Nitric oxide modulates capillary formation at the endothelial cell-tumor cell interface.

Nitric oxide synthase expression has been documented in lung tumors, but a potential role for nitric oxide (NO) in induction of capillary formation remains to be elucidated. The purpose of this report was to characterize the direct effects of NO at the level of the tumor-endothelium interface with respect to angiogenesis. A Transwell two-compartment culture system, human endothelial cells (EC), and two human non-small cell lung cancer (CA) lines that constitutively produce NO were used to simulate the EC-tumor cell interface. Both histological types of lung CA, squamous and adenocarcinoma, induced baseline capillary formation by EC within 3 days. This process was inhibited by NO in the microenvironment because decreasing NO production with 100 microM aminoguanidine (AG) significantly increased capillary formation, whereas coincubation with 100 microM AG plus 400 microM L-arginine returned angiogenesis to baseline values. We demonstrate further that NO may exert its inhibitory effects by influencing matrix metalloproteinase expression/activity and tyrosine phosphorylation of proteins in the sprouting tips of nascent capillaries.

Localization and activation of type IV collagenase/gelatinase at endothelial focal contacts.

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.

Prediction of posterior fossa tumor type in children by means of magnetic resonance image properties, spectroscopy, and neural networks.

Recent studies have explored characteristics of brain tumors by means of magnetic resonance spectroscopy (MRS) to increase diagnostic accuracy and improve understanding of tumor biology. In this study, a computer-based neural network was developed to combine MRS data (ratios of N-acetyl-aspartate, choline, and creatine) with 10 characteristics of tumor tissue obtained from magnetic resonance (MR) studies, as well as tumor size and the patient's age and sex, in hopes of further improving diagnostic accuracy. Data were obtained in 33 children presenting with posterior fossa tumors. The cases were analyzed by a neuroradiologist, who then predicted the tumor type from among three categories (primitive neuroectodermal tumor, astrocytoma, or ependymoma/other) based only on the data obtained via MR imaging. These predictions were compared with those made by neural networks that had analyzed different combinations of the data. The neuroradiologist correctly predicted the tumor type in 73% of the cases, whereas four neural networks using different datasets as inputs were 58 to 95% correct. The neural network that used only the three spectroscopy ratios had the least predictive ability. With the addition of data including MR imaging characteristics, age, sex, and tumor size, the network's accuracy improved to 72%, consistent with the predictions of the neuroradiologist who was using the same information. Use of only the analog data (leaving out information obtained from MR imaging), resulted in 88% accuracy. A network that used all of the data was able to identify 95% of the tumors correctly. It is concluded that a neural network provided with imaging data, spectroscopic data, and a limited amount of clinical information can predict pediatric posterior fossa tumor type with remarkable accuracy.

Hypoxia induces capillary network formation in cultured bovine pulmonary microvessel 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.

Thrombin-induced alterations in endothelial cell cytoarchitectural and functional properties.

alpha-Thrombin interacts with confluent bovine pulmonary artery endothelial cells to produce two types of actin microfilament rearrangements: (1) The loss of cortical actin correlates with interruption of barrier integrity, evident from increased permeability to 125I-albumin; and (2) an increase in actin stress fibers results in greater adherence of the cells to their extracellular substrate. Use of phallatoxin compounds that stabilize actin filaments and prevent their depolymerization prevents both the cytoarchitectural and functional changes resulting from thrombin challenge.

Tumor necrosis factor-alpha activates pulmonary artery endothelial protein kinase C.

We investigated the hypothesis that tumor necrosis factor-alpha (TNF) activates pulmonary endothelial protein kinase C (PKC). Confluent bovine pulmonary artery endothelial monolayers were exposed to recombinant human TNF, and the translocation of PKC, an indicator of enzyme activation, was studied using both slot immunoblotting and immunofluorescence. For slot immunoblot analysis, membrane and cytosol lysate fractions were prepared, and PKC antigen was assessed using MC5 monoclonal anti-PKC antibody. TNF (1,000 U/ml for 15 min) induced translocation of PKC into the membrane. Immunofluorescence analysis with the MC5 antibody was also used. Monolayers treated with culture medium showed diffuse cytoplasmic fluorescence. In contrast, treatment with either TNF (1,000 U/ml for 15 min) or 1,2-dioctanoylglycerol (4 x 10(-5) M for 5 min), a diacylglycerol that activates PKC, resulted in translocation of fluorescence to the cell periphery; fine, punctate PKC-associated fluorescence was localized to the margins of cells. The TNF-induced translocation of PKC was inhibited using either IP-300 polyclonal anti-TNF antibody (indicating that the TNF effect was not due to the vehicle or contaminating endotoxin) or calphostin C (10(-6) M for 15 min), which inhibits PKC activation by interacting with the regulatory diacylglycerol-binding domain. TNF treatment had no effect on either the content of PKC, or of total protein, in the membrane + cytosol, and cycloheximide (40 microM for 5 min) did not alter the translocation of PKC induced by TNF; these results indicate that the effect of TNF on PKC translocation was related to neither de novo membrane synthesis of PKC (as opposed to translocation per se) nor nonspecific augmentation of protein synthesis.(ABSTRACT TRUNCATED AT 250 WORDS)

Influence of extracellular matrix in tumor necrosis factor-induced increase in endothelial permeability.

We examined the possibility that alterations of the extracellular matrix (ECM) contribute to the tumor necrosis factor-alpha (TNF-alpha)-induced increase in endothelial monolayer permeability. Endothelial permeability to 125I-labeled albumin was determined using bovine pulmonary microvessel endothelial cell (BPMVE) monolayers grown to confluence on microporous (0.8 microns diam) gelatin- and fibronectin-coated polycarbonate filters. Treatment of BPMVE with TNF-alpha (10(2) to 10(4) U/ml for 4-24 h) produced concentration- and time-dependent increases in endothelial permeability that paralleled the changes in morphology from cobblestone to elongated cells and the formation of prominent intercellular gaps and actin stress fibers. We examined the role of ECM in these changes using filters coated with ECM made by the BPMVE. Fresh BPMVE seeded onto filters coated with ECM produced by TNF-alpha-treated BPMVE had two- to threefold higher 125I-albumin permeability values than BPMVE monolayers seeded onto filters coated with ECM from control cells (P < 0.05). BPMVE seeded onto ECM from TNF-alpha-treated BPMVE also developed intercellular gaps and centralized actin filaments characteristic of the TNF-alpha-treated BPMVE. This effect was not attributable to TNF-alpha adsorbed to ECM. Polyacrylamide gel electrophoresis of ECM extracted from BPMVE treated with TNF-alpha showed decreased fibronectin. These findings suggest that the TNF-alpha-induced increase in endothelial permeability involves the loss of fibronectin and remodeling of the ECM. The increase in endothelial permeability may be secondary to decreased endothelial cell-ECM contacts resulting in elongation of cells and formation of intercellular gaps.

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.

Hyperoxia increases plasminogen activator activity of cultured endothelial cells.

Confluent calf pulmonary artery endothelial monolayers exposed to 95% oxygen for 1, 2, or 3 days exhibit a time-dependent increase in adherence to substratum, which closely parallels changes in actin cytoarchitecture and the distribution of focal contact proteins vinculin and talin. Oxygen exposure also resulted in elevated plasminogen activator (PA) activity in conditioned media (CM) and in cytoskeletal protein- and focal contact protein-enriched fractions, with highest levels achieved in the latter two fractions at 48 h after oxygen exposure. PAs have been shown to participate in dismantling of extracellular matrix in a number of physiological and pathological situations. Immunocytochemical studies demonstrated extensive restructuring of matrix proteins collagen IV, laminin, and fibronectin, which correlated temporally with elevated PA levels. Further, when protease-containing cell fractions were used to study degradation of isolated matrices, those obtained from hyperoxia-exposed cells were substantially more active than those from normoxia-exposed cells. Our data suggest that hyperoxia-induced production of PA (and perhaps other proteases) may be partly responsible for degradation of the extracellular matrix of endothelial cells.

Mechanisms of pulmonary edema induced by tumor necrosis factor-alpha.

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.

The substrate-associated protein p45 of porcine endothelial cells: multiple isoforms, cytoskeletal-like properties and induction by hyperoxic stress.

1. Cultured mesenchymal cells respond to hyperoxic (hyper-O2) stress with increased cell flattening/substrate adhesion and overall 47-69% reductions in total matrix-associated (i.e. saponin-resistant [SAP fraction]) protein. 2. Electrophoretic analysis revealed a selective hyper-O2-related 2.7- to 4-fold increase in SAP and cytoskeletal fraction deposition of the protein p45 beginning early (within 12 hr) after initial exposure of porcine endothelial cells to hyper-O2 and increasing over a 48 hr period. 3. p45 consisted of 8 distinct isoforms differing only in pI; hyper-O2-augmented matrix deposition of 3. p45 consisted of 8 distinct isoforms differing only in pI; hyper-02-augmented matrix deposition of p45 involved all 8 isoforms with the more basic subtypes exhibiting slightly greater net increases. 4. Both the specificity and time course of p45 induction, relative to the onset of hyper-O2 cytoarchitectural remodeling, indicate that p45 up-regulation constitutes an early aspect of the hyper-O2 adaptive response.

Phallacidin prevents thrombin-induced increases in endothelial permeability to albumin.

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.

Effect of hyperoxia on the cytoarchitecture of cultured endothelial cells.

When confluent pulmonary artery endothelial cells in culture were exposed to hyperoxia (95% O2 and 5% CO2), they became enlarged and mean corpuscular volume increased 30-35%. Rhodamine-phalloidin staining of actin filaments demonstrated that hyperoxia was associated with a progressive alteration in the actin distribution. Three days after oxygen exposure, the number and thickness of cytoplasmic stress fibers were increased, while the peripheral bands were disrupted or absent. Sodium dodecyl sulfate polyacrylamide gel electrophoresis revealed that the amount of filamentous actin was increased in oxygen-exposed cells, while the total actin content remained unchanged, suggesting that oxygen exposure shifted the equilibrium from G actin to F actin.

Direct staining and visualization of endothelial monolayers cultured on synthetic polycarbonate filters.

Endothelial and epithelial cells cultured on synthetic filter supports have been used to study permeability and transport under various experimental conditions. However, because of the non-transparent nature of these filters, morphological studies using light microscopy are not possible. Presently, investigators circumvent this problem by using cells cultured on glass coverslips, extrapolating morphological data from a system clearly different from that used for functional studies. We describe here a useful technique for direct staining and visualization of cells grown on polycarbonate filter supports, using fluorochrome probes and fluorescence microscopy. We have utilized acridine orange, rhodamine phalloidin, and an anti-vimentin monoclonal antibody to provide information about cell shape, monolayer configuration, and cytoskeletal protein distribution in cultured calf pulmonary artery endothelial cell monolayers. Comparison staining of coverslip and filter preparations revealed a number of clear differences in these parameters. This technique should enable investigators to perform the necessary studies to obtain direct correlations between functional and morphological data.

L-2-oxothiazolidine-4-carboxylate protects cultured endothelial cells against hyperoxia-induced injury.

When bovine pulmonary artery endothelial monolayers were exposed to hyperoxia (95% O2 and 5% Co2), they responded by selectively elevating the intracellular concentration of glutathione without affecting the activities of glutathione peroxidase or glutathione reductase, L-2-Oxothiazolidine-4-carboxylate, an intracellular cysteine-delivering agent, further enhanced the intracellular concentration of glutathione in oxygen-exposed endothelial cells and protected them from the lethal effect of hyperoxia. In contrast, buthionine sulfoximine, a potent inhibitor of gamma-glutamylcysteine synthetase, reduced the glutathione concentration and rendered the cells more sensitive to the toxic effect of oxygen. Both L-2-oxothiazolidine-4-carboxylate and buthionine sulfoximine had no effect on the activities of glutathione peroxidase or glutathione reductase. Our results suggest that L-2-oxothiazolidine-4-carboxylate may have the potential of preventing oxygen toxicity.

Hyperoxia causes increased albumin permeability of cultured endothelial monolayers.

When confluent calf pulmonary arterial endothelial monolayers cultured on polycarbonate micropore membranes were exposed to hyperoxia (95% O2) for 3 days, endothelial cells became enlarged, and their permeability to 125I-labeled albumin was markedly increased. Similar changes were not observed when endothelial monolayers were exposed to hyperoxia for 1 or 2 days. Cell counting and acridine orange staining of endothelial monolayers revealed that the hyperoxia-induced increase in albumin permeability was not associated with a denuding injury or loss of cells from the monolayers. Vimentin filament staining of O2-exposed monolayers showed thickening of the perinuclear vimentin coil in some cells. Rhodamine-phalloidin staining demonstrated that hyperoxia caused a progressive alteration in the actin distribution. Two days after O2 exposure, peripheral actin bands became thinner, whereas the number of cytoplasmic stress fibers was increased. Three days after O2 exposure, peripheral actin bands of most cells were disrupted or absent. Because peripheral actin bands play an important role in maintaining the integrity of endothelial monolayers, disruption of peripheral bands by hyperoxia may in part be responsible for the observed change in permeability.

Cyclosporin-A for the treatment of pure red cell aplasia in a patient with chronic lymphocytic leukemia.

A 62-year-old man with B-cell chronic lymphocytic leukemia had three separate episodes of pure red cell aplasia (PRCA). The last episode was treated with cyclosporin-A (CyA) and prednisone. After the patient was on the therapy for 2 weeks, erythropoietic recovery was observed and with continued therapy the hematocrit (Hct) became normal. The PRCA remission was associated with a fall in the blood lymphocyte count, and a reduction in the spleen and lymph node size and bone marrow lymphocyte density. At diagnosis of PRCA the blood T-cells bearing IgG Fc receptors (T gamma cells) were increased, and the marrow contained very few or no late-stage erythroid progenitors. After remission of PRCA the T gamma cell fraction decreased, and the marrow erythroid progenitor's number became normal. We speculate that therapy with CyA and prednisone inhibited the production of interleukins-1 and -2 from monocytes and T-cells, respectively, and was responsible for the reduction of the T gamma cell fraction and B-cell leukemic mass in this patient. Further, we believe that normalization of T gamma cells in association with the therapy was responsible for the PRCA remission.

Alpha 2U-globulin: measurement in rat kidney and relationship to hyaline droplets.

The concentration of renal alpha 2U-globulin increased in a dose-dependent manner in adult male but not female rats which received a single dose of 2,2,4-trimethylpentane (TMP). After administration of a single dose of 12 mmol TMP/kg to adult male rats, the renal concentration of alpha 2U-globulin reached a peak at 48 hours and returned to near background level after 7 days. These changes in renal alpha 2U-globulin concentration were closely paralleled by changes in renal hyaline droplet formation. Renal alpha 2U-globulin and hyaline droplets were absent in normal pre-puberty male rats, and neither could be stimulated by a single dose of TMP. alpha 2U-Globulin was localised in the renal cortex of adult male rats, in particular the S2 segment of the proximal tubule. A greater staining intensity due to alpha 2U-globulin was seen in the S2 and adjacent segments after a single dose of TMP. A strong association is suggested between the presence of renal hyaline droplets and the occurrence of alpha 2U-globulin.

Alpha 2U-globulin: measurement in rat kidney following administration of 2,2,4-trimethylpentane.

Hyaline droplet formation was stimulated markedly in the kidneys of post-puberty male rats 24-48 h after a single oral dose of 12/24 mmol/kg 2,2,4-trimethylpentane [TMP]. Renal hyaline droplet formation could not be detected in female rats or in pre-puberty male rats following similar doses of TMP. A dose-dependent increase in the renal concentration of the androgen-dependent low molecular weight protein, alpha 2U-globulin was observed in post-puberty male rats 24 h after a single oral dose of TMP, over the range 0.3-12.0 mmol/kg. After administration of a single dose of 12 mmol/kg TMP to male rats, the renal concentration of alpha 2U-globulin rose steadily up to a peak after 48 h and then returned slowly to near normal after 7 days. Renal alpha 2U-globulin could not be detected in female rats and in pre-puberty male rats. An immunocytochemical assay was developed to examine the distribution of alpha 2U-globulin within the kidney. alpha 2U-Globulin was localised primarily in the S2 segment of renal proximal tubules in untreated male rats. Rats which received a single dose of 12 mmol TMP/kg showed not only a greater staining intensity, due to the presence of a higher concentration of alpha 2U-globulin, but also staining in adjacent segments of the renal cortex. Several urinary biochemical indicators of nephrotoxicity were measured daily in male rats for up to 72 h following a single dose of 12 mmol TMP/kg. Renal proximal tubular function was unimpaired by TMP treatment. On the basis of studies in untreated and TMP-treated rats, a strong association has been found between the presence of renal hyaline droplets and the occurrence of renal alpha 2U-globulin. The findings in the present study provide an explanation for the occurrence of renal hyaline droplets only in adult male rats, but do not, as yet, establish the toxicological significance of increases in renal hyaline droplet formation.