Abstract Body (Do not enter title and authors here): Introduction: Endothelial cell dysfunction causes recruitment of inflammatory cells to the intima, initiating atheromatous plaque build-up. Atherosclerosis underlies myocardial infarction and stroke which remain leading causes of death in the US. Identifying key regulators that limit endothelial dysfunction is critical. We uncovered a previously unrecognized role of an endocytic adaptor protein, Disabled homolog 2 (Dab2), which regulates endocytosis and lysosomal degradation of receptor tyrosine kinases such as VEGFR2. We found that endothelial Dab2 promoted VEGF signaling during angiogenesis in diabetic wound healing. However, the mechanism how Dab2 in the endothelial cells protected endothelium function in atherosclerosis is unknown.
Hypothesis: Endothelial Dab2 protects endothelial function under atherogenic conditions by promoting pulsatile shear (PS)-induced PI3K-Akt-eNOS signaling via endosomal trafficking. PS increases transcription factor KLF4 expression which activates Dab2 expression. Restoring Dab2 in the endothelium offers a therapeutic strategy to limit plaque progression.
Methods and Results: We found reduced endothelial Dab2 expression in human atherosclerosis and ApoE-/- mice. We generated endothelial-specific inducible Dab2 knockout mice (EC-Dab2iKO) and crossed to ApoE-null background (EC-Dab2iKO/ApoE^-/-). Western diet-fed EC-Dab2iKO/ApoE^-/- mice exhibited heightened arterial inflammation, more severe plaque formation, increased macrophage infiltration and reduced plaque stability. Single-cell RNA-seq, qPCR and western blot revealed that endothelial Dab2 depletion significantly upregulated pro-inflammatory markers and shear stress-related atherosclerotic pathways, indicating Dab2 protect against endothelial dysfunction by increasing PS-dependent eNOS activation via endosomal PI3K-Akt activation. ATAC-seq and ChIP-qPCR showed that KLF4 binds to Dab2 promoter. Using an in vitro PS flow channel to mimic atheroprotective flow, we found both Dab2 and KLF4 were increased. Using an endothelial-targeted engineered nanoparticle, we delivered Dab2 mRNA to the atherogenic endothelium to restore Dab2 function. It increased Dab2 level in the atheroma which restrains plaque progression in ApoE-/- mice.
Conclusions: Our study reveals Dab2 as a key protector of atherogenic endothelium, linking PS and endothelial homeostasis via KLF4 and PI3K-Akt-eNOS signaling. Targeted restoration of Dab2 may represent a promising therapeutic approach for atherosclerosis.