Deficiency of myeloid PHD proteins aggravates atherogenesis via macrophage apoptosis and paracrine fibrotic signaling
van Kuijk K, Demandt JAF, Perales-Patón J et al.
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Atherosclerotic plaque hypoxia is detrimental for macrophage function in the plaque. Prolyl hydroxylases (PHD) 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.
Two major tiers of data were generated to uncover the role of Myeloid PHD2 in atherogenesis for the bioinformatics analysis described here.
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In-vitro experiments with cell lines were profiled using conventional RNA-seq sequencing. Empirical observations suggested that the impact of PHD2 knock-out in Macrophages triggers self-controlled apoptosis but not collagen enhancement for atherogenesis. Thus, BMDM (macrophages) carrying PHD2cKO and PHD2-WT were compared to uncover potential inddirect impact of the gene perturbation in the myeloid cells in atherogenesis. This assay was followed by a second one that aimed to characterize mesenchymal cells that potentially could promote fibrosis (smooth muscle cells and fibroblasts). These cells were subjected of conditioned medium cultured by the BMDM with and without the functional knock-out of PHD2 to investigate its influence in pro-collagen producing cells in atherosclerotic plaques. Only 3T3 cells (fibroblast cell line) showed an increase in collagen production/secretion. Therefore, 3T3 cells stimulated with conditioned medium culture derived from BMDM cells with and without PHD2-KO were profiled for further investigation.
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In-vivo experiments with a generated model carrying KO and WT forms of PHD2 in the myeloid lineage (incl. macrophages) were subjected of a high cholesterol diet to promote atherosclerosis. Atherosclerotic plaques (pool per condition, n=9 and 11 mice for PHD2cKO and WT, respectively) from this model were subjected of single-cell RNA sequencing.