Kaushik Parthasarathi

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

Acute lung injury (ALI), which is associated with a mortality of 30-40%, is attributable to inflammation that develops rapidly across the lungs vast vascular surface, involving an entire lung or even both lungs. No specific mechanism explains this extensive inflammatory spread, probably because of the lack of approaches for detecting signal conduction in lung capillaries. Here, we addressed this question by applying the photolytic uncaging approach to induce focal increases in Ca2+ levels in targeted endothelial cells of alveolar capillaries. Uncaging caused Ca2+ levels to increase not only in the targeted cell, but also in vascular locations up to 150 microm from the target site, indicating that Ca2+ was conducted from the capillary to adjacent vessels. No such conduction was evident in mouse lungs lacking endothelial connexin 43 (Cx43), or in rat lungs in which we pretreated vessels with peptide inhibitors of Cx43. These findings provide the first direct evidence to our knowledge that interendothelial Ca2+ conduction occurs in the lung capillary bed and that Cx43-containing gap junctions mediate the conduction. A proinflammatory effect was evident in that induction of increases in Ca2+ levels in the capillary activated expression of the leukocyte adherence receptor P-selectin in venules. Further, peptide inhibitors of Cx43 completely blocked thrombin-induced microvascular permeability increases. Together, our findings reveal a novel role for Cx43-mediated gap junctions, namely as conduits for the spread of proinflammatory signals in the lung capillary bed. Gap junctional mechanisms require further consideration in the understanding of ALI.

Capillary recruitment in response to tissue hypoxia and its dependence on red blood cell deformability

The effect of reduced red blood cell (RBC) deformability on microvessel recruitment attendant to a reduction in tissue PO2 was studied in rat cremaster muscle using indicator-dilution techniques. Transit times (TT) of fluorescently labeled RBCs (TTRBC) and plasma (TTPl) between functionally paired arterioles and venules were obtained from their dispersion throughout the microvascular network. Changes in PO2 were effected by superfusing the tissue with Ringer solution deoxygenated to different levels. Arteriolar blood flow (Q) was measured with the two-slit technique, and the vascular volume (V) occupied by RBCs and plasma was computed from the product of Q x TT during bolus infusions of rat and less deformable human RBCs to obtain VRBC and fluorescently labeled albumin to obtain VPl. Measurements of TTRBC and TTPl permitted computation of an average flow-weighted tissue (microvascular) hematocrit (HM) relative to systemic values (HS). During infusions of autologous rat RBCs, Q and total V increased threefold in response to hypoxia, whereas normalized RBC TT (TTRBC/TTPl) and normalized tissue hematocrit (HM/HS) did not show a significant trend, indicating an increase in the number of pathways through which the RBCs can traverse the network because of spatial recruitment of capillaries. In contrast, during infusions of human RBCs, TTRBC/TTPl and HM/HS decreased significantly in response to hypoxia. Although Q exhibited an increase similar to that during rat RBC infusions, VRBC exhibited a smaller increase compared with VPl, suggesting that reduced RBC deformability leads to a redistribution of RBCs through larger-diameter pathways within the network and exclusion of these RBCs from pathways normally recruited during hypoxia. Hence, reduced RBC deformability may adversely affect capillary recruitment and physiological mechanisms that ensure adequate delivery of oxygen to tissue.

A novel signaling mechanism between gas and blood compartments of the lung

Propagation of inflammatory signals from the airspace to the vascular space is pivotal in lung inflammation, but mechanisms of intercompartmental signaling are not understood. To define signaling mechanisms, we microinfused single alveoli of blood-perfused rat lung with TNF-α, and determined in situ cytosolic Ca2+ concentration ([Ca2+]i) by the fura-2 ratio method, cytosolic phospholipase A2 (cPLA2) activation and P-selectin expression by indirect immunofluorescence. Alveolar TNF-α increased [Ca2+]i and activated cPLA2 in alveolar epithelial cells, and increased both endothelial [Ca2+]i and P-selectin expression in adjoining perialveolar capillaries. All responses were blocked by pretreating alveoli with a mAb against TNF receptor 1 (TNFR1). Crosslinking alveolar TNFR1 also increased endothelial [Ca2+]i. However, the endothelial responses to alveolar TNF-α were blocked by alveolar preinjection of the intracellular Ca2+ chelator BAPTA-AM, or the cPLA2 blockers AACOCF3 and MAFP. The gap-junction uncoupler heptanol had no effect. We conclude that TNF-α induces signaling between the alveolar and vascular compartments of the lung. The signaling is attributable to ligation of alveolar TNFR1 followed by receptor-mediated [Ca2+]i increases and cPLA2 activation in alveolar epithelium. These novel mechanisms may be relevant in the alveolar recruitment of leukocytes.

Mechano-oxidative coupling by mitochondria induces proinflammatory responses in lung venular capillaries.

Elevation of lung capillary pressure causes exocytosis of the leukocyte adhesion receptor P-selectin in endothelial cells (ECs), indicating that lung ECs generate a proinflammatory response to pressure-induced stress. To define underlying mechanisms, we followed the EC signaling sequence leading to P-selectin exocytosis through application of real-time, in situ fluorescence microscopy in lung capillaries. Pressure elevation increased the amplitude of cytosolic Ca(2+) oscillations that triggered increases in the amplitude of mitochondrial Ca(2+) oscillations and in reactive oxygen species (ROS) production. Responses to blockers of the Ca(2+) oscillations and of mitochondrial electron transport indicated that the ROS production was Ca(2+) dependent and of mitochondrial origin. A new proinflammatory mechanism was revealed in that pressure-induced exocytosis of P-selectin was inhibited by both antioxidants and mitochondrial inhibitors, indicating that the exocytosis was driven by mitochondrial ROS. In this signaling pathway mitochondria coupled pressure-induced Ca(2+) oscillations to the production of ROS that in turn acted as diffusible messengers to activate P-selectin exocytosis. These findings implicate mitochondrial mechanisms in the lungs proinflammatory response to pressure elevation and identify mitochondrial ROS as critical to P-selectin exocytosis in lung capillary ECs.

Mitochondrial Reactive Oxygen Species Regulate Spatial Profile of Proinflammatory Responses in Lung Venular Capillaries

Cytokine-induced lung expression of the endothelial cell (EC) leukocyte receptor P-selectin initiates leukocyte rolling. To understand the early EC signaling that induces the expression, we conducted real-time digital imaging studies in lung venular capillaries. To compare receptor- vs nonreceptor-mediated effects, we infused capillaries with respectively, TNF-α and arachidonate. At concentrations adjusted to give equipotent increases in the cytosolic Ca2+, both agents increased reactive oxygen species (ROS) production and EC P-selectin expression. Blocking the cytosolic Ca2+ increases abolished ROS production; blocking ROS production abrogated P-selectin expression. TNF-α, but not arachidonate, released Ca2+ from endoplasmic stores and increased mitochondrial Ca2+. Furthermore, Ca2+ depletion abrogated TNF-α responses partially, but arachidonate responses completely. These differences in Ca2+ mobilization by TNF-α and arachidonate were reflected in spatial patterning in the capillary in that the TNF-α effects were localized at branch points, while the arachidonate effects were nonlocalized and extensive. Furthermore, mitochondrial blockers inhibited the TNF-α- but not the arachidonate-induced responses. These findings indicate that the different modes of Ca2+ mobilization determined the spatial patterning of the proinflammatory response in lung capillaries. Responses to TNF-α revealed that EC mitochondria regulate the proinflammatory process by generating ROS that activate P-selectin expression.

IL-13 Regulates the Immune Response to Inhaled Antigens

The large inhibitory effect of IL-13 blockers on the asthma phenotype prompted us to ask whether IL-13 would play a role in regulating the allergic immune response in addition to its documented effects on structural pulmonary cells. Because IL-13 does not interact with murine T or B cells, but with monocytes, macrophages, and dendritic cells (DCs), we examined the role of IL-13 in the activation of pulmonary macrophages and DCs and in the priming of an immune response to a harmless, inhaled Ag. We found that a majority of cells called “alveolar or interstitial macrophages” express CD11c at high levels (CD11chigh) and are a mixture of at least two cell types as follows: 1) cells of a mixed phenotype expressing DC and macrophage markers (CD11c, CD205, and F4/80) but little MHC class II (MHC II); and 2) DC-like cells expressing CD11c, CD205, MHC II, and costimulatory molecules. Endogenous IL-13 was necessary to induce and sustain the increase in MHC II and CD40 expression by pulmonary CD11chigh cells, demonstrated by giving an IL-13 inhibitor as a measure of prevention or reversal to allergen-primed and -challenged mice. Conversely, IL-13 given by inhalation to naive mice increased the expression of MHC II and costimulatory molecules by CD11chigh cells in an IL-4Rα-dependent manner. We found that exogenous IL-13 exaggerated the immune and inflammatory responses to an inhaled, harmless Ag, whereas endogenous IL-13 was necessary for the priming of naive mice with an inhaled, harmless Ag. These data indicate that blockade of IL-13 may have therapeutic potential for controlling the immune response to inhaled Ags.

Paracrine purinergic signaling determines lung endothelial nitric oxide production

Although the vascular bed is a major source of nitric oxide (NO) production, factors regulating the production remain unclear. We considered the role played by paracrine signaling. Determinations by fluorescence microscopy in isolated, blood-perfused rat and mouse lungs revealed that a brief lung expansion enhanced cytosolic Ca(2+) (Ca(2+)cyt) oscillations in alveolar epithelial (AEC) and endothelial (EC) cells, and NO production in EC. Furthermore, as assessed by a novel microlavage assay, alveolar ATP production increased. Intra-alveolar microinfusion of the purinergic receptor antagonist, PPADS, and the nucleotide hydrolyzing enzyme, apyrase, each completely blocked the Ca(2+)cyt and NO responses in EC. Lung expansion induced Ca(2+)cyt oscillations in mice lacking the P2Y1, but not the P2Y2, purinergic receptors, which were located in the perivascular interstitium basolateral to AEC. Prolonged lung expansion instituted by mechanical ventilation at high tidal volume increased EC expression of nitrotyrosine, indicating development of nitrosative stress in lung microvessels. These findings reveal a novel mechanism in which mechanically induced purinergic signaling couples cross-compartmental Ca(2+)cyt oscillations to microvascular NO production.

Preexposure to hyperoxia causes increased lung injury and epithelial apoptosis in mice ventilated with high tidal volumes

Both high tidal volume mechanical ventilation (HV) and hyperoxia (HO) have been implicated in ventilator-induced lung injury. However, patients with acute lung injury are often exposed to HO before the application of mechanical ventilation. The potential priming of the lungs for subsequent injury by exposure to HO has not been extensively studied. We provide evidence that HO (90%) for 12 h followed by HV (25 μl/g) combined with HO for 2 or 4 h (HO-12h+HVHO-2h or -4h) induced severe lung injury in mice. Analysis of lung homogenates showed that lung injury was associated with cleavage of executioner caspases, caspases-3 and -7, and their downstream substrate poly(ADP-ribose) polymerase-1 (PARP-1). No significant lung injury or caspase cleavage was seen with either HO for 16 h or HV for up to 4 h. Ventilation for 4 h with HO (HVHO) did not cause significant lung injury without preexposure to HO. Twelve-hour HO followed by lower tidal volume (6 μl/g) mechanical ventilation failed to produce significant injury or caspase cleavage. We also evaluated the initiator caspases, caspases-8 and -9, to determine whether the death receptor or mitochondrial-mediated pathways were involved. Caspase-9 cleavage was observed in HO-12h+HVHO-2h and -4h as well as HO for 16 h. Caspase-8 activation was observed only in HO-12h+HVHO-4h, indicating the involvement of both pathways. Immunohistochemistry and in vitro stretch studies showed caspase cleavage in alveolar epithelial cells. In conclusion, preexposure to HO followed by HV produced severe lung injury associated with alveolar epithelial cell apoptosis.

Lipopolysaccharide Induces Endoplasmic Store Ca2+-Dependent Inflammatory Responses in Lung Microvessels

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 connexin43 mediates acid-induced increases in pulmonary microvascular permeability

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.