Regulation of Permeability by the Endothelial Cell Cytoskeleton

Because the actin microfilament system is well studied and known to be essential for EC barrier regulation, we will explore its functional role in greater detail in this section. The current paradigm of EC barrier regulation suggests that a balance exists between barrier-disrupting cellular contractile forces and barrier-protective cell-cell and cell-matrix tethering forces. As outlined in Figure 1, both competing forces in this model are intimately linked to the actin-based EC cytoskeleton by a variety of actin-binding proteins that are critical both to tensile force generation and to linkage of the actin cytoskeleton to adhesive membrane components. Although less structured than in skeletal or smooth muscle cells, ECs contain similar molecular machinery for generation of tension via an actomyosin motor (actin and myosin represent more than 15% of total EC protein). Focally distributed changes in tension within EC are accomplished by regulation of the level of myosin light chain (MLC) phosphorylation, which promotes actomyosin interaction, and subsequent actin stress fiber formation. There is a good association between the development of transcellular actin stress fibers, increased MLC phosphorylation, enhanced tension development, paracellular gap formation, and increased EC permeability. A key regulator of this EC contractile apparatus in this process is the Ca2+/calmodulin (CaM)-dependent enzyme myosin light chain kinase (MLCK).

Balancing these EC contractile forces are cell-cell and cell-matrix contacts that provide tethering forces essential for mechanical stability and barrier maintenance (Figure 1). Adherens junctions are the primary cell-cell contacts along the EC monolayer and are composed of cadherins bound together in a homotypic and Ca2+-dependent fashion to link adjacent ECs. Cadherins interact through their cytoplasmic tails with the catenin family of intracellular proteins, which provide anchorage directly to the actin cytoskeleton. The primary cadherin in human EC adherens junctions, vascular endothelial (VE)-cadherin, is critical to EC barrier integrity since infusion of VE-cadherin blocking antibody increases lung vascular permeability in cultured ECs and mice. Focal adhesions provide the primary tethering sites of ECs to the underlying extracellular matrix through transmembrane integrin receptors connected to the actin cytoskeleton via multiprotein focal adhesion plaques. These linkages are also essential for EC barrier integrity as blocking antibodies to Pj integrin alter EC attachment and permeability. Unliganded integrins are not associated with the cytoskeleton, but binding to the ECM induces the attachment of integrins to intracellular actin fibers and stimulates tyrosine phosphorylation of multiple proteins and Ca2+ influx. Moreover, intracellular signaling pathways that regulate cytoskeletal rearrangement can also modulate cell-matrix contacts. For example, inhibition of the small GTPase Rho dissociates stress fibers from focal adhesions, decreases phosphotyrosine content of focal adhesion proteins, and enhances EC barrier function. Thus, the tethering forces of both focal adhesion and adherens junction complexes are essential components of EC barrier integrity.

Barrier Disruption

Activation of the contractile apparatus is a critical step in many models of agonist-induced EC barrier dysfunction. For example, one well-characterized model utilizes the central coagulation regulatory protein, thrombin, to induce significant pulmonary vascular leakage. The observation that microthrombi occur in the pulmonary microvasculature of patients with acute lung injury suggests this model has biologic relevance to EC barrier regulation in human disease. Thrombin induces a profound increase in EC permeability through rapid actin cytoskeletal rearrangement and force generation dependent on actomyosin interaction catalyzed by the phosphorylation of regulatory MLC by MLCK. Direct activation of MLCK is sufficient to produce EC contraction and barrier disruption, whereas MLCK inhibition abolishes barrier dysfunction in rat lung models of ischemia-reperfusion injury and ventilator-induced lung permeability. Tyrosine phosphorylation status appears to play an important role in regulation of EC permeability since tyrosine phosphorylation of EC MLCK evokes significant increases in MLCK activity, EC contraction, and subsequent EC barrier dysfunction.

The small GTPase Rho also plays an important role in regulation of the EC contractile apparatus in several models of agonist-induced EC barrier dysfunction. Linking extracellular stimuli to dynamic actin cytoskeletal rearrangement, Rho activation induces increased MLC phosphorylation, actomyosin interaction, stress fiber formation, and subsequent EC barrier dysfunction. The relative contributions of the EC MLCK and Rho pathways in regulating EC permeability are not completely understood since inhibition of either MLCK or Rho activation attenuates

Barrier Disruption

Barrier Enhancement

Edg-1 Receptor

Cortical Actin

Edg-1 Receptor

Cortical Actin

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Extracellular Matrix

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Extracellular Matrix

Essentials of Human Physiology

Essentials of Human Physiology

This ebook provides an introductory explanation of the workings of the human body, with an effort to draw connections between the body systems and explain their interdependencies. A framework for the book is homeostasis and how the body maintains balance within each system. This is intended as a first introduction to physiology for a college-level course.

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