­­Deficiency of myeloid PHD proteins aggravates atherogenesis via macrophage apoptosis and paracrine fibrotic signaling [single-cell RNA-seq]

Atherosclerotic plaque hypoxia is detrimental for macrophage function. Prolyl hydroxylases (PHDs) initiate cellular responses to hypoxia and may therefore govern macrophage function in plaque hypoxia. PHD inhibitors are clinically investigated to treat anemia in patients with chronic kidney disease, but the consequences for cardiovascular disease are not clear. Hence, we studied the influence of myeloid specific PHD deficiency on atherosclerotic plaque development and stability. PHD mRNA and protein levels were expressed by human plaque macrophages, and murine bone-marrow-derived macrophages, thus myeloid specific PHD knockout (PHDko) mice were fed 0.25% cholesterol diet for 6-12 weeks to induce atherosclerosis. Aortic root plaque size was augmented 2.6fold in PHD2cko, and 1.4-fold in PHD3ko, but not in PHD1ko mice compared to controls, independent of plasma cholesterol levels. Macrophage apoptosis was promoted in PHD2cko and PHD3ko mice in vitro and in vivo via the HIF1α/BNIP3 axis as shown by pathway analysis of bulk and single cell RNA sequencing of murine bone-marrow-derived macrophages and plaque macrophages, respectively, and validated by siRNA silencing. Human plaque BNIP3 mRNA associated with carotid plaque necrotic core content, suggesting similar adverse effects in humans. Further, PHD2cko plaques -not PHD1 or PHD3ko- displayed enhanced fibrosis at two different disease stages, and across vascular beds, independent of macrophage function. We show PHD2cko bone-marrow-derived macrophages enhanced collagen secretion by fibroblasts in a paracrine manner, while smooth muscle cell function was unchanged. In silico analysis of macrophage-fibroblast communication predicted SPP1 signaling by PDH2cko macrophages to regulate the fibroblast transcriptome, in line with enhanced SPP1 protein content in plaques of PDH2cko mice