Pulmonary Microvascular Endothelial Cell Monolayers Research Articles

Bicarbonate disruption of the pulmonary endothelial barrier via activation of endogenous soluble adenylyl cyclase, isoform 10.

It is becoming increasingly apparent that cAMP signals within the pulmonary endothelium are highly compartmentalized, and this compartmentalization is critical to maintaining endothelial barrier integrity. Studies demonstrate that the exogenous soluble bacterial toxin, ExoY, and heterologous expression of the forskolin-stimulated soluble mammalian adenylyl cyclase (AC) chimera, sACI/II, elevate cytosolic cAMP and disrupt the pulmonary microvascular endothelial barrier. The barrier-disruptive effects of cytosolic cAMP generated by exogenous soluble ACs are in contrast to the barrier-protective effects of subplasma membrane cAMP generated by transmembrane AC, which strengthens endothelial barrier integrity. Endogenous soluble AC isoform 10 (AC10 or commonly known as sAC) lacks transmembrane domains and localizes within the cytosolic compartment. AC10 is uniquely activated by bicarbonate to generate cytosolic cAMP, yet its role in regulation of endothelial barrier integrity has not been addressed. Here we demonstrate that, within the pulmonary circulation, AC10 is expressed in pulmonary microvascular endothelial cells (PMVECs) and pulmonary artery endothelial cells (PAECs), yet expression in PAECs is lower. Furthermore, pulmonary endothelial cells selectively express bicarbonate cotransporters. While extracellular bicarbonate generates a phosphodiesterase 4-sensitive cAMP pool in PMVECs, no such cAMP response is detected in PAECs. Finally, addition of extracellular bicarbonate decreases resistance across the PMVEC monolayer and increases the filtration coefficient in the isolated perfused lung above osmolality controls. Collectively, these findings suggest that PMVECs have a bicarbonate-sensitive cytosolic cAMP pool that disrupts endothelial barrier integrity. These studies could provide an alternative mechanism for the controversial effects of bicarbonate correction of acidosis of acute respiratory distress syndrome patients.

The effects of ulinastatin on systemic inflammation, visceral vasopermeability and tissue water content in rats with scald injury

The aim of this study was to examine whether administration of ulinastatin inhibits pro-inflammatory mediators and ameliorate visceral vasopermeability both in a rat model of major burn, and also in rat cultured endothelial cells stimulated with permeability-evoking mediators. Plasma levels of tumor necrosis factor-alpha (TNF-α), C-reactive protein (CRP), myeloperoxidase (MPO), microvascular permeability, and water content of organ tissues were evaluated in a rodent model of a 55% TBSA full-thickness scald injury. Microvascular permeability was also evaluated with a cultured pulmonary microvascular endothelial cells (PMECs) monolayer after stimulation with trypsin, bradykinin, histamine, prostaglandin E2 and burn serum. We found that the plasma levels of TNF-α, CRP, MPO, vascular permeability and water content of heart, lung, kidney, and small intestine tissues were significantly increased in animals after scald injury, and administration of ulinastatin lowered the levels TNF-α, CRP, MPO, vascular permeability and water content of those organ tissues. In vitro, ulinastatin lowered the levels of TNF-α, interleukin-6 (IL-6) and attenuated permeability in PMEC monolayers after being stimulated with burn serum or trypsin, but not by bradykinin, histamine or prostaglandin E2. These results indicate that ulinastatin attenuates the systemic inflammatory response and visceral vasopermeability both in vivo and vitro, and may serve as a therapeutic agent for prevention of systemic inflammatory response and leakage of fluid into tissue after major burn.

Changes in lymph proteome induced by hemorrhagic shock

Damage-associated molecular patterns (DAMPs) released from host tissue after trauma and hemorrhagic shock (HS) have been shown to activate polymorphonuclear cells (PMNs) and lead to acute lung injury and systemic inflammatory response syndrome. The avenue by which DAMPs reach the circulation is unclear; however post-HS lymph has been shown to contain biologically active mediators. We therefore studied the time course of DAMP detection in systemic lymph and the effect of isotonic versus hypertonic resuscitation on DAMPs production and PMN activation in vitro. A canine HS/hind-limb lymph cannulation model was used. Animals were bled to a mean arterial pressure of 40 mm Hg and were resuscitated with shed blood plus equivalent amounts of Na+as either lactated Ringer's solution or 7.5% hypertonic saline solution (HSS). Lymph samples were collected at baseline, end-shock, and at various times after resuscitation. DAMPs were isolated from lymph samples and detected by Western blot for high-mobility group box 1 and mitochondrial DNA. Priming of naive PMNs was indexed by mitogen-associated protein kinase phosphorylation. Human pulmonary microvascular endothelial cell monolayers were established and exposed to the various lymph samples. Endothelial intracellular adhesion molecule expression, apoptosis, and monolayer permeability were determined. DAMPs were detected in lymph samples starting at the end of the shock period and peaking at 120 minutes after resuscitation. HSS resuscitation resulted in the highest levels of DAMPs detected in systemic lymph and plasma. PMN mitogen-associated protein kinase activation was noted during the resuscitation phase and peaked 120 minutes after resuscitation. Similar temporal changes in human pulmonary microvascular endothelial cell intracellular adhesion molecule expression and cellular injury were noted after shock with the greatest effect noted with the hypertonic saline resuscitation regimen. Lymph represents an important avenue for the delivery of DAMPs into the systemic circulation after HS. HSS lead to a significant increase in DAMPs production in the model. This finding may account for the conflicting data regarding the salutary effects of HSS resuscitation noted in clinical versus experimental shock studies. ).

Pulmonary Endothelial Barrier Disruption via Bicarbonate Stimulation of Endogenous Soluble Adenylyl Cyclase, AC10

Transmembrane adenylyl cyclase (AC) generates subplasma membrane cAMP that strengthens the pulmonary endothelial barrier. In contrast, P. aeruginosa inserts the toxic soluble AC, ExoY, into pulmonary microvascular endothelial cells (PMVECs) where it generates cytosolic cAMP that phosphorylates the microtubule associating protein, tau, destabilizes microtubules and disrupts the endothelial barrier. A mammalian soluble AC, AC10, has been identified, which also localizes to the cytosol. We tested the hypothesis that bicarbonate stimulation of AC10 increases pulmonary endothelial cell permeability via tau phosphorylation. Western analysis revealed PMVECs have greater AC10 expression than pulmonary artery endothelial cells (PAECs). While adjusting CO2 tension to maintain constant media pH, bicarbonate dose‐dependently increased the phosphodiesterase 4 sensitive cAMP pool in PMVECs, but not in PAECs. RT‐PCR and Western analysis revealed heterogeneous expression of carbonic anhydrase and bicarbonate cotransporters in pulmonary endothelial cells. Further, bicarbonate increased tau serine214 phosphorylation and decreased resistance across the PMVEC monolayer. These data reveal bicarbonate stimulates AC10 activity generating a phosphodiesterase 4 regulated cAMP pool in PMVECs, which increases tau phosphorylation and compromises PMVEC barrier integrity. Funding AHA11GRNT7430039

Methods for measurement of permeability coefficient of pulmonary microvascular endothelial cells in rat

To explore an optimal method for the measurement of pulmonary microvascular endothelial cell (PMVEC) permeability coefficient. A monolayer of rat PMVEC model was constructed by culturing a cell suspension on transwell filter or polycarbonate filter membrane. After the state of confluence of cells was affirmed with epithelial volt-ohm meter or inverted microscope, the permeability coefficient was determined by means of transendothelial electrical resistance (TER), fluoresceinisothiocyanate-dextran (Pd), and permeation of Hanks solution (Lp) across monolayers. Meanwhile,changes in PMVEC permeability expressed by the ratio of the observed value and the original value were observed after lipopolysaccharide (LPS) challenge for 0. 5 hour or 2 hours. The cells reached the state of confluence as observed under inverted microscope on the third day post-seeding, and the TER and Pdat this time-point were C(39. 45 ± 3. 96) ( 2 cm2] and [(8. 52 + 0. 50) X 10-6cm/s], respectively. After PMVEC were seeded on transwell filters, the TER increased steadily in a time-dependent manner after seeding of PMVEC, reaching the summit at the fourth day post-seeding C(49. 84 ± 3. 93)f " cm2].Under the natural state, the TER, Pd and Lp of confluent PMVEC monolayers were (49.84 ±3.93) ·.* cm2, (6.15±0.63) X 106 cm/s and (6.80 + 0.62) X10< cm * s-' * cm HZO-', respectively.After PMVEC monolayers were challenged with 10 mg/L LPS for both 0. 5 hour and 2 hours, there was significant decrease in the permeability coefficient as measured by TER (0. 87+ 0. 03, 0.45 0. 04 vs. 1.00+0.08, respectively, both P< 0.05), and an increase in the permeability coefficient measured by Pd (1.33±0. 11, 2.43±0. 14 vs. 1.00+0.10, respectively, both P<0. 05) and the permeability coefficient measured by Lp (1.30± 0.07, 2.38 0.15 vs. 1.00 + 0.11, respectively, both P< 0.05) when compared with the normal group. Three methods, namely TER, Pd and Lp are available to use to assess PMVEC permeability coefficient. The combination of an inverted microscope, TER and Pd enhances the accuracy in determining PMVEC permeability coefficient, and it provides an experimental technique for studying the pathogenesis of acute lung injury in vitro.

Urokinase-type Plasminogen Activator (uPA) Induces Pulmonary Microvascular Endothelial Permeability through Low Density Lipoprotein Receptor-related Protein (LRP)-dependent Activation of Endothelial Nitric-oxide Synthase

Urokinase plasminogen activator (uPA) and PA inhibitor type 1 (PAI-1) are elevated in acute lung injury, which is characterized by a loss of endothelial barrier function and the development of pulmonary edema. Two-chain uPA and uPA-PAI-1 complexes (1-20 nM) increased the permeability of monolayers of human pulmonary microvascular endothelial cells (PMVECs) in vitro and lung permeability in vivo. The effects of uPA-PAI-1 were abrogated by the nitric-oxide synthase (NOS) inhibitor L-NAME (N(D)-nitro-L-arginine methyl ester). Two-chain uPA (1-20 nM) and uPA-PAI-1 induced phosphorylation of endothelial NOS-Ser(1177) in PMVECs, which was followed by generation of NO and the nitrosylation and dissociation of β-catenin from VE-cadherin. uPA-induced phosphorylation of eNOS was decreased by anti-low density lipoprotein receptor-related protein-1 (LRP) antibody and an LRP antagonist, receptor-associated protein (RAP), and when binding to the uPA receptor was blocked by the isolated growth factor-like domain of uPA. uPA-induced phosphorylation of eNOS was also inhibited by the protein kinase A (PKA) inhibitor, myristoylated PKI, but was not dependent on PI3K-Akt signaling. LRP blockade and inhibition of PKA prevented uPA- and uPA-PAI-1-induced permeability of PMVEC monolayers in vitro and uPA-induced lung permeability in vivo. These studies identify a novel pathway involved in regulating PMVEC permeability and suggest the utility of uPA-based approaches that attenuate untoward permeability following acute lung injury while preserving its salutary effects on fibrinolysis and airway remodeling.

Adenosine monophosphate‐activated kinase α1 promotes endothelial barrier repair

The vascular endothelium responds to damage through activation of multiple signaling events that restore cell-cell adhesion and vascular integrity. However, the molecular mechanisms that integrate these events are not clearly defined. Herein, we identify a previously unexpected role for adenosine monophosphate-activated protein kinase (AMPK) in pulmonary microvascular endothelial cell (PMVEC) repair. PMVECs selectively express the AMPKα1 catalytic subunit, pharmacological and short hairpin RNA-mediated inhibition of which attenuates Ca(2+) entry in these cells induced by the inflammatory Ca(2+)-signaling mimetic thapsigargin. We find that AMPKα1 activity is required for the formation of PMVEC cell-cell networks in a prorepair environment and for monolayer resealing after wounding. Decreasing AMPKα1 expression reduces barrier resistance in PMVEC monolayers, results consistent with a role for AMPKα1 in cell-cell adhesion. AMPKα1 colocalizes and coimmunoprecipitates with the adherens junction protein N-cadherin and cofractionates with proteins selectively expressed in caveolar membranes. Assessment of permeability, by measuring the filtration coefficient (K(f)) in isolated perfused lungs, confirmed that AMPK activation contributes to barrier repair in vivo. Our findings thus provide novel evidence for AMPKα1 in Ca(2+) influx-mediated signaling and wound repair in the endothelium.

Effects of Preconditioning with Sevoflurane on TNF-α-Induced Permeability and Activation of p38 MAPK in Rat Pulmonary Microvascular Endothelial Cells

Preconditioning with sevoflurane (SPC) diminishes effusion of rat alveolar membrane during inflammation. It is not clear whether this preconditioning directly inhibits permeability of pulmonary microvascular endothelial cell (PMVEC) monolayer. In this article, we evaluated effects of SPC on permeability of PMVEC monolayer and identified signaling pathways involved in these effects. PMVEC monolayer was exposed to different conditions (5-hydroxydecanoate (5-HD), TNF-α, SPC, SPC with subsequent exposure to TNF-α and 5-HD, and SPC with subsequent exposure to TNF-α alone), and the permeability of PMVEC monolayer was assessed using FITC-bovine serum albumin (ELISA). Expression of ICAM-1 (Western blot and RT-PCR) and activation of p38 MAPK (Western blot) were also assessed. Compared to the TNF-α group, permeability of PMVEC monolayer in the SPC+TNF-α group was significantly lower. Activation of p38 MAPK was also diminished in the TNF-α group. Pre-treatment with 5-HD reverted beneficial effects of SPC. Expression of ICAM-1 was not modulated by any of the tested experimental exposures. The results of this study demonstrate that SPC is capable of diminishing the TNF-α-induced increase of permeability of PMVEC monolayer, and that this beneficial effect is partly reversed by 5-HD. Further, SPC suppresses activation of p38 MAPK.

Proteolytic Activity of Pseudomonas aeruginosa Isolates with TTSS-Mediated Cytotoxicity and Invasiveness to Host Cells

Over 100 of Pseudomonas aeruginosa isolates representing the two TTSS genotypes (exoU (-)/exoS (+) or exoU (+)/exoS (-)) were cultured in different media in order to evaluate their proteolytic activities and find a relationship between proteolytic activity and the cytotoxic and/or invasive phenotypes displayed by the strains upon infection of RAW 264.7 murine macrophage-like cells and pulmonary microvascular endothelial cells (PME). The elastolytic activity, protein concentration, and total proteolytic activity (TPA) were measured in culture supernatants. No significant differences were observed in the median elastolytic activities among cytotoxic/noninvasive, noncytotoxic/invasive, and cytotoxic/invasive phenotypes displayed by P. aeruginosa strains. The only significant difference was noted when isolates of the two different TTSS genotypes were grown in a calcium-depleted minimal medium for induction of TTSS (MI). The exoU (-)/exoS (+) isolates showed significant higher levels of the median elastolytic activity when compared to the exoU (+)/exoS (-) isolates. These two groups of isolates secreted the elastase B (LasB) with distinct molecular masses 158 or 116 kD, respectively. The strains of the two TTSS genotypes secreted similar amount of total proteins; however, the higher values of TPA were observed for the isolates of the exoU (+) /exoS (-) genotype when grown in MI medium. We concluded that there is no direct relationship between secretion of proteases with elastolytic activity and the cytotoxic and/or invasive phenotypes of the isolates observed upon infection of both RAW 264.7 and PME monolayers. Further studies are needed to find out whether others factors beside proteases could influence the mechanism of host cells intoxication mediated by the P. aeruginosa TTSS-delivered toxins.

P. aeruginosa ExoY Disrupts Microtubules and Induces Endothelial Cell Gap Formation

Microtubule architecture controls endothelial cell barrier function, as acute microtubule disruption leads to inter‐endothelial gap formation. The P. aeruginosa toxin, ExoY, is injected through a type III secretion system (TTSS) into the cytosol of endothelial cells, where it associates with microtubules, and produces a cytosolic cAMP pool that disrupts the endothelial barrier. However, it is unclear whether cytosolic cAMP reorganizes microtubules as a necessary prerequisite. To test this idea, confluent pulmonary microvascular endothelial cell monolayers were infected with two strains of P. aeruginosa that possess a: (1) functional TTSS, and only injects ExoY (ExoY+), and (2) functional TTSS, but injects a catalytically inactive ExoY (ExoYK81M) due to a single point mutation at the ATP binding site. After 4 hours of incubation, cells were stained for microtubule organization. Although microtubules were disrupted and inter‐endothelial gaps were formed in ExoY+‐treated cells, neither microtubules nor inter‐endothelial gaps were formed in ExoYK81M or vehicle control‐treated cells. However, stabilizing microtubules prior to infection prevented ExoY+‐induced microtubule disruption and inter‐endothelial gap formation. Collectively, these data suggest that ExoY generates a cytosolic cAMP pool that reorganizes microtubules and causes endothelial barrier disruption. [HL‐60024 &amp; HL‐66299]

Septic pulmonary microvascular endothelial cell injury: role of alveolar macrophage NADPH oxidase

A significant role for alveolar macrophages (AM) in the pathophysiology of sepsis-induced acute lung injury (ALI) has been shown; however, the mechanisms behind AM-related lung injury remain relatively uncertain. We examined the role of AM nicotinamide adenine dinucleotide phosphate (NADPH) oxidase in pulmonary endothelial cell septic injury. NADPH oxidase is one of the major sources of cellular reactive oxygen species and has been implicated in endothelial injury in ALI. Pulmonary microvascular endothelial cells (PMVEC) monolayers were grown on Transwell inserts and incubated with wild-type and NADPH oxidase-deficient AM in the presence or absence of cytomix (equimolar TNF-alpha, IL-1beta, and IFN-gamma). Injury to the monolayers was assessed by trans-PMVEC Evans blue (EB)-labeled albumin flux. We found AM under cytomix stimulation caused significant EB-albumin flux across the PMVEC monolayers, and this effect was attenuated by the genetic deletion of AM NADPH oxidase. The pharmacological inhibition of AM NADPH oxidase with apocynin and PR-39 also significantly reduced AM-dependent PMVEC injury. In the AM-PMVEC cocultures, we also assessed PMVEC injury through measurement of protein oxidation and lipid peroxidation. AM were shown to cause a significant increase in these markers of PMVEC injury, which was also attenuated by the inhibition of NADPH oxidase or through the use of NADPH oxidase-deficient AM. PMVEC NADPH oxidase was shown not to significantly contribute to PMVEC injury in our studies. From our findings we have concluded that AM NADPH oxidase is crucial for the septic increase in pulmonary vascular permeability.

Inducible NO synthase (iNOS) in human neutrophils but not pulmonary microvascular endothelial cells (PMVEC) mediates septic protein leak in vitro

Sepsis-induced acute lung injury (ALI) is characterized by injury of the pulmonary microvascular endothelial cells (PMVEC) leading to high-protein pulmonary edema. Inducible NO synthase (iNOS) mediates trans-PMVEC protein leak in septic mice in vivo and in murine PMVEC under septic conditions in vitro, but the role of iNOS in human PMVEC protein leak has not been addressed. We hypothesized that iNOS in human neutrophils, but not human PMVEC, mediates septic trans-PMVEC protein leak in vitro. We isolated human PMVEC from lung tissue using magnetic bead-bound anti-PECAM antibody and assessed Evans blue albumin leak across human PMVEC monolayers under septic conditions in the presence/absence of human neutrophils. PMVEC were used at passages 3–4, seeded on 3 μm Transwell inserts and grown to confluence. Cytomix-stimulated trans-PMVEC albumin leak was not attenuated by pre-treatment with 1400 W, a selective iNOS inhibitor, or l-NAME, a non-selective NOS inhibitor. In neutrophil-PMVEC co-culture, basal unstimulated trans-EB-albumin leak was 0.6 ± 0.3%, which was increased by cytomix stimulation to 11.5 ± 4.4%, p < 0.01. Cytomix-stimulated EB-albumin leak in neutrophil-PMVEC co-cultures was inhibited by pre-treatment with 1400 W (3.8 ± 1.0%, p < 0.05) or l-NAME (4.0 ± 1.1%, p < 0.05). Pre-treatment of neutrophil-PMVEC co-cultures with PEG-SOD (superoxide scavenger) and FeTPPS (peroxynitrite scavenger) also significantly attenuated neutrophil-dependent cytomix-stimulated leak (4.7 ± 3.0%, p < 0.05; 0.5 ± 1.0%, p < 0.01, respectively). In conclusion, trans-human PMVEC albumin leak under septic conditions is dependent on iNOS activity specifically in neutrophils, but not in PMVEC themselves. Septic neutrophil-dependent trans-PMVEC albumin leak may be mediated by peroxynitrite.

Alveolar macrophage induced septic pulmonary microvascular endothelial cell injury is inducible nitric oxide synthase‐dependent

We previously found that alveolar macrophage (AM) inducible nitric oxide synthase (iNOS) appeared essential for pulmonary microvascular protein leak in septic mice. However, the direct role of AM iNOS in septic lung injury or endothelial injury has not been addressed. Thus, we assessed the effects of AM iNOS on pulmonary microvascular endothelial cell (PMVEC) injury under septic conditions in vitro AM-PMVEC co-culture. Murine PMVEC were utilized as protein leak and neutrophil migration predominantly occur in the pulmonary microvasculature during septic lung injury. PMVEC were isolated and seeded on Transwell inserts (3.0ìm pore) and allowed to grow to confluence. In AM-PMVEC co-cultures stimulated with 3ng/ml cytomix (equal concentrations of TNF-á, IL-â and IFN-ã) for 9 hours, the permeability of the PMVEC monolayer was assessed by the degree of Evans blue labeled albumin leak across the monolayer in the final hour of stimulation. The presence of wildtype (iNOS+/+) AM in the upper compartment dose-dependently (1:1 and 3:1) increased trans-PMVEC albumin leak (80.7 ± 18% of control in 1:1 vs. 265.1 ± 28% of control in 3:1). In contrast, presence of AM in the lower compartment had no effect on cytomix-stimulated PMVEC albumin leak (128 ± 30% of control). When compared to co-culture of PMVEC with iNOS+/+ AM, presence of iNOS knockout (−/−) AM, in a 3:1 ratio of AM to PMVEC, had no effect on cytomix-stimulated trans-PMVEC albumin leak. These data are consistent with a direct role of AM iNOS in the septic injury of PMVEC. This suggests an important role for AM iNOS in septic lung injury, and a possible new therapeutic approach.

Effects of the very late adhesion molecule 4 antagonist WAY103 on human peripheral blood eosinophil vascular cell adhesion molecule 1–dependent functions

Eosinophil infiltration to the lung in allergic inflammation can be initiated by the tethering of circulating cells through very late adhesion molecule 4 (VLA-4; alpha4beta1, CD49d/CD29) to vascular cell adhesion molecule 1 (VCAM-1) expressed on pulmonary vascular endothelium. Small-molecule VLA-4 antagonists have been proposed as a therapeutic mechanism to prevent eosinophil infiltration in asthma; however, they might affect other eosinophil functions. The small-molecule VLA-4 antagonist (2S)-3-(4-Dimethylcarbamoyloxyphenyl)-2-{[(4R)-5,5-dimethyl-3-(1-methyl-1H-pyrazole-4 sulfonyl)thiazolidine-4-carbonyl]amino}propionic acid (WAY103) was assessed for its effects on eosinophil VLA-4-dependent functions, including adhesion, migration, respiratory burst, and degranulation. Human peripheral blood eosinophils were preincubated with WAY103, anti-alpha4, and/or anti-beta2 integrin mAbs and then assessed for adhesion to recombinant VCAM-1, intercellular adhesion molecule 1, and endothelial cell monolayers. Transmigration was measured by using human pulmonary microvascular endothelial cell monolayers and Transwell filters. Superoxide anion generation was determined by means of cytochrome C reduction and degranulation by means of eosinophil-derived neurotoxin release. WAY103 inhibition of eosinophil adhesion to recombinant VCAM-1 was dose dependent (63% inhibition with 100 nM WAY103, P < .04) and comparable with inhibition caused by anti-alpha4 mAb (60.1% inhibition). Although pretreatment with WAY103 also decreased eosinophil adhesion to TNF-alpha plus IL-4-activated human pulmonary microvascular endothelial cell monolayers, it did not prevent eosinophil transendothelial migration in response to RANTES. Finally, WAY103 inhibited VCAM-1-stimulated superoxide generation but enhanced cytokine-activated eosinophil-derived neurotoxin degranulation. Although small-molecule VLA-4 antagonists, such as WAY103, might reduce eosinophil adhesion, this approach might not be sufficient to eliminate this cell from in vivo allergic airway inflammatory participation and could even promote specific cell activation.

LPS induces permeability injury in lung microvascular endothelium via AT 1 receptor

Lipopolysaccharide (LPS) is known to stimulate the circulation and local production of angiotensin II (Ang II). To assess whether Ang II plays a role in LPS-induced acute lung injury, rats were injected with LPS, the microvascular endothelial permeability injury was evaluated by histological changes, increased pulmonary wet/dry weight ratio, and pulmonary microvascular protein leak. Besides, increased rat pulmonary microvascular endothelial cell monolayer permeability coefficient ( K f) was measured after treatment with LPS and/or Ang II, respectively. LPS/Ang II, treatment resulted in a significant increase in K f. Ang II cooperates with LPS to further increase K f. Hence, LPS increases pulmonary microvascular endothelial permeability both in vitro and in vivo. Local lung Ang II was increased in response to LPS challenge, and elevated Ang II ulteriorly exacerbates LPS-induced endothelium injury. [Sar 1,Ile 8]Ang II, a selective block of Ang II type 1 (AT 1) receptors, eliminated these changes significantly. Our conclusion is that the LPS-induced lung injury may be mediated by the AT 1 receptor.

Immunotherapy Attenuates Eosinophil Transendothelial Migration Induced by the Supernatants of Antigen-Stimulated Mononuclear Cells from Atopic Asthmatics

Background: Eosinophil transendothelilal migration across vascular endothelial cells is an initial step of eosinophil accumulation in allergic inflammation. There is increasing evidence that specific immunotherapy (SIT) modulates the production of inflammatory molecules from mononuclear cells. Objective: The present study was undertaken to examine whether SIT modifies eosinophil transendothelial migration induced by the supernatants of antigen-stimulated mononuclear cells from atopic asthmatics. Methods:Dermatophagoides farinae(Df)-sensitive mild persistent asthmatics were divided into a SIT-treated group and a control group. Peripheral blood mononuclear cells (PBMC) were isolated before and after SIT using the rush protocol, and cultured for 96 h at 37°C in the presence or absence of Df antigen. Eosinophils were isolated from the blood of healthy subjects, and put on transwell filters coated with pulmonary microvascular endothelial cell monolayers stimulated with IL-4 plus TNF-α. The supernatants of PBMC were applied to the lower compartment and the transmigration of eosinophils was examined. Results:Df stimulation of PBMC resulted in an augmentation of eosinophil transendothelial migration. This enhancement was abrogated following SIT. In the control group, the antigen-induced effect on eosinophil transmigration did not show an interval change. Conclusion: SIT attenuates eosinophil transendothelial migration induced by antigen-stimulated mononuclear cells.

Oxidized low-density lipoprotein activates migration and degranulation of human granulocytes.

Oxidized low-density lipoprotein (oxLDL) has been reported as a major participant in the pathogenesis of atherosclerosis. We hypothesized that oxLDL can also interact with granulocytes during inflammatory airway diseases, such as asthma. To test the chemotactic effect of oxLDL, isolated human peripheral granulocytes were added to the upper chambers of Transwell filters and migration in response to oxLDL was determined. Cu+2-oxidized LDL stimulated neutrophil (23.4 +/- 3.2% for 100 microg/ml oxLDL versus 2.9 +/- 1.1% for buffer, P < 0.05) and eosinophil (19.3 +/- 3.5% versus 0.6 +/- 0.02% for buffer, P < 0.05) chemotaxis in a concentration-dependent manner. The magnitude of chemotaxis was dependent on the degree of LDL oxidation. Granulocyte transmigration across IL-1beta-activated human pulmonary microvascular endothelial cell monolayers was similarly stimulated by oxLDL. OxLDL activated significant degranulation of both neutrophils (100.9 +/- 9.8 versus 49.6 +/- 8.4 ng lactoferrin released/5 x 105 neutrophils for buffer, P < 0.05) and eosinophils (342 +/- 115.4 versus 85.8 +/- 30.4 ng eosinophil-derived neurotoxin/1 x 106 eosinophils for buffer, P < 0.05). Therefore, in vivo influx and oxidation of LDL may be an important mediator for the initiation of bronchial inflammation where granulocytes are recruited to the lung.

Role of nitric oxide in human pulmonary microvascular endothelial cell adhesion

We examined the effect of nitric oxide (NO) on cell adhesion using cultured human pulmonary microvascular endothelial cells (PMVEC). Attachment of these cells to fibronectin was significantly inhibited by NO donors, spermine NONOate and S-nitroso-N-acetyl-penicillamine or L-arginine, but not 8-bromoguanosine-3′,5′-cyclic-monophosphate. Similar results were obtained with the electrical cell-substrate impedance sensor (ECIS) technique. Addition of NO donors or L-arginine, but not 8-bromoguanosine-3′,5′-cyclic-monophosphate or N 2,2′-O-dibutyrylguanosine-3′,5′-cyclic-monophosphate, to confluent PMVEC monolayers resulted in a transient decrease in cell adhesion, which was quantitated by the ECIS. Exposure to 1 U/ml α-thrombin reduced the monolayer electrical resistance by ∼50%. The observed response was significantly suppressed by pretreatment of cells with intracellular calcium chelator, 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid or NO synthase inhibitor, N G-nitro-L-arginine methyl ester, but not guanylate cyclase inhibitor, 6-anilino-5,8-quinoline-quinone. Selective knockout of endothelial NO synthase with antisense oligodeoxynucleotides also significantly reduced thrombin-induced decrease in monolayer resistance. Our findings indicate that thrombin stimulates calcium-dependent release of NO from PMVEC, which mediates the retraction of endothelial cells via a cGMP-independent pathway. Our results suggest that NO modulates cell-matrix and/or cell-cell adhesion in PMVEC and that this molecule might modify microvascular permeability in the human lung.

Albumin and Ricinus communis agglutinin decrease endothelial permeability via interactions with matrix

We studied the effects of albumin and the lectin Ricinus communis agglutinin (RCA) on hydraulic conductivity (Lp) of bovine pulmonary microvascular endothelial cell monolayers (BPMVEC) because of the evidence that albumin and RCA can interfere with transendothelial albumin permeability (Siflinger-Birnboim, A., J. Schnitzer, H. Lum, F. Blumenstock, C. Shen, P. Del Vecchio, and A. Malik. J. Cell. Physiol. 149: 575-584, 1991). BPMVEC were seeded on microporous polycarbonate filters, and the liquid flux was measured by collecting effluent into a tubing of known inner diameter at transendothelial hydrostatic pressures (P) ranging from 5 to 20 cmH2O. Lp was calculated as the slope of the relationship of liquid flux per unit surface area (Jv) vs. P. Addition of RCA (50 micrograms/ml) or albumin (5 mg/ml) to the endothelial cell medium containing albumin-free Hanks' balanced saline solution (HBSS) decreased total Lp (expressed x 10(-6) cm.s-1 x cmH2O-1) from 17.2 +/- 3.6 during HBSS to 4.7 +/- 0.9 during albumin and 5.7 +/- 1.6 during RCA (P < 0.01 for both). The RCA effect, but not that of albumin, was prevented by the addition of D-galactose (0.1 M) (the cognate hapten monosaccharide of RCA). We determined the contribution of the extracellular matrix (ECM) in decreasing the Lp by obtaining ECM after treatment of the monolayers with 0.025 M NH4OH to detach endothelial cells from the ECM. Basal ECM Lp (expressed x 10(-6) cm.s-1 x cmH2O-1) was 57.0 +/- 15.3, and it decreased to 19.7 +/- 4.3 and 17.5 +/- 2.9 during RCA and albumin, respectively (P < 0.01 for both). In contrast, RCA and albumin did not alter the filter Lp values. Another lectin, Ulex europaeus agglutinin, and the protein immunoglobulin G had no effect on Lp values.(ABSTRACT TRUNCATED AT 250 WORDS)