Mitochondrial-targeted DNA repair enzyme 8-oxoguanine DNA glycosylase 1 protects against ventilator-induced lung injury in intact mice.

This study tested the hypothesis that oxidative mitochondrial-targeted DNA (mtDNA) damage triggered ventilator-induced lung injury (VILI). Control mice and mice infused with a fusion protein targeting the DNA repair enzyme, 8-oxoguanine-DNA glycosylase 1 (OGG1) to mitochondria were mechanically ventilated with a range of peak inflation pressures (PIP) for specified durations. In minimal VILI (1 h at 40 cmH(2)O PIP), lung total extravascular albumin space increased 2.8-fold even though neither lung wet/dry (W/D) weight ratios nor bronchoalveolar lavage (BAL) macrophage inflammatory protein (MIP)-2 or IL-6 failed to differ from nonventilated or low PIP controls. This increase in albumin space was attenuated by OGG1. Moderately severe VILI (2 h at 40 cmH(2)O PIP) produced a 25-fold increase in total extravascular albumin space, a 60% increase in W/D weight ratio and marked increases in BAL MIP-2 and IL-6, accompanied by oxidative mitochondrial DNA damage, as well as decreases in the total tissue glutathione (GSH) and GSH/GSSH ratio compared with nonventilated lungs. All of these injury indices were attenuated in OGG1-treated mice. At the highest level of VILI (2 h at 50 cmH(2)O PIP), OGG1 failed to protect against massive lung edema and BAL cytokines or against depletion of the tissue GSH pool. Interestingly, whereas untreated mice died before completing the 2-h protocol, OGG1-treated mice lived for the duration of observation. Thus mitochondrially targeted OGG1 prevented VILI over a range of ventilation times and pressures and enhanced survival in the most severely injured group. These findings support the concept that oxidative mtDNA damage caused by high PIP triggers induction of acute lung inflammation and injury.

Augmentation of the Esophageal Sphincter Using LINX.

Gastroesophageal reflux disease (GERD) is the retrograde flow of gastric contents into the distal esophagus and may be treated medically or surgically. Magnetic sphincter augmentation surgery using LINX has recently demonstrated comparable results to Nissen fundoplication. We aimed to evaluate preoperative patient risk factors that were associated with LINX removal rates or postoperative EGD with dilation rates (POEGDD). This is a single institution retrospective review of patients undergoing LINX between 2015 and 2021. One hundred and twelve patients were reviewed, 106 included within the study; those excluded had prior foregut surgery or device fracture. Variables including age, sex, BMI, size of device, DeMeester score, manometry, GERD Health-Related Quality of Life (GERD HRQL) questionnaires, POEGDD, and removal rates were recorded. Comparing removal and dilation status, the chi-square or Fisher's exact test and the Mann-Whitney U test were used to analyze categorical and continuous variables, respectively. A P < .05 was considered to be statistically significant. Eleven LINX devices were removed (10%); of these, 9 (81%) underwent POEGDD (P = .0023). There was no difference in DeMeester scores, size of device, or BMI in patients requiring LINX removal compared to those not removed or POEGDD rates. Patients who required LINX removal had higher GERD HRQL scores both preoperatively (median 34 vs 28) and postoperatively at all visits compared to those patients who did not undergo removal (P = .032). Manometry and DeMeester scores were not associated with LINX removal suggesting a less invasive GERD HRQL questionnaire may be a better predictor of patients who will succeed with LINX surgery.

TRPV4 channels augment macrophage activation and ventilator-induced lung injury.

We have previously implicated transient receptor potential vanilloid 4 (TRPV4) channels and alveolar macrophages in initiating the permeability increase in response to high peak inflation pressure (PIP) ventilation. Alveolar macrophages were harvested from TRPV4(-/-) and TRPV4(+/+) mice and instilled in the lungs of mice of the opposite genotype. Filtration coefficients (K(f)) measured in isolated perfused lungs after ventilation with successive 30-min periods of 9, 25, and 35 cmH(2)O PIP did not significantly increase in lungs from TRPV4(-/-) mice but increased >2.2-fold in TRPV4(+/+) lungs, TRPV4(+/+) lungs instilled with TRPV4(-/-) macrophages, and TRPV4(-/-) lungs instilled with TRPV4(+/+) macrophages after ventilation with 35 cmH(2)O PIP. Activation of TRPV4 with 4-alpha-phorbol didecanoate (4alphaPDD) significantly increased intracellular calcium, superoxide, and nitric oxide production in TRPV4(+/+) macrophages but not TRPV4(-/-) macrophages. Cross-sectional areas increased nearly 3-fold in TRPV4(+/+) macrophages compared with TRPV4(-/-) macrophages after 4alphaPDD. Immunohistochemistry staining of lung tissue for nitrotyrosine revealed increased amounts in high PIP ventilated TRPV4(+/+) lungs compared with low PIP ventilated TRPV4(+/+) or high PIP ventilated TRPV4(-/-) lungs. Thus TRPV4(+/+) macrophages restored susceptibility of TRPV4(-/-) lungs to mechanical injury. A TRPV4 agonist increased intracellular calcium and reactive oxygen and nitrogen species in harvested TRPV4(+/+) macrophages but not TRPV4(-/-) macrophages. K(f) increases correlated with tissue nitrotyrosine, a marker of peroxynitrite production.

Pulmonary vascular heterogeneity and the Starling hypothesis.

It has generally been assumed that movement of fluid between the pulmonary microvasculature and surrounding tissues is governed by a "Starling" balance of hydrostatic and protein osmotic forces similar to that which prevails in the extremities. However, both recent and older observations suggest that the lungs are more resistant to edema formation than most other organs. Several structural aspects of the lung may account for protection of the airspaces from edema formation. The pulmonary microvasculature, which comprises >70% of the pulmonary circulatory bed, appears to be less permeable to fluid and electrolytes than the endothelium of the pulmonary arteries and veins and other microvascular exchange areas. This arrangement may help explain why early edema is confined to the perivascular and peribronchial regions and why lymphatics do not reach the alveoli. Unlike the peripheral vasculature, which is compressed by edema formation, the extra-alveolar vessels remain tethered open by airway distention, even when interstitial pressures rise above those in the vessels. This may also facilitate return of proteins to the circulation. Ultrafiltration of plasma may lower local protein concentrations in the interstitium, thereby slowing further edema formation. Transendothelial reabsorption of fluid may also be altered by vesicular transport.

Pulmonary microvascular endothelial cells form a tighter monolayer when grown in chronic hypoxia.

Unique among the vascular beds, loss of endothelial integrity in the pulmonary microcirculation due to injury can lead to rapidly fatal hypoxemia. The ability to regain confluence and re-establish barrier function is central to restoring proper gas exchange. The adult respiratory distress syndrome (ARDS) is a heterogeneous disease, however, meaning that endothelial cells within different regions of the lung do not likely see the same oxygen tension as they attempt to proliferate and re-establish an intact endothelial monolayer; the effect of hypoxia on the integrity of this newly formed endothelial monolayer is not clear. Immortalized human pulmonary microvascular endothelial cells (PMVEC) (ST1.6R cells) were sparsely plated and grown to confluence over 4 days in either normoxia (21% oxygen) or hypoxia (5% oxygen). Confluence attained in a hypoxic environment resulted in a tighter, less permeable endothelial monolayer (as determined by an increase in transendothelial electrical resistance, decreased permeability to fluorescently labeled macromolecules, and decreased hydraulic conductance). PMVEC grown to confluence under hypoxia had decreased RhoA activity; consistent with this finding, inhibition of Rho kinase, a well-described downstream target of RhoA, markedly increased electrical resistance in normoxic, but not hypoxic, PMVEC. These results were confirmed in primary human and rat PMVEC. These data suggest that PMVEC grown to confluence under hypoxia form a tighter monolayer than similar cells grown under normoxia. This tighter barrier appears to be due, in part, to the inhibition of RhoA activity in hypoxic cells.

Cytosolic phospholipase A2 and arachidonic acid metabolites modulate ventilator-induced permeability increases in isolated mouse lungs.

We previously reported that the cytosolic phospholipase A(2) (cPLA2) pathway is involved in ventilator-induced lung injury (VILI) produced by high peak inflation pressures (PIP) (J Appl Physiol 98: 1264-1271, 2005), but the relative contributions of the various downstream products of cPLA2 on the acute permeability response were not determined. Therefore, we investigated the role of cPLA2 and the downstream products of arachidonic acid metabolism in the high-PIP ventilation-induced increase in vascular permeability. We perfused isolated mouse lungs and measured the capillary filtration coefficient (K(fc)) after 30 min of ventilation with 9, 25, and 35 cmH2O PIP. In high-PIP-ventilated lungs, K(fc) increased significantly, 2.7-fold, after ventilation with 35 cmH2O PIP compared with paired baseline values and low-PIP-ventilated lungs. Also, increased phosphorylation of lung cPLA2 suggested enzyme activation after high-PIP ventilation. However, treatment with 40 mg/kg arachidonyl trifluoromethyl ketone (an inhibitor of cPLA2) or a combination of 30 microM ibuprofen [a cyclooxygenase (COX) inhibitor], 100 microM nordihydroguaiaretic acid [a lipoxygenase (LOX) inhibitor], and 10 microM 17-octadecynoic acid (a cytochrome P-450 epoxygenase inhibitor) prevented the high-PIP-induced increase in K(fc). Combinations of the inhibitors of COX, LOX, or cytochrome P-450 epoxygenase did not prevent significant increases in K(fc), even though bronchoalveolar lavage levels of the COX or LOX products were significantly reduced. These results suggest that multiple mediators from each pathway contribute to the acute ventilator-induced permeability increase in isolated mouse lungs by mutual potentiation.

Mitochondrial damage pathways in ventilator induced lung injury (VILI): an update.

Although reduced tidal volumes have improved patient survival during ventilation for acute lung injury, further improvements will require pharmacologic interventions of the cellular pathways for inflammation and injury. We previously reported that pretreatment with mitochondrial targeted mtDNA repair enzymes largely prevented lung injury and inflammation during a protocol for moderately severe ventilation induced lung injury. GSH/GSSG ratios indicated that free radical production had been reduced to baseline levels by treatment. The central role of the alveolar macrophages and cellular mechanisms of injury are discussed. This includes a rapid calcium entry and mitochondrial production of excessive reactive oxygen species. Excessive ROS can then result in activation of the NLRP3 inflammasome and secretion of IL-1 and IL-18 by caspase-1. A simultaneous activation of NFkB to transcribe pro forms of the cytokines is stimulated by damage associated molecular pattern (DAMP) recognition receptors. These are primarily TLR4 responding to various cellular damage products and TLR9 responding to mtDNA fragments that appear to be primarily involved. Intervention in these pathways could result in useful future clinical treatments.

Reduction in alveolar macrophages attenuates acute ventilator induced lung injury in rats.

OBJECTIVE: Alveolar macrophages are the sentinel cell for activation of the inflammatory cascade when the lung is exposed to noxious stimuli. We investigated the role of macrophages in mechanical lung injury by comparing the effect of high-volume mechanical ventilation with or without prior depletion of macrophages. DESIGN AND SETTING: Randomized sham-controlled animal study in anesthetized rats. METHODS: Lung injury was induced by 15 min of mechanical ventilation (intermittent positive pressure ventilation) using high peak pressures and zero end-expiratory pressure. The mean tidal volume was 40+/-0.7 ml/kg. One group of animals was killed immediately after this period of volutrauma (HV), while in a second group normoventilation was continued for 2 h at a tidal volume less than 10 ml/kg (HV-LV). One-half of the animals were depleted of alveolar macrophages by pretreatment with intratracheal liposomal clodronate (CL2MDP). MEASUREMENTS: Arterial blood gas, blood pressure. After kill: lung static pressure volume curves, bronchoalveolar fluid concentration for protein, macrophage inflammatory protein 2, tumor necrosis factor alpha, and wet/dry lung weight ratio (W/D). RESULTS: During HV and HV+LV oxygenation, lung compliance, and alveolar stability were better preserved in animals pretreated with CL2MDP. In both groups W/D ratio was significantly greater in ventilated than in nonventilated animals (4.5+/-0.6), but the increase in W/D was significantly less in CL2MDP treated HV and HV-LV groups (6.1+/-0.4, 6.6+/-0.6) than in the similarly ventilated nontreated groups (8.7+/-0.2 and 9.2+/-0.5). CONCLUSIONS: Alveolar macrophages participate in the early phase of ventilator-induced lung injury.

Clara cell secretory protein and phospholipase A2 activity modulate acute ventilator-induced lung injury in mice.

Lung vascular permeability is acutely increased by high-pressure and high-volume ventilation. To determine the roles of mechanically activated cytosolic PLA2 (cPLA2)and Clara cell secretory protein (CCSP), a modulator of cPLA2 activity, we compared lung injury with and without a PLA2 inhibitor in wild-type mice and CCSP-null mice (CCSP-/-) ventilated with high and low peak inflation pressures (PIP) for 2- or 4-h periods. After ventilation with high PIP, we observed significant increases in the bronchoalveolar lavage albumin concentrations, lung wet-to-dry weight ratios, and lung myeloperoxidase in both genotypes compared with unventilated controls and low-PIP ventilated mice. All injury variables except myeloperoxidase were significantly greater in the CCSP-/- mice relative to wild-type mice. Inhibition of cPLA2 in wild-type and CCSP-/- mice ventilated at high PIP for 4 h significantly reduced bronchoalveolar lavage albumin and total protein and lung wet-to-dry weight ratios compared with vehicle-treated mice of the same genotype. Membrane phospho-cPLA2 and cPLA2 activities were significantly elevated in lung homogenates of high-PIP ventilated mice of both genotypes but were significantly higher in the CCSP-/- mice relative to the wild-type mice. Inhibition of cPLA2 significantly attenuated both the phospho-cPLA2 increase and increased cPLA2 activity due to high-PIP ventilation. We propose that mechanical activation of the cPLA2 pathway contributes to acute high PIP-induced lung injury and that CCSP may reduce this injury through inhibition of the cPLA2 pathway and reduction of proinflammatory products produced by this pathway.

Mitochondrial Targeted Endonuclease III DNA Repair Enzyme Protects against Ventilator Induced Lung Injury in Mice.

The mitochondrial targeted DNA repair enzyme, 8-oxoguanine DNA glycosylase 1, was previously reported to protect against mitochondrial DNA (mtDNA) damage and ventilator induced lung injury (VILI). In the present study we determined whether mitochondrial targeted endonuclease III (EndoIII) which cleaves oxidized pyrimidines rather than purines from damaged DNA would also protect the lung. Minimal injury from 1 h ventilation at 40 cmH2O peak inflation pressure (PIP) was reversed by EndoIII pretreatment. Moderate lung injury due to ventilation for 2 h at 40 cmH2O PIP produced a 25-fold increase in total extravascular albumin space, a 60% increase in W/D weight ratio, and marked increases in MIP-2 and IL-6. Oxidative mtDNA damage and decreases in the total tissue glutathione (GSH) and the GSH/GSSH ratio also occurred. All of these indices of injury were attenuated by mitochondrial targeted EndoIII. Massive lung injury caused by 2 h ventilation at 50 cmH2O PIP was not attenuated by EndoIII pretreatment, but all untreated mice died prior to completing the two hour ventilation protocol, whereas all EndoIII-treated mice lived for the duration of ventilation. Thus, mitochondrial targeted DNA repair enzymes were protective against mild and moderate lung damage and they enhanced survival in the most severely injured group.

TRPV4 calcium entry and surface expression attenuated by inhibition of myosin light chain kinase in rat pulmonary microvascular endothelial cells.

In previous studies, blockade or gene deletion of either myosin light chain kinase (MLCK) or the mechanogated transient receptor potential vanilloid 4 (TRPV4) channel attenuated mechanical lung injury. To determine their effects on calcium entry, rat pulmonary microvascular endothelial cells (RPMVEC) were labeled with fluo-4 and calcium entry initiated with the TRPV4 agonist, 4α-phorbol 12, 13-didecanoate (4αPDD). Mean calcium transients peaked at ∼25 sec and persisted ∼500 sec. The 4αPDD response was essentially abolished in calcium-free media, or after pretreatment with the MLCK inhibitor, ML-7. ML-7 also attenuated the 4αPDD-induced inward calcium current measured directly using whole-cell patch clamp. Pretreatment with dynasore, an inhibitor of dynamin produced an initial calcium transient followed by a 4αPDD transient of unchanged peak intensity. Automated averaging of areas under the curve (AUC) of calcium transients in individual cells indicated total calcium activity with a relationship between treatment groups of ML-7 + 4αPDD < 4αPDD only < dynasore + 4αPDD. Measurement of biotinylated surface TRPV4 protein indicated a significant reduction after ML-7 pretreatment, but no significant change with dynasore treatment. RPMVEC monolayer electrical resistances were decreased by only 3% with 10 µmol/L 4αPDD and the response was dose-related. Dynasore alone produced a 29% decrease in resistance, but neither ML-7 nor dynasore affected the subsequent 4αPDD resistance response. These studies suggest that MLCK may inhibit mechanogated calcium responses through reduced surface expression of stretch activated TRPV4 channels in the plasma membrane.

Acute lung injury and pulmonary vascular permeability: use of transgenic models.

Acute lung injury is a general term that describes injurious conditions that can range from mild interstitial edema to massive inflammatory tissue destruction. This review will cover theoretical considerations and quantitative and semi-quantitative methods for assessing edema formation and increased vascular permeability during lung injury. Pulmonary edema can be quantitated directly using gravimetric methods, or indirectly by descriptive microscopy, quantitative morphometric microscopy, altered lung mechanics, high-resolution computed tomography, magnetic resonance imaging, positron emission tomography, or x-ray films. Lung vascular permeability to fluid can be evaluated by measuring the filtration coefficient (Kf) and permeability to solutes evaluated from their blood to lung clearances. Albumin clearances can then be used to calculate specific permeability-surface area products (PS) and reflection coefficients (σ). These methods as applied to a wide variety of transgenic mice subjected to acute lung injury by hyperoxic exposure, sepsis, ischemia-reperfusion, acid aspiration, oleic acid infusion, repeated lung lavage, and bleomycin are reviewed. These commonly used animal models simulate features of the acute respiratory distress syndrome, and the preparation of genetically modified mice and their use for defining specific pathways in these disease models are outlined. Although the initiating events differ widely, many of the subsequent inflammatory processes causing lung injury and increased vascular permeability are surprisingly similar for many etiologies.

Rapid cooling preserves the ischaemic myocardium against mitochondrial damage and left ventricular dysfunction.

AIMS: We investigated whether rapid cooling instituted by total liquid ventilation (TLV) improves cardiac and mitochondrial function in rabbits submitted to ischaemia-reperfusion. METHODS AND RESULTS: Rabbits were chronically instrumented with a coronary artery occluder and myocardial ultrasonic crystals for assessment of segment length-shortening. Two weeks later they were re-anaesthetized and underwent either a normothermic 30-min coronary artery occlusion (CAO) (Control group, n = 7) or a comparable CAO with cooling initiated by a 10-min hypothermic TLV and maintained by a cold blanket placed on the skin. Cooling was initiated after 5 or 15 min of CAO (Hypo-TLV and Hypo-TLV(15') groups, n = 6 and 5, respectively). A last group underwent normothermic TLV during CAO (Normo-TLV group, n = 6). Wall motion was measured in the conscious state over three days of reperfusion before infarct size evaluation and histology. Additional experiments were done for myocardial sampling in anaesthetized rabbits for mitochondrial studies. The Hypo-TLV procedure induced a rapid decrease in myocardial temperature to 32-34 degrees C. Throughout reperfusion, segment length-shortening was significantly increased in Hypo-TLV and Hypo-TLV(15') vs. Control and Normo-TLV (15.1 +/- 3.3%, 16.4 +/- 2.3%, 1.8 +/- 0.6%, and 1.1 +/- 0.8% at 72 h, respectively). Infarct sizes were also considerably attenuated in Hypo-TLV and Hypo-TLV(15') vs. Control and Normo-TLV (4 +/- 1%, 11 +/- 5%, 39 +/- 2%, and 42 +/- 5% infarction of risk zones, respectively). Mitochondrial function in myocardial samples obtained at the end of ischaemia or after 10 min of reperfusion was improved by Hypo-TLV with respect to ADP-stimulated respiration and calcium-induced opening of mitochondrial permeability transition pores (mPTP). Calcium concentration opening mPTP was, e.g., increased at the end of ischaemia in the risk zone in Hypo-TLV vs. Control (157 +/- 12 vs. 86 +/- 12 microM). Histology and electron microscopy also revealed better preservation of lungs and of cardiomyocyte ultrastructure in Hypo-TLV when compared with Control. CONCLUSION: Institution of rapid cooling by TLV during ischaemia reduces infarct size as well as other sequelae of ischaemia, such as post-ischaemic contractile and mitochondrial dysfunction.

Physiological determinants of the pulmonary filtration coefficient.

Current emphasis on translational application of genetic models of lung disease has renewed interest in the measurement of the gravimetric filtration coefficient (K(f)) as a means to assess vascular permeability changes in isolated perfused lungs. The K(f) is the product of the hydraulic conductivity and the filtration surface area, and is a sensitive measure of vascular fluid permeability when the pulmonary vessels are fully recruited and perfused. We have observed a remarkable consistency of the normalized baseline K(f) values between species with widely varying body weights from mice to sheep. Uniformity of K(f) values can be attributed to the thin alveolar capillary barrier required for gas exchange and the conserved matching of lung vascular surface area to the oxygen requirements of the body mass. An allometric correlation between the total lung filtration coefficient (K(f,t)) and body weight in several species (r(2)=1.00) had a slope that was similar to those reported for alveolar and pulmonary capillary surface areas and pulmonary diffusion coefficients determined by morphometric methods in these species. A consistent K(f) is dependent on accurately separating the filtration and vascular volume components of lung weight gain, then K(f) is a consistent and repeatable index of lung vascular permeability.

Hydraulic conductance of lung endothelial phenotypes and Starling safety factors against edema.

Recent permeability studies comparing endothelial cell phenotypes derived from alveolar and extra-alveolar vessels have significant implications for interpreting the mechanisms of fluid homeostasis in the intact lung. These studies indicate that confluent monolayers of rat pulmonary microvascular endothelial cells had a hydraulic conductance (L(p)) that was only 5% and a transendothelial flux rate for 72-kDa dextran only 9% of values determined for rat pulmonary artery endothelial cell monolayers. On the basis of previous studies partitioning the filtration coefficients between alveolar and extra-alveolar vascular segments in rat lungs and previous studies of lymph albumin fluxes and permeability, the contribution of the alveolar capillary segment to total albumin flux in lymph was estimated to be less than 10%. In addition, the Starling safety factors against the edema calculated for the alveolar capillaries are quite different from those estimated for whole lung. Estimates of the edema safety factor due to increased filtration across the alveolar capillary wall based on the low L(p) indicate it is quantitatively the greatest safety factor, although it would be a minor safety factor for extra-alveolar vessels. Also, a markedly higher effective protein osmotic absorptive force for plasma proteins must occur in the capillaries relative to extra-alveolar vessels. The lower L(p) for alveolar capillaries also has implications for the sequence of hydrostatic edema formation, and it also may have a role in preventing exercise-induced alveolar flooding.

Phosphoinositide 3-kinase, Src, and Akt modulate acute ventilation-induced vascular permeability increases in mouse lungs.

To determine the role of phosphoinositide 3-OH kinase (PI3K) pathways in the acute vascular permeability increase associated with ventilator-induced lung injury, we ventilated isolated perfused lungs and intact C57BL/6 mice with low and high peak inflation pressures (PIP). In isolated lungs, filtration coefficients (K(f)) increased significantly after ventilation at 30 cmH(2)O (high PIP) for successive periods of 15, 30 (4.1-fold), and 50 (5.4-fold) min. Pretreatment with 50 microM of the PI3K inhibitor, LY-294002, or 20 microM PP2, a Src kinase inhibitor, significantly attenuated the increase in K(f), whereas 10 microM Akt inhibitor IV significantly augmented the increased K(f). There were no significant differences in K(f) or lung wet-to-dry weight (W/D) ratios between groups ventilated with 9 cmH(2)O PIP (low PIP), with or without inhibitor treatment. Total lung beta-catenin was unchanged in any low PIP isolated lung group, but Akt inhibition during high PIP ventilation significantly decreased total beta-catenin by 86%. Ventilation of intact mice with 55 cmH(2)O PIP for up to 60 min also increased lung vascular permeability, indicated by increases in lung lavage albumin concentration and lung W/D ratios. In these lungs, tyrosine phosphorylation of beta-catenin and serine/threonine phosphorylation of Akt, glycogen synthase kinase 3beta (GSK3beta), and ERK1/2 increased significantly with peak effects at 60 min. Thus mechanical stress activation of PI3K and Src may increase lung vascular permeability through tyrosine phosphorylation, but simultaneous activation of the PI3K-Akt-GSK3beta pathway tends to limit this permeability response, possibly by preserving cellular beta-catenin.

Total liquid ventilation provides ultra-fast cardioprotective cooling.

OBJECTIVES: We tested whether total liquid ventilation (TLV) can be used to rapidly cool and protect the infarcting heart. BACKGROUND: Decreasing myocardial temperature during ischemia is a powerful cardioprotective strategy, but clinical application has been impaired by lack of practical methodology to quickly cool the heart. METHODS: We performed 30-min coronary artery occlusion/3-h reperfusion in rabbits. Upon occlusion, rabbits underwent either oxygen (Gas), normothermic liquid (Liquid Warm), or cold liquid (Liquid Cool) ventilation. RESULTS: Left atrial chamber temperature decreased to 32.4 degrees +/- 0.2 degrees C within 5 min of onset of cold TLV. Blood gases were within acceptable limits during TLV. In the Liquid Warm group, perfluorocarbon inhalation did not alter infarct size compared with Gas (37.7 +/- 1.3% and 42.5 +/- 4.9% of risk zone, respectively). However, infarction was significantly reduced in the Liquid Cool group (4.0 +/- 0.5%). Cooling only during the initial 30 min of reperfusion did not reduce infarction. CONCLUSIONS: Total liquid ventilation can elicit rapid cardioprotective cooling during ischemia.

TRPV4 initiates the acute calcium-dependent permeability increase during ventilator-induced lung injury in isolated mouse lungs.

We have previously implicated calcium entry through stretch-activated cation channels in initiating the acute pulmonary vascular permeability increase in response to high peak inflation pressure (PIP) ventilation. However, the molecular identity of the channel is not known. We hypothesized that the transient receptor potential vanilloid-4 (TRPV4) channel may initiate this acute permeability increase because endothelial calcium entry through TRPV4 channels occurs in response to hypotonic mechanical stress, heat, and P-450 epoxygenase metabolites of arachidonic acid. Therefore, permeability was assessed by measuring the filtration coefficient (K(f)) in isolated perfused lungs of C57BL/6 mice after 30-min ventilation periods of 9, 25, and 35 cmH(2)O PIP at both 35 degrees C and 40 degrees C. Ventilation with 35 cmH(2)O PIP increased K(f) by 2.2-fold at 35 degrees C and 3.3-fold at 40 degrees C compared with baseline, but K(f) increased significantly with time at 40 degrees C with 9 cmH(2)O PIP. Pretreatment with inhibitors of TRPV4 (ruthenium red), arachidonic acid production (methanandamide), or P-450 epoxygenases (miconazole) prevented the increases in K(f). In TRPV4(-/-) knockout mice, the high PIP ventilation protocol did not increase K(f) at either temperature. We have also found that lung distention caused Ca(2+) entry in isolated mouse lungs, as measured by ratiometric fluorescence microscopy, which was absent in TRPV4(-/-) and ruthenium red-treated lungs. Alveolar and perivascular edema was significantly reduced in TRPV4(-/-) lungs. We conclude that rapid calcium entry through TRPV4 channels is a major determinant of the acute vascular permeability increase in lungs following high PIP ventilation.

Restoration of mucociliary transport in the fluid-depleted trachea by surface-active instillates.

Severe impairment of mucociliary transport (MCT) is a hallmark of cystic fibrosis (CF) lung disease. Recent studies demonstrate that pharmacologic inhibition of anion and liquid secretion in pig tracheas models the MCT defect in CF through depletion of the periciliary fluid component of airway surface liquid. In the present study, the effectiveness of various aqueous instillates on rehydration of the airway surface and restoration of MCT was assessed in this model. Excised porcine tracheas were mounted in a chamber that permitted simultaneous measurement of MCT and adventitial exposure of the airways to Krebs solution. When anion and liquid secretion were inhibited by treatment with bumetanide and dimethylamiloride, MCT was greatly reduced. Luminal instillation of aqueous solutions containing surface-active substances (1% Tween80 or calfactant) transiently restored MCT to high rates in nearly all tissues. Mucosal treatment with only Krebs solution or hypertonic saline restored MCT in only one half of the tracheas. We conclude that aqueous salt solutions alone can hydrate airway surfaces and restore MCT in some tissues, but surface-active substances may provide additional benefit in restoring MCT in this model of mucociliary stasis. We speculate that administration of surface-active substances, by aerosol or lavage, might help to restore MCT in the airways of patients with CF.

Hydraulic conductance of pulmonary microvascular and macrovascular endothelial cell monolayers.

Endothelial cells isolated from pulmonary arteries (RPAEC) and microcirculation (RPMVEC) of rat lungs were grown to confluence on porous filters and mounted on an Ussing-type chamber. Transmembrane pressure (deltaP) was controlled by the reservoir height, and the filtration rate corrected for surface area (J(v)/A) was measured by timing fluid movement in a calibrated micropipette. These parameters were used to calculate hydraulic conductance (Lp) by using linear regression of J(v)/A on deltaP. Mean Lp values for newly confluent RPAEC monolayers were 22 times higher than those for RPMVEC monolayers (28.6 +/- 5.6 vs. 1.30 +/- 0.50 x 10(-7) cm x s(-1) x cmH2O(-1); P < or = 0.01). After confluence was reached, electrical resistance and Lp remained stable in RPAEC but continued to change in RPMVEC with days in culture. Both phenotypes exhibited an initial time-dependent sealing response, but Lp also had an inverse relationship to deltaP in RPMVEC monolayers > or = 4 days postconfluence that was attributed to cell overgrowth rather than junctional length. In a comparison of the cadherin contents, E-cadherin was predominant in RPMVEC, but VE-cadherin was predominant in RPAEC. At a constant deltaP of 40-45 cmH2O for 2 h, J(v)/A increased 225% in RPAEC monolayers but did not change significantly in RPMVEC monolayers. Significant decreases in Lp were obtained after treatment with 5% albumin, GdCl3, or isoproterenol plus rolipram in both phenotypes. Thus lung microvascular endothelial cells exhibited a significantly lower Lp than conduit vessel endothelium, which would limit alveolar flooding relative to perivascular edema cuff formation during increased pulmonary vascular pressures.