Human Pulmonary Vascular Endothelial Cells Research Articles

Human Placental Mesenchymal Stem Cells-Exosomes Alleviate Endothelial Barrier Dysfunction via Cytoskeletal Remodeling through hsa-miR-148a-3p/ROCK1 Pathway.

Endothelial barrier disruption of human pulmonary vascular endothelial cells (HPVECs) is an important pathogenic factor for acute lung injury (ALI)/acute respiratory distress syndrome (ARDS). Mesenchymal stem cells-exosome (MSCs-Exo) represents an ideal carrier for cell-free therapy. The therapeutic implication and underlying mechanism of human placental MSCs-Exo (HPMSCs-Exo) in ALI/ARDS need to be further explored. HPMSCs-Exo was extracted from HPMSCs and characterized. Then, the therapeutic effects of exosomes were evaluated in ALI mice and HPVECs. RNA-sequencing was applied to reveal the miRNA profile of HPMSCs-Exo and differentially expressed genes (DEGs) in HPMSCs-Exo-pretreated HPVECs. The targets of miRNAs were predicted by bioinformatics methods and correlated to DEGs. Finally, the role of hsa-miR-148a-3p/ROCK1 pathway in HPVECs has been further discussed. The results showed that HPMSCs-Exo could downregulate Rho-associated coiled-coil-containing protein kinase 1 (ROCK1), upregulate the expression of zonula occludens-1 (ZO-1) and F-actin, promote HPVECs migration and tube formation, reduce cytoskeletal disorders and cell permeability, and thus improve ALI/ARDS. RNA-sequencing revealed the DEGs were mainly enriched in cell junction, angiogenesis, inflammation, and energy metabolism. HPMSCs-Exo contains multiple miRNAs which are associated with cytoskeletal function; the expression abundance of hsa-miR-148a-3p is the highest. Bioinformatic analysis identified ROCK1 as a target of hsa-miR-148a-3p. The overexpression of hsa-miR-148a-3p in HPMSCs-Exo promoted the migration and tube formation of HPVECs and reduced ROCK1 expression. However, the overexpression of ROCK1 on HPVECs reduced the therapeutic effect of HPMSCs-Exo. HPMSCs-Exo represents a protective regimen against endothelial barrier disruption of HPVECs in ALI/ARDS, and the hsa-miR-148a-3p/ROCK1 pathway plays an important role in this therapeutics implication.

DDAH1 Overexpression in Human Pulmonary Vascular Endothelial Cells Co‐Cultured with Human Pulmonary Artery Smooth Muscle Cells

Bronchopulmonary dysplasia (BPD) is the most common chronic lung disease of preterm infants. Pulmonary hypertension (PH) develops in 25–40% of BPD patients as a result of pathologic vascular remodeling, and PH is an independent predictor of death in BPD patients. Nitric oxide (NO) is a potent vasodilator and apoptotic mediator made in the pulmonary endothelium by nitric oxide synthase (eNOS). Asymmetric dimethylarginine (ADMA) is an endogenous competitive NOS inhibitor. Dimethylarginine dimethylaminohydrolase (DDAH) metabolizes the majority of ADMA in vivo and there are two isoforms, DDAH1 and DDAH2. Translational studies from our group analyzed a cohort of BPD patients and identified a potential biomarker, DDAH1 single nucleotide polymorphism (rs480414), that decreased the risk of PH in BPD by 61%. In the present study, we hypothesized that overexpression of DDAH1 in human pulmonary microvascular endothelial cells (hPMVEC) would lead to greater apoptosis and lower proliferation of human pulmonary artery smooth muscle cells (hPASMC) via effects in hPMVEC. We utilized a co‐culture system where hPMVEC were transfected with AdDDAH1 or AdGFP control for 24h and then incubated in co‐culture with hPASMC for 24h. Analysis included 1) western blot analysis for cleaved and total caspase‐3, ‐8, ‐9, p21, PCNA, and β‐actin and 2) trypan blue exclusion for viable cell numbers. Transfection of hPMVEC with AdDDAH1 resulted in greater DDAH1 protein expression (n=6, p=0.003) as compared to control. After AdDDAH1 transfection of hPMVEC and co‐culture with hPASMC, we found greater cleaved caspase‐3 (n=6, p=0.01), greater cleaved caspase‐8 (n=6, p=0.03), similar cleaved caspase‐9 (n=6, p=0.94), similar p21 (n=6, p=0.7), and similar PCNA (n=3, p=0.82) protein expression as compared to control. After AdDDAH1 transfection of hPMVEC and co‐culture with hPASMC, we found lower hPASMC viable cell numbers (n=3, p=0.008) as compared to control. After AdDDAH1 transfection of hPMVEC and co‐culture with hPASMC treated with DETA NONOate 250μM or DETA NONOate 250μM + Hemoglobin 100μM, the hPASMC viable cell numbers were lower compared to control (n=3, p=0.001; n=3, p=0.003, respectively). There was no difference detected between DETA NONOate 250μM and DETA NONOate 250μM + Hemoglobin 100μM groups (n=3, p=0.429). In conclusion, DDAH1 overexpression in hPMVEC resulted in greater apoptosis and lower proliferation in hPASMC, consistent with our hypothesis. We propose that DDAH1 is a potential new therapy for BPD‐PH via relieving pulmonary vascular endothelial ADMA‐inhibition of eNOS, resulting in greater endogenous NO production, greater pulmonary vascular wall apoptosis, and potentially less pathologic vascular remodeling. Future studies will determine if DDAH1 endothelial knockout mice have worse lung function as compared to wild‐type, after exposure to BPD‐PH model of LPS and hyperoxia.Support or Funding InformationK08 HL129080 (PI: Trittmann)

Avian lungs: A novel scaffold for lung bioengineering.

Allogeneic lung transplant is limited both by the shortage of available donor lungs and by the lack of suitable long-term lung assist devices to bridge patients to lung transplantation. Avian lungs have different structure and mechanics resulting in more efficient gas exchange than mammalian lungs. Decellularized avian lungs, recellularized with human lung cells, could therefore provide a powerful novel gas exchange unit for potential use in pulmonary therapeutics. To initially assess this in both small and large avian lung models, chicken (Gallus gallus domesticus) and emu (Dromaius novaehollandiae) lungs were decellularized using modifications of a detergent-based protocol, previously utilized with mammalian lungs. Light and electron microscopy, vascular and airway resistance, quantitation and gel analyses of residual DNA, and immunohistochemical and mass spectrometric analyses of remaining extracellular matrix (ECM) proteins demonstrated maintenance of lung structure, minimal residual DNA, and retention of major ECM proteins in the decellularized scaffolds. Seeding with human bronchial epithelial cells, human pulmonary vascular endothelial cells, human mesenchymal stromal cells, and human lung fibroblasts demonstrated initial cell attachment on decellularized avian lungs and growth over a 7-day period. These initial studies demonstrate that decellularized avian lungs may be a feasible approach for generating functional lung tissue for clinical therapeutics.

Comparative Decellularization and Recellularization of Wild-Type and Alpha 1,3 Galactosyltransferase Knockout Pig Lungs: A Model for Ex Vivo Xenogeneic Lung Bioengineering and Transplantation.

A novel potential approach for lung transplantation could be to utilize xenogeneic decellularized pig lung scaffolds that are recellularized with human lung cells. However, pig tissues express several immunogenic proteins, notably galactosylated cell surface glycoproteins resulting from alpha 1,3 galactosyltransferase (α-gal) activity, that could conceivably prevent effective use. Use of lungs from α-gal knock out (α-gal KO) pigs presents a potential alternative and thus comparative de- and recellularization of wild-type and α-gal KO pig lungs was assessed. Decellularized lungs were compared by histologic, immunohistochemical, and mass spectrometric techniques. Recellularization was assessed following compartmental inoculation of human lung bronchial epithelial cells, human lung fibroblasts, human bone marrow-derived mesenchymal stromal cells (all via airway inoculation), and human pulmonary vascular endothelial cells (CBF) (vascular inoculation). No obvious differences in histologic structure was observed but an approximate 25% difference in retention of residual proteins was determined between decellularized wild-type and α-gal KO pig lungs, including retention of α-galactosylated epitopes in acellular wild-type pig lungs. However, robust initial recellularization and subsequent growth and proliferation was observed for all cell types with no obvious differences between cells seeded into wild-type versus α-gal KO lungs. These proof of concept studies demonstrate that decellularized wild-type and α-gal KO pig lungs can be comparably decellularized and comparably support initial growth of human lung cells, despite some differences in retained proteins. α-Gal KO pig lungs are a suitable platform for further studies of xenogeneic lung regeneration.

Abstract 3274: VEGF─receptor antagonist, Sugen 5416, sensitizes pulmonary endothelial stem-like cells to estrogens: A microvascular model for the progression of lung cancer

Abstract Proliferative vascular lesions are an aggressive angiogenic phenotype associated with a poor prognosis in non-small cell lung cancer (NSCLC). Recent studies suggest that CD133+ endothelial stem-like cells are recruited to the angiogenic vascular system of NSCLC; and are associated with tumor stage and progression. Epidemiological studies have reported that women who received hormone replacement therapy (HRT) show an overall lower survival to lung cancer especially NSCLC when compared to women with no HRT. Proliferative vascular lesions have been studied extensively using the VEGF─receptor antagonist, Sugen 5416 (SU5416), plus chronic hypoxia (SuHx) rodent model. Therefore, the aim of this study was to determine whether SU5416 exposure of human pulmonary vascular endothelial cells (ECs) become more sensitive to estrogen-induced cell growth. We exposed human pulmonary endothelial cell line HPMEC-ST1.6R to SU5416 to select for a sub-population of cells which were then treated with 17β-estradiol and/or estrogenic polychlorinated biphenyl 153 (PCB153). We observed a significant increase of 2-3 fold change in cell growth and proliferation as determined by MTT, SRB, BrdU, and FACS analysis when SU5416 treated cells were exposed to estrogenic chemicals. Sugen 5416 treatment of ECs showed an increase in the inhibitor of DNA binding and differentiation protein 3 (ID3) along with stem cell markers CD133, Oct-4, and Sox-2. Immunohistochemistry of lung tissue samples from SuHx rodent model showed increased expression of ID3 in pulmonary vascular lesions that correlated with an increase in stem cell markers Oct-4 and Nanog; and increased VEGFR3+ cells surrounding vessel obliterating lesions from SuHx lung tissue. Our findings demonstrate that SU5416 sensitized cells to estrogens resulting in higher cell growth compared to control. Since VEGFR3 expression levels are reported to correlate with metastasis in NSCLC patients, our findings may be used as a model to study how VEGFR3 signaling can promote the aggressive growth and spread of lung cancer when exposed to estrogenic chemicals. Furthermore, we showed that stem cell markers Oct-4, Sox-2, and Nanog are inducible molecular targets of Sugen 5416 treatment. This finding is significant in that it points toward a potential mechanism of chemical-induced pluripotent stem-like cells. A better understanding of how vascular lesions depend on SU5416 may open new avenues for the prevention and treatment of lung cancer. The presence of stem cell markers does not imply that these cells actually function as stem cells. Instead, we propose that the combination of stem cell marker expression and chemical exposure to Sugen 5416 re-program terminally differentiated ECs to a pluripotent cell that gives rise to a clonal pulmonary vascular lesion that supports the aggressive growth of NSCLC. Citation Format: Mayur Doke, Quentin H. Felty. VEGF─receptor antagonist, Sugen 5416, sensitizes pulmonary endothelial stem-like cells to estrogens: A microvascular model for the progression of lung cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3274.

BMPR2 gene delivery reduces mutation-related PAH and counteracts TGF-β-mediated pulmonary cell signalling.

Idiopathic, familial and secondary pulmonary arterial hypertension (PAH) are associated with reduced bone morphogenetic protein receptor type 2 (BMPR2) expression, and in some contexts, TGF-β upregulation. Our aims were to assess BMPR2 gene therapy in a PAH mouse model and to assess the impact on TGF-β signalling. Using a targeted in vivo gene delivery approach, we assessed the impact of BMPR2 gene delivery in a transgenic mouse model in which PAH was first induced by doxycycline driven expression of a dominant negative BMPR2 mutant (R899X). We also assessed the impact of BMPR2 gene delivery on TGF-β-induced changes in cell signalling in human pulmonary vascular endothelial and smooth muscle cells. In the mouse model, changes in TGF-β levels were not detected, but BMPR2 gene delivery reversed the increase in right ventricle systolic pressure (RVSP) and Fulton Index (FI), associated with a trend to increased pulmonary endothelial nitric oxide synthase (eNOS) gene expression. In vitro, BMPR2 gene transfer reduced TGF-β effects on Smad2, Smad1/5/8 and Erk1/2 phosphorylation in human pulmonary arterial smooth muscle cells (HPASMC). BMPR2 was also found to upregulate nitric oxide (NO) production in lung derived human microvascular endothelial cells (HMVEC-L). This study provides further evidence that BMPR2 modulation may have therapeutic potential. See Editorial, page 406.

Role of GDF15 (growth and differentiation factor 15) in pulmonary oxygen toxicity

GDF15 (growth and differentiation factor 15) is a secreted cytokine, a direct target of p53 and plays a role in cell proliferation and apoptosis. It is induced by oxidative stress and has anti-apoptotic effects. The role of GDF15 in hyperoxic lung injury is unknown. We tested the hypothesis that GDF15 will be induced in vitro, in a model of pulmonary oxygen toxicity, and will play a critical role in decreasing cell death and oxidative stress. BEAS-2B (human bronchial epithelial cells) and human pulmonary vascular endothelial cells (HPMEC) were exposed to hyperoxia, and expression of GDF15 and effect of GDF15 disruption on cell viability and oxidative stress was determined. Furthermore, we studied the effect of p53 knockdown on GDF15 expression. In vitro, both BEAS-2B and HPMEC cells showed a significant increase in GDF15 expression upon exposure to hyperoxia. After GDF15 knockdown, there was a significant decrease in cell viability and increase in oxidative stress compared to control cells transfected with siRNA with a scrambled sequence. Knockdown of p53 significantly decreased the induction of GDF15 by hyperoxia. In conclusion, we show that GDF15 has a pro-survival and anti-oxidant role in hyperoxia and that p53 plays a key role in its induction.

S38 The Brd4 Inhibitor, Jq1 Decreases Proliferation And Arrests The Cell Cycle Of Pulmonary Vascular Cells: Implications For Pulmonary Arterial Hypertension

Background Pulmonary arterial hypertension (PAH) is an incurable disease characterised by raised pulmonary resistance, resulting from vascular remodelling which leads to right heart failure and death. Recently NF-kB mediated inflammatory gene expression and vascular proliferation/remodelling have been shown to be involved in the pathogenesis of PAH. The expression of subsets of NF-kB-induced inflammatory genes is dependent upon the recruitment of the bromodomains and extra-terminal (BET) family of proteins to the transcriptional activation complex. We hypothesise that inhibition of BET proteins which bind acetylated lysine residues on histones and non-histone proteins will attenuate the hyperproliferative and proinflammatory phenotype of vascular cells. Methods Primary human pulmonary vascular endothelial cells (P-EC) were serum starved for 24 h prior to treatment with the Brd4 mimic JQ1+ or JQ1- (inactive enantiomer) in complete (5% FCS) media. P-EC cell proliferation was measured by BrdU incorporation and apoptosis was determined using caspase 3/7 activity. Cell cycle progression was determined by FACs analysis. mRNA levels of cell cycle genes and inflammatory cytokines were measured by RT-PCR. MTT assay was used to measure cell viability. Results JQ1+ caused a significant (p Conclusion Inhibition of Brd4 with JQ1 decreases remodelling and inflammation in P-EC via a decrease in proliferation, cell cycle arrest and an increase in apoptosis. Further work is required but Brd4 inhibition may provide therapeutic drugs for the treatment of PAH.

Human lung on a chip: innovative approach for understanding disease processes and effective drug testing

Human lung on a chip: innovative approach for understanding disease processes and effective drug testing

Type I interferon induces CX3CL1 (fractalkine) and CCL5 (RANTES) production in human pulmonary vascular endothelial cells

Type I interferon (IFN) medications cause various adverse reactions, including vascular diseases. Although an association between chemokines and vascular diseases has also been reported, the relationship between type I IFN and chemokines in vascular endothelial cells (VEC) remains unclear. To provide clues to pathogenesis of the diseases, we analysed the effects of type I IFN on chemokine production in human VEC. Type I IFN induced higher CX3CL1 (fractalkine) mRNA expression and protein secretion in pulmonary arterial VEC than in umbilical vein VEC. Type I IFN also induced CCL5 [regulated upon activation normal T cell expressed and secreted (RANTES)] production in VEC, especially in lung micro-VEC. IFN-β induced much higher chemokine production than IFN-α, and Janus protein tyrosine kinase (JAK) inhibitor I prevented type I IFN-induced chemokine secretion. Type I IFN-induced chemokines may be involved in the pathophysiology of pulmonary vascular diseases, and the JAK inhibitor may serve as a therapeutic option for these diseases.

Infection of Pulmonary Vascular Endothelial Cells by H5N1 Avian Influenza Virus and its Role in Pathogenesis

Evaluation of: Zeng H, Pappas C, Belser JA et al. Human pulmonary microvascular endothelial cells support productive replication of highly pathogenic avian influenza viruses: possible involvement in the pathogenesis of human H5N1 virus infection. J. Virol. 86(2), 667–678 (2012). The H5N1 highly pathogenic avian influenza (HPAI) virus is still a major threat for pandemic influenza. The severity of the infection, with mortality reaching up to 60%, is of particular concern, and the detailed mechanism of this high pathogenicity is not fully understood. While alveolar epithelial cells are considered to be the main target cells in humans, Zeng et al. reported that human pulmonary vascular endothelial cells were more susceptible to the H5N1 HPAI virus than to seasonal, pandemic or low pathogenic avian influenza viruses. Infection induced the endothelial cells to produce a number of proinflammatory cytokines, which raised the possibility of a new virulence mechanism that may link to the systemic dissemination of the infection in humans. However, there are still many unanswered questions regarding the determinants of the higher infectivity of H5N1 HPAI in this cell type and the role of the infection of this cell type in the pathogenesis of H5N1 HPAI.

Interactions between PBEF and oxidative stress proteins – A potential new mechanism underlying PBEF in the pathogenesis of acute lung injury

Identification of pre-B-cell colony-enhancing factor (PBEF) interacting partners may reveal new molecular mechanisms of PBEF in the pathogenesis of acute lung injury (ALI). The interactions between PBEF and NADH dehydrogenase subunit 1(ND1), ferritin light chain and interferon induced transmembrane 3 (IFITM3) in human pulmonary vascular endothelial cells were identified and validated. ND1, ferritin and IFITM3 are involved in oxidative stress and inflammation. Overexpression of PBEF increased its interactions and intracellular oxidative stress, which can be attenuated by rotenone. The interaction modeling between PBEF and ND1 is consistent with the corresponding experimental finding. These interactions may underlie a novel role of PBEF in the pathogenesis of ALI. Structured summary MINT- 6538697: PBEF (uniprotkb: P43490) physically interacts (MI: 0218) with NADH1 (uniprotkb: P03886) by two hybrid (MI: 0018) MINT- 6538811, MINT- 6538868: PBEF (uniprotkb: P43490) physically interacts (MI: 0218) with interferon-induced transmembrane protein 3 (uniprotkb: Q01628) by anti bait coimmunoprecipitation (MI: 0006) MINT- 6538787, MINT- 6538841: PBEF (uniprotkb: P43490) physically interacts (MI: 0218) with NADH1 (uniprotkb: P03886) by anti bait coimmunoprecipitation (MI: 0006) MINT- 6538755: PBEF (uniprotkb: P43490) physically interacts (MI: 0218) with γ- glutamyl-transferase (uniprotkb: P19440) by two hybrid (MI: 0018) MINT- 6538799, MINT- 6538862: PBEF (uniprotkb: P43490) physically interacts (MI: 0218)with Ferritin light chain (uniprotkb: P02792) by anti bait coimmunoprecipitation (MI: 0006) MINT- 6538769: PBEF (uniprotkb: P43490) physically interacts (MI: 0218) with E2L6 (uniprotkb: O14933) by two hybrid (MI: 0018) MINT- 6538741: PBEF (uniprotkb: P43490) physically interacts (MI: 0218) with Adenosine A2aR (uniprotkb: P29274) by two hybrid (MI: 0018) MINT- 6538727: PBEF (uniprotkb: P43490) physically interacts (MI: 0218) with interferon-induced transmembrane protein 3 (uniprotkb: Q01628) by two hybrid (MI: 0018) MINT- 6538712: PBEF (uniprotkb: P43490) physically interacts (MI: 0218) with Ferritin light chain (uniprotkb: P02792) by two hybrid (MI: 0018)

Flavone as PARP-1 inhibitor: Its effect on lipopolysaccharide induced gene-expression

The nuclear enzyme poly(ADP-ribose) polymerase-1 (PARP-1) which was initially known for its role in the repair of oxidative stress-induced DNA damage, has also been reported to play a mediating role in the inflammatory response. Studies with PARP-1 knockout models have shown that PARP-1 is a co-activator of Nuclear Factor-kappa B (NF-kappaB), although this appears not to require its enzyme activity. In addition, drug-induced inhibition of the enzyme activity of PARP-1 was observed to reduce the production of pro-inflammatory mediators. In this study, the flavonoid compound flavone was demonstrated to significantly inhibit the enzyme activity of PARP-1. Further evaluation of flavone in N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)-treated human pulmonary epithelial and vascular endothelial cells revealed that both the decrease in NAD(+) levels, as well as the formation of PAR-polymers was dose-dependently attenuated by flavone. In addition, flavone was found to reduce the lipopolysaccharide (LPS)-induced interleukin (IL)-8 production in pulmonary epithelial cells, which was confirmed by transcription analysis. Furthermore, the transcription Inhibitor kappa B alpha (of IkappaBalpha) was significantly increased by flavone. The results of the present study indicate that the flavonoid flavone could be a potential candidate for application in treatment of chronic inflammatory diseases. PARP-1 inhibition could have beneficial effects in such diseases as Chronic Obstructive Pulmonary Disease (COPD) and diabetes, by preservation of cellular NAD(+) levels and attenuating inflammatory conditions.

Apoptosis signal-regulating kinase 1-mediated signaling pathway regulates hydrogen peroxide-induced apoptosis in human pulmonary vascular endothelial cells.

Reactive oxygen species initiate pulmonary vascular endothelial cell damage leading to an increase in endothelial permeability resulting in the production of pulmonary edema. Apoptosis signal-regulating kinase (ASK)-1 is a ubiquitously expressed mitogen-activated protein kinase kinase kinase (MAPKKK) that activates the MKK3/MKK6-p38 MAPK and the SEK1-c-Jun N-terminal kinase (JNK) signaling cascade. ASK1 has been implicated in cytokine- and stress-induced apoptosis. However, little is known about the role of ASK1 in apoptosis in hydrogen peroxide (H2O2)-stimulated pulmonary vascular endothelial cells and how ASK1-mediated apoptosis is executed. To clarify this issue, we examined the role of ASK1-p38 MAPK/JNK cascade in apoptosis and caspase-3 activation in H2O2-stimulated pulmonary vascular endothelial cells. Experimental laboratory study. University laboratory. Normal human pulmonary artery endothelial cells. Western blot analysis and quantification of apoptosis in cells. The results showed that H2O2 induced ASK1 phosphorylation and concomitantly p38 MAPK and JNK phosphorylation as well as induced caspase-3 activation in pulmonary vascular endothelial cells. To further characterize the role of ASK1 cascade in H2O2-induced apoptosis of pulmonary vascular endothelial cells, the dominant negative form of ASK1-stably transfected porcine artery endothelial cells was used. p38 MAPK and JNK phosphorylation, caspase-3 activation, and apoptosis in the dominant negative form of ASK1-stably transfected porcine artery endothelial cells were depressed compared with those in the parental porcine artery endothelial cells. ASK1-p38 MAPK/JNK cascade regulates apoptosis of H2O2-stimulated human pulmonary vascular endothelial cells.

Dexamethasone Does Not Modulate Eosinophil Adhesion to Endothelial Cells

Background: Glucocorticoids are representative agents in the anti-inflammatory treatment of allergic airway diseases. Although they reduce the number of tissue eosinophils, its exact mechanism has not been completely established. Objective: The present study was undertaken to evaluate whether dexamethasone modulates the adhesive property of eosinophils. The effect on the expression of endothelial adhesion molecules was also evaluated. Methods: Blood eosinophils from healthy subjects were incubated in the presence or absence of 0.1–1 µM of dexamethasone for 6 h at 37°C. Eosinophil chemotaxis and adhesion to paraformaldehyde-fixed human pulmonary vascular endothelial cells (HPMEC) were then examined. The effects of dexamethasone on the expression of VCAM-1 and ICAM-1 on HPMEC were examined by cell ELISA. Results: 1 µM dexamethasone significantly inhibited the chemotactic response of eosinophils to 1 µM formyl-methionyl-leucyl-phenylalanine (FMLP). On the other hand, dexamethasone did not modify either the spontaneous or FMLP-stimulated adhesion of eosinophils to resting HPMEC. Similarly, dexamethasone did not alter either the spontaneous adhesion of eosinophils to or the expression of VCAM-1 or ICAM-1 on HPMEC stimulated with IL-4 + TNF-α. Conclusion: Dexamethasone does not modify the adhesive property of eosinophils or the expression of adhesion molecules on endothelial cells. Thus, it is unlikely that glucocorticoids directly modulate the adhesive interaction between eosinophils and endothelial cells.

N-acetylcysteine attenuates TNF-alpha-induced p38 MAP kinase activation and p38 MAP kinase-mediated IL-8 production by human pulmonary vascular endothelial cells.

1. We have previously shown that tumour necrosis factor-alpha (TNF-alpha) activates p38 mitogen-activated protein (MAP) kinase to produce interleukin-8 (IL-8) by human pulmonary vascular endothelial cells. Reactive oxygen species (ROS) including H(2)O(2) generated by TNF-alpha can act as signalling intermediates for cytokine induction; therefore, scavenging ROS by anti-oxidants is important for the regulation of cytokine production. However, the effect of N-acetylcysteine (NAC), which acts as a precursor of glutathione (GSH) synthesis, on TNF-alpha-induced activation of p38 MAP kinase pathway and p38 MAP kinase-mediated IL-8 production by human pulmonary vascular endothelial cells has not been determined. To clarify these issues, we examined the effect of NAC on TNF-alpha-induced activation of p38 MAP kinase, MAP kinase kinase (MKK) 3 and MKK6 which are upstream regulators of p38 MAP kinase, and p38 MAP kinase-mediated IL-8 production. 2. Human pulmonary vascular endothelial cells that had been preincubated with NAC were stimulated with TNF-alpha and then the activation of p38 MAP kinase and MKK3/MKK6 in the cells and IL-8 concentrations in the culture supernatants were determined. 3. Intracellular GSH levels increased in NAC-treated cells. 4. NAC attenuated TNF-alpha-induced activation of p38 MAP kinase and MKK3/MKK6. 5. NAC attenuated p38 MAP kinase-mediated IL-8 production by TNF-alpha-stimulated cells. 6. These results indicate that the cellular reduction and oxidation (redox) regulated by intracellular GSH is critical for TNF-alpha-induced activation of p38 MAP kinase pathway and p38 MAP kinase-mediated IL-8 production by human pulmonary vascular endothelial cells, and we emphasize that anti-oxidant therapy is an important strategy for the treatment of acute lung injury.

Regulation by intracellular glutathione of TNF-alpha-induced p38 MAP kinase activation and RANTES production by human pulmonary vascular endothelial cells.

We have previously shown that p38 mitogen-activated protein (MAP) kinase regulates tumor necrosis factor-alpha (TNF-alpha)-induced RANTES production by human pulmonary vascular endothelial cells, and that sensitivity to TNF-alpha is inversely correlated with cellular reduction and oxidation (redox) state. However, a regulatory role of intracellular glutathione (GSH) in TNF-alpha-induced p38 MAP kinase activation and p38 MAP kinase-mediated RANTES production has not been determined. In the present study, therefore, we extended our previous studies and focused on redox regulation on p38 MAP kinase activation. Human pulmonary vascular endothelial cells were exposed to N-acetylcysteine (NAC) or buthionine sulfoximine (BSO), and then TNF-alpha-induced p38 MAP kinase activation and p38 MAP kinase-mediated RANTES production were determined. The results showed that 1) NAC attenuated TNF-alpha-induced p38MAP kinase activation and RANTES production 2) SB 203580 as the specific inhibitor of p38 MAP kinase activity attenuated TNF-alpha-induced RANTES production 3) BSO facilitated TNF-alpha-induced p38 MAP kinase activation and RANTES production 4) SB 203580 attenuated BSO-mediated facilitation of TNF-alpha-induced RANTES production. These results indicated that TNF-alpha-induced p38 MAP kinase activation and p38 MAP kinase-mediated RANTES production by human pulmonary vascular endothelial cells are inversely regulated by intracellular GSH levels.

P38 MAP kinase regulates RANTES production byTNF‐α‐stimulated human pulmonary vascular endothelial cells

RANTES plays an important role in the production of allergic inflammation of the airway through its chemotactic activity for eosinophils. However, the intracellular signal regulating RANTES expression in human pulmonary vascular endothelial cells has not been determined. In the present study, therefore, we examined the role of p38 mitogen-activated protein (MAP) kinase in RANTES production by tumor necrosis factor (TNF)-alpha-stimulated pulmonary vascular endothelial cells in order to clarify the signal transduction pathway regulating RANTES production by pulmonary vascular endothelial cells. We examined p38 MAP kinase activation, and the effect of SB 203580, as the specific inhibitor for p38 MAP kinase, on p38 MAP kinase activity and RANTES production by TNF-alpha-stimulated human pulmonary vascular endothelial cells. The results showed that TNF-alpha induced RANTES production and p38 MAP kinase activity in human pulmonary vascular endothelial cells. Abrogation of p38 MAP kinase activity by SB 203580 repressed TNF-alpha-induced p38 MAP kinase activity and RANTES production. These results indicate that p38 MAP kinase plays an important role in the TNF-alpha-activated signaling pathway which regulates RANTES production by human pulmonary vascular endothelial cells.

P38 Mitogen-activated protein kinase regulates IL‐8 expression in human pulmonary vascular endothelial cells

The aim of this study was to examine the role of p38 mitogen-activated protein (MAP) kinase in interleukin (IL)-8 expression in tumour necrosis factor (TNF)-alpha- and IL-1alpha-stimulated human pulmonary vascular endothelial cells. To this end, the phosphorylation and activation of p38 MAP kinase and the effect of SB 203580, a specific inhibitor of p38 MAP kinase activity, on p38 MAP kinase activity and IL-8 expression in TNF-alpha- and IL-1alpha-stimulated human pulmonary vascular endothelial cells were examined. TNF-alpha- and IL-1alpha- induced phosphorylation and activation of p38 MAP kinase and IL-8 expression in human pulmonary endothelial cells. Inhibition of TNF-alpha- and IL-1alpha-induced p38 MAP kinase activity by SB 203580 inhibited TNF-alpha- and IL-1alpha-induced IL-8 protein production as well as IL-8 messenger ribonucleic acid (mRNA) expression, indicating that SB 203580 was effective at the transcriptional level. These results indicate that p38 mitogen-activated protein kinase plays an important role in the tumour necrosis factor-alpha- and interleukin-1alpha-activated signalling pathway which regulates interleukin-8 expression in human pulmonary vascular endothelial cells.

Short-term and long-term epoprostenol (prostacyclin) therapy in pulmonary hypertension secondary to connective tissue diseases: results of a pilot study

The aim of this study was to examine the role of p38 mitogen-activated protein (MAP) kinase in interleukin (IL)-8 expression in tumour necrosis factor (TNF)-alpha- and IL-1alpha-stimulated human pulmonary vascular endothelial cells. To this end, the phosphorylation and activation of p38 MAP kinase and the effect of SB 203580, a specific inhibitor of p38 MAP kinase activity, on p38 MAP kinase activity and IL-8 expression in TNF-alpha- and IL-1alpha-stimulated human pulmonary vascular endothelial cells were examined. TNF-alpha- and IL-1alpha- induced phosphorylation and activation of p38 MAP kinase and IL-8 expression in human pulmonary endothelial cells. Inhibition of TNF-alpha- and IL-1alpha-induced p38 MAP kinase activity by SB 203580 inhibited TNF-alpha- and IL-1alpha-induced IL-8 protein production as well as IL-8 messenger ribonucleic acid (mRNA) expression, indicating that SB 203580 was effective at the transcriptional level. These results indicate that p38 mitogen-activated protein kinase plays an important role in the tumour necrosis factor-alpha- and interleukin-1alpha-activated signalling pathway which regulates interleukin-8 expression in human pulmonary vascular endothelial cells.