Cellular Mechanisms of Permeability Edema Endothelial Barrier Dysfunction

Overview

Capillary endothelial cells form the primary barrier between the plasma and interstitial fluid. Intercellular contacts between endothelial cells and cellular adhesion to the underlying subendothelial matrix are responsible for the characteristic barrier properties of endothelium, including a low permeability to plasma proteins. The manner in which pathological conditions foster barrier dysfunction is not well understood. Barrier dysfunction results in a loss of contact between microvascular endothelial cells and/or weakening of their adhesion to the basement membrane. Certain substances elaborated during inflammatory processes such as thrombin, histamine, and bradykinin disrupt barrier function by a direct action on the endothelium to increase vascular permeability by opening intercellular junctions.

Activation Endothelial Retraction and Disruption of Cell-Cell Junctions

The endothelial cell is a target for many proinflammatory and thrombogenic mediators and growth factors. These agents may disrupt interendothelial junctions, increasing endothelial permeability and allowing the passage of plasma proteins through intercellular gaps. Inflammatory mediators such as thrombin result in increased endothelial permeability by causing an intense endothelial cell retraction and shape change. The signal transduction pathways activated by thrombin that promote loss of barrier function involve a complex series of biochemical events leading to a rapid and sustained phosphorylation of myosin light chain (MLC) and a simultaneous inhibition of MLC phosphatase that prevents dephosphorylation of MLC and prolongs the response. The disruption of endothelial junctions is likely precipitated by increased interaction of endothelial contractile proteins actin and myosin. Phosphorylation of MLC by calcium-dependent myosin light chain kinase (MLCK) is required for actin-myosin interaction. Filamentous actin within endothelial cells is known to associate with the cytoplasmic tail of the major adherens-junction protein VE-cadherin. Contractile force may "unhinge" adherens junctions between endothelial cells, resulting in formation of interen-dothelial gaps [7]. Such gaps, which thrombin induces within minutes in pulmonary endothelial cells in vitro, provide a plausible structural basis for increased paracellular permeability.

Ca2+-Dependent and Ca2+-Independent Permeability Increasing Mechanisms

Several key signaling and effector proteins in this important pathway have been identified (see Figure 1). The agonist thrombin activates PAR-1 (the proteinase activated receptor). GTP-binding protein Gq signals calcium release from intracellular stores, and calcium-store depletion, in turn, signals calcium entry (see later discussion). Free calcium in the cytosol is thought to bind to calmodulin (a calcium-binding protein); the calcium-calmodulin complex activates MLCK, which induces the phosphorylation of MLC. In parallel with Gq, the G12/13 G-protein pathway, acting through cytoplasmic Rho GTPase and its effector Rho kinase, inhibits MLC dephosphorylation. The combined effect of MLCK plus Rho kinase activity is to strongly induce and maintain MLC phosphorylation, resulting in formation of contractile units (stress fibers) that exert force upon interendothelial junctional complexes [8]. The model provided is probably an oversimplification of the biochemical process underlying endothelial cell retraction, since there is considerable cross talk and lateral regulation of the seemingly parallel pathways.

Considerable attention has been devoted to the regulation of endothelial calcium entry by thrombin. Inositol (1,4,5)-trisphosphate (IP3) formation induced by thrombin is known to cause release of sequestered calcium and elicit calcium entry via store-operated channels (SOC). Tiruppathi and associates have identified TRPC4 (Transient Receptor Potential Channel 4) as an essential constituent of the SOC in the mouse lung [9]. Their data support a causal relationship between increases in calcium entry and elevated pulmonary microvascular permeability. Thus, increased

Calcium Endothelial Cells

Figure 1 Signaling mechanism of thrombin-induced endothelial cell retraction and vascular barrier dysfunction. The protease thrombin activates the G protein coupled protease activated receptor 1 (PAR-1) on the endothelial cell surface. The Ca2+-dependent signaling pathway is activated by G protein Gq stimulation of phospholipase C, the generation of lipid intermediates that induce the release of Ca2+ from an intracellular storage compartment, and the regulation of myosin light chain kinase (MLCK) by Ca2+-calmodulin (Ca2+-CaM). Phosphorylated MLC interacts with F-actin, forming stress fibers that contract and pull apart the cell-cell junctions. The Ca2+-independent component of thrombin signaling is mediated by G12 and G13 activation of Rho and Rho kinase, which inhibits myosin light chain phosphatase (MLC-Pase), thereby preserving MLC phosphorylation and the contractile event in the face of declining Ca2+ levels. Thus, endothelial barrier dysfunction caused by inflammatory mediators such as thrombin is the result of Ca2+ induced actin-myosin based contraction of individual endothelial cells, which generates gaps between cells and increases albumin leakage into perivascular spaces.

Figure 1 Signaling mechanism of thrombin-induced endothelial cell retraction and vascular barrier dysfunction. The protease thrombin activates the G protein coupled protease activated receptor 1 (PAR-1) on the endothelial cell surface. The Ca2+-dependent signaling pathway is activated by G protein Gq stimulation of phospholipase C, the generation of lipid intermediates that induce the release of Ca2+ from an intracellular storage compartment, and the regulation of myosin light chain kinase (MLCK) by Ca2+-calmodulin (Ca2+-CaM). Phosphorylated MLC interacts with F-actin, forming stress fibers that contract and pull apart the cell-cell junctions. The Ca2+-independent component of thrombin signaling is mediated by G12 and G13 activation of Rho and Rho kinase, which inhibits myosin light chain phosphatase (MLC-Pase), thereby preserving MLC phosphorylation and the contractile event in the face of declining Ca2+ levels. Thus, endothelial barrier dysfunction caused by inflammatory mediators such as thrombin is the result of Ca2+ induced actin-myosin based contraction of individual endothelial cells, which generates gaps between cells and increases albumin leakage into perivascular spaces.

calcium influx leading to activation of endothelial retraction may be a fundamental, underlying cause of pulmonary edema.

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Essentials of Human Physiology

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