The P2Y2 receptor mediates uptake of matrix-retained and aggregated low density lipoprotein in primary vascular smooth muscle cells

Background and aims: The internalization of aggregated low-density lipoproteins (agLDL) mediated by low-density lipoprotein receptor related protein (LRP1) may involve the actin cytoskeleton in ways that differ from the endocytosis of soluble LDL by the LDL receptor (LDLR). This study aims to define novel mechanisms of agLDL uptake through modulation of the actin cytoskeleton, to identify molecular targets involved in foam cell formation in vascular smooth muscle cells (VSMCs). The critical observation that formed the basis for these studies is that under pathophysiological conditions, nucleotide release from blood-derived and vascular cells activates SMC P2Y(2) receptors (P2Y(2)Rs) leading to rearrangement of the actin cytoskeleton and cell motility. Therefore, we tested the hypothesis that P2Y(2)R activation mediates agLDL uptake by VSMCs. Methods: Primary VSMCs were isolated from aortas of wild type (WT) C57BL/6 and. P2Y(2)R -/- mice to investigate whether P2Y(2)R activation modulates LRP1 expression. Cells were transiently transfected with cDNA encoding a hemagglutinin-tagged (HA-tagged) WT P2Y(2)R, or a mutant P2Y(2)R that unlike the WT P2Y(2)R does not bind the cytoskeletal actin-binding protein filamin-A (FLN-A). Results: P2Y(2)R activation significantly increased agLDL uptake, and LRP1 mRNA expression decreased in P2Y(2)R-/- VSMCs versus WT. SMCs, expressing P2Y(2)R defective in FLN-A binding, exhibit 3-fold lower LDLR expression levels than SMCs expressing WT P2Y(2)R, while cells transfected with WT P2Y(2)R show greater agLDL uptake in both WT and P2Y(2)R-/- VSMCs versus cells transfected with the mutant P2Y(2)R. Conclusions: Together, these results show that both LRP1 and LDLR expression and agLDL uptake are regulated by P2Y(2)R in VSMCs, and that agLDL uptake due to P2Y(2)R activation is dependent upon cytoskeletal reorganization mediated by P2Y(2)R binding to FLN-A. Published by Elsevier Ireland Ltd.