Despite numerous advances in our understanding of the pathophysiology of ALI, specific molecular mechanisms responsible for this clinical syndrome remain poorly understood. In this report, we have characterized a novel and potentially critical role for integrin αvβ5 in the development of an important pathologic hallmark of ALI–increased lung vascular permeability. Furthermore, αvβ5 regulates increased permeability in human, bovine, and murine pulmonary endothelial cells induced by a range of different edemagenic agonists including VEGF, TGF-β, and thrombin. This is a particularly intriguing finding since a complex network of proinflammatory mediators, produced locally in the lung (by fibroblasts, inflammatory, epithelial, and endothelial cells), or derived from extrapulmonary sources, are thought to initiate and amplify the inflammatory response in ALI (31
Our studies have also identified αvβ5 as a specific regulator of induction of actin stress fibers, a well-described contributor to induced increases in pulmonary endothelial permeability. This finding suggests that αvβ5 might regulate changes in vascular permeability by coordinating interactions with the actin cytoskeleton. Integrins are known to be principal components of focal adhesions—multimolecular structures that link the extracellular matrix (ECM) to the intracellular cytoskeleton. Several reports have linked focal adhesions and regulation of the actin cytoskeleton to endothelial barrier function, but the precise molecular basis for this regulation remains unclear (62
). Butler and coworkers recently reported that isolated focal adhesion complexes can initiate actin polymerization of actin monomers de novo
). Moreover, these investigators showed that actin polymerization is dependent on physical clustering of integrins to focal adhesion structures (66
). Additional studies are required to identify the molecular mechanisms by which αvβ5 regulates stress fiber formation.
Physical passage of solutes through the endothelial barrier is thought to occur via paracellular pathways or through receptor-activated transcytosis (67
). The functional relevance, relative contribution, and molecular determinants of these distinct mechanisms remain incompletely understood, but it has been suggested that direct modification of the actin cytoskeleton in endothelial cells is important for increasing paracellular permeability. One frequently cited model describes paracellular gap formation as a consequence of imbalanced competition between cytoskeletal, adhesive cell–cell and cell–matrix forces (10
). In this model, F-actin polymerizes and bundles into morphologically distinct “stress fibers”. Actomyosin-mediated generation of tension leads to alteration of cell shape and formation of paracellular gaps. Stress fibers have been shown to form in endothelial cells stimulated by several vasoactive mediators (11
), including VEGF, TGF-β, and thrombin (45
). Although our studies did not directly distinguish between paracellular and transcellular pathways, the parallel ability of αvβ5 to regulate both stress fiber formation and transendothelial flux suggests that in our system the paracellular pathway may be the more relevant.
Use of cells derived from proximal pulmonary macrovascular endothelium is a limitation to our studies. Microvascular endothelial cells are thought to be a more anatomically and physiologically relevant model of pulmonary capillary leak and many studies have detailed significant physiologic differences between lung cells from microvascular and macrovascular bed origins (72
). Previously, the αvβ5-specific antibody P1F6 was shown to have no effect on ligand-induced increases in lung capillary hydraulic conductivity (79
). Our studies are different because we have focused on agonist-induced permeability events, rather than on effects of integrin ligand binding alone. In fact, we found no effect of αvβ5 blockade on baseline permeability or on lung permeability in uninjured animals. However, future studies using pulmonary microvascular endothelial cells, pulmonary microvascular endothelial and alveolar epithelial cell co-culture systems (80
), or perhaps capillary split-drop techniques (79
) would be necessary to address the important issue of what role αvβ5 might play in regulating capillary permeability.
The model of VILI we used uses relative tidal volumes substantially larger than any currently used for ventilation of people. Therefore, results of the current study cannot be directly extrapolated to suggest that αvβ5 blockade would diminish increased permeability induced by volutrauma in mechanically ventilated patients. Nonetheless, this model is widely used and likely does reflect the effects of excess stretch on alveolar units. Determination of the direct relevance of our findings to patients with VILI will need to await clinical studies with drugs designed to target this integrin.
Several important unanswered questions remain, including how actin stress fiber formation is regulated by αvβ5 ligation. Our observations that αvβ5 antibody produced identical results to both β5 knockout mice and β5 knockout cells strongly suggest that the antibody exerted its effect by specifically inhibiting αvβ5 function, rather than as a result of other antibody–integrin interactions. VEGF, TGF-β, and thrombin activate different families of receptors (tyrosine kinase, serine-threonine kinase, and G protein coupled, respectively) that initiate distinct proximal signaling pathways. It will be important to determine how these diverse pathways converge on αvβ5, and to identify common signaling intermediates. An example of such a signaling intermediate might be the RhoA small GTPase, which has been shown to be activated downstream to a variety of different agonist pathways (45
), and to be both a critical regulator of actin stress fiber formation (37
) and increased endothelial permeability (32
). Our findings in this report suggest that total cellular RhoA activation is not directly affected by αvβ5 blockade, implying that RhoA activation occurs upstream of αvβ5.
While we have focused on disruption of the pulmonary endothelial cell barrier as the main target of αvβ5 effects, there are alternative targets to consider. In vivo
increases in lung vascular permeability in ALI involve complex interactions between multiple cell types, including leukocytes and epithelial cells, as well as endothelial cells (82
). Since αvβ5 is widely expressed, it is possible that αvβ5-mediated effects on other cell types could contribute to the overall in vivo
role of αvβ5 in regulating pulmonary edema formation. Our in vivo
models were chosen primarily as models of increased lung vascular permeability. Relevance to ALI, and even to their specific clinical correlates may be questioned, for example, with experimental ischemia and reperfusion times (for IR) and brief relative ventilation periods and extreme tidal volume settings (for VILI). The complexities of ALI mandate that other models be tested. Ultimate proof of relevance will only come from clinical studies in patients at risk for or affected by ALI.
Finally, although our results demonstrate an important role for αvβ5 ligation in regulating pulmonary endothelial permeability, they do not identify the relevant in vivo
ligand. Our cell culture studies were performed by seeding cells onto nonspecific collagen substrates in the presence of fetal calf serum, a rich source of vitronectin, and growing them in serum-enriched media over several days. This protocol allowed ample time for vitronectin to bind to the cells and substrate and for the cells to secrete additional ECM proteins. Our αvβ5 blocking antibody, ALULA, specifically recognizes αvβ5 and blocks adhesion to vitronectin in vitro
. However, it is certainly plausible that, in vivo
, other ligands are critical for the functions we have described. While vitronectin knockout mice have been observed to be viable and healthy (83
), and therefore, would be a good model system to determine relevance of vitronectin, these studies might potentially be confounded by effects exerted by the integrin αvβ3, which shares vitronectin as a common ECM protein ligand.
Despite these gaps in our current understanding, the findings reported here have potential clinical relevance. Given the robust regulatory effects of blocking αvβ5 in two quite different in vivo models of increased lung vascular permeability and in the pulmonary endothelial permeability response to multiple biologically relevant agonists, αvβ5 appears to be an attractive therapeutic target for ALI, a substantial cause of morbidity and mortality that is currently largely untreatable.