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Granger DN, Senchenkova E. Inflammation and the Microcirculation. San Rafael (CA): Morgan & Claypool Life Sciences; 2010.

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Inflammation and the Microcirculation.

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Chapter 10Endothelial Barrier Dysfunction

ECs normally serve as a barrier to the movement of fluid and proteins from the intravascular compartment to the interstitium. When this barrier function is diminished, either as a consequence of endothelial cell damage or contraction of adjacent ECs, plasma proteins gain greater access to the interstitial compartment, resulting in an elevated oncotic pressure and the withdrawal of fluid from the intravascular compartment, an increased interstitial fluid volume, and interstitial edema. While capillaries are the major source of fluid that is filtered into the interstitial spaces, postcapillary venules represent the major site of vascular protein leakage (extravagation). The role of venules in protein extravasation is particularly evident in inflamed tissue because the accumulated inflammatory mediators and immune cells can act on venular ECs to diminish barrier function [12,15,19,22,151,268270].

10.1. Site Of Inflammation-Induced Barrier Failure

There are several characteristic features of postcapillary venules that enables this segment of the microvasculature to regulate vascular permeability and the rate of egress of plasma proteins into the interstitium. Ultrastructural analyses of the pathways for transvascular exchange have revealed that both the size and frequency of interendothelial junctions and endothelial fenestrae are higher in postcapillary venules than in either arterioles or capillaries. These pathways are normally large enough to allow a low basal amount of plasma protein leakage that is driven by both diffusive and convective (coupled to fluid filtration) mechanisms. Venular endothelium also appears to possess a higher density of cell surface receptors for inflammatory mediators than their counterparts in arterioles and capillaries. Engagement of certain inflammatory mediators (e.g., histamine, PAF) with their receptors on venular ECs elicits subtle changes in the fine structure of the endothelial monolayer, such as a widening of the endothelial paracellular junctions, which results from the dissociation of junctional proteins and/or cytoskeletal contraction, and a consequent increase in the rate of protein extravasation. Furthermore, since postcapillary venules are the preferred site for leukocyte and platelet adhesion, venular ECs are more frequently and directly exposed to products of leukocyte (e.g., elastase, ROS) and platelets (e.g., RANTES, CD40L), which can diminish barrier function, compared to ECs in arterioles and capillaries [15,22,268270].

The permeation of albumin across an intact endothelial barrier can occur through transcellular as well as paracellular pathways (Figure 10.1). Protein transcytosis via plasmalemmal vesicles containing caveoli-1 has been demonstrated in ECs of different vascular beds, and it has been ascribed a significant role in the transport of macromolecules across continuous type capillaries. The vesicles shuttle plasma proteins between blood and the interstitium, producing a diffusive flux that favors net transport into the interstitium. While the quantitative importance of vesicular transport to total protein transport remains controversial, recent attention on vesicles has been directed toward their ability to interconnect with each other to produce grape cluster-like structures (vesiculovacuolar organelles, VVOs) that function as open channels that connect (and transport proteins between) the blood and interstitial compartments. Since the increased vascular permeability induced by some inflammatory mediators, including histamine and VEGF, is associated with an increased number of VVOs, it has been proposed that this pathway may contribute to the endothelial barrier dysfunction that accompanies inflammation [22,271,272].

FIGURE 10.1. Routes of solute exchange across the inflamed endothelial barrier.

FIGURE 10.1

Routes of solute exchange across the inflamed endothelial barrier. Albumin and other plasma proteins can gain access to the interstitial compartment from plasma via either a paracellular ( junctional) or intracellular (vesicular) transport pathway. The (more...)

The exchange of plasma proteins through interendothelial cell junctions (the paracellular pathway) is generally considered the primary target of the chemical mediators that elicit the endothelial barrier failure associated with inflammation. The restrictive properties (barrier function) of the endothelial cell junctions (adherens junctions) in most vascular beds result from the homophilic binding of VE cadherin expressed on the surface of two adjacent EC (Figure 10.1). The intracellular domains of VE cadherin molecules are anchored to the EC cytoskeleton via actin-binding proteins called catenins. The more highly restrictive cerebral microvessels (blood brain barrier) possess specialized (tight) junctions wherein the cell–cell contact area is maintained by other junctional proteins including claudins, junctional adhesion molecules, occludins, and zonular occludins. Inflammatory mediators that increase vascular permeability in microvessels with adherens junctions exert this effect by disrupting junctional complex assembly via the phosphorylation, internalization, and/or degradation of junctional molecules. The engagement of these mediators with their EC receptors elicits an increased cytosolic calcium and/or the activation of different signaling cascades, leading to cytoskeletal contraction, a loss of junctional protein stability and binding interactions, junctional complex disassembly, and ultimately endothelial barrier failure. Adherens junctions also support the transendothelial migration of leukocytes during inflammation. Consequently, once the inflammatory response resolves, i.e., mediator release subsides and leukocyte transmigration ceases, resealing of the junctional pathways is initiated to restore normal endothelial barrier properties [268270,273].

A variety of different experimental approaches have been used to assess endothelial barrier dysfunction in vivo and in vitro models of inflammation. Changes in the restrictive properties of monolayers of cultured ECs have been monitored using measurements of transendothelial resistance (cerebral ECs) or the permeability coefficient for albumin or other solutes. Isolated perfused venules have also been used to monitor changes in albumin permeability coefficients and consequently barrier function. In vivo approaches include measurements of the osmotic reflection coefficient for plasma proteins using lymph-to-plasma protein concentration ratios, single vessel estimates of hydraulic conductivity, electron microscopic evaluation of horseradish peroxidase leakage, and quantifying the extravasation of Evans blue dye or fluorescently labeled macromolecules [49]. Data generated with these methods are generally interpreted as reflecting changes in vascular permeability; however, caution should be given to the possibility that, under some circumstances, increased solute extravasation may reflect an increased diffusive exchange due to capillary recruitment and/or increased exchange due to convection, without a corresponding change in the restrictive properties of the endothelial barrier [274278].

10.2. Role Of Circulating Blood Cells

10.2.1. Leukocytes

Leukocytes produce and secrete a variety of factors that are capable of increasing vascular permeability (Table 10.1). The adhesion and transendothelial migration of leukocytes in inflamed venules have been linked to endothelial barrier dysfunction in both acute and chronic models of inflammation. There are several lines of evidence implicating neutrophils in inflammation-induced permeability responses. For example, animals that are rendered neutropenic or that receive blocking antibodies that prevent neutrophil–endothelial cell adhesion exhibit a blunted vascular permeability response in different models of inflammation. Similarly, mice that are genetically deficient in key endothelial cell (e.g., ICAM-1, P-selectin) or leukocyte (CD11/CD18) adhesion molecules also show an improved endothelial barrier function during inflammation. Indirect evidence supporting a role for neutrophils is provided by reports describing a significant positive correlation between the magnitude of the inflammation-associated increase in vascular permeability and the number of adherent and/or emigrated neutrophils in/around postcapillary venules [204,275,279284].

TABLE 10.1. Activation products released by leukocytes and platelets that may impair endothelial barrier function.

TABLE 10.1

Activation products released by leukocytes and platelets that may impair endothelial barrier function.

Whether transendothelial cell migration per se is a requirement for the leukocyte-dependent permeability response remains unclear. However, there is evidence suggesting that proteases released from activated adherent leukocytes can degrade (and promote the internalization) of EC junctional proteins. Furthermore, leukocyte adhesion-induced signaling events in ECs (e.g., increased intracellular calcium) have been implicated in the barrier dysfunction induced by inflammatory mediators. For example, engagement of leukocyte integrins with ICAM-1 or VCAM-1 on EC can result in junctional protein phosphorylation and internalization as well as stimulate the production of ROS through activation of endothelial cell NADPH oxidase. Since transendothelial leukocyte migration is not always associated with endothelial barrier dysfunction, whether an inflammatory mediator elicits an increased vascular permeability is likely determined by the level of activation (release of proteases and ROS) of the leukocyte as it traverses the junction and if adhesion-dependent signaling mechanisms cause EC activation, junctional protein disassembly, and cytoskeletal contraction [24,177,270,285,286].

While neutrophils have received more attention as mediators of endothelial barrier dysfunction during inflammation, there is also growing evidence for the involvement of T-lymphocytes, which are recruited into inflamed microvessels hour to days after the appearance of neutrophils. The pathophysiological relevance of the T-cell recruitment is evidenced by the observation that immunodeficient SCID mice (which lack T-cells) exhibit a significantly blunted vascular permeability response to acute or chronic inflammation and that the permeability response can be restored to WT levels when SCID mice are reconstituted with T-cells from WT mice [11]. Antibody-induced depletion of either CD4+ or CD8+ T-cells has also been used to implicate these T-cell populations in inflammation-induced vascular permeability responses. In some inflammation models, the permeability response appears to be dependent on T-cells, yet there is no evidence for increased trafficking of the immune cells through the affected microvessels. This suggests that T-cell activation products (e.g., cytokines) may mediate the barrier dysfunction via a mechanism that does not require T-cell–endothelial cell adhesion. The T-cell-derived mediators may directly act on the EC to cause barrier failure or may promote the recruitment and/or activation of other cells (e.g., neutrophils, mast cells) that exert a more direct influence on EC [287289].

10.2.2. Platelets

Like leukocytes, platelets produce a large number of chemical agents (e.g., VEGF, ROS, thrombin) that have the potential to impair endothelial barrier function (Table 10.1). Nonetheless, exposing monolayers of cultured ECs to platelets or platelet-conditioned media typically results in enhanced barrier function, i.e., a reduction in vascular permeability. Experimental evidence suggests that a sphingolipid (sphingosine-1-phosphate, S1P), which is released by activated platelets, accounts for this barrier protective effect of platelets and it acts by reorganizing the actin cytoskeleton of EC. While there are few reports that directly address the role of platelets in inflammation-induced barrier dysfunction, some of these studies suggest that the presence of platelets either does not alter or improves endothelial barrier function during acute inflammation. For example, one study of the postischemic coronary vasculature indicates that platelets are not requisite for the barrier dysfunction, while another reports describes an improved barrier function following the addition of platelets. A protective effect of platelets has also been reported for the lung. A limitation of all these studies is that the organs used to assess the permeability responses to the inflammatory insult were perfused with artificial solutions, to which platelets are added. Since the attachment of platelets to neutrophils is known to enhance the capacity of the leukocytes to produce ROS, it is conceivable that the vascular permeability enhancing potential of platelets is not realized in the absence of other blood cells [290294].

10.3. Role Of Perivascular Cells

Mast cells residing in the perivascular space have also been implicated in the endothelial barrier dysfunction that accompanies inflammation. Mast cell degranulation, a common feature of the inflammatory response, is characterized by the production and release of superoxide, amines (histamine, serotonin), leukotrienes, proteases, and cytokines (e.g., TNF-α, IL-1), all of which can diminish endothelial barrier function. A role for mast cell degranulation in inflammation-induced endothelial barrier failure is supported by reports describing an attenuating influence of mast cell stabilizing drugs or a genetic deficiency of mast cells on the vascular permeability response during acute and chronic inflammation. The microvasculature in mast cell-rich tissues, such as the intestine and lung, typically exhibit a dependence on mast cells in the increased vascular permeability response to inflammation. Since the myriad of factors released during mast cell degranulation can exert an influence on the recruitment of other inflammatory cells into inflamed microvessels, it is often difficult to distinguish between a direct effect of mast cell products on endothelial barrier function vs. the actions of secondary cells (e.g., leukocytes) that are recruited or activated by those products. However, some mediators that are relatively unique to mast cells (e.g., histamine, mast cell-specific serine proteases) have been shown to contribute to inflammation-induced permeability responses in a variety of animal models. Histamine typically exerts its barrier-altering action on EC by engaging the Gq-coupled H1 receptor, which elevates intracellular calcium, and triggers actin–myosin contraction. Mast cell-specific tryptases and chymases promote vascular permeability via indirect (enhancing bradykinin production) and possibly direct mechanisms. Even tissues with a relatively low population of mast cells, such as the brain, show evidence of mast cell dependent barrier failure. For example, the impaired blood brain barrier function that occurs in brain following focal ischemia/reperfusion is significantly blunted in WT rats treated with a mast cell stabilizer (cromoglycate) and in mast cell deficient rats [17,57,58,295298].

Macrophages that reside in the perivascular compartment have also been implicated in inflammation-induced endothelial barrier failure. Activated macrophages also produce and release a variety of cytokines, chemokines as well as ROS and NO, which can impair endothelial barrier function. Macrophage depletion can be achieved in vivo by intravascular injection of agents that are selectively cytotoxic to macrophages, such as clodronate liposomes or 2-chloroadenosine. Using these reagents, a dependency of endothelial barrier function on macrophages has been described in tissues with a large resident population of macrophages (e.g., lung, gut) and in tissues with smaller resident populations (e.g., nerves) [299303].

10.4. Reactive Oxygen And Nitrogen Species

ECs, leukocytes, platelets, macrophages, and mast cells are activated and produce ROS at an accelerated rate during inflammation. The elevated ROS levels have been proposed as potential mediators of inflammation-induced endothelial barrier failure. ROS can exert this effect in a variety of ways. Direct mechanisms include phosphorylation of catenins with the subsequent dissociation of VE-cadherins, eliciting actin–myosin cytoskeletal contraction, and degradation of the endocapillary layer (glycocalyx). Indirect actions of ROS include enhanced leukocyte adhesion and transendothelial migration via oxidant sensitive, transcription-dependent up-regulation of EC adhesion molecules, and activation of latent proteases such as MMPs. Lipid peroxidation-mediated cell membrane damage induced by the elevated ROS fluxes is an unlikely contributor to the inflammation-mediated changes in barrier function [13,39,40,268,270,273].

A role for ROS in the vascular permeability responses to inflammation is supported by studies that have focused on inhibiting the production of ROS by targeting enzymatic sources such as xanthine oxidase or NADPH oxidase, as well as studies that examine the influence of ROS scavengers (SOD) on the permeability response. These approaches have been used to implicate xanthine oxidase-derived ROS in the enhanced microvascular protein and water permeability induced by I/R. Since xanthine oxidase inhibition or ROS scavenging has also been shown to largely prevent the adherence and transendothelial migration of leukocytes in I/R and other inflammatory states, it remains unclear from these observations whether the ROS directly alter impair barrier function or do so indirectly by limiting the adhesive interactions between leukocytes and ECs [13,175,304,305].

Another source of ROS that has received considerable attention in inflamed vessels is NADPH oxidase. Nonspecific NADPH oxidase inhibitors as well as mice that are genetically deficient in critical protein subunits (e.g., p47phox, gp91phox) of the enzyme complex have been used to implicate this enzyme as a major source of ROS that mediates endothelial barrier failure. Chimeras produced by the transplantation of bone marrow from p47phox deficient into WT recipients (or vice versa) have revealed that bone marrow-derived cells, rather than ECs, mediate the NADPH oxidase-dependent barrier alterations elicited in the pulmonary microvasculature. Focal brain I/R also results in impaired endothelial barrier function that is dependent on NADPH oxidase, as evidenced by experiments showing reduced blood brain barrier dysfunction after focal ischemia stroke and reperfusion in NADPH oxidase (gp91phox) deficient mice and in WT mice treated with apocynin [306309].

An important pathophysiological consequence of increased superoxide production in inflamed ECs is inactivation of NO. As detailed above, physiologic levels of NO appear to play an important role in preventing leukocyte–endothelial cell, platelet–endothelial cell, and platelet–leukocyte adhesion in the normal noninflamed microvasculature, and it serves to stabilize mast cells in the perivascular space. Upon inactivation of NO by superoxide, the cell–cell interactions are elicited, mast cell degranulate, and there is a reduction in endothelial barrier function. NO donors have been shown to blunt all of these responses to increased ROS production. Similar protection against ROS-induced endothelial barrier failure has been noted in mice that genetically overexpress endothelial NO synthase (eNOS). In contrast, nonselective inhibition of NOS in otherwise healthy tissue results in oxidative stress and increased vascular permeability. While these studies suggest that NO normally protects against endothelial barrier failure, there is also evidence indicating that NO directly diminishes barrier function. The incongruent permeability responses to NO can be explained by a study that compared the water permeability responses of mesenteric venules perfused with or without blood-borne constituents to NOS inhibition. It was noted that in the absence of blood-borne constituents, permeability was reduced by approximately 50%, while a >75% increase in permeability was detected in vessels perfused by blood during exposure to the NOS inhibitor. Hence, these findings suggest that the major pathophysiological consequence of a reduction in NO bioavailability in inflamed microvessels is the loss of a critical anti-adhesion molecule that serves to limit the barrier compromising effects of leukocyte adhesion and transendothelial migration [310314].

Copyright © 2010 by Morgan & Claypool Life Sciences.
Bookshelf ID: NBK53374

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