Abstract 12597: Phenotypic Drug Screen Reveals That Lacidipine, a Calcium Channel Blocker, Enhances the Vascular Lumen Formation

Background: Vascular lumen formation is a critical process of angiogenesis towards establishing a functional circulatory system capable of delivering oxygen and nutrients to tissues. Targeting lumen formation is required for the development of efficient therapeutic angiogenesis. Objective: Perform a drug screen to identify compounds regulating the vascular lumen formation using a novel 2D angiogenesis assay that mimics the lumen formation of blood vessels and suitable for high throughput applications. Methods and Results: First, we optimized the phenotypic angiogenesis assay that captures the cellular process of lumen formation. Human Umbilical Vein Endothelial Cells (HUVEC) were seeded on Matrigel-coated wells. A mixture of matrix gel was added on top of adhered HUVEC. HUVECs form luminized vascular-like structures in 48 hours. To identify modulators of lumen formation, we conducted a high content screen of a library containing 150 FDA-approved cardiovascular drugs. The screening resulted in five calcium channel blockers being able to increase the lumen area compared to controls. Lacidipine was selected for further mechanistic studies. We found the cells in the luminal space stained positively for activated caspase-3 at 24 hours using immunofluorescence. Pharmacological inhibition of RAC1 or caspase activity abolished the Lacidipine-enhancing effect on lumen formation, suggesting the involvement of apoptosis and cell polarization. Next, we found that the addition of ML7, an MLCK inhibitor, enhances the lumen formation by inducing apoptosis. In contrast, the addition of the ROCK inhibitor GSK429286 or blebbistatin, an inhibitor of non-muscle myosin II ATPase, did not affect the lumen formation. Using a Ca2+ biosensor imaging, Lacipidine reduces the intracellular Ca2+ oscillations amplitude in the migrating cells at the early stage (6 hours), whereas Lacidipine blocked these Ca2+ oscillations completely at the late stages (24-48 hours). Conclusions: This study highlights a novel high throughput phenotypic assay to evaluate vascular lumen formation. Our findings suggest that calcium signaling plays a vital role during lumen morphogenesis, and calcium channel blockers could be used for more efficient angiogenesis-mediated therapies.