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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
Chem Biol Interact. Author manuscript; available in PMC 2010 December 12.
Published in final edited form as:
PMCID: PMC3001132
NIHMSID: NIHMS243060

Regulation of Endothelial Permeability by Src Kinase Signaling

Vascular leakage versus transcellular transport of drugs and macromolecules

Abstract

An important function of the endothelium is to regulate the transport of liquid and solutes across the semi-permeable vascular endothelial barrier. Two cellular pathways have been identified controlling endothelial barrier function. The normally restrictive paracellular pathway, which can become “leaky” during inflammation when gaps are induced between endothelial cells at the level of adherens and tight junctional complexes, and the transcellular pathway, which transports plasma proteins the size of albumin via transcytosis in vesicle carriers originating from cell surface caveolae. During non-inflammatory conditions, caveolae-mediated transport may be the primary mechanism of vascular permeability regulation of fluid phase molecules as well as lipids, hormones, and peptides that bind avidly to albumin. Src family protein tyrosine kinases have been implicated in the upstream signaling pathways that lead to endothelial hyperpermeability through both the paracellular and transcellular pathways. Endothelial barrier dysfunction not only affects vascular homeostasis and cell metabolism, but also governs drug delivery to underlying cells and tissues. In this review of the field, we discuss the current understanding of Src signaling in regulating paracellular and transcellular endothelial permeability pathways and effects on endogenous macromolecule and drug delivery.

Keywords: Src tyrosine kinases, endothelium, vascular permeability, inflammation, drug delivery, caveolae

1. Introduction

The vascular endothelium lining the blood vessels functions as a barrier between the blood and interstitial compartments that controls and restricts the transendothelial flux of fluid and macromolecules [1]. Increased endothelial permeability to plasma proteins resulting from endothelial barrier dysfunction leads to an abnormal extravasation of blood components and accumulation of fluid in the extravascular space. Vascular leakage not only causes multi-organ dysfunction, but also compromises the normal pharmacokinetics of therapeutic drugs. In such areas with increased vascular permeability, drugs can extravasate and accumulate inside the interstitial space. The bioavailability and effectiveness is therefore reduced and systemic toxicity can increase. Strategies which prevent vascular leakage therefore reduce drug dosages and side effects, and improve the efficacy of therapeutic interventions.

The pathological process of endothelial hyperpermeability is a common characteristic feature of many diseases, including inflammation, trauma, sepsis, ischemia-reperfusion injury, diabetes, and atherosclerosis. The homeostatic barrier function of the endothelium is ultimately maintained by the dynamic regulation of endothelial cell shape, endothelial cell-cell adherence, and endothelial-extracellular matrix adherence [2]. Compromised barrier function of the endothelium in response to proinflammatory mediators is accompanied by intercellular gap formation, which is the main mechanism of vascular leakage. Recently, evidence has emerged to support a role for the vesicular pathway in mediating macromolecular transport across the endothelium [1]. In particular, protein transport via caveolae has been reported to play a key role in maintaining endothelial barrier function and normal oncotic pressure gradient across the vessel wall [1,2].

Multiple signaling molecules have been identified in the mechanism of vascular endothelial permeability regulation [1] which trigger structural changes in endothelial barrier and/or induce transcellular protein transport by endothelial cells. Src protein tyrosine kinases regulate many cellular processes, such as cell morphology, motility, proliferation, and survival. Intracellular signal transduction via Src protein tyrosine kinases is also involved in acute inflammatory responses [1]. Recent experimental evidence points to the importance of Src family protein tyrosine kinases (SFK) signaling in the regulation of microvascular barrier function and various endothelial responses including hyperpermeability to different proinflammatory mediators [3-6]. Src family protein tyrosine kinases have been implicated in upstream signaling pathways that lead to endothelial hyperpermeability through both intercellular gap formation and increased transendothelial protein transport [1]. Elevated SFK activaty results in changes in gene expression which also affects endothelial permeability [7]. Over the last decade, some exhaustive and basic reviews addressing the regulation of endothelial permeability have been published [1]. However, a comprehensive review on the role of SFK signaling in modulation of endothelial barrier is still lacking. This review addresses the potential mechanisms of Src protein tyrosine kinases in regulating endothelial permeability and microvascular barrier function.

2. Basics of Src family tyrosine kinases

SFKs are nonreceptor, cytoplasmic, protein tyrosine kinases. They have been implicated in the regulation of diverse processes including cell growth and differentiation, cell adhesion and motility, carcinogenesis, immune cell function, and endothelial permeability. This broad-spectrum role of SFKs in regulating biological responses is associated with their ability to interact with a large number of different receptors and many distinct cellular targets [8]. The structural and functional interaction between SFKs and cellular receptors integrates a large amount of upstream signaling that coordinately regulates cellular activities.

2.1 The structure of SFKs

The SFKs are 52-62 kDa enzymes composed of eight distinct functional regions (Figure 1). From the N- to C-terminus, these regions include a myristylated site, Src homology (SH)4 domain, unique region, SH3 domain, SH2 domain, linker, the kinase/catalytic domain (SH1 domain), and regulatory domain. The glycine at position 2 is important for addition of a myristic acid moiety, and the myristoylated site along with the SH4 domain are associated with cell membrane binding. The unique region is specific for different Src family members and it may mediate the interaction between SFKs and other proteins. The three major domains, the kinase/catalytic domain (SH1 domain), SH2 domain, and SH3 domain, represent the modular structure of Src family kinases. SH3 and SH2 domains are protein-protein interaction domains shared not only with other Src family kinases but also with many other signaling proteins. The SH2 domain binds phosphotyrosine motifs in either an inter- or intramolecular fashion. The SH1 domain is the site of tyrosine kinase activity. There are two major phosphorylation sites on Src: on Tyr 416 located in the SH1 domain and at Tyr 527 in the regulatory domain near the carboxyl terminus. Tyr 416 can be auto-phosphorylated, whereas Tyr 527 can be phosphorylated and dephosphorylated by various proteins, such as Csk (carboxy-terminal Src kinase) which phosphorylates Src, and SHP-1 (Src-homology 2 domain containing phosphatase 1), SHP-2, or PTP1 (protein tyrosine phosphatase 1) which dephosphorylate Src (8,12). Both phosphorylation sites play a key role in regulating the activity of Src family kinases [7].

Figure 1
Src family kinase domain structure

2.2 Activation of Src family kinases

SFKs are phosphorylated on tyrosine residues, suggesting that Src activity and biological function might be regulated by phosphorylation. The inactive state of the Src kinases is maintained by an autoinhibitory interaction between the SH2 domain and Tyr527 (chicken c- Src) and also the interaction of the SH3 domain and a polyproline type II helix in the SH2 to SH1 linker domain (7). SFKs can switch from an inactive to an active state through control of its phosphorylation state, or through protein-protein interactions. They are activated by phosphorylation at Tyr 416 and dephosphorylation at Tyr 527 [9-11]. In contrast, the activity of SFKs is decreased by dephosphorylation at Tyr416 and phosphorylation at Tyr 527 (Figure 2). Under physiological conditions, 90–95% of c-Src is phosphorylated at Tyr 527 [12], and phosphotyrosine 527 binds intramolecularly with the SH2 domain [13], indicating that SFKs have low basal activity. This inhibitory interaction can also be displaced by a phosphotyrosine ligand with a higher affinity for the SH2 domain (7).

Figure 2
Activation of c-Src

Protein interactions also act to regulate Src by either directly activating them, or by moving SFKs to sites of action. SFKs can be activated by receptor protein-tyrosine kinases, integrin receptors, G-protein coupled receptors (GPCR), antigen- and Fc-coupled receptors, cytokine receptors, and steroid hormone receptors [10]. Many proinflammatory cytokines activate SFKs via different GPCR signaling pathways that include Gi- and Gq-coupled receptors. Stimulation of Gi-coupled receptor is known to activate Src in a Gβγ-dependent manner [14].

2.3 Expression and substrates of Src family kinases in endothelial cells

There are nine members of the Src family including c- Src, Fyn, Yes, Yrk, Lyn, Lck, Hck, Fgr and Blk (Table 1). c-Src, Fyn, Yes and Yrk are widely coexpressed in many cell types, including vascular endothelial cells [9,15,16], whereas Lyn, Lck, Hck, Fgr and Blk are found primarily in hematopoietic cells [8]. SFKs localize to numerous areas of the cell rather than in any one particular subcellular location. It appears that the subcellular location of SFKs can affect their function. SFKs can associate with cellular membranes, such as the plasma membrane, the perinuclear membrane, and the endosomal membrane. SFKs are also found in the cytoplasm and at adherens junctions, where they take on different roles. The function of SFKs in endothelial cells is complicated by the pleiotropic activities, as well as their targeting molecules. A variety of SFK target molecules (substrates) are related to the regulation of endothelial permeability (Table 2).

Table 1
Expression of Src family kinases
Table 2
Src family kinase target proteins that regulate endothelial permeability

3. Src family kinase signaling in vascular endothelial permeability

The route of solute flux across the vascular endothelium has been debated for decades. The transendothelial movement of solutes, ions and water can occur via both transcellular and paracellular pathways, through or between cells, respectively. The transcellular pathway consists of a highly mobile set of vesicles that shuttle across the endothelial barrier from its luminal aspect to the abluminal side [52]. The paracellular pathway, in contrast, offers a purely passive pathway for the diffusion of protein and other small solutes. Vascular permeability is determined primarily by multi-protein complexes, the tight and adherens junctions, that link adjacent endothelial cells. In absence of a pathological insult, these junctions are normally impermeable to albumin and other plasma proteins. Electron micrographic studies have shown that this pathway is closed (restricted) and excludes macromolecule tracers [53-57]. The transport of albumin and other macromolecules across the endothelium under non-inflammatory physiological conditions can be fully explained by transcytosis involving the plasma membrane vesicular structures or caveolae [54, 58].

SFKs have been implicated in upstream signaling pathways that lead to endothelial hyperpermeability [4,9,20]. The regulatory role of SFKs in endothelial permeability is two fold. SFK phosphorylation of proteins may directly modulate the function of these proteins. It phosphorylates substrates in the cytosol and at the inner face of the plasma membrane, or at cell–matrix or cell–cell adhesions. In addition, phosphotyrosyl residues serve as docking sites for the binding of signaling proteins containing SH2 domains. These signaling complexes initiate pathways that regulate protein synthesis, gene expression, cytoskeletal assembly, and many other aspects of cell function. Low levels of SFK activity is required in normal tissues to maintain integrity of the endothelial barrier. However, elevated Src activity induced by a wide spectrum of inflammatory mediators causes a marked increase in endothelial permeability [59-61].

3.1 Role of Src in paracellular permeability

3.1.1 Structural basis of paracellular permeability

The microvascular barrier consists of the endothelial monolayer, intercellular contacts between adjacent endothelial cells, and focal adhesions anchoring the endothelial lining to its surrounding matrices in the vascular wall. The integrity of these structural elements is necessary to maintain normal barrier function. The disintegration of endothelial cell-cell contact (junctions) and cell-matrix contact (focal adhesions) leads to increased endothelial permeability through the opening of paracellular pathways, enhancing macromolecular transport [62-68]. Paracellular permeability is regulated by interendothelial junctional complexes, the adherens junctions (AJ) and tight junctions (TJ), and through interaction of these complexes with the actin cytoskeleton [2].

Inter-endothelial cell contacts

Endothelial cells form junctional complexes consisting of TJs and AJs, which are the sites of diffusional transport of solutes. The integrity of interendothelial junctions can be impaired by endothelial cell retraction and shape change. Actin and myosin are the major contractile components in the cytoskeleton [63]. The signal transduction pathways that disrupt interendothelial junctions involve a complex series of signaling events leading ultimately to rapid and sustained phosphorylation of myosin light chain (MLC) and simultaneous inhibition of MLC-associated phosphatase (the function of which is to prevent dephosphorylation of MLC and prolong the contractile response) [46,69,70]. Phosphorylation of MLC by Ca2+-calmodulin dependent myosin light chain kinase (MLCK) is required for actin-myosin interaction and engagement of the endothelial contractile apparatus. Endothelial cell retraction is likely precipitated by disruption of endothelial AJs [46,71]. Filamentous actin within endothelial cells also associates with the cytoplasmic tail of the major AJ protein vascular endothelial (VE)-cadherin [72]. Contractile force may “unhinge” AJs resulting in formation of gaps [71]. VE-cadherin is localized in intercellular AJs where they are linked in the cytoplasm to β-, γ-, and p120-catenins, and in turn to α-catenin and the actin cytoskeleton [25,26]. Dissociation of VE-cadherin from the catenins can cause intercellular gap formation leading to an increase in endothelial permeability [27,73].

Endothelial cell-matrix contacts

Focal adhesions are mainly composed of integrins, transmembrane receptors that facilitate the actin cytoskeleton connection to the extracellular matrix (ECM) via cytoplasmic linker proteins. The cell-matrix interaction is dynamically controlled through assembly and disassembly of focal adhesions [74]. The linkage between proteins of the ECM with the cell is mediated mainly by transmembrane integrins which not only function as adhesion receptors but also transmit chemical signals and mechanical forces between the matrix and cytoskeleton [75,76]. The adhesive interactions between integrins and their extracellular ligands at focal adhesion complexes regulate endothelial cell shape and serves to maintain endothelial barrier properties [2]. Integrin-mediated attachment of endothelial cells to the substratum is an important component of paracellular permeability. A recent study demonstrated that inhibition of integrin binding to either fibronectin or vitronectin with specific peptides containing the arginine-glycine-aspartate (RGD) sequence motif increased venular permeability 2- to 3-fold in a concentration-dependent manner [77].

Focal adhesion kinase (FAK) is a protein tyrosine kinase which is recruited at an early stage to focal adhesions and which mediates many of the downstream signaling reactions leading to integrin engagement and focal adhesion assembly that ultimately affects barrier function [78-80]. The modulatory effect of FAK on endothelial permeability involves complex mechanisms depending on the chemical/physical states of the endothelium. In the basal condition, the constitutive activity of FAK is an essential component of the barrier structure. However, FAK can be further activated in response to inflammatory signals and stimulate paracellular transport of fluid and macromolecules through cell contraction and intercellular gap formation.

3.1.2 Src regulation in intercellular junctions

SFK-dependent tyrosine phosphorylation is considered to play an important role in regulating structural changes occurring in the endothelium [20,81]. The integrity of intercellular junctions can be regulated through phosphorylation of MLCK and AJ protein VE-cadherin by c-Src kinase [64,82-84]. Recent studies have identified sites of Src tyrosine phosphorylation in the unique N-terminus of endothelial MLCK-1. Phosporylation of MLCK-1 by Src results in a 2-3 fold increase in MLCK activity. MLCK activation is linked to increased MLCK tyrosine phosphorylation and stable association of MLCK with Src in pulmonary endothelial cells [9,23, 85]. Thus, Src binding to MLCK causes the activation of MLCK under submaximal calcium concentrations, providing a mechanism to orchestrate critical cytoskeletal rearrangements and cellular contraction [85]. Src regulates endothelial monolayer permeability at the cytoskeletal level by affecting myosin light chain phosphorylation [81]. These biochemical events induce actin-myosin contractility that leads to shape change of endothelial cells and interendothelial gap formation resulting in endothelial hyperpermeability. Alternatively, Src phosphorylation of both β-catenin and VE-cadherin can serve as important signaling mechanisms altering interactions between junctional and cytoskeletal proteins. Src tyrosine phosphorylation can also cause the dissociation of these junctional proteins from their cytoskeletal anchors [27,77,86,87]. Src kinase was found constitutively associated with VE-cadherin in both quiescent and angiogenic tissues [81]. VE-cadherin may serve as an anchor to maintain Src at endothelial cell junctions, where it could exert its activity on junctional components [81]. Src-VE-cadherin association in cultured endothelial cells is independent of VE-cadherin phosphorylation state and Src activation.

3.1.3 Src regulation at endothelial cell-matrix contacts

Both FAK and paxillin located in focal adhesion complexes are Src substrates. The activity of FAK and paxillin are mainly regulated through phosphorylation by the SFKs [10,17,84]. Association of c-Src with FAK may facilitate Src-mediated phosphorylation of tyrosine residues on FAK, some of which serve as binding sites for additional SH2-containing proteins [18,79]. Src is also involved in integrin-induced tyrosine phosphorylation [3]. Integrin engagement induces tyrosine phosphorylation of focal adhesion proteins found in focal adhesion complexes [4]. Src-dependent tyrosine phosphorylation is a critical requirement for the functional formation of integrin-dependent focal adhesion attachment to actin stress fibers [88]. Crosstalk between Src and focal adhesion kinase regulates vascular permeability by interfering with integrin adhesion and signaling [19,84].

3.2 Role of Src in transcellular permeability

3.2.1 Vesicle transport and transcellular permeability

Transport of the plasma protein albumin from the blood to underlying tissues is an important function of the endothelium. Under physiological conditions, the microvascular endothelium establishes a tight barrier (semipermeable cell-cell junctions) via AJs and TJs between neighboring cells. This keeps paracellular permeability of macromolecules, such as albumin, very low. Movement of these macromolecules does occur, however, through the vesicular or transcellular pathway involving caveolae. Recent data have shown convincingly that uptake and transport of albumin across the endothelial barrier in situ can be fully accounted for by the formation, fission and transport of caveolae [89-91].

Transendothelial transport is rapid (~30 sec), the cargo is predominantly in the fluid phase rather than receptor-bound, and requires SFK signaling to activate vesicle shuttling between apical and basal surfaces [92]. Transcytosis can be regulated by albumin via both constitutive (eg, fluid phase transport) or receptor mediated processes (the molecule transported requires the presence of its cognate receptor in caveolae) [93]. Caveolin-1, an integral membrane protein (20-22 kDa), is a specific marker and the primary structural component of endothelial caveolae. Evidence has accumulated suggesting that caveolin-1 regulates endothelial transcellular transport of albumin. First, the recent generation of caveolin-1 null mice has revealed the absence of caveolae and defective uptake and transport of albumin, which could be reversed by transduction of caveolin-1 cDNA [33-35]. Furthermore, we [36-38] and others [39-43] have demonstrated that phosphorylation of caveolin-1 on tyrosine residue 14 by SFKs initiates plasmalemmal vesicle fission and transendothelial vesicular transport, and that this facilitates the uptake and transport of albumin through endothelial cells (Figure 3).

Figure 3
Src signaling mechanism regulating transcytosis of albumin

3.2.2 Src regulation of transcellular permeability

The mechanism by which endothelial cells internalize and transport albumin from the luminal to abluminal side is not completely understood. Studies demonstrated that phosphorylation of caveolin-1 on tyrosine 14 by c-Src is a key switch initiating caveolar fission from the plasma membrane [36-41,43]. It is known that albumin binding to the 60 kDa glycoprotein (gp60) on the endothelial cell surface induces clustering of gp60 and its physical interaction with caveolin-1 [36]. c-Src can bind to the caveolin-1 scaffolding domain [41], palmitoylated C-terminal cysteine residue, and N-terminal phosphorylated tyrosine residue [36,41], and Src is activated upon albumin binding to cell surface gp60 [39]. Activated Src, in turn, phosphorylates caveolin-1, gp60, and dynamin-2 to initiate plasmalemmal vesicle fission and transendothelial vesicular transport of albumin (Figure 3) [37-39].

4. Role of Src signaling in proinflammatory mediator- and neutrophil-induced vascular hyperpermeability

4.1 Oxidants

Studies have shown that H2O2 increases the activity of c-Src and other SFKs, including Lck [94-96]. H2O2 directly activates Src via oxidization at two cysteine residues and indirectly through the dephosphorylation of Tyr 527 [97,98]. Exposure of endothelial cells to H2O2 increased Src activity in association with increased endothelial permeability [99]. Src kinase inhibitors, herbimycin A and PP1, prolonged the onset of increased permeability and attenuated H2O2-mediated increase in endothelial permeability [99]. However, Src family kinases do not appear to be involved in H2O2-mediated rearrangement of junctional proteins since H2O2-induced loss of VE-cadherin junctional staining along with concomitant gap formation was not affected by PP1 [100]. Although Src kinase activation has been shown to phosphorylate β-catenin and result in disorganization of the adherens junction complex [6,28,29], H2O2-induced decrease in the amount of β-catenin associated with the actin cytoskeleton was not blocked by PP1, suggesting that Src kinase activity is not involved in H2O2-mediated dissociation of β-catenin from the endothelial cell cytoskeleton. These findings raise the possibility that H2O2-mediated permeability stimulates both endothelial junctional disorganization and increased caveolae-mediated transcellular transport, and that inhibition of Src kinase ablates the vesicle trafficking-mediated permeability pathway [36].

4.2 TNFα

Tumor necrosis factor-α (TNFα) can induce increased endothelial permeability via intercellular gap formation [101]. A potential target for TNFα-induced endothelial permeability is VE-cadherin, a major component of endothelial AJs. TNFα activates Src kinases which results in tyrosine phosphorylation of VE-cadherin, redistribution of VE cadherin, and gap formation [27,87,102]. Confocal studies indicated that Src inhibitor PP2 prevented TNFα-induced phosphorylation of VE cadherin and intercellular gap formation, suggesting that a SFK activated by TNFα acts upstream of VE cadherin to affect changes in endothelial permeability [102]. The mechanism of Src activation stimulated by TNFα is unclear. It was suggested that TNFα-mediated oxidant generation in endothelial cells induces Src activation [103-106].

4.3 VEGF

Recent studies demonstrated that vascular endothelial growth factor (VEGF)-induced increased vascular permeability requires SFKs [107,108]. Mice lacking c-Src or Yes (but not Fyn) lacked a VEGF-mediated vascular permeability response [108]. The mechanism by which VEGF increases endothelial permeability through Src remains poorly understood. Unstimulated blood vessels contain a protein complex composed of VEGF receptor-1 (Flk-1), VE-cadherin, and β-catenin that is involved in maintenance of endothelial barrier integrity [31,39]. This molecular complex immediately dissociates following VEGF stimulation, an event that depends on Src kinase activity [109,110]. Src in its active form is recruited to Flk-1 following VEGF stimulation [111]. Therefore, it is conceivable that active Src associated with Flk-1 may account for the tyrosine phosphorylation of VE-cadherin and β-catenin, leading to dissociation of the junctional complex [112]. VEGF also promotes VE-cadherin endocytosis by regulating Vav2, a GEF, through c-Src [113]. VEGF stimulation results in the enhanced tyrosine phosphorylation of Vav2, together with Src and VEGF receptor-2, which was abolished by VEGF receptor-2 and SFK inhibitors. Src has an important function in linking VEGF receptor-2 activation to the stimulation of Vav2, thereby activating Rac and resulting in the endocytosis of VE-cadherin and the disruption of endothelial junctions. In addition, β-arrestin-2 may also aid VE-cadherin endocytosis based on its ability to interact with Src [114]. In this scenario, β-arrestin-2 may recruit Src to the vicinity of VE-cadherin, thus facilitating Src-dependent phosphorylation of cadherin–catenin complexes [110,115]. Therefore, the tyrosine phosphorylation of VE-cadherin and its associated molecules may be coordinated with the Src-dependent activation of Vav2 and Rac to regulate the dynamic disassembly and reassembly of adherens junctions. This process leads to the disassembly of endothelial-cell junctions, resulting in the enhanced permeability of the blood-vessel wall. In addition, Src kinase also regulates VEGF-induced assembly of a FAK/αvβ5 integrin complex in cultured endothelial cells. This complex was significantly reduced in endothelial cells from c-Src-deficient mice [2]. Pharmacological inhibition of SFKs with PP1 or retroviral delivery of kinase-defective c-Src suppressed VEGF-induced assembly of the FAK/αvβ5 complex. These findings indicate that the VEGF-induced formation of the FAK/αvβ5-complex via Src may be an important mechanism for coordinating growth factor-dependent integrin signaling in the regulation of vascular permeability [2, 116].

4.4 Thrombin

Thrombin, a pro-coagulant serine protease, is well known to increase vascular endothelial permeability [1]. Thrombin-induced Ca2+ influx is regulated by Src activation. Ca2+ signaling is critical in the mechanism of thrombin-induced myosin light chain phosphorylation and subsequent actinomyosin cross bridging (which induces actin stress fiber formation) [62, 65,71, 116-118]. The mechanism by which Src regulates Ca2+ influx is unclear. Src may phosphorylate plasma membrane transient receptor potential channels expressed in endothelial cells [119,120] that mediate Ca2+ influx during inositol trisphosphate-sensitive intracellular store depletion [121,122]. Thrombin increased the tyrosine phosphorylation of junctional proteins and the formation of interendothelial gaps that are characteristically associated with the loss of barrier function [2,11,66,67,123]. Tyrosine phosphorylation of adherens junction proteins is dependent on the augmented Ca2+ influx. These results suggest that the Src activation-dependent Ca2+ influx is an important factor signaling thrombin-induced endothelial barrier dysfunction [124]. Src is also involved in thrombin-mediated changes in endothelial cell adherens junctions. Thrombin treatment of human umbilical vein endothelial cells promotes Src-dependent SHP-2 phosphorylation and dissociation from VE-cadherin complexes. The loss of SHP-2 from the cadherin complex correlates with a dramatic increase in the tyrosine phosphorylation of β-, γ-, and p120-catenins complexed with VE-cadherin. Thrombin regulates the tyrosine phosphorylation of VE-cadherin-associated β-catenin, γ-catenin, and p120-catenin by modulating the quantity of SHP-2 associated with VE-cadherin complexes. This event promotes cell-cell junction disassembly and intercellular gap formation, detected in endothelial cell monolayers after thrombin treatment, and the resulting increase in monolayer permeability [48].

4.5 Neutrophils

It is well known that activated polymorphonuclear neutrophils (PMNs) increase the permeability of the endothelium to albumin, thus promoting fluid loss into the interstitial space. Although the precise mechanisms have not been completely elucidated, studies have implicated an increase in paracellular permeability via opening of interendothelial junctions caused by PMN adherence and oxidant generation by PMNs and endothelial cells which leads to increased solute (mainly albumin) and fluid transport across the vessel wall [1,125,126]. Accumulating evidence has demonstrated that Src activation is linked to the mechanism of increased endothelial permeability caused by PMNs [4,23,127]. Activation of PMNs with complement peptide C5a induced endothelial cell Src activation and increased endothelial permeability. This PMN-induced hyperpermeability in both microvessels and endothelial cells could be greatly attenuated by Src inhibition [127]. Moreover, cross-linking of endothelial cell surface intercellular adhesion molecule (ICAM)-1 with a monoclonal antibody also increased the activity of Src kinase (128-131), suggesting that PMN adhesion via CD18/ICAM-1 interaction may be important in the regulation of Src activity. The mechanism by which activated PMNs increase Src activity is not clear. Activation of PMNs may increase Src Tyr416 phosphorylation and reduce Src Tyr527 phosphorylation [127]. Src and β-catenin interaction and phosphorylation are necessary for PMN-induced endothelial barrier dysfunction. The inhibition of Src caused Src/β-catenin disassociation and blocked PMN-induced β-catenin tyrosine phosphorylation in cultured endothelial cells. Src kinase may directly phosphorylate β-catenin in response to activated PMNs; this event leads to the disorganization of cell-to-cell adherens junction and ultimately endothelial barrier dysfunction [127]. Although Src activation is involved in increased endothelial permeability [4,23,127], its role in activating endothelial transcytosis following PMN activation remains unclear. Since Src-dependent caveolin-1 phosphorylation is a key switch in albumin endocytosis and transcytosis through the endothelium, it is likely that activation of PMNs may stimulate transcellular albumin transport via the Src dependent pathway. The contribution of endothelial transcytosis in the mechanism of increased lung microvessel permeability remains to be addressed.

5. Pharmacological perspectives and conclusion

In recent years, investigations of Src signaling in vascular endothelial permeability regulation have led to newer and more sophisticated methods to probe the molecular mechanisms involved. Indeed, as discussed above, there is now evidence to support the concept that SFKs are key regulators of the vascular endothelial barrier. This area of research warrants further investigation, as pharmacological inhibitors that selectively block individual Src family members may represent novel therapeutic approaches for limiting vascular leakage. However, due to the lack of selectivity of inhibitors of SFKs and the involvement of SFKs in many cellular activities, a strategy for the treatment of Src-mediated vascular leakage is not yet available. For instance, c-Src-and Yes-deficient mice show a negligible VEGF-induced vascular permeability response, yet Fyn-deficient mice display normal permeability responses [132]. Gene knockout and selective siRNA targeting of different isoforms of Src are needed to elucidate the role of SFKs in different types of inflammatory vascular leakage and in various cell types.

Transcellular transport is the primary mechanism by which albumin, lipids, steroid hormones, fat-soluble vitamins, and other substances that bind avidly to albumin cross the normally restrictive microvessel barrier lined with continuous endothelia. However, the importance of this pathway as a mechanism of protein leakage in pathological conditions remains to be investigated. Src signaling may play a critical role in proinflammatory mediator-induced transvascular hyperpermeability. In this regard, strategies directed against preventing Src-mediated increase in transcellular permeability via caveolae may be useful in reversing the accumulation of protein-rich fluid in the lung extravascular space. These studies could also lead to novel drug therapies for treatment of many diseases including acute lung injury and ARDS that target the transcellular permeability pathway in endothelial cells.

In summary, we believe that further insight into the regulatory mechanisms of Src signaling that contribute to endothelial hyperpermeability will help us to understand how this pathologic process can be treated. Understanding the role of Src in the various forms of vascular leakage that occurs during the different stages of inflammation will provide novel targets against increased paracellular and/or transcellular permeability for therapeutic intervention in inflammatory diseases.

6. Acknowledgements

This work was supported by NIH National Heart, Lung, and Blood Institute (HL71626 and HL60678), and the American Heart Association (0730331N).

Footnotes

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