Deficiency of myeloid prolyl hydroxylase domain proteins aggravates atherogenesis via macrophage apoptosis and paracrine fibrotic signaling

Abstract

Abstract Background Atherosclerotic plaque hypoxia is detrimental for macrophage function. Prolyl hydroxylases (PHDs) initiate cellular hypoxic responses, possibly influencing macrophage function in plaque hypoxia. Thus, we aimed to elucidate the role of myeloid PHDs in atherosclerosis. Methods Myeloid specific PHD knockout (PHDko) mice were fed high cholesterol diet for 6–12 weeks to induce atherosclerosis. Plaque parameters, e.g. plaque size and macrophage content, were analyzed. Bulk and single cell RNA sequencing was performed on PHD2 BMDMs and plaque macrophages, respectively. Results Aortic root plaque size was augmented 2.6fold in PHD2cko, and 1.4-fold in PHD3ko, but not in PHD1ko mice compared to controls. Macrophage apoptosis was promoted in PHD2cko and PHD3ko mice in vitro and in vivo, via the HIF1α/BNIP3 axis. Bulk and single cell RNA data of PHD2cko bone-marrow-derived macrophages (BMDM) and plaque macrophages, respectively, confirmed these findings and were validated by siRNA silencing. Human plaque BNIP3 mRNA associated with plaque necrotic core, suggesting similar adverse effects. Further, PHD2cko plaques displayed enhanced fibrosis, independent of macrophage MMP activity, collagen secretion or proliferation and of SMC collagen production, or proliferation. Rather, PHD2cko BMDMs enhanced fibroblast collagen secretion in a paracrine manner. Nichenet in silico analysis of macrophage-fibroblast communication predicted SPP1 signaling as regulator, in line with enhanced plaque SPP1 protein content, and SPP1 mRNA in TREM2-foamy plaque macrophages, but not in neutrophils. Conclusion Myeloid PHD2cko and PHD3ko enhanced plaque growth, macrophage apoptosis, and PHD2cko activated paracrine collagen secretion by fibroblasts. Funding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): NWO, Leducq