Abstract 4369233: Endothelial-specific Suppression of Canonical Wnt Signaling Promotes Fatty Acid Metabolism and Protects Against Endothelial Hyperpermeability

Introduction: Dysregulated endothelium contributes to the pathogenesis of cardiovascular disease. Endothelial cell (EC) metabolic pathways are pivotal regulators of inflammation and neovascularization, which promote atherosclerotic plaque formation and instability. Data suggest reprogramming of fatty acid oxidation (FAO) in ECs activates endothelial-to-mesenchymal transition (EndMT), which is linked to the pathogenesis of atherosclerosis. Prior studies show that augmented Wnt signaling in ECs exacerbates atherosclerosis through disruption of EC homeostasis and reduced FAO. Global Wnt suppression using a small molecule inhibitor can partially reverse this phenotype. However, the effects of EC-specific Wnt inhibition are unknown. We hypothesize that suppression of canonical Wnt signaling in ECs preserves cell metabolism and diminishes EndMT, thus mitigating dysregulated EC behavior. Methods: Mice with EC-specific deletion of key canonical Wnt co-receptors LRP5 and LRP6 were generated. ECs were isolated from both males and females. The qPCR, western blot, seahorse analysis, electron microscopy, and electric cell-substrate impedance sensing (ECIS) were used in this study. One/two-way ANOVA was used to get statistical significance. Results: Cells from mutant mice showed higher levels of the metabolic genes CPT1a , PPARα , PGC-1α , and SIRT3 compared to wild-type (WT) (p=.003, .002, .04, .0001, respectively; WT vs LRP5 KO). Analysis of FAO in ECs from WT and LRP5 ECKO mice showed higher maximal (p=.003) oxygen consumption rates in LRP5 KO ECs compared to WT, indicating that mutant ECs are more reliant on oxidative phosphorylation. Higher spare respiratory capacity in LRP5 KO ECs (p=.04) was also observed. Mutant ECs exhibited elongated mitochondria compared to those from WT ECs (p<.0001), suggesting higher metabolic activity. TNFα (10ng/ml) treatment significantly induced mRNA expression of αSMA (p=.04) and increased Snail protein (p=.04) in WT ECs; however, these effects were not observed in TNFα-treated mutant ECs. In addition, mutant ECs demonstrate higher endothelial integrity, as measured by ECIS, after either TNFα (30ng/ml) or LPS (0.2µg/ml) treatment compared to WT ECs. Conclusions: Suppression of canonical Wnt signaling in ECs upregulates FAO and key metabolic regulators, thus lessening EndMT. Inhibition of EC-specific canonical Wnt signaling may be a novel therapeutic approach for EC metabolic disturbances in cardiovascular disease, including atherosclerosis.