CAT-1 as a novel CAM stabilizes endothelial integrity and mediates the protective actions of L-Arg via a NO-independent mechanism.

Interendothelial junctions play an important role in the maintenance of endothelial integrity and the regulation of vascular functions. We report here that cationic amino acid transporter-1 (CAT-1) is a novel interendothelial cell adhesion molecule (CAM). We identified that CAT-1 protein localized at cell-cell adhesive junctions, similar to the classic CAM of VE-cadherin, and knockdown of CAT-1 with siRNA led to an increase in endothelial permeability. In addition, CAT-1 formed a cis-homo-dimer and showed Ca(2+)-dependent trans-homo-interaction to cause homophilic cell-cell adhesion. Co-immunoprecipitation assays showed that CAT-1 can associate with ß-catenin. Furthermore, we found that the sub-cellular localization and function of CAT-1 are associated with cell confluency, in sub-confluent ECs CAT-1 proteins distribute on the entire surface and function as L-Arg transporters, but most of the CAT-1 in the confluent ECs are localized at interendothelial junctions and serve as CAMs. Further functional characterization has disclosed that extracellular L-Arg exposure stabilizes endothelial integrity via abating the cell junction disassembly of CAT-1 and blocking the cellular membrane CAT-1 internalization, which provides the new mechanisms for L-Arg paradox and trans-stimulation of cationic amino acid transport system (CAAT). These results suggest that CAT-1 is a novel CAM that directly regulates endothelial integrity and mediates the protective actions of L-Arg to endothelium via a NO-independent mechanism.

Enhanced phosphorylation of caveolar PKC-α limits peptide internalization in lung endothelial cells.

We previously reported that the vasoactive peptide 1 (P1, "SSWRRKRKESS") modulates the tension of pulmonary artery vessels through caveolar endothelial nitric oxide synthase (eNOS) activation in intact lung endothelial cells (ECs). Since PKC-α is a caveolae resident protein and caveolae play a critical role in the peptide internalization process, we determined whether modulation of caveolae and/or caveolar PKC-α phosphorylation regulates internalization of P1 in lung ECs. Cell monolayers were incubated in culture medium containing Rhodamine red-labeled P1 (100 µM) for 0-120 min. Confocal examinations indicate that P1 internalization is time-dependent and reaches a plateau at 60 min. Caveolae disruption by methyl-ß-cyclodextrin (CD) and filipin (FIL) inhibited the internalization of P1 in ECs suggesting that P1 internalizes via caveolae. P1-stimulation also enhances phosphorylation of caveolar PKC-α and increases intracellular calcium (Ca(2+)) release in intact cells suggesting that P1 internalization is regulated by PKC-α in ECs. To confirm the roles of increased phosphorylation of PKC-α and Ca(2+) release in internalization of P1, PKC-α modulation by phorbol ester (PMA), PKC-α knockdown, and Ca(2+) scavenger BAPTA-AM model systems were used. PMA-stimulated phosphorylation of caveolar PKC-α is associated with significant reduction in P1 internalization. In contrast, PKC-α deficiency and reduced phosphorylation of PKC-α enhanced P1 internalization. P1-mediated increased phosphorylation of PKC-α appears to be associated with increased intracellular calcium (Ca(2+)) release since the Ca(2+) scavenger BAPTA-AM enhanced P1 internalization. These data indicate that caveolar integrity and P1-mediated increased phosphorylation of caveolar PKC-α play crucial roles in the regulation of P1 internalization in lung ECs.

Beta-actin association with endothelial nitric-oxide synthase modulates nitric oxide and superoxide generation from the enzyme.

Protein-protein interactions represent an important post-translational mechanism for endothelial nitric-oxide synthase (eNOS) regulation. We have previously reported that beta-actin is associated with eNOS oxygenase domain and that association of eNOS with beta-actin increases eNOS activity and nitric oxide (NO) production. In the present study, we found that beta-actin-induced increase in NO production was accompanied by decrease in superoxide formation. A synthetic actin-binding sequence (ABS) peptide 326 with amino acid sequence corresponding to residues 326-333 of human eNOS, one of the putative ABSs, specifically bound to beta-actin and prevented eNOS association with beta-actin in vitro. Peptide 326 also prevented beta-actin-induced decrease in superoxide formation and increase in NO and L-citrulline production. A modified peptide 326 replacing hydrophobic amino acids leucine and tryptophan with neutral alanine was unable to interfere with eNOS-beta-actin binding and to prevent beta-actin-induced changes in NO and superoxide formation. Site-directed mutagenesis of the actin-binding domain of eNOS replacing leucine and tryptophan with alanine yielded an eNOS mutant that exhibited reduced eNOS-beta-actin association, decreased NO production, and increased superoxide formation in COS-7 cells. Disruption of eNOS-beta-actin interaction in endothelial cells using ABS peptide 326 resulted in decreased NO production, increased superoxide formation, and decreased endothelial monolayer wound repair, which was prevented by PEG-SOD and NO donor NOC-18. Taken together, this novel finding indicates that beta-actin binding to eNOS through residues 326-333 in the eNOS protein results in shifting the enzymatic activity from superoxide formation toward NO production. Modulation of NO and superoxide formation from eNOS by beta-actin plays an important role in endothelial function.

Hypoxic upregulation of arginase II in human lung endothelial cells.

Activated arginase has been implicated in many diseases including cancer, immune cell dysfunction, infections, and vascular disease. Enhanced arginase activity has been reported in lungs of patients with pulmonary artery hypertension. We used hypoxia as a model for pulmonary hypertension and studied the effect of exposure to hypoxia on arginase activity in human lung microvascular endothelial cells (HMVEC). Hypoxia induces upregulation of arginase activity as well as mRNA and protein levels of arginase II (Arg II), the only arginase isoform we were able to identify in HMVEC. In endothelial cells, arginase shares and competes for the substrate l-arginine with nitric oxide (NO) synthase (NOS). Through regulation of substrate availability for NOS, arginase is able to modulate NO production. To evaluate the role of Arg II in regulation of NO production under hypoxia, we compared NO output (RFL-6 reporter assay) in cells with normal and silenced Arg II. Exposure to hypoxia led to an increase in NO levels produced by HMVEC. Inhibition of Arg II by specific small interfering RNA or by the pharmacological inhibitor BEC additionally enhanced the levels of NO. Another possible role for activated arginase is involvement in regulation of cell proliferation. However, we showed that hypoxia decreased cell proliferation and upregulated Arg II did not have an effect on cell proliferation. Since hypoxia-inducible factors (HIF) are a family of transcriptional factors activated by hypoxia, we tested the possibility of involvement of HIF-1 and HIF-2 in regulation of Arg II under hypoxia. The silencing of HIF-2 but not HIF-1 prevented the activation of Arg II by hypoxia.

Endothelial arginase II responds to pharmacological inhibition by elevation in protein level.

Arginase is an enzyme which converts arginine to ornithine and urea. Recently, arginase has been implicated in many physiological and pathological processes including vascular diseases. Inhibition of arginase activity by pharmacological inhibitors is a useful tool to study the biology of arginases and their possible role in therapy. There are several arginase-specific inhibitors commercially available. Herein, we show that some of these inhibitors lead to an increase in arginase II protein level and activity. These effects should be anticipated when these inhibitors are in use or during the testing of new arginase inhibitors.

Peptide-stimulation enhances compartmentalization and the catalytic activity of lung endothelial NOS.

We reported that an 11 amino acid synthetic peptide (P1) activates lung endothelial cell nitric oxide synthase (eNOS) independent of its change in expression and/or phosphorylation. Since caveolae/eNOS dissociation is known to enhance the catalytic activity of eNOS, we examined whether P1-mediated increase of eNOS activity is associated with caveolae/cholesterol modulation, increased caveolin-1 phosphorylation, and intracellular compartmentalization of eNOS in pulmonary artery endothelial cells (PAEC). PAEC were incubated with or without (control) P1 or cholesterol modulators/caveolae disruptors, cholesterol oxidase (CHOX) and methyl-beta-cyclodextrin (CD), for 1 h at 37 degrees C. After incubation cells were used for: i) immunoprecipitation, ii) isolation of plasma membrane (PM)-, Golgi complex (GC)-, and non-Golgi complex (NGC)-enriched fractions, iii) immunofluorescence confocal imaging, and iv) electron microscopy for localization and/or eNOS activity. P1, CHOX, and CD-stimulation caused dissociation of eNOS from PM with increased localization to GC and/or NGC. P1 and CHOX significantly increased eNOS activity in PM and GC and CD-stimulation increased eNOS activity localized only in GC. P1 increased phosphorylation of caveolin-1 in intact cells and GC fraction. Immunofluorescence and/or immunogold labeled imaging/electron microscopy analysis of P1-, CHOX-, and CD-stimulated intact cells confirmed eNOS/caveolae dissociation and translocation of eNOS to GC. These results suggest that: i) P1-stimulation translocates eNOS to GC and enhances the catalytic activity of eNOS in both the PM and GC fractions of PAEC, ii) CHOX- but not CD-mediated caveolae and/or cholesterol modulation mimics the effect of P1-stimulated compartmentalization and activation of eNOS in PAEC, and iii) P1-stimulated caveolae/cholesterol modulation, phosphorylation of caveolin-1, and activation of eNOS is physiologically relevant since P1 is known to enhance NO/cGMP-dependent vasorelaxation in the pulmonary circulation.

Beta-actin: a regulator of NOS-3.

Beta-actin is traditionally considered a structural protein that organizes and maintains the shape of nonmuscle cells, although data now indicate that beta-actin is also a signaling molecule. beta-actin is directly associated with nitric oxide synthase type 3 (NOS-3) in endothelial cells and platelets, and this interaction increases NOS-3 activity and the affinity of NOS-3 for heat shock protein 90 kD (Hsp90). The beta-actin-induced increase in NOS-3 activity may be caused directly by beta-actin, the binding of Hsp90 to NOS-3, or both. Alterations in the interaction between beta-actin and NOS-3 could be caused by changes either in the availability of beta-actin or in the affinity of NOS-3 for beta-actin, and these alterations probably contribute to vascular complications and platelet aggregation. Studies examining the interactions between NOS-3, beta-actin, and Hsp90 could potentially lead to the discovery of effective peptides for the treatment of diseases associated with impaired NOS-3 activity and nitric oxide release, such as systemic and pulmonary hypertension, atherosclerosis, and thrombotic diseases.

Calpain-2 regulation of VEGF-mediated angiogenesis.

Angiogenesis is a complex process involving endothelial cell migration, proliferation, and differentiation as well as tube formation. These processes are stimulated by a variety of growth factors such as vascular endothelial growth factor (VEGF). VEGF-induced cytoskeletal reorganization plays a crucial role in the angiogenic processes. In the present study, we evaluated the role of calpain in VEGF-induced angiogenesis in vitro and in vivo. Human pulmonary microvascular endothelial cells (PMEC) were incubated with VEGF (10-60 ng/ml) for 2-24 h, after which we measured calpain activities, protein contents of the calpain subunits and of calpastatin, endothelial monolayer wound repair, tube formation, and actin cytoskeleton changes. Incubation of PMEC with VEGF resulted in dose- and time-dependent increases in calpain activity and protein content of calpain-2. VEGF did not change the protein contents of calpain-1 and the small subunit or of calpastatin. Incubation of PMEC with a VEGF receptor blocker prevented the VEGF-induced increase in calpain activity. Inhibition of calpain activity by siRNA directed against calpain-2 and by overexpression of calpastatin prevented VEGF-induced increases in actin stress fibers in endothelial cells and angiogenesis. Overexpression of calpastatin also inhibits vessel formation in subcutaneous (s.c.) matrigel plugs in mice. These results indicate that calpain mediates VEGF-induced angiogenic effects by modulating actin cytoskeletal organization.

Peptides modified by myristoylation activate eNOS in endothelial cells through Akt phosphorylation.

1. Myristoylated pseudosubstrate of PKCzeta (mPS) - a synthetic myristoylated peptide with a sequence (13 amino acids) mimicking the endogenous PKCzeta pseudosubstrate region -- is considered a selective cell-permeable inhibitor of PKCzeta. We present strong evidence that in endothelial cells the action of mPS is not limited to inhibition of PKC activity and that myristoylation of certain peptides can activate eNOS (endothelial nitric oxide synthase) through Akt phosphorylation. 2. mPS at micromolar concentrations (1-10 microM) induced profound phosphorylation of eNOS, Akt, ERK 1/2, and p38 MAPK in cultured pulmonary artery endothelial cells (PAEC). The same changes were observed after treatment of PAEC with a myristoylated scrambled version of mPS (mScr), whereas a cell-permeable version of PKCzeta pseudosubstrate fused to the HIV-TAT membrane-translocating peptide did not induce analogous changes, suggesting that myristoylation confers new properties on the peptides consisting of activation of different signaling pathways in endothelial cells. 3. In addition to mPS and mScr, a number of other myristoylated peptides induced phosphorylation of eNOS suggesting that myristoylation of peptides can activate eNOS by mechanisms unrelated to inhibition of PKC. All active myristoylated peptides contained basic amino acids motif and were longer than six amino acids. 4. Activation of eNOS by myristoylated peptides was dependent on the PI3K/Akt pathway and the rise of intracellular calcium and was associated with an elevation of cGMP levels in PAEC and with relaxation of precontracted isolated pulmonary artery segments. 5. Myristoylated peptides can be considered a new class of activators of NO production in endothelial cells and that using mPS as a specific inhibitor of PKC should be done with caution, especially in endothelial cells.

Cytoskeletal regulation of nitric oxide synthase.

The three isoforms of nitric oxide synthase (NOS)--endothelial NOS (eNOS), inducible NOS (iNOS), and neural NOS (nNOS)--colocalize with the cytoskeleton including actin microfilaments, microtubules, and intermediate filaments directly or indirectly. These colocalizations enable optimal nitric oxide production and help NOS exert their functions. The reorganization of cytoskeletal polymerization state induced by extracellular stimuli such as shear stress, hypoxia, and drugs regulates eNOS, nNOS, and iNOS. Alterations of nitric oxide production caused by cytoskeletal reorganization play an important role in physiological and pathophysiological conditions. This review focuses on recent data regarding the regulation of NOS by the cytoskeleton at transcriptional, posttranscriptional, and posttranslational levels.

Enhanced apoptosis in prolonged cultures of senescent porcine pulmonary artery endothelial cells.

Senescent or aged endothelial cells in culture remain metabolically active after cessation of division, and are generally believed to eventually die. However, mechanisms underlying the terminal aging of cultured cells, i.e. from senescence to death, are poorly understood. Here, we report that culturing of replicative senescent endothelial cells for a prolonged period of time without passaging leads to enhanced programmed cell death or apoptosis. Senescent (passage 45) and young (passage 3) porcine pulmonary artery endothelial cells (PAEC) were cultured for 0-42 days post confluence. The cells attached to culture dishes and floating in medium were collected at 0, 7, 14, 21, 28, 35 and 42 days post confluence and were assessed for markers of apoptosis. Morphology studies showed that ratios between senescent and young cells attached to dishes declined to 45% after 42 days postconfluence. Apoptotic cells in prolonged cultures of senescent PAEC increased from 5 to 35% as determined by protein mass, DNA breakage, and caspase-3 activation. Steady state levels of Bcl-2, an anti-apoptotic protein, in senescent prolonged cultures decreased to less than 20% for all time points compared with young cells. Relative levels of Bad, a pro-apoptotic protein, in senescent cells were elevated from 60 to 130% during prolonged culturing. These results indicate that terminal cellular aging enhances apoptosis and the levels of Bcl-2/Bad may be associated with the apoptotic process in porcine lung endothelial cells.

Down-regulation of mitochondrial cytochrome c oxidase in senescent porcine pulmonary artery endothelial cells.

Cellular aging is associated with dysfunction of the mitochondrial respiration chain. Deficiency of mitochondrial cytochrome c oxidase (complex IV) plays a critical role in aging-induced mitochondrial dysfunction. We investigated whether in vitro cellular aging causes the downregulation of complex IV activity and gene expression using senescent (passage 45) and young (passage 3) pulmonary artery endothelial cells (PAEC). In senescent PAEC, the catalytic activity of complex IV decreased 84%, compared to that in young cells. Relative protein levels of complex IV subunits I and IV (complex IV S1 and S4) in senescent cells decreased 91%, compared to those in young cells. This suggests that lack of complex IV S1 and S4 in senescent cells may contribute to the deficiency of complex IV. Total steady state levels of mRNA for complex IV S1 and S4 in senescent cells were decreased to 20% and 18% of those in young cells. The relative rates of mRNA synthesis of complex IV S1 and S4 were decreased 46% and 37% in senescent cells, respectively, compared to young cells. The degradation of complex IV S1 and S4 was increased 76% and 64% in senescent cells, compared to young cells. These data indicate that mitochondrial DNA-encoded subunit I and nuclear DNA-encoded subunit IV of complex IV are downregulated through reduced synthesis and enhanced degradation of their mRNA, which may be responsible for the deficiency of complex IV in replicative senescent PAEC.

Senescence-enhanced oxidative stress is associated with deficiency of mitochondrial cytochrome c oxidase in vascular endothelial cells.

Cellular senescence-elevated oxidative stress plays a critical role in age-associated vascular endothelial dysfunction. We investigated whether deficiency of mitochondrial cytochrome c oxidase (complex IV) is causally linked to increased oxidant generation during cellular aging using senescent (passage 45) and young (passage 3) pulmonary artery endothelial cells (PAEC). In senescent PAEC, levels of O2- and H2O2 were elevated onefold, respectively, compared to those in young cells. Lipid peroxidation and protein carbonyl contents in aged cells were increased more than twofold compared to young cells. To determine whether lack of complex IV in senescent cells contributed to the increased oxidant generation, complex IV activity in young cells was specifically inhibited using antisense oligonucleotides directed against the mRNA of complex IV subunits. Levels of O2- and H2O2 in PAEC treated with antisense oligonucleotides were elevated onefold, respectively, which correlated with a similar increase in lipid (110%) and protein (20%) oxidation, compared to control oligonucleotides-transfected cells. Moreover, levels of nitrosylated proteins in antisense-transfected cells were increased 30%, compared to controls. These data demonstrate that deficiency of complex IV in senescent cells enhances oxidative and nitrosative stress, which may be responsible for senescence-induced endothelial cell loss and dysfunction.

Metabolism of dynorphins by peptidases of pulmonary artery endothelial cells.

Degradation of several dynorphins by peptidases expressed in cultured porcine pulmonary artery endothelial cells was studied by incubation of the peptide in cell suspensions followed by electrospray ionization and tandem mass spectrometric analyses. Under the in vitro conditions applied, only the metabolism of dynorphin A1-8 occurred in a significant extent. Studies involving specific peptidase inhibitors indicated that mainly bestatin-sensitive aminopeptidases, thiorphan-sensitive endopeptidases, and cFPAAF-pAB-sensitive endopeptidases expressed by the endothelial cells were involved in the process that converted dynorphin A1-8 to dynorphin A2-8, dynorphin A1-6, and leucine enkephalin (dynorphin A1-5), respectively. These peptidases may form a metabolic barrier for the cellular penetration of intact dynorphin A1-8 and/or control effects of the circulating peptide on endothelial opioid receptors of the cells.

Could uric acid be a modifiable risk factor in subjects with pulmonary hypertension?

A high serum uric acid is common in subjects with pulmonary hypertension. The increase in serum uric acid may be a consequence of the local tissue ischemia and/or hypoxia, and it may also result from other factors independent of ischemia or hypoxia that occur in various forms of pulmonary hypertension. While classically viewed as a secondary phenomenon, recent studies suggest that hyperuricemia may also have a role in mediating the local vasoconstriction and vascular remodeling in the pulmonary vasculature. If uric acid does have a contributory role in pulmonary hypertension, we may see an increasing prevalence of pulmonary hypertension as hyperuricemia is common in subjects with obesity and metabolic syndrome. We propose studies to investigate the role of uric acid in pulmonary hypertension and to determine if lowering serum uric acid may have clinical benefit in this condition.

Priming donor lungs with thioredoxin-1 attenuates acute allograft injury in a rat model of lung transplantation.

BACKGROUND: Lung graft dysfunction and rejection are significant causes of morbidity and mortality in transplant recipients. Thioredoxin-1, a redox-regulatory protein, functions as an antioxidant in multiple organs, including lungs. We examined whether priming of the donor lungs with thioredoxin-1 before transplantation attenuates acute lung injury. METHODS: Orthotopic left lung transplantation was performed from Lewis (donor) to Sprague-Dawley (recipient) rats. Donor lungs were perfused and stored in Perfadex solution (Vitrolife, Uppsala, Sweden), with or without purified thioredoxin-1. Changes in bronchoalveolar lavage (BAL) analysis, allograft oxygen exchange function, nuclear factor kappaB (NF-kappaB)/DNA binding, myeloperoxidase activities, and immunohistologic evaluation of neutrophils, macrophages, and cytotoxic T-cells (CD8(+)) infiltration were examined in post-transplant allograft (left) and native (right) lungs at Days 1 and 5. RESULTS: BAL cell differential analysis showed significant increases in macrophages and neutrophils in allografts at Day 1 post-transplant. At Days 1 and 5, lymphocyte infiltration was significantly increased and myeloperoxidase and NF-kappaB/DNA binding activities were increased vs basal activities. Immunohistology staining revealed increased infiltration of macrophages, neutrophils, and CD8(+) T cell sub-sets. Pre-transplant priming of donor lungs with thioredoxin-1 improved oxygen exchange and attenuated NF-kappaB/DNA binding activity, and infiltration of macrophages, neutrophils, and CD8(+) T cell sub-sets in allografts at Days 1 and 5 post-transplant. CONCLUSIONS: Priming of donor lungs with thioredoxin-1 before transplant attenuates acute allograft injury in a rat model of lung transplantation, and appears to be associated with the antioxidant function of thioredoxin-1 that limits early ischemia-reperfusion injury, NF-kappaB activation, and progressive infiltration of inflammatory and immune cells in allografts.

Uric acid decreases NO production and increases arginase activity in cultured pulmonary artery endothelial cells.

Elevated levels of serum uric acid (UA) are commonly associated with primary pulmonary hypertension but have generally not been thought to have any causal role. Recent experimental studies, however, have suggested that UA may affect various vasoactive mediators. We therefore tested the hypothesis that UA might alter nitric oxide (NO) levels in pulmonary arterial endothelial cells (PAEC). In isolated porcine pulmonary artery segments (PAS), UA (7.5 mg/dl) inhibits acetylcholine-induced vasodilation. The incubation of PAEC with UA caused a dose-dependent decrease in NO and cGMP production stimulated by bradykinin or Ca(2+)-ionophore A23187. We explored cellular mechanisms by which UA might cause reduced NO production focusing on the effects of UA on the l-arginine-endothelial NO synthase (eNOS) and l-arginine-arginase pathways. Incubation of PAEC with different concentrations of UA (2.5-15 mg/dl) for 24 h did not affect l-[(3)H]arginine uptake or activity/expression of eNOS. However, PAEC incubated with UA (7.5 mg/dl; 24 h) released more urea in culture media than control PAEC, suggesting that arginase activation might be involved in the UA effect. Kinetic analysis of arginase activity in PAEC lysates and rat liver and kidney homogenates demonstrated that UA activated arginase by increasing its affinity for l-arginine. An inhibitor of arginase (S)-(2-boronoethyl)-l-cysteine prevented UA-induced reduction of A23187-stimulated cGMP production by PAEC and abolished UA-induced inhibition of acetylcholine-stimulated vasodilation in PAS. We conclude that UA-induced arginase activation is a potential mechanism for reduction of NO production in PAEC.

Thioredoxin as a biomarker for graft rejection in lung transplant recipients.

Primary graft dysfunction and rejection are common complications in lung transplant recipients. Increased expression of thioredoxin-1 (Trx), a 12-kDa redox-regulatory protein, has been reported in multiple lung pathophysiological conditions involving oxidative and inflammatory mediated injury including graft rejection in canine and rat models of lung transplantation. Our objective was to determine whether increased Trx expression is associated with progression of rejection pathophysiology in human lung transplant recipients. Bronchoalveolar lavage (BAL) fluid and transbronchial biopsy samples were collected as a routine part of post-transplant clinical care from 18 lung transplant patients from our adult lung transplant programme. Lung transplant recipient profile included age/sex, ethnic background, days on ventilator, total ischaemic time, and cytomegalovirus (CMV) status. Based on histopathological grading criteria, patients were divided into two groups, rejecting (A1/A2 or B1) and non-rejecting (A0/B0). Rejecting and non-rejecting group total BAL cell counts and differential cell counts for neutrophils, macrophages, lymphocytes and eosinophils as well as total BAL cell Trx levels were analysed. Total BAL cell counts were significantly (p <0.05) elevated in graft rejecting versus non-rejecting patients. Differential BAL macrophage counts were comparable in rejection and non-rejection groups, whereas there were significant increases in neutrophils and lymphocytes but not eosinophils in patients with rejection versus non-rejection pathology (p <0.05). Total ischaemic time and days on ventilator in rejection and non-rejection groups were comparable. However, Trx levels were significantly elevated in BAL cells from graft-rejecting patients compared with non-rejecting patients (p <0.05). These data suggest that surveillance monitoring of BAL Trx levels after lung transplantation can serve as a biomarker to assess severity of graft rejection.

Nitric oxide upregulation of caspase-8 mRNA expression in lung endothelial cells: role of JAK2/STAT-1 signaling.

We recently reported that nitric oxide (NO) modulates expression of multiple genes associated with apoptotic pathways, including expression of caspase-8. The objective of the present study is to determine whether the NO-induced expression of the caspase-8 gene is regulated via signal transducers and activators of transcription-1 (STAT-1) signaling. The confluent monolayers of pulmonary artery endothelial cells (PAEC) were incubated with or without (control) 1 mM NOC-18, a NO donor, at 37 degrees C for 0-24 h. In some experiments PAEC were pretreated with a Janus kinase (JAK-2) inhibitor, AG490 (20 microM). Exposure of PAEC to NO-increased relative levels of caspase-8 mRNA as determined using quantitative real time PCR. Relative levels of phosphorylated STAT-1 at Serine (Ser)-727, but not total STAT-1 expression in NO-exposed cells, were upregulated significantly compared to control cells. AG490 attenuated NO-induced phosphorylation of STAT-1 at Ser 727 and expression of caspase-8 mRNA, suggesting JAK2 plays a role in the induction of caspase-8 mRNA. The promoter of caspase-8 has four gamma-activated sequence (GAS) and two interferon-stimulated response element (ISRE) transcription factor-binding sites. NO enhanced the STAT-1 binding activity to GAS/ISRE. Suppression of STAT-1 expression attenuated NO-induced elevation of caspase-8 mRNA. These studies demonstrate that a NO-dependent increase in caspase-8 mRNA levels is associated with phosphorylation of STAT-1 at Ser-727 and STAT1 binding to the caspase-8 promoter in cultured PAEC.

Use of recombinant calpain-2 siRNA adenovirus to assess calpain-2 modulation of lung endothelial cell migration and proliferation.

In this study, we developed an adenoviral vector harboring calpain-2 siRNA expression unit in which sense and anti-sense strands composing the siRNA duplex were connected by a loop and transcribed into a siRNA in porcine pulmonary artery endothelial cells (PAEC). We screened one efficient adenoviral vector Ad/si-m187 and found that Ad/si-m187 successfully exerted a gene knockdown effect on calpain-2 mRNA transcription and protein expression levels. The protein content of calpain-2 was reduced by 30%-80% in PAEC infected with Ad/si-m187 in comparison to a control adenoviral vector Ad/si-luc. The mRNA levels of calpain-2 were measured by real-time PCR and were decreased by 60%-100% and in a dose dependent manner. In correspondence to silencing calpain-2 gene expression, calpain-2 activity was decreased significantly. We further evaluated the role of calpain-2 in endothelial cell migration and proliferation. PAEC infected with Ad/si-m187 displayed impaired migration and cell proliferation in comparison to cells infected with control adenoviral vector (Ad/si-luc). These results indicate that adenoviral vector harboring calpain-2 siRNA expression unit is a valuable tool to study the biology of calpains and that calpain-2 plays an important role in lung endothelial cell migration and proliferation.