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,2
* Laboratory of Surgical Research, Departments of Anesthesia, Surgery, and
Medicine and Cardiovascular Research Institute, University of California, San Francisco, California, USA
2Correspondence: Department of Anesthesia, Rm. 3C-38, San Francisco General Hospital, 1001 Potrero Ave., San Francisco, CA 94110, USA. E-mail: pittetj{at}anesthesia.ucsf.edu
SPECIFIC AIMS
ISCHEMIA-REPERFUSION (I/R) LUNG injury is a common complication seen after hemorrhagic shock, lung transplantation, or pulmonary embolism. Recent experimental studies have implicated vascular endothelial growth factor (VEGF) as an important factor that increases vascular permeability in the lung. The first objective was to test the hypothesis that VEGF cell signaling mediates the increase in vascular permeability associated with the early phase of I/R lung injury. Because previous studies have shown that the activation of the stress response (SPR) was associated with a decrease in lung endothelial permeability in experimental animal models of I/R lung injury, the second objective was to determine whether SPR activation could attenuate the increase in lung vascular permeability induced by VEGF cell signaling in an experimental model of lung ischemia-reperfusion in the rat. The last objective was to determine whether VEGF-mediated increase in protein permeability, VE/E-cadherin phosphorylation, and actin stress fiber formation in primary cultures of macro- and microvascular lung endothelial cell monolayers was inhibited by the activation of the stress protein response.
PRINCIPAL FINDINGS
1. VEGF cell signaling mediates I/R lung injury in rats
I/R lung injury was characterized by a sixfold increase in lung endothelial permeability in the ischemic compared with control lung. There was also a 55% increase in the amount of water that accumulated in the extravascular spaces of the lungs of rats with I/R injury. The accumulation of pulmonary edema fluid was associated with a progressive decrease in the arterial PO2.
To test the hypothesis that VEGF cell signaling mediates the increase in vascular permeability associated with the early phase of lung I/R injury, rats were pretreated with an adenovirus encoding the extracellular portion of the murine VEGF receptor type 2 fused to sequences encoding murine IgG2
-Fc. These rats showed a sixfold reduction in the quantity of plasma proteins that accumulated in the extravascular spaces of the ischemic lung (Fig. 1
A), as well as a significant decrease in the accumulation of pulmonary edema fluid compared with the rats that were treated with the control adenovirus encoding the GFP protein. There was no difference in the amount of VEGF protein extracted from the lung tissue of ischemic lungs between sham rats and those that underwent I/R lung injury (Fig. 1B
). In contrast, I/R was associated with a sustained phosphorylation of the VEGF receptor II that was not observed in the lungs of sham rats or when rats were pretreated with Ad-Flk1-Fc virus (Fig. 1C
).
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2. SPR activation protects lungs from I/R injury in rats
Rats pretreated for 4 h with geldanamycin or PDTC demonstrated an increase expression of hsp72 in the lungs, an indicator of SPR activation. (Fig. 2
A, B). Furthermore, SPR activation caused a twofold decrease in the amount of proteins that accumulated in the extravascular spaces of the lung after ischemia/reperfusion (Fig. 2C, D
) and a significant decrease in the accumulation of pulmonary edema fluid measured by the gravimetric method and a corresponding attenuation of the decrease in the arterial PO2 caused by I/R lung injury (data not shown). Finally, SPR activation with geldanamycin prevented the phosphorylation of the VEGF receptor II (Figure 2E
).
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3. Effect of SPR activation on VEGF-mediated increase in permeability across lung endothelial cell monolayers
Bovine macrovascular lung endothelial cell monolayers were exposed to VEGF (50 ng/ml) for 1 h and protein permeability was measured with 14C-albumin. In some experiments, cells were stress preconditioned with heat (90 min at 43°C, then recovered overnight at 37°C) or pretreated with geldanamycin (10 ng/ml) for 6–8 h before the exposure to VEGF. VEGF induced a greater than twofold increase in the protein permeability, as well as VE-cadherin phosphorylation and actin stress fiber formation in these cell monolayers. SPR activation prevented VEGF-dependent increase in protein permeability, VE-cadherin phosphorylation, and actin stress fiber formation. Similar results were obtained in microvascular lung endothelial cells.
CONCLUSIONS AND SIGNIFICANCE
Previous studies have reported that the zinc-finger transcription factor EGR-1, HIF-1, and PKCIIßbeta; are important for coordinating the up-regulation of divergent gene families after a hypoxemic and ischemic stress in the lung. However, none of these factors has been shown to have a direct effect on the vascular permeability in the lung. In contrast, there is experimental evidence that VEGF expression is up-regulated after I/R in the lung, suggesting that VEGF, a mediator known to increase vascular permeability, could play an important role in the development of pulmonary edema associated with I/R in the lung. Therefore, the first objective of these studies was to test the hypothesis that VEGF cell signaling would mediate increased permeability of the lung endothelium early after onset of I/R in rats.
To answer this question, we developed an experimental model of I/R lung injury in rats by clamping the hilum of the left lung for 30 min and reperfusing it for 1 to 3 h. Some of the rats were pretreated with the Ad-Flk1-Fc adenovirus that has previously been shown to block VEGF cell signaling in different organs. Our results indicated that pretreatment with this adenovirus prevented the development of pulmonary edema associated with ischemia and reperfusion in rats. These results are the first to demonstrate that VEGF cell signaling is directly implicated in the changes in vascular permeability associated with I/R in the lung. They are also in accordance with our previously published work that demonstrated that the overexpression of the VEGF gene in the lung caused pulmonary edema.
The second objective was to determine whether SPR activation would affect the increase in lung vascular permeability induced by the activation of VEGF cell signaling that is associated with I/R injury. SPR activation resulted in a significant decrease in the accumulation of pulmonary edema and in the phosphorylation of the VEGF receptor type 2 associated with I/R lung injury. Our in vivo data in these studies suggest that SPR activation either prevented the release of biologically active VEGF from cells and/or attenuated VEGF cell signaling in endothelial cells.
To answer this question, we developed an in vitro cell model to determine the effect of VEGF on protein permeability across primary cultures of bovine macrovascular and microvascular lung endothelial cell monolayers. We found that exposure to VEGF caused a significant increase in the protein permeability across these monolayers that was associated with phosphorylation of VE-cadherin and actin stress fiber formation. Prior SPR activation inhibited increase in protein permeability, VE-cadherin phosphorylation, and actin stress fiber formation induced by exposure to VEGF. Overall, these data support the hypothesis that SPR activation inhibits VEGF-mediated cell signaling responsible for the increase in lung vascular permeability.
In summary, these results provide the first in vivo evidence that VEGF cell signaling mediates the development of pulmonary edema caused by lung ischemia and reperfusion in rat. This effect is prevented by the prior activation of the stress protein response, in part by inhibiting the VEGF-mediated actin stress fiber formation and phosphorylation of VE- and E-cadherins; crucial components of the adherens junction protein complex controlling paracellular permeability in lung endothelial cells.
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FOOTNOTES
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.05-4708fje
1 These authors equally contributed to this work. 
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