Albumin transport across pulmonary capillary-interstitial barrier in anesthetized dogs

To evaluate albumin transport across the pulmonary capillary endothelial and interstitial barriers, we simultaneously measured blood-to-tissue (QA,t) and blood-to-lymph (QA,l) clearances of 125I-radiolabeled albumin as well as endogenous albumin clearance (Qa,l) in the canine lung in vivo (n = 10). Steady-state prenodal lung lymph flows (Qw,l) and protein clearances were measured over a 2-h period at a constant capillary pressure (Pc, 13-33 cmH2O). Comparison between QA,t and QA,l as a function of Pc suggests that little of the albumin that crossed the capillary wall remained in the lung tissue, with most leaving in the lymph. Qw,l increased significantly as Pc increased, but lung tissue water was minimally affected. From the ratio of the clearance-Pc slopes for albumin and water, the albumin reflection coefficient was estimated to be 0.81 using QA,l and Qw,l and 0.56 using Qa,l and Qw,l. The permeability surface area product for the sum of blood-to-tissue and blood-to-lymph fluxes of labeled albumin (QA,t + QA,l) was 31 +/- 9 microliters/min, whereas that calculated from the blood-to-lymph flux of endogenous albumin (Qa,l) was 97 +/- 22 microliters/min. These data suggest that 1) both tissue and lymph accumulations of albumin must be considered when microvascular permeability is evaluated using protein tracers; 2) lymph clearance, but not tissue accumulation of albumin, was filtration dependent; and 3) lymph flow was an important contributor to the safety factor against edema formation over a moderate range of capillary pressures.     

Evans blue dye as a marker of albumin clearance in cultured endothelial monolayer and isolated lung.

Determination of protein transfer across the endothelial barrier or the entire alveolar capillary membrane is critical for investigation of mechanisms leading to pulmonary edema. The purpose of this study was to evaluate Evans blue dye for determination of protein clearance across cultured bovine pulmonary artery endothelial cell monolayers and as a quantitative marker for albumin leakage to the air spaces in isolated perfused rat lungs. Evans blue dye bound tightly to albumin (EBA) as determined by lack of transfer through dialysis membranes and specific elution with albumin from a molecular exclusion column. EBA was equivalent to 125I-labeled albumin for calculation of albumin clearance rates (Calb) across intact and challenged monolayers [Calb (+ vehicle) = 0.12 microliters/min; Calb (+10 nM alpha-thrombin) = 0.47 microliters/min; Calb (+5 mg/ml trypsin) = 1.29 microliters/min]. Transfer of EBA was linear with time in both the endothelial cell monolayer model and the perfused lung. EBA was a sensitive marker for early edema in the perfused lung (before detectable weight gain) as well as for severe edema in the oxidant-injured lung (marked EBA accumulation in lavage fluid) and was a more specific marker for protein transfer than lavage fluid protein. EBA transfer is a convenient, reproducible, and accurate means to assess alterations in vascular permeability.     

Pulmonary edema: ischemia reperfusion endothelial injury and its reversal by c-AMP.

A review of the factors that oppose pulmonary edema formation (alveolar flooding) when capillary pressure is elevated are presented for a normal capillary endothelial barrier and for damaged endothelium associated with ischemia/reperfusion in rabbit, rat, and dog lungs. Normally, tissue pressure, the plasma protein osmotic pressure gradient acting across the capillary wall and lymph flow (Edema Safety Factors) increase to prevent the build-up of fluid in the lung's interstitium when capillary pressure increases. No measureable alveolar edema fluid accumulates until capillary pressure exceeds 30 mmHg. When the capillary wall has been damaged, interstitial edema develops at lower capillary pressures because the plasma protein osmotic pressure will not change greatly to oppose capillary filtration, but lymph flow increases to very high levels to remove the increased filtrate and the result is that capillary pressures can increase to 20-25 mmHg before alveolar flooding results. In addition, the mechanisms responsible for producing pulmonary endothelial damage with ischemia/reperfusion are reviewed and the effects of O2 radical scavengers, neutrophil depletion or altering their adherence to the endothelium, and increasing cAMP on reversing the damage to the pulmonary endothelium is presented.     

Regulation of capillary hydraulic conductivity in response to an acute change in shear

The effects of mechanical perturbations (shear stress, pressure) on microvascular permeability primarily have been examined in micropipette-cannulated vessels or in endothelial monolayers in vitro. The objective of this study is to determine whether acute changes in blood flow shear stress might influence measurements of hydraulic conductivity (L(p)) in autoperfused microvessels in vivo. Rat mesenteric microvessels were observed via intravital microscopy. Occlusion of a third-order arteriole with a micropipette was used to divert and increase flow through a nonoccluded capillary or fourth-order arteriolar branch. Transvascular fluid filtration rate in the branching vessel was measured with a Landis technique. Flow (shear)-induced increases in L(p) disappeared within 20-30 s of the removal of the shear and could be eliminated with nitric oxide synthase inhibition. The shear-induced increase in L(p) was greater in capillaries compared with terminal arterioles. An acute change in shear may regulate L(p) by a nitric oxide-dependent mechanism that displays heterogeneity within a microvascular network.     

Pulmonary microvascular response to LTB4: effects of perfusate composition

We examined the effects of leukotriene B4 (LTB4) on pulmonary hemodynamics and vascular permeability using isolated perfused guinea pig lungs and cultured monolayers of pulmonary arterial endothelial cells. In lungs perfused with Ringer solution, containing 0.5 g/100 ml albumin (R-alb), LTB4 (4 micrograms) transiently increased pulmonary arterial pressure (Ppa) and capillary pressure (Pcap). Pulmonary edema developed within 70 min after LTB4 injection despite a normal Pcap. The LTB4 metabolite, 20-COOH-LTB4 (4 micrograms), did not induce hemodynamic and lung weight changes. In lungs perfused with autologous blood hematocrit = 12 +/- 1%; protein concentration = 1.5 +/- 0.2 g/100 ml), the increases in Ppa and Pcap were greater, and both pressures remained elevated. The lung weight did not increase in blood-perfused lungs. In lungs perfused with R-alb (1.5 g/100 ml albumin) to match the blood perfusate protein concentration, LTB4 induced similar hemodynamic changes as R-alb (0.5 g/100 ml) perfusate, but the additional albumin prevented the pulmonary edema. LTB4 (10(-11)-10(-6) M) with or without the addition of neutrophils to the monolayer did not increase endothelial 125I-albumin permeability. Therefore LTB4 induces pulmonary edema when the perfusate contains a low albumin concentration, but increasing the albumin concentration or adding blood cells prevents the edema. The edema is not due to increased endothelial permeability to protein and is independent of hemodynamic alterations. Protection at higher protein-concentration may be the result of LTB4 binding to albumin.     

Polymorphonuclear leukocyte: arachidonate edema

Polymorphonuclear leukocytes (PMN) are important participants in many models of acute lung edema. Enhanced metabolism of arachidonate is also characteristic of many of these models. We found that PMN and arachidonate, but neither alone, increased alveolar capillary permeability of isolated perfused lungs and increased transfer of albumin across monolayers of endothelial cells cultured on micropore filters. Inhibition of PMN, but not endothelial cyclooxygenase, blunted the edematous process. Neither PMN proteases nor PMN-derived oxidants were involved. The edemagenic activity was not found in supernatants of PMN and arachidonate, and unstable prostaglandins did not alter endothelial albumin transfer. The edemagenic process was not inhibited by blocking leukotriene synthesis, and endothelial albumin transfer was not increased by direct addition of leukotrienes to endothelium. These data demonstrate that PMN and arachidonate can interact to increase endothelial permeability and that PMN cyclooxygenase activity is important for this process. This interaction is of potential significance to the acute inflammatory process in the lung vasculature.     

Keratinocyte growth factor attenuates hydrostatic pulmonary edema in an isolated, perfused rat lung model

Hydrostatic pulmonary edema is a common complication of congestive heart failure, resulting in substantial morbidity and mortality. Keratinocyte growth factor (KGF) is a mitogen for type II alveolar epithelial and microvascular cells. We utilized the isolated perfused rat lung model to produce hydrostatic pulmonary edema by varying the left atrial and pulmonary capillary pressure. Pretreatment with KGF attenuated hydrostatic edema formation. This was demonstrated by lower wet-to-dry lung weight ratios, histological evidence of less alveolar edema formation, and reduced alveolar accumulation of intravascularly administered FITC-labeled large-molecular-weight dextran in rats pretreated with KGF. Thus KGF attenuates injury in this ex vivo model of hydrostatic pulmonary edema via mechanisms that prevent increases in alveolar-capillary permeability.     

Nitric oxide-dependent inhibition of alveolar fluid clearance in hydrostatic lung edema

Formation of cardiogenic pulmonary edema in acute left heart failure is traditionally attributed to increased fluid filtration from pulmonary capillaries and subsequent alveolar flooding. Here, we demonstrate that hydrostatic edema formation at moderately elevated vascular pressures is predominantly caused by an inhibition of alveolar fluid reabsorption, which is mediated by endothelial-derived nitric oxide (NO). In isolated rat lungs, we quantified fluid fluxes into and out of the alveolar space and endothelial NO production by a two-compartmental double-indicator dilution technique and in situ fluorescence imaging, respectively. Elevation of hydrostatic pressure induced Ca(2+)-dependent endothelial NO production and caused a net fluid shift into the alveolar space, which was predominantly attributable to impaired fluid reabsorption. Inhibition of NO production or soluble guanylate cyclase reconstituted alveolar fluid reabsorption, whereas fluid clearance was blocked by exogenous NO donors or cGMP analogs. In isolated mouse lungs, hydrostatic edema formation was attenuated by NO synthase inhibition. Similarly, edema formation was decreased in isolated mouse lungs of endothelial NO synthase-deficient mice. Chronic heart failure results in endothelial dysfunction and preservation of alveolar fluid reabsorption. These findings identify impaired alveolar fluid clearance as an important mechanism in the pathogenesis of hydrostatic lung edema. This effect is mediated by endothelial-derived NO acting as an intercompartmental signaling molecule at the alveolo-capillary barrier.     

Extra-alveolar vessel fluid filtration coefficients in excised and in situ canine lobes

We continuously weighed fully distended excised or in situ canine lobes to estimate the fluid filtration coefficient (Kf) of the arterial and venous extra-alveolar vessels compared with that of the entire pulmonary circulation. Alveolar pressure was held constant at 25 cmH2O after full inflation. In the in situ lobes, the bronchial circulation was interrupted by embolization. Kf was estimated by two methods (Drake and Goldberg). Extra-alveolar vessels were isolated from alveolar vessels by embolizing enough 37- to 74-micron polystyrene beads into the lobar artery or vein to completely stop flow. In excised lobes, Kf's of the entire pulmonary circulation by the Drake and Goldberg methods were 0.122 +/- 0.041 (mean +/- SD) and 0.210 +/- 0.080 ml X min-1 X mmHg-1 X 100 g lung-1, respectively. Embolization was not found to increase the Kf's. The mean Kf's of the arterial extra-alveolar vessels were 0.068 +/- 0.014 (Drake) and 0.069 +/- 0.014 (Goldberg) (24 and 33% of the Kf's for the total pulmonary circulation). The mean Kf's of the venous extra-alveolar vessels were similar [0.046 +/- 0.020 (Drake) and 0.065 +/- 0.036 (Goldberg) or 33 and 35% of the Kf's for the total circulation]. No significant difference was found between the extra-alveolar vessel Kf's of in situ vs. excised lobes. These results suggest that when alveolar pressure, lung volume, and pulmonary vascular pressures are high, approximately one-third of the total fluid filtration comes from each of the three compartments.     

Reversible alterations in cultured pulmonary artery endothelial cell monolayer morphology and albumin permeability induced by ionizing radiation

The effects of ionizing irradiation (0, 600, 1,500, or 3,000 rads) on the permeability of pulmonary endothelial monolayers to albumin were studied. Pulmonary endothelial cells were grown to confluence on gelatin-coated polycarbonate filters, placed in serum-free medium, and exposed to a 60Co source. The monolayers were placed in modified flux chambers 24 hours after irradiation; 125I-albumin was added to the upper well, and both the upper and lower wells were serially sampled over 4 hours. The amount of albumin transferred from the upper well/hour over the period of steady-state clearance (90-240 min after addition of 125I-albumin) was 2.8 +/- 0.2% in control monolayers and was increased in monolayers exposed to 1,500 or 3,000 rads (increase of 63 +/- 10% and 61 +/- 10%, respectively, P less than 0.01). No increase was found in monolayers exposed to 600 rads. The increases in endothelial albumin transfer rates were associated with morphologic evidence of monolayer disruption and endothelial injury which paralleled the changes in albumin permeability. Dose-dependent alterations in endothelial actin filament organization were also found. Incubation of the monolayers exposed to 3,000 rads with medium supplemented with 10% fetal calf serum for 24 hours resulted in normalization of albumin permeability, improvement in morphologic appearance of the monolayers, and reorganization of the actin filament structure. These studies demonstrate that ionizing radiation is an active principle in the reversible disorganization of cultured pulmonary endothelial cell monolayers without the need of other cell types or serum components.     

Extra-alveolar veins are contiguous with, and leak fluid into, periarterial cuffs in rabbit lungs.

We previously observed physiological evidence that arterial and venous extra-alveolar vessels shared a common interstitial space. The purpose of the present investigation was to determine the site of this continuity to improve our understanding of interstitial fluid movement in the lung. Orange G and Evans blue dyes were added to the arterial and venous reservoirs, respectively, of excised rabbit lungs as they were placed 20 cmH2O into zone 1 (pulmonary arterial and venous pressures = 5 cmH2O, alveolar pressure = 25 cmH2O). After 10 s or 4 h the lungs were fixed by immersion in liquid N2, freeze-dried, cut into 5-mm serial slices, and examined by light macroscopy. Serial sections of 0.25-0.5 mm were subsequently examined by scanning electron microscopy. In the animals subjected to the zone 1 stress for 4 h, arterial and venous extra-alveolar vessels were surrounded by cuffs of edema. The edema ratio (cuff area divided by vessel lumen area) was greater around arteries than veins and decreased with increasing vessel size. Periarterial cuffs usually contained orange dye and frequently contained both orange and blue dye. Lymphatics containing orange or blue dye were frequently seen in periarterial cuffs. Scanning electron microscopy demonstrated that extra-alveolar veins of approximately 100 microns diameter were anatomically contiguous with arterial extra-alveolar vessel cuffs. In rabbit lungs, both arterial and venous extra-alveolar vessels (and/or alveolar corner vessels) leak fluid into perivascular cuffs surrounding arterial extra-alveolar vessels, and lymphatics located in the periarterial cuff contain fluid that originates from both the arterial and venous extra-alveolar vessels.     

Fibrin contact increases endothelial permeability to albumin.

We studied the effects of contact of bovine pulmonary artery endothelial cell monolayers with fibrin on the endothelial barrier function. Fibrin formed by clotting purified fibrinogen (0.5 to 3.0 mg/ml) with alpha-thrombin (1 U/ml) was added to endothelial monolayers and permeability measurements were made after fibrin removal. Fibrin incubation for 3 hours resulted in 2- to 5-fold increases in transendothelial 125I-albumin permeability. Permeability returned to baseline value within 3 hours after fibrin removal. Direct contact with fibrin was necessary for the response, since fibrin separated from the endothelium did not increase permeability. Contact with agarose (2 mg/ml) or fibrinogen (0.5 to 3.0 mg/ml) also did not increase endothelial permeability. Transmission electron microscopic examination indicated normal appearance of interendothelial junctions at a time when albumin permeability was increased and no overt evidence of endothelial injury. Incubation of fibrin with endothelial monolayers at 4 degrees C prevented the increase in albumin permeability. We examined the possibility that increased albumin transcytosis was responsible for fibrin's effect using 14C-sucrose (Mr = 342D), a lipid insoluble tracer. Fibrin increased sucrose flux by 1.5-fold compared to 2- to 5-fold increases in albumin flux. The results indicate that fibrin contact with the endothelial cell increases endothelial permeability. The effect of fibrin may involve activation of temperature-sensitive bulk phase transcytosis of albumin.     

Blockade of platelet endothelial cell adhesion molecule-1 (PECAM-1) protects against ischemia-reperfusion injury in muscle flaps at microcirculatory level

Several lines of evidence show that platelet endothelial cell adhesion molecule-1 (PECAM-1), a component of endothelial cell junctions, is required for leukocyte transmigration through endothelial cell monolayers. Polymorphonuclear leukocytes play an important role in ischemia-reperfusion injury. We sought to determine whether administering an anti-PECAM-1 antibody would prevent or attenuate ischemia-reperfusion injury in a rat cremaster muscle flap injury model. Eighteen male Sprague-Dawley rats were divided into three groups. Group I (control): Cremaster muscle island flaps were dissected for baseline measurements of eight indicators: numbers of rolling, sticking, and transmigrating neutrophils, numbers of rolling and sticking lymphocytes, number of perfused capillaries, endothelial edema, and vessel permeability. Group II: The prepared cremaster flap was subjected to 4 hours of ischemia and 24 hours of reperfusion. Group III: The muscle flap was subjected to ischemia and reperfusion as in group II, and anti-PECAM-1 antibodies (1 mg/kg) were injected subcutaneously 15 minutes before reperfusion. Blood vessels were observed in vivo under an intravital microscopy system. Microvascular permeability was made visible with injected fluorescein isothiocyanate-labeled albumin and evaluated with Kontron Elektronik computer software. The ischemia-reperfusion-alone group (group II) presented a 225-percent increase in the activation of sticking leukocytes (2.4 +/- 0.4 to 7.8 +/- 0.8, p < 0.05) (p < 0.01). This leukocyte activation was reduced by 83 percent following anti-PECAM-1 monoclonal antibody treatment (1.3 +/- 0.5 per 100 microm) (p < 0.01). At 24 hours, endothelial injury in group II was confirmed by a 4-fold increase in the number of transmigrating leukocytes into the interstitial space (7.6 +/- 1.2 per field versus 1.9 +/- 0.4 per field in controls). This phenomenon was reduced by 85 percent following anti-PECAM-1 monoclonal antibody treatment (1.1 +/- 0.2 per field) (p < 0.01). Analysis showed that the number of flowing capillaries was 67 percent lower in group II (6.8 +/- 0.3 to 2.2 +/- 0.7, p < 0.01). Anti-PECAM-1 antibody treatment caused a 2.5-fold increase in this number (5.6 +/- 0.5, p < 0.01). Microcirculatory permeability index showed a 180-percent increase in group II (p < 0.05) when compared with baseline values. This increased albumin leakage was effectively attenuated by antibody treatment (p < 0.05). Blocking the action of PECAM-1 in vivo by administering monoclonal antibodies significantly attenuated ischemia-reperfusion injury, presumably by inhibiting transendothelial migration of neutrophils and by increasing capillary perfusion at a muscle flap microcirculatory level.     

Role of Rho-kinase in reexpansion pulmonary edema in rabbits

Reexpansion of a collapsed lung increases the microvascular permeability and causes reexpansion pulmonary edema. Neutrophils and their products have been implicated in the development of this phenomenon. The small GTP-binding proteins Rho and its target Rho-kinase (ROCK) regulate endothelial permeability, although their roles in reexpansion pulmonary edema remain unclear. We studied the contribution of ROCK to pulmonary endothelial and epithelial permeability in a rabbit model of this disorder. Endothelial and epithelial permeability was assessed by measuring the tissue-to-plasma (T/P) and bronchoalveolar lavage (BAL) fluid-to-plasma (B/P) ratios with (125)I-labeled albumin. After intratracheal instillation of (125)I-albumin, epithelial permeability was also assessed from the plasma leak (PL) index, the ratio of (125)I-albumin in plasma/total amount of instilled (125)I-albumin. T/P, B/P, and PL index were significantly increased in the reexpanded lung. These increases were attenuated by pretreatment with Y-27632, a specific ROCK inhibitor. However, neutrophil influx, neutrophil elastase activity, and malondialdehyde concentrations in BAL fluid collected from the reexpanded lung were not changed by Y-27632. In endothelial monolayers, Y-27632 significantly attenuated the H(2)O(2)-induced increase in permeability and mitigated the morphological changes in the actin microfilament cytoskeleton of endothelial cells. These in vivo and in vitro observations suggest that the Rho/ROCK pathway contributes to the increase in alveolar barrier permeability associated with reexpansion pulmonary edema.     

Filtration profile in isolated zone 1 and zone 3 dog lungs at constant high alveolar pressure

We measured the rate of liquid filtration in isolated dog lung lobes inflated to a constant alveolar pressure of 25 cmH2O and with all open vessels filled with plasma. We measured lung weight gain at vascular pressures ranging from 5 to 40 cmH2O relative to pleural pressure. We confirmed that under zone 1 conditions the "arterial" and "venous" extra-alveolar segments have essentially the same filtration characteristics. Using the combined extra-alveolar vascular system, we determined when recruitment of filtration surface area occurred as we increased vascular pressure from 0 to 40 cmH2O. Based on an abrupt increase in filtration rate as vascular pressure approached the zone 1/3 boundary, we infer that a sudden recruitment of exchange surface area occurred at that point. Based on the slopes of the zone 1 and zone 3 filtration profiles, we conclude that extra-alveolar vascular segments contribute approximately 25% of total to filtration in the lung under zone 3 conditions, although the exact vessels filtering under zone 1 conditions have yet to be determined. Our analysis of the data supports the concept that there is a difference in the perimicrovascular pressure around alveolar and extra-alveolar vessels, which in part may account for the apparent high filtration fraction apportioned to extra-alveolar vessels.     

Protein tyrosine phosphatase activity regulates endothelial cell-cell interactions,the paracellular pathway,and capillary tube stability

Protein tyrosine phosphorylation is tightly regulated through the actions of both protein tyrosine kinases and protein tyrosine phosphatases. In this study, we demonstrate that protein tyrosine phosphatase inhibition promotes tyrosine phosphorylation of endothelial cell-cell adherens junction proteins, opens an endothelial paracellular pathway, and increases both transendothelial albumin flux and neutrophil migration. Tyrosine phosphatase inhibition with sodium orthovanadate or phenylarsine oxide induced dose- and time-dependent increases in [14C]bovine serum albumin flux across postconfluent bovine pulmonary artery endothelial cell monolayers. These increases in albumin flux were coincident with actin reorganization and intercellular gap formation in both postconfluent monolayers and preformed endothelial cell capillary tubes. Vanadate (25 microM) increased tyrosine phosphorylation of endothelial cell proteins 12-fold within 1 h. Tyrosine phosphorylated proteins were immunolocalized to the intercellular boundaries, and several were identified as the endothelial cell-cell adherens junction proteins, vascular-endothelial cadherin, and beta-, gamma-, and p120-catenin as well as platelet endothelial cell adhesion molecule-1. Of note, these tyrosine phosphorylation events were not associated with disassembly of the adherens junction complex or its uncoupling from the actin cytoskeleton. The dose and time requirements for vanadate-induced increases in phosphorylation were comparable with those defined for increments in transendothelial [14C]albumin flux and neutrophil migration, and pretreatment with the tyrosine kinase inhibitor herbimycin A protected against these effects. These data suggest that protein tyrosine phosphatases and their substrates, which localize to the endothelial cell-cell boundaries, regulate adherens junctional integrity, the movement of macromolecules and cells through the endothelial paracellular pathway, and capillary tube stability.     

The effects of ionizing radiation on the pulmonary vasculature of intact rats and isolated pulmonary endothelium

We studied the effects of ionizing radiation on the morphology of the pulmonary circulation using an in vivo rat model and an in vitro pulmonary artery endothelial cell model. Gamma radiation was given as either an acute (30 Gy) or fractionated (5 X 6 Gy) dose to one hemithorax of rats. An acute 30-Gy dose delivered resulted in a 70% decrease in pulmonary arterial perfusion, using technetium-99m microaggregated albumin (99mTc-MAA), in the irradiated lung by 2-3 weeks after irradiation. Pulmonary microradiographs, using a barium sulfate perfusion method, obtained 2-3 weeks after irradiation demonstrated widespread loss of capillary filling and segmentation of the vessels. Histologic examination demonstrated intact capillaries, suggesting that the alterations in pulmonary perfusion were at the precapillary level. Similar abnormalities in lung perfusion and morphology were found after delivery of fractionated doses of radiation, but the onset of the changes was delayed, occurring 4-6 weeks postirradiation. Using cultured pulmonary endothelial cell monolayers, cell sloughing and retraction from the surface substrate were observed within 24 h after in vitro delivery of 30 Gy. Similar findings occurred in monolayers given fractionated doses (5 X 6 Gy) of radiation 2-3 days after the final dose. The in vivo animal and in vitro endothelial cell models offer a useful means of examining the morphologic alterations involved in radiation lung vascular damage.     

Roles of tumor necrosis factor p55 and p75 receptors in TNF-alpha-induced vascular permeability

We have investigated the role of p55 andp75 tumor necrosis factor receptors 1 and 2 (TNFR1 and TNFR2,respectively) in TNF-induced alteration of endothelial permeability invitro and in vivo. Stimulation of TNFR1 with an agonist antibody or areceptor-selective TNF mutein increased the flux of¹²⁵I-albumin through endothelial cell monolayers. Anantagonist anti-TNFR1 antibody, but not antagonist anti-TNFR2antibodies, blocked the activity of TNF in vitro. Stimulation of TNFR1,but not TNFR2, induced cytoskeletal reorganization associated withincreased permeability. SB-203580, a p38 mitogen-activated proteinkinase inhibitor, blocked TNFR1-induced cytoskeletal reorganization and permeability. A selective mouse TNFR1 agonist and human TNF, which binds to murine TNFR1, increased the leakage of trypan blue-albumin from liver vessels in mice. These results indicate that stimulation ofTNFR1 is necessary and sufficient to increase endothelial permeability in vitro and in vivo. However, an antagonist anti-murine TNFR2 antibodypartially inhibited the effect of murine TNF on liver vessels,suggesting that TNFR2 also plays a role in the regulation ofTNF-induced vascular permeability in vivo.

     

Indirect estimate of perimicrovascular pressure rise in edema in isolated zone 1 lung

To determine how liquid accumulation affects extra-alveolar perimicrovascular interstitial pressure, we measured filtration rate under zone 1 conditions (25 cmH2O alveolar pressure, 20 or 10 cmH2O vascular pressure) in isolated dog lung lobes in which all vessels were filled with autologous plasma. In the base-line condition, starting with normal extra-alveolar water content, filtration rate decreased by about one-half over 1 h as edema liquid slowly accumulated. We repeated each experiment after inducing edema (up to 100% lung weight gain). The absolute values and time course of filtration in the edema condition did not differ from base-line, i.e., the edema did not affect the time course of filtration. To compute the maximal initial and maximal change in extra-alveolar perimicrovascular pressure that occurred over each 1-h filtration study, we first assumed that the reflection coefficient is 0 in the Starling equation, then calculated perimicrovascular pressure and filtration coefficient from two equations with two unknowns. The mean filtration coefficient in 10 lobes is 0.063 g/(min X cmH2O X 100 g wet wt), and the initial perimicrovascular pressure is 3.9 cmH2O, rising by 4-7 cmH2O at 1 h. Finally we tested low protein perfusates and found the filtration rate was higher. We calculated an overall reflection coefficient = 0.44, a decrease in the initial perimicrovascular pressure to 1.9 cmH2O and a slightly lower increase after 1 h of edema formation, 2.2-6.6 cmH2O.