Lamellipodia dynamics and microrheology in endothelial cell paracellular gap closure.

Vascular endothelial cells (ECs) form a semipermeable barrier separating vascular contents from the interstitium, thereby regulating the movement of water and molecular solutes across small intercellular gaps, which are continuously forming and closing. Under inflammatory conditions, however, larger EC gaps form resulting in increased vascular leakiness to circulating fluid, proteins, and cells, which results in organ edema and dysfunction responsible for key pathophysiologic findings in numerous inflammatory disorders. In this study, we extend our earlier work examining the biophysical properties of EC gap formation and now address the role of lamellipodia, thin sheet-like membrane projections from the leading edge, in modulating EC spatial-specific contractile properties and gap closure. Micropillars, fabricated by soft lithography, were utilized to form reproducible paracellular gaps in human lung ECs. Using time-lapse imaging via optical microscopy, rates of EC gap closure and motility were measured with and without EC stimulation with the barrier-enhancing sphingolipid, sphingosine-1-phosphate. Peripheral ruffle formation was ubiquitous during gap closure. Kymographs were generated to quantitatively compare the lamellipodia dynamics of sphingosine-1-phosphate-stimulated and -unstimulated ECs. Utilizing atomic force microscopy, we characterized the viscoelastic behavior of EC lamellipodia. Our results indicate decreased stiffness and increased liquid-like behavior of expanding lamellipodia compared with regions away from the cellular edge (lamella and cell body) during EC gap closure, results in sync with the rapid kinetics of protrusion/retraction motion. We hypothesize this dissipative EC behavior during gap closure is linked to actomyosin cytoskeletal rearrangement and decreased cross-linking during lamellipodia expansion. In summary, these studies of the kinetic and mechanical properties of EC lamellipodia and ruffles at gap boundaries yield insights into the mechanisms of vascular barrier restoration and potentially a model system for examining the druggability of lamellipodial protein targets to enhance vascular barrier integrity.