Pulmonary Protein Leak Research Articles

L‐Arginine Prevents Heme‐Induced Acute Lung Injury (ALI) in a Rat Model of Trauma/Hemorrhage and Resuscitation with Transfusion

Resuscitation with long (> 28 days) stored packed red blood cells (PRBC) promotes acute lung injury (ALI) in our rat model of trauma/hemorrhage. Low nitric oxide (NO) production has been implicated in the development of ALI. Arginase can decrease NO production by decreasing L‐arginine availability for endothelial NO synthase (eNOS). We found that arginase, released from red blood cells (RBC) during prolonged storage, contributes to ALI in our rat model of trauma/hemorrhage. Prolonged storage also increases levels of free hemoglobin (Hb). Heme, a breakdown product of Hb, is metabolized by heme oxygenase to form carbon monoxide (CO). We were the first to report that heme‐derived CO inhibits endothelial nitric oxide (NO) synthase and promotes vasoconstriction in a manner that can be prevented by L‐arginine administration. This study tests the hypothesis that the hemolysis product, heme, promotes ALI in our rat model of trauma/hemorrhage and resuscitation with transfusion in a manner that is exacerbated by limited L‐arginine availability. Fresh PRBC were prepared from rat whole blood. Male Lewis rats were implanted with femoral arterial and venous catheters (trauma), subjected to a 30% hemorrhage and resuscitated with crystalloids and fresh PRBC containing heme alone or with L‐arginine or L‐lysine (competitive inhibitor of L‐arginine uptake). ALI was assessed by pulmonary protein leak (Evans blue dye ‐ EBD) and cytokine‐induced neutrophil chemoattractant‐1 (CINC‐1; rat homologue of human interleukin (IL)‐8) accumulation in bronchoalveolar lavage fluid (BALF). Heme alone caused a 5.6‐fold increase in pulmonary protein leak and 3.5‐fold increase in CINC‐1 accumulation. Co‐administration of L‐arginine decreased heme‐induced pulmonary protein leak by 77% and CINC‐1 accumulation by 32.4%. In contrast, L‐lysine, a competitive inhibitor of L‐arginine uptake, increased heme‐induced pulmonary protein leak by 102% and CINC‐1 accumulation by 106.4%. These data show that the Hb breakdown product, heme, promotes ALI in our rat model of trauma/hemorrhage and resuscitation with transfusion. L‐Arginine co‐administration protects against heme‐induced ALI, while blocking L‐arginine transport by L‐lysine exacerbates heme‐induced ALI. These results suggest that the other hemolysis product, heme, also contributes to ALI in a manner that is greatly exacerbated by decreased L‐arginine bioavailability. We conclude that hemolysis products arginase and heme work in concert to promote ALI during trauma/hemorrhage and resuscitation with transfusion.Support or Funding InformationSupported by grants AHA 0865241F (FKJ), NIH HL074966 (WD) and USAMRC DOD W81XWH‐07‐1‐0717 (RAJ, RMS).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Abstract 20531: C-Abl Mediated Tyrosine Phosphorylation of Paxillin is Essential for LPS-Induced Endothelial Barrier Dysfunction and Acute Lung Injury

Paxillin, a multi-domain scaffold-adapter focal adhesion (FA) protein, plays an important role in facilitating protein networking and efficient signaling transduction. Paxillin is phosphorylated at multiple serine/threonine and tyrosine residues; however, the role of tyrosine phosphorylation of paxillin in endothelial barrier dysfunction and the acute respiratory distress syndrome (ARDS) remains unclear. In this study, we used paxillin-specific siRNA and site-specific non-phosphorylatable mutants of paxillin to abrogate the function of paxillin, both in vitro and in vivo, to determine its role in the regulation of lung endothelial permeability and ARDS. In vitro, lipopolysaccharide (LPS) challenge of human lung microvascular endothelial cells (ECs) resulted in paxillin accumulation at focal adhesions, enhanced tyrosine phosphorylation of paxillin at Y31 and Y118, and significant endothelial barrier dysfunction. However no significant changes in Y181 phosphorylation by LPS challenge was observed. Paxillin silencing (siRNA) attenuated LPS-induced endothelial barrier dysfunction and dissociation of VE-cadherin from adherens junctions. LPS-induced paxillin phosphorylation at Y31 and Y118 was mediated by c-Abl tyrosine kinase and not by Src or focal adhesion kinase (FAK) in human lung microvascular ECs. Furthermore, down-regulation of c-Abl (siRNA) significantly reduced LPS-mediated endothelial barrier dysfunction. Transfection of human lung microvascular ECs with paxillin Y31, Y118 and Y31/Y118 mutants mitigated LPS-induced barrier dysfunction and VE-cadherin destabilization at adherens junctions. In vivo, knockdown of paxillin with siRNA in mouse lungs ameliorated LPS-induced pulmonary protein leak and lung inflammation. Together, these results suggest that c-Abl-mediated tyrosine phosphorylation of paxillin at Y31 and Y118 regulates LPS-mediated pulmonary vascular permeability and injury.

Ibuprofen protects ventilator-induced lung injury by downregulating Rho-kinase activity in rats.

Background. Ventilator-induced lung injury-(VILI-) induced endothelial permeability is regulated through the Rho-dependent signaling pathway. Ibuprofen inhibits Rho activation in animal models of spinal-cord injury and Alzheimer's disease. The study aims to investigate ibuprofen effects on high tidal volume associated VILI. Methods. Twenty-eight adult male Sprague-Dawley rats were randomized to receive a ventilation strategy with three different interventions for 2 h: (1) a high-volume zero-positive end-expiratory pressure (PEEP) (HVZP) group; (2) an HVZP + ibuprofen 15 mg/kg group; and (3) an HVZP + ibuprofen 30 mg/kg group. A fourth group without ventilation served as the control group. Rho-kinase activity was determined by ratio of phosphorylated ezrin, radixin, and moesin (p-ERM), substrates of Rho-kinase, to total ERM. VILI was characterized by increased pulmonary protein leak, wet-to-dry weight ratio, cytokines level, and Rho guanine nucleotide exchange factor (GEF-H1), RhoA activity, p-ERM/total ERM, and p-myosin light chain (MLC) protein expression. Results. Ibuprofen pretreatment significantly reduced the HVZP ventilation-induced increase in pulmonary protein leak, wet-to-dry weight ratio, bronchoalveolar lavage fluid interleukin-6 and RANTES levels, and lung GEF-H1, RhoA activity, p-ERM/total ERM, and p-MLC protein expression. Conclusion. Ibuprofen attenuated high tidal volume induced pulmonary endothelial hyperpermeability. This protective effect was associated with a reduced Rho-kinase activity.

Plasma arginase promotes acute lung injury (ALI) in a rat model of trauma/hemorrhage and resuscitation

Red blood cells (RBC) and liver contain arginase that competes for L‐arginine with nitric oxide (NO) synthase. Low NO levels promote neutrophil activation which is a key mediator of ALI. Arginase is released during RBC storage and ischemic liver damage. Massive hemorrhage promotes liver ischemia and is treated by RBC transfusion. This study tests the hypothesis that increased plasma arginase activity contributes to ALI following resuscitation with stored PRBC in a rat model of trauma/hemorrhage. PRBC were prepared from rat whole blood. Male Lewis rats were implanted with femoral arterial and venous catheters (trauma), subjected to a 30% hemorrhage and resuscitated with crystalloids and fresh or stored (28 days) PRBC. Plasma arginase activity (0.2±0.1 vs 3.1±1.1 U/g protein), CINC‐1 (rat homologue of human IL 8) accumulation (1.5±0.5 vs 66.9±22.6pg/min) and pulmonary protein leak (3.4±1.4 vs 115.3±48.7μg/min) were higher in rats transfused with stored PRBC. In contrast, plasma aspartate aminotransferase activity was not different between rats transfused with fresh or stored PRBC. Transfusion with bovine arginase added to fresh PRBC increased plasma arginase activity (3.4±0.5 U/g protein), pulmonary CINC‐1 accumulation (13.9±7.7μg/min) and protein leak (41.1±8.8μg/min). Conversely, the arginase inhibitor, Nω‐hydroxy‐nor‐L‐arginine (100mg/kg IV) during resuscitation with stored PRBC decreased pulmonary CINC‐1 accumulation by 27% and protein leak by 30%. These data suggest that resuscitation with stored PRBC increases plasma arginase activity which contributes to ALI. Supported by grants AHA 0865241F (FKJ) and NIH HL074966 (WD).

Mild hypothermia reduces ventilator–induced lung injury, irrespective of reducing respiratory rate

In the era of lung-protective mechanical ventilation using limited tidal volumes, higher respiratory rates are applied to maintain adequate minute volume ventilation. However, higher respiratory rates may contribute to ventilator-induced lung injury (VILI). Induced hypothermia reduces carbon dioxide production and might allow for lower respiratory rates during mechanical ventilation. We hypothesized that hypothermia protects from VILI and investigated whether reducing respiratory rates enhance lung protection in an invivo model of VILI. During 4 h of mechanical ventilation, VILI was induced by tidal volumes of 18 mL/kg in rats, with respiratory rates set at 15 or 10 breaths/min in combination with hypothermia (32°C) or normothermia (37°C). Hypothermia was induced by external cooling. A physiologic model was established. VILI was characterized by increased pulmonary neutrophil influx, protein leak, wet weights, histopathology score, and cytokine levels compared with lung protective mechanical ventilation. Hypothermia decreased neutrophil influx, pulmonary levels, systemic interleukin-6 levels, and histopathology score, and it tended to decrease the pulmonary protein leak. Reducing the respiratory rate in combination with hypothermia did not reduce the parameters of the lung injury. In conclusion, hypothermia protected from lung injury in a physiologic VILI model by reducing inflammation. Decreasing the respiratory rate mildly did not enhance protection.

Effects of macrophage inducible nitric oxide synthase in murine septic lung injury

Inducible nitric oxide synthase (iNOS) contributes importantly to septic pulmonary protein leak in mice with septic acute lung injury (ALI). However, the role of alveolar macrophage (AM) iNOS in septic ALI is not known. Thus we assessed the specific effects of AM iNOS in murine septic ALI through selective AM depletion (via intratracheal instillation of clodronate liposomes) and subsequent AM reconstitution (via intratracheal instillation of donor iNOS+/+ or iNOS-/- AM). Sepsis was induced by cecal ligation and perforation, and ALI was assessed at 4 h: protein leak by the Evans blue (EB) dye method, neutrophil infiltration via myeloperoxidase (MPO) activity, and pulmonary iNOS mRNA expression via RT-PCR. In iNOS+/+ mice, AM depletion attenuated the sepsis-induced increases in pulmonary microvascular protein leak (0.3 +/- 0.1 vs. 1.4 +/- 0.1 microg EB.g lung(-1).min(-1); P < 0.05) and MPO activity (37 +/- 4 vs. 67 +/- 8 U/g lung; P < 0.05) compared with that shown in non-AM-depleted mice. In AM-depleted iNOS+/+ mice, septic pulmonary protein leak was restored by AM reconstitution with iNOS+/+ AM (0.9 +/- 0.3 microg EB.g lung(-1).min(-1)) but not with iNOS-/- donor AM. In iNOS-/- mice, sepsis did not induce pulmonary protein leak or iNOS mRNA expression, despite increased pulmonary MPO activity. However, AM depletion in iNOS-/- mice and subsequent reconstitution with iNOS+/+ donor AM resulted in significant sepsis-induced pulmonary protein leak and iNOS expression. Septic pulmonary MPO levels were similar in all AM-reconstituted groups. Thus septic pulmonary protein leak is absolutely dependent on the presence of functional AM and specifically on iNOS in AM. AM iNOS-dependent pulmonary protein leak was not mediated through changes in pulmonary neutrophil influx.

Role of Inducible Nitric Oxide Synthase in Pulmonary Microvascular Protein Leak in Murine Sepsis

The effects of nitric oxide (NO) from calcium-independent NO synthase (iNOS) on microvascular protein leak in acute lung injury (ALI) are uncertain, possibly because of disparate effects of iNOS-derived NO from different cells. We assessed the contribution of iNOS from inflammatory versus parenchymal cells to pulmonary protein leak in murine cecal ligation and perforation-induced ALI. We studied iNOS+/+, iNOS-/-, and two reciprocally bone marrow-transplanted iNOS chimeric mice groups: + to - (iNOS+/+ donor bone marrow-transplanted into iNOS-/- recipient mice) and - to +. Sepsis-induced ALI was characterized by pulmonary leukocyte infiltration, increased pulmonary iNOS activity, and increased pulmonary microvascular protein leak, as assessed by Evans blue (EB) dye. Despite equal neutrophil infiltration, sepsis-induced EB-protein leak was eliminated in iNOS-/- mice and in - to + iNOS chimeras (parenchymal cell-localized iNOS) but was preserved in + to - chimeric mice (inflammatory cell-localized iNOS). EB-protein leak was also prevented by pretreatment with allopurinol and superoxide dismutase. Microvascular protein leak in sepsis-induced ALI is uniquely dependent on iNOS in inflammatory cells with no obvious contribution of iNOS in pulmonary parenchymal cells. Pulmonary protein leak is also dependent on superoxide, suggesting an effect of peroxynitrite rather than NO itself.