The Interplay Between mTOR Inhibitors and Endothelial Cell-to-Cell Junction Dynamics: Implications for Vascular Barrier Dysfunction

PROJECT SUMMARY Cell growth and proliferation are regulated by signaling pathways, notably the mechanistic Target of Rapamycin (mTOR). Inhibitors of mTOR signaling protein complexes (mTORC1, mTORC2), such as everolimus (EVL), are commonly used in transplant medicine and coronary stent intervention. Despite its widespread use, EVL induces endothelial barrier dysfunction (EBD) in over 28% of transplant patients and increases the rate of heart attacks in those undergoing stent intervention by 5-fold. EBD arises from disrupted stable endothelial cell junctions (ECJs), which are crucial for regulating paracellular permeability between neighboring endothelial cells. Our limited understanding of ECJ protein regulation, exacerbated by a lack of quantitative tools for assessing their structure, underscores significant deficits in comprehending EVL's effects on EBD. FKBP12 is recognized for its dual role in stabilizing calcium (Ca2+) channels and as a receptor for immunosuppressive drugs like EVL. Together, FKBP12 and EVL form a complex that inhibits mTORC1. While pre-clinical studies suggest that EVL- induced EBD may occur due to FKBP12 displacement, triggering the endocytic turnover of vascular endothelial (VE)-cadherin—a critical protein in ECJs—this mechanism alone cannot fully explain EVL's effect on EBD. Notably, leakage occurs shortly after EVL treatment, preceding changes in VE-cadherin expression levels observed at 24 hours. Related drugs, such as tacrolimus, utilize FKBP12 to target alternative signaling pathways without inducing endothelial permeability. Recent advancements in mTOR inhibitors, such as Torin-2, show promise in mitigating EBD in pre-clinical studies. Unlike EVL, Torin-2 directly inhibits both mTORC1 and mTORC2. Our preliminary data demonstrate that EVL inhibition of mTORC1 disrupts ECJ proteins within 4 hours, but the precise impact of mTORC2 inhibition on EBD and ECJ stability remains uncertain. We hypothesize that direct inhibition of mTORC1 and mTORC2 will effectively preserve ECJ integrity, thereby reducing vascular leakage. Aim 1 will investigate how inhibiting mTORC1 and mTORC2 affects the integrity of endothelial barriers. It will analyze the impact of two inhibitors, EVL and Torin-2, on ECJs, morphology, and cell-cell adhesion strength to understand their role in maintaining barrier function. Aim 2 seeks to explore the relationship between ECJ dynamics and monolayer permeability under mTOR inhibition, employing live-imaging techniques and local permeability assays to address the limitations of previous studies that overlooked time-dependent changes in permeability. Successful completion of this proposal will clarify the impact of mTORC1 and mTORC2 on ECJ structures, providing insights into their vascular effects. This research will enhance our understanding of EBD and mTOR inhibitors at a fundamental level, potentially informing translational applications. Along with my detailed training plan, the research planned in this F31 award period will advance my ultimate goal of becoming an independent scientist focused on improving vascular health.