Hazel Lum

Reoxygenation of endothelial cells increases permeability by oxidant-dependent mechanisms.

We investigated the effects of hypoxia/reoxygenation exposure on the barrier function of endothelial cell monolayers. Bovine pulmonary microvessel endothelial cells were grown to confluence on microporous filters (0.8-microns pore diameter) and exposed to hypoxia (0.1% O2 or PO2 approximately 1 mm Hg) for 2, 4, 12, or 24 hours, followed by reoxygenation with room air for a period ranging from 16 seconds to 2 hours. The transendothelial clearance rate of 125I-albumin was measured to determine the permeability of endothelial monolayers. Permeability increased twofold or fivefold over control values after 1 hour of reoxygenation in monolayers that had been exposed to either 12 or 24 hours of hypoxia. The response occurred within 5 minutes of reoxygenation, increased maximally by 40 minutes, and remained elevated with continuous reoxygenation for up to 2 hours. The increase in permeability was associated with F-actin reorganization, a change to spindlelike cells, and injured mitochondria. Immunoblot analysis indicated that neither hypoxia alone nor reoxygenation changed CuZn superoxide dismutase (SOD), MnSOD, and catalase levels. However, release of superoxide anions (O2-) into the extracellular medium increased by twofold within 40-60 minutes of reoxygenation. Treatment of endothelial cells with CuZnSOD (100 units/ml) for the 24-hour hypoxia period prevented O2- generation and approximately 50% of the increase in permeability. Higher CuZnSOD concentrations (greater than or equal to 200 units/ml) were not protective. Treatment with catalase (100-1,000 units/ml) inhibited the reoxygenation-induced increase in permeability at the highest catalase concentration (1,000 units/ml), suggesting a critical role of hydrogen peroxide in mediating the response.(ABSTRACT TRUNCATED AT 250 WORDS)

Time course of thrombin-induced increase in endothelial permeability: relationship to Ca2+i and inositol polyphosphates

The temporal relationship between the alpha-thrombin-induced increase in transendothelial permeability and the alpha-thrombin-mediated changes in several key transmembrane signaling events was examined in confluent monolayers of bovine pulmonary artery endothelial cells (BPAEC). The time courses of inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] generation, changes in cytosolic [Ca2+] ([Ca2+]i), and reorganization of cytoskeletal F-actin were determined to assess the relationship between these events and the onset of the alpha-thrombin-induced increase in endothelial permeability. alpha-Thrombin (10(-7) M) increased the transendothelial 125I-albumin clearance rate half-maximally by approximately 1 min and maximally by approximately 2 min (160% over control level). The increase in permeability occurred concomitantly with reorganization of F-actin cytoskeleton (i.e., loss of peripheral band and increased stress fiber density) and increased actin polymerization. Stimulation of fura-2-loaded BPAEC with 10(-7) M alpha-thrombin produced a typical biphasic rise in [Ca2+]i. The initial rapid increase in [Ca2+]i peaked by approximately 16 s after thrombin challenge and the [Ca2+]i response showed a slow decrease to half-maximal within 50 s. The alpha-thrombin-induced increase in permeability as well as the increase in [Ca2+]i were consistently preceded by increased Ins(1,4,5)P3 generation detectable within 10 s after thrombin challenge. These results indicate that alpha-thrombin triggers a cascade of events (i.e., Ins(1,4,5)P3 generation and the ensuing rise in [Ca2+]i), which may comprise the second messengers that mediate F-actin reorganization and the increase in endothelial permeability.

Culture and characterization of pulmonary microvascular endothelial cells.

SummarySurface proteins were compared in endothelial cells (EC) obtained from bovine peripheral lung, pulmonary artery and vein, and dorsal aorta using sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis. Galactose-containing glycoproteins [molecular weight (Mr) 160–220 and 40 kDa] binding to theRicinus communis agglutinin (RCA) and peanut agglutinin (PNA) were selectively observed on pulmonary microvessel EC as compared to EC from pulmonary artery, pulmonary vein, and dorsal aorta. The unique RCA- and PNA-binding profiles of EC from the pulmonary artery and microvessels may be important in characterizing EC from different sites in the pulmonary circulation. The pulmonary microvessel EC monolayer was also 15-fold more restrictive to transendothelial flux of [14C]sucrose (Mr=342 Da) than the pulmonary artery EC monolayer. In contrast, the microvessel EC were only six- and twofold more restrictive to the flux of larger tracer molecules, ovalbumin (Mr 43 kDa) and albumin (Mr=69 kDa) than pulmonary artery EC. The greater restrictiveness of pulmonary microvessel EC monolayer indicates a major phenotypic difference in the cultured pulmonary microvessel EC barrier function.

Pulmonary edema induced by phagocytosing neutrophils. Protective effect of monoclonal antibody against phagocyte CD18 integrin.

We studied the changes in pulmonary hemodynamics and lung wet weight induced with opsonized zymosan (OZ) in isolated guinea pig lungs perfused with Ringer-albumin solution containing neutrophils (PMNs). Addition of OZ to the PMN-perfused lungs caused pulmonary vasoconstriction and weight gain; neither OZ nor PMNs added individually to the perfusate altered pulmonary vasomotor tone or wet weight. The steady gain in lung weight by 1,588 +/- 464 mg over the 45-minute study period was associated with pulmonary capillary hypertension and an increase in the capillary filtration coefficient, indicative of increased lung vascular permeability. These responses may not be due to generation of oxygen radicals, because the alterations in pulmonary hemodynamics and lung weight were not reduced by addition of superoxide dismutase, catalase, or superoxide dismutase plus catalase. We examined the basis of the PMN-mediated effects by layering PMNs on bovine pulmonary artery endothelial monolayers. Challenge with OZ resulted in increased endothelial permeability to 125I-albumin. The monoclonal antibody IB4 (directed against CD18, the common beta-subunit of structurally related adhesion receptors on phagocytes, LFA-1, Mac-1, and P150,95) prevented the OZ-mediated increase in PMN adherence to endothelial cells and the increase in endothelial permeability to 125I-albumin. IB4 also inhibited the lung weight gain mediated by the OZ-stimulated PMNs in intact lungs. The protective effect of IB4 could be ascribed neither to inhibition of uptake of OZ by PMNs nor to inhibition of release of oxygen radicals, myeloperoxidase, and elastase.(ABSTRACT TRUNCATED AT 250 WORDS)

Serum albumin decreases transendothelial permeability to macromolecules

We examined the effects of serum albumin and other serum proteins on the fluxes of tracer 125I-albumin (MW 69 kDa) and 125I-haptoglobin (MW 100 kDa) across the pulmonary artery endothelial monolayer in vitro to test the role of serum proteins in modulating the endothelial barrier function. Replacement of control complete culture medium (20% fetal calf serum in DMEM) with DMEM alone increased the transendothelial 125I-albumin clearance rate (a measure of 125I-albumin permeability) by 83% of the control value. Repletion with 50% calf serum or with 2.0 g% albumin (i.e., the albumin concentration in 50% serum) decreased 125I-albumin permeability to the control value. This effect of serum or albumin was concentration-dependent since neither 12.5% serum nor 0.5 g% albumin (i.e., albumin concentration in 12.5% serum) altered 125I-albumin permeability from control values. The ammonium sulfate-precipitated serum protein fraction rich in albumin decreased 125I-albumin permeability from the control DMEM value, whereas serum fractions containing predominantly gamma-globulin or depleted of protein did not significantly alter 125I-albumin permeability. Other serum proteins that have been proposed to reduce endothelial permeability, alpha 1-acid glycoprotein (0.035-0.14 g/100 ml) and fibronectin (5 mg/100 ml), did not decrease 125I-albumin permeability from DMEM values. The endothelial permeability of 125I-haptoglobin of 4.63 +/- 0.53 x 10(-6) cm/sec in the presence of DMEM was 30% of the 125I-albumin permeability value. The addition of 2.0 g% albumin or 50% serum decreased 125I-haptoglobin permeability to 57 and 31%, respectively, of the DMEM value. These results indicate the critical role of serum albumin in regulating the restrictiveness of the endothelial barrier to macromolecules.

Selectivity of the endothelial monolayer: effects of increased permeability.

We investigated the mechanism of thrombin-induced increases in endothelial monolayer permeability by examining the effect of thrombin on the molecular sieving characteristics of the endothelial monolayer and comparing the responses of arterial- and venous-derived endothelial cell lines. Bovine pulmonary artery (BPA) and pulmonary vein (BPV) endothelial cells were similarly harvested and cultured. The endothelial cells were grown to confluence on gelatinized polycarbonate filters and the permeabilities to sucrose, albumin, and IgG were measured and corrected for effects of unstirred layers. The control permeabilities of BPA and BPV were similar with both monolayers, demonstrating selectivity to different sized tracer molecules. alpha-Thrombin (10(-6) M) increased the permeability of both BPA and BPV to albumin and sucrose. The permeability of BPA was increased to a greater extent than BPV, perhaps due to phenotypic differences. In both cell lines, the permeability increase was most pronounced for albumin, which by pore theory is best described by an increase in the radius of the small pore pathway for diffusion.