Cpla2 and Lrp1 Deficiency Promote Severe Atherosclerosis Driven by LXRa/ABCA1 Dysfunction in Smooth Muscle Cells

Abstract

Our group has identified smooth muscle cell targeted deficiency of LDL Receptor related protein 1 (LRP1), smLRP1-/-, resulting in accelerated aortic atherosclerosis. Activation of cytoplasmic phospholipase A2 (cPLA2) in these mice leaded to abolished LXRa/ABCA1 in vascular smooth muscle cells (VSMCs) and increased intracellular cholesterol accumulation. Based on our previous work we hypothesized that deficiency of cPLA2 would impede atherogenesis in the smLRP1-/- mouse model. Methods: Adult male smLRP1-/-;cPLA2-/-;LDLR-/- (Triple knockout) mice were placed on a high cholesterol diet (HCD) for 16 weeks and compared to age- and diet- matched sibling control smLRP1+/+;cPLA2-/-;LDLR-/- (Double KO) mice. Primary VSMC were explanted for in vitro migration, cell viability and proliferation studies when stimulated w/PDGF-BB. Histologic analysis was performed using en face whole aorta Oil red O (ORO) staining, as well as cross-sectional analysis of the aortic root with ORO, Picro Sirius Red, Alizarin Red and immunofluorescence. Immunoblot protein analysis was performed using lysed whole aortas. Data is represented as mean±SEM. Statistical analysis was performed using one- and two-way ANOVA with Tukey's correction. Results: Triple KO VSMC have decreased viable and proliferating cells but increased VSMC migration both compared to unstimulated and double KO controls. En face ORO analysis revealed increased lipid accumulation in Triple Knockout mice as compared to controls (60+/-3% vs 13+/-2%, p<0.001) (Figure 1). Uniquely, Triple knockout mice develop extensive necrotic cores and thin fibrous caps in atherosclerotic lesions in the aortic root (Figure 1). We found increased LXRa aggregates in triple KO mice and paradoxically ABCA1 is increased compared to controls observed by immunoblot and immunofluorescence tissue analysis of VSMC atherosclerotic tissues. Conclusions: Deficiency of cPLA2 in the smLRP1-/- mouse model induces LXRa increase and aggregation leading to rescue of ABCA1, but unexpectedly increases VSMC dysfunction and lipid accumulation within the plaque and generates more vulnerable plaque. Future studies will underpin the exact molecular mechanism that guide severe disease development in our Triple knockout mice via regulation of ABCA1.