Vascular Filtration Coefficient Research Articles

Mitochondria Amplify TRPV4‐dependent Calcium Dynamics within the Pulmonary Microvascular Endothelium

Acute respiratory distress syndrome (ARDS) is an inflammatory lung disease common in critically ill patients marked by diffuse alveolar damage and increased pulmonary endothelial barrier permeability. A large body of evidence supports the role of mitochondrial dysfunction in ARDS, which has been shown to mediate endothelial permeability. For example, in cultured rat pulmonary microvascular endothelial cells (PMVECs), the mitochondrial complex I inhibitor rotenone increases endothelial permeability and in isolated perfused lung studies, rotenone increases the pulmonary vascular filtration coefficient. In both cell culture and isolated perfused lungs, the amphipathic quinone coenzyme Q1 (CoQ1) rescues rotenone‐dependent increases in endothelial permeability by bypassing complex I to restore mitochondrial bioenergetics.The transient receptor potential vanilloid protein 4 (TRPV4), a mechanically gated calcium channel, has also been implicated in mediating endothelial permeability by increasing intracellular calcium in response to high pressure ventilation. The phorbol ester 4α‐phorbol‐12,13‐didecanoate (4αPDD) provides a pharmacological means of TRPV4 activation and stimulates endothelial permeability responses in both cell culture and animal models of lung injury. Given that mitochondria actively participate in calcium buffering, we hypothesized that TRPV4‐dependent calcium signals may be compartmentalized by mitochondria. To test this hypothesis, we used confocal microscopy to measure calcium signals in PMVECs in response to TRPV4 activation via 4αPDD (5 μM) after treatment with vehicle, rotenone (0.5 μM), or rotenone in combination with CoQ1 (10 μM), and developed a sensitive, automated technique for intracellular calcium signal transient analysis.Using our novel analysis approach, we found that rotenone significantly decreased the number of calcium signal transients stimulated by 4αPDD over a 15 minute interval (mean ± SEM: 22 ± 4 rotenone vs 96 ± 14 vehicle, p <0.01), and that CoQ1 in combination with rotenone reversed this effect (60 ± 15, p = n.s. vs vehicle). Rotenone also dampened the amplitude of calcium responses to TRPV4, an effect that was partially restored by CoQ1. These findings support the hypothesis that mitochondria regulate TRPV4‐mediated calcium dynamics by amplifying calcium signals, and that mitochondrial dysfunction impairs this phenomenon. This suggests that mitochondrial regulation of endothelial calcium signaling is essential to the maintenance of barrier integrity and provides a possible therapeutic target in ARDS.Support or Funding InformationNIH K25HL136869 and NIH P01 HL066299‐17

Targeting F-Box Protein Fbxo3 Attenuates Lung Injury Induced by Ischemia-Reperfusion in Rats.

Background: Increasing evidence suggests that Fbxo3 signaling has an important impact on the pathophysiology of the inflammatory process. Fbxo3 protein inhibition has reduced cytokine-driven inflammation and improved disease severity in animal model of Pseudomonas-induced lung injury. However, it remains unclear whether inhibition of Fbxo3 protein provides protection in acute lung injury induced by ischemia-reperfusion (I/R). In this study, we investigated the protective effects of BC-1215 administration, a Fbxo3 inhibitor, on acute lung injury induced by I/R in rats.Methods: Lung I/R injury was induced by ischemia (40 min) followed by reperfusion (60 min). The rats were randomly assigned into one of six experimental groups (n = 6 rats/group): the control group, control + BC-1215 (Fbxo3 inhibitor, 0.5 mg/kg) group, I/R group, or I/R + BC-1215 (0.1, 0.25, 0.5 mg/kg) groups. The effects of BC-1215 on human alveolar epithelial cells subjected to hypoxia-reoxygenation (H/R) were also examined.Results: BC-1215 significantly attenuated I/R-induced lung edema, indicated by a reduced vascular filtration coefficient, wet/dry weight ratio, lung injury scores, and protein levels in bronchoalveolar lavage fluid (BALF). Oxidative stress and the level of inflammatory cytokines in BALF were also significantly reduced following administration of BC-1215. Additionally, BC-1215 mitigated I/R-stimulated apoptosis, NF-κB, and mitogen-activated protein kinase activation in the injured lung tissue. BC-1215 increased Fbxl2 protein expression and suppressed Fbxo3 and TNFR associated factor (TRAF)1–6 protein expression. BC-1215 also inhibited IL-8 production and NF-κB activation in vitro in experiments with alveolar epithelial cells exposed to H/R.Conclusions: Our findings demonstrated that Fbxo3 inhibition may represent a novel therapeutic approach for I/R-induced lung injury, with beneficial effects due to destabilizing TRAF proteins.

Analysis of pulmonary vascular injury and repair during Pseudomonas aeruginosa infection-induced pneumonia and acute respiratory distress syndrome.

Herein we describe lung vascular injury and repair using a rodent model of Pseudomonas aeruginosa pneumonia-induced acute respiratory distress syndrome (ARDS) during: 1) the exudative phase (48-hour survivors) and 2) the reparative/fibro-proliferative phase (1-week survivors). Pneumonia was induced by intratracheal instillation of P. aeruginosa strain PA103, and lung morphology and pulmonary vascular function were determined subsequently. Pulmonary vascular function was assessed in mechanically ventilated animals in vivo (air dead space, PaO2, and lung mechanics) and lung permeability was determined in isolated perfused lungs ex vivo (vascular filtration coefficient and extravascular lung water). At 48 hours post infection, histological analyses demonstrated capillary endothelial disruption, diffuse alveolar damage, perivascular cuffs, and neutrophil influx into lung parenchyma. Infected animals displayed clinical hallmarks of ARDS, including increased vascular permeability, increased dead space, impaired gas exchange, and decreased lung compliance. Overall, the animal infection model recapitulated the morphological and functional changes typically observed in lungs from patients during the exudative phase of ARDS. At 1 week post infection, there was lung histological and pulmonary vascular functional evidence of repair when compared with 48 hours post infection; however, some parameters were still impaired when compared with uninfected controls. Importantly, lungs displayed increased fibrosis and cellular hyperplasia reminiscent of lungs from patients during the fibro-proliferative phase of ARDS. Control, sham inoculated animals showed normal lung histology and function. These data represent the first comprehensive assessment of lung pathophysiology during the exudative and reparative/fibro-proliferative phases of P. aeruginosa pneumonia-induced ARDS, and position this pre-clinical model for use in interventional studies aimed at advancing clinical care.

Mitochondrial DNA Damage Initiates Acute Lung Injury and Multi-Organ System Failure Evoked in Rats by Intra-Tracheal Pseudomonas Aeruginosa.

Although studies in rat cultured pulmonary artery endothelial cells, perfused lungs, and intact mice support the concept that oxidative mitochondrial (mt) DNA damage triggers acute lung injury (ALI), it has not yet been determined whether enhanced mtDNA repair forestalls development of ALI and its progression to multiple organ system failure (MOSF). Accordingly, here we examined the effect of a fusion protein construct targeting the DNA glycosylase, Ogg1, to mitochondria in a rat model intra-tracheal Pseudomonas aeruginosa (strain 103; PA103)-induced ALI and MOSF. Relative to controls, animals given PA103 displayed increases in lung vascular filtration coefficient accompanied by transient lung tissue oxidative mtDNA damage and variable changes in mtDNA copy number without evidence of nuclear DNA damage. The approximate 40% of animals surviving 24 h after bacterial administration exhibited multiple organ dysfunction, manifest as increased serum and tissue-specific indices of kidney and liver failure, along with depressed heart rate and blood pressure. While administration of mt-targeted Ogg1 to control animals was innocuous, the active fusion protein, but not a DNA repair-deficient mutant, prevented bacteria-induced increases in lung tissue oxidative mtDNA damage, failed to alter mtDNA copy number, and attenuated lung endothelial barrier degradation. These changes were associated with suppression of liver, kidney, and cardiovascular dysfunction and with decreased 24 h mortality. Collectively, the present findings indicate that oxidative mtDNA damage to lung tissue initiates PA103-induced ALI and MOSF in rats.

Mitochondrial DNA damage associated molecular patterns in ventilator-associated pneumonia: Prevention and reversal by intratracheal DNase I.

Previous studies in isolated perfused rat lungs have revealed that endothelial barrier disruption after intratracheal administration of Pseudomonas aeruginosa (strain 103; PA103) only occurs after accumulation of extracellular mitochondrial DNA (mtDNA) damage-associated molecular patterns (DAMPs) in the perfusate and is suppressed by addition of DNase to the perfusion medium. Herein, we tested the hypothesis that intratracheal DNase-a route of administration readily translatable to patient with ventilator-associated pneumonia (VAP)-also enhances degradation of mtDNA and prevents bacteria-induced lung injury. Intratracheal DNase was administered to isolated rat lungs either before or after intratracheal challenge with PA103 to determine if bacteria-induced mtDNA DAMP-dependent lung injury could be prevented or reversed by enhanced mtDNA degradation. To explore whether this concept is translatable to patients with VAP, consecutive patients suspected of VAP were prospectively enrolled. All patients suspected of VAP received a bronchoalveolar lavage (BAL) with quantitative culture for the diagnosis of VAP. Mitochondrial and nuclear DNAs were measured from the BAL. MtDNA DAMPs (i.e., ND6) were measured from serum at time of suspected diagnosis and at 24 to 48 hours afterward. Intratracheal PA103 caused significantly increased the vascular filtration coefficient (Kf) and perfusate mtDNA DAMPs. In contrast, lungs pretreated or posttreated with intratracheal DNase were protected from increases in Kf and mtDNA DAMPs. Patients with the diagnosis of VAP had significantly higher mtDNA DAMPs in the BAL (248.70 ± 109.7 vs. 43.91 ± 16.61, p < 0.05, respectively) and in the serum at 24 hours (159.60 ± 77.37 vs. 10.43 ± 4.36, p < 0.05; respectively) when compared with patients that did not have VAP. These findings in isolated perfused rat lungs and a cohort of severely injured patients reveal an association between bacterial pneumonia and accumulation of mtDNA DAMPs in the lung and serum. Furthermore, administration of intratracheal DNase I prevented and reversed pulmonary endothelial dysfunction evoked by PA103.

Mitochondrial DNA damage-associated molecular patterns mediate a feed-forward cycle of bacteria-induced vascular injury in perfused rat lungs.

Fragments of the mitochondrial genome released into the systemic circulation after mechanical trauma, termed mitochondrial DNA damage-associated molecular patterns (mtDNA DAMPs), are thought to mediate the systemic inflammatory response syndrome. The close association between circulating mtDNA DAMP levels and outcome in sepsis suggests that bacteria also might be a stimulus for mtDNA DAMP release. To test this hypothesis, we measured mtDNA DAMP abundance in medium perfusing isolated rat lungs challenged with an intratracheal instillation of 5 × 10(7) colony-forming units of Pseudomonas aeruginosa (strain 103; PA103). Intratracheal PA103 caused rapid accumulation of selected 200-bp sequences of the mitochondrial genome in rat lung perfusate accompanied by marked increases in both lung tissue oxidative mtDNA damage and in the vascular filtration coefficient (Kf). Increases in lung tissue mtDNA damage, perfusate mtDNA DAMP abundance, and Kf were blocked by addition to the perfusion medium of a fusion protein targeting the DNA repair enzyme Ogg1 to mitochondria. Intra-arterial injection of mtDNA DAMPs prepared from rat liver mimicked the effect of PA103 on both Kf and lung mtDNA integrity. Effects of mtDNA and PA103 on Kf were also attenuated by an oligodeoxynucleotide inhibitor of Toll-like receptor 9 (TLR-9) by mitochondria-targeted Ogg1 and by addition of DNase1 to the perfusion medium. Collectively, these findings are consistent with a model wherein PA103 causes oxidative mtDNA damage leading to a feed-forward cycle of mtDNA DAMP formation and TLR-9-dependent mtDNA damage that culminates in acute lung injury.

Alveolar macrophages mediate release of mtDNA damage‐associated molecular patterns in Pseudomonas aeruginosa‐challenged rat lungs (145.6)

Oxidative mitochondrial DNA (mtDNA) damage and release of mtDNA damage‐associated molecular patterns (mtDNA DAMPs) may contribute to bacteria‐induced lung injury. The cellular mechanism by which bacteria trigger mtDNA DAMP formation is unknown. We tested whether P. aeruginosa causes alveolar macrophage‐dependent release of mtDNA DAMPs and attendant degradation of alveolar‐capillary barrier function. To deplete alveolar macrophages, rats were given an intratracheal injection of liposome‐encapsulated clodronate or liposomes alone 24 hours prior to experiments. Lungs isolated, mechanically ventilated, and buffer‐perfused were challenged with intratracheal instillation of P. aeruginosa (Strain 103). The vascular filtration coefficient was measured 15 min after challenge. Amplification of 200 bp sequences via RT‐PCR was used to measure accumulation of mtDNA DAMPs or bacterial DNA in perfusion medium. Rat lungs treated with vehicle had increased permeability, whereas those treated with clodronate showed no increases in the vascular filtration coefficient or accumulation of mtDNA DAMPs in perfusate 15 minutes after PA103 challenge. These observations suggest that alveolar macrophages are critical for P. aeruginosa‐induced formation and release of mtDNA DAMPs into the pulmonary circulation and attendant changes in lung endothelial permeability.Grant Funding Source: Supported by NIH

Lung Endothelial Ca2+ and Permeability Response to Platelet-Activating Factor Is Mediated by Acid Sphingomyelinase and Transient Receptor Potential Classical 6

Platelet-activating factor (PAF) increases lung vascular permeability within minutes by activation of acid sphingomyelinase (ASM) and a subsequent nitric oxide (NO)-inhibitable and Ca(2+)-dependent loss in barrier function. To elucidate the molecular mechanisms underlying this response. In isolated perfused rat and mouse lungs, endothelial Ca(2+) concentration ([Ca(2+)](i)) was quantified by real-time fluorescence imaging, and caveolae of endothelial cells were isolated and probed for Ca(2+) entry channels. Regulation of transient receptor potential classical (TRPC) 6-mediated currents in lung endothelial cells was assessed by patch clamp technique. PAF increased lung weight gain and endothelial [Ca(2+)](i). This response was abrogated by inhibitors of ASM or in ASM-deficient mice, and replicated by lung perfusion with exogenous ASM or C2-ceramide. PAF increased the caveolar abundance of TRPC6 channels, which was similarly blocked by ASM inhibition. PAF-induced increases in lung endothelial [Ca(2+)](i), vascular filtration coefficient, and edema formation were attenuated by the TRPC inhibitor SKF96365 and in TRPC6-deficient mice, whereas direct activation of TRPC6 replicated the [Ca(2+)](i) and edema response to PAF. The exogenous NO donor PapaNONOate or the cyclic guanosine 3',5'-monophosphate analog 8Br-cGMP blocked the endothelial [Ca(2+)](i) and permeability response to PAF, in that they directly blocked TRPC6 channels without interfering with their PAF-induced recruitment to caveolae. The present findings outline a new signaling cascade in the induction of PAF-induced lung edema, in that stimulation of ASM causes recruitment of TRPC6 channels to caveolae, thus allowing for Ca(2+) influx and subsequent increases in endothelial permeability that are amplified in the absence of endothelial NO synthesis.

Mitochondrial DNA integrity may be a determinant of endothelial barrier properties in oxidant-challenged rat lungs

In cultured pulmonary artery endothelial cells and other cell types, overexpression of mt-targeted DNA repair enzymes protects against oxidant-induced mitochondrial DNA (mtDNA) damage and cell death. Whether mtDNA integrity governs functional properties of the endothelium in the intact pulmonary circulation is unknown. Accordingly, the present study used isolated, buffer-perfused rat lungs to determine whether fusion proteins targeting 8-oxoguanine DNA glycosylase 1 (Ogg1) or endonuclease III (Endo III) to mitochondria attenuated mtDNA damage and vascular barrier dysfunction evoked by glucose oxidase (GOX)-generated hydrogen peroxide. We found that both Endo III and Ogg1 fusion proteins accumulated in lung cell mitochondria within 30 min of addition to the perfusion medium. Both constructs prevented GOX-induced increases in the vascular filtration coefficient. Although GOX-induced nuclear DNA damage could not be detected, quantitative Southern blot analysis revealed substantial GOX-induced oxidative mtDNA damage that was prevented by pretreatment with both fusion proteins. The Ogg1 construct also reversed preexisting GOX-induced vascular barrier dysfunction and oxidative mtDNA damage. Collectively, these findings support the ideas that mtDNA is a sentinel molecule governing lung vascular barrier responses to oxidant stress in the intact lung and that the mtDNA repair pathway could be a target for pharmacological intervention in oxidant lung injury.

Role of cyclooxygenase metabolites in the increase in pulmonary vascular permeability caused by mechanically activated white blood cells

To study the role of cyclooxygenase metabolites in changes in the pulmonary vasculature induced by mechanically activated white blood cells (WBCs), the effects of activated and inactive WBCs, and of a cyclooxygenase inhibitor, were studied in isolated perfused lungs from Sprague-Dawley rats. WBCs were activated by gentle agitation in a glass container for 10s. Baseline measurements were made, and then activated or inactive WBCs were added to the perfusate. Perfusion was stopped for 90 minutes, and then started again. The effects of the cyclooxygenase inhibitor meclofenamate on the pulmonary vascular filtration coefficient and on pulmonary vascular resistance were also measured. In the group that received activated WBCs, the pulmonary vascular filtration coefficient and the pulmonary vascular resistance were about 2.5 times and 3.3 times higher, respectively, than those in the group that received inactive WBCs. However, this apparent increase in the filtration coefficient caused by activated WBCs was partly blocked by meclofenamate. Histological examination indicated that meclofenamate did not prevent the adhesion of WBCs to the pulmonary vascular endothelium. These date indicate that WBCs that have been made to adhere to vessel walls can induce pulmonary vascular injury via cyclooxygenase products.

Effect of adhesiveness of white blood cells on pulmonary vascular permeability and resistance

We studied pulmonary vascular injury evoked by mechanically activated white blood cells (WBCs). In isolated perfused rat lungs, changes in pulmonary vascular resistance were measured, and a gravimetric method was used to measure the pulmonary vascular filtration coefficient, which was taken as an index of pulmonary vascular injury. WBCs were activated by gentle agitation in a glass container for 10 sec. The filtration coefficient was measured under baseline conditions, and then inactive or activated WBCs were added to the perfusate. The perfusion was stopped for 90 minutes and then the lungs were reperfused. The expression of CD18 on the surface of WBCs was measured by flow cytometry. The filtration coefficient and the pulmonary vascular resistance in lungs exposed to activated WBCs were about 2.5 times and 3.3 times higher, respectively, than those in the lungs exposed to inactive WBCs. These results indicate increases in resistance and in pulmonary vascular permeability. The results of flow cytometry indicated that the mechanical agitation increased the expression of CD18 on the surface of WBCs. In conclusion, WBCs in which adhesiveness has been increased can induce pulmonary vascular injury and can increase pulmonary vascular resistance.

Cyclooxygenase metabolites possibly produced by endothelial cells mediate the lung injury caused by mechanically stimulated leukocytes.

To determine whether mechanically stimulated leukocytes increase pulmonary vascular permeability and resistance and, if so, whether cyclooxygenase metabolites mediate the increase, we assessed the effects of stimulated and unstimulated leukocytes, and of a cyclooxygenase inhibitor on pulmonary vascular permeability and resistance in isolated perfused lungs from Sprague-Dawley rats. Leukocytes were stimulated by gentle agitation in a glass container for 10 seconds. After baseline measurements were made, stimulated or unstimulated leukocytes were added to the perfusate. The effects of the cyclooxygenase inhibitor, meclofenamate, on the pulmonary vascular filtration coefficient and pulmonary vascular resistance were measured. In the rats that received stimulated leukocytes, the pulmonary vascular filtration coefficient and the vascular resistance were about 2.5 times and 3.3 times higher, respectively, than those in the rats that received unstimulated leukocytes. These increases were completely and partly blocked by meclofenamate. Histological examination indicated that meclofenamate did not prevent the adhesion of leukocytes to the pulmonary vascular endothelium. These findings suggest that mechanically stimulated leukocytes increase pulmonary vascular permeability and that cyclooxygenase metabolites produced by endothelial cells may injure the cells.

Effect of urodilatin on platelet-activating factor-induced bronchoconstriction, vasoconstriction and edema formation in isolated rat lung.

In the isolated perfused rat lung, perfusion with platelet-activating factor causes bronchoconstriction, vasoconstriction and edema formation. The bronchoconstriction and vasoconstriction are largely mediated by thromboxane, whereas the edema formation is due to enhanced vascular permeability unrelated to eicosanoids. Since natriuretic peptides are known to relax smooth muscle and were suggested to attenuate enhanced vascular permeability, we investigated the effect of urodilatin on the PAF-induced alterations in lung function. Pretreatment with urodilatin (0.25 microM or 0.75 microM) reduced the PAF-induced increase in airway and vascular resistance by approximately 50%. Urodilatin pretreatment, however, was completely ineffective against the PAF-induced increase in weight gain and in vascular permeability, as assessed by the vascular filtration coefficient. Furthermore, urodilatin failed to affect the release of thromboxane into the perfusate in PAF-exposed lungs. Thus, urodilatin relaxes airway and vascular smooth muscle, but fails to reduce edema formation in PAF-perfused rat lungs.

Inhaled nitric oxide prevents the increase in pulmonary vascular permeability caused by hydrogen peroxide

Given the interest in using inhaled nitric oxide (NO.) to treat acute lung injury and the importance of oxygen radicals in its pathogenesis, we studied the effects, in buffer-perfused isolated rabbit lungs, of inhaled NO. (24 ppm) on the injury caused by generating hydrogen peroxide with glucose and glucose oxidase (GOX). Experiments were performed at a constant pulmonary arterial pressure. GOX substantially augmented vascular permeability, as demonstrated by an increase in the lung-to-perfusate 125I-labeled albumin ratio, lavage-to-perfusate 125I-albumin ratio, wet-to-dry lung weight ratio, and pulmonary vascular filtration coefficient. Lungs treated with inhaled NO. before perfusion with GOX had lung-to-perfusate and lavage-to-perfusate 125I-albumin ratios that were not significantly different from control values and intermediate between the control and GOX groups. Inhaled NO. also prevented the increase in wet-to-dry lung weight ratio and pulmonary vascular filtration coefficient caused by GOX.. Thus inhaled NO. substantially reduced in the isolated lung the increase in pulmonary vascular permeability produced by the intravascular generation of hydrogen peroxide.

シェアストレスによる白血球の接着能こう進と透過性こう進 再伸展性肺水腫のラット摘出肺モデルでの検討

We investigated pulmonary vascular injury caused by mechanically activated white blood cells (WBCs) . We assessed the pulmonary vascular injury by measuring pulmonary vascular filtration coefficient using the gravimetric method and changes in the pulmonary vascular resistance, in the isolated perfused rat lungs. We activated WBCs by gently shaking in a glass container for 10 sec. After measuring the filtration coefficient under baseline conditions, we added inactive or activated WBCs into the pulmonary artery and then stopped the perfusion for 90 minutes and then reperfused. The filtration coefficient and the perfusate flow in activated WBC group was changed to about 2.5 times and 1/3 as that in the inactive WBC group, respectively. These results indicate the increase in the pulmonary vascular permeability and resistance. We conclude that WBCs which are increased in adhesiveness induce pulmonary vascular injury and increase in pulmonary vascular resistance. These results suggest that WBC activation by mechanical share stress may be partly involved in the pathogenesis of reexpansion pulmonary edema.