Sequence of Endothelial Signaling during Lung Expansion

Abstract

Although high tidal volume ventilation exacerbates lung injury, the mechanisms underlying the inflammatory response are not clear. Here, we exposed isolated lungs to high or low tidal volume ventilation, while perfusing lungs with whole blood, or blood depleted of leukocytes and platelets. Then, we determined signaling responses in freshly isolated lung endothelial cells by means of immunoblotting and immunofluorescence approaches. In depleted blood perfusion, high tidal volume induced modest increases in both P-selectin expression on the endothelial surface, and in endothelial protein tyrosine phosphorylation. Both high tidal volume-induced responses were markedly enhanced in the presence of whole blood perfusion. However, a P-selectin-blocking antibody given together with whole blood perfusion inhibited the responses down to levels corresponding to those for depleted blood perfusion. These findings indicate that the full proinflammatory response occurs in two stages. First, lung distension causes modest endothelial activation. Second, subsequent endothelial-inflammatory cell interactions augment P-selectin expression and tyrosine phosphorylation. We conclude that interactions of circulating inflammatory cells with P-selectin critically determine proinflammatory endothelial activation during high tidal volume ventilation.Keywords: leukocytes; lung; mechanical; P-selectin; phosphorylation Although mechanical ventilators provide essential respiratory support in lung injury, a potential difficulty is that the high tidal volumes delivered by mechanical ventilation may independently exacerbate lung injury (1). Since high tidal volume (HV) causes excessive expansion of pulmonary alveoli, and since stretching cultured endothelial cells activates kinases (2), it is proposed that alveolar overexpansion stretches endothelial cells, causing inflammatory responses. However, no direct data in situ support this view.Because endothelial cells are critical for initiating lung inflammation, it is important to understand the endothelial signaling sequence activated by HV ventilation. An important but unanswered question is whether lung expansion is the predominant factor that induces proinflammatory endothelial signaling, or whether secondary inputs are required. The stretch hypothesis is derived largely from findings in cultured cells (2). Hence, the possibility exists that even under HV conditions, lung endothelial cells in situ may not stretch sufficiently to account entirely for the induced inflammatory consequences.To test this hypothesis, we exposed lungs to HV ventilation. Our aim was to determine the extent to which alveolar stretch, (Received in original form April 11, 2005 and in final form July 19, 2005) This study was supported by grant HL54157 to S.B. from the National Institutes of Health.Correspondence and requests for reprints should be addressed to Sunita Bhattacharya, M.D., St. Luke's-Roosevelt Hospital Center, AJA #510, 1000 10th Avenue, New York, NY 10019. E-mail: sb80@columbia.edu as opposed to inflammato...

Connexin 43 mediates spread of Ca2+ -dependent proinflammatory responses in lung capillaries

“…Increased endothelial P-selectin expression and an increase in microvascular permeability are potentially proinflammatory events, since they may underlie, respectively, leukocyte recruitment (6,22) and lung water increase (23). Our findings indicate that such proinflammatory events are regulated by Cx43-determined mechanisms.…”

Platelets Enhance Endothelial Adhesiveness in High Tidal Volume Ventilation

“…These studies indicate that despite the lack of overt lung injury, as indicated by the absence of microvascular hyperpermeability, FLEC showed proinflammatory responses, including increased tyrosine phosphorylation, and expression of P-selectin (19,20). Importantly, these findings indicate that HVinduced EC stretch is itself sufficient to activate an adhesive phenotype on EC.…”

“…Using our reported procedures (19,20), we isolated and blood-perfused rat (Sprague-Dawley, Taconic Farms, Germantown, NY) and mouse lungs. Mice were wild type (WT, C57BL/6) or genetically deficient in P-selectin (P-selectin knockout [P sel 2/2 ]) (Jackson laboratories, Bar Harbor, ME).…”

“…In recent reports, we determined the proinflammatory phenotype of freshly recovered lung EC (FLEC) from lungs exposed to HV (19,20). These studies indicate that despite the lack of overt lung injury, as indicated by the absence of microvascular hyperpermeability, FLEC showed proinflammatory responses, including increased tyrosine phosphorylation, and expression of P-selectin (19,20).…”

Phosphoinositide 3-kinase, Src, and Akt modulate acute ventilation-induced vascular permeability increases in mouse lungs

“…Immunoblotting of pathway signal molecules in both isolated lungs and lungs of intact mice was used to measure protein quantities and their phosphorylation state. An intact animal preparation was used because the presence of whole blood and leukocytes in the lung increased endothelial phosphorylation of intracellular proteins by more than 10-fold and better represents in vivo effects of high pressure ventilation without additional insults such as surgical manipulation of the lung (73). Acute changes in vascular permeability were evaluated using the capillary filtration coefficients (K f ) in isolated perfused lungs and using bronchoalveolar lavage (BAL) fluid albumin concentrations in intact mice.…”