Role of actin and myosin in the control of paracellular permeability in pig, rat and human vascular endothelium.

Abstract

SUMMARY1. We have investigated the endothelial actomyosin system with particular emphasis on its possible role in actively opening a paracellular route for permeability.2. Actin and myosin comprised 16% of total endothelial protein with a molar actin/myosin ratio of 16-2 which is close to the actin/myosin ratio of muscle (studies on freshly isolated pig pulmonary arterial endothelial cells, PAEC).3. By immunocytochemistry at the light and electron microscope levels the bulk of actin and myosin was colocalized in close vicinity to the intercellular clefts of both micro-and macrovascular endothelial cells in situ and in vitro.4. Calcium-ionophore-induced rise in permeability of human umbilical venous endothelial cells (HUVEC) and PAEC monolayers grown on filters in a two-chamber permeability system was caused by opening of intercellular gaps. Gap formation depended on the rise in intracellular Ca2+ and could be blocked by the calmodulinbinding drugs trifluperazine (TFP) and W7.5. In skinned monolayers of cultured PAEC and in isolated sheets of HUVEC gap formation was shown to require ATP and occurred only when free myosin binding sites were available on endothelial actin filaments (experiments with myosin subfragment 1 modified by N-ethylmaleimide, S1-NEM).6. These experiments suggest that actin and myosin in endothelial cells play a central role in regulating the width of the intercellular clefts, thereby controlling the paracellular pathway of vascular permeability.

Evidence of a Tubular System for Transendothelial Transport in Arterial Capillaries of the Rete Mirabile

“…In keeping with this, we have demonstrated the presence of actin associated with the tubular and vesicular membranes. Indeed, actin was detected not only in close association with the plasmalemmal membrane corresponding to the cortical filamentous cell web and the junctional complex, as reported previously (Schnittler et al 1990), but also with the limiting membrane of vesicular and tubular profiles. These cytochemical results are consistent with the previous demonstration of actin and actin binding proteins associated with plasmalemmal vesicles or caveolae (Izumi et al 1988;Lisanti et al 1994) and with the requirement of an intact actin network for the functional properties of caveolae (Parton et al 1994).…”

“…This milder condition of fixation and the embedding in Lowicryl are required for optimal detection of cytoskeletal protein antigenic sites but lead, however, to sub-optimal preservation of cellular structures (Bendayan 1983;Schnittler et al 1990). For the protein A-gold immunolabeling, a rabbit anti-actin antibody (Bendayan 1983) was used at 1:50 dilution according to the protocol described above.…”

Dynamics between actin and the VE-cadherin/catenin complex

“…Non-muscle actin, particular β-actin and γ-actin, 38 comprises about 10% of the total endothelial protein, 39,40 and thus, is a major protein in endothelium. Actin filaments (F-actin) polymerize from actin monomers (globular or G-actin) forming filaments between 5-7 nm.…”

“…13,111 These stress fibers are contractile as they consist of polarized actin filaments, myosin II, and α-actinin. 39,43 Together, the appearance and dynamics of actin filaments in endothelium critically depend on the physiological and pathological conditions in their environment. 37,41,42 Thus, both the VE-cadherin and the actin patterning in growing culture differ from the actin and VE-cadherin patterning seen physiologically in endothelial cells in vivo (see above).…”

“…Actin bundles consist of polarized short actin filaments that are associated with myosin II and α-actinin; proteins required for development of contractile forces in endothelium. 39,43 Actin bundles include both the circumferential actin filaments along the junctions and the cytoplasm traversing stress fibers. The circumferential junction-associated actin bundles are characteristic for intact endothelium in vivo, while stress fibers preferentially appear when endothelium becomes activated e.g., under inflammatory conditions and wound healing; conditions that are generally associated with cell proliferation and migration.…”