CD36 down regulation by the macrophage antioxidant 7,8-dihydroneopterin through modulation of PPAR-γ activity.

CD36 is the key scavenger receptor driving the formation of cholesterol-loaded foam cells, the principal cellular component of atherosclerotic plaques. CD36 is down regulated by 7,8-dihydroneopterin, a potent superoxide and hypochlorite scavenging antioxidant generated by interferon-γ stimulated macrophages. 7,8-dihydroneopterin downregulates CD36 mRNA and protein levels so inhibiting macrophage foam cell formation in vitro. We examined the mechanism of 7,8-dihydroneopterin downregulation of CD36 by measuring CD36 and PPAR-γ levels by Western blot analysis, in the monocyte-like U937 cells with a range of PPAR-γ stimulants and inhibitors. Lipoxygenase activity was measured by monitoring linoleic acid oxidation at 234 nm for diene formation. Between 100 and 200 µM, 7,8-dihydroneopterin decreased CD36 levels by 50% within 12 h with levels dropping below 25% by 24 h. CD36 levels returned to basal levels after 24 h. Inhibition of protein synthesis by cycloheximide shows 7,8-dihydroneopterin had no effect on CD36 degradation rates. PPAR-γ levels were not altered by the addition of 7,8-dihydroneopterin. MAP Kinase, P38 and NF-κB pathways inhibitors SP600125, PD98059, SB202190 and BAY 11-7082, respectively, did not restore the CD36 levels in the presence of 7,8-dihydroneopterin. The addition of the lipophilic PPAR-γ activators rosiglitazone and azelaoyl-PAF prevented the CD36 downregulation by 7,8-dihydroneopterin. 7,8-dihydroneopterin inhibited soybean lipoxygenase and reduced U937 cell basal levels of cellular lipid oxides as measured by HPLC-TBARS analysis. The data show 7,8-dihydroneopterin down regulates CD36 expression by decreasing the level of lipid oxide stimulation of PPAR-γ promotor activity, potentially through lipoxygenase inhibition.

Foam cell formation but not oxLDL cytotoxicity is inhibited by CD36 down regulation by the macrophage antioxidant 7,8-dihydroneopterin.

BACKGROUND AND AIMS: Cluster of differentiation 36 (CD36) is a key scavenger receptor in the control of macrophage uptake of oxidised low-density lipoproteins (oxLDL). CD36 expression levels are not down regulated by intracellular cholesterol but are upregulated by oxidised low density lipoprotein (oxLDL) leading to the formation of lipid loaded foam cells, a major constituent of atherosclerotic plaques. We have previous shown that CD36 is down regulated by 7,8-dihydroneopterin, an antioxidant generated by γ-interferon activated macrophages. How CD36 down regulation affects oxLDL induced cytotoxicity, CD36 oxLDL upregulation and foam cell formation is examined using human monocyte like U937 cell line as a model system of human macrophages. METHODS: Low density lipoprotein (LDL) was prepared by ultracentrifugation from human plasma and oxidised in copper chloride. CD36 levels in U937 cells were measured by western blot analysis. and lipid accumulation was measured by oil red-O staining and 7-ketocholesterol accumulation by high performance liquid chromatography. Cell viability was measured by flow cytometry analysis after propidium iodide staining. RESULTS: 7,8-dihydroneopterin concentrations above 100 µM caused a concentration and time dependent decrease in cellular CD36 levels to 20 % of the untreated cells after 24 h. Upregulation of CD36 by oxLDL was inhibited by 7,8-dihydroneopterin treatment. The CD36 down regulation was associated with decrease in foam cell formation but not a reduction on oxLDL cytotoxicity. CONCLUSIONS: 7,8-dihydroneopterin down regulated CD36 in U937 cells, inhibiting foam cell formation but not oxLDL mediated cell death. 7,8-dihydroneopterin may modulate foam cell formation in atherosclerotic plaques.