Protein interactions at endothelial junctions and signaling mechanisms regulating endothelial permeability

Abstract

The monolayer of endothelial cells lining the vessel wall forms a semi-permeable barrier (in all tissue except the relatively impermeable blood-brain and inner retinal barriers) that regulates tissue-fluid homeostasis, transport of nutrients, and migration of blood cells across the barrier. Permeability of the endothelial barrier is primarily regulated by a protein complex called adherens junctions (AJs). AJs are not static structures: they are continuously remodeled in response to mechanical and chemical cues in both physiological and pathological settings. Here we discuss recent insights into the post-translational modifications of junctional proteins and signaling pathways regulating plasticity of AJs and endothelial permeability. We also discuss in the context of what is already known and newly defined signaling pathways that mediate endothelial barrier leakiness (hyper-permeability) that are important in the pathogenesis of cardiovascular and lung diseases and vascular inflammation.

1. Endothelial barrier integrity in health and disease

The endothelium lining the intima of all blood and lymphatic vessels forms a semi-permeable barrier between circulating plasma and the interstitium. Inter-endothelial junctions connect endothelial cells into a contiguous monolayer to restrict the transport of proteins across the endothelial barrier in a size-selective manner ^1–3. Molecules of 3 nm radii or less passively diffuse through junctions while high molecular weight proteins such as albumin (67 kD), constituting 75% of protein in the plasma, and blood cells are largely retained in the circulation ^2–4.

Inter-endothelial junctions are the main structures maintaining tissue-fluid homeostasis. The plasma oncotic pressure derived from the circulating albumin is the main factor contributing to passive re-absorption of fluid and solutes back into the circulation ⁵. Loss of inter-endothelial junctions as the result of an acute or chronic process leads to flux of proteinaceous fluid into the interstitium causing tissue edema. This is a common cause of a broad range of pathological conditions in humans including systemic capillary leak syndrome ⁶, angioedema ⁷, anaphylaxis ⁸, acute respiratory distress syndrome ^{9, 10}, and age-related and diabetic-associated eye diseases ^11–13, various disorders of central nervous system ^14–17. Hence, elucidating signaling mechanisms responsible for control of junction integrity is of fundamental importance to developing novel therapeutic strategies for treating edema.

2. Role of inter-endothelial junctions in regulating endothelial barrier function

Inter-endothelial junctions are composed of protein complexes of adherens junctions (AJs), tight junctions (TJs), and gap junctions (GJs) ^18–21 (Figure 1a). Both AJs and TJs form pericellular zipper-like structures along endothelial cell borders through adhesion of distinct adhesive proteins ^18–20. In contrast, gap junctions are intercellular channels enabling direct electrical and chemical communication between endothelial cells through the passage of ions and signaling molecules with a size of 1kD or less ²².

Figure 1. Composition of inter-endothelial junctions.

A) Schematic representation of inter-endothelial junctions comprised of tight junctions (TJs), adherens junctions (AJs), and gap junctions (GJs). TJs are mediated by adhesion proteins such as claudins, occludin, and junctional adhesion molecules (JAMs) whereas the zona occludin proteins (ZO-1, ZO-2 and ZO-3) connect adhesion molecules to the actin cytoskeleton. AJs are comprised of VE-cadherin and associated β- and p120-catenins. α-catenin binds β-catenin to connect AJs to the actin cytoskeleton. GJs are comprised of two connexin hexamers forming hemichannels.

B) VE-cadherin mediates adhesion by trans-dimerization of tryptophan 2 and tryptophan 4 residues in a hydrophobic pocket of the opposing VE-cadherin molecule. Ribbon presentations of VE-cadherin trans-dimer. Adapted with permission from Brasch et al., Trends in Cell Biology, 2012.

C) Strand swap dimerization occurs through the insertion of a tryptophan residue into the hydrophobic binding pocket of the opposing cadherin. Adapted with permission from Brasch et al., Trends in Cell Biology, 2012.

D) Trans dimerization (between EC1 and EC1 of opposing cadherins) orients VE-cadherin molecules and facilitates cis interactions (between EC1 and EC2 of neighboring cadherins). Adapted with permission from Brasch et al., Trends in Cell Biology, 2012.

Gap junctions

A functional gap junction is comprised of two hemichannels aligned in the plasma membrane of adjacent endothelial cells ²³. Each hemichannel consists of six connexin molecules, assembled within the ER or trans-Golgi ^24–26. The hemichannel can be either homomeric or a heteromeric; i.e., assembled by the same or distinct connexin isoforms, respectively ²⁷. Channels comprised of different isoforms might exhibit altered activities in respect to ion selectivity and permeability as compared to homomeric channels ^28–30.

The three major connexin isoforms expressed in systemic arteriolar endothelial cells are Cx37, Cx40, Cx43 ^{31, 32}. These gap junctions are responsible for communication between endothelial and endothelial-smooth muscle cells ^{33, 34}. In animal models, deletion of Cx43 in endothelial cells causes hypotension ³⁵, whereas deletion of Cx40 leads to hypertension associated with dysregulation of renin system ^36–38. Interestingly, Cx43-mediated gap junctions elicited distinct functions in pulmonary circulation ³⁹. These junctions contribute to conduction of Ca²⁺ between endothelial cells in lung capillaries and induce the expression of P-selectin, the cell surface adhesion molecule involved in the recruitment of leukocytes to sites of injury in post-capillary venules ³⁹. Hence, Cx43-mediated gap junctions are critical for regulation of vascular tone in the systemic circulation and contribute to the propagation of pro-inflammatory signaling in pulmonary capillary beds.

Tight junctions

The architecture and composition of endothelial TJs varies in different vascular beds ^{19, 40–42}. For example, TJs are more developed in small arterioles whereas AJs are more predominant in post-capillary venules ^{19, 40}. TJs are localized at the outermost part of inter-endothelial junctions but can also be intermingled with AJs ^{40, 43–45}. In contrast to the peripheral microcirculation, highly specialized vascular beds such as the blood brain barrier (BBB) and the inner blood–retinal barrier (iBRB) where exchange of solutes between microvessel and brain is restricted ^{18, 46}, TJs are predominant in forming extensive networks at the apical side of inter-endothelial junctions (for reviews, see ⁴⁷). Disruption of TJs is associated with BBB and iBRB leakage, a characteristic of multiple human diseases including diabetic and oxygen-induced retinopathy ⁴⁸ and disorders of the central nervous system such as stroke ^49–51.

TJs are comprised of several adhesive proteins including occludin, claudins, and junctional adhesion molecules (JAMs) ^52–57 (for reviews, see ⁵⁸). Claudin 5 is ubiquitously expressed in all vascular beds whereas claudin 1, 3, and 12 are specific to the brain microvasculature ^59–61. Claudin-1, -2, and -5 are found in TJs of retinal vessels ⁴⁸. Claudins and occludins, in association with cytosolic Zonula occludens (ZO)-1, 2 and 3 proteins assemble “zipper-like” structures along the rim of endothelial cells ^{62, 63}. The role of JAM-A in the organization of TJs is less understood.

The integrity of the BBB is crucial to the proper functioning of the central nervous system. Disruption of the BBB associated with trauma, hemorrhagic stroke, rupture of cerebral aneurysm, and inflammation leads to serious consequences ranging from progressive neuronal dysfunction, sclerosis, brain edema, paralysis, and death ⁶⁴ (for reviews, see ^{65, 66}). Studies in animal models demonstrate that deletion of the claudin-5 gene cldn5 causes death in newborn animals due to increased permeability of the BBB in a size-selective manner ⁵⁹. Claudin-5 is targeted for degradation by matrix metalloproteinase after an ischemic insult, and loss of claudin-5 is responsible for disruption of the BBB in ischemic stroke ⁵¹. In contrast, loss of claudin-3 but not claudin-5 or occludin accounted for breakdown of the BBB in experimental models of allergic encephalomyelitis and glioblastoma multiforme ⁶⁰ suggesting that claudin proteins might have distinct and indispensable roles in regulating the organization of TJs in brain circulation. Deletion of the occludin gene ocln causes no apparent defects in the organization or strength of TJs ⁶⁷ suggesting that its function is compensated by other adhesive proteins.

In contrast to the BBB, neither claudin nor ocludin proteins are downregulated in the iBRB using experimental models of diabetic and oxygen-induced retinopathy ^{48, 68}. In fact, expression of claudin-2 and -5 is upregulated in oxygen-induced retinopathy ⁴⁸ suggesting that breakdown of the iBRB is associated with post-translational modifications of adhesion proteins of TJs. PKCζ is known to facilitate the formation of TJs through phosphorylation of occludin ⁶⁹. Hyper-activation of PKCζ observed in type 2 diabetic induced the mis-localization of occludin and disruption of TJs resulting in increased leakage of plasma proteins into the retina ^{70, 71}.

TJs are linked to the actin cytoskeleton through the Zonula Occludens proteins ZO-1, ZO-2, and ZO-3 expressed in endothelial cells ^{62, 63}. They interact with adhesive proteins of TJs and anchor the actin cytoskeleton with TJs (62, 63). ZO-1 plays a crucial role in the assembly of functional TJs and AJs ^{62, 72, 73}. As discussed below, ZO-1 might regulate the cross-interaction between TJs and AJs through control of intracellular tension and assembly of the VE-cadherin mechanosensory complex 73. Decreased expression of ZO-1 is associated with severe plasma leakage observed in multiple sclerosis ⁷⁴ and diabetic rats ^{75, 76}.

In a study in mice with inducible endothelial cell-restricted disruption of β-catenin it is shown that endothelial β-catenin signaling was essential for maintaining BBB integrity through regulation of claudin-1 and claudin -3 in adult brain endothelial cells ⁷⁷. These mice developed multiple brain petechial hemorrhages accompanied by neuronal injury and CNS inflammation. Thus, nuclear β-catenin is an essential mechanism in regulating BBB via the expression of claudin-1 and claudin-3. This conclusion is supported by the evidence that Wnt /β-catenin signaling regulates expression of claudin-3 ⁷⁸.

Adherens junctions

AJs are comprised of Vascular Endothelial (VE)-cadherin and associated α-, β- and p120-catenin adhesion complexes ^79–81. In addition, there is also a variety of other recently described junctional proteins, i.e. vinculin, N-WASP and Arp2/3, which interact with catenins involved primarily in stabilizing VE-cadherin-mediated adhesion (discussed below). Multiple lines of evidence showed that VE-cadherin adhesion is the primary adhesion event during vascular development ⁸². VE-cadherin-mediated adhesion promotes activation of forkhead box transcriptional factor FoxO1, which is also required for claudin-5 expression ⁸³. Knockout of β-catenin in endothelial cells leads to disruption of TJs ⁷⁷ indicating the importance of AJs in assembly and maintenance of TJs. Disassembly of AJs compromised by the integrity of the VE-cadherin adhesion complex is the leading cause of tissue edema associated with a broad range of pathological conditions ^84–87.

Another major cadherin present in endothelial cells, Neural (N)-cadherin ^{88, 89} has been shown to mediate the interaction between endothelial cells and the surrounding mural cells (smooth muscle cells and pericytes), and is critical for endothelial vessel maturation and stabilization ^90–92. N-cadherin adhesions are excluded from AJs both in vitro and in vivo ^{87, 93, 94}. Deletion of N-cadherin gene cdh2 in endothelial cells causes embryonic lethality due to severe vascular defects ⁹⁵. Multiple lines of evidence indicate a specific role of pericytes in the formation of the BBB and iBRB ^96–99. The study demonstrated that interaction between pericytes and endothelial cells was required for the formation of TJs in iBRB and BBB ^100–102. An attractive hypothesis is that N-cadherin adhesion, which is involved in the recruitment of pericytes ^{90, 92}, also contributes to the assembly of TJs (although the mechanisms of this unknown, this idea deserves scrutiny). Another study demonstrating that N-cadherin adhesion-induced signaling contributes to the resolution of lung vascular injury through an AMP kinase dependent mechanism ¹⁰³ is consistent with this concept.

T-cadherin (cadherin 13) is also highly expressed in the vasculature ¹⁰⁴. Unlike most cadherins, T-cadherin lacks a transmembrane as well as cytoplasmic region, and is not involved in cell-cell adhesion or anchorage to the actin cytoskeleton ¹⁰⁵. T-cadherin is anchored to lipid raft regions via a glycosylphosphatidylinositol anchor, where it acts as a signaling molecule ¹⁰⁶. T-cadherin has been suggested to act as a receptor for LDL, and may play a role in angiogenesis by a yet undefined mechanism ¹⁰⁷. Furthermore, T-cadherin enhances endothelial barrier function in monolayers, but appears to negatively regulate the barrier when challenged with thrombin ¹⁰⁸.

Retinal (R)-cadherin is critical for retinal vascular formation, and relies on a similar network pattering as found in neurons ¹⁰⁹. It has also been reported that R-cadherin forms functional, heterotypic interactions with N-cadherin, suggesting a possible role for R-cadherin in endothelial-mural cell interactions ¹¹⁰.

VE-cadherin 2 (protocadherin 12, PCDH12) is also localized to endothelial cell-cell junctions, and while sharing a common extracellular cadherin sequence it has a cytosolic region with unknown homology to typical cadherins ^{111, 112}. VE-cadherin 2 does not bind catenins and is only weakly associated with the cytoskeleton. VE-cadherin 2 does not seem to affect endothelial permeability, and seems to be only involved in cell-cell adhesion ¹¹². Transgenic mice deficient in VE-cadherin 2 had no gross morphological defects ¹¹¹. However recent studies showed that arteries lacking VE-cadherin2 had medial elastic lamellae, increased inner-diameter and circumferential mid-wall stress indicating it is required for both the structure and function of arteries ¹¹³.

3. Mechanisms of VE-cadherin cis- and trans-interaction

VE-cadherin homophilic dimerization

VE-cadherin is a member of the classical cadherin family that possess a modular structure of five ectodomains, a transmembrane domain, and a cytoplasmic tail ¹¹⁴. VE-cadherin displays characteristics of both type I and type II cadherins ¹¹⁵. Like type I cadherins, it lacks the hydrophobic non-swapped region that extends the hydrophobicity of the docking surface. Similar to type II cadherins, it contains two conserved tryptophans, Trp2 and Trp4, important for its adhesive property. Anchorage of these tryptophans to a hydrophobic pocket of the partner ectodomain 1 induces “strand-swap” binding mode, resulting in the so-called trans-dimerization of VE-cadherin ^116–118 (Figure 1b–c). Trans-interaction reduces the flexibility of the extracellular domain, which enables a secondary adhesion event between ectodomains 1 and 2 of two cadherins on the same side of an endothelial cell (Figure 1d). This low-affinity cis-interaction is proposed to be responsible for lateral clustering of VE-cadherin, which may increase the strength of adhesive bonds ^{116, 119}. Formation of both trans- and cis-interactions is an intrinsic property of the extracellular moiety of VE-cadherin that does not require the intracellular portion of the protein or assembly of the cadherin-catenin complex ¹¹⁶.

Tethering of VE-cadherin adhesion complex to actin cytoskeleton

The strength of adhesive bonds, defined specifically as the ability of VE-cadherin adhesion to sustain mechanical stresses from blood flow and pressure, is regulated through attachment of the adhesion complex to the actin cytoskeleton ^{114, 120–122}. The actin cytoskeleton contributes to the strength of AJs) by several fundamental mechanisms. It generates intracellular tension and clustering of VE-cadherin at AJs ^121–124, and facilitates assembly of the VE-cadherin mechanosensory complex ^{73,125, 126}.

α-catenin is the only member of the cadherin-associated catenin proteins that contains an actin-binding domain ^{120, 127} enabling the direct association between VE-cadherin adhesion and the actin cytoskeleton ^127–129. α-catenin can either tether pre-existing actin filaments to the VE-cadherin complex ¹³⁰, or alternatively, induce de novo polymerization of actin filaments at sites of AJs ¹³⁰. The latter mechanism involves recruitment of actin binding proteins such as α-actinin, Epithelial Protein Lost in Neoplasm (EPLIN), and vinculin to VE-cadherin adhesion in the presence of intracellular tension ^{123, 130} (for review, see ¹³¹).

α-catenin and vinculin are allosteric molecules that undergo a rapid and reversible switch between conformational states depending on the applied tension ^{128, 132–134}. α-catenin-mediated recruitment of vinculin, along with N-WASP, VASP, and myosin II to AJs enhances the strength of VE-cadherin adhesion by promoting Arp2/3-dependent polymerization of de novo actin filaments ^{131, 135–137}. Recent work from our group has shown that p120-catenin forms a complex with Arp2/3 and N-WASP ¹³⁸. Knock down of Arp2 did not inhibit N-WASP interaction with p120 catenin, suggesting that N-WASP binds directly to p120 catenin and induces organization of cortical actin ¹³⁸. Stabilization of F-actin filaments occurs through the binding of a variety of capping proteins including Capping Protein (CP, aka β-actinin), CapZ, FSGS3/CD2-associated protein (FSGS3/CD2AP) to the barbed (plus) end of F-actin, and is required for actin assembly ¹³⁹. Hence, the strength of VE-cadherin adhesive bonds and therefore integrity of the endothelial barrier is regulated by a complex network involving the aforementioned regulators of actin-polymerization.

Role of intracellular tension in regulating VE-cadherin adhesion

Intracellular tension is a critical component regulating stable anchorage of VE-cadherin to the actin cytoskeleton (for review, see ¹⁴⁰). Simultaneous binding of α-catenin to both β-catenin and F-actin filament occurs only in the presence of tension ^{129, 130, 141}. Tension of up to 10 pN induces stable bond formation between the β-catenin/α-catenin complex and F-actin in vitro ¹²⁹. VE-cadherin adhesion at AJs are formed in a tension-dependent manner ¹²⁶ indicating an important role of the acto-myosin apparatus at AJs. Endothelial cell monolayers generate an intercellular tugging force of ~40 nN ¹²⁶ with an average tension on a VE-cadherin molecule from the actin cytoskeleton ranging from 1.8 to 2.4 nN per molecule ¹⁴². Pro-inflammatory mediators such as α-thrombin increase traction forces and the resultant mechanical stress at AJs (up to ~ 8 nN/μm2) that uncouples the VE-cadherin complex from the actin cytoskeleton ¹²⁶.

Intracellular tension is generated by the acto-myosin contractile apparatus ^{126, 143} (Figure 2a–b). The ubiquitously expressed non-muscular actin motor myosin-IIA and B ^{133, 144} are central to control of intracellular tension at endothelial AJs ¹²⁶. Myosin II binds to F-actin filaments and generates tension by sliding these filaments along each other ¹³². The ability of myosin II to assemble antiparallel filaments consisting of 10–30 motors ^{135, 145} is the main determinant of the magnitude of intracellular tension (Figure 2a).

Figure 2. Role of acto-myosin apparatus in stabilizing AJs.

A) Domain structure of non-muscle myosin II (NM-II). The NM-II consists of a globular head domain containing both actin-binding and motor domains, essential light chains (ELCs), regulatory light chains (RLCs), and heavy chains. NM-II possesses a head to tail interaction in the absence of phosphorylation. Phosphorylation of regulatory light chain at Thr18/Ser19 by myosin light chain kinase (MLCK) unfolds the molecule, enabling assembly of anti-parallel filaments through interactions between their rod domains. Activation of Rho-associated kinase (ROCK), which inhibits phosphatase activity of myosin light chain phosphatase (MLCP) in a phosphorylation-dependent manner, also favors RLC phosphorylation. NM-II filaments bind to actin filaments, which slide along each other, and cause a cell contraction. Adapted with permission from (Vicente-Manzanares et al., Nature Reviews Molecular Cell Biology, 2009).

B) Proposed mechanism of regulation of NM-II activity at AJs in confluent endothelium. NM-II regulates attachment of the VE-cadherin adhesion complex to the actin cytoskeleton, thereby generating mechanical tension required for binding of α-catenin to both β-catenin and f-actin. NM-II phosphorylation is controlled by MLCK and MLCP activities. In the model, we propose that Src and Cdc42 pathways cooperate in regulating NM-II activity at AJs. Cdc42 facilitates activation of NM-II through myotonic dystrophy kinase-related Cdc42-binding kinase (MRCK)-dependent phosphorylation of MLCP whereas Src phosphorylates MLCK at sites of VE-cadherin adhesion. CaM = calmodulin; GEF = guanine nucleotide exchange factor; GAP = GTPase activating protein; GTP = guanosine triphosphate; GDP = guanosine diphosphate.

Phosphorylation of regulatory myosin light chain (MLC) at Thr18 and Ser19 is a prerequisite for motor activity ^{132, 136} (Figure 2a). The activity of myosin-II in endothelial cells is finely regulated by a variety of intracellular signals ¹⁴⁶. The canonical pathway involves phosphorylation of MLC by endothelial-specific myosin light-chain kinase (MLCK), which is commonly activated by Ca²⁺/calmodulin binding ¹⁴⁷ or Src-dependent phosphorylation at Tyr464 and Tyr471 ¹⁴⁸. Myosin light chain phosphatase (MLCP) counteracts MLCK activity by dephosphorylating MLC ¹⁴⁶. Therefore, a fine balance between MLCK and MLCP is essential for limiting myosin II phosphorylation and thereby magnitude of contractile forces at endothelial AJs.

Activity of MLCP (PP1, type 1 protein phosphatase), is downregulated by RhoA signaling ¹⁴⁹. RhoA activates Rho-associated coiled-coil forming protein kinase (ROCK), which in turn, elicits its effect through phosphorylation of PP1 at Thr-695, Ser-894, and Thr-850 ^{149, 150}. The latter inhibits PP1 activity, allowing for myosin II phosphorylation by MLCK and assembly of the acto-myosin apparatus ^{149, 150}.

In endothelial monolayers, myosin-II activity is finely tuned at VE-cadherin adhesions by a yet unknown mechanism. A basal level of ROCK activity appears to be essential for the maintenance of endothelial AJs ¹⁵¹. Recent studies utilizing the RhoA/B/C biosensors show that both RhoA and RhoB are constitutively activated at AJs ^{152, 153}. It remains unclear, however, how the basal level of Rho activity is maintained at AJs. To date, we have a better understanding of the regulation of RhoA signaling at epithelial AJs. In epithelial cells, the RhoA zone at E-cadherin adhesion represents the main molecular mechanism for generation of apical-lateral patterns of junctional contractility ^154–156. Both p120-catenin and myosin-IIA recruit ROCKI to nascent adhesions and provide positive feedback regulation of RhoA activity at E-cadherin adhesions ^{154, 156}. ROCKI phosphorylates Rnd3 and prevents cortical recruitment of the GTPase-activating protein (GAP), p190RhoGAP to AJs, and hence preserves RhoA from inactivation ¹⁵⁶.

It is unlikely that a similar mechanism operates in endothelial cells where p190RhoA accumulation and activity at AJs is required for maintenance of stable AJs ^157–159. A possible mechanism of myosin-II might involve Cdc42 signaling as evident by the finding that Cdc42 also activates myosin II (Figure 2b) ^{160, 161}. Cdc42 mediates the assembly of myosin-II filaments through its effectors Pak2, Pak4 (162), and myotonic dystrophy kinase-related Cdc42-binding kinase (MRCK) ¹⁶⁰. MRCK phosphorylates and thereby inhibits MLCP, although it is less potent than ROCK in activating myosin-II ¹⁶⁰. This makes MRCK the best candidate for induction of low-grade tension at endothelial AJs. Therefore, it is possible that Cdc42 coordinates N-WASP-mediated polymerization of actin filaments with that of p120-catenin ^{138, 163}, such that activation of myosin II at VE-cadherin adhesion is able to strengthen AJs (Figure 2b).

Mechanosensing at the level of AJs

Cells experience external mechanical forces from neighboring cells and the extracellular matrix (ECM) as well as the internal force generated by the actomyosin contractile machinery. In the vascular system, endothelial cells are also exposed to hemodynamic forces resulting from hydrostatic pressure in vessels and blood flow ¹⁶⁴. VE-cadherin forms a mechanosensory complex with platelet endothelial cell adhesion molecule 1 (PECAM-1) along with VEGFR2 and VEGFR3 enabling the endothelium to sense changes in hemodynamics and thus activate a variety of signaling pathways ^{165, 166} (Figure 3). These signaling pathways in turn orchestrate a coordinated cellular response resulting in reorganization of the actin cytoskeleton, redistribution of intracellular tension, and a shift in phosphorylation of VE-cadherin and associated catenins ^{142, 167–169}. Using a recently developed biosensor that measures actomyosin-mediated tension across VE-cadherin adhesion and PECAM-1, it was shown that shear stress applied to an endothelial monolayer reduces tension across VE-cadherin adhesion concomitant with a decrease in total cell-cell force ^{142, 170}. Thus, it appears that the distribution of intracellular tension is tightly regulated in response to external mechanical forces thus allowing AJs to align in the direction of flow.

Figure 3. Mechanotransduction at AJs.

The mechanosensory complex in endothelial cells is comprised of vascular endothelial (VE)-cadherin, platelet endothelial cell adhesion molecule (PECAM)-1 and vascular endothelial growth factor receptor (VEGFR)2. Mechanosensing of shear stress occurs through PECAM-1-dependent activation of Fyn, which in turn facilitates VEGFR2-mediated signaling in a ligand-independent manner and activates PI3K. PI3K activates both Rac1 and eNOS signaling pathways. Rac1 relieves tension at AJs whereas NO concomitantly promotes vasorelaxation of smooth muscle cells. PECAM-1-dependent sensing of shear stress also promotes α2β1 integrin signaling and consequently activation of PKA in atheroresistant regions. PKA phosphorylates RhoA and decreases RhoA-dependent cellular stiffness allowing the endothelial cell to align in the direction of blood flow. PI3K = phosphatidylinositol-4,5-bisphosphate 3-kinase; PKA = protein kinase A; VSMC = vascular smooth muscle cell; NO = nitric oxide; eNOS = endothelial nitric oxide synthase; cGMP = cyclic guanosine monophosphate; sGC = soluble guanylyl cyclase.

The current concept of mechanosensing at AJs involves a series of distinct (and perhaps linear) sequences of signal transduction events (Figure 3). Signaling is initiated with conformational changes in PECAM-1 followed by activation of Src family kinase Fyn at AJs ¹⁷¹. Fyn in turn phosphorylates PECAM-1 and activates the receptor tyrosine kinase VEGFR2 in a ligand independent manner ¹⁷¹. VEGFR2, in turn, induces phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K), Akt, and endothelial nitric oxide synthase (eNOS) ^{120, 165, 172}. PI3K subsequently activates Rac1, relieving the tension at AJs ^{173, 174} whereas eNOS mediates vasorelaxation through the effect of nitric oxide on vascular smooth muscle cells ^{120, 172}.

Interestingly, the response to shear stress varies depending on the composition of the ECM and corresponding integrin signaling ^175–177. Shear stress induces stiffening of endothelial cells adherent to fibronectin (through α5/β1 integrin signaling) but not on collagen (through α2/β1 integrin singling) ^{175, 178} signifying the importance of cross-talk between AJs and focal adhesions (FAs) in the mechanosensory response. In cells adherent to collagen, mechanosensing at AJs resulted in Gαs-dependent protein kinase A (PKA) signaling and subsequent phosphorylation of RhoA ^{175, 179}. PKA phosphorylates RhoA at serine 188 and promotes the association of RhoA with Rho-guanine dissociation inhibitor, which sequesters RhoA in the cytosol ^{180, 181}. PKA-mediated inhibition of RhoA signaling is responsible for blunted stiffening of endothelial cells in response to hemodynamic shear stress ¹⁷⁵. Hence, specifics of ECM composition might permit a differential response of endothelial cells to shear stress ¹⁷⁵. In the fibronectin-rich aortic arch, which is prone to atherosclerosis, endothelial cells are stiffer and more permeable to protein-rich fluids and leukocytes. Failure to activate PKA and reduce stiffness of endothelial cells in these regions of the aorta might contribute to the development of atherosclerosis. In this context, remodeling of the ECM, itself a function of endothelial cell activation is likely a key determinant of change in endothelial barrier integrity at the level of AJs.

4. Signaling mechanisms mediating stability and remodeling of VE-cadherin adhesion

VE-cadherin adhesion as a “gatekeeper” of endothelial barrier

The steady-state dynamics of VE-cadherin at AJs is a critical determinant of AJ integrity. This includes several interdependent events concerning both biophysical properties of VE-cadherin adhesive bonds and the integration of intracellular proteins within VE-cadherin. VE-cadherin adhesive bonds undergo continuous assembling, disassembling, and remodeling at AJs; the kinetics of these events are defined by the affinity of trans-dimerization ^{114, 115}. This primary adhesion event requires neither energy nor attachment of the VE-cadherin complex to the actin cytoskeleton^{114, 115}.

In contrast, turnover of VE-cadherin molecules at AJs, specifically the exchange between junctional and intracellular pools, is tightly regulated by the interaction of VE-cadherin with associated catenin proteins and the actin cytoskeleton ^{123, 126, 182}. The steady-state kinetics of VE-cadherin at AJs is controlled through the stability of the cadherin-catenin complex, intracellular tension, and organization of the actin cytoskeleton ^{126, 182}. The disassembly of VE-cadherin adhesion in response to extracellular stimuli is triggered by phosphorylation of VE-cadherin and associated catenins and the re-distribution of the actin cytoskeleton to the sites of FAs. Depending on the duration and magnitude of the intracellular response, changes in VE-cadherin dynamics at AJs can lead to weakening or disassembly of AJs, causing either transient or prolonged increase in junctional permeability. For example, tumor vessels represent a case of chronic vascular leakage that is associated with downregulation of VE-cadherin expression ¹⁸³.

Multiple lines of evidence suggest that the hyper-permeability response to pro-inflammatory mediators can be mitigated if the integrity of VE-cadherin internalization is preserved. Various strategies have been developed to stabilize VE-cadherin adhesion. They include overexpression of p120-catenin, which blocks clathrin-mediated VE-cadherin internalization ^184–186; expression of a VE-cadherin-α-catenin chimera ¹⁶⁶, which directly tethers adhesion to the actin cytoskeleton; and artificial bridging of opposing VE-cadherin molecules at AJs with a cyclic peptide ¹⁸⁷. This evidence suggests that it is possible to manipulate the integrity of VE-cadherin adhesion, the main gatekeeper of the endothelial barrier.

Kinase-mediated regulation of VE-cadherin turnover at AJs

The spatio-temporal control of VE-cadherin turnover at AJs is an integral part of the intracellular response to environmental cues. Destabilization of VE-cadherin adhesion occurs during trans-endothelial migration of leukocytes ^{161, 164–166} and in response to extracellular stimuli associated with opening AJs and increased barrier permeability ^{165, 166, 188–190}. Intracellular signaling such as phosphorylation of VE-cadherin and associated catenin proteins (summarized in Table 1) induce disassembly of the VE-cadherin-catenin complex. In particular, dissociation of p120-catenin from the juxtamembrane region of VE-cadherin unmasks the binding site for AP2, an adaptor protein complex of the endocytic machinery, and primes VE-cadherin for internalization ^{159, 184, 185, 189} (Figure 4). VEGF promotes VE-cadherin internalization via β-arrestin2-mediated endocytosis ¹⁸⁸. In this context, VEGF induces cSrc-dependent phosphorylation of the guanine nucleotide exchange factor Vav2, which in turn activates Rac1 and p21-activated kinase PAK ¹⁸⁸. PAK phosphorylates VE-cadherin at S665 and targets VE-cadherin for β-arrestin2-mediated internalization ¹⁸⁸. Moreover, VEGF signaling decreases VE-cadherin/p120-catenin association, promoting clathrin-dependent VE-cadherin endocytosis ¹⁸⁴. Multiple pro-inflammatory mediators including thrombin, histamine, platelet-activating factor, Vascular Endothelial Growth Factor (VEGF), and tumor necrosis factor (TNF)-α facilitate disassembly of VE-cadherin adhesion, although they do not seem to function by inducing a similar pathway. This is evident by the finding that they induce differential phosphorylation of VE-cadherin and p120-catenin ^{165, 166, 188–190}. Some mediators, such as thrombin, function through phosphorylating VE-cadherin at Tyr 658 by c-Src ^{170, 172} or p120 catenin at Ser879 by protein kinase C (PKC)α resulting in decreased binding of VE-cadherin to p120-catenin ^{185, 189}. Indeed, leakage of tumor vessels is associated with c-Src-dependent phosphorylation of VE-cadherin and β- and p120-catenin proteins ¹⁹¹. Other studies showed that phosphorylation of β-catenin at Tyr654 and Tyr489 by c-Src and Abelson, respectively, reduced β-catenin affinity to VE-cadherin allowing dissociation of β-catenin from VE-cadherin ^{192, 193}. The latter events uncouple VE-cadherin from the actin cytoskeleton, and thereby reduce VE-cadherin adhesion strength ¹²⁰. In addition, phosphorylation of β-catenin at Tyr142 by Fer or Fyn interferes with the formation of the β-catenin/α-catenin complex and detaches VE-cadherin adhesion from the actin cytoskeleton ¹⁹³. An explanation for the complexity of regulation of VE-cadherin adhesion is that endothelial permeability is a fundamental evolutionarily conserved process requiring activation of multiple “phospho-switches”.

Table 1.

The role of kinases at AJs.

Kinases	Activity within VE-cadherin complex
c-Src (p60 Src, tyrosine kinase)	Phosphorylates VE-cadherin at Y658 and reduces binding to p120-cat; Phosphorylates VE-cadherin Y685 and increases binding to CSK Phosphorylates β-catenin Y654 and reduces binding to VE-cadherin
CSK (cytosolic C-terminal Src kinase, tyrosine kinase)	Phosphorylates c-SRC Y530 and inhibits c-Src activity at AJs
Fer (tyrosine kinase)	Phosphorylates PTP1B Y152 and induces binding to VE-cadherin
Fyn (SFK, tyrosine kinase)	Phosphorylates β-catenin Y142 and reduces binding to VE-cadherin
Yes (SFK)	Phosphorylates β-catenin Y142 and reduces binding to VE-cadherin
Abelson (tyrosine kinase)	Phosphorylates β-catenin Y489 and reduces binding to VE-cadherin
PYK2 (proline-rich tyrosine kinase)	Phosphorylates VE-cadherin Y731 and reduces binding to β-catenin
PAK (Ser/Thr kinase)	Phosphorylates VE-cadherin S665 and targets VE-cadherin for β-arrestin-mediated internalization

Figure 4. Role of specialized kinases and phosphatases in stabilizing AJs.

A) Stable adherens junctions are characterized by low phosphorylation of VE-cadherin and associated catenin proteins. Protein tyrosine phosphatases DEP1, VE-PTP, PTPμ, SHP2, and PTP1B at AJs counteract the effect of tyrosine kinases (Src, Fen, Fyn, and Ableson) to stabilize the VE-cadherin-catenin complex. FAK also stabilizes VE-cadherin adhesion by inhibiting RhoA signaling through phosphorylation-dependent activation of p190RhoGAP. Ang1 = angiopoietin 1; VEGF = vascular endothelial growth factor ; VEGFR2 = vascular endothelial growth factor receptor 2; DEP1 = density enhanced phosphatase 1; VE-PTP = vascular endothelial protein tyrosine phosphatase; PTPμ = protein tyrosine phosphatase μ; SHP2 = Src homology phosphatase; PTP1B = protein tyrosine phosphatase 1 B; FAK = focal adhesion kinase; RhoGAP = Rho GTPase activating protein; RhoGEF = Rho guanine nucleotide exchange factor; GDP = guanosine diphosphate; GTP = guanosine triphosphate.

B) Phosphorylation-dependent activation of kinases by VEGF, histamine, thrombin, PAF, and TNF-α leads to phosphorylation of VE-cadherin, β-catenin, and p120-catenin (residues are indicated) by distinct kinases. This results in destabilization of the VE-cadherin complex. Dissociation of p120-catenin due to phosphorylation of VE-cadherin at Y658 or p120-catenin at S879 exposes a VE-cadherin binding site for AP2 to facilitate VE-cadherin endocytosis via clathrin coated pits. Phosphorylation of β-catenin induces the uncoupling of VE-cadherin adhesion from the actin cytoskeleton. Activation of RhoA leads to phosphorylation of MLC, formation of stress fibers, and increased tension across VE-cadherin adhesion. TNF-α = tumor necrosis factor alpha; PAF = platelet-activating factor; AP2 = adaptor protein 2; MLC = myosin light chain; PKCα = protein kinase C alpha.

In contrast to c-Src and Yes, intriguingly, the other Src family kinases Lyn and Fyn stabilize VE-cadherin adhesion by inducing phosphorylation of focal adhesion kinase (FAK) at Tyr 576/577 ^{194, 195}. FAK, in turn, inhibits RhoA activity at AJs basally and during reannealing of AJs after challenge with α-thrombin through phosphorylation-dependent activation of p190RhoGAP ¹⁹⁶. Inducible deletion of FAK in endothelial cells impairs the balance between RhoA and Rac1 activities leading to hyper activation of RhoA signaling, disruption of AJs, and endothelial barrier leakage ^{196, 197}.

Interestingly, FAK can also contribute to destabilization of VE-cadherin adhesion during angiogenesis when activation of FAK converges with VEGF-activated signaling pathway ¹⁹⁸. FAK contributes to increased endothelial permeability of glioma tumor vessels ¹⁹⁹. FAK appears to facilitate translocation of c-Src to AJs where Src induces the phosphorylation of VE-cadherin at Tyr658 ²⁰⁰. In addition, activation of FAK downstream of VEGF induces the phosphorylation of β-catenin at Y142 and its subsequent dissociation from the VE-cadherin complex ¹⁹⁸. Deficiency of FAK in endothelial cells decreases extravasation of tumor cells and prevents spontaneous orthotopic melanoma metastasis ¹⁹⁸. Together these studies suggest a critical role of FAK in regulating endothelial barrier integrity; however, this function of FAK may either be through direct interaction or upstream of another kinase such as Src.

It is also important to note that many of these kinases are themselves constituents of the VE-cadherin adhesion complex in the resting endothelium ^{201, 202}. Many of them are basally inactive because their activity is suppressed by phosphatases and other kinases at AJs ²⁰². This negative feed-back regulation is disrupted in response to pro-inflammatory mediators allowing fast phosphorylation of proteins within the junctional complex ^{190, 203–205}. Activation of c-Src by Gα13 in response to oxidative stress, a common signal activated by multiple pro-inflammatory stimuli ¹⁹⁰, is one such example. Whereas c-Src activity is basally suppressed by Csk at AJs, this suppression is relieved by Gα13 interaction with VE-cadherin downstream of oxidative redox signaling ¹⁹⁰.

Role of AJ localized phosphatases in regulating junctional integrity

Protein tyrosine phosphatases (PTP) such as PTP1B, PTPμ, PTPβ (also known as Vascular Endothelial [VE]-PTP), Src homology 2-domain containing tyrosine phosphatase (SHP2), and density-enhanced phosphatase-1 (DEP1), are also constituents of the VE-cadherin adhesion complex ^{203, 204, 206, 207} (Figure 4). They stabilize the cadherin-catenin complex by opposing the barrier-disruptive action of kinases (summarized in Table 2) ^{204, 207}. PTP1B is required for continuous dephosphorylation of β-catenin at Tyr654, thus preventing the dissociation of β-catenin from AJs ²⁰⁶. SHP2 also induces dephosphorylation of β-catenin and promotes re-assembly of AJs after inflammatory insult ²⁰⁷.

Table 2.

The role of phosphatases at AJs.

Phosphatases	Activity within VE-cadherin complex
PTP1B	Dephosphorylates β-catenin Y654 and increases binding to VE-cadherin
SHP2 (tyrosine phosphatase)	Dephosphorylates β-, γ- and p120-catenins
PTPμ (tyrosine phosphatase)	Dephosphorylates VE-cadherin
DEP1 (tyrosine phosphatase)	scaffold function
VE-PTP (tyrosine phosphatase)	Dephosphorylates VE-cadherin and γ-catenin

VE-PTP, the most studied of the AJ-associated phosphatases in endothelial cells, interacts with VE-cadherin through the membrane proximal fibronectin (FN)-like extracellular domain ²⁰³. It stabilizes basal VE-cadherin adhesion by decreasing the rate of VE-cadherin internalization ^{190, 165}. Phosphatase activity per se may not be required for this effect since inhibition of VE-PTP activity with a small molecule inhibitor stabilizes AJs and restores tissue-fluid balance in eye and lung vascular inflammation models ^{177, 208}. The therapeutic effect of VE-PTP inhibitor has been explained by angiopoietin-1 signaling, which is suppressed by VE-PTP-dependent dephosphorylation of Tie-2 ^{177, 208} (Figure 4). Angiopoietin-1 elicits a barrier protective effect in the endothelium by activating Tie-2 receptor signaling that uncouples Src kinase from VEGFR2 and inactivates VEGFR2 signaling ¹⁷⁹. The angiopoietin-1/Tie-2 axis also triggers sequential activation of Rap1 and Rac1 in the endothelium ^{177, 208}. Rac1, in turn, causes dissolution of actin stress fibers and stabilizes VE-cadherin trans-interaction by preventing RhoA-mediated intracellular tension at AJs ¹⁸². As discussed below, this “tug of war” between RhoA and Rac1 at AJs is a major determinant of the stability and plasticity of VE-cadherin adhesion both basally and in response to permeability-increasing stimuli.

Role of nitric oxide synthases

Endothelial nitric oxide synthase (eNOS) is responsible for constitutive synthesis of nitric oxide (NO) in the resting endothelium ^210–212. Basal production of NO controls vascular tone and vasorelaxation in response to increased blood flow, whereas hyper-activation of eNOS in response to VEGF or pro-inflammatory stimuli such as Platelet-Activating factor (PAF) triggers S-nitrosylation of VE-cadherin, β-catenin, and p120 catenin ^213–215. S-nitrosylation, the covalent attachment of S-nitrosothiol to a cysteine thiol ²¹⁶, represents another regulatory pathway of AJ stability. Similar to phosphorylation, S-nitrosylation reversibly modulates affinity of β- and p120- catenin proteins to VE-cadherin ^{213–215, 217}. S-nitrosylation of β-catenin on the Cys619 residue promotes dissociation of β-catenin from VE-cadherin causing destabilization of AJs and resultant hyper-permeability of the endothelial barrier ^{213–215, 217}. Deletion of eNOS causes a blunted VEGF-mediated permeability response ²¹⁷, further supporting the role of NO redox signaling in regulating endothelial barrier function.

PAF induces S-nitrosylation of p120-catenin on multiple cysteine residues, Cys579, Cys429, Cys450, Cys618, and Cys692 ²¹⁴. S-nitrosylation of Cys579, located within the VE-cadherin-interacting domain, might represent a critical event associated with NO redox signaling in regulating endothelial hyper-permeability ²¹⁴. PAF can also induce S-nitrosylation of VE-cadherin and consequent disruption of AJs ²¹⁵. In this context, S-nitrosylation of VE-cadherin is required for tyrosine phosphorylation and internalization of VE-cadherin ²¹⁵. These data indicate that S-nitrosylation of junctional proteins is an important mechanism for destabilization of AJs.

Another post-translational modification induced by reactive nitrogen species such as anion (ONOO-) is nitraton of tyrosine residues ²¹⁸. Nitration of junctional proteins, such as p190RhoGAP, which is associated with p120-catenin ¹⁵⁷, and β-catenin itself ²¹⁹, facilitates disassembly of the VE-cadherin adhesion complex. The pro-inflammatory mediator serine protease α-thrombin triggers nitration of p190RhoGAP on Tyr1105 downstream of eNOS-mediated NO redox signaling ¹⁵⁷. This inhibits GAP activity, consequently activating RhoA signaling at AJs and corresponding acto-myosin cell contraction ¹⁵⁷. Hence, nitration of p190RhoGAP represents a crucial mechanism in the activation of RhoA signaling implicated in hyper-permeability of the endothelial barrier during inflammation.

Nitration of β-catenin occurs in the context of chronic or acute inflammation associated with the expression of inducible iNOS. Many inflammatory processes including diabetes, atherosclerosis, and systemic inflammation are associated with protein nitration due to activation of iNOS in the endothelium ^220–225. Induction of iNOS in macrophages triggers nitration of β-catenin in endothelial cells and the resultant dissociation of VE-cadherin adhesion-mediated complex ²¹⁹. Nitration of β-catenin also promotes its translocation to the nucleus where it is associated with T-cell factor (TCF)/Lef transcription factors ²¹⁹. This ultimately leads to vascular remodeling after injury ²²⁶. In this context, nitration of β-catenin induces vascular leakage, but at the same time limits endothelial injury by promoting pro-survival pathways ^{219, 226}.

Role of acetyltransferases

A growing body of evidence suggests that lysine acetylation of β-catenin might also provide an important mechanism for regulating endothelial barrier permeability ²²⁷. Acetylation is a reversible process controlled by acetyltransferases and deacetylases. Acetylation of β-catenin induces its association with the plasma membrane ²²⁸ and modulates β-catenin activity towards specific genes ^{229, 230}. Acetylation of β-catenin at Lys49 is mediated by CREB-binding protein (CBP) acetyltransferase ²²⁹ and is known to modulate Wnt signaling in a promoter-specific fashion. In contrast, acetylation of β-catenin at Lys345 by the transcriptional coactivator p300 increases its affinity for TCF4 ²³⁰, suggesting that acetylation might differentially modulate β-catenin transcriptional activity.

In contrast, deacetylation of β-catenin at Lys49 is controlled by a member of class II histone deacetylase HDAC6 ²²⁸. Knockout of HDAC6 or treatment of animals with the specific HDAC6 inhibitor tubastatin A protects against endotoxin-induced pulmonary edema and acute lung injury and improves survival of mice in septic shock ^{227, 231}.

5. Role of endothelial cells expressed transient receptor potential (trp) channels

Multiple pathological conditions are associated with calcium signaling, which represent a crucial pathway in mediating hyper-permeability of endothelial barrier ^232–236. A superfamily of transient receptor potential (TRP) channel that are responsible for regulation of Ca²⁺ entry in endothelial cells presented by TRPC (Canonical), TRPV (Vanilloid), TRPM (Melastatin) ^234–236, and therefore, have been extensively studied for their role in mediating hyper-permeability response.

TRPC

Endothelial cells express 5 non-selectively permeable transient receptor potential cation channels (canonical), TRPC1, TRPC3, TRPC4, TRPC5, TRPC6, and TRPC7 ^234–241. These channels mediate store- and receptor-operated Ca²⁺ entry from extracellular spaces in response to edemagenic and pro-angiogenic mediators.

Store-operated calcium entry (SOCE) involves TRPC1 and TRPC4. These channels are activated by G protein-coupled receptors (GPCR), and receptor tyrosine kinases coupled to activation of phospholipase C (PLC). Multiple pro-inflammatory mediators such as serine protease α-thrombin, histamine and PAF induce disruption of the endothelial barrier through binding to GPCRs on the surface of endothelial cells. This triggers activation of PLC, which catalyzes phosphoinositol 4,5-bisphosphate (PIP2) to inositol 1,4,5-trisphosphate (IP₃) and diacylglycerol (DAG) (for review, see ^{244, 245}). IP₃ activates Ca²⁺ release from intracellular stores and consequent Ca²⁺ entry through SOCE.

The critical role of SOCE in the hyper-permeability response is evident from genetic deletion of murine trpc1 and trpc4 genes. These animals demonstrate markedly reduced hyper-permeability responses in lungs and reduced mortality induced by endotoxins ^{240, 246}. Intriguingly, full activation of TRPC1 is required for RhoA-mediated reorganization of the actin cytoskeleton, enabling the interaction of TRPC1 with IP3Rs, calcium channels on the endoplasmic reticulum membrane responsible for Ca²⁺ release from stores ^{236, 238}, indicative of a positive amplification loop. Other studies demonstrate that TRPC1 downregulates both the expression and activity of sphingosine kinase 1 (SPHK1), the kinase responsible for production of the barrier enhancing mediator Sphingosine-1-phosphate (S1P), thereby weakening AJs in both resting and inflammatory (activated) endothelium ²⁴⁶. This function of TRPC1 appears to be independent of Ca²⁺ entry since expression of a TRPC1 pore-defective mutant is sufficient to limit expression of SPHK1 and restore the permeability response of the endothelial barrier ²⁴⁶.

Receptor-activated calcium entry (ROCE) is mediated through TRPC6 and TRPC7 in endothelial cells (234–236). These channels are activated by a diacylglycerol (DAG) dependent mechanism and are independent of intracellular store depletion ^{247, 248}. Multiple lines of evidence show that TRPC6 promotes both histamine- and lipopolysaccharide (LPS)-mediated increases in endothelial permeability ^{236, 235, 249}. Importantly, TRPC6 co-localizes with PECAM-1 at AJs during trans-endothelial migration of leukocytes ²⁵⁰. In this context, activation of TRPC6 is mediated through homophilic PECAM-1 adhesion between endothelial cells and leukocytes ²⁵⁰. Furthermore, TRPC6-mediated ROCE is required for neutrophil transendothelial migration (TEM), since expression of a pore-defective channel or knockdown of TRPC6 in endothelial cells arrests neutrophils within AJs ²⁵⁰.

TRPV

The subfamily members of TRPV induce Ca²⁺ entry in response to osmolar, thermal, mechanical, and chemical stimuli ^{251, 252}. In endothelial cells, TRPV4 is activated by heat and endogenous lipid mediators such as epoxyeicosatrienoic acids (14,15-EET) and phorbol ester 4a-phorbol 12,13-didecanoate (4αPDD) ^252–255. In mice, knockout of trpv4 gene inhibits permeability responses of lung microvasculature to both 4αPDD and 14,15-EET without affecting SOCE ²⁵³. Other studies indicate that TRPV4 might also be involved in the mechanism of Ca²⁺ entry in response to shear stress ²⁵⁶.

TRPM

The TRPM family is presented by TRPM2 and TRPM4 in endothelial cells ^257–260. TRPM2 is activated by intracellular ADP-ribose, hydrogen peroxide, and Nicotinamide adenine dinucleotide ^{257, 261}. TRPM2 has been shown to induce Ca²⁺entry in response to H₂O₂ in a dose-dependent manner ²⁵⁸. It is plausible that TRPM2 may serve as a cellular redox sensor in endothelial cells.

6. Role of RhoGTPases in Regulating Integrity of AJs

The subfamily of Rho (Ras homologous) RhoGTPases belongs to the Ras-sarcoma (Ras)-related superfamily of low molecular weight monomeric G proteins with highly conserved sequence homology ^{262, 263}. RhoA, Rac1, and Cdc42 are the best-studied members of the RhoGTPases sub-family due to their critical role in organization of the actin cytoskeleton as well as profoundly affecting the integrity of AJs ^264–268.

A fine balance among RhoA, Rac1, and Cdc42 at AJs is regulated by VE-cadherin “outside-in” signaling ^{133, 269}. Formation of nascent VE-cadherin adhesions activates Rac1 ²⁷⁰. Rac1, in turn, induces polymerization of actin filaments specifically at sites of VE-cadherin adhesion and contributes to the stabilization of AJs ²⁷⁰. Rac1 also stabilizes VE-cadherin trans-interaction by counteracting RhoA activity and suppressing acto-myosin tension ¹⁸². Hence, a subtle balance between RhoA and Rac1 activities is a critical control point of VE-cadherin turnover at AJs ¹⁸².

RhoGTPases are also involved in destabilization and reannealing of AJs in response to mechanical and humoral stimuli. The net effect of RhoGTPases on barrier integrity depends on the nature of extracellular stimuli and activation of convergent signaling pathways that are able to re-wire RhoGTPase signaling to specific intracellular locations and establish their interactions with particular downstream effectors. As described below, the complexity of these biological outcomes can be explained by the combinatorial effects of activation of multiple RhoGTPases.

Sub-family of RhoGTPases

Monomeric RhoGTPases cycle between active (GTP-bound) and inactive (GDP-bound) states and thus act as binary molecular switches ^{263, 271, 272}. In the GTP-bound state, they interact with the downstream effectors to elicit a physiological response ^{181, 184, 186}. RhoGTPases interact with a wide spectrum of downstream effectors that are structurally different from each other ^{186, 205}, and yet the RhoGTPase domain structure itself is highly conserved. All members of the RhoGTPase sub-family contain a G domain structure at the N-terminal, which is comprised of 5 sets of G box binding motifs (for review, see ²⁷³) (Figure 5A). The G domain consists of the nucleotide binding site (also called the p-loop), core effector domain, and switch regions (I and II) forming the interface for interaction with GEFs (Figure 5B). The p-loop motif inside the switch I and switch II regions represents the site of GDP to GTP exchange as well as the interface for interaction with downstream effectors upon binding to GTP ^274–276. This ability to interact with effectors is lost when the switch region possesses a conformational change due the release of the hydrolyzed phosphate ²⁷³.

Figure 5. Regulation of RhoGTPase activity.

A) Schematic representation of general domain structure for RhoGTPases. The 5 G-box motifs (green) represent nucleotide binding motifs whereas the switch I and Switch II are the region of GDP/GTP exchange. C-terminus (red) undergoes post translational modification required for modulating the membrane-targeting of RhoGTPases.

B) Conformational changes within Switch I and II regions upon GTP hydrolysis and exchange. The closed GTP-bound conformation has a higher affinity for GAP binding. Cleavage of hydrolyzed phosphate by GAPs put the switch regions into a relaxed, open conformation. The open GDP-bound conformation has a high affinity for GEF binding. GAP = GTPase activating protein; GEF = guanine nucleotide exchange factor; GDP = guanosine diphosphate; GTP = guanosine triphosphate; GNBP = guanine nucleotide binding protein. Adapted with permission from Vetter and Wittinghofer, Science, 2001.

C) Regulation of RhoGTPase cycle. In the GDP-bound state, RhoGTPases are prevented from interacting with downstream effectors. Release of GDP is facilitated by GEFs allowing exchange for GTP. GAPs catalyze the hydrolysis of GTP resulting in inactivation of the GTPase. GDIs prevent GTP exchange by binding to the GDP bound state. GDI = guanosine nucleotide dissociation inhibitor. Adapted with permission from Etienne-Manneville and Hall; Nature, 2002.

Because of the high binding affinity of GTPases for both GDP and GTP and slow rate of intrinsic GTP hydrolysis, the GTPase cycle is controlled by upstream regulators; specifically GTPase Activating Proteins (GAPs), Guanine Nucleotide Exchange Factors (GEFs), and Guanine Nucleotide Dissociation Inhibitors (GDIs) (Figure 5C). GAPs accelerate the rate of GTP hydrolysis and switch “off” RhoGTPase activity, whereas GEFs promote GDP to GTP exchange, thus turning RhoGTPases “on” ^277–281. The latter is a multi-step process involving formation of a ternary complex between the GTPase, GEF, and nucleotide followed by nucleotide release (Figure 5B). Rebinding of GTP, predominantly due to higher concentration in the cell, restores GTPase activity. GEFs promote GTP exchange by increasing the rate of GDP release ^{282, 283}. Another regulator, GDI interacts with the GDP-bound form and prevents GTP exchange ^{280, 281}. GDIs shield the hydrophobic tail by binding to a prenylated COOH-terminus, and hence sequesters GTPase from the membrane compartment ^{284, 285}.

The interaction between RhoGTPases and downstream effectors requires translocation of GTPases from the cytosol to the plasma membrane ^{284, 285}. This is controlled by post-translational modifications (PTMs) by the lipids farnesylpyrophosphate (FPP) and geranylgeranylpyrophosphate (GGPP). CAAX sequence at the C-terminus serves as both membrane targeting signal and a recognition motif for farnesylation and geranylgeranylation ^286–288. Some members of the RhoGTPase family such as RhoA and RhoC are only geranylgeranylated and are localized in the cytoplasm, whereas others such as RhoB possess geranylgeranylated, farnesylated, or palmitoylated sites and can be localized at the plasma membrane or in the cytoplasm (localized to endosomes) ^288–290.

The recruitment of RhoGTPases to membranes, as demonstrated for Rac1, occurs preferentially at the boundaries between the cholesterol-rich, ordered domains (i.e., lipid rafts) and the liquid disordered phase ²⁹¹. Rac1 then diffuses into both raft and non-raft domains, where it interacts with either downstream effectors inside of ordered domains or can be selectively inactivated by GAPs that prefer non-raft regions ²⁹¹. Hence, on one hand, PTMs target small RhoGTPases to distinct sub-cellular localizations, allowing them to interact with a specific set of downstream effectors and thus elicit distinct biological outputs through spatially-regulated signaling networks. On the other hand, the organization of plasma membrane domains modulates RhoGTPase signaling by limiting their activities in the non-raft regions.

Rac1 and Cdc42 signaling pathways regulate stability of VE-cadherin adhesion

The role of Rac1 and Cdc42 on assembly and maturation of VE-cadherin adhesion is predominantly associated with their ability to induce nucleation, polymerization, and organization of the actin cytoskeleton through interactions with actin binding proteins ^{186, 292, 293}. Whereas Rac1 promotes polymerization of branched actin network within lamellipodia protrusions ^{187, 189, 294, 270}, Cdc42 facilitates polymerization of linear F-actin filaments into filopodia ^{295, 296}. Upon activation, Rac1 interacts with several downstream effectors including the WASP-family verprolin-homologous protein (WAVE), IQRas GTPase-activating proteins (IQGAPs), partitioning-defective polarity protein PAR6, and members of p21 Activated Kinase (Pak) family ¹⁸⁶ (Figure 6). Among the members of Pak family, Pak1 facilitates actin polymerization through activation of Lin1, Isl-1, and Mec-3 Kinase (LIMK) ²⁹⁷. The latter phosphorylates the actin binding protein cofilin at Ser3 and consequently blocks actin monomer de-polymerization ²⁹⁸.

Figure 6. RhoA, Rac1 and Cdc42 regulation of endothelial AJs.

Rac1 and Cdc42 promote organization of the actin cytoskeleton into lamellipodia and filopodia protrusions resulting in re-annealing and stabilization of AJs. Regardless of differential effect on actin organization, Rac1 and Cdc42 share common downstream effectors such as PAR6 and IQGAP1. These effectors serve as scaffolds by recruiting active Rac1 and Cdc42 to AJs. Cdc42 can also generate low grade tension at AJs through activation of non-muscle myosin II. In contrast to Rac1 and Cdc42, RhoA activity is basally suppressed at AJs. Activation of RhoA is associated with formation of stress fibers, increased intracellular tension, and destabilization of AJs. MRCK = myotonic dystrophy kinase-related Cdc42-binding kinase; WASP = Wiskott-Aldrich Syndrome protein; IRSp53 = insulin receptor tyrosine kinase substrate p53; mDia = mammalian Diaphanous; Pak = p21 activated kinase; IQGAP = IQ motif containing GTPase activating protein; PAR6 = partitioning defective protein 6; WAVE = Wasp family verproline-homologue; MLCP = myosin light chain phosphatase; Arp2/3 = Actin-related proteins 2 and 3; LIMK = LIM (Lin1, Isl-1, & Mec-3) kinase; Isl-1 = Insulin gene enhancer protein; Lin1 = CD2 cytoplasmic tail binding protein 2.

The Cdc42 downstream effectors include Wiskott–Aldrich Syndrome protein (WASP), neuronal (N)-WASP, Diaphanous-related formin-1 (mDia1), IQGAPs, PAR6, and MRCK (Figure 6) ¹⁸⁶. Cdc42 induces nucleation and polymerization of actin filaments through WASP and mDia pathways ¹⁶³. It can also bind to the insulin receptor substrate p53 (IRSp53) that coordinates actin nucleation and polymerization through binding to both WASP and mDia at the plasma membrane ^{299, 300}. The Cdc42-MRCK pathway activates myosin II and strengthens AJs by generating low magnitude intracellular tension ¹⁶⁰. Hence, in addition to nucleation, polymerization, and stabilization of the actin cytoskeleton at AJs, the Cdc42 signaling pathway is also capable of generating intracellular tension independent of RhoA signaling.

Cdc42 plays a crucial role in assembly and maintenance of AJs ³⁰¹. Deletion of Cdc42 in endothelial cells results in loss of apical-basal polarity and disrupted AJs (163). Consistent with the proposed role of Cdc42 in activating both actin polymerization and stabilization, these defects are associated with formation of aberrant filopodia as well as impaired assembly of the acto-myosin apparatus ¹⁶³. The current model suggests a critical role of Cdc42 signaling in the assembly and maturation of AJs via effectors Pak2, Pak4, and N-WASP (Figure 2) ¹⁶³. Cdc42 signaling thus elicits an endothelial barrier protective effect in inflammatory lung injury ³⁰² and also promotes re-annealing of the barrier in inflammatory endothelium through N-WASP-mediated actin polymerization ^{138, 301}. Moreover, Cdc42 can also act as a competitive inhibitor of Rac1 and thereby counteract the barrier-disruptive effect of p67phox signaling and ROS production ^{303, 304}.

In contrast to Cdc42 that promotes AJ assembly, the outcome of Rac1 signaling on endothelial barrier integrity highly depends on intracellular context ³⁰⁵. In some cases, in response to shear stress or the bioactive lipid mediator Sphingosine-1-phosphate (S1P), the activation of Rac1 signaling enhanced endothelial barrier function ^306–309. In other cases, such as stimulation of endothelial cells with TNFα, Platelet-activating factor (PAF), or VEGF, activation of Rac1 caused disruption of the endothelial barrier ^{296, 310–312}. Recent work utilizing a photo-activatable Rac1 probe sheds light on the biological outcome of Rac1 signaling at AJs independent of convergent signaling events ¹⁸². Rac1 counterbalanced RhoA activity at mature AJs and promoted stabilization of VE-cadherin trans-interactions ¹⁸². This mechanism of RhoA inhibition appears to rely on junctional localization and activity of p190RhoGAP ³¹³. Recruitment of p190RhoGAP to AJs is mediated through its direct interaction with p120-catenin, whereas p190RhoGAP activity is regulated by binding to Rac1 as well as Src- and FAK-mediated phosphorylation ¹⁵⁹. Rac1 signaling through the effector Pak1 also suppresses MLCK-dependent phosphorylation of myosin II ²⁹². Hence, activation of Rac1 at mature AJs is a pivotal mechanism for balancing the opposing RhoA signaling and suppressing intracellular tension at AJs ¹⁸².

Rac1 signaling may also cause disassembly of VE-cadherin adhesion and disruption of the endothelial barrier ¹⁸⁸. This is evident by the finding that the pro-inflammatory mediator TNFα leads to a transient and robust increase in Rac1 activity ³¹⁰ through phosphatidylinositol (3,4,5)-trisphosphate – dependent Rac exchanger 1 (P-Rex1) ²⁹⁶. In this case, Rac1 signals through p67phox effector leading to production of ROS, and subsequent activation of Src and VE-cadherin phosphorylation ¹⁷⁴. Another pro-inflammatory mediator PAF also induces Rac1 signaling through T-lymphoma invasion and metastasis-inducing protein 1 (Tiam-1) ³¹¹. PAF-induced activation of Rac1 is associated with profound reorganization of the actin cytoskeleton and vascular leakage ^314–315. Furthermore, VEGF activates Rac1 through Src-dependent phosphorylation of Vav2 and causes Pak-mediated phosphorylation of VE-cadherin at Serine 665 and subsequent VE-cadherin internalization by β-arrestin ^{188, 316}. In conclusion, it appears that Rac1 signaling can have divergent effects on AJs ranging from stabilization to disassembly of VE-cadherin adhesions. These responses exemplify the central importance of intracellular environment, localized signaling, and interaction with specific partners in the net biological outcome of Rac1 signaling.

RhoA signaling pathway

In contrast to Rac1 and Cdc42 that mediate the assembly, stabilization, and maturation of AJs ^{182, 317–319}, RhoA signaling mainly contributes to destabilizing AJs and increasing endothelial permeability ^320–323. RhoA promotes the formation of actin stress fibers and acto-myosin contraction through activation of downstream effectors such as ROCK and mDia (Figure 6). The reorganization of the actin cytoskeleton via the mDia pathway and concurrent assembly of the contractile apparatus through activation of ROCK signaling leads to the generation of intracellular tension at junctions that disassembles AJs ¹⁵¹.

The mDia and ROCK pathways demonstrate a cooperative behavior downstream of RhoA activation ^{300, 324}. mDia promotes the assembly of actin stress fibers, which are re-enforced by ROCK-mediated activation of myosin II ³²⁴. ROCKI and ROCKII are differentially regulated in endothelial cells ^{325, 326}. ROCKI is basally active ³²⁵ and contributes to early responses of endothelial cells to pro-inflammatory mediators such as TNFα and Lipopolysaccharide (LPS) ^{194, 327}. In contrast, activation of ROCKII in response to pro-inflammatory stimuli is required for the long-term effects of LPS and TNFα in disrupting endothelial barrier integrity ^{325, 328}. Evidence also indicates that ROCKII maintains baseline junctional tension and primes the endothelium for hyperpermeability responses such as during thrombin challenge, independent from subsequent ROCKI-mediated contractile stress-fiber formation ³²⁶. Both ROCKs maintain MLC in a phosphorylated state through interaction with the PI3K/AKT pathway ³²⁹. ROCKs also block PI3K/AKT signaling, and thus limit the activation of Rac1 at AJs ¹⁷³. Protracted RhoA signaling leads to persistent disruption of AJs and promotes sustained endothelial leakage ³³⁰, which may be important in the initiation and progression of chronic inflammatory diseases.

7. Spatial control of RhoGTPases at inter-endothelial junctions

Spatial control of RhoGTPases at AJs

VE-cadherin adhesion modulates the organization of the actin cytoskeleton at AJs through the recruitment of signaling and scaffolding proteins such as upstream regulators and downstream effectors of RhoGTPases ^{304, 331, 332}. Engagement of VE-cadherin at cell-cell contacts initiates spatial activation of Rac1 and Cdc42 signaling ^{302, 304, 333}. Rac1 signaling is induced through the activation of phosphatidylinositol 3-kinases (PI3K) ³³⁴ as well as recruitment of the RhoGEFs Tiam1, Vav2, and Triple functional domain protein (TRIO) to AJs ^{174, 270}. Tiam1 serves as the scaffold for Rac1 at AJs ³³⁵ whereas Vav2, a common GEF for RhoA, Rac1, and Cdc42 ³³⁶ promotes Rac1 GTP loading and hence facilitates activation of Rac1 signaling ^{174, 337}. Some evidence suggests that triple functional domain protein TRIO, a GEF for both RhoA and Rac1, is also recruited to nascent VE-cadherin adhesion where it activates Rac1 signaling and promotes the formation of AJs ³³8. IQGAP1, which stabilizes both Cdc42 and Rac1 in the GTP-bound state and protracts the activity of these GTPases ^{308, 339}, is also recruited to AJs through binding to β-catenin ^{308, 340}. Recent data suggest that IQGAP1 is responsible for Rac1 activity at the sites of AJs and hence is an important regulator of AJ integrity and vascular leakage in acute lung injury ^{341, 342}.

In contrast to Rac1 and Cdc42, RhoA activity is suppressed at endothelial AJs by multiple convergent pathways ^{180, 343}. RhoA activity is finely counterbalanced by Rac1 signaling ^{182, 323}. Rac1-mediated activation of p190RhoGAP, a RhoA specific GAP, as well as phosphorylation of p190RhoGAP by Src and FAK ^{157, 196, 344} play a central role in inhibiting RhoA signaling at endothelial AJs. Whether Cdc42 can also counteract RhoA signaling remains unclear. One tenable mechanism involves Cdc42/MRCK-dependent assembly of myosin-IIB filaments, which can then bind to and suppress activities of Dbl family GEFs containing a DH-PH module at AJs ³⁴⁵. It is an attractive possibility that the interaction between myosin-IIB and the RhoGEFs expressed in endothelial cells (TRIO, GEF-H1, Dbl, LARG, Tiam1 and Vav2) might provide a mechanism for switching small RhoGTPases ‘on’ and ‘off’ at AJs.

Spatial control of RhoGTPases at TJs

In contrast to endothelial AJs, which are characterized by low RhoA activity, TJs are shown to be sites of RhoA activation ^{73, 346}, however there are important differences. RhoA activity is induced by p114GEF at endothelial TJs ³⁴⁷. ZO-1, an adaptor protein of TJs, scaffolds a complex consisting of junction-associated coiled-coil protein (JACOP) and p114GEF to provide spatial activation of RhoA ⁷³. Conversely, both Rac1 and Cdc42 activities are suppressed at TJs. Rich1, a GAP for both Cdc42 and Rac1, is associated with angiomotin, a scaffolding protein of TJs, where it controls cell polarity and endothelial junction integrity through inhibition of Rac1 and Cdc42 ³⁴⁸. High activity of RhoA and low activities of Rac1 and Cdc42 are required for generation of intracellular forces at the level of TJs that are transmitted to VE-cadherin adhesion allowing formation of stable AJs ⁷³. This finely compartmentalized regulation of RhoGTPase signaling in endothelial cells might be critical for the stability of VE-cadherin adhesion. Activation of RhoA at the level of TJs rather than AJs might be beneficial for achievement of a proper balance, magnitude and directionality of mechanical forces across VE-cadherin adhesion, the main gatekeeper of junctional permeability in endothelial cells.

8. Role of RhoGTPases in response of endothelium to mechanical and humoral stimuli

Endothelial cells express at least 17 different RhoGAPs and 20 RhoGEFs at high levels (for review, see ³⁴9). This broad spectrum of upstream regulators of RhoGTPases might be important for spatio-temporal control of intracellular tension at endothelial AJs exposed to pulsatile blood pressure and blood flow ³⁵⁰. Both RhoA and Rac1 contribute to cell responses induced by mechanical forces ^351–355. At least 11 different GEFs including Abr, alsin, ARHGEF10, Bcr, GEF-H1, LARG, p190RhoGEF, PLEKHG1, P-REX2, Solo, and a-PIX mediate endothelial cell adaptation to the cyclic stretch response to pulsatile blood pressure and flow ³⁵⁶. In particular, a GEF for RhoA, Solo, transduces mechanical force at cell–cell adhesion sites ³⁵⁶ whereas Leukemia-associated Rho GEF (LARG) and GEF-H1 are involved in integrin-dependent mechotransduction ¹⁷⁶. Both responses contribute to cell alignment and stress fiber reorientation in the endothelium exposed to cyclic stretch.

Conversely, mechanosensing of laminar shear stress and resultant stabilization of VE-cadherin adhesion occurs through activation of Rac1 at AJs ^{174, 357, 358}. Mechanotransduction emanates at the level of the sensory complex comprised of VE-cadherin/PECAM-1/VEGFR2. PECAM-1 induces activation of Src, which in turn promotes phosphorylation of Vav2 and hence Vav2-mediated activation of Rac1 signaling at AJs ¹⁷⁴. Rac1 functions as a reversible modulator of intracellular tension at mature AJs and induces stabilization of VE-cadherin adhesion without notable reorganization of the actin cytoskeleton ¹⁸².

It is important to note that the vast majority of responses to humoral stimuli involve a broad spectrum of RhoGTPases expressed in endothelial cells. Activation of RhoA signaling in response to pro-inflammatory stimuli is induced by p115RhoGEF ¹⁹⁷, GEF-H1 ³⁵⁹, and TRIO ^{360, 361}. In contrast, secondary messengers such as cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP) elicit a barrier protective effect by inhibiting RhoA signaling. cGMP blocks RhoA signaling through PKG-mediated phosphorylation of RhoA ^{362, 363} whereas cAMP inhibits RhoA activity by both activating p190RhoGAP ¹⁵⁸ and preventing RhoA dissociation from RhoGDI ¹⁸⁰. Hence, modulation of RhoA activity might represent an attractive strategy for preventing or treating vascular leakage in disease states.

9. RhoGTPases as therapeutic targets in vascular inflammation

As discussed above, RhoGTPases are fundamental to the biology of endothelial AJs. They serve as a control point for many signaling pathways, making them ideal targets for ameliorating inflammatory disease. Thus, a possible therapeutic approach for treating vascular inflammation may depend on “rewiring” of signaling pathways to restore AJs quickly by shifting the balance from RhoA towards Rac1 and Cdc42 activities. In the following sections, we provide an overview of agents and describe newer targets preventing or resolving inflammation due to leaky AJs.

Small molecule inhibitors

Rho kinase inhibitors

While a number of targets downstream of RhoA have been identified, Rho kinase, a serine/threonine protein kinase of ~160 kDa (also referred to as ROCK) is the major RhoA downstream effector ³⁶⁴. Two isoforms of ROCK, namely ROCKI (also known as ROCKβ) and ROCKII (ROCKα), are encoded by two independent genes ³⁶⁵. Kinase activities of both ROCKs are autoregulated by the COOH-terminal domain, which folds into the active site and inhibits kinase activity. Despite their structural similarity, the two proteins have distinct functions in endothelial cells. ROCKII but not ROCKI regulates basal tension across AJs and shifts the endothelium towards hyper-permeability ³²⁶. The deletion of either ROCKI or ROCKII genes in mice has no apparent phenotype except for a defective placenta–embryo interaction ^{366, 367}. The above results suggest that ROCK potentially can be used as a drug target.

The small-molecule inhibitors targeting ROCK kinase activity can be grouped into four classes: isoquinolines, 4-aminopyridines, indazoles, and amide and urea derivatives. Fasudil, dimethyl-fasudil (H-1152), and compound 4 belong to the isoquinoline series of Rho kinase inhibitors. Fasudil shows higher potency towards ROCKI (Ki of 330 nM) ³⁶⁸ whereas H-1152 and compound 4 inhibited both ROCKs with Ki values of 1.6 to 23 nM, respectively ^{369, 370}. Fasudil has demonstrated high efficacy in pre-clinical models of pulmonary hypertension, pulmonary fibrosis, and vascular leakage ^371–373. Fasudil is approved in Japan for the treatment of cerebral vasospasm after aneurysm rupture ³⁷⁴, and has been tested in US clinical trials of angina and pulmonary hypertension ³⁷⁵. Y-27632, a member of the 4-aminopyridine series, binds the ATP binding pocket of ROCK and has a Ki of 220–300 nM ³⁷⁶. Like fasudil, Y-27632 has been widely used in animal models of hypoxia-induced pulmonary hypertension, lung injury, and cerebral vasospasm ^{197, 377}. However, Y-27632 has been much less investigated clinically.

Statins

Statins can influence endothelial function by virtue of their direct effect on HMG-CoA reductase, a rate limiting enzyme which generates cholesterol by converting HMG-CoA to mevalonic acid. However, statins can block the synthesis of isoprenoid intermediates such as farnesyl pyrophosphate (FPP) and geranylgeranyl pyrophosphate (GGPP), which are important lipid attachments for post-translational modification of a variety of proteins including attachment of small GTP-binding proteins at the plasma membrane. Statins are shown to prevent thrombin- and LPS-induced loss of endothelial barrier function using in vitro and in vivo models of vascular injury through their “pleiotrophic” effects on RhoA- and Rac1-mediated cytoskeletal arrangements and ROS generation ³⁷⁸. Interestingly, simvastatin reduces pulmonary and systemic inflammatory responses in healthy human patients following LPS inhalation ³⁷⁹ and improves organ function in a small, single-center, randomized study on ALI patients ³⁸⁰. However, a recent large clinical trial failed to demonstrate such a protective effect of statins in ARDS patients ³⁸¹.

Sphingosine-1-phosphate receptor agonists and antagonists

S1P acts as a ligand in an autocrine or paracrine manner for the G protein coupled receptor (GPCR) S1P receptors (S1PR1–5; formerly termed the endothelial differentiation gene [Edg] receptors). In several studies, S1P enhances endothelial barrier function ^382–384 through ligating its high affinity receptor, S1PR1 ³⁸⁵. S1PR1 functions in endothelial cells through interaction with heterotrimeric Gi proteins and downstream activation of Rac1 ^382–384. FTY720 has a barrier enhancing effect both in vitro and in vivo ^386–388. However, FTY720P induces vascular leak in a mouse model through promoting phosphorylation of S1PR1 at several serine residues, which trigger receptor ubiquitination and degradation ^{385, 389}. In contrast, newly developed and modified FTY720, (R)-methoxy-FTY720 ((R)-OMe-FTY), (R)/(S)-fluoro-FTY720 (FTY-F), and β-glucuronide-FTY720 (FTY-G) compounds were shown to display in vitro barrier-enhancing properties ³⁹⁰. Tyrosine phosphorylation of S1PR1 at Y143 in endothelial cells also regulates receptor expression at the cell surface and hence the responsiveness to S1P ³⁸⁴. Thus, newer S1P analogs may be efficacious in repairing endothelial AJ integrity.

In contrast to S1PR1, S1PR3 has a barrier disruptive role ³⁹¹. Lung endothelial cells shed S1PR3 in microparticles following activation with LPS or low-molecular-weight hyaluronan ³⁹². Exposure of normal endothelial cells to S1PR3-containing microparticles significantly reduces AJ integrity, consistent with increased permeability response ³⁹². These changes are attenuated by RNAi-mediated depletion of S1PR3. Intriguingly, elevated S1PR3 plasma concentration has been linked to sepsis and ALI mortality ³⁹¹, indicating S1PR3 antagonists as novel therapeutics targeting AJ integrity ³⁹¹.

Growth factors

Angiopoietin (Ang1 and 2) stabilize developing vessels by ligating the endothelial Tie2 receptor (393). However, both have different effects on mature endothelium. While Ang1 promotes endothelial survival, migration, and barrier formation, Ang2 induces vascular leak by disrupting AJs through activation of actin-myosin induced stress fiber formation ^394–396. Ang2 has emerged as a predictor of patient mortality from ARDS and sepsis, as circulating levels of Ang2 are consistently greater in patients who died of ARDS or sepsis as compared to control groups ^{397– 401}. Ang1 infusion has been thought to be a therapeutic approach to counteract Ang2 disruption of the endothelial barrier ⁴⁰² but its use has been limited due to its side effect of inducing pulmonary hypertension ^{403– 405}.

microRNAs

miRNAs are small (19–23 nucleotides) non-coding RNAs which can suppress or augment cellular signaling in several cell types, including endothelial cells, based on their ability to target mRNA ⁴⁰⁶. miR-27a targets VE-cadherin and induces vascular leak during ischemia and reperfusion injury ⁴⁰⁶, indicating inhibition of miR-27a may be a useful approach for preventing vascular barrier disruption. The expression of mature miR-150-5p but not miR-150-3p is induced during recovery from endothelial injury post LPS challenge ⁴⁰⁷. Loss of miR-150 does not alter AJ organization or barrier function under basal conditions but markedly impairs AJ re-annealing after LPS challenge leading to persistent vascular injury ⁴⁰⁸. miR-150 restores endothelial barrier function post injury by suppressing Ang2 generation through targeting the transcription factor EGR2 ⁴⁰⁹. Conversely, depletion of Ang2 in miR-150 null endothelial cells rescues AJ re-annealing and barrier function ⁴⁰⁹ demonstrating that miR-150 functions by suppressing Ang2 generation.

Cellular therapy

Recent evidence suggests that trafficking and differentiation of non-resident and resident stem cells facilitates the repair of injured vessels ^{309, 410}. Stem cells can be mobilized from the bone-marrow to the damaged tissue where they proliferate and function in the same manner as the original cell type ⁴¹¹. Hematopoietic and non-hematopoietic stem cells are located in the bone marrow and are critical in tissue repair ^{309, 410}. Mesenchymal stem cells have been shown to prevent lung vascular injury by secreting growth factors such as keratinocyte growth factor (KGF) or S1P which may alter Rho-Rac1 signaling ⁴¹². Studies also demonstrated that transplanted bone-marrow derived hematopoietic stem cells are detected in several organs including lungs ⁴¹³. A number of studies have shown the so called endothelial progenitor cells (EPCs) to be pro-angiogenic ⁴¹⁴. Wary et al ⁴¹⁰ further showed that VE-cadherin+/Flk1+ EPCs promote endothelial barrier function and that loss of the barrier is prevented by activation of α4 and α5 integrins in an LPS mouse lung vascular injury model. Additionally, Zhao et al ³⁰⁹ have demonstrated that bone marrow derived MSCs can be directed to AJs where they restore normal endothelial permeability through the generation of S1P and thereby prevent LPS-induced lung injury.

10. Concluding remarks and future directions

Here we have discussed the current view on the organization and dynamics of VE-cadherin adhesion, the main gatekeeper of AJs, in resting and activated (or inflamed) endothelium. Although the list of constituents of the VE-cadherin complex is incomplete, it is apparent that it forms an extraordinary well-organized network of signaling molecules at AJs. VE-cadherin assembles a mechanosensory complex with another adhesion molecule PECAM-1 and receptor tyrosine kinases VEGFR2 and VEGFR3 at AJs enabling sensing and adaptation of endothelial cells to rapid shifts in local perfusion and pressure. The signals sensed by this complex are tuned and then transmitted to integrins at the sites of FAs to elicit coordinated responses to modify barrier properties of the junctions.

Small RhoGTPases at the level of AJs are key molecular switches that play a fundamental role in regulating the plasticity of VE-cadherin adhesion, and hence endothelial permeability. They are essential for signaling endothelial responses to both humoral and mechanical stimuli making. They are therefore potential drug targets in a variety of inflammatory disorders. The endothelium expresses numerous upstream regulators of RhoGTPases that regulate GTPase activation in space and time. These are also drug targets that might be exploited.

Several fundamental questions remain unanswered, such as how PECAM1, VE-cadherin, and VEGFR2 organize themselves to serve as a unified mechanosensor, and whether the mechanosensing function is different in the quiescent endothelium as opposed to the activated endothelium. Perturbation of the mechanosensory complex has consequences in disease pathogenesis. Another question is what dictates the balance between RhoA, Rac1, and Cdc42 activities. It is clear that Rac1 and Cdc42 activities (as opposed to RhoA activity) need to be exquisitely balanced, but how this is achieved remains unknown. Another question is how inflammatory mediators in disease states disrupt the sensing function of AJs and how this leads to short- or long-term disruption of AJs. Hence, better understanding of the formation of the mechanosensory complex in the endothelium and the function of RhoGTPases at AJs will be a critical for development of novel therapeutic targets for treating inflammatory diseases.

VE-cadherin also assembles a complex with signaling molecules comprising kinases (e.g., Src kinases), phosphatases (e.g., VE-PTP), and RhoGTPases, which in turn, provide spatial control of VE-cadherin adhesion under physiological and pathological conditions. An increasing body of evidence suggests that VE-cadherin adhesion undergoes continuous reorganization resulting in on-demand remodeling at AJs. Exchange of VE-cadherin molecules between junctional and cytosolic pools is a constitutive process accounting from the permeable nature of the endothelial barrier. VE-cadherin adhesion is finely regulated by specific intracellular signaling pathways that assemble and disassemble AJs. Depending on the stimuli, these signals can also enhance or weaken the endothelial barrier. Disruption of VE-cadherin adhesion triggers increased endothelial permeability and tissue edema, a central feature of human diseases ranging from cancer to acute inflammatory disorders such as ARDS. Thus, an important concept evolving from these studies is whether a cross-talk mechanism exists between phosphatases such as VE-PTP and myosin phosphatase (PP1), which stabilize AJs.

It is also apparent that the tyrosine kinases Fyn, Src, and FAK differentially regulate AJs depending on the context and their mode of activation. For example, as described above, FAK is involved in both formation and disruption of AJs. FAK also influences RhoGTPases. What determines whether FAK functions one way or the other? It will be important in future studies to understand how the interplay between the underlying extracellular matrix and AJs dictates the function of FAK, Fyn, or Src and how the function of these kinases influences the endothelial barrier. It is likely that further clarification of how mechanical stimuli sensed by the AJ mechanosensor influence endothelial permeability in response to inflammatory mediators will advance our understanding of mechanisms regulating endothelial permeability.

Non-standard Abbreviations and Acronyms

AJ

adherens junction

ARDS

acute respiratory distress syndrome

BBB

blood brain barrier

cAMP

cyclic adenosine monophosphate

CBP

CREB-binding protein

cGMP

cyclic guanosine monophosphate

CNS

central nervous system

CP

capping protein

DAG

diacylglycerol

DEP1

density-enhanced phosphatase-1

ECM

extracellular matrix

eNOS

endothelial nitric oxide synthase

EPC

endothelial progenitor cell

EPLIN

epithelial protein lost in neoplasm

FA

focal adhesion

FAK

focal adhesion kinase

FN

fibronectin

FPP

farnesylpyrophosphate

GAP

GTPase-activating protein

GDI

guanine nucleotide dissociation inhibitors

GDP

guanosine diphosphate

GEF

guanine nucleotide exchange factor

GGPP

geranylgeranylpyrophosphate

GJ

gap junction

GPCR

G protein-coupled receptors

GTP

guanosine triphosphate

HDA

histone deacetylase

iBRB

inner blood retinal barrier

IQGAP

IQRas GTPase-activating protein

IRSp53

insulin receptor substrate p53

JACOP

junction-associated coiled-coil protein

JAM

junctional adhesion molecules

KGF

keratinocyte growth factor

LDL

low density lipoprotein

LIMK

Lin1, Isl-1, and Mec-3 Kinase

LPS

lipopolysaccharide

MLC

myosin light chain

MLCK

myosin light-chain kinase

MLCP

myosin light-chain phosphatase

MRCK

myotonic dystrophy kinase-related Cdc42-binding kinase

MSC

mesenchymal stem cell

NO

nitric oxide

N-WASP

Neural Wiskott-Aldrich syndrome protein

PAF

platelet-activating factor

PAK

p21-activated kinase

PAR6

partitioning-defective polarity protein

PECAM-1

platelet endothelial cell adhesion molecule 1

PI3K

phosphatidylinositol-4,5-bisphosphate 3-kinase

PKA

protein kinase A

PKC

protein kinase C

PLC

phospholipase C

PTM

post-translational modification

PTP

protein tyrosine phosphatase

ROCE

receptor-activated calcium entry

ROCK

Rho-associated coiled-coil forming protein kinase

ROS

reactive oxygen species

S1P

sphingosine-1-phosphate

SHP2

Src homology 2-domain containing tyrosine phosphatase

SOCE

store-operated calcium entry

SPHK1

sphingosine kinase 1

TCF

T-cell factor

TEM

transendothelial migration

TJ

tight junction

TNF

tumor necrosis factor

TRIO

triple functional domain protein

TRP

transient receptor potential

VASP

vasodilator-stimulated phosphoprotein

VE

vascular endothelial

VEGF

vascular endothelial growth factor

VEGFR

vascular endothelial growth factor receptor

WASP

Wiskott-Aldrich syndrome protein

WAVE

WASP-family verprolin-homologous protein

ZO

zonula occludens