Absence Of CD36 Research Articles

CD36 Plays A Key Role As A Cell Cycle Regulator In Skeletal Muscle Cells

As a fatty acid transporter, CD36 plays an important role in regulating glucose and lipid metabolism. However, in addition to the regulation of skeletal muscle metabolism, whether it can regulate skeletal muscle cell cycle and proliferation is still unclear. PURPOSE: To investigate the role of CD36 in regulating skeletal muscle cell cycle. METHOD: Cells at 80%–90% confluence were differentiated, then transfected with siCtrl or siCD36 using Lipofectamine RNAiMAX when 80% ~ 90% of cells were in long shape. Skeletal muscle cells were harvested after 72 hours of transfection. The cell cycle of differentiated skeletal muscle cells was monitored by flow cytometry. Real-time fluorescent quantitative PCR was used to detect the cell cycle-related genes of skeletal muscle cells. Cyclin D1 protein level was determined by Western blot analysis. RESULT: Significant cell cycle arrest at G0/G1 phase occurred in the absence of CD36 (83.81% ± 0.59% vs. 53.25% ± 4.94%, p < 0.001), and fewer cells entered S phase(7.11% ± 0.82% vs. 36.49% ± 3.72%, p < 0.001). The mRNA expression levels of Mcm5, Orc1, cdkn1c and Skp2 increased significantly in CD36 deficiency (2.06% ± 0.17% vs. 1% ± 0.30%, p < 0.05, 1.82% ± 0.15% vs. 1% ± 0.17%, p < 0.05, 2.39% ± 0.37% vs. 1% ± 0.34%, p < 0.05, 2.05% ± 0.29% vs. 1% ± 0.35%, p < 0.05). The expression of Cyclin D1 in CD36 dysfunction group was decreased(83.31% ± 3.06% vs. 134.41% ± 6.57%, p < 0.01). CONCLUSION: Loss of CD36 leads to cell cycle arrest, implicating a promising target for normal progression of muscle progenitors from proliferation to differentiation. Supported by the National Natural Science Foundation of China (No. 32071172, 31600966).

MMP-9-Dependent Serum-Borne Bioactivity Caused by Multiwalled Carbon Nanotube Exposure Induces Vascular Dysfunction via the CD36 Scavenger Receptor.

Inhalation of multiwalled carbon nanotubes (MWCNT) causes systemic effects including vascular inflammation, endothelial dysfunction, and acute phase protein expression. MWCNTs translocate only minimally beyond the lungs, thus cardiovascular effects thereof may be caused by generation of secondary biomolecular factors from MWCNT-pulmonary interactions that spill over into the systemic circulation. Therefore, we hypothesized that induced matrix metalloproteinase-9 (MMP-9) is a generator of factors that, in turn, drive vascular effects through ligand-receptor interactions with the multiligand pattern recognition receptor, CD36. To test this, wildtype (WT; C57BL/6) and MMP-9(-/-)mice were exposed to varying doses (10 or 40 µg) of MWCNTs via oropharyngeal aspiration and serum was collected at 4 and 24 h postexposure. Endothelial cells treated with serum from MWCNT-exposed WT mice exhibited significantly reduced nitric oxide (NO) generation, as measured by electron paramagnetic resonance, an effect that was independent of NO scavenging. Serum from MWCNT-exposed WT mice inhibited acetylcholine (ACh)-mediated relaxation of aortic rings at both time points. Absence of CD36 on the aortic rings (obtained from CD36-deficient mice) abolished the serum-induced impairment of vasorelaxation. MWCNT exposure induced MMP-9 protein levels in both bronchoalveolar lavage and whole lung lysates. Serum from MMP-9(-/-)mice exposed to MWCNT did not diminish the magnitude of vasorelaxation in naïve WT aortic rings, although a modest right shift of the ACh dose-response curve was observed in both MWCNT dose groups relative to controls. In conclusion, pulmonary exposure to MWCNT leads to elevated MMP-9 levels and MMP-9-dependent generation of circulating bioactive factors that promote endothelial dysfunction and decreased NO bioavailability via interaction with vascular CD36.

English

We have previously shown that CD36 is a membrane protein that facilitates long chain fatty acid (FA) transport by muscle tissues. We also documented the significant impact of muscle CD36 expression on heart function, skeletal muscle insulin sensitivity as well as on overall metabolism. To identify a comprehensive set of genes that are differentially regulated by CD36 expression in the heart, we used two microarray technologies (Affymetrix and Agilent) to compare gene expression in heart tissues from CD36 KnocK-Out (KO-CD36) versus wild type (WT-CD36) mice. The obtained results using the two technologies were similar with around 35 genes differentially expressed using both technologies. Absence of CD36 led to down-regulation of the expression of three groups of genes involved in pathways of FA metabolism, angiogenesis/apoptosis and structure. These data are consistent with the fact that the CD36 protein binds FA and thrombospondin 1 invoved respectively in lipid metabolism and anti-angiogenic activities. In conclusion, our findings led to validate our data analysis workflow and identify specific pathways, possibly underlying the phenotypic abnormalities in CD36 Knock -Out hearts.

Endoplasmic Reticulum Stress Controls M2 Macrophage Differentiation and Foam Cell Formation

Macrophages are essential in atherosclerosis progression, but regulation of the M1 versus M2 phenotype and their role in cholesterol deposition are unclear. We demonstrate that endoplasmic reticulum (ER) stress is a key regulator of macrophage differentiation and cholesterol deposition. Macrophages from diabetic patients were classically or alternatively stimulated and then exposed to oxidized LDL. Alternative stimulation into M2 macrophages lead to increased foam cell formation by inducing scavenger receptor CD36 and SR-A1 expression. ER stress induced by alternative stimulation was necessary to generate the M2 phenotype through JNK activation and increased PPARγ expression. The absence of CD36 or SR-A1 signaling independently of modified cholesterol uptake decreased ER stress and prevented the M2 differentiation typically induced by alternative stimulation. Moreover, suppression of ER stress shifted differentiated M2 macrophages toward an M1 phenotype and subsequently suppressed foam cell formation by increasing HDL- and apoA-1-induced cholesterol efflux indicating suppression of macrophage ER stress as a potential therapy for atherosclerosis.

Fever induction by systemic stimulation with macrophage-activating lipopeptide-2 depends upon TLR2 but not CD36

This study was designed to test the responses of TLR2-knockout mice (TLR2-KO) and wild- type mice (C57/BL-6), and of CD36 deficient spontaneously hypertensive rats (SHR) and their genetic controls [Wistar Kyoto (WKY) rats] to systemic stimulations with the TLR2/6 agonist MALP-2 and the TLR4 agonist LPS. Fever and formation of TNF-α and IL-6 induced by intraperitoneal injections of MALP-2 (1000 µg/kg) were completely blunted in TLR2-KO, while LPS (100 µg/kg)-induced responses were not abolished in these animals. In SHR lacking CD36, a reduction of fever was observed in response to MALP-2 (100 µg/kg), but LPS-fever was even more attenuated in SHR when compared with WKY controls. Concentrations of circulating IL-6 tended to be lower in SHR after stimulation with both pyrogens. However, the IL-6-mediated activation of the transcription factor STAT3 in the brain was identical in both strains, indicating that the brain-controlled inflammatory response to MALP-2 (and LPS) is not impaired in the absence of CD36. In addition, stimulation of peritoneal macrophages with LPS and MALP-2 (10 µg/ml) caused the appearance of similar concentrations of bioactive cytokines in the supernatants from cells of both rat strains. These results demonstrate that TLR2 is essential for the manifestation of MALP-2, but not LPS-induced inflammatory responses. A moderate participation of CD36 in MALP-2-induced sickness- and cytokine-responses can not be ruled out but is unlikely as LPS-induced inflammatory responses were also attenuated in SHR.

Circulating monocytes and atherogenesis: From animal experiments to human studies

Despite significant improvements in the prevention and management of atherosclerosis, its consequences still remain a major cause of disability and mortality. Better understanding of pathophysiological processes implicated in the initial stages of atherogenesis is critical for the discovery of effective approaches to prevent ischaemic stage of the disease and to reduce huge economic and social burden currently associated with atherosclerosis (1). According to the current paradigm the atherosclerotic process is primarily initiated in the vascular wall itself where monocyte-derived macrophages transform to ‘foam’ cells by accumulating lipids (2). Consequent apoptosis or death of macrophages is largely responsible for necrotic core generation, progressive accumulation of free cholesterol and expansion of the necrotic core within plaques. Often, unbalanced generation of inflammatory cytokines by plaque monocyte-derived macrophages (e.g. via up-regulation of Toll-like receptors) as well as angiogenic promoting pathological plaque neovascularisation, and enzymes degrading extracellular matrix results in destabilisation of atherosclerotic plaques (3–5). Whilst a critical role of vascular wall monocyte-derived macrophages in atherosclerotic plaque formation is widely recognised, the impact of circulating monocytes on atherogenesis is less established. For example, accumulation of modified (mainly oxidised) lipids via scavenger receptors was, until recently, exclusively attributed to vascular wall resident macrophages rather than circulating monocytes (6). Oxidised low-density lipoproteins (LDL) taken up via scavenger receptors (e.g. CD36 and CD204) are delivered to lysosomes, where they undergo esterification by cholesterol esterase and are largely converted into free cholesterol and fatty acids (6). It has been reported that CD36 contributes 60–70% of cholesterol ester accumulation in macrophages exposed to oxidised LDL (7). Deletion of the genes encoding CD36 and CD204 retards the development of atherosclerotic lesions in animal models of atherosclerosis (6). However, circulating monocytes are equipped with these same scavenger receptors and may thus start accumulating lipids even prior to their migration to tissues and differentiation to macrophages (8). Exposure to modified LDL induces rapid ‘foam’ cell formation from freshly isolated peripheral blood monocytes, accompanied by upregulation of CD204 and CD36 (8). The clinical relevance of these findings is supported by the observation that an absence of CD36 on circulating monocytes in individuals from a Japanese population results in 40–50% reduction in oxidised LDL binding to monocyte-derived-macrophages compared to normal subjects (9). Certainly, monocyte counts are increased in patients with stenotic coronary artery disease and the Intermountain Heart Collaborative Study Group recently reported a population of 3,227 patients with or without coronary artery disease whereby high monocyte counts were independent predictors of future myocardial infarction and death (10). However, the impact of monocytes in atherogenesis may go far beyond direct lipid accumulation as they are also actively involved in inflammatory responses attributable to their principal function within the innate immune system. Indeed, Correspondence to: Dr. Eduard Shantsila University of Birmingham Centre for Cardiovascular Sciences, City Hospital Birmingham, B18 7QH, UK Tel: +44 121 554 3801, Fax: +44 121 554 4083 E-mail: e.shantsila@bham.ac.uk

A CD36-dependent signaling cascade is necessary for macrophage foam cell formation

Accumulation of macrophage foam cells in atherosclerotic blood vessel intima is a critical component of atherogenesis mediated by scavenger receptor-dependent internalization of oxidized LDL. We demonstrated by coimmunoprecipitation and pull-down assays that the macrophage scavenger receptor CD36 associates with a signaling complex containing Lyn and MEKK2. The MAP kinases JNK1 and JNK2 were specifically phosphorylated in macrophages exposed to oxLDL. Using cells isolated from SRA, TLR2, or CD36 null mice, and phospholipid ligands specific for either SRA or CD36, we showed that JNK activation was mediated by CD36. Both foam cell formation and activation of JNK2 in hyperlipidemic mice were diminished in the absence of CD36. Furthermore, inhibition of Src or JNK blocked oxLDL uptake and inhibited foam cell formation in vitro and in vivo. These findings show that a specific CD36-dependent signaling pathway initiated by oxLDL is necessary for foam cell formation and identify potential targets for antiatherosclerosis therapy.

Absence of CD36 is Protective at Late Time Points in the apoE Knockout (KO), and Additional Absence of Scavenger Receptor A I/II (SRA) Provides no Added Benefit

Previously, absence of the scavenger receptor CD36 or SRA was shown to be protective against atherosclerosis in the apoE KO after 12 weeks on western diet. A recent report (JCI 115. 2192 (2005)) showed no effect for SRA, and protection in females only for CD36 by en face analysis after 8 weeks on western diet. Here we determined if combined absence of CD36 and SRA had greater effect in the apoE KO. We analyzed lesions from CD36/apo E double KOs (dkos) and SRA/apoE dkos as controls. These mice are derived from those used in the JCI study. Because atherosclerosis is a multistep process involving many different factors over time, we also investigated whether absence of CD36 was protective at later time points in the apoE KO. Methods For the tko study, mice were on western diet for 12 weeks; for the long term study, mice were on diet for 20 and 35 weeks. Aortas were dissected and subjected to en face analysis. After 12 weeks on diet, tko males had 58% less lesion area compared with apoE KO males; tko females had 63% less lesion area. Male CD36/apoE dkos had 61% less lesion compared with apoE KO males; CD36/apoE dko females had 81% less lesion. These results indicate no added benefit to absence of SRA compared with absence of CD36 alone in this model. At 20 and 35 weeks, lesions were 25–28% smaller in the CD36/apoE dkos compared to apoE KOs. However, most impressive here was the difference in the gross appearance of the aortas: the apoE KO aortas were sclerotic and nearly occluded by lesion, whereas in the CD36/apoE dkos, the lesions were more punctate. Funding: NIH HL70083, HL072942, & HL46403

Th-W61:7 Absence of CD36 is protective at late time pointsin the apoE KO model

Th-W61:7 Absence of CD36 is protective at late time pointsin the apoE KO model

CD36 deficiency in mice impairs lipoprotein lipase-mediated triglyceride clearance

CD36 is involved in high-affinity peripheral FFA uptake. CD36-deficient (cd36(-)(/)(-)) mice exhibit increased plasma FFA and triglyceride (TG) levels. The aim of the present study was to elucidate the cause of the increased plasma TG levels in cd36(-)(/)(-) mice. cd36(-)(/)(-) mice showed no differences in hepatic VLDL-TG production or intestinal [(3)H]TG uptake compared with wild-type littermates. cd36(-)(/)(-) mice showed a 2-fold enhanced postprandial TG response upon an intragastric fat load (P < 0.05), with a concomitant 2.5-fold increased FFA response (P < 0.05), suggesting that the increased FFA in cd36(-/-) mice may impair LPL-mediated TG hydrolysis. Postheparin LPL levels were not affected. However, the in vitro LPL-mediated TG hydrolysis rate as induced by postheparin plasma of cd36(-)(/)(-) mice in the absence of excess FFA-free BSA was reduced 2-fold compared with wild-type plasma (P < 0.05). This inhibition was relieved upon the addition of excess FFA-free BSA. Likewise, increasing plasma FFA in wild-type mice to the levels observed in cd36(-)(/)(-) mice by infusion prolonged the plasma half-life of glycerol tri[(3)H]oleate-labeled VLDL-like emulsion particles by 2.5-fold (P < 0.05). We conclude that the increased plasma TG levels observed in cd36(-)(/)(-) mice are caused by decreased LPL-mediated hydrolysis of TG-rich lipoproteins resulting from FFA-induced product inhibition of LPL.

Loss of receptor-mediated lipid uptake via scavenger receptor A or CD36 pathways does not ameliorate atherosclerosis in hyperlipidemic mice

Macrophage internalization of modified lipoproteins is thought to play a critical role in the initiation of atherogenesis. Two scavenger receptors, scavenger receptor A (SR-A) and CD36, have been centrally implicated in this lipid uptake process. Previous studies showed that these receptors mediated the majority of cholesterol ester accumulation in macrophages exposed to oxidized LDL and that mice with deletions of either receptor exhibited marked reductions in atherosclerosis. This work has contributed to an atherosclerosis paradigm: scavenger receptor-mediated oxidized lipoprotein uptake is required for foam cell formation and atherogenesis. In this study, Apoe-/- mice lacking SR-A or CD36, backcrossed into the C57BL/6 strain for 7 generations, were fed an atherogenic diet for 8 weeks. Hyperlipidemic Cd36-/-Apoe-/- and Msr1-/-Apoe-/- mice showed significant reductions in peritoneal macrophage lipid accumulation in vivo; however, in contrast with previous reports, this was associated with increased aortic sinus lesion areas. Characterization of aortic sinus lesions by electron microscopy and immunohistochemistry showed abundant macrophage foam cells, indicating that lipid uptake by intimal macrophages occurs in the absence of CD36 or SR-A. These data show that alternative lipid uptake mechanisms may contribute to macrophage cholesterol ester accumulation in vivo and suggest that the roles of SR-A and CD36 as proatherosclerotic mediators of modified LDL uptake in vivo need to be reassessed.

Hypercholesterolemia Promotes a CD36-dependent and Endothelial Nitric-oxide Synthase-mediated Vascular Dysfunction

Numerous studies have implicated either the presence or absence of CD36 in the development of hypertension. In addition, hypercholesterolemia is associated with the loss of nitric oxide-induced vasodilation and the subsequent increase in blood pressure. In the current study, we tested the hypothesis that diet-induced hypercholesterolemia promotes the disruption of agonist-stimulated nitric oxide generation and vasodilation in a CD36-dependent manner. To test this, C57BL/6, apoE null, CD36 null, and apoE/CD36 null mice were maintained on chow or high fat diets. In contrast to apoE null mice fed a chow diet, apoE null mice fed a high fat diet did not respond to acetylcholine with a decrease in blood pressure. Caveolae isolated from in vivo vessels did not contain endothelial nitric-oxide synthase and were depleted of cholesterol. Age-matched apoE/CD36 null mice fed a chow or high fat diet responded to acetylcholine with a decrease in blood pressure. The mechanism underlying the vascular dysfunction was reversible because vessels isolated from apoE null high fat-fed mice regained responsiveness to acetylcholine when incubated with plasma obtained from chow-fed mice. Further analysis demonstrated that the plasma low density lipoprotein fraction was responsible for depleting caveolae of cholesterol, removing endothelial nitric-oxide synthase from caveolae, and preventing nitric oxide production. In addition, the pharmacological removal of caveola cholesterol with cyclodextrin mimicked the effects caused by the low density lipoprotein fraction. We conclude that the ablation of CD36 prevented the negative impact of hypercholesterolemia on agonist-stimulated nitric oxide-mediated vasodilation in apoE null mice. These studies provide a direct link between CD36 and the early events that underlie hypercholesterolemia-mediated hypertension and mechanistic linkages between CD36 function, nitric-oxide synthase activation, caveolae integrity, and blood pressure regulation.

Role of CD36, the Macrophage Class B Scavenger Receptor, in Atherosclerosis

Recent work in the field of atherosclerosis has greatly expanded our knowledge of the pathogenesis of this disease. Scavenger receptors, including CD36, are thought to be most important early in the disease progression during macrophage uptake of modified LDL and foam cell formation. Genetically engineered murine models have been used to elucidate the contribution of the different scavenger receptors, to identify specific ligands related to LDL modifications, and to assess the possible therapeutic ramifications of targeting scavenger receptors. We have demonstrated a major role for CD36 in macrophage foam cell development and subsequent lesion development in vivo. Absence of CD36 in an atherogenic Apo E null background resulted in a 70% decrease in total lesion area in Western diet-fed mice. We have also made significant progress in our understanding of the regulation of expression of CD36 and have demonstrated that OxLDL can stimulate its own uptake by induction of CD36 gene expression. The mechanism by which OxLDL upregulates CD36 involves activation of the transcription factor, PPAR-gamma.

Chondroitin-4-Sulfate Impairs In Vitro and In Vivo Cytoadherence of Plasmodium falciparum Infected Erythrocytes

Chondroitin-4-sulfate (CSA) was recently described as a Plasmodium falciparum cytoadherence receptor present on Saimiri brain microvascular and human lung endothelial cells. To specifically study chondroitin-4-sulfate-mediated cytoadherence, a parasite population was selected through panning of the Palo-Alto (FUP) 1 P. falciparum isolate on monolayers of Saimiri brain microvascular endothelial cells (SBEC). Immunofluorescence showed this SBEC cell line to be unique for its expression of CSA-proteoglycans, namely CD44 and thrombomodulin, in the absence of CD36 and ICAM-1. The selected parasite population was used to monitor cytoadherence inhibition/dissociating activities in Saimiri sera collected at different times after intramuscular injection of 50 mg CSA/kg of body weight. Serum inhibitory activity was detectable 30 min after injection and persisted for 8 hr. Furthermore, when chondroitin-4-sulfate was injected into monkeys infected with Palo-Alto (FUP) 1 P. falciparum, erythrocytes containing P. falciparum mature forms were released into the circulation. The cytoadherence phenotype of circulating infected red blood cells (IRBC) was determined before and 8 hr after inoculation of CSA. Before inoculation, in vitro cytoadherence of IRBCs was not inhibited by CSA. In contrast, in vitro cytoadherence of circulating infected erythrocytes obtained 8 hr after CSA inoculation was inhibited by more than 90% by CSA. In the squirrel monkey model for infection with P. falciparum, chondroitin-4-sulfate impairs in vitro and in vivo cytoadherence of parasitized erythrocytes.