Calf pulmonary artery endothelial monolayers cultured on polycarbonate filters were utilized to study 125I-labeled albumin permeability and actin filament distribution in response to thrombin challenge. Thirty-minute exposure to alpha-thrombin (10(-7) M) significantly increased albumin clearance rates. These changes were associated with marked alterations in actin filament distribution, resulting in loss of peripheral actin bands and an increase in the number of cytoplasmic stress fibers. Because the actin peripheral filaments are thought to play an important role in junctional stability, we postulated that stabilization of actin filaments should protect against thrombin-induced barrier disruptions. Pretreatment of cells with 0.3 microM 7-nitrobenz-2-oxa-1,3-diazole (NBD)-phallacidin, a specific actin-stabilizing agent, prevented the changes in actin filament distribution and markedly attenuated the increase in albumin permeability. Because of the potential toxicity of phallatoxins, we evaluated the effects of pretreatment on cell viability and growth parameters. There were no differences in viability, seeding efficiency, or doubling times in cells treated with 0.3 microM NBD-phallacidin in comparison to controls. Our data support the hypothesis that actin filaments, particularly peripheral bands, contribute significantly to the maintenance of barrier function in cultured endothelial cells.

The development of new vessels (angiogenesis) is essential to wound healing. The center of a wound space is hypoxic, a condition that has been shown to stimulate angiogenesis in animal models of coronary artery occlusion. Because the mechanisms involved in this complex process are difficult to study in situ, an in vitro model would provide a useful complement to in vivo studies. This laboratory has developed and characterized calf pulmonary microvessel endothelial cell (PMVEC) cultures and an in vitro model system of angiogenesis using collagen three-dimensional gels that permit migration of cells into vessel networks. This system was used to study the direct effect of normoxia (20% O2) or hypoxia (5% O2) on PMVEC ability to undergo angiogenesis in vitro. Major changes leading to formation of capillary-like networks occurred during the first 3 days of hypoxic exposure only and included restructuring of actin filament networks, focal changes in distribution of basic fibroblast growth factor, and orientation and migration of cell tracts into a collagen gel matrix to form vessel networks.

We investigated the effect of H2O2 (92 microM) in isolated guinea pig lungs perfused with a buffered Ringer solution. Pulmonary arterial pressure (Ppa), pulmonary capillary pressure (Ppc), and change in lung weight (delta W) were recorded at 0 min and at 15, 30, and 60 min after the H2O2. The capillary filtration coefficient (Kfc) was measured at 0 and 30 min. The perfusion of H2O2 increased the Ppa, Ppc, delta W, and Kfc. The thromboxane synthetase inhibitor Dazoxiben, or the vasodilator papaverine, prevented the increases in Ppa and Ppc. The protein kinase C (PKC) inhibitor H7 [1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride] prevented the increases in Ppa, Ppc, delta W, and Kfc, whereas the inactive isoquinoline HA1004 [N-(2-guanidinoethyl)-5-isoquinolinesulfonamide hydrochloride] had little effect on the H2O2 response. H2O2 increased the number of stress fibers and disrupted the peripheral band of cultured confluent endothelial cells, changes that were prevented with pretreatment with H7. PKC may mediate the increases in vascular permeability and pulmonary edema that occur in response to H2O2.

The cell-surface localization and site of activation of type IV collagenases/gelatinases (matrix metalloproteinases, MMP) in bovine pulmonary microvascular endothelial (BPMVE) cells was examined. Sucrose density centrifugation of plasma membranes and immunofluorescent staining of whole cells indicated association of 72 kDa (MMP-2) and 96 kDa (MMP-9) type IV collagenase/gelatinases with the plasma membrane. Incubation of the BPMVE cells with rhodaminated MMP-9 demonstrated colocalization with beta 1-integrin, indicating incorporation into the focal contacts. The focal contacts were extracted with saponin, and associated proteolytic activity was examined by zymography. The focal contacts contained latent MMP-2, and stimulation of the cells with cytochalasin D or with 8-bromoadenosine 3',5'-cyclic monophosphate with 3-isobutyl-1-methylxanthine increased both latent and activated MMP-9 in the focal contacts. Addition of these stimuli in unconditioned culture medium did not produce this effect, indicating that the MMP-9 in focal contact extracts was derived from previously secreted enzyme. The activated metalloproteinase degraded extracellular matrix collagens and was inhibited by 1,10-phenanthroline. These findings indicate that endothelial cells release MMP into the extracellular milieu and then concentrate and activate MMP-9 from medium at the focal contacts.

We tested the hypothesis that human recombinant tumor necrosis factor-alpha (TNF) promotes pulmonary edema by neutrophil-dependent effects on the pulmonary vasculature. The isolated guinea pig lung was perfused with phosphate-buffered Ringer's solution with or without human neutrophils. The infusion of neutrophils (9 x 10(6) total) into lungs isolated after the in vivo administration of TNF (3.2 x 10(5) units/kg) resulted in weight gain (+1.951 +/- 0.311 g versus -0.053 +/- 0.053 g in control) and an increase in the lung (wet-dry)-to-dry weight ratio (8.3 +/- 0.5 versus 6.0 +/- 0.2 in control), indicating the formation of pulmonary edema. The neutrophil-dependent pulmonary edema induced by TNF was associated with a combination of increased capillary permeability (capillary filtration coefficient [Kf,c], 0.170 +/- 0.048 g/min/cm H2O/g at 30 minutes versus 0.118 +/- 0.008 g/min/cm H2O/g at baseline) and increased pulmonary capillary pressure (Ppc, 12.8 +/- 0.8 cm H2O at 60 minutes versus 6.0 +/- 0.3 cm H2O at baseline). The Ppc increase was mediated by thromboxane A2 (TXA2) because the TXA2 synthetase inhibitor Dazoxiben (0.5 mM) prevented the effect (Ppc, 6.7 +/- 0.6 cm H2O at 60 minutes with Dazoxiben), and thromboxane B2 (TXB2) levels were increased in the pulmonary venous effluent (5,244 +/- 599 pg/ml at 60 minutes versus 60 +/- 13 pg/ml at baseline). Studies using WEB-2086 (37 microM), a platelet activating factor (PAF) receptor antagonist, indicated that PAF mediated the increased vascular permeability (Kf,c, 0.107 +/- 0.014 g/min/cm H2O/g at 30 minutes using WEB-2086) and, in part, the increased Ppc (Ppc, 8.4 +/- 0.7 cm H2O at 60 minutes using WEB-2086). In addition, alterations of endothelial peripheral actin bands were noted after TNF administration. The data indicate that TNF induces neutrophil-dependent pulmonary edema associated with increased Ppc (mediated by TXA2 and PAF), increased Kf,c (mediated by PAF), and changes in endothelial peripheral actin bands.