Equivalent pore estimate for the alveolar-airway barrier in isolated dog lung

In high-pressure pulmonary edema, lung interstitial and air space edema liquids have equal protein concentrations (Am. J. Physiol. 231: 1466, 1976). This suggests that the alveolar-airway barrier separating the air and interstitial spaces is relatively unrestrictive, even without apparent epithelial injury. To estimate the equivalent pore population of the alveolar-airway barrier we inflated each of 18 isolated dog lung lobes for 1 h with a solution of colored tracer of uniform radius. Tracer radii ranged from 1.3 to 405 nm. After freezing the lobes in liquid N2, we measured interstitial tracer concentrations in frozen perivascular cuffs or in samples thawed after dissection from frozen cuffs. Relative to the concentrations instilled, interstitial concentrations ranged from 0.34 for the smallest particles (1.3 and 3.5 nm radius) to zero for particles with radii of 405 nm. From the results we designed a pore model of the alveolar-airway barrier to reproduce the concentrations we measured. No single-pore model could be obtained, although a three-pore model fit the data well. The model results predict that pores with radii of 1, 40, and 400 nm would account for 68, 30, and 2% of total liquid flux, respectively. The majority of liquid flux (68%) would occur through passageways smaller than the smallest tracer we used (1.3 nm radius). We believe the alveolar-airway barrier consists not only of tight intercellular junctions that allow passage of only water and electrolytes but also of a smaller number of large leaks that allow passage of particles up to nearly 400 nm in radius.     

Effect of lung inflation on diaphragmatic shortening

The effect of lung inflation on chest wall mechanics was studied in 11 vagotomized pentobarbital sodium-anesthetized dogs. Diaphragmatic shortening (percent change from initial length at functional residual capacity, %LFRC) and transdiaphragmatic pressure swings (delta Pdi) were compared with control values over a range of positive-pressure breathing that produced a maximum increase in lung volume to 40% of inspiratory capacity. There was no change in the electromyogram of the diaphragm or parasternal intercostals during positive-pressure breathing. delta Pdi and tidal volume (VT) fell to 52 +/- 3.3 and 42.5 +/- 5% (SE) of control. This was associated with a reduction in the initial resting length of 13 +/- 1.9 and 21 +/- 2.2%LFRC (SE) in the costal and crural diaphragms, respectively. Tidal diaphragmatic shortening, however, decreased to 66 +/- 7 and 57 +/- 7 and the mean velocity decreased to 78 +/- 10 and 63 +/- 8% (SE) of control for the costal and crural diaphragms, respectively. We conclude that the reduction in diaphragmatic shortening is the main determinant of the reduced delta Pdi and VT during lung inflation and relate this to what is currently known about diaphragmatic contractile properties.     

Effect of elevated vascular pressure transients on protein permeability in the lung

Neurogenic pulmonary edema (NPE) may develop in individuals with head trauma or seizures and is generally thought to have a hydrostatic basis in the severe degree of pulmonary hypertension that occurs. Recently, it has been suggested that vascular pressures may rise to levels that damage the vessels, leaving the patient at risk for further edema development. The objective of this study was to determine if pulmonary vascular protein permeability is increased in a canine isolated perfused left lower lung lobe (LLL) preparation by pressure transients that may occur in NPE. Venous pressure (Pv) was transiently raised to values ranging from 8 to 102 Torr in 19 LLL. One Pv transient was studied per LLL. After Pv was returned to normal, the osmotic reflection coefficient (sigma d) for total proteins was determined by the hematocrit-protein double indicator technique. No reduction in sigma d was observed until microvascular pressure exceeded 70 Torr. The average sigma d for the 11 LLL in which the peak microvascular pressure was less than 70 Torr was 0.74 +/- 0.03 (SE). Above this level sigma d fell linearly with increasing Pv, with a value of 0.26 being observed after the highest Pv transient. These results suggest that protein permeability may increase in patients with NPE who develop very large increases in pulmonary vascular pressures but may not be a universal occurrence in this disorder.     

Effect of high transcapillary pressures on capillary ultrastructure and permeability coefficients in dog lung.

To determine the correlation between ultrastructural and physiological changes in blood-gas barrier function in lungs transiently exposed to very high vascular pressures, we increased capillary transmural pressure (Ptm) of 6 canine isolated perfused left lower lung lobe preparations (high-pressure group) to 80.3 Torr for 3.8 min and then determined the capillary filtration (K(fc)) and osmotic reflection (sigma(d)) coefficients at a Ptm of 19.1 Torr in the ventilated lung lobes. This was followed by perfusion fixation of the lobes at a Ptm of 20.5 Torr for ultrastructural analysis. These data were compared with those obtained in six lobes in which Ptm was not transiently elevated before K(fc), sigma(d), and ultrastructural evaluation. K(fc) was higher [0.249 +/- 0.042 (SE) vs. 0.054 +/- 0.009 g. min(-1). Torr(-1). 100 g(-1); P < 0.01] and sigma(d) was lower (0.52 +/- 0.07 vs. 0.85 +/- 0.08; P < 0.01) in the high-pressure group. In contrast, although endothelial and epithelial breaks were occasionally observed in some experiments, their incidence was not increased in the high-pressure group. These data suggest that the increased transvascular water and protein flux occurred through pathways of a size not resolvable by electron microscopy after vascular perfusion-fixation at a Ptm of 20.5 Torr.     

Zinc modulates cytokine-induced lung epithelial cell barrier permeability

Apoptosis plays a causative role in acute lung injury in part due to epithelial cell loss. We recently reported that zinc protects the lung epithelium during inflammatory stress whereas depletion of intracellular zinc enhances extrinsic apoptosis. In this investigation, we evaluated the relationship between zinc, caspase-3, and cell-to-cell contact via proteins that form the adherens junction complex. Cell adhesion proteins are directly responsible for formation of the mechanical barrier of the lung epithelium. We hypothesized that exposure to inflammatory cytokines, in conjunction with zinc deprivation, would induce caspase-3, leading to degradation of junction proteins, loss of cell-to-cell contact, and compromised barrier function. Primary human upper airway and type I/II alveolar epithelial cultures were obtained from multiple donors and exposed to inflammatory stimuli that provoke extrinsic apoptosis in addition to depletion of intracellular zinc. We observed that zinc deprivation combined with tumor necrosis factor-alpha, interferon-gamma, and Fas receptor ligation accelerates caspase-3 activation, proteolysis of E-cadherin and beta-catenin, and cellular apoptosis, leading to increased paracellular leak across monolayers of both upper airway and alveolar lung epithelial cultures. Zinc supplementation inhibited apoptosis and paracellular leak, whereas caspase inhibition was less effective. We conclude that zinc is a vital factor in the lung epithelium that protects against death receptor-mediated apoptosis and barrier dysfunction. Furthermore, our findings suggest that although caspase-3 inhibition reduces lung epithelial apoptosis it does not prevent mechanical dysfunction. These findings facilitate future studies aimed at developing therapeutic strategies to prevent acute lung injury.     

Effect of lung inflation on regional lung expansion in supine and prone rabbits

At functional residual capacity, lung expansion is more uniform in the prone position than in the supine position. We examined the effect of positive airway pressure (Paw) on this position-dependent difference in lung expansion. In supine and prone rabbits postmortem, we measured alveolar size through dependent and nondependent pleural windows via videomicroscopy at Paw of 0 (functional residual capacity), 7, and 15 cmH2O. After the chest was opened, alveolar size was measured in the isolated lung at several transpulmonary pressures (Ptp) on lung deflation. Alveolar mean linear intercept (Lm) was measured from the video images taken in situ. This was compared with those measured in the isolated lung to determine Ptp in situ. In the supine position, the vertical Ptp gradient increased from 0.52 cmH2O/cm at 0 cmH2O Paw to 0.90 cmH2O/cm at 15 cmH2O Paw, while the vertical gradient in Lm decreased from 2.17 to 0.80 microns/cm. In the prone position, the vertical Ptp gradient increased from 0.06 cmH2O/cm at 0 cmH2O Paw to 0.35 cmH2O/cm at 15 cmH2O Paw, but there was no change in the vertical Lm gradient. In anesthetized paralyzed rabbits in supine and prone positions, we measured pleural liquid pressure directly at 0, 7, and 15 cmH2O Paw with dependent and nondependent rib capsules. Vertical Ptp gradients measured with rib capsules were similar to those estimated from the alveolar size measurements. Lung inflation during mechanical ventilation may reduce the vertical nonuniformities in lung expansion observed in the supine position, thereby improving gas exchange and the distribution of ventilation.     

Effect of atrial natriuretic factor on blood-brain barrier permeability

Recent studies have demonstrated receptors for atrial natriuretic factor on endothelium of intracerebral vessels. The physiological role of these receptors is not known. The present study was undertaken to determine whether atrial natriuretic factor has an effect on blood-brain barrier permeability to protein and ions using horseradish peroxidase and lanthanum as markers of permeability alterations. This study does not demonstrate a significant effect of atrial natriuretic factor on blood-brain barrier permeability mechanisms in steady states.     

Effect of lung inflation on lung blood volume and pulmonary venous flow

Phasic changes in lung blood volume (LBV) during the respiratory cycle may play an important role in the genesis of the respiratory wave in arterial pressure, or pulsus paradoxus. To better understand the effects of lung inflation on LBV, we studied the effect of changes in transpulmonary pressure (delta Ptp) on pulmonary venous flow (Qv) in eight isolated canine lungs with constant inflow. Inflation when the zone 2 condition was predominant resulted in transient decreases in Qv associated with increases in LBV. In contrast, inflation when the zone 3 condition was predominant resulted in transient increases in Qv associated with decreases in LBV. These findings are consistent with a model of the pulmonary vasculature that consists of alveolar and extra-alveolar vessels. Blood may be expelled from alveolar vessels but is retained in extra-alveolar vessels with each inflation. The net effect on LBV and thus on Qv is dependent on the zone conditions that predominate during inflation, with alveolar or extra-alveolar effects being greater when the zone 3 or zone 2 conditions predominate, respectively. Lung inflation may therefore result in either transiently augmented or diminished Qv. Phasic changes in left ventricular preload may therefore depend on the zone conditions of the lungs during the respiratory cycle. This may be an important modulator of respiratory variations in cardiac output and blood pressure.     

Endotoxin increases pulmonary vascular protein permeability in the dog

Endotoxin increases pulmonary vascular permeability consistently in some species but fails to reliably cause injury in the dog. We wondered whether this phenomenon depended on the method of injury assessment, as others have relied on edema measurement; we quantified injury by monitoring the rate of extravascular protein accumulation. 113mIn-labeled protein and 99mTc-labeled erythrocytes were injected into anesthetized dogs and monitored by an externally placed lung probe. A protein leak index, the rate of extravascular protein accumulation, was derived from the rate of increase in lung protein counts corrected for changes in intravascular protein activity. After administration of Salmonella enteriditis endotoxin (4 micrograms/kg), the protein leak index was elevated 2.5-fold (41.1 +/- 4.6 X 10(-4) min-1) compared with control (16.0 +/- 2.8 X 10(-4) min-1). In contrast, wet-to-dry weight ratios failed to increase after endotoxin (4.6 +/- 0.8 vs. control values of 4.2 +/- 0.5 g/g dry bloodless lung). However, we observed that endotoxin increased lung dry weight (per unit body weight), which may have attenuated the change in wet-to-dry weight ratios. To determine whether low microvascular pressures following endotoxin attenuated edema formation, we increased pulmonary arterial wedge pressures in five dogs by saline infusion, which caused an increase in wet-to-dry weight ratios following endotoxin but no change in the five controls. We conclude that low dose endotoxin causes pulmonary vascular protein leak in the dog while edema formation is minimal or absent.     

Effect of lung inflation on fluid flux in zone 1 lungs

Because of conflicting data in the literature, we studied the effect of positive-pressure inflation on transvascular fluid filtration in zone 1 lungs. Lungs from New Zealand White rabbits (n = 10) were excised, perfused with saline and autologous whole blood (1:1), ventilated, and continuously weighed. Pulmonary arterial and venous pressures (Pvas) were referenced to the most dependent part of the lung. A change in vascular volume (delta Vvas) and a fluid filtration rate (FFR) were calculated from the change in lung weight that occurred from 0 to 30 s and from 3 to 5 and 5 to 10 min, respectively, after changing alveolar pressure (PA). FFR's and delta Vvas's were measured with Pvas equal to 2 or 10 cmH2O and PA changing from 15 to 30 cmH2O when the lungs were normal and after they were made edematous. When Pvas = 2 cmH2O, increasing PA increased the Vvas and the FFR in both normal and edematous lungs. However, when Pvas = 10 cmH2O, increasing PA only slightly changed the Vvas and reduced the FFR in the normal lungs, and decreased Vvas and markedly decreased the FFR in the presence of edema. Inflating zone 1 lungs by positive pressure has an effect on transvascular fluid flux that depends on the Pvas. The results suggest that the sites of leakage in zone 1 also vary depending on Pvas and PA.     

Effect of cyclosporin-A on the blood--retinal barrier permeability in streptozotocin-induced diabetes

BACKGROUND: Our previous results showed that in retinas from streptozotocin (STZ)-induced diabetic rats there is an increased level of interleukin-1beta (IL-1beta). This cytokine may be involved in the expression of the inducible isoform of the nitric oxide synthase (iNOS), with consequent synthesis of large amounts of NO and blood-retinal barrier (BRB) breakdown. AIMS: The aim of this work was to examine whether the administration of cyclosporin-A (Cs-A) to STZ-induced diabetic rats inhibits the synthesis of IL-1beta and the expression of the inducible proteins, iNOS and cyclo-oxygenase-2 (COX-2) in retinal cells, and whether the activity of these proteins contribute to BRB breakdown. METHODS: The level of IL-1beta was evaluated by ELISA and the NO production by L-[3H]-citrulline formation. Expression of iNOS and COX-2 proteins was determined by two methods, western blot and immunohistochemistry. The permeability of the BRB was assessed by quantification of the vitreous protein. RESULTS AND DISCUSSION: Our results indicated that the levels of IL-1beta and NO in retinas from Cs-A-treated diabetic rats are significantly reduced, as compared to that in non-treated diabetic rats. The treatment of diabetic rats with Cs-A also significantly inhibited the expression of the inducible proteins, iNOS and COX-2. The evaluation of the vitreous protein content revealed that Cs-A also reduces the BRB permeability. Taken together, these results suggest that the increased production of the inflammatory mediators, IL-1beta and NO, in diabetes may affect the BRB permeability and therefore contribute to the development of diabetic retinopathy.     

Asef controls vascular endothelial permeability and barrier recovery in the lung

Increased levels of hepatocyte growth factor (HGF) in injured lungs may reflect a compensatory response to diminish acute lung injury (ALI). HGF-induced activation of Rac1 GTPase stimulates endothelial barrier protective mechanisms. This study tested the involvement of Rac-specific guanine nucleotide exchange factor Asef in HGF-induced endothelial cell (EC) cytoskeletal dynamics and barrier protection in vitro and in a two-hit model of ALI. HGF induced membrane translocation of Asef and stimulated Asef Rac1-specific nucleotide exchange activity. Expression of constitutively activated Asef mutant mimicked HGF-induced peripheral actin cytoskeleton enhancement. In contrast, siRNA-induced Asef knockdown or expression of dominant-negative Asef attenuated HGF-induced Rac1 activation evaluated by Rac-GTP pull down and FRET assay with Rac1 biosensor. Molecular inhibition of Asef attenuated HGF-induced peripheral accumulation of cortactin, formation of lamellipodia-like structures, and enhancement of VE-cadherin adherens junctions and compromised HGF-protective effect against thrombin-induced RhoA GTPase activation, Rho-dependent cytoskeleton remodeling, and EC permeability. Intravenous HGF injection attenuated lung inflammation and vascular leak in the two-hit model of ALI induced by excessive mechanical ventilation and thrombin signaling peptide TRAP6. This effect was lost in Asef^−/− mice. This study shows for the first time the role of Asef in HGF-mediated protection against endothelial hyperpermeability and lung injury.