title: Endothelial barrier disruption and recovery is controlled by substrate stiffness.
abstract: Circulating barrier disruptive agonists bind specific cell membrane receptors and trigger signal transduction pathways leading to the activation of cell contractility and endothelial cell (EC) permeability. Although all cells in tissues including vascular EC are surrounded by compliant extracellular matrix, the impact of matrix stiffness on agonist-induced signaling, cytoskeletal remodeling and EC barrier regulation is not well understood. This study examined agonist-induced cytoskeletal and signaling changes associated with EC barrier disruption and recovery using pulmonary EC grown on compliant substrates of physiologically relevant (8.6 kPa) stiffness, very low (0.55 kPa) and very high (42 kPa) stiffness. Human pulmonary microvascular and macrovascular EC grown on 0.55 kPa substrate contained a few actin stress fibers, while stress fiber amount increased with increasing matrix stiffness. Thrombin-induced stress fiber formation was maximal in EC grown on 42 kPa substrate, diminished on 8.6 kPa substrate, and was minimal on 0.55 kPa substrate. These effects were linked to a stiffness-dependent increase in thrombin-induced phosphorylation of the Rho kinase target, myosin light chain phosphatase (MYPT1), and regulatory myosin light chains (MLC). Surprisingly, EC barrier recovery and activation of Rac GTPase-dependent barrier protective signaling reached maximal levels in EC grown on 8.6 kPa, but not on 0.55 kPa substrate. In conclusion, these data show a critical role of extracellular matrix stiffness in the regulation of the Rac/Rho signaling balance during onset and resolution of agonist-induced EC permeability. The optimal conditions for the Rho/Rac signaling switch, which provides an effective and reversible EC cytoskeletal and permeability response to agonist, are reached in cells grown on the matrix of physiologically relevant stiffness.
authors.affiliation: Lung Injury Center, Section of Pulmonary and Critical Medicine, Department of Medicine, University of Chicago, Chicago, IL 60637, USA. kbirukov@medicine.bsd.uchicago.edu
authors.forename: Anna A
authors.initials: AA
authors.lastname: Birukova
authors.forename: Xinyong
authors.initials: X
authors.lastname: Tian
authors.forename: Ivan
authors.initials: I
authors.lastname: Cokic
authors.forename: Yvonne
authors.initials: Y
authors.lastname: Beckham
authors.forename: Margaret L
authors.initials: ML
authors.lastname: Gardel
authors.forename: Konstantin G
authors.initials: KG
authors.lastname: Birukov
mesh_terms: D000180:Acrylic Resins / Q000737:chemistry*; D002199:Capillary Permeability / Q000187:drug effects; D018929:Cell Culture Techniques; D002478:Cells, Cultured; D004548:Elasticity; D042783:Endothelial Cells / Q000187:drug effects / Q000378:metabolism*; D006801:Humans; D020100:Hydrogels; D008168:Lung / Q000098:blood supply*; D018994:Myosin Light Chains / Q000378:metabolism; D043263:Myosin-Light-Chain Phosphatase / Q000378:metabolism; D010766:Phosphorylation; D015398:Signal Transduction; D022502:Stress Fibers / Q000378:metabolism; D013917:Thrombin / Q000494:pharmacology; D013997:Time Factors; D020744:rac GTP-Binding Proteins / Q000378:metabolism; D020741:rho GTP-Binding Proteins / Q000378:metabolism; D000099166:Polyacrylamides
chemical_list: D000180:Acrylic Resins; D020100:Hydrogels; D018994:Myosin Light Chains; D043263:Myosin-Light-Chain Phosphatase; D013917:Thrombin; D020744:rac GTP-Binding Proteins; D020741:rho GTP-Binding Proteins; D000099166:Polyacrylamides; C490372:PPP1R12A protein, human