In this study, we demonstrate that Cav-1-/-
is required for increased endothelial permeability in ALI induced by mechanical ventilation. In addition to being the major coat protein of caveolar vesicles, Cav-1 binds and inhibits several components of key cell signaling pathways[20
] (p44/42, Src, eNOS) and is thus an important regulator of fundamental cellular processes.[7
] Several lines of evidence point to a possible involvement of endothelial Cav-1 in the pathological increase in permeability. For example, tyrosine kinases, for which Cav-1 is a substrate, are activated in white blood cells by VILI;[21
] Src is a major activator of caveolar albumin uptake triggered by activated neutrophils bound to ICAM-1,[16
] and Cav-1 expression can be upregulated in response to bacterial endotoxin stimulation.[20
] Here we show that lung distention activates Cav-1, an event important in the generation of lung injury.
In our experiments, Src was phosphorylated within 30 minutes of onset of high tidal volume ventilation which was accompanied by synchronous phosphorylation of Cav-1 at Tyr14. In previous work, it was shown that Cav-1 phosphorylation is a crucial step in endothelial dysfunction induced by oxidants.[8
] To determine the functional significance of Cav-1 activation in vivo, we subjected Cav-1-/-
mice and appropriate controls to VILI. We found that Cav-1-/-
mice were protected from lung permeability increases using two different protocols: (1) by ventilating with 21 mL/Kg and measuring 121
I-BSA uptake in the lung; and (2) by ventilating with 30 cmH 2
O pressure and measuring capillary filtration coefficient. Using these complementary approaches, we found that Cav-1-/-
mouse lungs were less susceptible to pulmonary endothelial hyperpermeability induced by injurious ventilation. It seems therefore evident that Cav-1 expression is required for microvascular endothelial cell injury to occur.
The presence of lung injury by our volume-cycled experimental protocol was documented in histologic sections from mouse lungs stained with hematoxylin-eosin. While mice ventilated with protective settings showed no significant histologic abnormalities, use of the injurious protocol was associated with substantial congestion of pulmonary capillaries, focal intraalveolar hemorrhage, and mononuclear cell infiltration.
To assess the degree of inflammatory response to alveolar over-distention, we measured the levels of the inducible cytokines IL-6 and CXCL1 as well as the BAL neutrophil fraction. Both cytokines were substantially elevated in WT mice ventilated with high tidal volumes; however in Cav-1-/- mice, the levels were significantly lower, indicating a blunted inflammatory response. Contrary to WT, which had a marked BAL neutrophilia, Cav-1-/- mice subjected to injurious ventilation had near-normal BAL neutrophil counts.
The observed attenuation in permeability and innate immune reaction observed in Cav-1-/- is suggestive of an important role of this protein in initiating these fundamental responses. To confirm this result, we re-established Cav-1 expression in Cav-1-/-, which were then exposed to high lung inflation volumes. We observed that rescue of Cav-1 expression restored the hyperpermeability response. This is in line with the initial findings in Cav-1-/- mice and also indicates that these were not due to the known chronic pulmonary alterations of these mice, e.g. increased interstitial matrix deposition, which could make the lung more resistant to distention. The reversal of the phenotype within 48 hours of restoring Cav-1 expression in lung endothelia supports the direct role of Cav-1 as a regulator of immediate permeability responses to acute insults.
We next sought to determine the importance of Cav-1 expression in the permeability response of ECs. To this end, we used cultured pulmonary vascular ECs isolated from WT and Cav-1-/- mice. To increase endothelial permeability in vitro, cells were incubated with thrombin, a serine protease with procoagulant properties, and an extremely potent vasoactive agent known to induce endothelial barrier disruption via ligation of protease-activated receptor-1. Endothelial cells exposed to thrombin contracted resulting in the loosening of interendothelial junctions and underlying focal contacts with the matrix, a conformation which favors leakage of plasma water, solutes, and proteins into the interstitial space. This process can, to some extent, be duplicated in vitro by culturing cells on microporous filters by measuring the rate of labeled albumin tracer flux across this endothelial monolayer. In the presence of thrombin, we observed increased EC permeability only in the presence of Cav-1, indicating that this protein somehow triggers or is required for thrombin-induced signaling events that result in increased monolayer permeability.
In this and other experimental systems, thrombin results in activation of MAP-kinases, most notably p38, p44/42, and c-Jun N-terminal kinase.[22
] We observed time-dependent p44/42 activation in ECs induced by thrombin which was substantially reduced in cells lacking Cav-1, providing evidence that Cav-1 is an important regulator of thrombin signaling and possibly of other vasoactive mediators as well. Putative mechanisms of Cav-1 regulation of stress-induced endothelial pathways include control of Ca++
influx, spatial organization of signaling pathways, and regulation of nitric oxide release. Recently, a role for Cav-1 in the rearrangement processes taking place in endothelial adherence junctions, which are required for the permeability-enhancing effect of thrombin, has also been recently described.[23
Our results are consistent with other models of lung inflammation and fibrosis which favor a role of Cav-1 in the innate immune response.[24
] A notable exception is the study by Hoetzel et al
., which, by employing an injurious ventilation model of lower intensity than ours, found that Cav-1-/-
mice were actually more susceptible to lung injury.[15
] This discrepancy illustrates the importance of experimental design among animal studies, in particular with respect to the dose of the applied stressor. In our experimental system, a relatively high tidal volume was delivered using two different strategies, one being volume-controlled and other via pressure-controlled ventilation, and we observed that Cav-1-/-
mice were less susceptible to lung injury. This approach of ventilating experimental animals with tidal volumes that far exceed what is used in clinical practice is commonly the subject of criticism. However, in patients requiring respiratory support because of lung injury, large areas of the lung are collapsed and not accessible to ventilation, while less affected alveoli receive the bulk of delivered gas volume. Under these circumstances, distention of alveoli to an unknown extent may still occur, even if protective ventilation with low tidal volume is applied.
In summary, we present evidence that Cav-1 is required for endothelial permeability increases in response to insults relevant to acute lung injury, including mechanical stretch and thrombin signaling.