Modulating atherosclerotic plaque formation by targeting plasminogen receptor PlgRKT

Background Macrophages are essential for atherosclerotic plaque progression. Recently a plasminogen receptor, Plg-RKT was shown to modulate monocyte and macrophage migration and lipid metabolism. However, its function on atherosclerotic development is not described yet. Purpose The aim of this study was to define the effect of Plg-RKT-mediated leukocyte function on atherosclerosis. Methods Mice deficient for Plg-RKT and their littermates on a low-density lipoprotein (LDL)-receptor-deficient C57Bl6/J background were challenged with high fat diet (HFD) for 6 (n=10-12) or 16 weeks (n=6-11). En face preparation of isolated aortas and histological assessment of paraffin-embedded hearts were performed. Single cell RNA sequencing according to Cell Fixation Protocol for GEM-X Single Cell 3' & 5' Assays by 10x genomics was performed and analysed by gene set enrichment analysis using hallmark gene set. Results Comparing Plg-RKT-/- mice with littermates revealed significantly smaller atherosclerotic lesion areas in the aorta (3.0%±2.2 vs. 6.8%±3.6, p=0.0068), especially in the aortic arch after 6 weeks HFD. Plaques in the aortic root of Plg-RKT-/- mice exhibited increased collagen content (0.38±0.10 vs. 0.57±0.19, p=0.006) indicating more stable plaques. Plasma parameters revealed reduced cholesterol and LDL levels in Plg-RKT-/- mice having a beneficial effect on atherosclerosis. However, prolonged feeding with HFD over 16 weeks overruled initial protection and led to similar sizes of aortic lesion areas. Although plaque development equalized, Plg-RKT-/- mice were protected from hepatic steatosis after 16 weeks HFD. Single cell RNA sequencing of the liver demonstrated that Plg-RKT-/-- mice had more Zone 1 A periportal hepatocytes with upregulated cholesterol homeostasis, while Zone 1 B periportal hepatocytes were reduced, which exhibited reduced fatty acid metabolism. Moreover, Kupffer cells and their inflammatory pathways were diminished. Thus, this shift towards highly active hepatocytes and reduced inflammation in the liver upon Plg-RKT-deficiency led to a delay in HFD-induced atherosclerosis and hepatic steatosis. Conclusions We found that Plg-RKT enhances plasma lipids exacerbating early atherosclerotic lesion development associated with less stable plaques as lack of Plg-RKT boosts collagen deposition. Moreover, hepatic steatosis is diminished in Plg-RKT -deficient mice likely due to changes in the cellular composition of the liver associated with increased cholesterol homeostasis and reduced inflammation.