechanical stimuli activate signaling pathways leading to a dysfunctional endothelium lining that is barrier compromised, prothrombotic, and proinflammatory (9). In atherosclerosis susceptible regions, the endothelial cells have cuboidal morphology, a thin glycocalyx layer, and a disordered alignment (8,10,11). In addition, these regions have increased endothelial cell senescence and apoptosis as evidenced by ER stress markers (12-14).In contrast, less atherosclerosis prone endothelium is exposed to laminar shear stress causing activation of signaling pathways that maintain endothelial cell coaxial alignment, proliferation, (13,14)glycocalyx layer, (15)and survival (12,16). In atherosclerosis resistant regions, the transcription factors, Kruppel-like factors (KLF) 2 and 4, are activated via MEK5/ERK5/MEF2 signalingwhich enhances expression of endothelial nitric oxide synthase (eNOS) (17-19). The increased nitric oxide (NO) production promotes endothelial cell migration and survival thereby maintaining an effective barrier (20). In addition, the expression of superoxide dismutase (SOD) is increased to reduce cellular oxidative stress (18). In atherosclerosis susceptible regions, reduced expression of eNOS and SOD leads to compromised endothelial barrier integrity (Figure 1), leading to increased accumulation and retention of subendothelial atherogenic apolipoprotein B (apoB)-containing lipoproteins (low-density lipoproteins (LDL)) and remnants of very low-density lipoproteins (VLDL) and chylomicrons) (21,22). KLF2, KLF4, and NO production inhibit activation of the nuclear factor kappa B (NF‐κB) pathway.Increased NF‐κBactivation in atherosclerosis susceptible areas leads to endothelial cell activation (Figure 1), as evidenced by increased expression of monocyte adherence proteins (VCAM-1, ICAM-1,and P-selectin) and proinflammatory receptors (toll-like receptor 2, TLR2) and cytokines (MCP-1 and IL-8) (19,23,24). In addition, endothelial cell activationleads to increased production of reactive oxygen species (25)that can cause oxidative modification of apoB-containing lipoproteins (26). Besides mechanical stimuli, endothelial cell activation is increased by various molecular stimuli, including oxidized LDL, cytokines,advanced glycosylation end products, and pathogen-associated molecules (27-30). In contrast, an atheroprotective function of HDL is to prevent endothelial activation and enhance NO production to maintain barrier integrity (see details below) (31).

Immune Cell Recruitment and Foam Cell Formation

 

Activation of endothelial cells causes a monocyte recruitment cascade involving rolling, adhesion, activation and transendothelial migration (Figure 1). Selectins, especially P-selectin, mediate the initial rolling interaction of monocytes with the endothelium (32). Monocyte adherence is then promoted by endothelial cell immunoglobulin-G proteins including VCAM-1 and ICAM-1(32). Potent chemoattractant factors such as MCP-1 and IL-8 then induce migration of monocytes into the subendothelial space (33-35). Ly6^himonocytes, versus Ly6^lo, preferentially migrate into the subendothelial space to convert to proinflammatory macrophages in mice (36-38). The enhanced migration of Ly6^hiversus Ly6^lomonocytes likely results from increased expression of functional P-selectin glycoprotein ligand-1 (39). In addition, the number of blood monocytes originating from the bone marrow and spleen, especially Ly6^hicells, increases in response to hypercholesterolemia (36). Furthermore, hypercholesterolemia and atherosclerosis increase monocytosis in humans (40,41). Importantly, increased numbers of inflammatory CD14⁺⁺CD16⁺monocytes independently predicted cardiovascular death, myocardial infarction, and stroke in patients undergoing elective coronary angiography (42). Intimal macrophages also result from proliferation of monocyte/macrophages, especially in more advanced lesions (43). During the initial fatty streak phase of atherosclerosis (Figure 1), the monocyte-derived macrophages internalize the retained apoB-containing lipoproteins, which are degraded in lysosomes, where excess free cholesterol is trafficked to the endoplasmic reticulum (ER) to be esterified by acyl CoA:cholesterol acyltransferase (ACAT), and the resulting cholesteryl ester (CE) is packaged into cytoplasmic lipid droplets, which are characteristic of foam cells (42)(Figure 2) (44,45). Modification of apoB lipoproteins via oxidation and glycation enhances their uptake through a number of receptors not down-regulated by cholesterol including CD36, scavenger receptor A, and lectin-like receptor family (see details below) (Figure 2) (46,47). Enzyme-mediated aggregation of apoB lipoproteins enhances uptake via phagocytosis (Figure 2) (48,49). In addition, native remnant lipoproteins can induce foam cell formation via a number of apoE receptors (LRP1 and VLDLR) (Figure 2) (50,51). Uptake of native LDL by fluid phase pinocytosis may also contribute to foam cell formation (Figure 2) (52,53).

 

The triggering of macrophage inflammatory pathways is also a critical event in lesion development. Inflammatory M1 phenotype macrophages exhibit increased oxidative stress, impaired cholesterol efflux and enhanced cytokine/chemokine secretion, leading to more LDL/remnant oxidation, endothelial cell activation, monocyte recruitment, and foam cell formation (54-59). Oxidative stress, modified lipoproteins, and other lesion factors (bioactive lipids, pattern recognition molecules, cytokines) are capable of inducing inflammation via receptors (54,55,60). In addition, plasma membrane cholesterol in macrophage foam cells enhances signaling via inflammatory receptors (61,62). Recently, inflammasome activation of IL-1β and IL-18 has been implicated in atherogenesis (63,64). Indeed, a recent clinical trial showed that subjects treated with the IL-1β monoclonal antibody, canakinumab, had a significantly lower rate of recurrent cardiovascular events which were independent of cholesterol lowering (65). Macrophage foam cell formation and cholesterol dependent inflammatory receptor signaling can be reduced by the removal of cholesterol by atheroprotective HDL and apoA-I via a number of mechanisms including ABCA1, ABCG1, SR-BI, and aqueous diffusion (Figure 2) (61,66-68)(see details below). Lipid-poor apoA-I stimulates efflux via ABCA1, whereas lipidated apoA-I or mature HDL are the main drivers of efflux via ABCA1, ABCG1, SR-BI, and aqueous diffusion (Figure 2) (61,69-71). Cytoplasmic CE is cleared by two major pathways. One route involves the hydrolysis of cytoplasmic CE by neutral cholesterol esterase and the resulting free cholesterol is mobilized away from the ACAT pool (72,73)and made available for efflux via ABCA1, ABCG1, SR-BI, and aqueous diffusion (Figure 2). Alternatively, cytoplasmic CE is packaged into autophagosomes, which are trafficked to lysosomes, where the CE is hydrolyzed by acid lipase(73,74), generating free cholesterol that is made available for efflux mainly via ABCA1(Figure 2) (73,74). Furthermore, HDL and apoA-I protect against atherosclerosis by reducing inflammation via mechanisms independent of cholesterol efflux (31,75)(see details below). In addition, small non-coding RNAs have been found to impact atherosclerosis development by regulating inflammation and/or cholesterol homeostasis in different cell types in lesions (76,77). MiR-33a and MiR-33b promote atherosclerosis by impairing cholesterol efflux and promoting inflammatory M1 macrophage conversion (78-80).Other microRNAs including MiR-223 and MiR-93 exhibit atheroprotective effects by increasing cholesterol efflux and conversion to the anti-inflammatory M2 macrophage phenotype (76,81-83). HDL carry small non-coding RNAs (77), which can also reduce or promote atherosclerosis development depending upon composition of individual non-coding RNAs (see details below).

 

Although macrophages are the main infiltrating cells, other cells contribute to the development of lesions including dendritic cells (84,85), mast cells, T cells, and B cells (Figure 1) (86,87). Dendritic cells promote the priming of reactive T cell clones and secrete cytokines, functioning in a largely pro-inflammatory capacity(88). They also take up lipid, which leads to inflammasome activation and increased pro-inflammatory cytokine secretion (89). Mast cells produce interferon-g(IFNg) and IL-6 and appear to promote lesion development(90). Atherosclerotic plaques also contain a significant number of adaptive immune cells, including T and B lymphocytes. The role of T cells is subset-dependent and atherosclerotic plaques have been shown to contain CD4⁺and CD8⁺effector T cells as well as T helper 1 (Th-1), Th-2, Th-17, and regulatory T (T-reg) cells. Antigen-specific Th-1 cells produce IFNgthat converts macrophages to a proinflammatory M1 phenotype. Th-17 cells have also been identified in atherosclerotic plaques and have been shown to produce IFNg. However, their specific role in atherosclerosis has not yet been elucidated (91). Classical T-reg cells produce anti-inflammatory cytokines (TGF-β and IL-10) and inhibit activation of Th-1 cells, leading to more anti-inflammatory M2 macrophages. As atherosclerosis progresses, T effector cell numbers increase or remain constant, while T-reg numbers decline. This reduction in T-regs is due in part to their heightened susceptibility to cell death as well as their impaired trafficking into lesions (91). Further, T-regs may appear fewer in number because they undergo phenotypic switching into other T-reg subtypes. Several subclasses of these ‘former’ T-regs have been identified in the atherosclerotic lesions of mice, including Th1-Tregs (CD4⁺CCR5⁺IFN-g⁺FoxP3⁺T-bet⁺) and T follicular helper cells (CXCR5⁺PD1⁺Bcl6⁺CD62L^loCD44^hiCD4⁺Foxp3^-), and these have been shown to have both impaired regulatory and enhanced inflammatory function, therefore contributing to atheroprogression (91,92). B cells preferentially reside in the adventitial layer of arteries neighboring sites of plaque, in regions known as tertiary lymphoid organs (TLOs). The function of B lymphocytes is also subset dependent, with B-1 cells being atheroprotective and B-2 cells being atherogenic. B-1 cells undergo limited or no affinity maturation and produce natural antibodies (NAbs) that have broad specificity and low binding affinity. Among these are NAbs, found within atherosclerotic plaques, that can bind to oxidation motifs in LDL and block the uptake of oxLDL by macrophages (93). Mice engineered to overexpress a single-chain variable fragment of E06, an IgM NAb directed against oxidized phospholipids (oxPL), were found to have reduced atherosclerosis and features consistent with greater overall plaque stability, confirming the atheroprotective nature of these B-1 cell-derived antibodies (94). B-2 cells produce high-affinity IgA, IgE and IgG antibodies. While the role of IgA in atherosclerosis remains controversial, IgG and IgE are atherogenic. IgG forms immune complexes with oxLDL and promotes an inflammatory macrophage phenotype while IgE also stimulates macrophages and mast cells to produce proatherogenic cytokines (95).

 

ApoE in Atherosclerosis

 

In addition to apoA-I and HDL, the endogenous production of apoE by macrophages is critical in preventing atherosclerotic lesion formation. The majority of apoE in plasma is produced by the liver, but macrophages are responsible for producing 5 -10% of apoE in plasma (96). ApoE serves as the ligand for clearance of all of the apoB containing lipoproteins from the blood by the liver except for LDL. Gene knockout of apoE in mice results in hypercholesterolemia and spontaneous atherosclerotic lesion development (97,98). Hence, ApoE deficient mice have been used widely to study mechanisms of atherosclerotic lesion development. Bone marrow transplantation studies were used to examine the role of macrophage apoE in lipoprotein metabolism[Linton, 1998 #5676]. Transplantation of Apoe^-/-mice with wildtype bone marrow, resulted in normalization of plasma cholesterol levels and protection from atherosclerosis (99), demonstrating the ability of macrophage apoE to exchange between lipoproteins and to serve as a vehicle for cellular gene therapy of atherosclerosis. Furthermore, reconstitution of wildtype (100)or LDL receptor deficient mice(Ldlr^-/-)(101)with Apoe^-/-bone marrow accelerates atherosclerotic lesion development without affecting plasma cholesterol levels, demonstrating an atheroprotective role for macrophage apoE. Interestingly, ApoE protects against atherosclerosis via several mechanisms. Expression of apoE by hematopoietic stem cells reduces monocyte proliferation and infiltration into the intima (102). In addition, apoE on apoB lipoproteins reduces the lysosomal accumulation of cholesterol by enhancing the expression of acid lipase (103). Importantly, secretion of apoE by macrophages stimulates efflux in the absence and presence of exogenous acceptors, including HDL and lipid-free apoA-I (Figure 2) (104-107). Recent studies demonstrated that macrophage apoE facilitates reverse cholesterol transport in vivo(108). Macrophage apoE stimulates phospholipid and cholesterol efflux via ABCA1, and the apoE particles formed then promotecholesterol efflux through ABCG1, SR-BI, and aqueous diffusion (104,109-111). Endogenous apoE is required for efficient formation of the most buoyant, cholesterol-enriched particles by macrophages (Figure 2) (104,112-116). In addition to cholesterol efflux, macrophage apoE prevents inflammation (117-120)and oxidative stress (121-124). The local production of apoE is likely a critical atheroprotective mechanism considering that areas of atherosclerotic lesions have limitedaccessibilityto plasma apoA-1 and HDL (100,101,125). Humans express three common apoE polymorphisms that predict CAD rates independently from plasma cholesterol levels (126). ApoE3 (C112, R158) is the most common isoform and is functionally similar to mouse apoE. Compared to apoE3 and apoE2 (C112, C158), apoE4 (R112, R158) are impaired in stimulating cholesterol efflux (127-130)and in preventing inflammation and oxidation (117,124,131). Consistent with the compromised function of apoE4, human carriers exhibit increased risk of CAD compared to humans expressing apoE3 or apoE2 (heterozygous) (126,132,133).

 

Progression to Advanced Atherosclerotic Lesions

 

Fatty streaks do not result in clinical complications and can even undergo regression. However, once smooth muscle cells infiltrate, and the lesions become more advanced, regression is less likely to occur (134,135). Small populations of vascular smooth muscle cells (VSMCs) already present in the intima proliferate in response to growth factors produced by inflammatory macrophages (136). In addition, macrophage-derived chemoattractants cause tunica media smooth muscle cells to migrate into the intima and proliferate (Figure 3). Critical smooth muscle cell chemoattractants and growth factors include PDGF isoforms, (137)matrix metalloproteinases, (138)fibroblast growth factors, (139)and heparin-binding epidermal growth factor (Figure 3) (140). HDL prevents smooth muscle cell chemokine production and proliferation. The accumulating VSMCs produce a complex extracellular matrix composed of collagen, proteoglycans, and elastin to form a fibrous cap over a core comprised of foam cells (Figure 4) (141). A vital component of the fibrous cap is collagen, and macrophage-derived TGF-bstimulates its production (Figure 4) (142). In addition, HDL maintains plaque stability by inhibiting degradation of the fibrous cap extracellular matrix through its anti-elastase activity (143).A subset of VSMCs accumulates CE and resides in the lesion core (Figures 3 and 4). This smooth muscle cell phenotype produces less a-actin and expresses macrophage markers, including CD68, F4/80 and Mac2 (144-146). While studies have shown that VSMCs express the VLDL receptor and various scavenger receptors, (145,147,148)data showing that these cells robustly load with CE, (147)similar to macrophages via these mechanisms is lacking. As lesions advance, substantial extracellular lipid accumulates in the core, in part due to large CE-rich particles arising from dead macrophage foam cells (149,150). Earlier in vitrostudies showed that these CE-rich particles effectively cholesterol load VSMCs (151,152). Regardless of the mechanisms of cholesterol enrichment, VSMCs compared to macrophages are inefficient at lysosomal processing and trafficking of cholesterol (152,153)and express much less ABCA1(154), which all contribute to impaired cholesterol efflux (155). However, macrophages in more advanced plaques also have reduced lysosome function and trapping of free and esterified cholesterol within their lysosomes contributes to the overall sterol accumulation in the lesion (156-158). The reduced lysosome function appears multifactorial but includes direct and indirect inhibition of lysosomal acid lipase, the enzyme responsible for hydrolysis of cholesteryl esters in lysosomes, and a reduced capacity for transferring cholesterol from lysosomes (159-162). In cell culture models of human macrophage foam cells, the inability to clear cholesterol from macrophages with compromised lysosome function continues even in the presence of compounds that stimulate efflux (161,163). Proteomic analysis of foam cells shows that changes in a number of lysosome proteases are related to macrophage sterol accumulation (164). Thus, at least in the advanced stages of atherosclerosis, lysosome dysfunction contributes to the overall lesion severity. As the intimal volume enlarges due to accumulating cells, there is vascular remodeling to lessen protrusion of the lesion into the lumen (Figure 4), thereby decreasing occlusion and the appearance of clinical symptoms for much of the life of the lesion (165-167).

Vulnerable Plaque Formation and Rupture

 

The advanced atherosclerotic lesion is essentially a nonresolving inflammatory condition leading to formation of the vulnerable plaque, increasing the risk of plaque rupture. The vulnerable plaque is characterized by two fundamental morphological changes: 1) Formation of a necrotic core and 2) Thinning of the fibrous cap. Sections of the atheroma with a deteriorated fibrous cap are subject to rupture (Figures 4 and 5) (168,169). A recent lipidomics study showed that stable versus unstable plaques have different lipid subspecies profiles (170). Compared to plasma and control arteries, stable plaques have increased CE containing polyunsaturated fatty acids (170), which have increased susceptibility to oxidation. The CE containing polyunsaturated fatty acids are decreased in unstable plaques compared to stable plaques of the same subjects (170). In addition, 18:0 containing lysophosphatidylcholine is increased in unstable plaques indicating enhanced oxidation (170). Plaque rupture leads to acute exposure of procoagulant and prothrombotic factors from the necrotic core of the lesion to platelets and procoagulant factors in the lumen, thereby causing thrombus formation (Figure 5) (168,169). Thrombus formation at sites of plaque rupture accounts for the majority of clinical events with acute occlusive luminal thrombosis causing myocardial infarction, unstable angina, sudden cardiac death, and stroke (168,169).

Macrophage Cell Death and Efferocytosis Influence Plaque Stability

 

The necrotic core results from a combination of accelerated macrophage death and impaired efferocytosis (receptor-mediated phagocytosis of apoptotic cells) (Figure 5) (171,172). As apoptotic cells accumulate and fail to be internalized by phagocytes, they undergo secondary necrotic death leading to the leakage of intracellular oxidative and inflammatory components, which then propagate more inflammation, oxidative stress, and death in neighboring cells (Figure 5) (173). Multiple triggers likely occur in lesions to accelerate macrophage death, including oxidative stress, death receptor activation, and nutrient deprivation (174). Prolonged ER stress and activation of the unfolded protein response (UPR) contribute to macrophage apoptosis as substantiated by studies showing that apoptosis and the UPR effector, CHOP, increase with each stage of atherosclerosis in humans, but the largest increase is observed in the vulnerable plaque (175). In diabetes and obesity, accelerated formation of an enlarged necrotic core is likely instigated by defective macrophage insulin signaling (176)and saturated fatty acids (177,178), which are potent inducers of ER stress. In addition, other triggers act in tandem with ER stress to accelerate apoptosis. In particular, activation of toll-like receptors (TLR) (TLR2 and TLR4) and scavenger receptors (CD36 and SR-A) by oxidized phospholipids induces apoptotic signaling (178-181). Death is also accelerated by simultaneous suppression of survival pathways such as pAkt and NF‐κB via these same receptors. Accelerated apoptotic macrophage death is not sufficient to promote necrosis. Apoptotic cells undergo secondary necrotic death if they are not internalized by phagocyte efferocytosis receptors. Necrotic death leads to the leakage of intracellular oxidative and inflammatory components, which then propagate more inflammation, oxidative stress, and death in neighboring cells (Figure 5) (173). The presence of necrotic tissue together with apoptosis is consistent with defective efferocytosis in human plaques. Studies have shown that the majority of apoptotic cells are free in advanced human lesions, whereas in tonsils apoptotic cells are macrophage-associated (182). Efferocytosis also becomes defective in advanced atherosclerosis through several different mechanisms. First, accumulating evidence has shown that the expression and function of key efferocytosis receptors,MerTK (183),  LRP1 (184), and SR-BI (185)are impaired in advanced atherosclerosis. These receptors recognize apoptotic cell ligands such as phosphatidylserine (185,186). and efferocytosis efficiency is enhanced by bridging molecules such as apoE and MFG-E8 that interact with efferocytosis receptors to enhance their efficiency and also have reduced expression in advanced lesions (186-190). Compared to apoE3, apoE4 is defective at facilitating efferocytosis of apoptotic cells (191). Efferocytosis via LRP1 (187)and SR-BI (185)also stimulates signaling pathways leading to pAkt production to promote phagocyte survival. In addition, anti-inflammatory signaling (185,192)is activated so that phagocytes secrete TGF-β and IL-10 (Figures 4 and 5). In addition, efferocytosis may be limited by competition for apoptotic cell binding. For example, oxPLs bind efferocytosis receptors and effectively compete for apoptotic cell recognition. In addition, lesional autoantibodies to oxPL and oxLDL are able to bind to ligands on the apoptotic cell themselves in order to prevent their binding and ingestion. Finally, apoptotic cells in advanced lesions appear to become poor substrates for efferocytosis. CD47, which typically acts as a “don’t eat me” signal expressed by live cells, is upregulated by apoptotic cells within human and murine atherosclerotic plaques, allowing them to evade uptake by phagocytes. When given to atheroprone Apoe^-/-mice, a CD47-blocking antibody enhanced lesional efferocytosis and resulted in smaller necrotic cores(193). Similarly, mice that express low levels of the “eat me” signal, calreticulin, have increased necrotic cores compared to control mice and apoptotic cells from these mice demonstrate resistance to uptake by phagocytes (194).

 

Components of the necrotic core promote thinning of the fibrous cap. Loss of extracellular matrix is in part due to death of fibrous cap smooth muscle cells, resulting from macrophage-derived Fas receptor ligand (195), inflammatory cytokines (196), and oxidation products (Figure 5) (197,198). Smooth muscle cells are inefficient at efferocytosis (199)relying on macrophages to internalize apoptotic smooth muscle cells. As such, the impaired efferocytosis by lesional macrophages likely leads to uncontrolled VSMC death (Figure 5). In addition, impaired production of TGF-β by phagocytes (185,200)reduces collagen production by healthy smooth muscle cells (Figures 4 and 5). The extracellular matrix components are degraded by macrophage-derived matrix metalloproteinases, (201-203)elastases, and cathepsins (Figure 5) (204). HDL can reduce VSMC apoptosis and elastin degradation induced by elastases (143,205).

 

Importantly, HDL can prevent efferocyte apoptosis via ER stress by its cholesterol efflux and anti-oxidant functions (179,206,207). Furthermore, HDL drives conversion to the anti-inflammatory M2 macrophages which have enhanced efferocytosis ability compared to inflammatory M1 macrophages (56,208)leading to increased plaque stability. Once plaque rupture occurs, critical HDL functions may also include prevention of platelet activation and thrombus formation. In addition to the role of HDL in stabilizing plaques, recent studies have focused on the lesional loss of specialized proresolving mediators (SPM) versus proinflammatory factors (i.e. leukotriene B₄)in promoting uncontrolled inflammation and formation of vulnerable plaques (209). Studies on human atherosclerotic lesions have shown that unstable versus stable plaques have decreased lipid-derived SPM including resolvin D1 and lipoxin A₄ (210). In addition, resolving D1 treatment ofLdlr^-/-mice with established atherosclerosis increased lesional efferocytosis and collagen content and reduced the necrotic area and reactive oxygen species content (210). Similar results were observed in Apoe^-/-micetreated with the phospholipase D derived proresolving lipid, palmitoylethanolamide(211).Other lipid derived resolving mediators which impact atherosclerotic plaques include maresin 1 and resolvin D2 (212). Protein SPM have also been identified including annexin 1 and IL-10 (209). Administration of lesion targeting nanoparticles containing the bioactive annexin 1 peptide, Ac2-26, to Ldlr^-/-mice with atherosclerosis reduced both lesional oxidative stress and necrosis while increasing collagen content and fibrous cap thickness (213). Enhancing the lesional IL-10 content also improved atherosclerotic lesion stability (214). In addition, Treg cells likely control atherosclerotic lesion inflammation resolution as recent studies demonstrated that Treg cells regulate efferocytosis in atherosclerotic lesions by secreting IL-13 to stimulate macrophage production of IL-10 to induce Vav-1 activation of Rac1 and increased efferocytosis (215).

 

Summary

 

Atherosclerotic lesions initiate with endothelial cell dysfunction causing modification of apoB containing lipoproteins (LDL, VLDL, remnants) and infiltration of immune cells, particularly monocytes, into the subendothelial space (Figure 1). The macrophages internalize the retained apoB containing lipoproteins to become foam cells forming the fatty streak (Figure 1). Macrophage inflammatory pathways are also activated leading to increased oxidative stress and enhanced cytokine/chemokine secretion, causing more LDL/remnant oxidation, endothelial cell activation, monocyte recruitment, and foam cell formation (Figure 1). HDL, apoA-I, and endogenous apoE reduce lesion formation by preventing endothelial cell activation, inflammation, and oxidative stress and also by promoting cholesterol efflux from foam cells. As the lesion progresses to fibrotic plaques as a result of continued inflammation, macrophage chemoattractants stimulate infiltration and proliferation of smooth muscle cells (Figure 3). Smooth muscle cells produce the extracellular matrix providing a stable fibrous barrier between plaque prothrombotic factors and platelets (Figure 4). Unresolved inflammation results in formation of vulnerable plaques, which have large necrotic cores and a thinning fibrous cap (Figure 5). Enhanced macrophage apoptosis and defective efferocytosis of apoptotic cells results in necrotic cell death causing heightened inflammation leading to increased smooth cell death, decreased extracellular matrix production, and collagen degradation by macrophage proteases. An imbalance between inflammatory factors and SPMs is prominent in facilitating formation of the vulnerable plaque.  Rupture of the thinning fibrous cap promotes thrombus formation resulting in clinical ischemic cardiovascular events (Figure 5).

 

 

 

THE ROLE OF CHOLESTEROL AND LIPOPROTEINS IN ATHEROGENESIS

 

Metabolism of ApoB Containing Lipoproteins

 

Apolipoprotein B (apoB) occurs in two isoforms, apoB100 and apoB48. ApoB100 is the main structural apolipoprotein of low-density lipoproteins (LDL), and there is only one molecule of apoB100 per LDL particle (216). ApoB100 is produced mainly by the liver, where it is required for the synthesis and secretion of triglyceride-rich very low-density lipoprotein (VLDL) particles (Figure 6). In the circulation, VLDL is metabolized to the cholesteryl ester-enriched intermediate low-density lipoprotein (IDL) and LDL particles through the progressive hydrolysis of triglycerides by lipoprotein lipase (LPL) and hepatic lipase (Figure 6). In humans, apoB48 is produced exclusively in the intestine through an unique RNA editing mechanism by the apobec-1 enzyme complex (217). ApoB100 is the full-length protein, which contains 4536 amino acids, whereas apoB48 contains the first 48% of the amino terminal amino acids. ApoB48 is required for the synthesis and secretion of triglyceride-rich chylomicrons, which play a critical role in the intestinal absorption of dietary fats and fat-soluble vitamins. Similar to the metabolism of VLDL, chylomicrons are metabolized in the circulation through the hydrolysis of triglycerides by LPL and hepatic lipase to form cholesteryl ester-enriched chylomicron remnants, which release free fatty acids that can be used for energy by the tissues.

Static measurements of cholesterol in the LDL pool (LDL-C) represent the steady state of production of VLDL, its metabolism to LDL, and the receptor-mediated clearance of LDL by the LDL receptor (LDLR). Mutations in the Ldlrgene are the most common cause of familial hypercholesterolemia (FH), an autosomal dominant disorder associated with elevated levels of LDL-C and increased risk for premature cardiovascular disease (218). ApoB100 serves as the ligand for receptor-mediated clearance of LDL by the liver (Figure 6). In contrast, apoE mediates the clearance of triglyceride-rich remnants (IDL and chylomicron remnants) either through the LDLR or the remnant receptor pathway (Figure 6). The existence of the remnant receptor pathway was suggested by the fact that patients with homozygous FH, who completely lack LDLR function, have severely elevated levels of LDL-C but normal blood levels of triglycerides. The clearance of these remnant lipoproteins involves binding to heparin sulfate proteoglycans and the LDLR like protein -1 (LRP1) in the hepatic space of Disse, in a process called secretion capture that requires local enrichment by hepatic expression of apoE

(96,219).

 

The Cholesterol Hypothesis

 

Studies by Anitschkow showing that feeding cholesterol in oil to rabbits caused the formation of atheroma, similar to those seen in humans, demonstrated a causal role of cholesterol in the pathogenesis of atherosclerosis in 1913 (220). In 1939, Muller described families with inherited high cholesterol and increased risk for cardiovascular disease (221). Yet it would take several decades before compelling evidence from epidemiological studies, such as Framingham (221)and MRFIT (222), demonstrated that elevated blood cholesterol levels were associated with increased risk of cardiovascular events (CVE). Subsequently, LDL-C levels were found to be directly associated with CVE (223); whereas HDL-C levels were shown to be inversely related to risk of CVE (224). The Seven Countries Studies by Ancel Keys showed that coronary heart disease (CHD) mortality rates were higher in countries with higher blood levels of cholesterol (e.g. Finland, Norway, and the USA) than in countries of southern Europe and Japan with lower blood levels of cholesterol (225). The high levels of cholesterol were proposed to be associated with the amount of saturated fat in the diet. As such, the cholesterol hypothesis was born, proposing that lowering LDL-C would reduce CVE(226).

 

Response to Retention Hypothesis for the Initiation of Atherosclerosis

 

The response to retention hypothesis holds that retention of atherogenic lipoproteins in the artery wall is a critical initiating event that sparks an inflammatory response and promotes the development of atherosclerosis (Figure 1). First articulated in 1995 by Williams and Tabas (227), the hypothesis was based on more than two decades of work demonstrating that apoB-containing lipoproteins are retained in the artery wall by interaction with proteoglycans (228,229). Proteoglycans consist of a protein core bound covalently to one or more glycosaminoglycans (GAGs). The most common proteoglycans in the artery wall are decorin, biglycan, perlecan, versican, and syndecan-4 (230). There is ionic binding between the positively charged GAGs and negatively charged amino acids of apoB100 (229). Boren et al.identified the principal proteoglycan-binding site in LDL and showed that a single point mutation in apoB100 impaired binding to proteoglycans (231). The major proteoglycan binding site consists of residues 3359-3369 in apoB100 (site B), which is in the C-terminal half of apoB100. Furthermore, mutation of “site B” in mice resulted in reduced retention of apoB100 in the artery wall and reduced atherosclerosis, providing in vivosupport for the response to retention hypothesis (232). Subsequently, proteoglycan binding sites were identified for apoB48 (233)and a second site (site A) on apoB100, which is exposed when LDL is modified by secreted phospholipase A2 (sPLA2), forming a small dense LDL particle (234).

 

Surprisingly, native LDL, despite the strong evidence for its critical role in promoting atherosclerosis, does not induce macrophage foam cell formation or much in the way of inflammation in vitro. These observations led to the hypothesis that LDL has to be modified to promote foam cell formation and induce inflammation. Binding of proteoglycans induces structural changes in LDL impacting both the configuration of apoB100 and the lipid composition (234). Hence, the binding of LDL to proteoglycans makes the LDL more susceptible to oxidation and aggregation, which promotes foam cell formation and a proinflammatory response, and the process is self-perpetuating. Oxidized LDL (oxLDL) can induce further production of proteoglycans by vascular smooth muscle cells, retaining more LDL in the arterial wall. Furthermore, macrophages express LPL, which can serve as bridging molecules, binding both lipoproteins and proteoglycans (235,236). Consistent with an important role for LPL in atherogenesis, the loss of macrophage LPL expression protects mice from atherosclerosis (237,238). In addition, macrophages secrete sphingomyelinase, which has been reported to act synergistically with LPL to promote binding of LDL and lipoprotein (a) (Lp(a)) to vascular smooth muscle cells (VSMC) and the extracellular matrix promoting their retention in the artery (239,240). Furthermore, sphingomyelinase induces aggregation and fusion of LDL particles, promoting increased binding to proteoglycans and induces foam cell formation (241). Thus, interfering with the retention of apoB-containing lipoproteins in the artery wall is a potential strategy for preventing atherosclerosis.

 

OXIDATION OF PHOSPHOLIPIDS AND PROTEINS IN LIPOPROTEINS AND THEIR ROLE IN ATHEROSCLEROSIS

 

Overview

 

The response to retention hypothesis for the initiation of atherosclerosis posits that retention of LDL in the artery wall leads to its modification into highly atherogenic particles that initiate inflammatory responses. A key overall point is that retention of LDL leads to oxidative modification of LDL, allowing this oxidized LDL (oxLDL) to be recognized by scavenger receptors on macrophages and other cells. Uptake of oxLDL by macrophages leads to marked accumulation of cholesterol, converting them to foam cells and initiating development of atherosclerotic lesions. In addition to serving as a substrate for cholesterol accumulation, oxLDL exerts a wide range of bioactivities that are consistent with it being critical for driving atherogenesis (Table 1). In mouse models, loss of enzymes that modulate LDL oxidation increases atherosclerosis, and dietary antioxidants that reduce levels of oxLDL also inhibit atherosclerosis. Although human trials with dietary antioxidants have failed to reduce disease outcomes, it is important to recognize that these interventions are less efficacious in reducing oxLDL levels in humans than in rodent models. Additional studies are needed to determine optimal interventions for lowering oxLDL levels and whether such interventions will be effective for preventing or treating atherosclerosis.

 

Table 1-Potential Atherogenic Activities of Oxidized LDL (oxLDL)
Macrophages	Smooth muscle cells
Serves as ligand for recognition by scavenger receptors 256, 257, 258	Induces proliferation, migration, and transition to inflammatory phenotype 276, 277, 278, 279
Serves as substrate for unregulated cholesterol uptake 262
Induces expression and secretion of inflammatory cytokines 280, 281, 282, 283	Lymphocytes
Induces polarization to M1 (minimally oxidized LDL) or M2-phenotype (highly oxidized LDL) 284	Serves as a neo-antigen 274
Inhibits egress from atherosclerotic lesions 289	Induces chemotaxis 275
Induces macrophage apoptosis and rupture of atherosclerotic plaques 290, 291, 292	Increases antibody production 275
	Other cell types
Endothelial cells	Induces chemotaxis of monocytes, PMN, and eosinophils 285, 286, 287, 288
Induces surface expression of adhesion molecules 266, 268, 269, 270	Increases platelet aggregation 293, 294, 295, 296
Induces inflammatory genes including cytokine release 271, 272	Activates dendritic cells and induces their release of T cells stimulating cytokines 284

 

Peroxidation of Polyunsaturated Fatty Acids Generates Oxidatively Modified Lipoproteins

 

The outer shell of lipoproteins is composed of phospholipids with polyunsaturated fatty acid (PUFA) side chains. These PUFAs (and to a lesser extent the PUFAs of cholesterol esters and triglycerides in the lipoprotein core) are highly vulnerable to oxidation by free radical species, particularly hydroxyl radicals (^●OH). This vulnerability results from the relatively low energy required for free radicals to abstract hydrogen atoms located between two adjacent double bonds (bis-allelic hydrogens). Hydrogen abstraction by free radicals creates a lipid radical that reacts nearly instantaneously with any molecular oxygen present in the environment.

 

The resulting lipid peroxide radical (LOO^●) can then propagate the radical reaction by abstracting hydrogens from neighboring phospholipids or can react with itself to create a large number of secondary peroxidation products (Figure 7). Secondary products that may be relevant to atherogenesis can be thought of in two broad classes: oxidized lipids (primarily oxidized phospholipids but also oxidized cholesterol esters) and reactive lipid aldehydes that exert their effects by modifying proteins and other macromolecules. Oxidized phospholipids (oxPL) include chain shortened oxPL such as 1-palmitoyl-2-oxovaleroyl-sn-glycero-3-phosphorylcholine (POVPC)(242), 1-O-alkyl-2-azelaoyl-sn-glycero-3-phophorylcholine (azPAF) (243), and 1-(Palmitoyl)-2-(5-keto-6-octene-dioyl) phosphatidylcholine (KOdiA-PC)  and cyclized oxPL such as  1-palmitoyl-2-(5,6)-epoxyisoprostane E2-sn-glycero-3-phosphocholine (PEIPC) (244)and 1-palmitoyl-2-F2-isoprostane-sn-glycero-3-phosphocholine (F2IsoP-PC) (245). Reactive lipid species include malondialdehyde  (246), 4-hydroxynonenal (246), and isolevuglandins (247)that modify proteins associated with lipoprotein particles including ApoB100 (Figure 7).

It is critical to keep in mind that oxidatively modified LDLs (oxLDLs) are in fact highly heterogeneous and complex particles, even though oxLDL is usually referred to as a discrete entity. Oxidation of LDL in vitro has been used extensively to study the biological activities of oxLDL, but, even here, the actual species present varies significantly based on the oxidation method (exposure to air, to copper, or to oxidases) and length of oxidation. Many of the methods commonly used to measure the concentration of oxLDL in vivo only measure general characteristics of oxLDL. For instance, because the reaction of reactive lipid species with lysine residues of ApoB100 converts LDL from a positively charged particle to a negatively charged particle, oxLDL is often detected by increased mobility during agarose gel electrophoresis. Alternatively, oxLDL in plasma and other tissues can be quantified by the immunoreactivity of the natural IgM autoantibody E06. However, while E06 recognizes a variety of oxidized phosphatidylcholines, it does not necessarily recognize all oxPL equally. Therefore, equivalent E06 immunoreactivity does not necessarily mean exposure to identical oxLDL particles. While modification of LDL with malondialdehyde (MDA-LDL) (248)is often used as a model of oxLDL for bioactivity assays, modification of LDL by other reactive lipid species can exert unique effects from MDA-LDL, and MDA-LDL does not include any of the various oxPL species. Therefore, it is important to keep in mind that in vivo oxLDL is a mixture of many different compounds and that the atherogenic activities of oxLDL represent the net cellular responses to the full range of compounds present.

 

While oxLDL has been studied in greatest detail, all lipoproteins are vulnerable to oxidation at least in vitro, and this oxidative modification alters their biological activities in ways that may be atherogenic. The species of plasma lipoprotein that has the highest content of oxidized phospholipids (oxPL) depends on the species of oxPL under consideration. This suggests that not all oxPL are formed in situ on the lipoprotein where they are found and might instead be transferred from other lipoproteins or tissues. Lp(a) is the major carrier in plasma of oxPLs that are detected by E06 immunoreactivity (249)and these oxPLs associate with Lp(a) in preference to native LDL particles in human plasma (250). E06 immunoreactive oxPL generated in chemically oxidized LDL can rapidly transfer to Lp(a) (249), so the high content of these lipids in Lp(a) isolated from human plasma may be due either to direct oxidation of Lp(a) or by transfer of the oxPL from oxLDL to Lp(a). In LDL and Lp(a) isolated from human plasma, levels of MDA-modified lysine (based on E014 immunoreactivity) are higher in LDL than Lp(a), while E06 immunoreactivity is much greater for Lp(a) than for LDL (249). Because MDA-modified proteins do not readily transfer between particles, these findings suggest that oxidation initially occurs in LDL with subsequent transfer of oxPL to Lp(a). Thus, a physiological role has been proposed for Lp(a) in binding and transporting oxPL in the plasma (251). Unlike oxPL detected by E06 immunoreactivity that are highest in Lp(a), the F₂-IsoP-phospholipids forms of oxPL are highest in HDL (252). As with Lp(a), the high levels of these oxPLs in HDL may well be the result of transfer from other oxidized lipoproteins and tissues. Because oxidation is unlikely to occur in the circulation, the rate that oxPL are transferred from tissue to various plasma lipoproteins could potentially be an important determinant of the risk for atherosclerosis.

 

Significant correlations have been found between levels of oxLDL and extent of atherosclerosis in human patients. Measurement of oxLDL using E06 antibody showed that: 1) significant elevation of oxLDL in acute coronary syndromes (250), 2) treatment with a statin markedly reduced these levels (253), 3) oxLDL levels are higher in children with familial hypercholesterolemia compared to their siblings (254), and 4) oxLDL levels predict the presence and progression of atherosclerosis and symptomatic cardiovascular disease (255). Measurement of oxLDL using antibodies against MDA-LDL found that oxLDL were elevated in patients with coronary artery disease (CAD) (256), that elevated levels of oxLDL predicted future cardiac events in diabetic patients with CAD, that oxLDL were particularly elevated in patients with rheumatoid arthritis and CAD compared to either alone (257), and that treatment with fibrates decreased levels of oxLDL (258). Thus, there is a clear correlation between the presence of oxLDL and cardiovascular disease.

 

Mechanisms of Lipoprotein Oxidation In Vitro And In Vivo

 

The precise mechanisms that generate oxidized lipoproteins in vivo are still only partially understood. LDL circulating in the plasma appears to be protected from oxidation, both by dietary antioxidants such as vitamin E and C (259)and by protective enzymes including glutathione peroxidases (260,261), peroxiredoxins, PAF-acetylhydrolase (also known as lipoprotein-PLA2) (262,263), and paraoxonases (PON) (264,265). Penetration of LDL into the artery wall occurs at branch points in the aorta and other places with turbulent flow and shear stress. Retention of LDL in the intima, due to interactions with extracellular matrix such as chondroitin sulfate-rich proteoglycans, sequesters LDL away from the antioxidant environment of the plasma and exposes LDL to oxidation. A variety of oxidases and peroxidases generate strong oxidants that can readily oxidize LDL. These include myeloperoxidase (MPO) (266), xanthine oxidase (XO) (267), NADPH oxidases (NOXs) (268), and inducible nitric oxide synthase (iNOS) (269). Oxygenases such as lipoxygenases (LOX) have also been shown to oxidize LDL in vitro (270,271). The extent that each of these enzymes contributes to lipoprotein oxidation in vivo and thus to atherosclerosis remains to be fully elucidated, and there is much we do not understand about these individual processes. This is illustrated by studies on MPO and the 12/15-Lipoxygenase, the two enzymes most closely linked to lipoprotein oxidation.

 

MYELOPEROXIDASE (MPO)

 

MPO released from activated neutrophils (and to a lesser extent from monocytes/macrophages) can accumulate in the subintimal space of the artery wall(272), so neutrophil activation indirectly increases the chance for lipoprotein oxidation. Increased plasma levels of MPO correlate with increased levels of oxLDL in hypercholesterolemic children (273). Increased MPO blood levels also associate with increased risk for atherosclerosis (274-277)and polymorphisms in the MPO gene that lower MPO activity reduce the risk for atherosclerosis (278,279).

 

Incubation of lipoproteins with MPO generates oxidized phospholipids that serve as ligands for CD36(280). A putative binding site for MPO with the apoB of LDL has been identified (281), although further verification is needed. Of interest, MPO also associates with HDL via binding to ApoAI and PON1 in a ternary complex(282), so that the binding of MPO to HDL and subsequent generation of reactive oxygen species may account for the high levels of oxidized lipids carried by HDL. Association of MPO with HDL leads to modification of tyrosine 71 of paraoxonase, reducing PON1 activity (282). It also generates reactive lipid dicarbonyls such as isolevuglandins that modify ApoAI (283)and phosphatidylethanolamine (284). In the presence of small molecules that scavenge lipid dicarbonyls, the ability of MPO to crosslink ApoAI is markedly reduced (283).  Modification of HDL by lipid dicarbonyls such as isolevuglandins and MDA reduce its ability to drive cholesterol efflux from macrophages and protect against inflammatory stimuli such as LPS (283,285).

 

Mouse models have been used to directly examine the contribution of MPO to atherosclerosis, although these studies carry the caveat that mouse MPO levels are only 10-20% that of humans (286,287). Transplantation of bone marrow from genetically altered mice into atherosclerosis susceptible strains (e.g. Ldlr^-/- and Apoe^-/- mice) after lethal irradiation to ablate host hematopoietic cells is commonly used to study the effect of specific genes expressed by macrophages and other hematopoietic cells on atherogenesis (99). Reconstitution of Ldlr^-/- mice with macrophages overexpressing MPO markedly increased their susceptibility to atherosclerosis (288). However, transplantation of MPO^-/- macrophages into Ldlr^-/- mice, also markedly increased atherosclerosis, and this was confirmed in MPO^-/-/Ldlr^-/- double knockout mice compared to MPO^+/+/Ldlr^-/- controls (289). The reasons for these paradoxical findings with both MPO overexpression and deletion remain unclear. Perhaps the complete lack of MPO activity is harmful because it allows overgrowth of specific microbes that incite atherosclerosis via alternative mechanisms. In contrast to effects of complete ablation, a recently developed selective MPO inhibitor (e.g. INV315) that only partially reduces MPO activity markedly reduced atherosclerosis in Apoe^-/- mice (290). Thus, clinical studies with selective MPO inhibitors are needed to determine if this will be a meaningful therapeutic approach to the treatment of atherosclerosis in humans.

 

12/15-LIPOXYGENASES

 

Although the primary substrates for lipoxygenases are non-esterified fatty acids, exposure of LDL to 15-LOX also leads to oxidation of phospholipids and cholesterol esters (270,271). In mice, the gene analogous to the human 15-LOX encodes a lipoxygenase that converts arachidonyl chains to both 12-HPETE and 15-HPETE and is thus a 12/15-LOX. 12/15-LOX^-/-  mice on Apoe^-/-  background have reduced atherosclerosis compared to Apoe^-/- mice (291). Importantly, they also have lower levels of autoantibodies against oxLDL and MDA-LDL (291). These results support the notion that 12/15-LOX can directly contribute to atherosclerosis via LDL oxidation. Nevertheless, the role of 15-LOX in human atherogenesis is less clear-cut. While homozygotes of an Alox15 variant that almost completely ablates 15-LOX activity tended to have a reduced risk for coronary artery disease, heterozygotes paradoxically have increased risk of disease (292). Other polymorphisms in the Alox15 gene encoding 15-LOX increase risk for coronary artery calcification (293), yet others have no effect (294). Direct correlations between Alox15 polymorphisms and biochemical measurements of oxidized lipoproteins or oxPL and oxidized cholesterol esters have not been reported to date in humans, but are clearly needed.

 

Biological Activities of OxLDL And Receptors That Mediate These Activities

 

Perhaps the most important atherogenic effect of LDL oxidation is that this modification of LDL shifts recognition and internalization of the lipoprotein from the LDL receptor (LDLR) to scavenger receptors (295-297). While internalization of LDL by the LDLR in hepatocytes downregulates cholesterol synthesis to maintain cholesterol homeostasis, internalization of oxLDL by scavenger receptors fails to trigger this inhibition (298,299,300). Thus, cholesterol synthesis continues unabated despite the fact that peripheral cells are accumulating large amounts of cholesterol. In particular, macrophages express scavenger receptors and gluttonously take up large quantities oxLDL to form foam cells in the initial atherosclerotic lesion (301).

 

OxLDL also activates a number of cellular responses in macrophages, dendritic cells, endothelial cells, T cells, and smooth muscle cells that in aggregate promote inflammation, lesion formation, atherogenesis, and unstable atherosclerotic plaques (302-304). OxLDL induces surface expression of adhesion molecules and the release of chemokines from endothelial cells (305-311),  all of which are important steps in recruitment of leukocytes to sites of lesions. Exposure to oxLDL activates dendritic cells so that they induce T-cell proliferation and production of IL-17 (312). OxLDL itself also serves as a neo-antigen (313). OxLDL also induces increased antibody generation by lymphocytes (314). OxLDL also promotes smooth muscle cell proliferation, migration, and transition to a proinflammatory phenotype (315-318). OxLDL induces secretion by macrophages of inflammatory cytokines (e.g. TNF, IL-1, MCP-1, and IL-8) that activate other inflammatory cell types (319-322). OxLDL polarizes macrophages towards the M1-like phenotype or M2-like phenotype depending on its extent of oxidation (323). OxLDL promotes the chemotaxis of monocytes, neutrophils,  eosinophils, and T cells (314,324-327), bringing them into the arterial wall. In contrast, oxLDL inhibits macrophage emigration out of atherosclerotic lesions, because it induces netrin-1 (328). OxLDL induces apoptosis of macrophages and development of unstable plaques prone to rupture (329-331). Thrombotic arterial occlusion in the aftermath of plaque rupture is a critical cause of mortality, therefore the fact that oxLDL increases platelet aggregation (332-335)suggests an additional mechanism whereby elevated circulating oxLDL may increase risk of mortality during acute coronary events (336). As discussed in detail below, identification of cognate receptors for various components of oxLDL and other oxidized lipoproteins has provided important insight into the mechanisms by which these oxidized lipoproteins exert their pathophysiological effects.

 

MACROPHAGE SCAVENGER RECEPTOR (SR-AI)

 

In 1979, Brown and Goldstein demonstrated that macrophages had specific binding sites for acetylated LDL (AcLDL) that allowed uptake of this modified LDL even in the presence of high cellular cholesterol levels (298). This was in contrast to LDL uptake by the LDLR, which is markedly downregulated when cellular cholesterol levels rise (Figure 2). Cholesterol synthesis is also downregulated by LDL uptake by LDLR (337). The lack of feedback inhibition during uptake of modified LDL by this unidentified receptor suggested a plausible mechanism for the massive accumulation of cholesterol in macrophages that generates foam cells. The putative receptor mediating this binding was named the macrophage scavenger receptor (MSR). Later, oxLDL (338)and MDA-LDL (248)were shown to compete with AcLDL for binding and uptake by macrophages, suggesting they were native ligands for MSR. In 1990, Kodama et al. purified and sequenced this scavenger receptor, allowing identification of the MSRgene (339). Through alternative gene splicing, this gene gives rise to Scavenger Receptor A–I (SR-AI), SRA-II, and SRA-III.  Deletion of the MSR gene in C57BL6 mice fed butterfat diet substantially reduced atherosclerotic lesions and deletion of MSR in Ldlr^-/-mice also reduced lesion formation (340).

 

CD36 AND OTHER SCAVENGER RECEPTORS

 

Subsequent work has shown that in addition to SR-AI, macrophages express a wide range of scavenger receptors that recognize oxidized lipoproteins including MARCO, scavenger receptor-B1, -B2, -B3 (CD36), and Lectin-like oxLDL Receptor-1 (LOX-1) (341). These scavenger receptors belong to a larger family of pattern recognition receptors, all of which are individually capable of binding to a wide spectrum of ligands. Quantitatively, SR-A1 and CD36 account for the vast majority of all oxLDL uptake by macrophages (342). The specific ligands of the two receptors on oxLDL appear to diverge (342).  SR-AI appears to preferentially recognize more rigorously oxidized LDL and seems to primarily recognize modified lysine residues like MDA-lysines. In contrast, the primary ligands of CD36 on oxLDL appear to be oxidized phospholipids, in particular fragmented phosphatidylcholine including azPAF (243), POVPC (343)and KOdiA-PC (280). Apoe^-/- mice lacking CD36 are more vulnerable to some bacterial infections (344)but also have less atherosclerosis when fed a high cholesterol diet (345).

 

Recent findings suggest that SR-AI and other scavenger receptors have both pro- and anti-atherosclerotic effects, depending on the context. For instance, deletion of the MSR gene actually increased lesion size in male Apoe^-/-mice (346); however, deletion of both MSR and CD36 greatly reduces lesion complexity and vulnerable plaques, the most critical aspect of lesion development (347). The complex results of scavenger receptor deletion should not be surprising given that scavenger receptors have multiple ligands and that an important role of scavenger receptors expressed by macrophages is to allow these macrophages to remove bacteria and damaged cells from surrounding tissues. Under normal physiological conditions, uptake of oxLDL by macrophages is probably generally protective, because subsequent efflux of the cholesterol from the macrophages to HDL via reverse cholesterol transport as well as emigration of these macrophages from the arterial wall to lymph nodes serves to minimize the accumulation of cholesterol-laden macrophages in the arterial wall. However, under conditions where reverse cholesterol transport capacity is reduced or where emigration of macrophages is inhibited, uptake of oxLDL by macrophages leads to its accumulation and initiation of pathophysiological processes.

 

TOLL-LIKE RECEPTORS AND OTHER TARGETS OF OXLDL

 

In addition to scavenger receptors, other pattern recognition receptors also recognize components of oxLDL. Perhaps most important among these are the Toll-like Receptors (TLR) including TLR-2 (348,349), TLR-4 (350), TLR-6 (351), TLR-7 (352), and TLR-9 (352).  TLRs can interact with scavenger receptors, for instance, CD36 forms complexes with TLR4 and TLR6 that recognize oxLDL and activate NFkappaB (351).While bacterial components such as bacterial lipopolysaccharide (LPS) are full agonists for TLRs, oxLDL components like POVPC often appear to act functionally as partial agonists of TLRs, so that activation of macrophages and dendritic cells by full agonists like LPS is reduced in the presence of oxLDL (353,354).

 

In addition to TLRs, another important pattern recognition receptor for oxLDL is the receptor for advanced glycation end-products (RAGE) (355). Other factors of the innate immune response that bind oxidized phospholipids including C-reactive protein(CRP) (356,357)and natural IgM antibodies like E06 (358,359). While scavenger and pattern recognition receptors tend to recognize broad classes of compounds, a number of G-protein coupled receptors (GPCRs) recognize specific oxidized phospholipids. These include the receptor for platelet-activating factor (PAFR) (360-362), prostaglandin receptor EP2 (363,364), and sphingosine-1-phosphate receptor 1 (S1P1) (365). Intracellular receptors for oxidized phospholipids include nuclear hormone receptors PPAR alpha (366)and PPAR gamma (243). Non-receptor, intracellular targets for oxLDL include c-SRC (367)and NRF-2 (368,369).

 

Mechanisms Protecting Against LDL Oxidation In Vivo

 

Given the susceptibility of LDL to oxidation, it is perhaps not surprising that a number of mechanisms appear to exist in order to protect LDL from oxidation. These include small molecule antioxidants circulating in plasma and enzymes that catabolize oxidized lipids. How essential each of these mechanisms are to the control of oxLDL levels and preventing the development of atherosclerosis remains an area of active investigation. Obviously, a better understanding of the relationship between changes in protective mechanism and atherogenesis might allow identification of particularly vulnerable individuals and the development of novel therapeutic approaches.

 

SMALL MOLECUE ANTIOXIDANTS

 

Circulating small molecule antioxidants such as ascorbate (vitamin C), alpha-tocopherol (vitamin E), urate, and bilirubin serve as sacrificial targets reacting with free radicals and reactive oxygen species to prevent lipid and protein oxidation. Thus, even when strong oxidants are added to plasma ex vivo, there is relatively little generation of oxLDL until the oxidants have depleted these small molecule antioxidants, most specifically ascorbate (370). Depletion of vitamin C and vitamin E increase atherosclerosis in Apoe^-/-mice(371). Importantly, plasma ascorbate levels inversely correlate with prevalence of cardiovascular disease in humans (372). Supplementation with vitamin C appears to play a role in preventing endothelial dysfunction in humans (373). However, it is not clear that supplementing dietary antioxidants beyond those typically obtained in a well-balanced diet endows any additional atheroprotective effects. Supplementation with dietary antioxidants inhibits development of atherosclerosis in susceptible mice (374-378). While a few human trials with dietary antioxidants have demonstrated reduced atherosclerosis and cardiovascular disease (379-382), most large-scale trials have failed to demonstrate any disease reduction (383-387). The reasons underlying these failures continue to be investigated and debated (388,389). Because it had not been fully appreciated that relatively high doses of these antioxidants were needed to markedly alter lipid peroxidation rates in humans (390), one possibility is that the doses used in most large scale prevention trials were simply insufficient (390,391) . However, the ability to use very high doses of small molecule antioxidants like vitamin E for extended periods of times may be limited by the toxicity of these high doses (392).

 

ANTIOXIDANT ENZYMES

 

Antioxidant enzymes appear to play a more critical role than dietary antioxidants in limiting lipoprotein oxidation. Two families of nonheme peroxidases, the glutathione peroxidases and the peroxiredoxins, appear to be the most critical. Glutathione peroxidases (Gpx) 1-4 are selenoproteins that convert glutathione to glutathione disulfide while reducing peroxides (including lipid peroxides) to water (393,394). Polymorphisms in glutathione peroxidase 1 (Gpx1) are associated with increased risk for atherosclerosis in various human populations (395-397). Furthermore, genetic deletion of Gpx1 markedly exacerbates atherosclerosis in Apoe^-/- mice(398,399), while overexpression of Gpx4 in Apoe^-/-mice inhibits atherogenesis (400). Peroxiredoxins (Prdx) are cysteine containing proteins where the cysteine is oxidized to sulfenic acid during reduction of peroxides (401). Deletion of either Prdx1 or Prdx2 increases atherosclerosis in Apoe^-/- mice(402,403). Overexpression of Prdx4 inhibits atherosclerosis in Apoe^-/- mice (404). In contrast, overexpression of Prdx6 failed to inhibit atherosclerosis in C57BL6 mice fed an atherogenic diet (405).

 

In general, studies looking for associations between risk for atherosclerosis and polymorphisms or deficiencies in other major antioxidant genes including catalase, SOD-1, -2, and -3, and glutathione S-transferase have been negative (406,407). In fact, SOD-1 overexpression may even increase fatty streak lesions in mice (408). However, SOD-1 does inhibit proliferation and migration of smooth muscle cells induced by oxLDL in vitro   (315), and overexpression of both SOD-1 and catalase reduce atherosclerosis in Apoe^-/-mice (409). Sod2^+/-mice crossed with Apoe^-/-mice have increased atherosclerosis compared to control Apoe^-/-mice (410), but there is little effect on atherosclerosis of crossing Sod3^-/-mice with Apoe^-/-mice (411).  Several studies have demonstrated an association between SOD2and hypertriglyceridemia (412,413).

 

ENZYMES THAT CATABOLIZE LIPIDS

 

In addition to anti-oxidant enzymes, several enzymes specifically catabolize oxidized phospholipids including secreted Platelet-Activating Factor Hydrolases (sPAF-AH) and Paraoxonases (PON). sPAF-AH, also known as lipoprotein associated PLA2 (LP-PLA2) is a calcium independent PLA₂secreted by macrophages that primarily circulates on LDL and to a lesser extent on HDL (414,415). sPAF-AH does not hydrolyze phospholipids with the typical long-chain fatty acids, but efficiently cleaves phospholipids with oxidatively fragmented (e.g. azPAF and POVPC) (362,416,417)or oxidatively cyclized (e.g. F2-isoprostane-PC) sn-2 chains (418). Whether this effect results in a net gain of pro- or anti-inflammatory lipids is controversial, because only some of these oxPL are highly potent inflammatory mediators, while others are partial agonists that might therefore antagonize inflammatory responses to other mediators like LPS. Furthermore, this hydrolysis generates lysoPC and lysoPAF, which are proinflammatory at high concentrations. This ambivalent effect is also seen in vivo. While a large number of clinical studies have found that increased sPAF-AH predicts increased risk for atherosclerosis (419,420), whether increased sPAF-AH actually contributes to atherogenesis or simply reflects a compensatory increase in response to elevated oxLDL is unclear (421,422). Some gene polymorphisms in sPAF-AH that reduce its activity (i.e. Val279Phe) appear to increase the risk of myocardial infarction (423), yet another polymorphism (i.e. Ala379Val) appears to have little effect (424). The interpretation that increased sPAF-AH activity caused an increased risk of atherosclerotic cardiovascular disease (ASCVD) led to the development of selective sPAF-AH inhibitors and their clinical trials (425). However, two recently completed phase III trials with one such inhibitor, darapladib, found that while this drug significantly reduced circulating PAF-AH activity, it had no effect on ASCVD events (426,427).

 

Paraoxonases (PONs) were originally named for their ability to hydrolyze the neurotoxin paraoxon and this activity is still routinely used to assay paraoxonase activity in plasma. However, in terms of atherosclerosis, the most important physiological function of PONs appears to be their ability to protect against LDL oxidation (428). PON-1 and PON-3 circulate bound to HDL. HDL treated with specific inhibitors of PON fails to protect LDL from oxidation (429). Treatment of oxLDL with purified PON1 markedly decreases its ability to induce endothelial cell activation and monocyte binding (264). Genetic deletion of PON1 markedly increases atherosclerosis in C57BL6 mice(430), and this is further exacerbated in Apoe^-/-mice (431). Conversely, overexpression of PON-1 reduces atherosclerotic lesions in both wild-type mice fed high cholesterol diets and Apoe^-/- mice (432). Adenovirus expression of PON-2 and PON-3 also inhibits atherosclerosis in Apoe^-/-mice (433,434), indicating that all three PON enzymes have protective effects. However, transgenic Apoe^-/- mice overexpressing the entire gene cluster of PON genes (PON-1, -2, -3) were not further protected compared to Apoe^-/-mice with transgenic expression of PON-1 or PON-3 alone (435), suggesting these effects are redundant rather than additive. These mouse studies appear relevant to human disease, as a large number of studies have shown that polymorphisms in PON1 are associated with increased risk for atherosclerosis (265). It should be noted that PON activity varies greatly even in persons with the same polymorphism, suggesting that environmental factors leading to PON inactivation may also be important in determining disease risk.

 

Summary for Oxidized Lipoproteins

 

In summary, substantial evidence has accumulated over the past several decades for a causative role for oxidized lipoproteins in the initiation and progression of atherosclerosis and the need to reduce lipoprotein oxidation in order to reduce disease burden. Nevertheless, significant questions remain including which mechanisms are most important for driving lipoprotein oxidation, what treatment strategies can effectively reduce lipoprotein oxidation, and what are the key components of oxidized lipoproteins that drive atherogenesis?

 

ELEVATED LDL-C AND RISK FOR ASCVD

 

Genetic Causes of Elevated LDL-C

 

As described above, FH is an autosomal dominant inherited disorder associated with elevated levels of LDL-C and premature ASCVD, and provides some of the most compelling evidence for a causal role for LDL-C in atherosclerosis. Brown and Goldstein discovered the LDLR pathway and found that mutations in the Ldlrgene cause FH (300). Heterozygotes for loss-of-function mutations have cholesterol levels that are about twice normal, and these subjects are at increased risk of premature CVE. In contrast, individuals with homozygous FH have extremely high levels of LDL-C (> 500 mg/dL) and often develop severe coronary atherosclerosis and supravalvular aortic stenosis in early childhood. The prevalence of heterozygous FH is around 1/200-250 in the USA, whereas homozygous FH is extremely rare affecting only about 1/160,000 to 1/250,000 individuals (436). Nonetheless, about 20% of people having myocardial infarctions (MI) before 40 years of age have heterozygous FH. Thus, FH offers an important opportunity to target therapies to prevent atherosclerosis (437), but FH remains under recognized with recent evidence suggesting that only 1-10% of subjects with FH have been identified (438). Most individuals with significant hypercholesterolemia do not have classic monogenic autosomal dominant inherited dyslipidemias, but polygenic factors contributing to susceptibility to environmental factors underlie the observed increase in LDL-C levels.  A recent study suggests that among individuals with LDL cholesterol ≥190 mg/dl, gene sequencing identified a monogenic FH mutation in only <2%of subjects (439). However, for any observed LDL cholesterol, FH mutation carriers are at substantially increased risk for CAD (439). Pathogenic variants in three genes (LDLR, APOB, and PCSK9) account for the majority of monogenic FH cases. Recent genome-wide association studies (GWAS) have identified more than 50 discrete genetic loci that are associated with an increased risk of CVE(440,441). Many of these genetic loci are associated with genes previously known to impact LDL-C levels and cardiovascular risk (e.g. Ldlr, APOB, PCSK9), but novel loci that impact both LDL-C levels and risk for MI have also been identified, e.g. sortilin-1 (SORT1)(442,443). Most importantly, inherited low levels of LDL-C due to loss-of-function mutations in the PCSK9gene have been shown to be associated with dramatic reductions in risk for ASCVD events in the Atherosclerosis Risk in Communities study (444). Hence, genetic disorders of lipoprotein metabolism provide strong evidence that the impact of LDL-C on the development of atherosclerosis is dose- and time-dependent (445), supporting a causal role for LDL-C in atherosclerosis.

 

Lowering LDL-C Reduces ASCVD

 

Large randomized outcomes trials of cholesterol lowering drugs have provided critical proof of the cholesterol hypothesis (446). The Coronary Drug Project, conducted between 1966 and 1975, found niacin treatment showed modest benefit in decreasing definite nonfatal recurrent myocardial infarction by 26% (10.2% for niacin group vs 13.8% for placebo group) (447). However, there was no benefit in primary endpoint, total mortality. Impressively, with a mean follow-up of 15 years, nearly 9 years after termination of the trial, all-cause mortality was 11% lower in niacin group than in the placebo group (448). The Lipid Clinics Research trial was another early major outcomes trial to show that lowering cholesterol reduced cardiovascular events. Treatment with cholestyramine, a bile acid binding inhibitor, resulted in a 12% reduction in LDL-C levels and a 19% reduction in CHD events (449). The early lipid lowering cardiovascular outcomes trials were limited by a lack of highly effective approaches for lowering LDL-C levels, and several trials raised concerns that cholesterol lowering did not reduce total mortality and might increase the risk of cancer, accidental death and suicide (446). The advent of the statin drug class (HMG-CoA reductase inhibitors) provided a much more effective approach to lowering LDL-C and laid to rest the concerns raised by the earlier trials. The 4S trial was a landmark clinical trial of cholesterol lowering with simvastatin in patients with coronary artery disease (CAD) and severely elevated levels of LDL-C that was designed to look at total mortality as the primary endpoint (450). The 4S showed for the first time that lowering LDL-C levels by 35% with simvastatin resulted in a 30% reduction in total mortality with a 42% reduction in CHD deaths and a 34% reduction in the risk of Major Coronary Events (450). A large number of subsequent trials extended these results to populations with CHD with low levels of LDL-C and to subjects without known CAD (primary prevention) with high or low levels of LDL-C (451). It is important to note that the relationship between on-treatment LDL-C lowering and reduction in cardiovascular events in secondary prevention trials was similar for both statin and non-statin approaches to lowering LDL-C levels. A large meta-analysis of 26 statin trials involving over 170,000 subjects demonstrated that statin treatment for 5–years reduced the combined incidence of major coronary events, coronary revascularization, and stroke by 20% per every 1 mmol/l (38.7 mg/dL) reduction in LDL-C (452). These results have been extended by a recent large meta-analysis of 49 trials involving 9 different interventions to lower LDL that included more than 300,000 patients and approximately 40,000 major vascular events, each 1mmol/l (38.7mg/dl) reduction in LDL-C was associated with 23% relative reduction in the risk of major vascular events (453). This raised the question of whether further lipid lowering would be of additional benefit. With the recent development of proprotein convertase subtilisin/kexin type 9 (PCSK9) inhibitors, dramatic additional LDL lowering up to 50 -70% is now possible. In the FOURIER trial, patients with prior stable CAD who received the PCSK9 inhibitor, evolocumab, in combination with statin therapy achieved median LDL-C levels of 30 mg/dl. This was associated with a 15% reduction in the composite endpoint of cardiovascular death, MI, stroke, hospitalization for unstable angina or coronary revascularization (454). Similarly, ODYSSEY demonstrated that administration of alirocumab to acute coronary syndrome patients already on maximally tolerated statin therapy led to LDL-C values <50 mg/dl and was associated with a 15% reduction in the composite endpoint of death from coronary heart disease, nonfatal MI, ischemic stroke or unstable angina requiring hospitalization, and this benefit approached 24% in the subgroup of patients with initial LDL-C values >100 mg/dL (455). Together, the results of these PCSK9 trials reinforce the “lower is better” hypothesis.

 

Although statins are very effective in preventing CVE, many patients on statins do still have CVE, a phenomenon referred to as residual risk (456). This residual risk is likely attributable at least in part to inflammation. Indeed, definitive support for this hypothesis recently came from the Canakinumab Anti-inflammatory Thrombosis Outcome Study (CANTOS), where administration of an anti-IL1bantibody to patients with prior MI and elevated serum hsCRP successfully reduced recurrent CVE independent of lipid lowering (>90% of patients were receiving concurrent statin therapy) (457). A secondary analysis of the FOURIER trial also demonstrated that, while relative risk for the primary cardiovascular endpoint was consistent across groups, the absolute risk reduction with evolocumab was greatest in patients with elevated hsCRP (458). These results suggest that targeting both LDL and inflammation will provide the most robust strategy for lowering ASCVD risk.

 

Levels of LDL-C, ApoB-100, Non-HDL Cholesterol and LDL-P as Markers for ASCVD Risk.

 

Based on the strength of the direct association of LDL-C levels and risk for ASCVD, the guidelines for treatment of hypercholesterolemia have focused on LDL-C levels for risk assessment, stratification and treatment recommendations. Indeed, the terms LDL-C and LDL are often, though incorrectly, used interchangeably in practice. It is important to understand that LDL is a collection of particles defined by density (d = 1.019 – 1.063 g/ml) that are heterogeneous, consisting of a large variety of lipids and proteins (459). In addition, LDL particles vary in size and cholesterol content. The relationship between LDL-C levels and risk for ASCVD is “J-shaped”, and the predictive value of LDL-C levels is better at higher levels of LDL-C. Surprisingly, the majority of subjects presenting to the hospital with acute coronary artery syndrome do not have elevated levels of LDL-C, but tend to have low levels of HDL-C and elevated triglycerides (460). There has been tremendous interest in whether other measures of LDL, including subpopulations, apoB100, or particle number, might serve as a better predictors of CVE than quantifying LDL cholesterol content.

 

Groundbreaking studies by Krauss and co-workers (461)described two major patterns for LDL subpopulations based on size and density of the LDL particles. Pattern A is characterized by large buoyant LDL (lbLDL) particles, whereas Pattern B is associated with small dense LDL (sdLDL). Importantly, sdLDL is associated with increased triglyceride levels and low HDL-C, which is referred to as the lipid triad, a phenotype common in insulin resistance. Hence, Pattern B is commonly seen in subjects with obesity, metabolic syndrome and type 2 diabetes mellitus. A number of studies have reported that Pattern B is associated with an increased risk of CVE (462). Several different approaches have been used to characterize LDL phenotypes, including gradient gel electrophoresis, ultracentrifugation (sequential and vertical), ion mobility and nuclear magnetic resonance (NMR) (462,463). A number of mechanisms have been proposed to underlie the proatherogenic properties of sdLDL, including increased susceptibility of oxidation (464)and glycation (465), promoting arterial retention and increased macrophage foam cell formation. Cholesteryl ester transfer protein (CETP), which transfers CE from HDL to VLDL/LDL and triglycerides in the opposite direction, and hepatic lipase, which hydrolyses triglycerides, impacts the lipid composition and size of sdLDL. As such, increased levels of sdLDL have the potential to provide additional information regarding risk of CVE in individuals with normal LDL-C levels but elevated triglycerides and low HDL. Alternatively, it has been proposed that the real impact of sdLDL is due to increased LDL particle number.

 

Each LDL particle contains one molecule of apoB100, and the majority of apoB100 in plasma is on LDL particles (466). Hence, levels of apoB100 correlate directly with LDL particle (LDL-P) number. A large number of studies have shown that levels of apoB100 are superior markers of ASCVD risk compared to LDL-C (467). Because the mass of cholesterol in LDL particles varies, LDL-C levels will result in overestimation apoB levels and the number of LDL particles, when LDL particles are cholesterol-enriched (Figure 8) and underestimate apoB and LDL particle number when the particles are cholesterol depleted (Figure 8) (468).

Furthermore, all of the major atherogenic lipoproteins contain apoB (LDL, triglyceride rich remnants of VLDL, IDL, chylomicron remnants, and Lp(a)). LDL-C is routinely calculated using the Friedewald formula (LDL-C = TC – HDL-C – TG/5), but this formula is not accurate when serum TG levels are > 400 mg/dl. It has long been recognized that LDL-C underestimates risk of ASCVD in the setting of hypertriglyceridemia (467). Non-HDL cholesterol is the mass of cholesterol in all of the apoB-containing particles: Non-HDL-C = TC – HDL-C. The ATPIII guidelines recommended using Non-HDL-C to estimate risk of ASCVD, when TG > 200 mg/dL (469,470). A meta-analysis by Sniderman et al.found that Non-HDL-C was a slightly better marker of ASCVD risk than LDL-C, but apoB was far superior to Non-HDL-C (471). NMR spectroscopy is another way to measure LDL-P concentrations. Table 2 includes selected percentiles for mean levels for the various LDL-related markers from the Framingham Offspring Study (472). In an analysis of the Framingham Offspring Study, LDL-P determined by NMR was more strongly related to incident CVD events than LDL-C levels, and the ability of Non-HDL-C to predict risk was less than LDL-P, but better than LDL-C (473). In addition, they found that low LDL-P numbers were a better index of low CVD risk than low LDL-C (473). In contrast, an earlier meta-analysis from the Emerging Risk Factors Collaboration found LDL-C, Non-HDL and apoB to be equivalent markers of CVE (474). The lack of difference may relate to the population studied. When the LDL particles have normal cholesterol content, then LDL-C, Non-HDL and apoB are equivalent markers (Figure 8) of risk (471,473). Interestingly, data from the Multi-Ethnic Study of Atherosclerosis (MESA) demonstrated that when LDL-C and LDL-P are discordant (Figure 8), then LDL-P proves to be a better predictor of risk for incident CVD events than LDL-C (475).

 

Table 2. Equivalent Percentiles in the Framingham Offspring Study
Percentile %	LDL-C mg/dL	Non-HDL-C mg/dL	ApoB mg/dL	LDL-P nmol/L
2	70	83	54	720
20	100	119	78	1100
50	130	153	97	1440
80	160	187	118	1820
95	191	224	140	2210
Adapted from Contois JH, et al. Clinical Chemistry 2009; 55:407-419

 

For several decades the guidelines for treatment of hypercholesterolemia have focused on LDL-C levels both for risk stratification and as the principal target of therapy to prevent ASCVD. Indeed, therapeutic goals for LDL-C of < 100 mg/dL and < 70 mg/dL for subjects at high-risk and very high risk of CVE, respectively, were recommended by the 2004 update of the NCEP ATPIII, guidelines (469,470). The 2013 ACC/AHA guidelines for treatment of hypercholesterolemia abandoned these targets in favor of recommending the use of high-intensity statins in high risk individuals (476), there are numerous sets of guidelines that have maintained the recommendation for LDL-C targets, including those of the National Lipid Association (NLA) (477), the American Association for Clinical Endocrinologists (478), and the European Guidelines (479). The Canadian guidelines include targets for levels of apoB(480), and the NLA guidelines include targets for both LDL-C and Non-HDL-C(477). Table 2 includes the percentiles for mean levels for these various LDL markers from the Framingham Offspring Study. The levels of LDL-C shown in Table 2 closely coincide with levels that have been widely used in guidelines for lipid management for decision-making regarding levels at which to initiate therapy and goals of therapy. The recent NLA guidelines recommend using both LDL-C and Non-HDL-C as targets of therapy with only two sets of targets: LDL-C < 70 mg/dL and Non-HDL-C < 100 mg/dL for very high-risk subjects and LDL-C < 100 mg/dL and Non-HDL-C < 130 mg/dL for high, moderate or low risk subjects (who qualify for drug therapy). Most recently, AHA/ACC published a new version of guidelines for cholesterol management (481). These guidelines included evidence from recent 2 large randomized clinical end-point trials of PCSK9 inhibitors (454,455)and a long-awaited ezetimibe trial in patients with recent acute coronary syndromes (482). The new guidelines re-introduced LDL-C treatment goals in some high-risk patient groups, such as those with high risk of ASCVD and those with very high baseline LDL-C. The new AHA/ACC guidelines also stated that elevated apoB particle number, elevated Lp(a) and hypertriglyceridemia are all additional risk factors for ASCVD.

 

Lp(a)

 

Lp(a) has been shown to be an independent risk factor for atherothrombotic events, including heart attack, stroke and peripheral vascular disease, in multiple prospective studies (451,483). The new AHA/ACC guidelines list elevated Lp(a) a one of the risk-enhancing factors for developing ASCVD (481). Lp(a) consists of an LDL particle in which apoB100 is covalently linked via a disulfide bridge to apo(a), a glycoprotein with repeating Kringle units that share homology with plasminogen. Although apo(a) is synthesized by the liver, the Lp(a) particles are not formed in the liver but in the plasma. Despite being a modified LDL particle, Lp(a) levels are independent of LDL-C levels. The catabolism of Lp(a) is poorly understood, but Lp(a) is not cleared by the LDLR (484).  The number of repeating Kringle units is highly variable but largely genetically determined, and this contributes to tremendous heterogeneity in size of Lp(a). The plasma levels of Lp(a) vary tremendously in humans, and plasma Lp(a) levels are generally inversely related to the size of the apo(a) isoform (485). Thus, smaller Lp(a) particles with fewer Kringle repeats are present at higher levels in the plasma. In American Caucasians, the increased levels of smaller Lp(a) particles is largely explained by the size of the LPAgene, based on the size of the repeated KIV₂domain (486), which is believed to be due to difficulty of hepatic secretion of larger apo(a) isoforms. Nevertheless, this relationship varies in different ethnic populations. Early studies suggested that even though Lp(a) levels are higher in African Americans that Lp(a) levels did not appear to be an independent risk factor for cardiovascular events in this group(487). However, by determining allele specific Lp(a) concentrations, a larger more recent analysis demonstrated that elevated Lp(a) levels associated with small apo(a) isoform sizes serve as an independent risk factor for CHD in both African Americans and Caucasians (488). Similarly, a 20 year follow up study of the ARIC cohort found that elevated levels of Lp(a) are associated with a similar degree of risk in in both African Americans and Caucasians (489). A recent meta-analysis by the Emerging Risk Factors Collaboration evaluated 36 prospective studies with 126,634 subjects found that Lp(a) is an independent risk factor for CHD (490). In contrast to previous studies that suggested Lp(a) was only relevant as a risk factor when levels were extremely elevated, the meta-analysis demonstrated that risk and that Lp(a) levels are continuously associated with CHD risk (490). The Ile4399Met polymorphism (rs3798220) in the protease-like domain of apo(a) is particularly associated with increased risk for severe CAD (491). Subsequently, Clarke et al.found that the rs3798220 and rs10455872 variants were associated with small apo(a) isoform size, increased Lp(a) levels and substantially increased risk of CAD (492). Furthermore, a Mendelian randomization study by Kamstrup et al.demonstrated that a genetically determined doubling of Lp(a) plasma levels leads to a 22% increase in the risk of MI, strongly supporting a causal role for elevated levels of Lp(a) and risk for MI (493).

 

The proatherogenic mechanisms for Lp(a) remain incompletely understood, but recent studies suggest an important role for oxidative modification of Lp(a) by oxidized phospholipids (OxPL) (251). Mounting evidence supports an important role for OxPLs in the development of atherosclerosis (251). Interestingly, OxPLs associate with Lp(a) in preference to native LDL particles in human plasma (250). Hence a physiological role has been proposed for Lp(a) for binding and transporting OxPL in the plasma (251). Although, Lp(a) is found only in humans and Old-World monkeys, mice expressing human Lp(a) have been developed to examine the role of Lp(a) in atherogenesis and lipoprotein metabolism. The first transgenic mice expressing high levels of human apoB100 were created using a 79.5-kb human genomic DNA fragment containing the entire human APOBgene that was isolated from a P1 bacteriophage library, and crossing these mice with apo(a) transgenic mice produced high levels of human Lp(a) in plasma (494). In a study of transgenic mice expressing high and low concentrations of Lp(a), high levels of OxPLs were found in transgenic mice with very high levels of Lp(a), but not in LDL of apoB transgenic control mice (495). These studies support the concept of preferential transfer of OxPL to Lp(a). In the Dallas Heart Study, levels of OxPL on apoB were strongly correlated with Lp(a) levels, and inversely related to the size of the apo(a) isoforms (496). In the European Prospective Investigation of Cancer (EPIC)–Norfolk prospective study the impact of OxPL and Lp(a) levels on CHD risk was additive (497). Further studies are needed to define the extent to which the preferential binding of OxPL by Lp(a) is responsible for mediating the increased risk of atherothrombotic events attributable to Lp(a).

 

Lp(a) is considered an emerging risk factor, but the approach to managing patients with elevated levels of Lp(a) has not been well established. Elevated levels of Lp(a) do not respond well to changes in diet or statin therapy. Analysis of data from the Familial Atherosclerosis Treatment Study (FATS) showed that substantial lowering of LDL-C (with lovastatin plus colestipol or niacin plus colestipol) in subjects with CAD and high apoB100 eliminated the increased risk attributable to having very high Lp(a) (498). The JUPITER trial showed that treatment of subjects with low levels of LDL-C, but increased hsCRP, with rosuvastatin (20 mg) reduced CVE. In JUPITER, elevated Lp(a) was a significant determinant of residual risk, but the reduction in relative risk with rosuvastatin was similar among participants with high or low Lp(a) (499,500). Treatment with niacin reduces Lp(a) by 20-30%, and the European guidelines recommend treating patients with elevated Lp(a) who are at intermediate to high risk of CVD with extended release niacin to obtain levels of Lp(a) < 50 mg/dL (501). Nonetheless, the recent failure of the AIM-HIGH and HPS-2 THRIVE studies have cast doubt on the use of extended release niacin in subjects fitting the profile of those studies (CAD with LDL well treated on a statin). LDL apheresis is approved and effective for lowering Lp(a) in individuals with recurring CVE in the setting of very high levels of Lp(a). There are a number of new therapies that may prove useful in treating patients with elevated levels of Lp(a). The recently approved monoclonal antibodies to PCSK9 significantly lower Lp(a) by around 30% in addition to lowering LDL-C by 30-50%. Furthermore, a Phase 1 clinical trial of a second-generation antisense to apo(a) has recently reported potent, dose-dependent, selective reductions of plasma Lp(a) (502). This approach has the appeal of specifically targeting apo(a) to reduce Lp(a) levels. Hopefully, these new approaches will ultimately yield an effective approach to lower levels of Lp(a) that translates into reduced cardiovascular events.

 

INTESTINAL LIPID METABOLISM AND CHYLOMICRON ASSEMBLY

 

Intestinal Lipid Absorption

 

Through absorption of dietary lipids, the intestine is a key regulator of stored and circulating lipids. Primarily it is enterocytes in the small intestine that actively regulate the release of dietary lipids into circulation (503-505). The predominant lipids derived from diet are triglycerides, phospholipids and cholesteryl esters. In the intestinal lumen, ingested lipids are emulsified by bile salts to enhance their hydrolysis by lipases (Figure 9) (506-509). Triglycerides make up the largest percentage of the intestinal lipids. Lipolysis of triglycerides releases free fatty acids (non-esterified fatty acids) and monoacylglycerides (Figure 9). These are absorbed on the luminal surface of the enterocytes both by free diffusion and actively by protein-mediated transport into the enterocyte cytosol (Figure 9) (508-510). The principal transporters identified to date are CD36 (now known as SR-B2 (511)) and several fatty acid binding and transport proteins (512-514).

Chylomicron Assembly and Secretion

 

In the enterocyte, the free fatty acids and monoacylglycerides are used to synthesize triglycerides, phospholipids, and cholesteryl esters (Figure 9) (508,509,513,515-517). The majority of the triglycerides formed in the enterocytes are repackaged into large, buoyant lipoproteins, called chylomicrons, and secreted from the basolateral surface of the cell (Figure 9). These particles play a central role in the transport of triglycerides and fat-soluble vitamins to the rest of the body (518).

 

The assembly of the chylomicron particle from precursors is a complex process. Each particle contains a single copy of apolipoprotein B48 and assembly begins with the synthesis of this protein in the rough endoplasmic reticulum. Apolipoprotein B48 is a truncated form of apolipoprotein B100 that is formed by posttranscriptional editing (519,520). As apolipoprotein B48 is synthesized and translocated across the endoplasmic reticulum membrane, it becomes lipidated to form a phospholipid-rich, dense primordial chylomicron in the lumen of the endoplasmic reticulum (Figure 9). The primordial chylomicron contains apolipoprotein B48, phospholipid, cholesterol and minor amounts of cholesteryl ester and triglyceride (513,521,522). The assembly process requires microsomal triglyceride transfer protein(523). In the absence of sufficient lipid, or if microsomal triglyceride transfer protein function is impaired, apolipoprotein B48 is ubiquitinated and targeted for proteasome degradation (524). The importance of this initiating assembly step is seen in patients with a defect in the MTP gene leading to the rare recessive disorder abetalipoproteinemia. Individuals with abetalipoproteinemia have almost undetectable levels of apoB or and very low total cholesterol levels in their plasma because of the inability to assemble apoB-containing lipoproteins in their enterocytes or hepatocytes. Among the sequelae experienced by these patients are accumulation of triglycerides in their intestines and livers and a deficiency of lipid-soluble vitamins in their plasma (525,526). If untreated, these patients develop severe neurological problems; mostly related to vitamin E and A deficiency.

 

After formation, the initial primordial particle expands by the acquisition of additional triglyceride and cholesteryl ester (Figure 9). The additional lipid is acquired by fusion with non-apolipoprotein B48 containing particles that are rich in triglyceride and cholesteryl ester. The exact origin of these lipid particles and their precise composition is currently actively debated (504,505,513,527,528), but the fusion of the primordial chylomicron with the apolipoprotein B48-free particles occurs in the endoplasmic reticulum (513). The resulting particle is a prechylomicron (Figure 9).  In addition to apolipoprotein B48, the prechylomicron surface can contain multiple copies of other small, exchangeable apoproteins including apolipoprotein A-IV and apolipoprotein C-III. Exchangeable apoproteins are soluble proteins that are not as tightly adherent to the particle surface and so can be exchanged between lipid particles.

 

Prechylomicrons are transported out of the endoplasmic reticulum and delivered to the Golgi apparatus for further processing (Figure 9). Transport occurs in specialized vesicles that can accommodate their large size. The unique vesicles contain a number of specific proteins necessary for the transport and docking process. Vesicle-associated membrane protein-7, coatomer protein II and Sar1b, a small GTPase component of the coatomer protein II vesicle assembly machinery (Figure 9)  are among the specialized proteins on the lipid transport vesicles (505,529-531). The maturation of the particle in the Golgi apparatus includes further glycosylation of apolipoprotein B48 and the addition of apolipoprotein A-I to the surface (505,532,533). After processing, the mature chylomicron is packaged into Golgi-derived secretory vesicles and transported to the basolateral surface and exocytosed into the lymph (Figure 9) (527,534,535).

 

The assembly of chylomicrons in enterocytes is a complex process requiring a number of coordinated steps and specific factors to work in unison. A failure in any of these can lead to lipid-related disease states. For instance, mutations in the SAR1B gene lead to retention of prechylomicrons within membrane-bound structures in the enterocytes (529). The condition is marked in childhood by decreased blood cholesterol levels, lipid accumulation in the enterocytes, chronic fat malabsorption with steatorrhea, and deficiencies in fat-soluble vitamin and essential fatty acids.

 

Chylomicron Cholesterol

 

Although chylomicrons are triglyceride-rich, they also carry substantial amounts of cholesterol (536,537). The cholesterol in chylomicrons comes from the general pool of enterocyte cholesterol. Enterocytes acquire cholesterol by uptake at the luminal surface, acquisition from lipoproteins at the basal lateral surface, and by de novo synthesis within the enterocyte. Niemann-Pick C1-Like 1 protein is a key component of the luminal acquisition machinery (Figure 9) (538), while the low density lipoprotein receptor appears to be a major mediator of cholesterol acquisition at the basolateral surface (539,540). The incorporation of cholesterol into chylomicrons contributes to the circulating levels of cholesterol, and increases in intestinal synthesis of chylomicrons due to increased dietary lipids contributes to cardiovascular risk and atherosclerosis, albeit by complex mechanisms (516,541,542).

 

Non-Chylomicron Intestinal Lipid Metabolism

 

Enterocytes can also regulate circulating lipids by means other than chylomicron secretion.  In the presence of excess fatty acids or cholesterol, the enterocyte can store excess lipid in their esterified forms (triglycerides and cholesteryl esters, respectively) within cytoplasmic lipid droplets (543-545). The neutral lipids in the droplets can subsequently be mobilized by hydrolysis as needed by the cell. The free fatty acids liberated from storage droplets can be incorporated into the chylomicron production pathway to become part of secreted chylomicrons.

 

Finally, the intestine also regulates circulating cholesterol levels by taking up excess circulating cholesterol and excreting it into the intestinal lumen for clearance in the feces. This process is known as trans-intestinal cholesterol excretion. It acts as an adjunct to liver biliary secretion and can account for as much as 30% of neutral sterol excretion (546). Trans-intestinal cholesterol excretion occurs at the luminal surface of the enterocytes by a process that primarily utilizes the ATP-binding cassette transporter pair ABCG5/G8 (Figure 9) but can use other pathways as well (547).

 

Summary

 

It is clear that intestinal lipid processing is a key contributor to the circulating levels of both triglyceride and cholesterol. Dietary, genetic and metabolic factors that disrupt the process of enterocyte lipid metabolism potentially can alter lipid homeostasis and produce disease states.

 

 

 

TRIGLYCERIDES, CARDIOVASCULAR DISEASE AND ATHEROSCLEROSIS

 

Causes of Hypertriglyceridemia

 

The prevalence of high circulating triglyceride levels is increasing worldwide, particularly in developed countries. In the United States there has been a greater than 7 fold increase in average plasma triglyceride concentration over the last 30 years (548). This increase coincides, in part, with increased instances of obesity and type 2 diabetes (T2DM) although the relationship of these conditions to hypertriglyceridemia is complex (549-553). Most classifications of hypertriglyceridemia are based, at least in part, on the Third Report of the National Cholesterol Education Program (NCEP) Expert Panel on Detection, Evaluation, and Treatment of High Blood Cholesterol in Adults (Adult Treatment Panel III) (469). These guidelines classified circulating triglyceride levels <150 mg/dL as normal. Values between 150 mg/dL and 199 mg/dL are considered borderline high and anything above 200 mg/dL are classified as high, with those above 500 mg/dL deemed to be very high (469,470). Hypertriglyceridemia is generally the result of increases in one or more of the triglyceride-rich lipoproteins; chylomicrons, VLDL, or their remnants. The increase occurs because of increased synthesis, decreased catabolism or both, with the underlying cause generally being the result of alterations in metabolic factors such as apolipoprotein C-II, apolipoprotein C-III, CETP and lipoprotein lipase. However, hypertriglyceridemia can also be secondary to other disease states (e.g. diabetes mellitus, hypothyroidism, renal disease, and nephrotic syndrome) (548,554). Not surprisingly, environmental conditions, particularly a diet with high fat or high glycemic index content and in which energy intake is out of balance with energy utilization, are associated with hypertriglyceridemia as is excess alcohol consumption (548,554). In fact, dietary choices and lack of exercise are widely held to be a major contributor to the recent rise in circulating triglyceride levels in developed countries.

 

Hypertriglyceridemia as an Independent Risk Factor for Cardiovascular Disease

 

Individuals with elevated triglyceride levels are at increased risk for cardiovascular complications, particularly atherosclerosis (555,556). The Framingham Study was one of the first large studies to associate hypertriglyceridemia with cardiovascular disease, particularly in women (557). However, many other studies before and since have also shown a univariate association of high triglycerides and increased risk of cardiovascular disease. In many of these studies, however, the affect went away after accounting for other major risk factors (558-561),calling into question whether triglycerides represent an independent risk factor. For instance, a meta-analysis of the Emerging Risk Factors Collaboration data revealed triglycerides as a strong risk factor for cardiovascular disease and stroke, but, after adjusting for standard risk factors (primarily lipoprotein-associated cholesterol), the researchers concluded that triglyceride levels provided no additional predictive value (474). The authors did note, as have other studies (562-565), that patients with triglyceride levels above 500 mg/dL are at increased risk of pancreatitis; providing impetus for measuring triglyceride levels in patients and treating those with high levels irrespective of cardiovascular risk. The lack of strong association of triglyceride concentration with cardiovascular disease (after accounting for other risk factors such as elevated LDL-C and low HDL-C) has led some to question whether measuring triglyceride levels has any utility for cardiovascular patient management. In contrast, we argue below that there are a number of important reasons for evaluating triglyceride levels in patients, particularly those with cardiovascular disease, metabolic syndrome or diabetes.

 

First, we would point out that the difficulty in substantiating an independent association of triglycerides with cardiovascular disease may simply reflect the fact that a number of interrelated risk factors make it difficult to determine to what extent triglycerides independently contribute to cardiovascular events. One key issue is that, even in studies suggesting independence, the effect size has been small compared to traditional risk factors like LDL-C (548). Therefore, independence is very hard to detect in small studies. There are also issues regarding the way triglyceride levels are determined, the high variability of triglyceride concentrations in a single individual, and the association of triglyceride levels with other atherogenic conditions such as low HDL-C, obesity and T2DM (548,566-571). These confounding issues are not always considered by authors when drawing conclusions. Moreover, endpoints have differed widely among studies. Despite the confounding issues, an increasing number of case control studies do indicate triglycerides as an independent risk factor for cardiovascular disease even when adjusting for total cholesterol, LDL-C and HDL-C (572-578). The PROCAM study, for instance, found increases in risk for cardiovascular events as triglyceride levels increased and residual risk remained after accounting for other major risk factors (579), and the PROVE IT-TIMI 22 study revealed that triglyceride levels had a substantial impact on cardiovascular outcomes in patients with acute coronary syndrome that was independent of LDL-C (580). Moreover, Mendelian randomization studies strongly suggest a causal relationship between factors involved in regulating triglyceride rich lipoprotein levels and cardiovascular disease (581-583). For instance, analysis of data from the Copenhagen City Heart Study showed that genetic variants of lipoprotein lipase that resulted in reduced circulating triglyceride levels also reduced all-cause mortality (583).

 

Meta-analyses of randomized, prospective trials probably provide the strongest evidence for triglyceride levels as an independent risk factor. One such analysis assessing the effects of lowering circulating cholesterol levels with statins, indicated that in patients with preexisting coronary heart disease, there was a reduction in residual risk not associated with lowering LDL-C that could be related to other lipoproteins, such as triglyceride-rich lipoproteins (584). Most convincingly, a recent meta-analysis of 29 prospective studies showed that considering triglyceride concentrations yielded an adjusted odds ratio of 1.72 (95% Confidence interval=1.56-1.90) for those in the top tertile of triglyceride levels even after adjusting for other common risk factors (556). A similar odds ratio was reported in a meta-analysis that included data from 26 prospective studies in Asian and Pacific populations (585).

 

Given the increasing evidence that hypertriglyceridemia is indicative of increased cardiovascular disease risk, a key question is whether reducing triglyceride levels are protective. The results of several studies do suggest that reducing TG levels can reduce risk of cardiovascular events. An analysis of two secondary prevention trials of pravastatin suggests that high HDL-C and low triglycerides were significant predictors of reduced risk for CHD events (586). A recent meta-analysis of 18 trials evaluating the effects of fibrates on cardiovascular outcomes reported a 10% relative risk reduction for major cardiovascular events in individuals with hypertriglyceridemia alone or in combination with low HDL-C (587). Other meta-analyses have generally shown small but significant associations of low triglycerides and protection from cardiovascular events independent of other major risk factors (588).

 

Thus, the evidence is mounting for an independent role of circulating triglyceride levels in mediating cardiovascular risk and certainly has established the utility of determining triglyceride levels in at-risk patients. However, the studies also suggest that the association between high triglycerides and cardiovascular disease is complicated, multidimensional, and possibly indirect.

 

Is There a Direct Role for Triglycerides in Promoting Cardiovascular Disease?

 

If hypertriglyceridemia does directly affect cardiovascular disease, the mechanism(s) remain to be fully elucidated. Nonetheless, several hypotheses have been put forward. As the most prevalent form of cardiovascular disease, atherosclerosis has been the target for most explorations of a direct role for triglycerides in cardiovascular disease, and there is growing evidence, albeit circumstantial, that triglycerides can directly influence specific aspects of atherosclerotic lesion development. Many of the hypotheses are based on the fact that triglyceride rich lipoproteins (VLDL, chylomicron) also contain significant amounts of cholesterol (536)and could promote foam cell formation by contributing cholesterol to the lesion. Remnants of VLDL and chylomicrons are created by partial hydrolysis of their triglycerides through the action of lipoprotein lipase. These particles have an increased percentage of cholesterol(537,589)and can acquire additional cholesterol by transfer from HDL through the action of cholesterol ester transfer protein(CETP) (590). In hypertriglyceridemia, there is increased VLDL synthesis, delayed clearance and often increases in remnant particles (591,592). In fact, it has been argued that nonfasting triglyceride levels primarily reflect remnant lipoproteins, particularly in hypertriglyceridemia, and these particles may be the atherogenic moiety (593). Although chylomicrons and, to some extent, very low density lipoproteins are generally too large to cross the endothelial layer and invade the arterial intima, conversion to remnants allows these particles to accumulate within atherosclerotic lesions and to deposit their cholesterol (594-596). This would imply that levels of lipoprotein lipase, by increasing remnants, could influence atherosclerotic lesion development and there are animal studies showing just such a correlation (237,238,597). Evidence for the importance of remnants in atherogenesis also comes from individuals with type III hyperlipoproteinemia. Patients with type III hyperlipoproteinemia have decreased clearance of remnant lipoproteins and develop premature atherosclerosis (598).  ApoE is crucial for the normal clearance of chylomicrons and VLDL remnants, but the ApoE-2 isoform has reduced ability to bind to lipoprotein receptors and mediate clearance (599). Type III hyperlipoproteinemia occurs most often in subjects who are homozygous for APOE2, but the majority of E2/E2 individuals do not have the Type III phenotype, suggesting that a second hit is required to express the phenotype (600). Interestingly, rare genetic variants of APOE have been described that cause an autosomal dominant form of Type III hyperlipoproteinemia (601,602)and  ApoE deficiency in humans is extremely rare but is associated with the Type III phenotype  (600,603).

 

One mitigating factor in evaluating how much delivery of cholesterol in triglyceride-rich particles contributes to atherosclerosis is the fact that, although triglyceride-rich particles and their remnants contain large amounts of cholesterol, they also contain significant amounts of triglyceride. At least with respect to cellular cholesterol accumulation in macrophage foam cells (a hallmark of atherosclerosis), the presence of triglyceride in cells actually promotes the hydrolysis of cholesteryl esters to cholesterol (604,605). Cholesterol stored in foam cells is primarily in the form of cholesteryl esters. In order to be removed from the cell and eventually from the plaque, esterified cholesterol must first be converted to unesterified cholesterol (606). The presence of triglyceride intermixed with cholesteryl esters in foam cells facilitates the hydrolysis and removal of cholesterol (604,605,607). The differing effects of circulating triglyceride levels on cardiovascular disease risk and their cellular effects on cholesterol metabolism have yet to be reconciled.

 

There are mechanisms other than cholesterol delivery by which triglycerides could influence atherosclerosis. Lipolysis of triglyceride rich particles not only concentrates cholesterol in the particles it also produces free fatty acids and monoglycerides. Cell culture studies have demonstrated that long-chain fatty acids, particularly saturated fatty acids like palmitate and stearate, are cytotoxic (608-610). Thus, the presence of triglyceride lipolysis within atherosclerotic lesions could raise toxic free fatty acid levels in cells of the arterial wall, which would promote cell death and resulting inflammation. Both increased cell death and increased inflammatory signaling are key attributes of atherogenesis (611-614). In support of triglyceride lipolysis as an atherogenic driver, macrophages make and secrete lipoprotein lipase (lipoprotein lipase) and it is estimated that macrophages are the primary source of lipoprotein lipase in atherosclerotic plaques (615). Localized lipolysis of triglyceride-rich lipoproteins and their remnants can also liberate other oxidized fatty acids, which can promote cytotoxicity and inflammation (616-619); key players in atherosclerotic lesion development. Increases in macrophage lipoprotein lipase do stimulate macrophage cytotoxicity (620), while diminution of macrophage lipoprotein lipase in mice reduces atherosclerotic plaque size (237,621,622). Thus, localized hydrolysis of triglyceride-rich particles by macrophages have the potential to produce cytotoxic and inflammatory effects.

 

It is also becoming clear that the dietary fatty acid composition of lipoproteins, including triglyceride-rich lipoproteins, affects their metabolism in complex and not completely understood ways. The fatty acid composition of lipoproteins (as well as phospholipids and cholesteryl esters) is strongly influenced by dietary intake of fatty acids. Although dietary intake of saturated fatty acids is popularly believed to be bad, whether consuming saturated fat, per se, increases cardiovascular risk is somewhat controversial based on available evidence (623,624). However, in subjects with FH, increased saturated fat in the diet clearly increases LDL-C levels. What also appears clear is that replacing saturated fatty acids in the diet with polyunsaturated fatty acids (PUFA) reduces cardiovascular events (623-627). Omega-6 PUFA are the primary PUFA found in western diets. There is evidence these lower triglyceride levels, in part, by increasing lipolysis of triglyceride-rich lipoproteins (628). Omega-3 PUFA are the other major source of dietary PUFA. Fish are a rich source of long-chain omega-3 PUFA, and there is compelling evidence that omega-3 PUFA (at least from marine sources) reduce both triglyceride levels and cardiovascular risk (629-631). A recent large scale randomized controlled trial (REDUCE-IT) using an EPA only fish oil product reduced major cardiovascular event by 25% in patients who have hypertriglyceridemia (632). Replacing saturated fat with monounsaturated fatty acids may provide some reduction in cardiovascular events, but PUFA appear to have a stronger correlation with improved cardiovascular risk compared to monounsaturated fatty acids (633-635). In contrast to cis fatty acids, trans unsaturated fatty acids, which are common in processed foods, have been convincingly associated with increased cardiovascular risk (623,636,637). Given this and other evidence, a recent report from the National Lipid Association’s Expert Panel recommends, for patients with low or moderate risk for cardiovascular disease, that intake of saturated fatty acids be reduced to <7% of total energy and trans fatty acids should be avoided (638). The reduction in saturated and trans fats should be replaced with PUFA, protein and carbohydrate (638). The guidelines also suggest eating fish twice weekly. For individuals with high triglyceride levels, the Expert Panel also recommends supplementation with omega-3 polyunsaturated fatty acids from marine sources (638). The 2018 AHA/ACC listed persistent hypertriglyceridemia as a risk enhancer for developing ASCVD and recommend using omega-3 fish oil for individuals with high triglyceride levels to prevent pancreatitis. However, the AHA/ACC guidelines did not include the evidence of the REDUCE-IT trial. Therefore, in these individuals with hypertriglyceridemia and other risk factors for ASCVD, one should consider initiating omega-3 fish oil or intensifying statin therapy(481).

 

Lipoprotein lipase-mediated hydrolysis of triglyceride is not the only mechanism in the artery wall for the metabolism of triglyceride rich particles to produce potentially atherogenic compounds. The foam cell macrophages are also capable of the endocytic uptake of VLDL and remnant particles, which can then be catabolized in the lysosome (Figure 2) (639-642). Interestingly, there is evidence that under atherogenic influences, including macrophage sterol engorgement, the route of triglyceride metabolism in macrophages can shift to favor endocytic delivery of triglyceride-rich lipoproteins rather than surface hydrolysis (641,643). Whereas surface hydrolysis of triglycerides by surface lipases primarily delivers only free fatty acids to cells, endocytic uptake of particles would include the delivery of the particle’s full content, including its sterol, which would exacerbate foam cell sterol accumulation.

 

Another potential way that triglyceride-rich lipoproteins could influence atherosclerosis focuses on the apolipoprotein CIII content of VLDL and remnants. ApoCIII inhibits lipoprotein lipase, inhibits remnant uptake by the liver, and its levels are associated with hypertriglyceridemia (644-648). Thus, high apolipoprotein CIII concentrations could promote arterial retention of VLDL and remnants making them more atherogenic, suggesting apolipoprotein CIII as a therapeutic target. In fact, individuals with certain mutations in APOC3 have low triglycerides and LDL-C (649,650). Two recent studies show that loss-of-function mutations in apoCIII lowered serum triglycerides by >39%, significantly reduced LDL-C and raised HDL-C, and lowered the incidence of cardiovascular events by >36% (651,652). An antisense oligonucleotide selective inhibitor of apoCIII has been developed that lowers serum apoCIII and triglycerides in mice, non-human primates, and humans and is currently in a phase 2 clinical trial (653).  These studies indicate that reduction of apoCIII by antisense oligonucleotide inhibition significantly reduces circulating triglyceride levels (654,655). Besides their effects on circulating lipids, Apo CIII-containing lipoproteins also stimulate a range of processes including activation of monocytes, inflammation, endothelial cell NO production resulting in vascular dysfunction and increased lipid oxidation and binding of lipoproteins to PG which can stimulate macrophage foam cell formation (227,239,656-658).

 

A final way in which triglyceride levels could influence atherogenesis is related to the finding that patients with hypertriglyceridemia also tend to have increased circulating levels of thrombotic factors such as fibrinogen and plasminogen activator inhibitor and inflammatory mediators (TNF-alpha, IL-6, VCAM-1 and MCP-1) (659-661). Thrombosis and inflammation are key factors in atherosclerosis and its progression to heart attack and stroke.

 

Reducing Circulating Triglyceride Levels

 

It is clear, therefore, that there are a variety of ways in which the triglyceride-containing particles in hypertriglyceridemic plasma could contribute either directly or indirectly to multiple aspects of atherosclerotic lesion development. Regardless of whether triglycerides are directly causative of cardiovascular disease, the evidence is mounting that assessment of triglyceride levels has an important role in evaluating and managing cardiovascular risk, and treating elevated triglyceride levels may reduce risk for cardiovascular events (548,662). This is particularly true for patients with coronary heart disease or diabetes (548,662-664). Several agents have shown efficacy in reducing triglyceride levels and also in reducing cardiovascular disease risk. The reduced risk is thought to occur to a large extent by reducing atherosclerosis. Currently, therapeutic agents recommended for treating hypertriglyceridemia are fibrates, statins, niacin and omega-3 PUFA but others are being developed. Unfortunately, clinical trials of the impact of triglyceride lowering medications on cardiovascular events in subjects with severe hypertriglyceridemia have not been undertaken.

 

Fibrates are the most effective approach for directly lowering triglyceride levels. Fibrates have been shown to lower triglyceride levels by 30%-50% depending on the baseline levels (548). More importantly, fibrate therapy with gemfibrozil has been shown to reduce cardiovascular risk in patients with elevated triglycerides (665-667). Unfortunately, the trials of combination therapy of statins with fenofibrate have failed to meet their primary endpoints in terms of reducing cardiovascular events (668,669). However, posthoc analysis of all of the fibrate trials show significant benefits in terms of reducing CVD events, when looking at the subgroup of patients with elevated triglycerides and low HDL-C and features of the metabolic syndrome or diabetes (670,671).

 

Statins inhibit HMG-CoA reductase, the rate-limiting enzyme in cholesterol biosynthesis, and produce dramatic reductions in LDL-C levels; typically from 20%-60% depending upon the particular statin used and dosage (672). However, they also reduce circulating levels of triglycerides (673)and Non-HDL-C (477). Levels of LDL-C are often low in the setting of hypertriglyceridemia, so Non-HDL-C levels are a more useful measure of the burden of atherogenic apoB-containing lipoproteins than LDL-C in patients with hypertriglyceridemia. Indeed, the National Lipid Association recommends using goals for Non-HDL-C levels of < 130 mg/dl or < 100 mg/dl, in subjects at high- and very-high risk of cardiovascular events, respectively (477).  Niacin is a B vitamin that can lower overall circulating lipid levels when given in high doses. The mechanism of action is not entirely clear but niacin reduces VLDL production by the liver. Unfortunately, clinical trial data regarding the use of niacin on cardiovascular outcomes in severe hypertriglyceridemia is lacking. Although in a subgroup analysis of the AIM-HIGH trial, niacin showed a trend toward benefit in the tertile of subjects with the highest triglycerides (>198mg/dl) and lowest HDL-C (<33mg/dl), which is consistent with the post hoc analysis of fibrate trials (674). Finally, evidence indicates that daily intake of omega-3 PUFA from marine sources (which primarily containing eicosapentaenoic and docosahexaenoic acids) can significantly reduce circulating triglyceride levels (675-678). Treatment with 3 – 4 grams a day of omega-3 PUFA (EPA+DHA) is effective in lowering triglycerides. A large meta-analysis of omega-3 FA in 20 studies including 63,000 participants did not see an impact on a combined cardiovascular endpoint or coronary events, but there was a reduction in vascular death (679). However, most omega-3 outcome trials used less than one gram of omega-3 PUFA, which was probably too low of a dose to have meaningful triglyceride lowering effects that could yield clinical efficacy. The JELIS trial, compared the effect of 1.8 g EPA vs. placebo on top of statin in a hypercholesteremic but relatively normal triglycerides patient population (mean LDL-C 182 mg/dl and triglycerides 151 mg/dl) (95). JELIS found a 19% relative risk reduction in CV events but a more pronounced 53% reduction in the subgroup with mixed dyslipidemia, specifically the subgroup with triglycerides >150mg/dl and HDL<40 mg/dl(680). Most recently, a large randomized controlled trial (REDUCE-IT) of an EPA only fish oil product reduced major cardiovascular event by 25% in patients who have hypertriglyceridemia (632). Whether the clinical benefit was confined to EPA only or it can be generalized to all omega-3 PUFA is still yet to be determined. In contrast to marine-derived omega-3 PUFA, plant-derived omega-3 PUFA have generally not shown efficacy for lowering triglycerides(681,682). A number of new approaches for treating hypertriglyceridemia are in development including antisense oligonucleotides to ApoC3(654,655).

 

Summary

 

The association of elevated triglyceride levels and cardiovascular disease has been well established (555,556,579,588). What remains a subject of ongoing debate is the extent to which triglycerides directly promote atherogenesis or, alternatively, simply represent a biomarker for other processes that influence cardiovascular risk. (542,548,554,561,570,592,683,684). Nonetheless, the evidence supports measuring triglycerides and including triglyceride and Non-HDL-C reduction in treatment regimens is strengthening especially in patients with metabolic syndrome, diabetes, or cardiovascular disease.

 

HDL METABOLISM AND ATHEROSCLEROSIS

 

HDL and Reverse Cholesterol Transport

 

Apolipoprotein A-I (apoA-I) is the major protein on HDL and provides both structure and function. Lipid-poor apoA-I and mature HDL both contribute to removing cholesterol from macrophages and prevent foam cell formation (Figure 2). Although cholesterol flux from macrophages to HDL (or apoA-I) alleviates cholesterol-accumulation in lesions, the net flux of cholesterol from the lesion has little to no effect on systemic cholesterol levels. Nevertheless, macrophage cholesterol efflux to HDL reduces inflammation and the atherosclerotic burden, and is the first step in reverse cholesterol transport (RCT) (Figure 10) (685-687). This pathway was first described in 1966 (688). The rate at which cholesterol flows through the RCT pathway is of greater importance than steady state levels of HDL-cholesterol (HDL-C). Interestingly, cholesterol movement from macrophages to HDL occurs through at least 4 routes (70). First, lipid-poor apoA-I stimulates the efflux of phospholipid and free cholesterol through interaction with ATP-binding cassette transporter A1 (ABCA1) (Figure 2), which generates pre-beta HDL and nascent discoidal particles (689). The more lipidated the apoA-1 becomes, the discoidal HDL particles transition into a spherical structure and lose their ability to interact with ABCA1 and stimulate cholesterol efflux through ABCA1. Both discoidal HDL particles and mature spherical HDL particles can also promote free cholesterol efflux from another transporter, ATP-binding cassette transport G1 (ABCG1), which is thought to reside on sub-cellular organelles as opposed to the plasma membrane (Figure 2) (690,691). This transporter is a critical regulator of intracellular cholesterol trafficking cellular cholesterol availability, and cholesterol export (690,692). HDL’s primary receptor for cholesteryl ester (CE) uptake, scavenger receptor BI (SR-BI), is also a bidirectional free cholesterol transporter in that it facilitates the efflux and influx of free cholesterol between cells and mature HDL (693-695)(Figure 2). The net direction of cholesterol flux is determined by the cholesterol concentration gradient (plasma membrane and HDL ratio of free cholesterol to phospholipid) (696)as well as by the phospholipid subspecies (697,698). Finally, cholesterol can simply move from the plasma membrane to HDL through passive aqueous diffusion, which is a major route of cholesterol efflux from macrophages (Figure 2) (70,687,695). On HDL free cholesterol is solubilized in the phospholipid surface layer and is rapidly esterified by lecithin:cholesterol acyltransferase (LCAT) (Figure 6), and the hydrophobic CE is then mobilized to HDL’s core (699,700).

Spherical mature HDL then transports CE to peripheral cells and tissues, and back to the liver as part of the RCT pathway (Figure 10). HDL delivers CE to the liver through 2 primary routes. HDL delivers CE to the liver through binding to SR-BI (Figure 6), which drives selective uptake of core lipids (694). Another major route of cholesterol delivery to the liver is mediated through LDL and the LDL receptor (LDLR) (Figure 6) (701). In the circulation, HDL exchanges CE for TG from VLDL and LDL through cholesteryl ester transfer protein (CETP) activity (Figure 6), and this action is responsible for directing CE through the LDL receptor pathway (702). Besides these major routes holoparticle uptake of HDL may also contribute to delivery of HDL-CE to the liver. Hepatocytes, and many other cell types in other tissues, likely participate in HDL retro-endocytosis where apoA-I or HDL particles are taken up by endocytosis and resecreted without degradation in late endosomes and lysosomes (703,704). SR-BI and CD36 may participate in this process as well as other potential HDL receptors (705-707). For example, the F₀F₁ATPase and P2Y₁₃receptor have been reported to facilitate the uptake of the entire HDL particle(703,704,708,709). The liver then excretes both cholesterol and bile acids-derived from cholesterol into the bile which are removed from the body in feces, thus completing RCT from peripheral macrophages to bile through HDL and the liver (710). Recent evidence suggests there is also likely an HDL-independent pathway for systemic cholesterol removal through transintestinal cholesterol excretion (TICE) (711). Historically, HDL’s anti-atherogenic properties were largely attributed to HDL’s role in RCT and removing excess cholesterol from macrophages and peripheral tissues; however, continually emerging alternative HDL functions likely significantly contribute to HDL’s protection against CVD.

 

HDL Levels and Risk of CVD

 

Historically, HDL-C was synonymous with the term HDL; however, the amount of cholesterol in the HDL pool (HDL-C) and the number and quality of HDL particles (HDL-P) are independent concepts that are important to consider in the context of HDL function. Several decades of high-quality epidemiological studies have clearly shown that HDL-C levels are inversely correlated to CVD risk and events, independent of race, gender, and ethnicity (712). In well-controlled studies assessing CVD risk using multivariate approaches to adjust for covariates, both apoA-I and HDL-C are strong independent predictors of CVD risk (474). Nonetheless, HDL-C levels are also inversely correlated to insulin resistance, obesity, and triglycerides. As such, HDL-C’s causality in protection from CVD is difficult to define and is somewhat controversial, mainly due to epidemiological discrepancies between the dose-response of HDL-C levels to CVD outcomes. It is possible that HDL-C levels may simply be a biomarker for CVD and not play a causal role in atherosclerosis; however, an increasing number of functional studies clearly support HDL’s functional relevance in biochemical mechanisms of atherosclerosis. In any case, epidemiological studies over the past 50 years have provided many insights into HDL-C and CVD risk. The first evidence came from the Framingham Heart Study in 1966 demonstrating a link between HDL-C and ASCVD (713). In 1975, HDL-C levels were found to be inversely associated with CVD in a Norwegian trial (Tromso Heart Study) (714). In subsequent years, the Honolulu Heart Study (1976) (715)and Framingham Heart Study (1977) (559)both reported that many CVD patients had low HDL-C levels. Over the years, low HDL-C levels have consistently been reported to be associated with increased risk of ASCVD and events (716-718). By the late 1980s and early 1990s, the relationship between HDL-C and CVD was generally accepted, as studies during this period established that low HDL-C levels were associated with CVD risk independent of other risk factors even in patients with normal total cholesterol levels (719-721).

 

Clinical Outcomes Trials

 

Prior to the statin-era, results from randomized controlled clinical trials suggested that increasing HDL-C levels 1 mg/dL or 1% reduces mortality from CVD by 3-4% (722,723). In the Air Force/Texas Coronary Atherosclerosis Prevention Study (AFCAPS/TexCAPS), treatment of men and women withaverage TC and LDL-C levels and below-average HDL-C levels with lovastatin (20-40 mg) reduced LDL-C by 25% and raised HDL-C 6%, resulting in a 37% reduction in the risk for the first major acute coronary event (724). These results showed that statin therapy was effective in reducing risk for CVE in subjects with low-HDL-C. The extent to which the benefit came from HDL-C raising is unclear. Studies completed in subjects on statins have yielded inconsistent results with regard to the importance of raising HDL-C partly due to evidence suggesting that statin (fluvastatin) use in low HDL-C subjects decreased coronary artery disease (CAD) with little to no increase in HDL-C levels (725-727). In the fluvastatin regression study, low HDL-C subjects on placebo showed increased disease (angiographic) progression compared to subjects with high HDL-C levels (727). Collectively, evidence from these and a large number of epidemiological studies overwhelmingly support a clear inverse association between HDL-C levels and CVD risk. This is demonstrated clinically as raising HDL levels through injections of reconstituted HDL (rHDL) resulted in atherosclerotic plaque regression, as determined by intravascular ultrasound (728). A number of animal studies clearly support the HDL-C hypothesis. For example, raising HDL in mice and rabbits consistently blocks atherogenesis( 729-731). However, raising HDL-C levels by mono- or combined therapy to reduce risk and events has proven challenging. Two major clinical outcomes trials of raising HDL with niacin failed to show a benefit. In subjects with CAD with LDL-C levels well controlled with a statin, the addition of extended release niacin in AIM-HIGH (732)and extended release niacin plus laropiprant (prostaglandin D2 receptor blocker to inhibit flushing) in HPS-2THRIVE (733)failed to reduce cardiovascular outcomes. However, structural limitations of the two Niacin trials design complicated their interpretation (734). In addition, major cardiovascular outcomes trials of 3 CETP inhibitors torcetrapib (735), dalcetrapib (736), evacetrapib have now failed to show a benefit in reducing cardiovascular events. More recently, the CETP inhibitor (anacetrapib) was tested in the REVEAL trial, which was a positive outcomes trial (737). However, the benefit of anacetrapib in reducing CVE seems to be largely explained by lowering of non-HDL, rather than increases in HDL-C (738). More recently, two recombinant apoA-I products MDCO-216 and CER001 showed no benefit in imaging studies (739,740). Collectively, the failure of these clinical studies has raised doubts about the HDL hypothesis.  Indeed, raising HDL-C is presently not a primary target for therapeutic intervention. Nevertheless, HDL infusion in humans has been reported to improve endothelial function, which should contribute to inhibiting atherogenesis (741). At this time, HDL particle infusion therapies have not been proven to be an effective approach to reduce cardiovascular events (742); however, clinical trials with reconstituted HDL are still ongoing. Furthermore, recent studies indicate that HDL particle number and cholesterol efflux capacity are better indicators of CHD risk than HDL-C levels (743,744). The therapeutic targeting of HDL non-cholesterol cargo, quality, and function are emerging and gaining support, as HDL have many other biological properties that likely contribute to prevention of atherosclerosis and CVD (745). In addition, quantifying HDL function, including cholesterol efflux capacity, will provide a better risk index than steady-state HDL-C levels (746).

 

Particle Number and Cholesterol Efflux

 

A major blow to HDL causality in atherosclerosis comes from genetic studies. Mendelian disorders resulting in very low HDL-C levels have yielded conflicting data, as mutations to critical lipoprotein genes (e.g. apoA-I) were found to be associated with protection from atherosclerosis in one study (747)and increased risk in another study (748). The ApoA-I Milano mutation is associated with low levels of HDL-C and reduced risk of CVD (747). Infusion of recombinant apoA-I Milano was reported to induce regression of atherosclerosis (749), but there has not been clear progress in developing it as an approach to therapy since the initial regression study was published. The evidence that some genetic causes of low HDL-C are associated with increased risk for premature atherosclerosis, whereas others are not, supports the notion that HDL function may be more important than HDL-C levels. Nonetheless, Mendelian disorders of low HDL-C levels are rare, and thus the sample sizes in these studies are limited and it is difficult to draw accurate conclusions. To address this issue, genome-wide association studies were completed to attempt to resolve if HDL-C is a risk index or causal factor. These studies are limited in that many variants that raise or lower HDL-C levels also affect other lipoproteins, namely LDL-C levels. For example, variants in CETPraise HDL-C levels and reduce LDL-C levels, which complicates risk prediction based on HDL-C levels (750). Nevertheless, studies have found that variance solely associated with HDL-C levels is not linked to cardiovascular events. For example, single nucleotide polymorphisms (SNPs) in endothelial lipase (LIPG), which raises HDL-C levels, are not associated with decreased CVD(751).

 

As failed clinical trials aimed at raising HDL-C levels and genetic studies do not uniformly support causality for HDL-C in CVD, HDL functional tests in future prospective studies will likely provide more resolution to HDL’s causal role in CVD. Cholesterol efflux capacity, a marker of HDL function, has been reported to be inversely associated with CVD risk independent of HDL-C levels (744,746). This was first demonstrated in a cross-sectional study using radio-tracing of cholesterol efflux (746). A subsequent study also found an inverse association between HDL efflux capacity and atherosclerosis, but reported a positive link to cardiovascular events (752). In a third study assessing HDL cholesterol efflux in a US cohort using a fluorescence method, efflux was again linked to decreased risk of CVD (743). Recently, HDL cholesterol efflux capacity was found to be inversely associated with CVD risk and events in a large nested case-control prospective study (n=3,494 subjects) from the EPIC-Norfolk Study (744,753). These associations were independent of many other co-founding factors, including HDL-C, T2DM, obesity, LDL-C, and age amongst others (744).

 

In addition to HDL cholesterol efflux and functional indices as risk predictors, HDL particle number (HDL-P) has also been reported to provide biomarker potential. HDL-P numbers can be quantified using nuclear magnetic resonance (754)or calibrated ion mobility assays (755). HDL-P was found to be inversely associated with carotid intima medial thickness (cIMT) and coronary heart disease (CHD) independent of LDL particle numbers and HDL-C levels in the large multi-ethnic study of atherosclerosis (MESA) (756). Importantly, HDL-P remains inversely associated to CHD after adjusting for triglycerides and apolipoprotein B (apoB), thus suggesting that HDL-P is far superior to HDL-C levels as a biomarker of ASCVD and events (757,758). Furthermore, neither HDL-C levels nor HDL-P levels correlate to cholesterol efflux from macrophages; therefore, the rate of cholesterol efflux is still critical to understanding RCT and HDL function. Likewise, HDL quality is more important than apoA-I levels, which also do not correlate with HDL function, e.g. RCT (759). Serum samples with identical apoA-I and HDL-C levels were found to have differing cholesterol acceptance capacities, mostly due to pre-beta HDL levels, which contributed to altered ABCA1-mediated cholesterol efflux (759). These studies strongly suggest that HDL function (cholesterol efflux capacity), as opposed to HDL-C, HDL-P, and apoA-I levels, provide a more important risk assessment and better predictor of future events as well as a more reasoned therapeutic target for reducing CVD risk and events. However, clinical assays for apoA-I and HDL-P are widely available and well-established, whereas assays for cholesterol efflux capacity have not been standardized and remain a research tool at present.

 

HDL Composition and Analysis

 

Historically, HDL have been isolated by density-gradient ultracentrifugation (DGUC) based on isopycnic equilibrium, and HDL have been defined by their density 1.063-1.21 g/mL since the 1950s (760,761). Based on mass, HDL can also be separated from other lipoproteins by size-exclusion chromatography (fast protein liquid chromatography, FPLC), and HDL’s molecular weight ranges from 175,000 - 360,000 Da (762). In addition to DGUC and FPLC, affinity chromatography can also be used to purify HDL from plasma using antibodies against apoA-I (763)or apoA-II, as HDL heterogeneity includes particles containing apoA-I:apoA-II (75%) or apoA-I only (25%) (763,764). Furthermore, asymmetric flow field-flow fractionation is now being used to isolate and characterize HDL (765). HDL can also be separated by non-denaturing gradient gel electrophoresis, e.g. polyacrylamide gel electrophoresis. Large HDL (HDL₂, 8.8-12.9 nm in diameter) and small HDL (HDL₃, 7.2-8.8 nm) are both α migrating particles (high negative charge), whereas pre-β HDL (5.4-7 nm) are β migrating particles for which they are defined. To quantify pre-β HDL particles, 2-D gel electrophoresis is often used to separate pre-β from mature HDL (766). HDL-P numbers can be quantified by either nuclear magnetic resonance spectroscopy or calibrated ion mobility assays. HDL can also be quantified and qualified by other methods, including vertical rotor ultracentrifugation, and transmission electron microscopy.

 

HDL are very dynamic and should be acknowledged as a heterogeneous pool of sub-classes with differing sizes, shapes, densities, protein compositions, and lipid diversity. Lipid-free apoA-I is secreted from the liver and small intestine as an amphipathic helix, and it quickly becomes lipidated by ABCA1 to form pre-β HDL, which then becomes discoidal after accepting phospholipid and free cholesterol from hepatocytes and peripheral cells. Upon further lipidation and cholesterol accumulation and esterification, nascent spherical HDL forms that range 7-12 nm in diameter. Mature HDL contains 3-4 apoA-I molecules of which 1 remains on the particle and the other apoA-I are free to (dis)associate (exchange) on and off the particle with other HDL. This is predominantly associated with rearrangement of HDL’s aqueous phase and surface area (767). As such, HDL are in a constant state of remodeling and interconversion. Each spherical HDL particle has approximately 50-130 phospholipids, 10-50 free cholesterol molecules, 30-90 CE molecules, and 10-20 triglyceride (TG) molecules (536). Phosphatidylcholine makes up the largest amount of lipid on HDL (approximately 90%); however, over 200 species of lipids have been reported, including sphingolipids, acylglycerols, isoprenoids, glycerophospholipids, and vitamins (768,769). The HDL proteome has been extensively studied and there is a general consensus of approximately 80 proteins (770,771). In addition to apoA-I and apoA-II, HDL transports over a dozen other apolipoproteins, as well as many enzymes and other factors. HDL have also been found to transport small RNAs, namely microRNAs (miRNA), which were found to be altered in hypercholesterolemia and atherosclerosis (772,773). Most interestingly, HDL have been demonstrated to transport a wide-variety of exogenous non-host small RNAs, including rRNA and tRNA fragments derived from bacterial and fungal species present in the microbiome and environment (774).The size of HDL is determined by the amount of CE and triglyceride (TG) in the hydrophobic core, and HDL is generally separated into 5 sub-classes based on size. Distinct HDL sub-species have been associated with CVD risk, and the sub-species have differential biological functions, e.g. large HDL are less anti-inflammatory (775-777). Many of the cargo or components of HDL are enriched in the small HDL sub-class which provides many of the alternative functions to the total HDL pool (778,779). The concentration of all HDL particles in plasma is approximately 20 umol/L; however, small HDL particles are the most abundant sub-class at approximately 10 umol/L. HDL are heterogeneous particles that transport a wide-variety of proteins, lipids, and nucleic acids, which confer many of HDL’s biological properties and beneficial functions in health and dysfunction in specific diseases.

 

HDL Cell Signaling

 

Many of HDL’s cellular functions – cell survival, proliferation, vasodilation -- are mediated by HDL-induced cell signaling cascades (780). As such, HDL can be characterized as hormone-like agonists. Although substantial work still remains in identifying HDL binding proteins and receptors on the cell surface, HDL have been found to activate many signaling cascades through various receptors. The most studied example of this is HDL’s ability to bind to the plasma membrane and through cell signaling mobilize cholesterol from intracellular stores in organelles to the plasma membrane for efflux. This has been attributed to HDL-induced activation of protein kinase C (PKC) (781). Specifically, apoA-I binds to ABCA1 and activates phosphatidylcholine lipases, which activate PKC leading to the movement of cellular cholesterol from intracellular stores to the plasma membrane for efflux, as well as PKC-mediated phosphorylation of ABCA1, which increases the transporter’s stability and efflux activity (782-784). This is a prime example of HDL-induced cell signaling that contributes to HDL cholesterol efflux capacity, which reduces the cholesterol burden for macrophages in the lesion, prevents foam cell formation, and antagonizes atherogenesis. Other HDL-induced signaling pathways that result in increased cholesterol and lipid efflux include protein kinase A (PKA) (785,786), cell division control protein 42 (Cdc42) (787), and Janus kinases-2 (JAK2) (788,789)cascades. HDL (i.e. apoA-I)-induced cell signaling through ABCA1 also suppresses macrophage M1 phenotype activation and pro-inflammatory cytokine production (Figure 10), and promotes M2 phenotype anti-inflammatory cytokine secretion (e.g. interleukin 10 (IL-10)) through JAK2 signaling and activation of signal transducer and activator of transcription 3 (STAT3) (75). In addition, the apoA-I:ABCA1:JAK2 axis was reported to suppress inflammation in endothelial cells through cyclooxygenase-2 (COX-2) activation leading to increased prostaglandins (PGI₂), which also suppresses atherogenesis (790). HDL have also been reported to induce cell signaling through SR-BI. HDL binding to SR-BI’s extracellular loop was reported to trigger activation of SR-BI’s cytoplasmic C terminal domain leading to the phosphorylation of protein kinase Src and activation of both liver kinase B1 (LKB1) and calmodulin-dependent protein kinase (CAMK) (791,792). This results in cell signaling through downstream kinases – AMP-activated protein kinases (AMPK) (792), protein kinase Akt (791), and mitogen-activated protein kinase (MAPK)(791)– which ultimately regulates angiogenesis (ubiquitin ligase Siah (Siah1/2) and hypoxia-inducible factor 1α (HIF1α) (793)), insulin sensitivity (glucose transporter 4 (Glut4)(794)), re-vascularization (Rac1(795)), and vasodilation (COX(796), endothelial nitric oxide synthase (eNOS)(797,798)). Interestingly, macrophage SR-BI has recently been shown to mediate efferocytosis (phagocytosis of dead cells) in the setting of atherosclerosis via a Src/Akt/Rac1 signaling pathway, reducing necrosis in lesions (185). All of these downstream effects contribute to HDL function, and to a lesser degree atherogenesis.

 

The most robust HDL signaling activation is mediated by bioactive lipids on HDL, namely the lysosphingolipid sphingosine-1-phosphate (S1P). A majority of S1P in circulation is associated with HDL, and HDL-S1P activates the G-coupled S1P receptors (S1P_1-5) on the surface of many vascular cell types, including macrophages, endothelial cells, and smooth muscle cells. Activation of S1P₁and S1P₂ receptors turns on a host of signaling cascades and factors that directly contribute to the many anti-atherogenic properties of HDL, including increasing endothelial barrier function (799)and angiogenesis (800,801)while decreasing inflammation (802)and apoptosis (803). HDL were also found to inhibit smooth muscle migration through S1P signaling, a key factor in restenosis and plaque development (804). All of these are critical processes to atherogenesis. In support of these studies, subjects with CAD were found to have decreased HDL-S1P levels (805). The key terminal effector factors in these G-protein receptor signaling cascades are focal adhesion kinase (FAK), nuclear factor κ beta (NF-κB), nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, eNOS, STAT3, and B-cell lymphoma-extra-large (Bcl-xl) (780). This HDL-S1P signaling pathway has also been linked to vasorelaxation (806)and cytoprotection (e.g. cardiomyocytes) (807). In addition to these direct pathways, HDL also likely activates cell signaling indirectly through ATP (β-ATPase/P2Y_12/13)(808)or toll-like receptors (809). Collectively, HDL induced cell signaling in vascular and inflammatory cells underlies HDL’s anti-atherogenic properties in health, and deficits in HDL signaling likely link HDL dysfunction in metabolic diseases to increased risk of atherosclerosis.

 

Anti-Inflammatory HDL

 

Outside reverse cholesterol transport, HDL’s anti-inflammatory properties have been the most extensively studied HDL function and likely play a large role in HDL’s anti-atherogenicity (Figure 10). HDL’s anti-inflammatory properties are conferred by numerous mechanisms in many types of cells. In addition to providing the vascular barrier, endothelial cells control vascular inflammation through expressing adhesion molecules that aid in monocyte adhesion and ultimate migration into the atherosclerotic lesion. Moreover, activated endothelial cells secrete cytokines and recruit monocytes through chemokine release. The induction of adhesion molecules, cytokines, and chemokines in activated endothelial cells is largely due to NF-B transcriptional activation. In humans, injection of apoA-I resulted in decreased adhesion molecule expression in atherosclerotic plaques (810). One mechanism by which HDL suppresses endothelial cell and monocyte activation is through inhibiting NF-kB activity by attenuating IkB kinase activity (811). Nonetheless, HDL decreases adhesion molecule expression through multiple mechanisms. Cells pre-treated with HDL or apoA-I are protected from TNFα or oxidized LDL (oxLDL)-induced adhesion molecule expression. In addition, HDL binding to SR-BI may also contribute to inhibition of adhesion molecule expression, as SR-BI-mediated Akt activation promoted heme oxygenase-1 expression. In addition, up-regulation of 3-beta-hydroxysteroid-delta 24 (DHCR24) by HDL binding to SR-BI was reported to underlie HDL’s ability to suppress adhesion molecules (812). Furthermore, HDL suppression of intracellular adhesion molecule-1 (ICAM-1) in endothelial cells was found to be mediated, in part, through the transfer of miR-223 to recipient cells (773). Recent studies also suggest that TGFβ and AMPK also contribute to HDL’s suppression of adhesion molecule expression (813).

 

In addition to HDL’s profound effects on vascular endothelium, HDL suppresses myelopoiesis, monocyte recruitment, macrophage activation, proliferation, and emigration from atherosclerotic lesions. Similar to its impact on endothelial cells, HDL also suppresses adhesion molecule expression in monocytes, which inhibits monocyte adhesion and migration to atherosclerotic lesions (814). HDL and apoA-I were demonstrated to suppress CD11b expression on human monocytes through both ABCA1-dependent and independent mechanisms (814). HDL inhibition of monocyte activation, which includes suppression of cytokines and adhesion molecules, is mediated through both peroxisome proliferator-activated receptor gamma (PPARγ) and NF-kB transcription factors (815). Suppression of chemokine and cytokines in myeloid cells inhibits infiltration and migration of circulating monocytes, and thus antagonizes atherosclerosis. HDL have also been reported to mediate macrophage reprogramming through the transcription factor ATF3 that reduces Toll-like receptor signaling (816). Importantly, much of HDL’s (and apoA-I’s) inhibition of macrophage activation is mediated through altering cholesterol levels in plasma membrane lipid rafts through cholesterol efflux mediated by ABCG1/SR-BI and ABCA1; however, apoA-I induced signaling through ABCA1 and the JAK/STAT pathway independent of cholesterol efflux may also contribute to HDL’s effect, as described above (75,817)(814,818). HDL have also been demonstrated to promote macrophage emigration through removing excess cholesterol and induction of signaling pathways (208). In addition to HDL’s impact on monocytes and macrophages, HDL also strongly suppresses neutrophil activation and vascular smooth muscle cell secretion of monocyte chemoattractant protein-1 (MCP1) (819).

 

In addition to HDL’s roles in innate immunity, recent evidence suggests that HDL play multiple roles in adaptive immunity (820). Mice lacking apoA-I develop autoimmunity when challenged with a high cholesterol (diet and background, Ldlr^-/-), which includes T cell activation and production of autoantibodies (821,822). This phenotype was rescued by apoA-I injections. HDL have also been reported to repress both antigen-presenting cell (APC) activation of T cells and T cell activation of monocytes, thus preventing the secretion of proinflammatory cytokines and chemokines (Figure 10) (823,824). ApoA-I also prevents the phenotypic switching of T-regs into pro-inflammatory follicular helper T cells during atheroprogression (92). Moreover, cholesterol efflux to HDL and apoA-I have been reported to suppress myelopoiesis and proliferation of myelopoietic stem and progenitor cells, as loss of function for both Abca1and Abcg1in mice resulted in increased myelopoiesis (820). Injection of apoA-I was also found to rescue this phenotype (825). In addition, HDL and cholesterol efflux were reported to suppress megakaryocyte progenitor proliferation, platelet levels, and thrombocytosis (826). Collectively, HDL and apoA-I inhibit circulating levels of hematopoietic progenitor cells, monocytes, neutrophils, and platelets all of which contribute to HDL’s capacity to limit inflammation and atherosclerosis.

 

Antithrombotic HDL

 

Another anti-atherogenic function of HDL is the capacity to directly and indirectly inhibit platelet activation, aggregation, and thrombus formation (Figure 10). HDL-C levels were found to be inversely associated with thrombus formation in humans (827). HDL is required to remove excess cholesterol from the plasma membrane of platelets for proper function, and platelets isolated from mice lacking SR-BI to mediate cholesterol efflux to HDL were found to be more susceptible to activation (828,829). Both HDL and cyclodextrin-mediated cholesterol efflux were found to inhibit platelet aggregation (828). However, HDL-induced cell signaling through binding to glycoprotein IIb/IIIa on the surface of platelets was reported to activate phospholipase C (PLC) and PKC, thus leading to flux through the Na+/H+ antiport system (717). This pathway can result in alkalization of the cytoplasm and calcium release, which can reduce platelet activation (830). Furthermore, HDL dose-dependently inhibits stimulated platelet activation, which leads to reduced platelet aggregation, granule secretion and fibrinogen binding. In rats, apoA-I injections inhibited thrombus formation and reduced thrombus mass (831). HDL’s anti-thrombotic effects are also mediated, in part, through HDL’s ability to inhibit tissue factor and factors X, Va, and VIIIa (Figure 10)(832). HDL also prevents thrombus formation through cell signaling and nitric oxide (NO) production in endothelial cells (828), and suppression of tissue factor and platelet-activating factor expression in endothelial cells (833,834). HDL also reduces erythrocyte influence on thrombus formation (835). Collectively, HDL has multiple biological mechanisms that inhibit thrombus formation, and thus, contribute to HDL’s anti-atherogenic properties.

 

Pro-Vasodilatory HDL

 

The endothelium significantly contributes to vascular tone, and HDL confer protection against endothelial cell activation, apoptosis, and loss of barrier function, which is critical to atherogenesis. HDL have been reported to induce endothelium-dependent vasodilation in aortic rings (806), and individuals with low HDL have reduced endothelium-dependent vasorelaxation (Figure 10) (741). HDL’s benefit to endothelial cells is largely mediated by cell signaling through phosphatidylinositol 3-kinase (PI3K) and Akt and is induced by bioactive lipids and associated proteins on HDL, including lysosulfatide, S1P, and sphingosylphosphorylcholine (SPC) (791,798,806). A key outcome of HDL-induced cell signaling is the production of NO (Figure 10) through both signaling induced phosphorylation of eNOS and increased eNOS expression (791,836). HDL can trigger eNOS-phosphorylation through SR-BI, S1P receptor (S1P_1-5), and ABCG1-mediated cholesterol efflux (806,837). HDL-induced NO underlies many of HDL’s beneficial properties to endothelial cells, including HDL-induced vasodilation, tightening of cell-to-cell junctions and increased barrier function, differentiation of endothelial progenitor cells, cell survival and proliferation, cell migration, inhibition of apoptosis, and suppression of adhesion molecule expression. In addition, HDL also has NO-independent properties on endothelial cells, including induced proliferation, increased barrier function, suppressed inflammation and decreased apoptosis (838). These studies clearly define a beneficial role for HDL in vascular integrity, which underlies HDL protection against atherosclerosis.

 

Anti-Apoptotic HDL

 

HDL have multiple anti-apoptotic properties that enhance cell survival (Figure 10). By various metrics, HDL support mitochondrial function and prevent the release of apoptotic signals, including cytochrome C (205,839). Moreover, HDL drives the expression of Bcl-xl, which is a strong anti-apoptotic factor and suppresses Bid, which is a pro-apoptic protein (839,840). HDL mediates these gene expression changes through cell signaling and NO production through activation of surface receptors by HDL-associated proteins and bioactive lipids, including apolipoprotein J (apoJ) and S1P (803,840). In addition, there are likely alternative anti-apoptotic mechanisms resulting from HDL-induced signaling. Nonetheless, HDL has been demonstrated to suppress apoptosis in endothelial cells (Figure 10) activated with tumor necrosis factor (TNFα) and oxLDL (839,841,842). HDL proteins (apolipoprotein M, apoM) and apoM-binding lipids (S1P) contribute to HDL’s ability to increase tight junctions and endothelial cell survival (843). Mice deficient in apoM have reduced S1P levels and loss of endothelium barrier function (843). HDL’s ability to support the endothelium barrier function is a key feature of its anti-atherosclerosis properties and represents a classic example of HDL’s control of cellular gene expression and phenotype that are beneficial to vascular health. However, HDL also have many capacities in the extracellular space (e.g. plasma) that protect against atherosclerosis.

 

Anti-Oxidative HDL

 

A key factor in monocyte activation and chemotaxis in the vascular wall is the accumulation of oxLDL, which is more pro-inflammatory and pro-atherogenic than unmodified LDL. LDL can become oxidized by a variety of endogenous mechanisms (844). In the vascular wall, LDL can be modified (oxidized) by many cell types, including vascular smooth muscle cells, endothelial cells, and macrophages (776). Remarkably, HDL prevents the oxidation of LDL (Figure 10) and recent evidence suggests that this may occur through 4 distinct proteins circulating on HDL – apoA-I (845,846), LCAT (847), lipoprotein-associated phospholipase A2 (Lp-PLA2)(848,849), and paraoxonase 1 (PON1) (430,846). First, HDL can simply soak up oxidized lipids or oxidizing factors from cells preventing their association with LDL and their modification of LDL lipids and proteins. In addition, HDL removes lipid hydroperoxides from LDL particles (846). Specifically, small apoAI containing HDL particles are the most efficient at accepting lipid hydroperoxides, which are reduced to their inactive lipid hydroxides via oxidation of the methionine residues in apoA-I (850). Compared to apoA-II the methionine residues in apoA-I are more conformationally conducive to reducing lipid hydroperoxides (851,852). In addition, HDL with low surface free cholesterol and sphingomyelin are more efficient at accepting lipid hydroperoxides (745,853). The capacity of HDL to prevent oxidation via this mechanism is also maintained by the selective removal of HDL lipid hydroperoxides and hydroxides by hepatocyte SR-BI (854). In addition, ApoA-I methionine sulfoxide is reduced to methionine by methionine sulfoxide reductases.(850). LCAT circulates on HDL and has also been reported to block LDL oxidation, as LCAT over-expression in mice reduced LDL oxidation as determined by reduced LDL autoantibodies (855). Lp-PLA₂appears to be pro-atherogenic on LDL and anti-atherogenic on HDL (856). Its activity on HDL likely contributes to HDL’s anti-oxidative capacity, as inhibition of HDL-associated Lp-PLA₂attenuated HDL’s ability to block LDL oxidation (848). The strongest anti-oxidative HDL protein is likely PON1. Over-expression of PON1 in mice confers enhanced HDL anti-oxidative capacity, and PON1 itself prevents LDL oxidation in vitro (432). Most importantly, HDL isolated from mice lacking PON1 have reduced ability to prevent LDL oxidation. HDL’s anti-oxidative capacity likely plays a large role in preventing inflammation and atherogenesis, and like many of the other alternative functions, confer HDL’s beneficial role in health.

 

HDL Intercellular Communication

 

HDL also likely participate in intercellular communication through the transfer of nucleic acids between tissues. Recently, HDL have been reported to transport miRNA (Figure 10), which are small non-coding RNAs that suppress gene expression through binding to complimentary target sites in the 3’ untranslated region of mRNAs, and thus inhibit translation and induce mRNA degradation (772). Most interestingly, the HDL-miRNA profile is significantly altered in hypercholesterolemia and atherosclerosis (772). miRNAs have been reported to be exported from macrophages to HDL, and HDL has been demonstrated to transfer specific miRNAs to recipient hepatoma cells (Huh7) and endothelial cells, likely through HDL’s receptor SR-BI (773). In endothelial cells, HDL was found to deliver miR-223 to recipient cells, where it directly targeted intracellular adhesion molecule-1 (ICAM-1) expression (Figure 10), and thus inhibited neutrophil adhesion to the cells (773). miR-223 is not transcribed or processed in endothelial cells and HDL delivery of mature miR-223 to endothelium likely confers, in part, HDL’s anti-inflammatory capacity associated with adhesion molecule suppression. Future studies are needed to determine the physiological relevance and functional impact of HDL-miRNAs in humans and animal models in the context of atherosclerosis and other inflammatory diseases.

 

Anti-Infectious HDL

 

HDL also contributes to innate immunity by modulating immune cell function. However, this hypothesis has not been extensively studied in the context of atherosclerosis. HDL are anti-infectious, anti-parasitic, and anti-viral. HDL have the unique capacity to prevent endotoxic shock and readily binds to lipopolysaccharides (LPS) and contributes to removing LPS through biliary excretion thus aiding innate immunity (857-859). Amongst the many proteins that circulate on HDL, apolipoprotein L1 (apo-L1) (also known as trypanosome lytic factor) is present in specific sub-classes of HDL (860,861). This factor kills Trypanosome bruceiand Trypanosome brucei rhdesiense, parasites that cause sleeping sickness, through creating ionic pores in endosomes (860-862). Although promising, future studies are required to define how HDL regulation of innate immunity contributes to the inhibition of atherogenesis.

 

HDL Dysfunction

 

HDL confer many anti-atherogenic properties that are lost in atherosclerosis and other inflammatory and metabolic diseases. These include 9 key processes –

• Loss of cholesterol efflux capacity from macrophages
• Reduced ability to inhibit LDL oxidation
• Decreased vasodilation through reduced NO production in endothelial cells
• Reduced ability to inhibit monocyte chemotactic activity
• Loss of the ability to metabolize hydroperoxides on erythrocyte membranes
• Reduced ability to suppress TNFα-induced NF-κB activation and adhesion molecule expression
• Loss of anti-apoptotic capacity in endothelial cells
• Decreased capacity to block TNFα-induced NADPH oxidase activity and superoxide production
• Suppression of cytokine inhibition in activated inflammatory cells.

 

Many of these defects are due to changes in HDL cargo, e.g. decreased PON1 levels or increased serum amyloid A (SAA) levels. Moreover, changes in the content of bioactive lipids or increased oxidative modifications to HDL’s lipids and protein cargo likely confer dysfunction. HDL-miRNAs have been shown to be significantly altered in hypercholesterolemia and atherosclerosis (772). It is unknown how these changes contribute to HDL’s loss of anti-atherogenic properties, but they hold great potential for future studies. In CHD, acute coronary syndrome (ACS), and ischemic cardiomyopathy, HDL have reduced ability to inhibit oxidation of LDL, likely through reduced PON1 levels as reported in CHD (845,863,864). Loss of PON1 also reduces HDL’s ability to prevent oxidation of its own lipids and proteins, which has been reported in metabolic syndrome as oxidation of apoA-I impairs HDL’s RCT and anti-inflammatory functions (865). Reduced HDL-PON1 levels are also found in other cardiometabolic diseases, including type 2 diabetic mellitus (T2DM) (866,867), type 1 diabetes mellitus (T1DM) (868), rheumatoid arthritis (RA) (869,870), dyslipidemia (e.g. hyperalphalipoproteinemia (HALP) (871)), and patients after cardiac surgery (872). In subjects with ACS and CAD, HDL have been reported to have decreased ability to prevent endothelial cell apoptosis likely through decreased activation (phosphorylation) of Bcl-xl and increased activation of Bcl-2, which are anti-apoptotic and pro-apoptotic proteins, respectively (840). Loss of HDL’s anti-apoptotic capacity has been proposed to be due to increased apoCIII and possibly decreased clusterin levels on HDL (840). HDL from subjects with CHD also have decreased ability to prevent monocyte adhesion to endothelial cells and recruitment in arterial wall co-cultures, which could be associated with reduced PON1 levels amongst other cargo (845,873,874). HDL from ischemic cardiomyopathy and CAD subjects also have reduced cholesterol efflux acceptance capacity, which likely leads to increased foam cell formation in the atherosclerotic lesion and increased atherogenesis (746,863). Although the molecular basis for all of HDL’s loss of anti-atherogenicity in CHD is not known, other functions of HDL are compromised in these subjects, including the ability to reduce hydroperoxides on erythrocyte membranes (875). This loss of HDL’s anti-oxidant capacity is also found in T2DM (875)and T1DM (868,876). HDL in metabolic syndrome have been reported to have decreased capacity to prevent oxidation of LDL and inhibit endothelial cell apoptosis (877,878). This loss of anti-atherogenic properties is also found in hypertension(879), T2DM (867,880,881), end-stage renal disease (ESRD) (882,883), RA (869,870,884,885), systemic erythematosus lupus (SLE) (884), obstructive sleep apnea (886), and dyslipidemia (HALP) (871). Reduced ability of HDL to stimulate NO production from endothelial cells and decreased vasorelaxation properties are reported for T2DM (887,888), T1DM (889), mild chronic kidney disease (CKD) (890), and rare forms of autoimmunity (ALPS) (891). Loss of HDL-mediated cholesterol efflux capacity has been found in patients with hyperhomocysteinemia (892), sepsis (893), psoriasis (894), SLE (895), RA (885,896), ESRD (897,898), and T2DM (899,900). Not only does HDL dysfunction result from loss of key proteins and cargo, HDL can gain pro-atherogenic cargo and properties in cardiometabolic diseases. Due to loss of PON1, HDL accumulate malonaldehydes, which inhibits NO production through increased phosphorylation of eNOS through LOX-1 receptor signaling (901).

 

HDL Summary

 

Years of sound epidemiological studies have clearly established an inverse relationship between HDL-C levels and risk of CVD. Nevertheless, recent GWAS studies suggest that individuals with high HDL-C levels are not protected from CVD. Furthermore, clinical studies aimed at raising HDL-C levels through niacin and CETP inhibitors have failed to reduce risk of cardiovascular events and have been stopped prematurely due to lack of efficacy or increased number of events. Although they’re often lumped together, HDL-C levels do not represent HDL particle numbers or HDL function (e.g. cholesterol efflux capacity); both of which have been reported to be better indicators of CVD risk than HDL-C. In addition to HDL’s transport of cholesterol and lipids in the RCT pathway, HDL transports a wide-variety of cargo, including a diverse group of proteins, small RNAs, bioactive lipids, and many other small molecules. These alternative cargos may confer many of HDL’s alternative functions outside of RCT. In fact, HDL have many beneficial properties, including anti-inflammatory, anti-oxidative, anti-thrombotic, anti-infectious, anti-apoptotic, intercellular communication, and pro-vasodilatory capacities. Recently, HDL dysfunction has been reported in many cardiometabolic diseases, including CAD, T2D, and CKD. Current and future challenges include the need to better define HDL anti-atherogenic properties in health and pro-atherogenic influences in disease to better control HDL function to potentially prevent and treat CVD.

 

ACKNOWLEGEMENTS

:

This work was supported in part by National Institutes of Health grants HL116263 and HL127173.

 

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