Accumulation of Plasma-Derived Lipids in the Lipid Core and Necrotic Core of Human Atheroma: Imaging Mass Spectrometry and Histopathological Analyses

Objective: To clarify the pathogenesis of human atheroma, the origin of deposited lipids, the developmental mechanism of liponecrotic tissue, and the significance of the oxidation of phospholipids were investigated using mass spectrometry-aided imaging and immunohistochemistry. Approach and Results: Atherosclerotic lesions in human coronary arteries were divided into 3 groups: pathological intimal thickening with lipid pool, atheroma with lipid core, and atheroma with necrotic core. The lipid pool and lipid core were characterized by the deposition of extracellular lipids. The necrotic core comprised extracellular lipids and liponecrotic tissue. The proportion of cholesteryl linoleate in cholesteryl linoleate+cholesteryl oleate fraction in the extracellular lipid and liponecrotic regions differed significantly from that of the macrophage foam cell–dominant region, and the plasma-derived components (apoB and fibrinogen) were localized in the regions. The liponecrotic region was devoid of elastic and collagen fibers and accompanied by macrophage infiltration in the surrounding tissue. Non–oxidized phospholipid (Non-OxPL), OxPL, and Mox macrophages were detected in the three lesions. In the atheroma with lipid core and atheroma with necrotic core, non-OxPL tended to localize in the superficial layer, whereas OxPL was distributed evenly. Mox macrophages were colocalized with OxPL epitopes. Conclusions: In human atherosclerosis, plasma-derived lipids accumulate to form the lipid pool of pathological intimal thickening, lipid core of atheroma with lipid core, and necrotic core of atheroma with necrotic core. The liponecrotic tissue in the necrotic core appears to be developed by the loss of elastic and collagen fibers. Non-OxPL in the accumulated lipids is oxidized to form OxPL, which may contribute to the lesion development through Mox macrophages.

Cross-linking modifications of HDL apoproteins by oxidized phospholipids: structural characterization, in vivo detection, and functional implications

Apolipoprotein A-I (apoA-I) is cross-linked and dysfunctional in human atheroma. Although multiple mechanisms of apoA-I cross-linking have been demonstrated in vitro, the in vivo mechanisms of cross-linking are not well-established. We have recently demonstrated the highly selective and efficient modification of high-density lipoprotein (HDL) apoproteins by endogenous oxidized phospholipids (oxPLs), including γ-ketoalkenal phospholipids. In the current study, we report that γ-ketoalkenal phospholipids effectively cross-link apoproteins in HDL. We further demonstrate that cross-linking impairs the cholesterol efflux mediated by apoA-I or HDL3 in vitro and in vivo. Using LC-MS/MS analysis, we analyzed the pattern of apoprotein cross-linking in isolated human HDL either by synthetic γ-ketoalkenal phospholipids or by oxPLs generated during HDL oxidation in plasma by the physiologically relevant MPO-H2O2-NO2− system. We found that five histidine residues in helices 5–8 of apoA-I are preferably cross-linked by oxPLs, forming stable pyrrole adducts with lysine residues in the helices 3–4 of another apoA-I or in the central domain of apoA-II. We also identified cross-links of apoA-I and apoA-II with two minor HDL apoproteins, apoA-IV and apoE. We detected a similar pattern of apoprotein cross-linking in oxidized murine HDL. We further detected oxPL cross-link adducts of HDL apoproteins in plasma and aorta of hyperlipidemic LDLR−/− mice, including cross-link adducts of apoA-I His-165–apoA-I Lys-93, apoA-I His-154–apoA-I Lys-105, apoA-I His-154–apoA-IV Lys-149, and apoA-II Lys-30–apoE His-227. These findings suggest an important mechanism that contributes to the loss of HDL's atheroprotective function in vivo.

Skap2 Regulates Atherosclerosis through Macrophage Polarization and Efferocytosis

ABSTRACTRationaleAtherosclerosis causes more deaths than any other pathophysiologic process. It has a well-established inflammatory, macrophage-mediated component, but important and potentially protective intracellular macrophage processes in atherosclerosis remain enigmatic. Src Kinase-Associated Phosphoprotein 2 (Skap2) is a macrophage-predominant adaptor protein critical for cytoskeletal reorganization, and thereby, for macrophage migration and chemotaxis. The role of macrophage Skap2 in atherosclerosis is unknown and deserves exploration.ObjectiveTo establish the critical role of Skap2 in macrophage-mediated atherosclerotic plaque homeostasis.ResultsIn human arterial gene expression analysis, Skap2 expression is enriched in macrophage-containing areas of human atheroma, and the transcript level varies with plaque characteristics. We have discovered that deletion of Skap2 accelerates atherosclerosis by threefold in ApoE-/- mice on standard diet. Skap2 expression is switched on only as monocytes differentiate into macrophages, so Skap2-/- monocytes have no defect in infiltrating the atheroma. On the other hand, once they fully differentiate, Skap2-deficient macrophages cannot polarize efficiently into alternatively-activated, regulatory cells, and instead they preferentially polarize toward the classical pro-inflammatory phenotype both ex vivo and within the developing atheroma. This defect extends to polarized effector functions, as ex vivo analysis of macrophage phagocytosis of dying foam cells indicates that Skap2 is required for the regulatory process of efferocytosis.ConclusionsTaken together, our findings support a model in which Skap2 drives a regulatory, efferocytic mode of behavior to quell atherosclerosis.CONDENSED ABSTRACT / SUMMARYSkap2—a macrophage protein found in the human atheroma—is atheroprotective. Skap2-null mice, whose foam cells do not migrate well due to a defect in integrin-induced cytoskeletal rearrangement, have accelerated atherosclerosis. Skap2 is not expressed in monocytes but becomes important once they reach the atheroma and become macrophage foam cells, at which point it drives toward a regulatory, anti-inflammatory polarization state required for efficient efferocytosis of dying foam cells. Thus, Skap2 drives a protective, regulatory mode of behavior, supporting the fact that macrophages are not solely deleterious in atherosclerosis, and further pointing to efferocytosis as a target for therapy.There are no relationships to disclose.

Hydrogen Sulfide Abrogates Hemoglobin-Lipid Interaction in Atherosclerotic Lesion

The infiltration of red blood cells into atheromatous plaques is implicated in atherogenesis. Inside the lesion, hemoglobin (Hb) is oxidized to ferri- and ferrylHb which exhibit prooxidant and proinflammatory activities. Cystathione gamma-lyase- (CSE-) derived H2S has been suggested to possess various antiatherogenic actions. Expression of CSE was upregulated predominantly in macrophages, foam cells, and myofibroblasts of human atherosclerotic lesions derived from carotid artery specimens of patients. A similar pattern was observed in aortic lesions of apolipoprotein E-deficient mice on high-fat diet. We identified several triggers for inducing CSE expression in macrophages and vascular smooth muscle cells including heme, ferrylHb, plaque lipids, oxidized low-density lipoprotein, tumor necrosis factor-α, and interleukin-1β. In the interplay between hemoglobin and atheroma lipids, H2S significantly mitigated oxidation of Hb preventing the formation of ferrylHb derivatives, therefore providing a novel function as a heme-redox-intermediate-scavenging antioxidant. By inhibiting Hb-lipid interactions, sulfide lowered oxidized Hb-mediated induction of adhesion molecules in endothelium and disruption of endothelial integrity. Exogenous H2S inhibited heme and Hb-mediated lipid oxidation of human atheroma-derived lipid and human complicated lesion. Our study suggests that the CSE/H2S system represents an atheroprotective pathway for removing or limiting the formation of oxidized Hb and lipid derivatives in the atherosclerotic plaque.

Abstract 158: PPAP2B Expression Regulates the Development of Atherosclerosis

Coronary artery disease (CAD) is the leading cause of death in both men and women worldwide. The bioactive lipid lysophosphatidic acid (LPA) accumulates in human atheroma compared to levels in healthy control tissue. LPA can be degraded by the membrane protein lipid phosphate phosphatase 3 (LPP3), encoded by the gene PPAP2B. Noncoding polymorphisms in PPAP2B associate with CAD risk, and gene expression analysis indicates that individuals possessing the risk allele exhibit lower levels of PPAP2B mRNA in leukocytes and vascular cells. We hypothesize that decreased LPP3, as a result of low PPAP2B expression, accelerates experimental atherosclerosis. Murine models of tissue specific LPP3 knockdown were generated using the Cre-Lox system driven by either the MX-1 promoter (MX-1Δ) or the smooth muscle cell specific SM22 promoter (SM22Δ) in mice on the atherogenic LDLr -/- background. Following 12 weeks on Western diet, knockdown of LPP3 in both the MX-1Δ and the SM22Δ mice resulted in significantly more atherosclerosis by en face analysis compared to LPP3-fl/fl littermate controls. LPA content determined using LC/MS/MS tended to be higher in the proximal aortas of MX-1Δ and SM22Δ knockdown mice compared to controls. Significantly higher macrophage gene expression was observed in the MX-1Δ mice while IL-6 levels were significantly higher in the SM22Δ mice compared to controls. Finally, targeting LPP3 expression in the MX-1Δ, but not the SM22Δ, mice resulted in significantly higher circulating levels of LPA compared to controls. These results are consistent with accelerated atherosclerosis in LPP3 knockdown mice and suggest a protective role for LPP3 in CAD. Increased vascular LPA content, as a consequence of decreased LPP3 expression, may promote the infiltration of monocyte/macrophages into lesions as well as exacerbate inflammation to accelerate the development of CAD. Our findings provide mechanistic insight into the genome wide association studies that linked genetic variation in PPAP2B with risk of CAD and focus attention on the LPA/ LPP3 signaling nexus as a novel therapeutic strategy to prevent atherosclerosis.