Abstract 179: Rho Kinase Isoform Contribution to Endothelial Contractility
Background: Rho kinase (ROCK) is the major effector protein of RhoA and is known to mediate F-actin stress fibers. ROCK activates myosin motors. Actomyosin-generated contractile forces can be transmitted to cell-cell junctions, resulting in increased junctional tension, junctional disassembly and endothelial hyperpermeability. In apparent contrast, basal ROCK activity is required for maintenance of barrier integrity. We hypothesize that these dual roles of ROCK could be contributed to the two highly homologous expressed isoforms.
Methods: Traction force microscopy was utilized to study the endothelium-generated contractile forces after depletion of ROCK1 and/or ROCK2 by SiRNA approach in the absence and presence of the vaso-active agent thrombin.
Results: The traction force landscape showed that ROCK is a major contributor to endothelial contraction and permeability. Depletion of ROCK1 and ROCK2 resulted in a blockade of contractile response to thrombin and a marked reduction in endothelial hyperpermeability. Detailed force distribution maps revealed that the absence of ROCK1, but not of ROCK2, resulted in an increase in baseline contractile forces (siROCK1: 88.1 ± 4.1 Pa, siROCK2: 53.6 ± 3.4 Pa, Control: 56.2 ± 6.1 Pa), which was associated with the formation of numerous inter-endothelial gaps upon stimulation with thrombin. Knockdown of ROCK2 stabilized the endothelial barrier and largely prevented traction force enhancements. Exposure to thrombin resulted in all conditions in an increase of traction forces and hyperpermeability.
Conclusion: Both ROCK isoforms have a distinct function in endothelial contractility and permeability. ROCK1 is predominantly contributing to barrier maintenance under baseline conditions, whereas ROCK2 mediates the thrombin-induced contractile force enhancements and subsequent barrier dysfunction.
Funding: Dutch Heart Foundation 2011T072
Author Disclosures: E. Valent: None. R. Krishnan: None. V. van Hinsbergh: None. G.P. van Nieuw Amerongen: None.
- © 2014 by American Heart Association, Inc.