Linking Metabolic Dysfunction to Atherosclerosis via Activation of Macrophage CD36 Gene Transcription by Retinol Binding Protein 4

Differentiation of intimal macrophages into lipid-laden foam cells is a sine qua non of atherogenesis at all stages of plaque development – but atherogenesis remains an incompletely understood process. Many factors contribute to foam cell formation within the developing atheroma, but ultimately an imbalance between uptake of cholesterol from modified LDL (e.g. oxidized or aggregated LDL particles) via pattern recognition receptors of the innate immune system, such as CD36, and efflux of cholesterol to HDL or apoA via ABC-family transporters is the driving force.

Circulating levels of retinol binding protein-4 (RBP4), a plasma retinol transporter and adipokine, were found more than 10 years ago¹ by Barbara Kahn’s laboratory to correlate with insulin resistance, type 2 diabetes and metabolic syndrome; subsequent studies showed associations of RBP4 with cardiovascular disease (CVD)². The Kahn lab later showed that RBP4 promoted adipocyte inflammatory responses and endothelial dysfunction through a receptor-mediated mechanism independent of retinols but requiring TLR4³.

A paper published by Liu and colleagues in this issue of Circulation⁴ now extends this work by mechanistically linking RBP4 to macrophage foam cell formation, experimental atherosclerosis, and human CVD risk. Taking a comprehensive approach with elegant cell biology studies of murine macrophages, mouse genetic models, and human clinical studies, they showed the following:

1. Circulating plasma RBP4 levels in a cohort of middle aged and elderly subjects from southern China correlated positively with cardiovascular disease, with each quartile of RBP4 expression having higher risk. Subjects with the highest quartile of RBP4 level had hazard ratios of 1.5 compared to the lowest quartile even after correcting for other known risk factors;

2. Overexpressing RBP4 to levels 4 times normal in high fat diet fed apoE null mice increased atherosclerotic plaque burden by more than 2 fold; and knocking down RBP4 expression lowered plaque burden by 45% without affecting circulating cholesterol levels; and

3. RBP4 triggered a signaling pathway in murine macrophages and macrophage cell lines that involved phosphorylation and activation of the tyrosine kinase c-Src with subsequent phosphorylation and activation of the MAP kinase JNK, and then phosphorylation and nuclear translocation of the transcription factor STAT1. STAT1 then interacts with specific nucleotide sequences in the promoter of the CD36 gene, activating transcription and increasing its surface expression. Cholesterol efflux pathways were not changed, so the unbalanced upregulation of macrophage CD36 in the setting of hyperlipidemia and oxidant stress ultimately led to enhanced oxLDL uptake and foam cell formation.

This study – with its complementary genetic, pharmacologic, and cell biologic approaches – adds to a growing body of literature suggesting that signals generated in the setting of obesity and metabolic syndrome promote macrophage pro-atherogenic responses. Interestingly, RBP4 signaling was abrogated by knockdown of TLR4, suggesting that this pro-atherogenic pathway might be mediated by a retinol-independent receptor-mediated process.

This study also suggests that macrophage derived RBP4 acts as an autocrine factor in foam cell formation, and adds to the growing body of evidence that the atherogenic microenvironment promotes foam cell formation by facilitating production of specific endogenous danger signals including oxLDL⁵, RBP4, 15(S)-HETE⁶, and prostanoids⁷, that activate macrophage signaling pathways via innate immune receptors such as TLR4 and CD36, and nuclear hormone receptors such as PPARɤ and LXR. These endogenous signals converge on the CD36 promoter to create a positive feedback loop, accelerating lipid uptake and promoting foam cell formation.

This intriguing study also identifies a potential biomarker for CVD risk and several potential targets for therapeutic intervention. The study also raises interesting mechanistic questions worthy of further study, especially related to the initiating events in RBP4 signaling. It is also interesting to note that CD36-mediated oxLDL uptake and pro-atherogenic signaling also require c-Src-family kinase and JNK activation⁸, suggesting that oxLDL-CD36 signaling could also activate the STAT1 pathway in an additional feed forward regulatory loop to further increase CD36 expression and foam cell formation.