Pulmonary Artery Endothelial Monolayers Research Articles

Bone morphogenetic protein receptor II regulates pulmonary artery endothelial cell barrier function

AbstractMutations in bone morphogenetic protein receptor II (BMPR-II) underlie most heritable cases of pulmonary arterial hypertension (PAH). However, less than half the individuals who harbor mutations develop the disease. Interestingly, heterozygous null BMPR-II mice fail to develop PAH unless an additional inflammatory insult is applied, suggesting that BMPR-II plays a fundamental role in dampening inflammatory signals in the pulmonary vasculature. Using static- and flow-based in vitro systems, we demonstrate that BMPR-II maintains the barrier function of the pulmonary artery endothelial monolayer suppressing leukocyte transmigration. Similar findings were also observed in vivo using a murine model with loss of endothelial BMPR-II expression. In vitro, the enhanced transmigration of leukocytes after tumor necrosis factor α or transforming growth factor β1 stimulation was CXCR2 dependent. Our data define how loss of BMPR-II in the endothelial layer of the pulmonary vasculature could lead to a heightened susceptibility to inflammation by promoting the extravasation of leukocytes into the pulmonary artery wall. We speculate that this may be a key mechanism involved in the initiation of the disease in heritable PAH that results from defects in BMPR-II expression.

Janus-faced signaling of cGMP in acute lung injury

The effect of increasing pulmonary endothelial cGMP concentration on endothelial function in acute lung injury appears to depend on 1) the presence of specific cGMP targets, 2) intracellular cGMP compartmentalization and 3) the timing of the increase in cGMP relative to the injury onset [1-4]. For example, we recently showed that pretreatment of pulmonary artery endothelial monolayers with 8pCPT-cGMP attenuated oxidant-induced barrier dysfunction by a cGMP-dependent kinase-1 (cGKI)-dependent mechanism [1,2]. More recently, however, we found that the increase in endogenous lung cGMP resulting from increased NO production in a ventilator-induced lung injury (VILI) mouse model caused lung endothelial barrier dysfunction [4]. The injurious effect of sGC-derived cGMP in VILI was mediated by the simultaneous generation of phosphodiesterase 2A (PDE2A), which was stimulated by cGMP to hydrolyze cAMP. Interestingly, in the same model, pretreatment with BAY 41-2272 (1.5 μM) to stimulate sGC before injurious tidal volume ventilation attenuated VILI. Recent evidence suggests that endothelial apoptosis may contribute to VILI [5] so we wondered if the protective effect of increasing lung endothelial cGMP before injury could be mediated by an anti-apoptotic effect of cGMP signalling. Mouse lung microvascular endothelial cells (MLMVEC) were isolated and purified by flow cytometry and shown to express cGK1 by Western blot and phosphorylation of VASP Ser235. A 6 hr pretreatment with 8pCPT-cGMP (50 μM), significantly attenuated H2O2-induced cell death assessed by flow cytometry (Annexin-, 7AAD-) and nuclear condensation. A similar protection was not observed in human pulmonary artery endothelial cells (HPAEC) which lack cGK1 expression in vitro. Restoration of cGK1 expression in HPAEC resulted in cGMP-mediated protection against oxidant cell death suggesting a cGK1-mediated effect. To determine if this protective effect was upstream of apoptotic signaling, MLMVEC from C57BL6 mice were treated with 8p-CPT-cGMP (50 uM) before exposure to increasing concentrations of H2O2. The extracellular H2O2 concentration ([H2O2]ext) was continuously measured with a H2O2 electrode. Compared with untreated cells, wildtype MLMVEC pre-treated with 8p-CPT-cGMP for 2 or 4 hrs (but not 30 min) significantly decreased the maximal Δ[H2O2]ext by 33 ± 11, 32 ± 10 and 25 ± 10% in cells exposed to 20, 50, and 100 μM H2O2, respectively (N = 8, P < 0.01). Consistent with this effect, 8pCPT-cGMP pretreatment attenuated H2O2-induced H2DCF fluorescence as well as p38MAPK and Akt phosphorylation suggesting that intracellular H2O2 concentration was also decreased. MLMVEC isolated from cGK1-/- mice failed to enhance H2O2 uptake suggesting cGK1-mediated signaling was responsible. An assessment of the major H2O2 degrading enzyme systems revealed a significant cGMP-mediated increase in catalase expression without an increase in catalase mRNA suggesting a post-translational effect. We conclude that the effects of cGMP signalling on lung endothelial function in acute lung injury are complex and include both injurious and protective mechanisms depending on the specific downstream signalling pathways that are present. Activation of lung microvascular endothelial cell cGK1 by cGMP protects against oxidant-mediated cell death possibly through an increase in endothelial antioxidant function.

Involvement of microtubules, p38, and Rho kinases pathway in 2-methoxyestradiol-induced lung vascular barrier dysfunction

2-Methoxyestradiol (2ME), a promising anti-tumor agent, is currently tested in phase I/II clinical trial to assess drug tolerance and clinical effects. 2ME is known to affect microtubule (MT) polymerization rather than act through estrogen receptors. We hypothesized that 2ME, similar to other MT inhibitors, disrupts endothelial barrier properties. We show that 2ME decreases transendothelial electrical resistance and increases FITC-dextran leakage across human pulmonary artery endothelial monolayer, which correlates with 2ME-induced MT depolymerization. Pretreatment of endothelium with MT stabilizer taxol significantly attenuates the decrease in transendothelial resistance. 2ME treatment results in the induction of F-actin stress fibers, accompanied by the increase in myosin light chain (MLC) phosphorylation. The experiments with Rho kinase (ROCK) and MLC kinase inhibitors and ROCK small interfering RNA (siRNA) revealed that increase in MLC phosphorylation is attributed to the ROCK activation rather than MLC kinase activation. 2ME induces significant ERK1/2, p38, and JNK phosphorylation and activation; however, only p38 activation is relevant to the 2ME-induced endothelial hyperpermeability. p38 activation is accompanied by a marked increase in MAPKAP2 and 27-kDa heat shock protein (HSP27) phosphorylation level. Taxol significantly decreases p38 phosphorylation and activation in response to 2ME stimulation. Vice versa, p38 inhibitor SB203580 attenuates MT rearrangement in 2ME-challenged cells. Together, these results indicate that 2ME-induced barrier disruption is governed by MT depolymerization and p38- and ROCK-dependent mechanisms. The fact that certain concentrations of 2ME induce endothelial hyperpermeability suggests that the issue of the maximum-tolerated dose of 2ME for cancer treatment should be addressed with caution.

Protein Kinase G and p38 MAPK in H 2 O 2 ‐induced pulmonary endothelial barrier dysfunction

H2O2 causes pulmonary endothelial cell barrier dysfunction dependent on increases in [Ca2+]i and activation of p38 mitogen-activated protein kinase (MAPK). In bovine pulmonary endothelium, we previously showed that increasing endothelial cGMP prevented H2O2-mediated hyperpermeability but the role of cGMP-dependent protein kinase (PKGI) was not examined. To address this in the current study, we compared human pulmonary artery endothelial (HPAEC) monolayers found to lack PKGI expression with HPAEC monolayers infected with a recombinant adenovirus encoding PKGI (Ad. PKG) or control adenovirus (Ad. Bgal). In uninfected HPAEC, H2O2 (250 μM) rapidly increased [Ca2+]i, phosphorylated p38, and increased monolayer permeability as measured by transendothelial electrical resistance. The p38 inhibitor SB203580 (20 μM) significantly attenuated the H2O2-induced increases in p38 phosphorylation and permeability (P<0.02) whereas 8pCPT-cGMP (50 μM) had no effect. In contrast, 8pCPT-cGMP (50 μM) completely prevented H2O2-induced hyperpermeability in HPAEC infected with Ad. PKG (but not Ad. Bgal) but 8pCPT-cGMP had no inhibitory effect on H2O2-mediated increases in [Ca2+]i or p38 phosphorylation. These data confirm an injurious role for p38 MAPK in H2O2-mediated endothelial barrier dysfunction and suggest that the protection conferred by cGMP is mediated through PKGI acting downstream of [Ca2+]i and p38. Supported by HL67189.

Transendothelial migration of leukocytes and signalling mechanisms in response to the neuropeptide secretoneurin

Secretoneurin (SN), a newly discovered neuropeptide, may be implicated in inflammatory responses as it was shown to modulate leukocyte, endothelial and mesenchymal cell functions. Neutrophils placed above pulmonary arterial or venous endothelial monolayers migrated through this cellular barrier in response to apical or basal stimulation with SN in a dose-dependent manner. At optimal concentrations of 10 −6 to 10 −8 M, SN was nearly equally effective in stimulating neutrophil transmigration as was tumor necrosis factor-α at 10 ng/ml or a chemotactic gradient of formyl-Met-Leu-Phe (10 −8 M). Stimulation of transendothelial migration appears to be specific, since a trypsin digest of SN was ineffective and excess concentrations of anti-SN antibodies completely abolished the effect. Inhibition of cyclooxygenase or nitric oxide synthase did not affect the action of SN. Preincubation of endothelial cells with pertussistoxin (PTx) or choleratoxin (CTx), and the presence of staurosporine significantly inhibited transmigration, suggesting that SN uses a signalling pathway that is coupled to G-proteins and protein kinase C in endothelium. Moreover, SN treatment resulted in transient elevation of cytoplasmatic calcium concentration in endothelial cells. These data support the hypothesis that SN might contribute to neurogenic inflammation in vivo and reveal signalling mechanisms of SN in endothelial cells.

Increased recycling of (alpha)5(beta)1 integrins by lung endothelial cells in response to tumor necrosis factor.

Tumor necrosis factor (alpha) (TNF-(alpha) can change the interaction of lung endothelial cell monolayers with their extracellular matrix in association with an increase in endothelial monolayer protein permeability. Using immunofluorescence microscopy and flow cytometry, we determined if exposure of calf pulmonary artery endothelial monolayers to TNF-(alpha) may influence cell-matrix interactions by altering the clustering as well as internalization of the (&agr;)5(beta)1 integrins (or fibronectin receptors) on the surface of endothelial cells. Immunofluorescence microscopy revealed that TNF-(alpha) caused an increase in the intracellular staining of (alpha)5(alpha)1 integrins within structures similar to endocytic vesicles as well as an increase in antibody-induced clustering of the integrins at the cell periphery. Flow cytometric analysis of endothelial cells incubated at 37 degrees C after antibody-labeling of their surface (alpha)5(beta)1 integrins at 4 degrees C confirmed an increase in the rate of (alpha)5(beta)1 integrin internalization which was at least 3 times greater after TNF-(&agr;) exposure, based on the half-life for antibody-labeled surface integrins to reach equilibrium with non-labeled integrins within the intracellular pool. Interestingly, the total cell surface expression of (alpha)5(beta)1 integrins was relatively constant after TNF-(alpha) exposure despite the enhanced rate of internalization, suggesting an accelerated recycling of the internalized (alpha)5(beta)1 integrins back to the cell surface. This response was confirmed by the measurement of labeled integrin recycling, which showed a significant (P<0.01) increase in the rate of recycling of the internalized integrins in TNF-treated endothelial cells. Enhanced internalization and subsequent recycling of (alpha)5(beta)1 integrins by endothelial monolayers exposed to TNF-(alpha) may facilitate the redistribution of cell-surface integrins in response to this inflammatory cytokine and may also modify cell-matrix interactions leading to reduced integrity and increased protein permeability of the lung endothelial monolayers.

TNF-alpha-induced matrix Fn disruption and decreased endothelial integrity are independent of Fn proteolysis.

Exposure of confluent pulmonary arterial endothelial monolayers to tumor necrosis factor (TNF)-alpha causes both a reorganization and/or disruption of fibronectin (Fn) in the extracellular matrix and an increase in transendothelial protein permeability. However, the factors initiating this response to TNF-alpha have not been defined. Because TNF-alpha can induce proteinase expression in endothelial cells, we determined whether proteinases cause both the alteration of the Fn matrix and the permeability increase as is often speculated. Incubation of calf pulmonary arterial endothelial monolayers with TNF-alpha (200 U/ml) for 18 h caused a disruption of the Fn matrix and an increase in transendothelial protein permeability. A reduced colocalization of cell-surface alpha5beta1-Fn integrins with the Fn fibers in focal contacts was also observed. TNF-alpha treatment of endothelial monolayers with matrices prelabeled with 125I-human Fn (hFn) did not cause the release of Fn fragments or alter the content of Fn antigen in the medium as analyzed by SDS-PAGE coupled with autoradiography. Both the content and fragmentation pattern of Fn within the cell layer and the insoluble Fn matrix also appeared unchanged after TNF-alpha exposure as confirmed by Western immunoblot. Fn-substrate zymography revealed that TNF-alpha increased the expression of two proteinases within the conditioned medium in which activity could be blocked by aprotinin but not by EDTA, 1,10-phenanthroline, leupeptin, or pepstatin. However, inhibition of the Fn proteolytic activity of these two serine proteinases did not prevent either the TNF-alpha-induced disruption of the Fn matrix or the increase in permeability. Thus the reorganization and/or disruption of the Fn matrix and the temporally associated increase in endothelial permeability caused by TNF-alpha appear not to be due to proteolytic degradation of Fn within the extracellular matrix. In contrast, decreased alpha5beta1-Fn integrin interaction with Fn fibers in the matrix may be important in the response to TNF-alpha exposure.

Role for anions in pulmonary endothelial permeability.

beta-Adrenergic stimulation reduces albumin permeation across pulmonary artery endothelial monolayers and induces changes in cell morphology that are mediated by Cl- flux. We tested the hypothesis that anion-mediated changes in endothelial cells result in changes in endothelial permeability. We measured permeation of radiolabeled albumin across bovine pulmonary arterial endothelial monolayers when the extracellular anion was Cl-, Br-, I-, F-, acetate (Ac-), gluconate (G-), and propionate (Pr-). Permeability to albumin (Palbumin) was calculated before and after addition of 0.2 mM of the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX), which reduces permeability. In Cl-, the Palbumin was 3.05 +/- 0.86 x 10(-6) cm/s and fell by 70% with the addition of IBMX. The initial Palbumin was lowest for Pr- and Ac-. Initial Palbumin was higher in Br-, I-, G-, and F- than in Cl-. A permeability ratio was calculated to examine the IBMX effect. The greatest IBMX effect was seen when Cl- was the extracellular anion, and the order among halide anions was Cl- > Br- > I- > F-. Although the level of extracellular Ca2+ concentration ([Ca2+]o) varied over a wide range in the anion solutions, [Ca2+]o did not systematically affect endothelial permeability in this system. When Cl- was the extracellular anion, varying [Ca2+]o from 0.2 to 2.8 mM caused a change in initial Palbumin but no change in the IBMX effect. The anion channel blockers 4-acetamido-4'-isothiocyanotostilbene-2, 2'-disulfonic acid (0.25 mM) and anthracene-9-carboxylic acid (0.5 mM) significantly altered initial Palbumin and the IBMX effect. The anion transport blockers bumetanide (0.2 mM) and furosemide (1 mM) had no such effects. We conclude that extracellular anions influence bovine pulmonary arterial endothelial permeability and that the pharmacological profile fits better with the activity of anion channels than with other anion transport processes.

Endothelial barrier dysfunction and p42 oxidation induced by TNF-alpha are mediated by nitric oxide

We tested the hypothesis that nitric oxide (.NO) mediates tumor necrosis factor-alpha (TNF-alpha)-induced alterations in permeability and actin of pulmonary artery endothelial monolayers (PAEM). The permeability of PAEM was assessed by the clearance rate of albumin labeled with Evans blue dye. The PAEM Triton-soluble ("cytoskeletal-nonassociated") and -insoluble ("cytoskeletal-associated") lysates were analyzed by Western blot for actin and oxidized protein using polyclonal antibodies to the COOH terminus of actin and dinitrophenylhydrazone (DNP), respectively. PAEM were incubated with TNF-alpha (100 U/ml) for 4 h. Incubation of PAEM with TNF-alpha resulted in increases in 1) the .NO oxidation product nitrite (NO2-), 2) nitrotyrosine immunofluorescence, 3) the oxidation of p42 (tentatively identified as actin), and 4) permeability to Evans blue dye-albumin. The .NO synthase inhibitor aminoguanidine (100 microM) prevented the TNF-alpha-induced increase in NO2-, nitrotyrosine immunofluorescence, oxidized p42, and permeability. Coincubation with L-arginine (200 microM) or the .NO mimic spermine-NO (1 microM) prevented the ablation of the response to TNF-alpha by aminoguanidine. The data indicate that TNF-alpha-induced increases in endothelial permeability and oxidized protein are mediated by .NO in PAEM.

TNF-alpha induces peroxynitrite-mediated depletion of lung endothelial glutathione via protein kinase C.

We tested the hypothesis that tumor necrosis factor-alpha (TNF) induces a peroxynitrite (ONOO-)-mediated depletion of glutathione via a protein kinase C (PKC)-dependent mechanism in pulmonary artery endothelial monolayers (PAEM). PAEM were incubated with TNF (1,000 U/ml) for 6 and 18 h. The PAEM were assayed for ONOO(-)-dependent changes in the concentration of luminol, free glutathione [Gfree; i.e., reduced glutathione and oxidized glutathione (GSSG)] and GSSG. TNF treatment decreased luminol and Gfree, and increased GSSG and GSSG/Gfree, compared with treatment with control media. The TNF-induced effects were prevented by co-incubation with the nitric oxide synthase inhibitors NG-monomethyl-L-arginine (1 mM), NG-nitro-L-arginine methyl ester (1 mM), or NG-nitro-L-arginine (1 mM). In addition, the TNF-induced effects were prevented by superoxide dismutase (10 U/ml), which removes O2-, and by urate (0.5 mM) and L-cysteine (3 mM), putative scavengers of ONOO-. The treatment of PAEM with the PKC activator phorbol 12-myristate 13-acetate (PMA, 1 microM) induced similar alterations in luminol and glutathione as TNF. TNF and PMA induced a protein of similar molecular weight (approximately 90 kDa) in the focal contact-rich fraction of PAEM lysate. TNF- and PMA-induced effects were prevented with the specific PKC inhibitor calphostin C (1 microM). The data indicate that TNF-induced PKC activation mediates ONOO- generation, which results in the oxidation and depletion of glutathione in PAEM.

Pasteurella haemolytica Lipopolysaccharide-induced Cytotoxicity in Bovine Pulmonary Artery Endothelial Monolayers: Inhibition by Indomethacin

Exposure of bovine pulmonary artery endothelial cells to Pasteurella haemolytica lipopolysaccharide caused severe morphologic changes. Initially, there was dilatation of the rough endoplasmic reticulum and mitochondrial swelling followed by cell retraction, membrane bleb formation, and cell detachment. The affected endothelial cells had severe membrane damage resulting in the leakage of lactate dehydrogenase. Indomethacin in concentrations of 0.5 mM or greater caused marked decreases in the lipopolysaccharide-induced leakage of lactate dehydrogenase. Indomethacin at 5 mM also caused a marked reduction of the lipopolysaccharide-induced morphologic changes resulting in apparent maintenance of the monolayer integrity for 8 hours versus 1 hour in the lipopolysaccharide-treated control. A marked decrease in the cell and nuclear membrane effects resulted, but the rough endoplasmic reticulum dilatation and mitochondrial changes proceeded. These results indicate that indomethacin does not prevent lipopolysaccharide binding but interferes with later events in lipopolysaccharide-induced cytotoxicity in the bovine pulmonary endothelial cell. The concentration of indomethacin required to produce this inhibition suggests that the primary mechanism is not cyclooxygenase inhibition.

β2-Adrenoceptors mediate a reduction in endothelial permeability in vitro

The permeability of bovine pulmonary artery endothelial (CPAE) monolayers to Evans blue-labelled albumin (Evans blue-albumin) has been measured in vitro. Thrombin caused a concentration-dependent increase in Evans blue-albumin clearance across endothelial monolayers. Isoprenaline inhibited thrombin-induced Evans blue-albumin clearance in a concentration-dependent manner (EC 50 21 nM). This effect was mimicked by the selective β 2-adrenoceptor agonists salbutamol (EC 50 64 nM) and salmeterol (EC 50 2.7 nM), but not by the selective β 1-adrenoceptor agonist, RO-363 ((1-[3′,4′-dihydroxyphenoxy]-2-hydroxy-[3″,4″-dimethoxyphenethylamino]-propane)oxalate), nor by the selective β 3-adrenoceptor agonist, CL-316,243 (disodium ( R,R)-5-[2-[[2-(3-chlorophenyl)-2-hydroxyethyl]-amino]propyl]-1,3-benzodioxole-2,2-dicarboxylate). Isoprenaline, salbutamol and salmeterol, but not RO-363 or CL-316,243 produced small, but significant reductions in Evans blue-albumin clearance across unstimulated endothelial monolayers. Inhibition of the response to thrombin by isoprenaline was antagonised by the selective β 2-adrenoceptor antagonist, ICI-118,551 (( erythro- dl-1(7-methylindan-4-yloxy)3-isopropylaminobutan-2-ol), p K B 8.4). Salmeterol also inhibited hydrogen peroxide-stimulated Evans blue-albumin clearance. Hence, the widely used β 2-adrenoceptor agonists, salbutamol and salmeterol, are able to reduce endothelial permeability at nanomolar concentrations.

Tumor necrosis factor-alpha decreases pulmonary artery endothelial nitrovasodilator via protein kinase C

We postulated that tumor necrosis factor-alpha (TNF) decreases endothelium-derived nitrovasodilator(s) via a protein kinase C (PKC)-dependent pathway. Calf pulmonary artery endothelial monolayers (PAEM) were treated with TNF (10, 100, and 1,000 U/ml) for 15 min or 18 h during an 18-h incubation. At the end of the incubation, the cell lysate and supernatant were harvested. Compared with controls, an 18-h incubation with TNF (100 and 1,000 U/ml) resulted in a decrease in NO2- [the oxidation product of nitric oxide (NO)] in PAEM lysate and supernatant. TNF (100 U/ml) treatment for 15 min did not suppress NO2- levels. The decrease in NO2- and the increase in lipid peroxides in response to TNF were prevented by pretreatment (15 min prior to and throughout the incubation) with either calphostin C (1 microM; a specific PKC inhibitor) or the antioxidants N-acetylcysteine (1 mM), 4,5-dihydroxy-1,3-benzene disulfonic acid (Tiron) (10 mM), and superoxide dismutase (10 U/ml). Treatment with phorbol 12-myristate 13-acetate (PMA, 1 microM for 15 min), an activator of PKC, decreased NO2- similarly to TNF. Pretreatment with calphostin C or N-acetylcysteine prior to TNF (10 U/ml) revealed an increase in NO2- levels above control treatment. Treatment with the NO synthase antagonists NG-monomethyl-L-arginine (1 mM) and N-nitroso-L-arginine (1 mM) induced an L-arginine (1 mM)-dependent decrease in NO2- in control but not in TNF-treated PAEM. The induction of NO2- by calcium ionophore (A23187; 500 nM) was not affected by treatment with TNF.(ABSTRACT TRUNCATED AT 250 WORDS)

PH and temperature modulate norepinephrine-dependent changes in endothelial permeability.

To evaluate the role of pH and temperature in norepinephrine (NE)-mediated decreases in endothelial permeability, we studied bovine pulmonary arterial endothelial monolayers at pH values of 4-9 and at temperatures of 35-39 degrees C. Only extremes of pH-modulated endothelial permeability and temperature had no effect. Although both NE and 3-isobutyl-1-methylxanthine decreased endothelial permeability, their effects were diminished by low pH and high temperature. Fever and acidosis may contribute to the edema seen in septic shock by a novel mechanism: attenuation of the barrier-improving function of NE.

Thrombin receptor activating peptides induce Ca2+ mobilization, barrier dysfunction, prostaglandin synthesis, and platelet-derived growth factor mRNA expression in cultured endothelium.

Endothelial cell activation by thrombin is a key event in wound healing, inflammation, and hemostasis. To better define thrombin-endothelial cell interactions we synthesized several peptides of varying length corresponding to the initial 14 amino acid sequence of the cloned human platelet thrombin receptor after cleavage at an arginine41 site (R/SFLLRNPNDKYEPF). Thrombin receptor activating peptides (TRAPs) as short as 5 amino acids induced significant levels of PGI2 synthesis and expression of PDGF mRNA in human endothelium and produced dose-dependent cellular contraction and permeability of confluent human umbilical vein and bovine pulmonary artery endothelial monolayers. To explore whether TRAPs utilized similar signal transducing pathways as alpha-thrombin to accomplish endothelial cell activation, phospholipase C production of the Ca2+ secretagogue IP3 was measured and detected 10 seconds after either TRAP 7 or alpha-thrombin. Furthermore, TRAPs ranging from 5-14 residues induced significant dose-dependent increases in Fura-2 fluorescence indicative of Ca2+(1) mobilization. These results indicate that thrombin-mediated proteolytic cleavage of the human and bovine thrombin receptor initiates stimulus/coupling responses such phospholipase C activation, Ca2+ mobilization, and protein kinase C activation. The functional consequence of this cellular activation via the cleaved receptor is enhanced cellular contraction, barrier dysfunction, PGI2 synthesis, and expression of PDGF mRNA.

Incorporation of fibronectin into matrix decreases TNF-induced increase in endothelial monolayer permeability

Plasma fibronectin, a dimeric adhesive protein in blood, incorporates into the subendothelial and interstitial matrix in the lung especially during vascular injury. Fibronectin in the matrix is believed to influence cell-cell interaction and endothelial cell adhesion to the collagen-rich extracellular matrix. We previously observed that addition of purified soluble human plasma fibronectin (hFn) to cultured pulmonary endothelial monolayers attenuates the increase in protein permeability of such monolayers exposed to tumor necrosis factor-alpha (TNF-alpha). In the current study, we determined the specificity of this permeability response to fibronectin by comparing hFn to two other purified adhesive proteins in human plasma, i.e., vitronectin (Vn) and fibrinogen (Fg). We also determined whether matrix incorporation was essential for this hFn-mediated protective response by comparing normal intact hFn to either hFn alkylated with N-ethylmaleimide (NEM) or to purified 160/180-kDa hFn fragments, since these alternate forms of fibronectin are believed to exhibit limited ability to incorporate into matrix. Calf pulmonary artery endothelial (CPAE) monolayers (3-4 days postseeding) were exposed to human recombinant TNF-alpha for 18 h at a medium concentration of 200 U/ml followed by assessment of protein permeability using transendothelial 125I-labeled albumin clearance. Dimeric hFn (600 micrograms/ml) significantly (P < 0.05) reduced the TNF-induced increase in endothelial monolayer permeability. Vn or Fg, added at equal molar concentrations to the hFn, were unable to attenuate endothelial permeability. Immunofluorescent analysis utilizing antibodies specific to either hFn, human Vn, or human Fg revealed incorporation of the exogenous hFn into the extracellular matrix, but no matrix incorporation of Vn or Fg. Both NEM-treated dimeric hFn as well as purified 160/180-kDa fragments of hFn, which cannot incorporate into the matrix, were also unable to prevent the TNF-induced increase in protein permeability. Thus the ability for soluble hFn to reduce the TNF-induced increase in lung endothelial monolayer permeability was specific and dependent on its incorporation into the extracellular matrix.

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)

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.

Lectin binding to gp60 decreases specific albumin binding and transport in pulmonary artery endothelial monolayers

The effect of albumin binding to cultured bovine pulmonary artery endothelial cell (BPAEC) monolayers on the transendothelial flux of 125I-labelled bovine serum albumin (BSA) was examined to determine its possible role on albumin transcytosis. The transport of 125I-BSA tracer across BPAEC grown on gelatin- and fibronectin-coated filters (0.8 microns pore diam.) was affected by the presence of unlabelled BSA in the medium in that transendothelial 125I-BSA permeability decreased, reaching a 40% reduction at BSA concentrations equal to or greater than 5 mg/ml. BSA binding to BPAEC monolayers was saturated at concentration of 10 mg/ml with an apparent binding affinity of 6 x 10(-7) M. In contrast, gelatin added to the medium altered neither 125I-BSA binding nor transport. Several lectins were tested for their ability to inhibit 125I-BSA binding and transport. One lectin, Ricinus communis (RCA), reduced 125I-BSA binding by 70% and transport by 40%. Other lectins, Ulex europaeus, Triticum vulgare, and Glycine max decreased neither 125I-BSA binding nor transport. The reduction of 125I-BSA transport by RCA was not observed in the presence of saturating levels of BSA, indicating that RCA influenced only the albumin-dependent component of transport. RCA, but not other lectins, precipitated a 60 kDa plasmalemmal glycoprotein from cell lysates of surface radioiodinated BPAEC monolayers. This 60 kDa glycoprotein appears to be the equivalent of gp60 identified previously as an albumin binding glycoprotein in rat microvascular endothelium. In summary, approximately 40% of albumin transport across BPAEC monolayers is dependent on albumin binding. This component of albumin transport is inhibited by 80% by the binding of RCA to gp60. These results suggest that binding of albumin to gp60 on pulmonary artery endothelial cell membrane is a critical determinant of transendothelial albumin flux involving mechanisms such as plasmalemmal vesicular transcytosis.

Fibrinogen degradation product fragment D increases endothelial monolayer permeability

We assessed the effects of the two primary high-molecular-weight fibrinogen degradation products (FDP), fragments D and E, on the pulmonary vascular endothelial barrier function. Fragments D and E were purified to homogeneity by QAE Sephadex chromatography followed by gel filtration. Incubation of bovine pulmonary artery endothelial monolayers with 0.5-2.0 microM fragment D for 2 h caused a doubling of transendothelial 125I-albumin clearance rate (a measure of 125I-albumin permeability). Fragment E only produced a 0.6-fold increase in 125I-albumin clearance rate at concentration of 4.0 microM. Both FDP remained active in incubating media with serum. The permeability-increasing effect of fragment D was reversible and was not due to cell detachment or lysis. The fragment-D effect was time dependent and was associated with redistribution of endothelial F-actin microfilaments. The effect was independent of the carboxy-terminal sequence on gamma-chain of fragment D. Fragments D and E binding to pulmonary artery endothelial cells was specific and reversible, but fragment D binding was three-fold greater than fragment E, which may account for the greater permeability increase mediated by fragment D. The results indicate that FDP, especially fragment D, increase endothelial permeability to albumin. The response involves specific binding of fragment D to endothelial cells and redistribution of intracellular actin.