An Imbalance Between CD36 and ABCA1 Protein Expression Favors Lipid Accumulation

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Advanced atherosclerotic plaques in the internal carotid artery wall are considered as a clinical threat potentially leading to stroke. A spectrum of factors underlying the final destabilization and rupture of the plaques has been studied, but distinguishing between high-risk and low-risk plaques is still an enigma. However, one of the morphological features of high-risk plaques seems to be a vast lipid-filled core, the formation of which is fueled by the death of engorged lipid-filled macrophage foam cells. In a genome-wide microarray expression study of advanced carotid plaques (CP), we discovered that the transformation of a CP from nonsymptom-causing to symptomcausing coincided with increased expression of macrophage adipophilin,4 a lipid-loading marker. We also found that CD36 and ABCA1 genes were overexpressed in symptomcausing CP, and the result was replicated by real-time reverse-transcription polymerase chain reaction for CD36. Interestingly, both CD36 and ABCA1 seem to play an important role in balancing macrophage lipid intake and efflux. Oxidized low-density lipoprotein-scavenging CD36, a multifunctional membrane glycoprotein, is considered essential for foam cell formation,1 and its expression is further stimulated by its own ligand.2 This vicious cycle potentially leading to fatal cholesterol overload is partly inhibited by ABCA1, a membrane transporter protein of ATP-binding cassette (ABC) family, widely supported in its atheroprotectiveness, removing free cholesterol and phospholipids, and necessary for the initiation of macrophage reverse cholesterol transport.3 On the basis of the differential expression of these 2 proteins in our DNA microarray, we wanted to also study immunohistochemically the expression of CD36 and ABCA1 proteins and their relation to the presence and localization of lipids and lipid-filled foam cells, plaque ulceration, extravasated red blood cells (RBC), and atheromatous/necrotic areas in single CP. We also related the data to previous investigations of the same Helsinki Carotid Endarterectomy Study (HeCES) cohort.4–8 Materials and Methods Ninety-two patients underwent carotid endarterectomy because of high-grade (70%–99%) carotid artery stenosis in digital subtraction angiography according to the North American Symptomatic Carotid Endarterectomy Trial (NASCET) criteria between years 1997 and 2000. Details on patient selection, baseline characteristics of the HeCES cohort, and the carotid endarterectomy procedure have been described previously.4–8 Symptom status was based on clinical criteria. Stroke, TIA, or amaurosis fugax within 120 days before carotid endarterectomy was considered as a culprit symptom. Patients with other potential sources of cerebral embolism were excluded. From all the specimens, a subgroup (n14) of completely asymptomatic CP and another subgroup (n23) of stroke-causing CP were selected for immunohistochemical analysis. Light microscopy (Axioplan 2; Carl Zeiss) was performed by an investigator (P.M.I.) blinded to the clinical data. The amount of lipid deposits was evaluated by another investigator (K.N.). To obtain detailed distribution data from the protein expression, we analyzed separate regions of interest of the entire immunostained section, as described previously.4 For details on immunohistochemical methods, please see Supplemental Figure I. Total RNA was extracted from a longitudinal slice containing a section with tightest stenosis, as previously described.4,5 Quantitative real-time reverse-transcription polymerase chain reaction was performed using Assays-on-Demand Gene Expression Products and an ABI PRISM 7000 Sequence Detection System (Applied Biosystems). Relative CD36 and ABCA1 gene expression was determined using the comparative CT method and standard curve method, respectively, and expression of-actin was used for normalization. Because of the small sample size, we used nonparametric statistical tests. Ordinal variables were tested with Mann–Whitney U and Jonckheere-Terpstra tests, and continuous variables were tested with Spearman rank correlation. To adjust for confounding factors such as age, gender, body mass index, blood low-density lipoprotein concentration, and the amount of plaque macrophages,8 a multinomial logistic regression model was used to explore the association between regions of interest levels and ABCA1/CD36, as described in detail previously.4 Statistical analyses were performed using SPSS (version 14.0). Results The relative amount of CD36 mRNA correlated with mean CD36 protein expression within plaques (rs0.551; P0.001), with the latter being observed in a subgroup of macrophage foam cell-formed rims around the lipid cores. CD36 mRNA and protein levels were higher in ulcerated than in nonulcerated CP. The mRNA and protein expressions of adipophilin and CD36 were correlated. Within each region of interest, CD36 expression associated with the presence of RBC and atheromatous/necrotic areas. ABCA1 expression was mostly co-distributed with macrophages. The relative expressions of ABCA1 mRNA and protein were in good correlation (rs0.367; P0.05), but neither of them differed when ulceration (Figure 2B) or symptom status was compared. ABCA1 mRNA and protein correlated with increased CP adipophilin mRNA and protein expressions. In CP with Figure 1. Ninety an increased amount of intracellular lipids, the expression of ABCA1 protein was elevated (P0.005). The regional balance of CD36 and ABCA1 protein expressions was analyzed in separate regions of interest categorized into 3 groups according to their ratio. In ulcerated CP, there were significantly more regions of interest with higher CD36 than ABCA1 expression, whereas the opposite was true for nonulcerated CP. ABCA1 expression was increased when compared with CD36 expression in patients with higher-plasma high-density lipoprotein cholesterol concentrations. Moreover, the amount of adipophilin mRNA was significantly decreased in CP with higher ABCA1 than CD36 expression. Extracellular lipid deposits were significantly increased in symptom-causing and ulcerated CP. Intraplaque hemorrhages were associated with a higher amount of extracellular CP lipid. The intracellular lipid load associated with both Fas-receptor and Fas-ligand expression. Adipophilin protein expression was increased in plaques with more total lipid deposits. Neither the traditional cardiovascular risk factors nor medications had important associations to plaque CD36 or ABCA1 expression or CP lipid status. For more detailed analysis on immunohistochemical results, please see Figure I. Discussion Uptake of modified low-density lipoprotein particles by multiple mechanisms with ensuing foam cell formation is an essential part of the atherosclerotic process.1 We found that the amount of extracellular lipid was elevated in unstable CP, and that a shifted balance in the expression of 2 key lipid trafficking proteins, CD36 and ABCA1, may favor progressive lipid accumulation into macrophages and lead to a stroke-prone, ulcerated CP.