Measurement of albumin permeability across endothelial monolayers in vitro

We have developed an experimental system to measure the permeability of the cultured endothelial monolayer. The luminal-to-abluminal flux of 125I-albumin across cultured pulmonary endothelium was expressed as a clearance rate equal to the permeability-surface area product. After clearance rate measurement for a 30-min base-line period, a test agent was added to the luminal side, and the clearance rate was remeasured during a 30-min experimental period. In control studies the base-line clearance rate was 0.343 +/- 0.017 microliter/min. After correction for the diffusional resistances of the filter and unstirred layers, the calculated permeability of the endothelial monolayer was 1.2 X 10(-5) cm/s. When culture medium was the test agent, the experimental clearance rate was unchanged from the base-line value. After addition of 4 mM oleic acid to the luminal chamber, the clearance rate was 0.528 +/- 0.017 microliter/min compared with a base-line value of 0.330 +/- 0.008 microliter/min (P less than 0.005). This method allows the calculation of endothelial permeability with correction for unstirred layers and the use of each monolayer as its own control.     

Thrombin-induced adherence of neutrophils to cultured endothelial monolayers: increased endothelial adhesiveness

We examined the effects of alpha-thrombin on the adherence of neutrophils to endothelial cell monolayers. Endothelial cells derived from the ovine pulmonary artery and ovine neutrophils were used. Thrombin (10(-8) M) resulted in a time-dependent increase in neutrophil adherence to the endothelium. The response was concentration-dependent with a maximal response at 10(-8) M. Thrombin did not induce neutrophil adherence either to plastic or to endothelial cell-derived matrix. The adherence response was inhibited in the presence of alpha-thrombin that had been inactivated with anti-thrombin III (1U:1U) or with hirudin (1 U/ml). However, the addition of either anti-thrombin III or hirudin simultaneously with alpha-thrombin to the cultured endothelial monolayers did not prevent neutrophil adherence. The monoclonal antibody MoAb 60.3, which precipitates a complex of four neutrophil surface glycoproteins (CDw18) was used to further characterize the reaction. MoAb 60.3 decreased the thrombin-induced adherence of neutrophils to the endothelial monolayer. Addition of 10(-8) M thrombin to the endothelial monolayer for 60 min, followed by washing the endothelium with fresh medium, caused resting neutrophils to adhere to the endothelial monolayers. MoAb 60.3 decreased neutrophil adherence to the washed endothelium. The factor(s) responsible for adherence was partially transferable. Medium obtained from incubating endothelial monolayers with thrombin (10(-8) M) for 60 min, adding hirudin to the medium to inactivate thrombin, and transferring it to untreated endothelial monolayers, elicited neutrophil adherence. The response was less than that obtained with thrombin alone (22.9 +/- 2.3% vs. 12.9 +/- 3.3%). The results indicate that the catalytic site of the thrombin molecule is responsible for the adherent activity. Thrombin elicits a rapid activation of endothelial cells with a response that involves the expression of endothelial adhesion sites and sites that interact with the neutrophil CDw18 adhesive glycoprotein complex. In addition, soluble transferable factor(s) which are generated by the endothelium also contribute to thrombin-induced neutrophil adherence.     

Mechanism for the increased permeability in endothelial monolayers induced by elastase

The aim of this study was to investigate the mechanism for the increase in endothelial permeability induced by human neutrophil elastase (HNE). Pretreatment of bovine pulmonary artery endothelial cells (BPAEC) with HNE(0-30 mug/ml) for 1 h produced a concentration dependent increase in (125)I-albumin clearance. The effect was reversible and was not due to cytolysis. Pretreatment of BPAEC with sodium tungstate, which depletes xanthine oxidase, or with oxypurinol, did not prevent HNE induced increased permeability. Heparin, which neutralizes the cationic charge of HNE, also had no protective effect. Pretreatment with heat inactivated HNE, which still had positive charge sites, did not result in increased endothelial permeability. Also, ONO-5046, a novel specific inhibitor of HNE, did prevent increased permeability. These results suggest that elastase increases endothelial permeability mainly through its proteolytic effects.     

Linoleic acid hydroperoxide increases the transfer of albumin across cultured endothelial monolayers

Exposure to albumin-bound linoleic acid (60 to 150 microM) for 24 h significantly increased the rate of albumin transfer across cultured endothelial monolayers. The increase was dependent on the linoleic acid (18:2) concentration to which the cultures were exposed. Linoleic acid hydroperoxide (18:2-OOH) further accelerated the rate of albumin transfer over that of 18:2. A near maximum albumin transfer was observed after a 2-h incubation with 90 microM 18:2-OOH. Cells exposed to 18:2-OOH caused a marked release of lactate dehydrogenase into the media. On the other hand, 18:2 at concentrations as high as 150 microM, did not significantly affect lactate dehydrogenase release. These results suggest that exposure to 18:2, and in particular to 18:2-OOH, reduces the ability of the endothelium to act as a selective permeability barrier to plasma components.     

Protein kinase C phosphorylates caldesmon77 and vimentin and enhances albumin permeability across cultured bovine pulmonary artery endothelial cell monolayers.

Cytoskeletal protein (CSP) interactions are critical to the contractile response in muscle and non-muscle cells. Current concepts suggest that activation of the contractile apparatus occurs through selective phosphorylation by specific cellular kinase systems. Because the Ca(2+)-phospholipid-dependent protein kinase C (PKC) is involved in the regulation of a number of key endothelial cell responses, the hypothesis that PKC modulates endothelial cell contraction and monolayer permeability was tested. Phorbol myristate acetate (PMA), a direct PKC activator, and alpha-thrombin, a receptor-mediated agonist known to increase endothelial cell permeability, both induced rapid, dose-dependent activation and translocation of PKC in bovine pulmonary artery endothelial cells (BPAEC), as assessed by gamma-[32P]ATP phosphorylation of H1 histone in cellular fractions. This activation was temporally associated with evidence of agonist-mediated endothelial cell contraction as demonstrated by characteristic changes in cellular morphology. Agonist-induced activation of the contractile apparatus was associated with increases in BPAEC monolayer permeability to albumin (approximately 200% increase with 10(-6) MPMA, approximately 400% increase with 10(-8) M alpha-thrombin). To more closely examine the role of PKC in activation of the contractile apparatus, PKC-mediated phosphorylation of two specific CSPs, the actin- and calmodulin-binding protein, caldesmon77, and the intermediate filament protein, vimentin, was assessed. In vitro phosphorylation of both caldesmon and vimentin was demonstrated by addition of exogenous, purified BPAEC PKC to unstimulated BPAEC homogenates, to purified bovine platelet caldesmon77, or to purified smooth muscle caldesmon150. Caldesmon77 and vimentin phosphorylation were observed in intact [32P]-labeled BPAEC monolayers stimulated with either PMA or alpha-thrombin, as detected by immunoprecipitation. In addition, BPAEC pretreatment with the PKC inhibitor, staurosporine, prevented alpha-thrombin- and PMA-induced phosphorylation of both cytoskeletal proteins, attenuated morphologic evidence of contraction, and abolished agonist-induced barrier dysfunction. These results demonstrate that agonist-stimulated PKC activity results in cytoskeletal protein phosphorylation in BPAEC monolayer, an event which occurs in concert with agonist-mediated endothelial cell contraction and resultant barrier dysfunction.     

Permeability of endothelial monolayers to albumin is increased by bradykinin and inhibited by prostaglandins

Using monolayers of bovine aortic endothelial cells (BAEC) in modified Boyden chambers, we examined the role of prostaglandins (PGs) in the bradykinin (BK)-induced increase of albumin permeability. BK induced a concentration-dependent increase of the permeability of BAEC, which reached 49.9 +/- 1% at the concentration of 10(-8) M. Two inhibitors of the prostaglandin G/H synthase, indomethacin (2.88 microM) and ibuprofen (10 microM), potentiated BK-induced permeability 1.8- and 3.9-fold, respectively. Exogenously administered PGE2 and iloprost, a stable analog of prostacyclin, attenuated the effect of BK in a concentration-dependent manner. Butaprost equally reduced the effect of BK, suggesting the participation of the EP2 receptor in this phenomenon. However, the EP4-selective antagonist AH-23848 did not significantly inhibit the protective effect of PGE2. The inhibitory effect of PGE2 was reversed by the adenylate cyclase inhibitor MDL-12330A (10 microM). These results suggest that BK-induced increase of permeability of BAEC monolayer to (125)I-labeled albumin is negatively regulated by PGs. This postulated autocrine activity of PGs may involve an increase in the intracellular level of cAMP.     

Increased permeability of primary cultured brain microvessel endothelial cell monolayers following TNF-alpha exposure.

TNF-alpha is a cytokine that produces increased permeability in the peripheral vasculature; however, little is known about the effects of TNF-alpha on the blood-brain barrier (BBB). Using primary cultured bovine brain microvessel endothelial cells (BBMEC) as an in vitro model of the BBB, this study shows that TNF-alpha produces a reversible increase in the permeability of the brain microvessel endothelial cells. The BBMEC monolayers were pre-treated with 100 ng/ml of TNF-alpha for periods ranging from 2 to 12 hours. Permeability was assessed using three molecular weight markers, fluorescein (376 MW), fluorescein-dextran (FDX-4400; 4400 MW), and FDX-70000 (MW 70000). The permeability of BBMEC monolayers to all three fluorescent markers was increased two-fold or greater in the TNF-alpha treatment group compared to control monolayers receiving no TNF-alpha. Significant changes in permeability were also observed with TNF-alpha concentrations as low as 1 ng/ml. These results suggest that TNF-alpha acts directly on the brain microvessel endothelial cells in a dynamic manner to produce a reversible increase in permeability. Exposure of either the lumenal or ablumenal side of BBMEC monolayers to TNF-alpha resulted in similar increases in permeability to small macromolecules, e.g. fluorescein. However, when a higher molecular weight marker was used (e.g. FDX-3000), there was a greater response following lumenal exposure to TNF-alpha. Together, these studies demonstrate a reversible and time dependent increase in brain microvessel endothelial cell permeability following exposure to TNF-alpha. Such results appear to be due to TNF's direct interaction with the brain microvessel endothelial cell.     

Permeability characteristics of cultured endothelial cell monolayers

The purpose of this study was to characterize the permeability characteristics of an in vitro endothelial cell monolayer system and relate this information to available in vivo data. We cultured bovine fetal aortic endothelial cells on fibronectin-coated polycarbonate filters and confirmed that our system was similar to others in the literature with regard to morphological appearance, transendothelial electrical resistance, and the permeability coefficient for albumin. We then compared our system with in vivo endothelium by studying the movement of neutral and negatively charged radiolabeled dextran tracers across the monolayer and by using electron microscopy to follow the pathways taken by native ferritin. There were a number of differences. The permeability of our monolayer was 10-100 times greater than seen in intact endothelium, there was no evidence of "restricted" diffusion or charge selectivity, and ferritin was able to move freely into the subendothelial space. The reason for these differences appeared to be small (0.5-2.0 micron) gaps between 5 and 10% of the endothelial cells. Although the current use of cultured endothelial cells on porous supports may provide useful information about the interaction of macromolecules with the endothelium, there appear to be differences in the transendothelial permeability characteristics of these models and in vivo blood vessels.     

Protamine increases the permeability of cultured epithelial monolayers.

Polycations, including protamine, have been reported to decrease the barrier integrity of cultured rat pulmonary type II epithelial monolayers. In contrast, protamine has been reported to increase the transepithelial electrical resistance of gallbladder epithelium. The present study was done using Madin Darby canine kidney epithelial cells (MDCK) to determine whether the effect of protamine on type II epithelial monolayers was species or organ specific or was dependent on the presence of nonepithelial cells and to investigate the effect of protamine on the actin cytoskeleton. Exposure of MDCK monolayers to protamine resulted in decreased transepithelial electrical resistance (Rt), increased short-circuit current (Isc) across the monolayers, and increased mannitol permeability (Pmann) of the monolayers. The decrease in Rt and increase in Isc was seen only after the addition of protamine to the apical surface of the cells. The importance of charge in this action was supported by the fact that exposure of the monolayer to the polycation poly-L-lysine also resulted in increased Pmann, and both the decreased Rt and increased Pmann seen after the addition of protamine were prevented if the monolayers were exposed in the presence of the polyanions heparin or sulfated dextran. The increase in Pmann appeared to be the result of increased permeability in the paracellular pathway, because increased mannitol uptake by the cells represented only a fraction of the increase in Pmann. Subtle changes in the actin cytoskeleton were seen after exposure of the monolayers to protamine.(ABSTRACT TRUNCATED AT 250 WORDS)     

Thrombin's enzymatic activity increases permeability of endothelial cell monolayers

Human alpha-thrombin increases the permeability of bovine pulmonary artery endothelial cell (CCL-209) monolayers. To determine if this increase is via an enzymatic or receptor-mediated mechanism, enzymatically active forms of alpha-thrombin and enzymatically inactive forms with cell binding activity were incubated with the monolayers. Enzymatic forms included alpha-thrombin and two digestion products, zeta-thrombin (chymotryptic product with 89% clotting activity) and gamma-thrombin (tryptic product). Enzymatically inactive forms included D-Phe-Pro-Arg-chloromethylketone-(PPACK) alpha-thrombin and diisopropylphosphorofluoridate-(DIP) alpha-thrombin. Cell binding activity of alpha- and PPACK-alpha-thrombin was demonstrated to be similar to each other and comparable to that cited in the literature for DIP-alpha-thrombin. gamma-Thrombin, on the other hand, did not compete for binding of 125I-labeled alpha-thrombin. All enzymatic forms of alpha-thrombin increased endothelial permeability as assessed by the clearance of 125I-albumin across the monolayers. Coincubation of PPACK, an enzymatic site inhibitor, with alpha- or gamma-thrombin prevented the increase in permeability, further indicating that alpha-thrombin increased permeability by its enzymatic activity. Both enzymatically inactive forms of alpha-thrombin with high-affinity binding activity had no effect on permeability. To further examine whether cell binding activity of alpha-thrombin contributed to the increased permeability, a sulfated COOH-terminal fragment of hirudin (hirugen) that binds to the anion-binding site of alpha-thrombin but, unlike hirudin, does not interact with the catalytic site was coincubated with alpha-thrombin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Histamine type I receptor occupancy increases endothelial cytosolic calcium, reduces F-actin, and promotes albumin diffusion across cultured endothelial monolayers

Considerable evidence suggests that Ca2+ modulates endothelial cell metabolic and morphologic responses to mediators of inflammation. We have used the fluorescent Ca2+ indicator, quin2, to monitor endothelial cell cytosolic free Ca2+, [Ca2+]i, in cultured human umbilical vein endothelial cells. Histamine stimulated an increase in [Ca2+]i from a resting level of 111 +/- 4 nM (mean +/- SEM, n = 10) to micromolar levels; maximal and half-maximal responses were elicited by 10(-4) M and 5 X 10(-6) M histamine, respectively. The rise in [Ca2+]i occurred with no detectable latency, attained peak values 15-30 s after addition of stimulus, and decayed to a sustained elevation of [Ca2+]i two- to threefold resting. H1 receptor specificity was demonstrated for the histamine-stimulated changes in [Ca2+]i. Experiments in Ca2+-free medium and in the presence of pyrilamine or the Ca2+ entry blockers Co2+ or Mn2+, indicated that Ca2+ mobilization from intracellular pools accounts for the initial rise, whereas influx of extracellular Ca2+ and continued H1 receptor occupancy are required for sustained elevation of [Ca2+]i. Ionomycin-sensitive intracellular Ca2+ stores were completely depleted by 4 min of exposure to 5 X 10(-6) M histamine. Verapamil or depolarization of endothelial cells in 120 mM K+ did not alter resting or histamine-stimulated [Ca2+]i, suggesting that histamine-elicited changes are not mediated by Ca2+ influx through voltage-gated channels. Endothelial cells grown on polycarbonate filters restricted the diffusion of a trypan blue-albumin complex; histamine (through an H1- selective effect) promoted trypan blue-albumin diffusion with a concentration dependency similar to that for the histamine-elicited rise in [Ca2+]i. Exposure of endothelial cells to histamine (10(-5) M) or ionomycin (10(-7) M) was associated with a decline in endothelial F- actin (relative F-actin content, 0.76 +/- 0.07 vs. 1.00 +/- 0.05; histamine vs. control, P less than 0.05; relative F-actin content, 0.72 +/- 0.06 vs. 1.00 +/- 0.05; ionomycin vs. control, P less than 0.01). The data support a role for cytosolic calcium in the regulation of endothelial shape change and vessel wall permeability in response to histamine.     

Albumin permeability across endothelial monolayers under pulsatile shear stress

Recent studies suggest that the temporal gradient of shear stress that is generated by blood flow plays an important role in the pathology of arteriosclerosis. We focused on the temporal gradient of shear stress and measured the permeability of albumin under steady or pulsatile shear stress conditions. Porcine aortic endothelial cells were seeded on a membrane filter and subjected to steady or pulsatile shear stress (1 Hz) at 1 Pa for 48 h, and the permeability of albumin was measured over time. The permeability increased gradually under steady flow but increased acutely under pulsatile shear stress. In particular, the maximum permeability of albumin differed under these conditions. The value was 4.2 × 10^?5 cm/s at 18 h under pulsatile shear stress and 2.8 × 10^?5 cm/s at 48 h under steady shear stress. The permeable route of albumin was examined using isoproterenol, which decreases junctional permeability. The increase in albumin permeability with pulsatile shear stress was decreased by isoproterenol. These results suggest that the increased permeability of albumin with pulsatile shear stress was related to trafficking through paracellular junctions. Thus, pulsation may promote a mechanotransduction process that differs from that of steady shear stress, and these pulsation effects likely play an important role in the permeability of macromolecules.     

Hyperoxia causes decreased expression of vascular endothelial growth factor and endothelial cell apoptosis in adult retina

Mice or humans with photoreceptor degenerations experience permeability and dropout of retinal capillaries. Loss of photoreceptors results in decreased oxygen usage and thinning of the retina with increased oxygen delivery to the inner retina. To investigate the possibility that increased tissue oxygen plays a role in the vascular damage, we exposed adult mice to hyperoxia, which also increases oxygen in the retina. After 1, 2, or 3 weeks of hyperoxia, there was a statistically significant decrease in retinal vascular density that was not reversible, and endothelial cell apoptosis was demonstrated by TUNEL staining. Mice exposed to hyperoxia and mice with photoreceptor degeneration both showed decreased expression of VEGF in the retina. After complete or near-complete degeneration of photoreceptors, there was increased expression of VEGF in RPE cells, which may explain the association of photoreceptor degeneration and neovascularization in or around the RPE. Increased expression of VEGF in photoreceptors of transgenic mice failed to prevent hyperoxia-induced retinal capillary dropout. These data suggest that increased oxygen in the retina, either by increased inspired oxygen or by photoreceptor degeneration, results in endothelial cell death and dropout of capillaries. Decreased expression of VEGF may be a contributing factor, but the situation may be more complicated for mature retinal vessels than it is for immature vessels, because VEGF replacement does not rescue mature retinal vessels, suggesting that other factors may also be involved.     

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.     

Effects of specific signal transduction inhibitors on increased permeability across rat endothelial monolayers induced by neuropeptide Y or VEGF

Neuropeptide Y (NPY) elevates the permeability of cultured rat aortic endothelial cells (RAECs) in monolayer cultures under hypoxic conditions (5% O(2)) possibly by binding to the NPY Y(3) receptor. The present study evaluated the effects of NPY compared to vascular endothelial growth factor (VEGF). RAECs were cultured on the upper chamber base of a double-chamber culture system, FITC-labeled albumin was introduced into the chamber, and permeation into the lower chamber was measured. Treatment was with 3 x 10(-7) M NPY or 10(-7) g/ml VEGF for 2 h along with specific inhibitors. The VEGF receptor-2 tyrosine kinase inhibitor tyrphostin SU-1498 and the protein kinase C inhibitor bis-indolylmaleimide I (GF-109203X) suppressed the VEGF-induced increase in monolayer permeability but not that caused by NPY. Furthermore, although the action of NPY was blocked in a concentration-dependent manner by phospholipase C inhibitor 1-(6-[[(17beta)-3-methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl)-1H-pyrrole-2,5-dione (U-73122), it was less sensitive than VEGF. However, the effects of both NPY and VEGF on the permeability of the RAEC monolayer were blocked with equal concentration dependence by STI571 (imatinib mesylate), which is an inhibitor of Abl tyrosine kinase in the nucleus and/or cytoplasm. The myosin light-chain kinase inhibitor 1-(5-chloronaphthalene-1-sulfonyl)-1H-hexahydro-1,4-diazepine HCl (ML-9) suppressed both NPY- and VEGF-induced increment in permeability by approximately 70%, whereas the calmodulin-dependent kinase inhibitor DY-9760e could decrease to below the baseline. These results indicate that the NPY Y(3)-receptor subtype is specifically linked to the effects of STI571 on endothelial cells, and that NPY, a sympathetic coneurotransmitter, may increase vascular permeability in association with altered intracellular or nuclear signal transduction.     

Platelets attenuate oxidant-induced permeability in endothelial monolayers: glutathione-dependent mechanisms

Strange, Charlie, Andrew Gottehrer, Karen Birmingham, andJohn E. Heffner. Platelets attenuate oxidant-induced permeability in endothelial monolayers: glutathione-dependent mechanisms.J. Appl. Physiol. 81(4):1701-1706, 1996.We studied the effects of adding washedhuman platelets or platelets with nonintact glutathione redox cycles toendothelial cell monolayers treated with glucose oxidase to initiateoxidant stress and increase permeability. Changes in¹²⁵I-labeled albumintransmonolayer movement were used as the index of monolayerpermeability. Washed human platelets attenuated oxidant-induced increases in albumin flux. Platelets treated with1,3-bis(2-chloroethyl)-1-nitrosurea, 1-chloro-2,4-dinitrobenzene, orbuthionine sulfoximine to inhibit selective enzymatic steps inthe glutathione redox cycle decreased permeability to a lesser degree.We conclude that 1) washed human platelets attenuate monolayer permeability defects in aorticendothelial monolayers exposed to glucose oxidase and2) the protective effects ofplatelets are partially dependent on an intact plateletglutathione redox cycle.

     

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.