DCBLD2 regulates vascular hyperplasia by modulating the platelet derived growth factor receptor-β endocytosis through Caveolin-1 in vascular smooth muscle cells

DCBLD2 is a neuropilin-like transmembrane protein that is up-regulated during arterial remodeling in humans, rats, and mice. Activation of PDGFR-β via PDGF triggers receptor phosphorylation and endocytosis. Subsequent activation of downstream signals leads to the stimulation of phenotypic conversion of VSMCs and arterial wall proliferation, which are common pathological changes in vascular remodeling diseases such as atherosclerosis, hypertension, and restenosis after angioplasty. In this study, we hypothesized that DCBLD2 regulates neointimal hyperplasia through the regulation of PDGFR-β endocytosis of vascular smooth muscle cells (VSMCs) through Caveolin-1 (Cav-1). Compared with wild-type (WT) mice or control littermate mice, the germline or VSMC conditional deletion of the Dcbld2 gene resulted in a significant increase in the thickness of the tunica media in the carotid artery ligation. To elucidate the underlying molecular mechanisms, VSMCs were isolated from the aorta of WT or Dcbld2^-/- mice and were stimulated with PDGF. Western blotting assays demonstrated that Dcbld2 deletion increased the PDGF signaling pathway. Biotin labeling test and membrane-cytosol separation test showed that after DCBLD2 was knocked down or knocked out, the level of PDGFR-β on the cell membrane was significantly reduced, while the amount of PDGFR-β in the cytoplasm increased. Co-immunoprecipitation experiments showed that after DCBLD2 gene knock-out, the binding of PDGFR-β and Cav-1 in the cytoplasm significantly increased. Double immunofluorescence staining showed that PDGFR-β accumulated Cav-1/lysosomes earlier than for control cells, which indicated that DCBLD2 gene knock-down or deletion accelerated the endocytosis of PDGF-induced PDGFR-β in VSMCs. In order to confirm that DCBLD2 affects the relationship between Cav-1 and PDGFR-β, proteins extracted from VSMCs cultured in vitro were derived from WT and Dcbld2^-/- mice, whereas co-immunoprecipitation suggested that the combination of DCBLD2 and Cav-1 reduced the bond between Cav-1 and PDGFR-β, and DCBLD2 knock-out was able to enhance the interaction between Cav-1 and PDGFR-β. Therefore, the current results suggest that DCBLD2 may inhibit the caveolae-dependent endocytosis of PDGFR-β by anchoring the receptor on the cell membrane. Based on its ability to regulate the activity of PDGFR-β, DCBLD2 may be a novel therapeutic target for the treatment of cardiovascular diseases.