This book gives a better understanding of the pulmonary vasculature by providing a comprehensive review of its structure, function and pathophysiology

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 Ca2+ 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.

Pulmonary function is dependent upon the precise regulation of alveolar surfactant. Alterations in pulmonary surfactant concentrations or function impair ventilation and cause tissue injury. Identification of the molecular pathways that sense and regulate endogenous alveolar surfactant concentrations, coupled with the ability to pharmacologically modulate them both positively and negatively, would be a major therapeutic advance for patients with acute and chronic lung diseases caused by disruption of surfactant homeostasis. The orphan adhesion GPCR GPR116 (also known as Adgrf5) is a critical regulator of alveolar surfactant concentrations. Here, we show that human and mouse GPR116 control surfactant secretion and reuptake in alveolar type II (AT2) cells by regulating guanine nucleotide-binding domain α q and 11 (Gq/11) signaling. Synthetic peptides derived from the ectodomain of GPR116 activated Gq/11-dependent inositol phosphate conversion, calcium mobilization, and cortical F-actin stabilization to inhibit surfactant secretion. AT2 cell-specific deletion of Gnaq and Gna11 phenocopied the accumulation of surfactant observed in Gpr116-/- mice. These data provide proof of concept that GPR116 is a plausible therapeutic target to modulate endogenous alveolar surfactant pools to treat pulmonary diseases associated with surfactant dysfunction.

Pulmonary blood vessels act as a well-regulated barrier to the flux of fluid and solutes between the lumen and the air space. Perturbation of the barrier function results in excessive fluid leak into the interstitium and alveoli, and impairs gas exchange. Recent studies provide deeper insight into the precise control mechanisms involved in the regulation of the barrier. This chapter will highlight these mechanisms and discuss the current understanding on the fluid and solute transport pathways across the vascular endothelial layer. In addition, the chapter summarizes the contributions of extra-endothelial structures such as pericytes and glycocalyx in regulating fluid flux across pulmonary vessels. The chapter concludes with an analysis on the impact of pulmonary endothelial heterogeneity and experimental models on current interpretations of barrier function and regulatory mechanisms.

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 Ca 2+ . 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.

Diarrhea is one of the most common adverse side effects observed in ∼7% of individuals consuming Food and Drug Administration (FDA)-approved drugs. The mechanism of how these drugs alter fluid secretion in the gut and induce diarrhea is not clearly understood. Several drugs are either substrates or inhibitors of multidrug resistance protein 4 (MRP4), such as the anti-colon cancer drug irinotecan and an anti-retroviral used to treat HIV infection, 3'-azido-3'-deoxythymidine (AZT). These drugs activate cystic fibrosis transmembrane conductance regulator (CFTR)-mediated fluid secretion by inhibiting MRP4-mediated cAMP efflux. Binding of drugs to MRP4 augments the formation of MRP4-CFTR-containing macromolecular complexes that is mediated via scaffolding protein PDZK1. Importantly, HIV patients on AZT treatment demonstrate augmented MRP4-CFTR complex formation in the colon, which defines a novel paradigm of drug-induced diarrhea.

Increased microvessel permeability is a major characteristic of lung inflammation. We showed inhibiting connexin 43 (Cx43)‐containing gap junctions blunts permeability increases in microvessels due to an inflammatory insult. However, it is unclear whether Cx43 levels directly influence the lung endothelial barrier. Toward this, we cultured rat lung microvascular endothelial cells (RLMVEC) on transwell inserts and determined permeability via flux of FITC dextran 40 kd (FDx40) across the endothelial monolayer. In vehicle‐treated monolayers, FDx40 fluorescence in the bottom chamber was 92±2 gray levels (mean±SEM; n=3; measurements in duplicate). Treating RLMVEC with LPS (100 ng/ml) for 4 hours increased FDx40 permeability to 99±3 gray levels. To determine responses to reductions in Cx43 levels, we treated RLMVEC monolayers with Cx43 shRNA lentiviral vector for 24 hours. FDx40 permeability in Cx43 shRNA‐treated monolayers was 13% lower than control shRNA‐treated cells. Further, LPS‐treatment of Cx43 shRNA‐treated monolayers induced a more than 27% decrease in permeability compared to control (P<0.05). To determine responses to elevated levels of Cx43, we transfected RLMVEC monolayers with Cx43‐cDNA. FDx40 permeability in Cx43 cDNA‐treated monolayers was 76% higher than control cDNA‐treated monolayers. Further, LPS‐treatment of Cx43 cDNA‐treated monolayers induced a more than 45% increase in permeability compared to control. Together, these data show for the first time that reduction and increase in Cx43 levels,respectively blunts and augments endothelial monolayer permeability. Thus we interpret, endothelial Cx43 levels directly influence endothelial barrier leak.Grant Funding Source: HL75503

The isolated blood-perfused lung preparation is widely used to visualize and define signaling in single microvessels. By coupling this preparation with real time imaging, it becomes feasible to determine permeability changes in individual pulmonary microvessels. Herein we describe steps to isolate rat lungs and perfuse them with autologous blood. Then, we outline steps to infuse fluorophores or agents via a microcatheter into a small lung region. Using these procedures described, we determined permeability increases in rat lung microvessels in response to infusions of bacterial lipopolysaccharide. The data revealed that lipopolysaccharide increased fluid leak across both venular and capillary microvessel segments. Thus, this method makes it possible to compare permeability responses among vascular segments and thus, define any heterogeneity in the response. While commonly used methods to define lung permeability require postprocessing of lung tissue samples, the use of real time imaging obviates this requirement as evident from the present method. Thus, the isolated lung preparation combined with real time imaging offers several advantages over traditional methods to determine lung microvascular permeability, yet is a straightforward method to develop and implement.

Chemotactic migration of fibroblasts toward growth factors relies on their capacity to sense minute extracellular gradients and respond to spatially confined receptor-mediated signals. Currently, mechanisms underlying the gradient sensing of fibroblasts remain poorly understood. Using single-particle tracking methodology, we determined that a lysophosphatidic acid (LPA) gradient induces a spatiotemporally restricted decrease in the mobility of LPA receptor 2 (LPA(2)) on chemotactic fibroblasts. The onset of decreased LPA(2) mobility correlates to the spatial recruitment and coupling to LPA(2)-interacting proteins that anchor the complex to the cytoskeleton. These localized PDZ motif-mediated macromolecular complexes of LPA(2) trigger a Ca2+ puff gradient that governs gradient sensing and directional migration in response to LPA. Disruption of the PDZ motif-mediated assembly of the macromolecular complex of LPA(2) disorganizes the gradient of Ca2+ puffs, disrupts gradient sensing, and reduces the directional migration of fibroblasts toward LPA. Our findings illustrate that the asymmetric macromolecular complex formation of chemoattractant receptors mediates gradient sensing and provides a new mechanistic basis for models to describe gradient sensing of fibroblasts.

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''

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.

Patients with acute lung injury are administered high concentrations of oxygen during mechanical ventilation, and while both hyperoxia and mechanical ventilation are necessary, each can independently cause additional injury. However, the precise mechanisms that lead to injury are not well understood. We hypothesized that alveolar epithelial cells may be more susceptible to injury caused by mechanical ventilation because hyperoxia causes cells to be stiffer due to increased filamentous actin (f‐actin) formation via the GTPase RhoA and its effecter Rho kinase (ROCK). We examined cytoskeletal structures in cultured murine lung alveolar epithelial cells (MLE‐12) under normoxic and hyperoxic (48 h) conditions. We also measured cell elasticity (E) using an atomic force microscope in the indenter mode. Hyperoxia caused increased f‐actin stress fibers and bundle formation, an increase in g‐ and f‐actin, an increase in nuclear area and a decrease in nuclear height, and cells became stiffer (higher E). Treatment with an inhibitor (Y‐27632) of ROCK significantly decreased E and prevented the cytoskeletal changes, while it did not influence the nuclear height and area. Pre‐exposure of cells to hyperoxia promoted detachment when cells were subsequently stretched cyclically, but the ROCK inhibitor prevented this effect. Hyperoxia caused thickening of vinculin focal adhesion plaques, and inhibition of ROCK reduced the formation of distinct focal adhesion plaques. Phosphorylation of focal adhesion kinase was significantly reduced by both hyperoxia and treatment with Y‐27632. Hyperoxia caused increased cell stiffness and promoted cell detachment during stretch. These effects were ameliorated by inhibition of ROCK.

Sphingosine‐1‐Phosphate (S1P) plays a major role in lung vascular pathophysiology. While S1P receptor subtypes that mediate the signaling in lung vessels are being defined, the expression of the receptors in these vessels remains unclear. Toward this, isolated blood‐perfused rat lung were pump‐perfused at 14 ml/min with autologous blood. The pulmonary artery, left atrial, and airway pressures were maintained at 10, 3, and 5 cmH2O, respectively. Through a microcatheter inserted via the left atrial cannula, we cleared blood cells from a small lung region and determined expression of S1P receptor 1–4 in microvessels by indirect in situ immunofluorescence. In two lungs for each receptor, we captured images of surface microvessels using a confocal microscope and quantified fluorescence intensity along the wall of the vessels. S1P4 immunofluorescence in venules and capillaries was 76.7±3.7 (n=11) and 27.7±1.8 (n=36), respectively. Immunofluorescence of S1P1, S1P2, and S1P3 in venules was 33.9±3.4 (n=15), 27.0±5.1 (n=10), and 31±2.9 (n=9), respectively, and in capillaries was 15.4±0.7 (n=50),11.8±0.8 (n=50) and 11.4±1 (n=43), respectively. These data revealed that S1P4 immunofluorescence was higher compared to other subtypes (p< 0.001). Thus, we show for the first time that in lung venules and capillaries (1) S1P receptors 1–4 are expressed, and (2) S1P4 may be the predominant receptor subtype. NIH HL75503

Endotoxemia, a major feature of sepsis, is a common cause of acute lung injury and initiates rapid accumulation of leukocytes in the lung vasculature. Endothelial mechanisms that underlie this accumulation remain unclear, as current experimental models of endotoxemia are less suitable for targeted activation of the endothelium. Toward elucidating this, we used the isolated blood-perfused rat lung preparation. With a microcatheter inserted through a left atrial cannula, we cleared blood cells from a small lung region and then infused lipopolysaccharide (LPS) into microvessels. After a Ringer's wash to remove residual LPS, we infused fluorescently-labeled autologous leukocytes and imaged their transit through the treated microvessels. Image analysis revealed that leukocytes infused 90min after LPS treatment were retained more in treated venules and capillaries than untreated vessels. Further, pretreatment with either the intercellular adhesion molecule-1 (ICAM-1) mAb or polymyxin-B blunted LPS-induced leukocyte retention in both microvessel segments. In addition, retention of leukocytes treated ex vivo with LPS in LPS-treated microvessels was higher compared to retention of untreated leukocytes. In situ immunofluorescence experiments revealed that LPS significantly increased microvessel ICAM-1 expression at 90min post treatment. Polymyxin pretreatment inhibited this increase. Taken together, the data suggest that LPS increased leukocyte retention in both venules and capillaries and this response was mediated by the increased expression of endothelial ICAM-1. Thus, endothelial mechanisms may themselves play a major role in LPS-induced leukocyte retention in lung microvessels. Blunting the endothelial responses may mitigate endotoxin-induced morbidity.

In sepsis, ICAM‐1 expression is augmented in endothelial cells of lung microvessels. However, the role of endothelial cytosolic Ca2+ in the induction of ICAM‐1 expression is not known. Toward this, we blood‐perfused isolated rat lungs. We introduced a microcatheter through the left atrial cannula and then infused LPS (100μg/ml) into microvessels for 30 min. After 60 min, we determined ICAM‐1 expression by indirect in situ immunofluorescence. We recorded confocal images of surface microvessels using a LSM‐710 imaging system and quantified fluorescence intensity along the wall of microvessels. Intensity of ICAM‐1 expression was higher in LPS‐treated microvessels (venule 132.2±9.2; capillary 56.9±4.3, p< 0.001, n=3) compared to saline‐treated controls (12.6±1.8; 7.0±0.6 mean±SE, n=3). To establish the role of Ca2+, we infused Xestospongin‐C (25 μM), an inhibitor of inositol‐1,4,5‐trisphosphate (IP3) receptor on the endoplasmic reticulum (ER), into microvessels 10 min prior to LPS infusion. Xestospongin markedly reduced LPS‐induced lCAM‐1 expression in both microvessels (28.3±1.7; 8.2±0.4, n=2), indicating a role for IP3 in the process. We interpret from these data that LPS induction of ICAM‐1 expression in microvessels depends on IP3‐mediated ER Ca2+ release. Thus, we show for the first time that endothelial Ca2+ increase may be important in LPS‐induced responses in lung microvessels. (NIH HL75503)

Endothelial Cx43‐containing gap junctions play a major role in the expansion of inflammatory responses in lung. However, it is unknown whether Cx43 expression is modulated during an inflammatory response. To establish this in an LPS‐induced inflammatory model, we instilled LPS (2mg/kg) intratracheally in rats and allowed the animals to recover for either 1, 5, or 14 days. At the end of each recovery period, lungs of the animals were isolated and blood‐perfused. Via a microcatheter introduced through the left atrial cannula, we established blood cell‐free conditions in microvessels. In these microvessels, we determined Cx43 expression by indirect immunofluorescence using a confocal imaging system. Fluorescence intensity quantified along the wall of microvessels revealed that endothelial Cx43 expression declined at day 1 post‐LPS treatment (venule 46.2±6.6; capillaries 10.9±1.0, mean±SE, n=3, p<0.001) compared to untreated controls (83.9±8.2; 43.4±3.6), and was lowest at day 5 (22.2±1.9; 4.5±0.5, p<0.001). However, the expression returned to baseline levels at post‐treatment day 14 (97.5±8.1; 54.9±4.9). Thus, the data indicate a steady post‐insult decline in Cx43 expression with time, and a recovery to pre‐insult levels over a longer period. We interpret that mechanisms that initiate recovery from an inflammatory insult may downregulate Cx43 expression in lung microvessels. (NIH HL75503)

Both hyperoxia and mechanical ventilation can independently cause lung injury. In combination, these insults produce accelerated and severe lung injury. We recently reported that pre-exposure to hyperoxia for 12 hours, followed by ventilation with large tidal volumes, induced significant lung injury and epithelial cell apoptosis compared with either stimulus alone. We also reported that such injury and apoptosis are inhibited by antioxidant treatment. In this study, we hypothesized that apoptosis signal-regulating kinase-1 (ASK-1), a redox-sensitive, mitogen-activated protein kinase kinase kinase, plays a role in lung injury and apoptosis in this model. To determine the role of ASK-1 in lung injury, the release of inflammatory mediators and apoptosis, attributable to 12 hours of hyperoxia, were followed by large tidal volume mechanical ventilation with hyperoxia. Wild-type and ASK-1 knockout mice were subjected to hyperoxia (Fi(O(2)) = 0.9) for 12 hours before 4 hours of large tidal mechanical ventilation (tidal volume = 25 μl/g) with hyperoxia, and were compared with nonventilated control mice. Lung injury, apoptosis, and cytokine release were measured. The deletion of ASK-1 significantly inhibited lung injury and apoptosis, but did not affect the release of inflammatory mediators, compared with the wild-type mice. ASK-1 is an important regulator of lung injury and apoptosis in this model. Further study is needed to determine the mechanism of lung injury and apoptosis by ASK-1 and its downstream mediators in the lung.

Acid aspiration, a common cause of acute lung injury, leads to alveolar edema. Increase in lung vascular permeability underlies this pathology. To define mechanisms, isolated rat lungs were perfused with autologous blood. Hydrochloric acid and rhodamine-dextran 70 kDa (RDx70) were coinstilled into an alveolus by micropuncture. RDx70 fluorescence was used to establish the spatial distribution of acid. Subsequently, FITC-dextran 20 kDa (FDx20) was infused into microvessels for 60 min followed by a 10-min HEPES-buffered saline wash. During the infusion, FITC fluorescence changes were recorded to quantify the ratio of peak to postwash fluorescence. The ratio, termed normalized fluorescence, was low for acid compared with buffer instillation both in microvessels abutting acid-treated alveoli and those located more than 700 μm away. In contrast, the normalized fluorescence was similar to buffer controls when a higher molecular weight tracer (FITC-dextran 70 kDa) was infused instead of FDx20, suggesting that normalized FDx20 fluorescence faithfully represented microvascular permeability. Inhibiting endothelial connexin43 (Cx43) gap junction communication with Gap27 blunted the acid-induced reduction in normalized fluorescence, although scrambled Gap27 did not have any effect. The blunting was evident not only in microvessels away from the site of injury, but also in those abutting directly injured alveoli. Thus the new fluorescence-based method reveals that acid increases microvascular permeability both at acid-instilled and away sites. Inhibiting endothelial Cx43 blocked the permeability increase even at the direct injury sites. These data indicate for the first time that Cx43-dependent mechanisms mediate acid-induced increases in microvascular permeability. Cx43 may be a therapeutic target in acid injury.