2.3.1 Pathogenesis of atherosclerosis
Atherosclerosis is a progressive disease of arteries and the primary cause of myocardial infarction and stroke (168). Arteries are composed of three distinct layers:
tunica intima, tunica media and tunica adventitia (169) (Figure 6). Formation of plaques in artery walls (i.e. atherogenesis) takes place in arterial intima which is the innermost layer of an artery. The intima is separated from the vascular lumen by a single layer of endothelial cells and from the media by the internal elastic lamina (169,170). The outermost layer of an artery, the adventitia, is mainly composed of fibroblasts, connective tissue, perivascular nerves and lymphatic vessels (171). The adventitia is separated from the media by the external elastic lamina. The intima is divided into two layers: a proteoglycan-rich layer with synthetic smooth muscle cells and an underlying musculoelastic layer composed of smooth muscle cells (SMC), collagen and elastic fibers (170) (Figure 6). The proteoglycan-rich layer also contains
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isolated macrophages near the endothelium. Circulating lipoproteins up to 70 nm in diameter (small chylomicron remnants and VLDL, IDL, LDL and HDL particles) can be transported across the intact endothelium by transcytosis (7,172). Lipoproteins that have entered the intima may bind to proteoglycans in the extracellular matrix, such as biglycan, perlecan and versican. Proteoglycans are negatively charged due to glycosaminoglycans, which can interact with the positively charged aminoacyl residues of apoB. Retention of lipoproteins within the artery wall is considered the initiating event in atherogenesis (173).
Retained lipoproteins, mainly LDL particles, are exposed to several modifications, such as oxidation and lipolysis (174). Modifications of the LDL surface components may decrease its stability, which can lead to the interactions between LDL particles, i.e. aggregation or fusion (8) (Figure 6). Modified LDL particles trigger several cellular responses within the artery wall, such as the secretion of interleukin-8 (IL-8) from endothelial cells, which induce the chemotactic recruitment of monocytes to the arterial intima (7,175). Modifications also increase the binding strength of lipoproteins and prevent their exit back into the bloodstream (176–178). Modified particles are mainly cleared by macrophages, but as a result of extensive lipid accumulation, SMC also phagocytize these particles (179). Uptake of lipids by macrophages and SMC results in the formation of foam cells. Retained and modified lipoproteins and macrophages trigger a cascade of inflammatory responses, which leads to the release of pro-inflammatory cytokines and bridging molecules, SMC migration and proliferation, induced synthesis of proteoglycans and eventually further entrapment of lipoproteins (7,8) (Figure 6). Accumulation of foam cells leads to the formation of fatty streaks – the first recognizable atherosclerotic lesions (180).
Increasing accumulation of lipoprotein-derived lipids leads to the apoptosis and necrosis of foam cells (180). At the same time the clearance of apoptotic cells deteriorates and together with the release of the lipid-rich cargo and accumulation of necrotic debris causes the formation of a necrotic core. Lesion progression involves thickening of the intima due to extensive lipid accumulation and deposition of extracellular matrix components and eventually the development of a fibrous cap over the necrotic core. The fibrous cap is predominantly composed of SMC and extracellular matrix components. Several inflammatory processes may cause thinning of the cap, which makes it prone to rupture (181). For example, aggregated LDL particles can induce the foam cell secretion of matrix metalloproteinase 7 (182), a proteinase that could contribute to the rupture of atherosclerotic plaques (183).
Once the cap ruptures, the contents of the necrotic core are exposed to the blood circulation and its coagulation factors (184). The blood enters the plaque core from the lumen and activates platelets and the cascade of coagulation, causing thrombosis, which can occlude the arterial lumen.
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Figure 6. Lipid retention and cholesterol efflux in the atherosclerotic arterial intima.
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2.3.2 Anti-atherogenic lipoprotein functions
Like progression of atherogenesis, also anti-atherogenic functions of HDL occur at the arterial intima (185). RCT may counteract the pathogenic events of atherosclerosis by promoting HDL-mediated removal of cholesterol from macrophages in atherosclerotic plaques (Figure 6). This process, known as cholesterol efflux, is considered the primary antiatherogenic mechanism of HDL, and it has been found to be inversely associated with the risk of CHD (185–187). RCT consists of transport of cholesterol by HDL from macrophage foam cells via the lymphatic system to the bloodstream and ultimately to liver for final excretion into the feces (185). Excretion can occur either directly as cholesterol or after metabolic conversion into bile acids (185).
Accumulated cholesterol can be transferred from macrophages to various HDL subpopulations by multiple pathways (Figure 7). Transport can occur passively via diffusion or via scavenger receptor-B1 (SR-B1) and actively via adenosine triphosphate (ATP) binding cassette transporter A1 (ABCA1) or ATP binding cassette transporter G1 (ABCG1) (188). In addition to apoA-I, also other HDL apolipoproteins, such as apoE, can accept cholesterol via ABCA1-mediated transport. In cholesterol efflux assays, different cell lines, different macrophages and various acceptors have been used to quantify the rate of cholesterol efflux from cultured cells and to investigate the specific pathways related to this process (189,190). As donor cells for example, Fu5AH cells have been used to measure the SR-B1-dependent cholesterol efflux. Furthermore, J774 macrophages have been often used to study the mediated and THP-1 monocyte-macrophages the ABCA1-and ABCG1-mediated cholesterol efflux. As acceptors, HDL particles have often been used to determine the ABCG1- and SR-B1-mediated pathways, whereas apoA-I has been used to study the ABCA1-dependent pathway (189).
Figure 7. Cholesterol efflux pathways. ABCA1, adenosine triphosphate binding cassette transporter A1; ABCG1, adenosine triphosphate binding cassette transporter G1; apoA-I, apolipoprotein A-I; SR-B1, scavenger receptor-B1. Adapted from references (188,191).
47 In addition to cholesterol efflux, HDL particles have been found to have anti-atherogenic effects on several processes and functions critically involved in atherosclerosis (120,122). For example, HDL particles have oxidative and anti-inflammatory properties. Therefore, in atheroclerotic plaques, HDL particles may prevent the oxidation of LDL and suppress several pro-inflammatory factors, such as the expression of adhesion molecules, monocyte chemoattractants and inflammatory cytokine production of macrophages (122). Furthermore, HDL particles have the ability to inhibit the apoptosis of macrophage foam cells.
Alterations in the profile and structure of HDL particles due to pathologic conditions, such as diabetes, dyslipidemia or inflammation, may impair the normal functions of HDL and play a central role in the progression of atherosclerosis (192) (Figure 6). The formation of dysfunctional HDL comprises alterations in the enzymatic functions related to HDL metabolism and structural changes in HDL particles (192,193). Changes in the activities of enzymes involved with HDL metabolism, such as LCAT or CETP, affect the maturation and remodeling of HDL particles. Altered enzyme activities lead to the enrichment of HDL particles with TG and loss of CE, reduced concentration of total HDL cholesterol and accumulation of small HDL particles. A decreased CE/TG ratio leads to unstable HDL particles, which are rapidly cleared from the circulation. Furthermore, changes in the HDL lipidome, such as loss of PL or increase in TG, impair antioxidative and cholesterol efflux capacity (122,190). PL composition of HDL largely determines the cholesterol efflux capacity. Changes in HDL PL, such as increased content of sphingomyelin, makes the PL layer more rigid. In addition to alterations in the lipidome, changes in protein content of HDL occur in metabolic disturbances. During inflammation serum amyloid A (SAA) replaces apoA-I in HDL particles, which reduces the cholesterol efflux and antioxidative capacity of HDL (192). Cholesterol efflux capacity may be impaired also due to oxidation or glycation of HDL particles (192,193).
Apolipoprotein E (apoE) and SAA contain domains that can bind to proteoglycans (194,195). Therefore, also HDL has a potential to bind to proteoglycans. HDL particles containing apoE have been shown to interfere with the formation of LDL-proteoglycan complex by apoE-mediated binding (195). This competition of binding sites could potentially prevent LDL accumulation in the artery wall. However, retained HDL particles are susceptible to the same modifications as LDL particles in the intima (185). This could eventually increase the cholesterol burden in the arterial tissue.
2.3.3 The effects ofn-3 fatty acids on pro- and antiatherogenic lipoprotein functions
Several mechanisms in the development and progression of atherosclerosis have been shown to be modulated byn-3 PUFAs (196,197). For example,n-3 PUFAs have been found to reduce the production of pro-inflammatory cytokines, adhesion and migration of monocytes and proliferation and migration of SMC into arterial intima (196,197). Furthermore, several findings suggest that atherosclerotic plaques are
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responsive to dietary modification (198–200). Incorporation of long-chainn-3 PUFAs in the atherosclerotic plaques have been associated with a reduced number of foam cells and T cells, less inflammation in the plaque and increased thickness of fibrous cap (198–200). These changes have been found to stabilize the plaques making them less vulnerable to rupture. Several molecular pathways are also modulated by the incorporation ofn-3 PUFAs into cell membranes, which enhances the fluidity of the particles (196). The fluidity of HDL particles have been found to be directly associated with the cholesterol efflux capacity (201).
In human intervention studies, dietary fat has been found to affect cholesterol efflux capacity (Table 8). However, it remains unclear to what degreen-3 PUFAs mediate these effects. Even less is known about the effects of dietary fat orn-3 PUFAs on other lipoprotein functions related to atherosclerosis. However, diets enriched with high-oleic canola oil and corn/safflower oil have been found to decrease the binding of LDL particles to biglycan, a proteoglycan found in human atherosclerotic plaques (202). Furthermore, increased vitamin E and decreased sucrose in the diet has been found to be positively associated with decreased aggregation susceptibility (182).