Phorbol ester-mediated pulmonary artery endothelial barrier dysfunction through regulation of actin cytoskeletal mechanics

Abstract

English. Phorbol ester-mediated pulmonary artery endothelial barrier dysfunction through regulation of actin cytoskeletal mechanics. Am J Physiol Lung Cell Mol Physiol 287: L153-L167, 2004. First published March 5, 2004 10.1152/ajplung.00292.2003.-The mechanisms of phorbol ester-and thrombin-mediated pulmonary artery endothelial barrier dysfunction were compared. Phorbol ester dibutyrate (PDBU) mediated slow force velocity and less force than thrombin. Taxol did not attenuate PDBU-mediated tension, while it reversed nocodazole-mediated tension. PDBU-mediated tension was not affected by acrylamide; PDBU increased cell stiffness and produced greater declines in transendothelial resistance (TER) than acrylamide. Thus PDBU caused a net increase in tension and did not unload microtubule or intermediate filaments. Microfilament remodeling, determined on the basis of immunocytochemistry and actin solubility, lacked the sensitivity and specificity to predict actindependent mechanical properties. Thrombin increased myosin light chain (MLC) kinase site-specific MLC phosphorylation, according to peptide map analysis, whereas PDBU did not increase PKC-specific MLC phosphorylation. The initial PDBU-mediated tension development temporally correlated with PDBU-mediated decline in TER and increased low-molecular-weight caldesmon (l-CaD) phosphorylation. PDBU-mediated tension development and decreases in TER were associated with a temporal loss of endothelial cell-matrix adhesion, based on a numerical model of TER. Although, on the basis of immunocytochemistry, thrombin-mediated tension was associated with actin insolubility, actin reorganization, and gap formation, these changes did not predict thrombin-mediated gap formation, based on TER and time-lapse differential interference contrast microscopy. These data suggest that PDBU may disrupt endothelial barrier function through loss of cell-matrix adhesion through l-CaD-dependent actin contraction. phorbol ester dibutyrate; porcine pulmonary artery endothelial cells; myosin light chain; low-molecular-weight caldesmon IT HAS BEEN WELL DOCUMENTED that phorbol esters directly increase endothelial permeability through loss of cell adhesion in association with remodeling of the actin cytoskeleton (2, 27). Phorbol esters represent unique edemagenic molecules that allow greater insight into how signal transduction pathways remodel endothelial cell adhesion through actin-dependent mechanical forces (2,17,27,30). The precise mechanism by which phorbol esters regulate endothelial cell adhesion through remodeling of actin-dependent mechanical forces remains poorly understood.

Myosin II Activity Softens Cells in Suspension

“…Our observation of cell stiffening for myosin-inhibited cells deformed at timescales of seconds stands in clear contrast to data published on cells adherent to a substrate (26)(27)(28)(29). In these publications, the cells were reported to soften upon myosin inhibition.…”

“…In summary, cells, once suspended and free from any physical attachment to the surrounding, stiffen and become more solidlike after myosin II inhibition-contrary to reports of cell softening with adherent cells (26)(27)(28)(29). Measurements on adherent cells are generally done on cells adhering to plastic tissue culture dishes.…”

“…The opposite effect has also been observed for studies using phorbol 12,13-dibutyrate to activate myosin II activity. Moy et al (26) applied magnetic twisting rheology to adherent cells and found an increase in cell stiffness, also possibly attributed to the buildup of cellular prestress. In our study, all cell types tested (with the exception of macrophages) became less compliant and less fluidlike after inhibition of myosin II, and vice versa.…”

“…Phorbol 12,13-dibutyrate, on the other hand, activates the protein kinase C (24,25) and therefore increases myosin II activity. So far, there exists a range of data on mechanical property measurements of adherent cells treated with these drugs (26)(27)(28)(29), which have shown that inhibition of myosin activity leads to a loss of cell prestress and a subsequent lower measured elastic modulus of the cells.…”

Isoform-specific differences between Rap1A and Rap1B GTPases in the formation of endothelial cell junctions

“…2) is reminiscent of what has been seen in other scenarios of EC barrier disruption. Expression of constitutively active RhoA or treatment with permeability-inducing agents such as thrombin 52,53 Negative control shrNA-treated and rap1B knockdown cell monolayers are visually intact; knockdown of rap1A using two independent shrNAs causes disruption of the endothelial cell monolayer. Scale bar = 30 μm.…”