R12 - Exploring epidermal growth factor receptor inhibition and caveolin 1-dependent calcifying extracellular vesicle biogenesis in vascular calcification

Introduction: : Vascular calcification represents the most significant predictor of cardiovascular events with no current therapeutic options for prevention or treatment. Osteogenically-differentiated vascular smooth muscle cells (VSMCs) release calcifying extracellular vesicles (EVs), which nucleate nascent mineral. Caveolin-1 (CAV1), a plasma membrane scaffolding protein residing in caveolar domains, plays a critical role in the formation of calcifying EVs. Previous studies have reported interactions between CAV1 and epidermal growth factor receptor (EGFR) in cancer pathology. Given the nature of these reported interactions, we hypothesized EGFR inhibition and a deficiency in CAV1 may prevent the biogenesis of calcifying EVs by altering CAV1 trafficking.

Materials and

Methods: : We assessed the potential of EGFR tyrosine kinase inhibition (AG1478 and PD153035, 2.5 uM, N = 3) to prevent calcification in vitro using VSMCs cultured in osteogenic media (OS) for 7, 14, or 21 days. The dependence of CAV1 in vascular calcification was tested in vivo by placing global CAV1 knockout mice and smooth muscle cell (SMC) dependent CAV1 knockout mice on an adenine-induced chronic kidney disease and high phosphate diet.

Results, Conclusions, and Discussions:: In vitro, EGFR tyrosine kinase inhibition significantly prevented calcifications in OS cultures treated for 21 days (p < 0.0001 and p < 0.0001), 14 days (p < 0.0001 and p < 0.0001), and 7 days (p < 0.01 and p < 0.01). In vivo, global CAV1 knockout mice exhibit a decrease in vascular calcification in both males (p < 0.05) and females (p < 0.01) compared to wild type mice. However, there was no significant difference between groups with the SMC CAV1 knockout compared to controls. Our results suggest that EGFR interferes with trafficking mechanisms that are required for calcifying EV biogenesis no matter when administered in the progression of calcification and that CAV1 is required for EV biogenesis. However, our results also suggest in vivo, VMSCs are not the only cell type responsible for this mechanism. Future studies will investigate other cell types and the dependency of CAV1 in vascular calcification. Given that EGFR inhibitors exhibit clinical safety, the current data show that EGFR may be a propitious target in preventing vascular calcification.

Acknowledgements and/or References (Optional):: This work was supported by a grant from the National Heart, Lung, and Blood Institute of the National Institutes of Health (1R01HL160740) and the American Heart Association Predoctoral Fellowship (25PRE1374847).