Chapter 8 Endothelial-matrix interactions in the lung

Advances in Molecular and Cell Biology, Vol. 35, pages 237-250 © 2005 Elsevier B.V. All rights reserved. ISBN: 0-444-51834-7

Chapter 8 Endothelial-Matrix Interactions in the Lung Sunita Bhattacharya, M.S., 1'2 Sadiqa Quadri,Ph.D., 1,3 & Jahar Bhattacharya, M.D., Ph.D) '3. Lung Biology Laboratory, l Departments of Pediatrics 2 and Physiology & Cellular Biophysics, 3 College of Physicians & Surgeons, Columbia University, St. Luke's-Roosevelt Hospital Center, New York, NY 10019

CONTENTS: Introduction Integrins

Function and Composition Unique Properties of a v fl 3 in Lung Integrin Ligation and Calcium Signaling Inside-Outside Signaling Focal Adhesions-Structure

Formation Focal Adhesion Kinase Focal Adhesions in Endothelial Barrier Regulation

Agonists Effects Hyperosmotic Effects Focal Adhesions in Blood Vessels

Effects of Hyperventilation Effects of Hyperosmolarity Future Directions

237

238

Introduction

The lung microvascular bed supports not only the gas exchange function of the lung, but it also forms the major site both for lung liquid production and for rapid leukocyte recruitment. These non-gas exchange functions are critical. The liquid production maintains tissue hydration in the lung parenchyma and probably forms the source of airway liquid, while rapid leukocyte recruitment is essential for establishing the lung's innate immune defense. Exacerbation or dysregulation of these processes precipitates some of the most devastating forms of pulmonary disease including the acute lung injury syndrome and pulmonary edema. Endothelial cells are the primary cell type that determines these non-gas exchange functions in lung. Endothelial cells regulate the paracellular traffic of transvascular flux of liquid and inflammatory cells across intercellular junctions. The luminal endothelial membrane expresses leukocyte adhesion receptors that initiate the lung inflammatory processes. For these reasons, an understanding of endothelial mechanisms continues to be of interest and has been the focus of recent reviews on junctional mechanisms and leukocyte recruitment (Ulbrich et aL, 2003; Bazzoni and Dejana, 2004). Here we consider these issues in the context of endothelial-matrix interactions that have received less attention. The matrix role is clearly important since the bulk of the lung microvascular bed comprises vessels that lack smooth muscle and in which endothelial cells lie immediately apposed to the surrounding interstitial matrix. It is long suspected that the endothelial-matrix association in the lung microvascular bed is very well developed. Classical data report that in capillaries lying immediately outside the alveolar septum, in socalled extra-alveolar vessels, the buttressing effect of the matrix prevents capillary collapse in the face of major decreases of vascular pressure (Sobin et aL, 1978; Sadurski et aL, 1994). Increasing implication that the matrix plays a direct role in endothelial regulation of barrier properties and proinflammatory responses now supports the older phenomenological evidence. In this chapter, we consider these matrix-related signaling mechanisms to the extent that they are known to apply to endothelial function in the adult lung. lnteRrins Function and Composition

Cell-matrix adhesion is mediated by integrins. These are a family of transmembrane c~ and 13 heterodimers with extracellular segments that contain matrix binding sites for proteins such as fibronectin, laminin, or collagen (Katsumi et aL, 2004). Intracellularly, they interact with a number of adaptor and signaling molecules and are linked to the actin eytoskeleton. Currently, about 18 different a subunits and 8 13 subunits are identified. Some of the heterodimers in endothelial cells and fundamentals of structure were introduced in Chapter 2. The role of endothelial integrins has been discussed and recently reviewed largely in the context of cell adhesion, angiogenesis, wound healing, and mechanotransduction (Juliano, 2002; Ruegg and Mariotti, 2003; Katsumi et aL, 2004). However, their significance in the quiescent, non-proliferating lung vascular bed has received relatively less attention. Increasing evidence now implicates the integrin av133 in lung endothelial barrier regulation.

239 Unique Properties o f civil3 in Lung

Immunoelectronmicroscopy of lung capillaries indicates that the c~v133 integrin is expressed both on the abluminal and luminal aspects of endothelial cells (Singh et al., 2000). The evidence from immunohistochemistry study and the in situ polymerase chain reaction (in situ PCR) affirms that expressions for txv133 protein, and ccv and f~.3mRNAs are absent in systemic vessels (except liver), but present in lung in both microvascular and large vessel endothelium (Singh et al., 2000). Southern blots on equal amounts of mRNA indicated that lung expression of ccvf33is highest amongst major organs (Singh et al., 2000). These findings indicate that the lung vascular bed is a preferred site of constitutive c~vl33 expression as opposed to systemic beds in which the integfin is probably expressed only during vessel proliferation, as in wound healing or tumor formation. Since the luminal ctvf33integrin of lung capillaries is exposed to blood-borne ligands, it is capable of ligating circulating products that contain t~v133 ligands, such as vitronectin (Preissner, 1991). For example, the SC5b-9 complex that forms as an end-product of complement activation, and the thrombin-anti-thrombin-III complex that forms in clotting, both contain vitronectin (Preissner, 1991). Interactions of the c~vl33 integrin with these ligands could have pathological consequences, since exposure of lung capillaries to complement-activated serum, purified SC5b-9, or multimeric vitronectin, each increases capillary permeability, as quantified by the capillary hydraulic conductivity (Lp) (Ishikawa et al., 1993; Tsukada et al., 1995). Anti-c~v133 antibodies block the Lp increases, thereby implicating the endothelial c~vl33integrin as a barrier deteriorating receptor in lung capillaries. Blood levels of SC5b-9 increase in complement-activated states such as sepsis (Langlois and Gawryl, 1988), raising the possibility that SC5b-9 ligation to ctv133 may contribute to the pathological microvascular effects characteristic of sepsis. Vitronectin may also form the basis of ctv133 ligation by Gram-positive and Gram-negative bacteria that bind vitronectin (Chhatwal et al., 1987). Other inflammatory av[33 ligands include thrombospondin that is secreted by neutrophils and macrophages (Savill et al., 1992), and osteopontin and von Willebrand factor that are excessively secreted by endothelial cells during lung injury (Kasper et al., 1996; Berman et al., 2004). The extent to which these c~v133 ligations promote hyperpermeability in lung inflammatory diseases requires further understanding. Although it is being understood that integrins aggregate following ligation (Miyamoto et aL, 1995; Miyamoto et al., 1996), the physiological significance of integrin aggregation requires further clarification. Aggregation appears to be the critical step that initiates integrinmediated cell signaling. Monomeric vitronectin, a normal plasma constituent that is evidently not pathogenic, ligates but does not aggregate the c~v[33 integrin. Accordingly, multimeric, but not monomeric vitronectin increases Lp (Tsukada et al., 1995). Multivalent ligation of ctv133 by multimeric vitronectin probably aggregates several c~v[33 dimers. The Lp increase attributable to SC5b-9, which contains multimeric vitronectin, is also the consequence of ctv133 aggregation. Confocal images of lung endothelial cells reveal vitronectin- or SC5b-9mediated ccv133aggregation as fluorescent clumps that localize mainly at the cell periphery at the apical, but not the basal surface (Bhattacharya et al., 2001). The distribution of the fluorescent aggregates at the lung EC periphery indicates that c~vl33 aggregation localizes to inter-endothelial junctions where they may regulate endothelial barrier responses.

240 Integrin Ligation and Calcium Signaling

Endothelial hyperpermeability is attributed to CaZ+-dependent endothelial contraction that increases the paracellular flux by widening inter-endothelial junctions. The present relevance is that the ~xv133 ligands, multimeric vitronectin and SC5b-9, as well as crosslinking antibodies that aggregate the integrin, all increase cytosolic Ca2+ (CaZ+cyt)in lung endothelial cells (Bhattacharya et aL, 2000). Interestingly, the Ca2+cyt increase initiates at the cell periphery, the site of c~vl33 aggregation, and then spreads centripetally. In contrast, histamine. 24. . . . induced Ca cyt increases occur more globally and mmate at the cell center (Bhattacharya et al., 2000). External Ca2+-depletion blunts the etv~3-induced Ca2+cyt increase, while thapsigargin inhibits the response completely, indicating that ~v[33 ligation induces Ca2+ release from endosomal stores (Bhattacharya et al., 2000). Although ligation of the av[33 integrin does not directly activate a Ca2+ channel, the induced Ca2+cyt increase activates a hyperpolarizing outward K÷ current (Kawasaki et al., 2004). It is suggested that in lung endothelial cells, this c~v[33-induced hyperpolarization may contribute to sustained CaZ+cyt increases rectuired2+for NO release (Kawasakl' et al., 2004). The Ca cyt response to e~v133ligation is attributable to involvement of the phospholipase C-71 (PLC-7!-inositol (3,4,5) triphosphate (InsP3) pathway. Induced aggregation of the av133 integrin enhances tyrosine phosphorylation of PLC-71, which hydrolyses inositol bisphosphate • • to release InsP3. Llgatlon of InsP3 receptors releases Ca2 + from endosomal stores. The subsequent decrease in barrier properties may occur by mechanisms such as activation of Ca2 + -calmodulin dependent kinases that phosphorylate myosin light chain leading to actin. . . . . myosm dependent endothelial retraction. Activation of Ca2 + -dependent phosphollpase A2 (cPLA2) induces the arachidonate pathway. Several products of this pathway may exert barrier-deteriorating effects. Genistein, a broad-spectrum tyrosine kinase inhibitor, abrogates tyrosine phosphorylation of PLC-Tx, as well the av[33-induced Ca2+cyt increases in cultured endothelial cells and permeability increases in lung capillaries (Tsukada et aL, 1995; Bhattacharya et al., 2000). These findings indicate that aggregation of the endothelial c~vl33 integrin induces a rapid tyrosine phosphorylation-dependent increase of Ca2+cyt that may underlie the integrins inflammatory role in lung blood vessels• Inside-Out Signaling

According to the hypothesis of affinity-modulation of integrin-ligand binding (Shimizu et al., 1990; Damsky and Werb, 1992; Crowe et al., 1994), whieh is also known as the theory of

"inside-out" signaling, cell activation induces binding of specific cytoplasmic proteins to conserved sequences in the cytoplasmic domains of the a and [3 subunits. This increases the extracellular binding affinity of integrins to specific ligands, presumably because of induced spatial or conformational changes in the extracellular integrin domains. Inside-out signaling has been documented for integrins of blood cells, but not for vascular integrins. The theory may be relevant to endothelial c~v[33 integrin, because ligand binding to the platelet integrin cdIb133, a homologue of ccvl33, is affinity modulated (Chela et al., 1994)• Prolonged ctvf33 ligation may activate inside-out signaling; thereby causing time dependent increases of binding affinity and possibly, secondary endothelial responses in barrier regulation or inflammation.

241

Focal Adhesions

- Structure

Formation

On the cytosolic aspect, integrins form associations with multiple proteins to form the socalled focal complexes. A summary of the burgeoning literature on the dynamic properties and constitutive features of these structures is beyond the present scope, but is available in recent reviews (Bershadsky et aL, 2003; Schlaepfer and Mitra, 2004). Briefly, focal complexes are submicron sized structures that stabilize cells against the interstitial matrix. In cells exposed to mechanical stress, focal complexes mature into structures known as focal adhesions that may be several microns in size. The growth of focal complexes is attributable to integrin ligation and aggregation that recruits focal adhesion kinase (FAK) to the cytoplasmic tail of the integrin 13 subunit. Subsequently, FAK phosphorylates paxillin and a cascade of tyrosine phosphorylations occur on vinculin, talin, ezrin, et-actinin and cortactin. In lung endothelial cells, ctv133 ligation causes tyrosine phosphorylation on FAK, talin, ezrin, paxillin and cortactin, and the proteins co-localize with the clustered integrin (Bhattacharya et al., 1995; Guan, 1997). Tyrosine phosphorylation of FAK and cytoskeletal proteins may be causally related, and the phoshorylations may promote actin stabilization and tension transmission from actin cytoskeleton to plasma membrane. Focal Adhesion Kinase

Specific phosphorylated tyrosine residues in FAK are thought to be critical for signal relay by mediating complex formation between FAK and other signaling molecules. Y397 is the major FAK autophosphorylation site and phosphorylation at this residue creates a high affinity binding site for the SH2 domains of pp60c-src and pp59c-fyn (Schaller et al., 1994), and phosphatidylinositol-3-kinase (Chen et al., 1996). FAK is also phosphorylated in vitro by Src at tyrosine residues 407, 576, 577, 861, and 925 (Schlaepfer et al., 1994; Calalb et al., 1995; Calalb et al., 1996). Phosphorylation at Y576 and Y577 in the activation loop enhances FAK kinase activity (Calalb et al., 1995) and Y925 is a binding site for the SH2 domain protein GRB-2 (Schlaepfer et al., 1994). Phosphorylation of FAK at Y397 upon cell adhesion allows FAK to associate with Src, which triggers downstream signaling events such as phosphorylation of mitogen activated kinase (MAPK) (Schlaepfer et al., 1998). FAK also binds the tyrosine kinase She, which is tyrosine phosphorylated in endothelial cells exposed to ~v[33 ligands (Schlaepfer et al., 1999). Subsequent signaling events are significant for MAPK activation, since She recruits the adaptor protein Grb2, which binds nucleotide exchange factor, SOS. This allows conversion of Ras-GDP to Ras-GTP, hence activation of MAPK precursors and finally, MAPK. A potentially barrier-relevant consequence is that MAPK may phosphorylate and activate cPLA2. Barrier effects may then follow as discussed above for the PLCT-IP3 mechanism. The COOH-terminal domain of FAK is expressed in some tissues as an alternative transcript encoding a 41-43kDa protein called FRNK (for FAK-related non-kinase) (Schaller et al., 1993), and this domain antagonizes FAK signaling by competing for binding to focal ~

242 contacts (Richardson et al., 1996; Taylor et al., 2001). However, FRNK also binds the COOHterminal domain of the FAK-like focal adhesion protein, PYK2 (Heidkamp et aL, 2002), thereby introducing a certain amount of non-specificity in the interpretation of FRNK-induced inhibitory effects. FAK localization to focal adhesions is mediated primarily by the COOHterminal focal adhesion targeting (FAT, residues 840-1052) domain (Hildebrand et al., 1993). The non-catalytic NH2-terminal domain of FAK shares homology with the FERM domain of the ERM family of proteins including ezrin, radixin and moesin, which interact with the cortical cytoskeleton (Chishti et aL, 1998; Girault et al., 1998; Girault et aL, 1999). The proposed direct association of the FAK NH2-terminal domain with cytoplasmic domains ofintegrins (Schaller et aL, 1995) remains unconfirmed in intact cells in which the FAK motif responsible for this interaction has not been identified. Increasing evidence suggests that the FAK NH2-terminal domain may be functionally distinct from the kinase and FAT domains (Sieg et aL, 2000; Vial et al., 2000; Chen et al., 2001; Poullet et aL, 2001), although the function remains unknown. The intracellular targeting characteristics of the FAK NH2-terminal domain are not well understood. In HEK 293 and epithelial MDCK cells, the FAK NH2-terminal domain localizes to nuclei and cell-cell junctions with the tight junction integral membrane protein, occludin of (Stewart et aL, 2002), suggesting a possible role for FAK in occludin-induced cell adhesion. FAK also associates with activated growth factor receptors through its NH2 -terminal domain and the FAK-Src complex is important in the regulation of growth factor-stimulated cell migration (Sieg et al., 2000). Activation of the FAK-Src complex facilitates the association with and/or phosphorylation of multiple signaling proteins (Schlaepfer et al., 1999).

Focal Adhesions in Endothelial Barrier Regulation

Agonists Effects

In lung endothelial cells, focal adhesions typically form near intercellular junctions, suggesting a role for these structures in endothelial barrier regulation (Ayalon and Geiger, 1997; Yuan, 2000; Lee et aL, 2004). Studies in cultured endothelial cells indicate that permeability agonists such as thrombin, hydrogen peroxide, and vascular endothelial growth factor induce intercellular gaps that may account for the barrier deteriorative effects of these agents (Lum et aL, 1993; Garcia et aL, 1995; Abedi and Zachary, 1997; Schaphorst et aL, 1997; Vepa et al., 1999; Carbajal et al., 2000; Alexander et al., 2001). Several of these agents have been shown to activate FAK and induce focal adhesion formation in an actin-dependent manner, since cytochalasin, an inhibitor of actin polymerization, prevents the FAK activation (Abedi and Zachary, 1997; Vepa et al., 1999), as also the induced increase in endothelial permeability (Phillips et al., 1989; Haselton et al., 1996; Waters, 1996). These findings give rise to the notion that loss of endothelial barrier function results from retraction of the endothelial plasma membrane at intercellular junctions. Hence, focal adhesions formed near the cell periphery may act as rivets that stabilize the cell membrane on the matrix, thereby counteracting the actin-myosin-induced membrane retraction that leads to increases of endothelial permeability.

243

Support for this notion comes from several recent studies on the role of FAK in barrier regulation in cultured lung endothelial cells. Taking advantage of the fact that thrombin not only causes endothelial barrier deterioration, but that it also forms focal adhesion complexes, the permeability responses was investigated in human pulmonary artery endothelial cells transfected with an antisense oligonucleotide for FAK (Mehta et al., 2002). The resultant reduction in endothelial FAK expression increased both the extent as well as the duration of the thrombin-induced hyperpermeability. Thus, loss of FAK not only augmented barrier deterioration, but it also delayed barrier recovery in these endothelial monolayers. Several reports identify actin filament formation as the mechanism responsible for FAK translocation to focal adhesions (Abedi and Zachary, 1997; Vepa et al., 1999; Bang et al., 2000; Gerli et aL, 2000; Li et al., 2002; Shikata et al., 2003). Consistent with this view, barrier deteriorating effect of FAK depletion could be blocked by pre-treating endothelial cells with the inhibitor of actin polymerization, latrinculin A (Mehta et al., 2002). Using a different receptor-mediated approach, cultured pulmonary artery endothelial cells were exposed to the platelet secretion product, sphingosine-1-phosphate (S 1P), which induces cell signaling by ligating Gict-linked Edg (endothelial differentiation gene) receptors (Garcia et al., 2001). Interestingly, S 1P enhanced endothelial barrier properties. Associated responses included a Racl-GTPase- and cortactin-dependent recruitment of the actin regulatory protein, cofilin and assembly of cortical actin filaments (Garcia et al., 2001 ; Dudek et aL, 2004). From the standpoint of focal adhesion involvement, an important finding was that SIP caused tyrosine phosphorylation of the focal adhesion protein, paxillin and that the platelet product caused a major redistribution of focal adhesion proteins to the cell periphery (Shikata et al., 2003). Although a direct link between focal adhesion proteins and the S1P-selectin induced barrier enhancing response was not established in these studies, the remodeling of junctional sites lends notional support to the proposed role of focal adhesions in endothelial barrier stabilization.

H y p e r o s m o t i c Effects

Since translational displacement of the cell membrane against the matrix appears to be the initiating event for focal adhesion formation, the effect of passive membrane retraction was studied by exposing plated lung microvascular endothelial cells to sucrose-enriched hyperosmolar medium (Quadri et al., 2003). The endothelial cells remodeled focal complexes into large focal adhesions at the cell periphery. Concomitantly, endothelial FAK activity increased and a dense thickening of cortical actin developed through a Racl GTPase-induced mechanism. Endothelial shrinkage should widen endothelial junctions, thereby increasing permeability. Contrary to this expectation, a bi-phasic permeability response was recorded, in which a transient barrier decrease was followed by a prolonged barrier enhancement. None of these effects occurred in the presence urea-induced hyperosmolarity that does not cause cell shrinkage, or in suspended cells, indicating that cell-matrix association is altogether important for establishing protective barrier responses. These studies also revealed that increases in junctional E-cadherin content accompanied the hyperosmolarity-induced endothelial barrier enhancement (Quadri et al., 2003). Suppression of endothelial focal adhesion formation and of FAK activity through expression of a kinase domain deleted FAK mutant completely blocked the E-cadherin response, while

244 markedly blunting the barrier enhancement. These findings provide definitive evidence for a new role for focal adhesions, namely - as regulators of endothelial junctional proteins, hence of barrier properties. This relationship is interesting in light of the apparent reciprocal relationship of focal adhesions and cell-cell junctional integrity in agonists-induced barrier dysfunction in cultured pulmonary endothelium, as shown in Chapter 2, Figure 7 and reported previously (Alexander et aL, 2001). Further understanding is required to clarify the sequence of events occurring between the remodeling of focal adhesions and the increased insertion of E-cadherin in the endothelial membrane.

Focal Adhesions in Blood Vessels

Effects of Hyperventilation Although reports are still relatively few, the understanding of endothelial focal adhesion function in intact blood vessels continues to accrue increasing interest. The technical difficulty of directly accessing endothelial cell signaling responses under intact conditions has been addressed by freshly isolating lung endothelial cells by immunosorting. In a new approach, collagenase treatment was applied at 4°C to block metabolic processes followed by immunosorting to obtain primary endothelial cell isolates from lungs previously exposed to experimental conditions (Bhattacharya et al., 2003). These studies indicated that high volume mechanical ventilation, a procedure that imposes mechanical stretch on lung vessels, induces all features of focal adhesion activation, namely aggregation of av133 integrin, association of FAK with the c~vl33 integrin, and tyrosine phosphorylation of paxillin. The ventilation stimulus increases expression of the leukocyte adhesion receptor, P-selectin on the endothelial surface and augments association of paxillin with P-selectin. As P-selectin is not externally expressed in quiescent endothelial cells, P-selectin expression signifies proinflammatory endothelial activation. Similarly, focal adhesion proteins also associate with expression of the leukocyte adhesion receptor, E-selectin (Yoshida et aL, 1996). The increased association of paxillin with P-selectin in ventilation challenged lungs indicates that focal adhesion proteins play a role in promoting the proinflammatory response in lung vessels. Effects of Hyperosmolarity The focal adhesion responses to hyperosmolar exposure have been replicated in lung capillaries to the extent that infusions of hyperosmolar sucrose enhance lung capillary barrier properties in association with increased E-cadherin deposition at endothelial junctions and enhanced formation of cortical actin filaments (Safdar et aL, 2003). Direct evidence for the presence of endothelial focal adhesions is now available through in situ immunofluorescence of vinculin (Safdar and Bhattacharya, unpublished findings). This immunofiuorescence, though weakly evident under quiescent conditions, increases markedly after hyperosmolar sucrose infusion (Fig. 1), indicating that similar to cultured endothelial cell, hyperosmolarity increases focal adhesion formation in the intact lung capillary.

245

Figure 1. Lung venular capillary with evidence ofvinculin staining. Left: Lung venular capillaries viewed under baseline conditions show sparse vinculin staining. Right: After 15 minutes of infusion of hyperosmolar sucrose vinculin staining is increased. Note that the vinculin fluorescence is patchy along the vascular wall.

Images of the hyperosmolarity-stimulated capillary reveal that vinculin distribution is typically non-uniform, being higher at capillary branch-points than at mid-segment. Confocal microscope optical sections reveal this unique distribution in that the fluorescence is clearly more pronounced at the point of entry of tributaries into trunk capillaries (Fig. 2). Although further functional evidence is required, the branch-point focal adhesion distribution suggests that the increased inflammatory potential of branch-point endothelial cells is determined by the higher density of focal adhesion proteins (Parthasarathi et al., 2002; Ichimura et al., 2003).

246

Figure 2. Optical sections through a lung venular capillary showing vinculin staining. The same capillary was viewed by confocal microscopy at two levels 5 lim apart. A tributary (upper, arrow) entering the trunk capillary shows high vinculin fluorescence around the ostium in the deeper section (lower, arrow).

Future Directions This review summarizes recent research that has led to recognition that endothelial focal adhesions are essential to barrier and inflammatory responses of lung blood vessels. Many conditions that promote lung endothelial barrier deterioration also stimulate endothelial focal adhesion formation. This concomitant juxtaposition of a negative barrier effect with positive enhancement of matrix adhesive properties signifies the barrier protective function of focal adhesion formation. Clearly, endothelial cells employ enlarged focal adhesions to counteract membrane retraction at the junction. Such a strategy counteracts the hyperpermeability stimulus and promotes restoration of the barrier. Evidently, more needs to be learned regarding the interplay of signaling mechanisms between focal adhesion proteins and constitutive junctional proteins that delineate overall permeability responses in lung blood vessels. The intriguing association of focal adhesion proteins in endothelial inflammatory responses, namely in the expressions of P- and E-selectin, also requires further clarification at the level of regulatory protein-protein interactions. Better understanding of these signaling mechanisms may lead to focal adhesion enhancing therapy to counteract pathology resulting from barrier deterioration and inflammatory activation of lung endothelium.

247 ACKNOWLEDGEMENTS

Supported by NIH grants HL36024, HL57556, HL69514, and HL54157.

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