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Yuan SY, Rigor RR. Regulation of Endothelial Barrier Function. San Rafael (CA): Morgan & Claypool Life Sciences; 2010.

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Regulation of Endothelial Barrier Function.

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Chapter 4The Endothelial Barrier

The barrier function of exchange microvascular endothelium derives from the integrity of the endothelial structure, which undergoes moment-to-moment changes at the cytoskeleton, cell–cell junction complexes, and cell attachments to extracellular matrix and basement membrane. Appropriate regulation of these events maintains a low and selective permeability to fluid and solutes under normal physiological conditions. Endothelial barrier dysfunction occurs during stimulation by inflammatory agents, pathogens, activated blood cells, or disease states. The pathophysiology is characterized by excessive flux of plasma across the exchange microvessel wall into the surrounding tissues. Traditionally, compromised endothelial cell–cell junctional integrity is considered to account for the leak response. However, recent evidence demonstrates that blood fluid, solutes, and even circulating cells can cross the endothelium via two routes: through the cell body (transcellular), or between the cells (paracellular, or intercellular) (Figure 13). Here, we discuss the ultrastructural basis and function of the transcellular pathway vs. paracellular pathway.

FIGURE 13. Transcellular and paracellular permeability pathways across the microvascular endothelium.


Transcellular and paracellular permeability pathways across the microvascular endothelium. Barrier function of the microvasculature is provided by closely apposed endothelial cells of themicrovessel walls. The thin layer of endothelium is attached to (more...)


Transcytosis represents an important pathway of endothelial transcellular permeability to macromolecules [reviewed in 78, 239, 301, 305, 361]. The mechanism involves vesicle-mediated endocytosis at the endothelial luminal membrane, followed by transcytosis across the cell, and exocytosis at the basolateral membrane. This process can be completed by individual vesicles capable of shuttling from the apical to basolateral membrane of an endothelial cell, or by clusters of interconnected vesiculo-vacuolar organelles (VVOs) that form channel-like structures 80–200 nm in diameter, spanning the cell interior (Figure 13) [119, 237].

Vesicle-mediated transcytosis occurs when albumin binds to gp60 receptors on the endothelial cell surface [201, 312, 469]. Endocytosis and exocytosis have been visualized by electron microscopy in capillaries and postcapillary venules using tracer-labeled (e.g., gold-labeled) albumin [377], and other macromolecular markers [119]. EM micrographs reveal albumin apparently in various phases of transcytosis in endothelial cells: open and closed vesicles at the luminal and abluminal surfaces. Blood cells (leukocytes) may also be enveloped by endocytic vesicles and moved across the endothelial cell interior by transcytosis. This has been shown in microscope images of fluorescent-labeled leukocytes extending foot processes into membrane invaginations, and being internalized [68, 69].

Endocytosis and exocytosis are mediated by caveolae, lipid raft microdomains that form “cave-like” invaginations in the plasma membrane (Figures 13 and 14) [360, 361, 424]. The volume of fluid residing within caveolae constitutes approximately 15–20% that of the endothelial cell interior volume [140], and therefore by forming vesicles, caveolae are capable of moving substantial amounts of fluid and solutes across the cell interior [360]. Caveolae contain caveolin-1 (cav-1), a 22-kDa structural protein that is recruited to the membrane and forms an oligomeric assembly that is necessary for the characteristic shape and structure of caveolae [361, 397].

FIGURE 14. Vesicle endocytosis and transcytosis across endothelial cells.


Vesicle endocytosis and transcytosis across endothelial cells. Vesicle formation is triggered by Src kinase-mediated phosphorylation of caveolin-1 (cav-1) at the endothelial luminal membrane. Cav-1 subunits aggregate in lipid rafts and oligomerize to (more...)

Caveolae contain typical lipid raft components including cholesterol and sphingolipids, as well as scaffolding and signaling molecules that modify cav-1 function and initiate vesicle trafficking (Figure 14) [78, 362, 424]. Cholesterol is necessary for cav-1 recruitment to the membrane. Oligomerization of cav-1 and caveolae formation are then initiated by phosphorylation of cav-1 (by Src kinase) [260, 271, 311, 408]. Subsequent recruitment of dynamin (large GTPase) and intersectin (adapter protein) allow caveolae to fully form and to pinch off from the plasma membrane [363] forming endocytic vesicles approximately 70 nm in diameter (Figure 14). These vesicles remain docked to the plasma membrane through interactions of soluble N-ethylmaleimide-sensitive factor attachment receptors (SNAREs): vesicle-associated (v)-SNARE (vesicle-associated membrane protein (VAMP)) binds to membrane-associated target (t)-SNARE (25-kDa synaptosome-associated protein (SNAP-25) and syntaxin) [200, 362]. Dissociation of v-SNARE from t-SNARE and vesicle detachment are induced by N-ethylmaleimide-sensitive factor (NSF), a vesicle fusion protein with ATPase activity that is inhibited by the compound N-ethylmaleimide (NEM) [359]. Detached endocytic vesicles may recycle back to the apical cell membrane or may move across the cell interior to the basolateral side (transcytosis), likely mediated by interactions with cytoskeletal microtubules [277, 301]. Upon arrival at the target membrane, vesicles bind to docking proteins (v-SNARE to t-SNARE) and fuse with the basolateral (tissue side) membrane or at VVOs within the cell interior [361]. Multiple caveoli may be fused with VVOs in clusters in endothelial cells [372]. Once fused to VVOs or to the basolateral membrane, vesicles can perform exocytosis, releasing their contents into the surrounding tissue. However, it remains to be clarified what solutes are deposited by vesicles into VVOs, and to what extent this contributes to the basal or stimulated permeability for any particular solute [119].

The involvement of caveolin-1 in regulating cardiovascular functions associated with endothelial barrier properties has been demonstrated through studies using transgenic and knockout animals [35, 167, 275]. However, in knockout mice, absence of caveolin-1 leads to a compensatory increase in the paracellular permeability response to hyperpermeability-inducing agents [361]. Thus, while caveolin-1 and transcytosis contribute to regulation of endothelial permeability under normal circumstances, transcytosis is not strictly necessary for hyperpermeability responses because of compensatory hyperpermeability at cell–cell junctions. Vogel and coworkers have also shown that although albumin transcytosis occurs in the intact lungs, transcellular albumin flux does not contribute to fluid filtration [469]. Hence, the contribution of endothelial transcytosis to fluid homeostasis under physiological conditions or plasma leakage under pathophysiological conditions in most systems is not known. In contrast, investigators have reported that transcellular water flux accounts for as much as 50% of hydraulic conductivity in some endothelial systems [489]. An alternative mechanism for facilitating the transcellular passage of fluid is through the transmembrane water channel aquaporin [264]. Aquaporins are integral membrane proteins expressed in endothelial cells that permit the diffusive flux of water across the cell membrane. However in many tissues the contribution of aquaporin channels to Lp is believed to be minor or insignificant (<10%) [378].


The paracellular pathway is responsible for the majority of leakage of blood fluid and proteins across the microvascular endothelium under pathophysiological conditions. In the microvessels of certain types of tissues or organs, such as the kidney and liver, there are discontinuities or fenestrations between endothelial cells that are sufficiently large to permit the passage of large molecules or proteins [262, 305]. In other organs, most endothelial cell–cell interfaces are fused together by intercellular junctions or pores that selectively allow water, macromolecules, and even blood cells to pass through. The structural and functional integrity of these junctions is a major determinant of paracellular permeability.

Two types of intercellular junctions have been characterized as the cell–cell adhesive barrier structures in the microvascular endothelium: the adherens junction (AJ) and tight junction (TJ) (Figure 15) [239, 301]. AJs are found in most, if not all microvascular beds, and are the most ubiquitous type of endothelial cell–cell junction. AJs are impermeant to albumin (69 kDa; molecular radius 3.6 nm) and other large proteins, and thus the major determinant of endothelial barrier to macromolecules in many organs and tissues [301, 423]. Compared to AJs, TJs are less common in the peripheral microvasculature. TJs are mainly expressed in the microvascular endothelium of some specialized tissues, for example, the blood–brain or blood–retinal barriers [184, 340]. TJs impart additional barrier function, preventing the passage of much smaller molecules (<1 kDa), even restricting the flow of small inorganic ions (e.g., Na+).

FIGURE 15. Endothelial cell junctions and adhesions.


Endothelial cell junctions and adhesions. Endothelial cells of the microvessel wall are joined together by intercellular junction proteins: adherens junctions (AJs), tight junctions (TJs) and/or gap junctions. Barrier function in most vascular beds is (more...)

The barrier properties of endothelial adherens and tight junctions are due to interactions of integral membrane glycoproteins on neighboring endothelial cells (Figure 15). The extracellulardomains of junction proteins displayed on the surfaces of juxtaposed endothelial cells bind to each other and form a seal, restricting the passage of molecules between the cells (paracellular). The tightness of this barrier varies according to the types of junctions that are present in each tissue type, and subject to moment-to-moment changes in response to physical forces or biological signals. Deleterious effects on junction integrity may arise through degradation or dissociation of junction proteins, reorganization or internalization of junction structures, altered interactions with the cytoskeleton, or destabilization of attachments to the ECM that ultimately interfere with the sealing efficiency of intercellular junctions and cause endothelial hyperpermeability. The composition of cell–cell junctions, including the types of junction proteins present, is determined by junction type. In general, the type of junction dictates the pore size and, hence, the size of molecule that is permitted to pass. For example, the mean pore size of adherens junctions is approximately 3 nm [301, 423], whereas the mean pore size of tight junctions is approximately 1 nm [96, 452].

There are two additional structures related to endothelial cell–cell junctions that are not considered to be determinants of paracellular permeability. First is the gap junction (Figure 15). Gap junctions are found mainly in larger vessels and do not contribute significantly or directly to microvascular barriers. The molecular structure of gap junctions is characterized by six units of connexin forming a channel that connects the cytosols of adjacent endothelial cells, allowing rapid propagation of signaling molecules (e.g., Ca2+) between the cells [242]. Thus, although gap junctions may indirectly participate in the regulation of endothelial permeability by promoting cell–cell communication, they do not provide the barrier function per se. The second issue concerns fenestrations and discontinuous endothelium. A few tissues contain microvascular endothelium with open fenestrations ranging 50 to 60 nm in diameter (e.g., kidney, intestine or choroid plexus), or discontinuous endothelium with gaps as large as 100 nm (e.g., liver or spleen), permitting the passage of very large solutes [262]. These types of junctions appear to be specialized structures in tissues responsible for absorption of nutrients or detoxification and elimination of toxic wastes. Due to their minimal importance or noncommonality in permeability regulation, these structures are not emphasized in the following sections, which provide a detailed analysis of the molecular structure and functional regulation of AJs and TJs.

Adherens Junctions

The adherens junction (Figure 16) has been identified in nearly all types of vascular beds, especially in the peripheral microvasculature. Vascular endothelial (VE)–cadherin is believed to be the most important protein in forming the molecular basis, as well as regulating the function of AJs. VE–cadherin is a transmembrane receptor; its extracellular domain binds to the extracellular domain of another VE–cadherin expressed in the membrane of an adjacent endothelial cell. By forming a homotypic bond in this manner, VE–cadherin glues the neighboring cells together. The intermolecular binding of VE–cadherin extracellular domains is dependent upon extracellular calcium. Ca2+ binding to negatively charged amino acid residues on the extracellular domain of VE–cadherin promotes the protein conformation necessary for VE–cadherin to perform homotypic binding [386]. Intracellularly, VE–cadherin is connected to the actin cytoskeleton via a family of catenins (α-, β-, γ-, and p120-catenins). A current model of adherens junction structure is that VE–cadherin binds directly to β-catenin and γ-catenin, which in turn are connected to actin via binding to α-catenin [301]. Connection to the actin cytoskeleton is further stabilized by α-catenin binding to other proteins, including α-actinin, vinculin, vasodilator-stimulated phosphoprotein (VASP) and formin. VE–cadherin is also stabilized by binding to p120-catenin, though p120-catenin does not directly bind to actin. Rather, p120-catenin binds to protein kinases (Src kinases) and phosphatases, serving as a scaffold to bring these signaling molecules into proximity with adherens junction proteins for further interactions [350, 380]. Thus, the catenins not only serve as a structural linkage between VE–cadherin and the cytoskeleton, they also transduce biochemical signals for cell–cell communications. The stability of the VE–cadherin–catenin–cytoskeleton complex is essential to the maintenance of endothelial barrier function [15, 389, 465].

FIGURE 16. Adherens junctions.


Adherens junctions. Adherens junctions (AJs) are ubiquitous throughout the vasculature. The intercellular adhesion protein vascular endothelial (VE)-cadherin is principally responsible for barrier function. VE–cadherin homophilic intercellular (more...)

In addition to VE-cadherin and catenins, other proteins are present at cell–cell contacts and may associate or interact with adherens junctions, including E-cadherin, junctional adhesion molecules (JAMs), and platelet–endothelial cell adhesion molecule (PECAM-1). The specific contribution of these proteins to endothelial barrier properties is unclear. JAM proteins can bind to zona occludens 1 (ZO-1), a linker protein that connects tight junction proteins to α-catenin (described below), as well as to signaling molecules and proteins that stabilize the actin cytoskeleton. PECAM-1 binds to integrins on leukocytes and may facilitate leukocyte transmigration across the microvascular endothelium.

Tight Junctions

Endothelial tight junctions are similar to adherens junctions, but are composed of interactions of tight junction proteins: occludin, claudins (3/5), and JAM-A (Figure 17) [2, 184, 301]. Occludin and claudins are integral membrane proteins, each with four transmembrane domains and two extracellular loop domains. The extracellular loop domains of occludin or claudins form homotypic binding with the extracellular domains of like molecules on neighboring endothelial cells. JAM-A, a member of the immunoglobin superfamily of proteins, is also present in tight junctions, though the role of JAM-A in tight junctions is not understood. Occludin, claudins, and JAM-A are connected to the actin cytoskeleton via zona occludens proteins (ZO-1, ZO-2) and α-catenin. In addition to connecting junction proteins to the cytoskeleton, ZO proteins serve as signaling molecules (guanylate kinases) or scaffolding proteins that recruit other signaling molecules via PDZ and Src homology 3 (SH3) binding domains. Hence ZO proteins play both structural and signaling roles in tight junctions. The connection between tight junctions and the actin cytoskeleton is further stabilized by actin cross-linking proteins (e.g., spectrin or filamen) and accessory proteins (e.g., cingulin or AF-6) [184, 414].

FIGURE 17. Tight junctions.


Tight junctions. Tight junctions (TJs) are found in most vascular beds; however, TJs contribute to microvascular barrier function only in a few specialized tissues, including the brain, retina and testicles. The intercellular junction proteins mainly (more...)


Focal adhesions are points of attachment between the endothelial basolateral membrane and the surrounding extracellular matrix (ECM) of the microvascular wall [493] (Figure 18). The major structural components of focal adhesions are transmembrane receptors called integrins. Integrins are a family of glycoproteins expressed as α/β heterodimers (discussed further in the next chapter). Their intracellular domains interact with the cytoskeleton either directly or indirectly through the linker proteins paxillin, talin, vinculin, or α-actinin (Figure 18, top), and their large extracellular domains bind to respective matrix proteins, such as fibronectin, vitronectin, collagen, fibrinogen, and laminin (Figure 18, bottom) [197, 348]. The molecular organization of integrins varies depending on the chemical and physical states of extracellular matrices [106].

FIGURE 18. Focal adhesions.


Focal adhesions. Upper panel: endothelial cells are anchored to the basement membrane via focal adhesions. Focal adhesions are lipid raft microdomains in the endothelial basolateral membrane that are enriched in integrins, transmembrane heterodimeric (more...)

Vascular endothelial cells express multiple integrins with distinct combinations of α/β subunits, including α1β1, α1β2, α1β5, α2β1, α3β1, α5β1, α6β1, α6β4, αvβ3, and αvβ5. Typically, α1β1 and α1β2 bind to collagen; α3β1, α6β1, and α6β4 bind to laminin; α5β1 binds to fibronectin; and αvβ3 and αvβ5 bind to vitronectin [14, 287]. Many of these integrins recognize the arg-gly-asp (RGD) sequence in matrix proteins and thereby are able to interact with more than one extracellular ligand.

Integrin–matrix binding is essential to the establishment and stabilization of endothelial barriers [107]. Altering integrin-binding properties reduces focal adhesion strength [448] or causes cell detachment from the substratum [77, 106]. Synthetic peptides that compete the RGD-binding sequence or antibodies directed against the β1 subunit of integrins produce a dramatic increase in transendothelial flux of water and large solutes [98, 368, 483]. Direct evidence that underscores the physiological significance of integrin–matrix interactions comes from Wu's study in intact exchange microvessels, showing that inhibition of integrin binding to either fibronectin or vitronectin with synthetic RGD peptides dose-dependently increased albumin permeability by 2- to 3-fold [495]. The RGD-induced hyperpermeability was time-dependent and reversible upon clearance of the peptides, indicating that the effect was not merely due to a permanent disruption of the endothelium, consistent with the idea that endothelial cell–matrix adhesion is a dynamic process [509, 511].

Integrins provide an important structural support for maintaining endothelial barriers as well as their moment-to-moment changes in coordination with other barrier components. This structural support is multidirectional and may not be limited to the basolateral site of endothelial cells. Indeed, some members of the integrin family have been identified to be located at endothelial cell–cell borders [250]. It is suggested that these integrins collaborate with other intercellular molecules to form lateral junctions. Thus, blocking integrin function could alter the junctional connection leading to permeation of macromolecules across endothelial monolayers.

Not only do focal adhesions maintain normal physiological endothelial barrier properties, their assembly and activity also mediate hyperpermeability responses under stimulated conditions in the presence of angiogenic factors, inflammatory mediators, or physical forces. For example, integrins are essential to the mechanotransduction of endothelial responses to shear stress, a well-known modulator of vascular permeability [79, 211, 514]. The β5 subunit of integrins has been identified as a key molecule involved in the recruitment of kinases to focal adhesions in endothelial cells upon stimulation by vascular endothelial growth factor (VEGF) [29]. Mice deficient in integrin β5 expression display a reduced vascular permeability response to VEGF treatment [123]. In addition, it has been shown that thrombin, a well-characterized hyperpermeability factor, contains an RGD-binding sequence that interacts with αvβ3 on endothelial cells leading to enhanced angiogenesis [197]. Most recently, an in vivo experiment demonstrated that plasma leakage across microvessels caused by fibrinogen degradation products is greatly attenuated in integrin β1 knockout mice [170], further supporting the role of integrins in regulating endothelial hyperpermeability.

The precise mechanisms by which focal adhesions contribute to the maintenance of endothelial barrier function and hyperpermeability responses to stimulation are not completely understood [493]. It appears that various physical and chemical signals can be sensed and coordinated at the cell–matrix focal contact sites where integrins play a central role in transmembrane crosstalk between the cells and extracellular matrix. Within this context, focal adhesions are lipid raft domains containing scaffold proteins that bind to multiple intracellular signaling molecules (Figure 19). On one hand, ECM–integrin interactions induce “outside-in” signaling events that may contribute to the maintenance of endothelial barrier integrity. On the other hand, agonist-receptor binding triggers “inside-out” signaling events that modulate integrin–ECM adhesions. These responses alter the pattern or strength of integrin–ECM interactions, and in worse cases, cause disengagement of focal adhesions and even detachment of cells from ECM. Altered focal adhesions may also interfere with maintenance of normal cytoskeletal tension required for basal barrier properties. In response to certain stimuli, such as histamine, both the number and strength of integrin–matrix bonds are increased. This may contribute to hyperpermeability by transducing cytoskeletal contractile forces to cell–cell junctions leading to barrier opening.

FIGURE 19. Outside-in and inside-out signaling at endothelial focal adhesions.


Outside-in and inside-out signaling at endothelial focal adhesions. Binding of RGD proteins to integrins triggers intracellular signaling events (outside-in signaling) including activation and recruitment of kinases (e.g., Src kinase, focal adhesion kinase (more...)

The ECM also supports normal endothelial barrier function by preventing extravasation of circulating cells into the extravascular tissues [222, 490]. Under pathophysiological conditions, such as inflammation or metastatic cancer, activated leukocytes or invasive tumor cells secrete proteases and other enzymes capable of digesting ECM proteins and disengaging focal adhesions [43, 232, 384]. Certain inflammatory cells also bind to endothelial cell surface through integrins (e.g., leukocyte β2 integrins), which stabilizes leukocyte attachment to endothelium and facilitates transendothelial migration, enabling subsequent chemotactic migration across matrices. ECM breakdown permits migrating cells to enter the surrounding tissue [222]. A similar process occurs during angiogenesis, where ECM breakdown enables provisional cell migration along the leading edge of sprouting capillaries [189, 356].


Similar to many other cell types, the endothelial cytoskeleton is composed of microtubules, intermediate filaments and actin filaments (Figure 20) [358, 414]. These structures are important for endothelial cell morphology, adhesion, and barrier function. While the structural support provided by all cytoskeletal components is important for barrier integrity, the actin cytoskeleton is most centrally important for regulation of endothelial permeability.

FIGURE 20. The endothelial cell cytoskeleton.


The endothelial cell cytoskeleton. Polymeric components of the cytoskeleton: actin microfilaments, microtubules and vimentin intermediate filaments, stabilize endothelial cell structure. Cell peripheral (cortical) actin filaments stabilize intercellular (more...)


Microtubules are tubular structures formed of polymers of heterodimeric subunits of alpha and beta tubulin (Figure 20) [299, 358]. The tubular structure is formed of 13 parallel polymeric filaments arranged in a ring. Microtubules are important for cell mitosis, morphology, and intracellular protein trafficking. In endothelial cells, microtubules are cross-linked to actin filaments and can affect endothelial permeability through effects on actin filaments. The stability of microtubules is determined by dynamic polymerization and depolymerization. Microtubules can be further stabilized by capping or by other posttranslational modifications. Dynamic rearrangements of microtubules affect the organization of other cytoskeletal components, and stabilization of microtubules is shown to protect endothelium against actin stress fiber formation and hyperpermeability [420]. Depolymerization of microtubules activates guanine nucleotide exchange factors, and signaling through Rho family GTPases, leading to actin stress fiber formation [48]. Signaling through Rho kinases has further consequences for endothelial barrier function (discussed in subsequent sections). Microtubules are destabilized by treatment of endothelial cells with the inflammatory cytokine TNF-α [418], or with thrombin [46], and may therefore contribute to barrier dysfunction in response to inflammation. Stabilization of microtubules in endothelial cells by cAMP [419], or activation of protein kinase A (PKA) [47], may account for some of the barrier protective effects of this kinase.

Intermediate Filaments

Intermediate filaments are formed of heterogeneous polymeric protein arrangements [299, 474]. In most cells, the principle intermediate filament protein monomer is vimentin. Intermediate filaments are important for endothelial cell structure, and are expressed most abundantly in cells exposed to shear stress, e.g., aortic endothelial cells. Intermediate filaments are connected to cell–cell junction proteins and to focal attachments to the basement membrane (Figure 20). Intermediate filaments undergo structural rearrangements in response to shear stress and are known to transmit mechanical tension between cells joined by intercellular junctions [474]. The role of intermediate filaments in control of endothelial barrier function is not clear. It is believed that the principle function of intermediate filaments in endothelial cells is to provide redundant structural support and strength, stabilizing connections formed by the less resilient actin microfilaments [299]. For example, vimentin gene knock-out mice have no major vascular defects [89], indicating that vimentin filaments are not strictly necessary for normal physiological functions of the vascular endothelium. However, the connections of vimentin filaments to the cell junction protein VE–cadherin are disrupted during exposure to the hyperpermeability-inducing agent histamine [409], suggesting the potential involvement of intermediate filaments in stimulated permeability responses. Connections to focal adhesions suggest that vimentin filaments may play a role in organization or serve as scaffold for focal adhesion proteins. Alternatively, vimentin may be involved in integrin signaling at cell–matrix attachments.

Actin Filaments

Actin filaments are linear polymers of filamentous (F)-actin [299, 358, 414]. The stability of actin filaments is dependent upon the concentration of globular (G)-actin within the cell (Figure 20). Maintenance of the intracellular concentration of G-actin above the critical concentration (0.1 μM) favors actin polymerization and formation of actin filaments. Under normal physiological conditions, actin filaments are randomly distributed throughout the cell (short filaments and diffuse actin monomers) and at the cell peripheral band (cortical actin) [44, 299, 358]. Upon treatment of endothelial cells with hyperpermeability-inducing agents, such as thrombin or histamine, actin filaments organize into linear, parallel bundles across the cell interior (stress fibers). Stress fiber formation is often accompanied by a contractile cell morphology and formation of gaps between adjacent endothelial cells. In contrast, when endothelial cells are treated with barrier-protective agents such as sphingosine-1-phosphate, actin filaments re-organize and localize at the cell periphery, appearing to strengthen cell–cell contacts.

At least 80 proteins are known to bind actin and modify the organization or function of the actin cytoskeleton [299, 358]. Several proteins are known to regulate actin polymerization (cofilin, gelsolin, or 27-kDa heat shock protein) [413, 414]. In addition, the architecture of the actin cytoskeleton is modified by signaling molecules, i.e., Rho family small GTPases (Rac1, Cdc42, and RhoA), to form specialized structures, such as cortical actin of the cell periphery or actin stress fibers [44, 358]. Cytoskeletal rearrangement and stress fiber formation are known to induce endothelial hyperpermeability; however, the precise mechanisms and specific contribution of this process to permeability regulation are not well established. In most cases, cytoskeletal rearrangements are inseparable from other events such as actin–myosin binding and contraction.

Actomyosin Contractile Machinery

Actomyosin contraction and increased cytoskeletal tension is a central mechanism for inducing endothelial hyperpermeability [145, 161, 320, 358, 413, 414]. Because endothelial cell junctions are connected to focal adhesions via the actin cytoskeleton, changes in cytoskeletal tension directly affect the barrier structure and function. In endothelial cells, as in muscle cells, myosin is bound to cytoskeletal actin, and cytoskeletal tension is increased by actin–myosin-based contractile activity (Figure 21). Actomyosin contractility is increased by phosphorylation of myosin regulatory light chain (MLC-2) [411, 413, 414]. Phosphorylation of MLC-2 causes an ATP-dependent change in the tertiary protein folding structure of myosin and a shift in position relative to actin. This shift produces actomyosin contractile force, increasing tension on the actin cytoskeleton. Because the actin cytoskeleton is connected both to cell junction proteins and to focal adhesions, the focal adhesion connection acts as a fulcrum, allowing cytoskeletal tension to physically pull apart cell–cell junctions and increase endothelial paracellular permeability. Conversely, dephosphorylation of MLC-2 decreases actomyosin contractility, relaxes the actin cytoskeleton, and decreases endothelial permeability. Thus, actomyosin contraction and actin cytoskeletal tension are proportional to the net phosphorylation status of MLC-2 (Figure 21).

FIGURE 21. The endothelial cell contractile machinery.


The endothelial cell contractile machinery. Endothelial paracellular permeability/hyperpermeability is controlled by actin-myosin driven cytoskeletal tension and retraction at cell–cell junctions. Actomyosin contraction is increased by phosphorylation (more...)

MLC-2 is phosphorylated by myosin light-chain kinase (MLCK) [145, 161] and is dephosphorylated by myosin light-chain-associated phosphatase (MLCP) [464]. Normal physiological endothelial permeability is maintained and regulated by the activity of both MLCK and MLCP [413, 414]. The balance of MLCK/MLCP activity determines the steady-state phosphorylation status of MLC-2. Any stimulus that increases MLCK activity or decreases MLCP activity will increase MLC-2 phosphorylation and increase endothelial permeability. Many inflammatory agents and diseases cause endothelial hyperpermeability coinciding with or dependent on increased phosphorylation of MLC-2 [145, 195, 509].

Copyright © 2011 by Morgan & Claypool Life Sciences.
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