The pulmonary microvasculature plays a critical role in endotoxin-induced acute lung injury. However, the relevant signaling remain unclear. Specifically the role of endothelial Ca2+ in the induction of endotoxin-mediated responses in lung microvessels remains undefined. Toward elucidating this, we used the isolated blood-perfused rat lung preparation. We loaded microvessels with the Ca2+ indicator, Fura 2 AM and then determined Ca2+ responses to infusions of lipopolysaccharide (LPS) into the microvessels. LPS induced a more than two-fold increase in the amplitude of cytosolic Ca2+ oscillations. Inhibiting inositol 1,4,5 trisphosphate receptors on endoplasmic reticulum (ER) Ca2+ stores with Xestospongin C (XeC), blocked the LPS-induced increase in the Ca2+ oscillation amplitude. However, XeC did not affect entry of external Ca2+ via plasma membrane Ca2+ channels in lung microvascular endothelial cells. This suggested that LPS augmented the oscillations via release of Ca2+ from ER stores. In addition, XeC also blocked LPS-mediated activation and nuclear translocation of nuclear factor-kappa B in lung microvessels. Further, inhibiting ER Ca2+ release blunted increases in intercellular adhesion molecule-1 expression and retention of naïve leukocytes in LPS-treated microvessels. Taken together, the data suggest that LPS-mediated Ca2+ release from ER stores underlies nuclear factor-kappa B activation and downstream inflammatory signaling in lung microvessels. Thus, we show for the first time a role for inositol 1,4,5 trisphosphate-mediated ER Ca2+ release in the induction of LPS responses in pulmonary microvascular endothelium. Mechanisms that blunt this signaling may mitigate endotoxin-induced morbidity.

Endothelial barrier restoration reverses microvessel hyperpermeability and facilitates recovery from lung injury. Because inhibiting connexin 43 (Cx43)-dependent interendothelial communication blunts hyperpermeability in single microvessels, we determined whether endothelial Cx43 levels correlate with changes in microvessel permeability during recovery from lung injury. Toward this, bacterial endotoxin was instilled intratracheally into rat lungs, and at different durations postinstillation the lungs were isolated and blood perfused. Microvessel Cx43 expression was quantified by in situ immunofluorescence and microvessel permeability via a fluorescence method. To supplement the immunofluorescence data, protein levels were determined by immunoblots of lung tissue from endotoxin-instilled rats. Immunofluorescence and immunoblot together revealed that both Cx43 expression and microvessel permeability increased above baseline within a few hours after endotoxin instillation but declined progressively over the next few days. On day 5 postendotoxin, microvessel Cx43 declined to negligible levels, resulting in complete absence of intermicrovessel communication determined by photolytic uncaging of Ca2+. However, by day 14, both Cx43 expression and microvessel permeability returned to baseline levels. In contrast to Cx43, expression of microvessel vascular endothelial (VE)-cadherin, a critical determinant of vascular barrier integrity, exhibited an inverse trend by initially declining below baseline and then returning to baseline at a longer duration. Knockdown of vascular Cx43 by tail vein injection of Cx43 shRNA increased VE-cadherin expression, suggesting that reduction in Cx43 levels may modulate VE-cadherin levels in lung microvessels. Together, the data suggest that endotoxin challenge initiates interrelated changes in microvessel Cx43, VE-cadherin, and microvessel permeability, with changes in Cx43 temporally leading the other responses.

Activation of plasma membrane receptors initiates compartmentalized second messenger signaling. Whether this compartmentalization facilitates the preferential intercellular diffusion of specific second messengers is unclear. Toward this, the receptor-mediated agonist, thrombin, was instilled into microvessels in a restricted region of isolated blood-perfused mouse lungs. Subsequently, the thrombin-induced increase in endothelial F-actin was determined using confocal fluorescence microscopy. Increased F-actin was evident in microvessels directly treated with thrombin and in those located in adjoining thrombin-free regions. This increase was abrogated by inhibiting inositol trisphosphate-mediated calcium release with Xestospongin C (XeC). XeC also inhibited the thrombin-induced increase in the amplitude of endothelial cytosolic Ca oscillations. Instillation of thrombin and XeC into adjacent restricted regions increased F-actin in microvessels in the thrombin-treated and adjacent regions but not in those in the XeC-treated region. Thus, inositol trisphosphate, and not calcium, diffused interendothelially to the spatially remote thrombin-free microvessels. Thus, activation of plasma membrane receptors increased the ambit of inflammatory responses via a second messenger different from that used by stimuli that induce cell-wide increases in second messengers. Thrombin however failed to induce the spatially extensive response in microvessels of mice lacking endothelial connexin43, suggesting a role for connexin43 gap junctions. Compartmental second messenger signaling and interendothelial communication define the specific second messenger involved in exacerbating proinflammatory responses to receptor-mediated agonists.

We showed endothelial Cx43 gap junctions mediate permeability in lung microvessels. However, whether Cx43 gap junctions modulate lung microvessel permeability during recovery from an inflammatory insult is unknown. Toward this, we instilled rats with lipopolysaccharide (LPS; 2mg/kg; IT). Rats were allowed to recover for 1, 5 or 14 days. At each recovery period, the animals’ lungs were isolated and perfused with autologous blood. The pulmonary artery, left atrial and airway pressures were maintained at 10, 3 and 5 cmH2O, respectively. Via a left atrial microcatheter, we loaded microvessels with the Ca2+ indicator, fluo4 AM and Ca2+ cage, NP‐EGTA AM. Uncaging in microvessels of untreated control lungs revealed that, fluo4 fluorescence increased by 39±11% (mean±SEM; n=5 vessels) at 80 µm from the uncaging site compared to that at 0 µm. In contrast, at 5 days post‐LPS treatment the fluorescence increased only 6±2% (P<0.05, n=6). At day 14, fluorescence increase was back at control levels. Further, we determined single microvessel permeability via normalized fluorescence of FITC‐dextran (FDx20) infused into microvessels. The permeability was high at day 1 post LPS treatment (1.9±0.1; n=15 vessels) compared to control (3.6±0.2; P<0.05; n=15). In contrast, permeability recovered by day 5 (3±0.1; n=15) and returned to control levels by day 14. Together the data suggest both interendothelial communication and microvessel permeability were continuously modulated during recovery from LPS‐injury. Thus we speculate, changes in Cx43‐dependent interendothelial communication may underlie decrease in microvessel permeability during recovery from LPS‐injury. Grant Funding Source: '' Supported by NIH HL75503''