Roflumilast-N-oxide induces surfactant protein expression in human alveolar epithelial cells type II

Surfactant proteins (SPs) are important lipoprotein complex components, expressed in alveolar epithelial cells type II (AEC-II), and playing an essential role in maintenance of alveolar integrity and host defence. Because expressions of SPs are regulated by cyclic adenosine monophosphate (cAMP), we hypothesized that phosphodiesterase (PDE) inhibitors, influence SP expression and release. Analysis of PDE activity of our AEC-II preparations revealed that PDE4 is the major cAMP hydrolysing PDE in human adult AEC-II. Thus, freshly isolated human AEC-II were stimulated with two different concentrations of the PDE4 inhibitor roflumilast-N-oxide (3 nM and 1 μM) to investigate the effect on SP expression. SP mRNA levels disclosed a large inter-individual variation. Therefore, the experiments were grouped by the basal SP expression in low and high expressing donors. AEC-II stimulated with Roflumilast-N-oxide showed a minor increase in SP-A1, SP-C and SP-D mRNA mainly in low expressing preparations. To overcome the effects of different basal levels of intracellular cAMP, cyclooxygenase was blocked by indomethacin and cAMP production was reconstituted by prostaglandin E2 (PGE2). Under these conditions SP-A1, SP-A2, SP-B and SP-D are increased by roflumilast-N-oxide in low expressing preparations. Roflumilast-N-oxide fosters the expression of SPs in human AEC-II via increase of intracellular cAMP levels potentially contributing to improved alveolar host defence and enhanced resolution of inflammation.     

Effect of chronic hypoxia on proliferation, apoptosis, and HSP70 expression in mouse bronchiolar epithelial cells

Heat shock proteins (HSPs) can be induced by various stresses and play an important role in cell cycle progression. HSP70 has been shown to act as an inhibitor of apoptosis. We studied HSP70 expression in bronchial epithelial cells of C57BL/6 mice and homozygous HPS70 knockout mice (hsp70.1-/-) exposed to chronic hypoxic stress. We also investigated changes in cellular proliferation and apoptosis in relation to HSP70. Lungs were removed from mice after a three-week period of exposure to 10 % O(2). Immunoblots for HSP70 and immunohistochemical staining for HSP70 and Ki-67 were performed. Apoptosis was assessed using the TUNEL assay. The three-week period of hypoxic stress did not change HSP70 levels in total lung tissue, but a significant reduction in HSP70 expression was observed in bronchiolar epithelial cells. In wild type mice, both HSP70 and Ki-67 expression were significantly reduced in bronchiolar epithelial cells. In homozygous HPS70 knockout mice (hsp70.1-/-), apoptosis of bronchiolar epithelial cells was significantly increased. Our results suggest that HSP70 may exert anti-apoptotic effects in mouse bronchiolar epithelial cells.     

Effects of alveolar surfactant aggregates on T-lymphocyte proliferation

The effects of alveolar large aggregate (LA) and small aggregate (SA) surfactant subfractions isolated from healthy adult rats on mitogen-stimulated proliferative responses of human peripheral blood mononuclear cells (PBMC) was examined. Various concentrations of total surfactant suppressed proliferation of stimulated lymphocytes by up to 95% of mitogen-stimulated cells alone. LA subfractions of total surfactant had no effect on proliferation, whereas SA significantly enhanced the lymphocyte proliferation at lower concentrations (7.8 microg/ml) compared to mitogen-stimulated cells alone. Higher concentrations of SA (62.5 microg/ml) inhibited lymphocyte proliferation. This concentration-dependent effect of SA on proliferation of PBMC was also present when cells were stimulated with various lectins including anti-CD3, concanavalin A and phytohemagglutinin. Analysis of the supernatant of mitogen-stimulated cell cultures treated with inhibitory concentrations of SA showed decreased amounts of interleukin (IL)-2, compared to cells alone, which could be reversed by adding exogenous IL-2 to the cell cultures with the SA. These results suggest that alveolar surfactant subfractions have distinct functions within the alveoli, both biophysically and with respect to their effects on the host's immunomodulatory responses.     

Effects of regional alveolar hypoxia and hypercapnia on small pulmonary vessels in cats

Using an X-ray TV system, we analyzed responses in the internal diameter (ID), flow velocity, and volume flow in small pulmonary vessels (100-600 microns ID) during unilobar hypoxia and hypercapnia in cats. In the hypoxic and hypercapnic lobes, the ID reduced in proportion to the degree of hypoxia and hypercapnia, respectively. The ID reduction was larger in the arteries than in the veins for a given stimulus. In the arteries, the ID reduced nonuniformly in the series-arranged vessels in response to both stimuli. The percentage ID reduction was maximal in the arteries of 200-300 microns ID, in which it was 21, 26, 28, and 36% with 5% O2, 0% O2, 5% CO2, and 10% CO2 inhalations, respectively. On the other hand, in the veins, uniform ID reduction occurred for a given stimulus. In the contralateral normoxic lobe, the ID did not change significantly. In both hypoxic and hypercapnic lobes, the flow velocity and volume flow of the small arteries decreased, with 5% O2, by 18 and 40%, respectively, and, with 5% CO2, by 23 and 50%, respectively. In contrast, in the normoxic lobe, they increased significantly during 5% O2 and 5% CO2 inhalations. We concluded that regional alveolar hypoxia and hypercapnia induced a local vasoconstriction particularly in the small arteries of 200-300 microns ID and decreased the flow velocity and volume flow in the same lung region.     

低氧对胎盘基蜕膜间充质干细胞增殖、凋亡和血管内皮细胞生长因子表达的影响

本文旨在探讨生理低氧和无血清培养条件下人胎盘基蜕膜间充质干细胞（placental decidua basalis—mesenchymal stemcells,PDB-MSCs）的生物学特征和细胞因子的表达情况。采用密度梯度离心法培养获得PDB—MSCs,利用MTT法、流式细胞术检测PDB—MSCs在不同氧浓度（20％02和1％O2）、有无血清（10％FBS和0％FBS）条件下各个时间点（6h、12h、24h、48h、72h、96h）的增殖和凋亡情况,并采用酶联免疫吸附实验（enzyme linked immunosorbent assay,ELISA）检测无血清条件下各个时间点细胞上清液中血管内皮细胞生长因子（vascularendothelial growth factor,VEGF）含量。结果显示,在特定的时间内低氧可以促进PDB—MSCs增殖（P〈0．01,n=3）,而血清对PDB—MSCs增殖的影响与氧浓度关系密切;同时,在本实验条件下,低氧、无血清分别或联合培养不会导致PDB—MSCs凋亡（P〉0．05,n=3）;在无血清条件下,24h时低氧组PDB-MSCs表达较高水平的VEGF。以上结果提示,PDB—MSCs可能成为缺血相关组织工程产品种子细胞的一个新来源。     

香烟烟雾提取物抑制肺泡上皮细胞的增殖并诱导其凋亡

香烟烟雾提取物（cigarette smoke extract,CSE）中含有丰富的氧化剂和自由基,由它所引起的氧化应激可导致肺泡壁的损伤进而发展为肺气肿.近年来,围绕CSE损伤肺泡壁作用机制的研究较为活跃,但其结果却一直存在着分歧.本实验的目的是观察CSE对肺泡Ⅱ型上皮细胞的损伤作用并探讨与其相关的分子机制.MTT比色法的结果显示,CSE以时间和剂量依赖性的方式降低细胞的增殖活力,流式细胞术的分析结果表明细胞增殖周期被阻滞在G1/S期.Hoechst 33258染色以及透射电镜观察从形态上确认CSE诱导细胞凋亡的发生,DNA梯的出现和Annexin V-FITC/碘化丙啶双染色的结果从分子水平得到进一步的证实.同时,运用流式细胞术检测到CSE诱导的凋亡伴随着Fas受体的高表达和caspase-3的显著活化.另外,使用H2DCFDA染色,经激光共聚焦显微镜术测得细胞内氧自由基在细胞受到CSE刺激以后大量快速积累.结果表明CSE能够抑制肺泡Ⅱ型上皮细胞来源的A549细胞的生长和增殖,并诱导细胞凋亡,由Fas受体所介导的死亡受体途径参与此凋亡过程,而CSE所引起的氧化应激则可能是阻止肺泡上皮细胞生长增殖并诱导其凋亡的始动因素.     

Effects of respiratory syncytial virus infection and major basic protein derived from eosinophils in pulmonary alveolar epithelial cells (A549)

RSV (respiratory syncytial virus)-induced pneumonia and bronchiolitis may be associated with hyperresponsive conditions, including asthma. Eosinophilic proteins such as MBP (major basic protein) may also be associated with the pathophysiology of asthma. To elucidate the roles of RSV infection and MBP in the pathogenesis of pneumonia with hyperresponsiveness, we investigated the effects of RSV infection and MBP on A549 (alveolar epithelial) cells. CPE (cytopathic effects) in A549 cells were observed by microscopy. Apoptosis and cell death was evaluated by flow cytometric analysis and modified MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay. We also measured 15 types of cytokines and chemokines in A549 cell supernatants. Although RSV alone did not affect the CPE of A549, high concentrations of MBP resulted in cell death within 24 h. Combinations of RSV and MBP synergistically induced cell death. In A549 cells infected with RSV alone, the release of GM-CSF (granulocyte-macrophage colony-stimulating factor) was significantly enhanced compared with control cells (no infection). In the cells treated with MBP alone, the production of IL (interleukin)-2, 4, 5, 7, 10, 12, 13, 17, IFN (interferon)-γ, GM-CSF, G-CSF (granulocyte colony-stimulating factor) and MIP (macrophage inflammatory protein)-1β was significantly increased compared with control cells. Notably, the levels of GM-CSF and IL-17 in RSV/MBP-treated cells were significantly higher than those treated with MBP alone. These results suggest that MBP synergistically enhanced the release of various cytokines/chemokines and the cell death of RSV-infected A549 cells, indicating that MBP may be closely associated with the pathophysiology of allergic reactions in bronchiolitis/pneumonia due to RSV.     

Effects of transdifferentiation and EGF on claudin isoform expression in alveolar epithelial cells.

Rat alveolar epithelial type II cells grown on polycarbonate filters form high-resistance monolayers and concurrently acquire many phenotypic properties of type I cells. Treatment with EGF has previously been shown to increase transepithelial resistance across alveolar epithelial cell (AEC) monolayers. We investigated changes in claudin expression in primary cultured AEC during transdifferentiation to the type I cell-like phenotype (days 0, 1, and 8), and on day 5 in culture +/- EGF (10 ng/ml) from day 0 or day 4. Claudins 4 and 7 were increased, whereas claudins 3 and 5 were decreased, on later compared with earlier days in culture. Exposure to EGF led to increases in claudins 4 and 7 and decreases in claudins 3 and 5. Claudin 1 was only faintly detectable in freshly isolated type II cells and remained unchanged over time in culture and after exposure to EGF. These results suggest that increases in transepithelial resistance accompanying AEC transdifferentiation and/or EGF exposure are mediated, at least in part, by changes in the pattern of expression of specific claudin isoforms.     

Pollen grains bind to lung alveolar type II cells (A549) via lung surfactant protein A (SP-A)

Lung surfactant protein A (SP-A) is the most abundant surfactant-associated protein present in the lung. A receptor for SP-A has been shown to be present on A549 alveolar type II cells and on other cell types, including alveolar macrophage. The SP-A receptor on A549 cells has been identified as the collectin receptor, or C1q receptor, which binds several structurally-related ligands. SP-A contains C-type lectin domains, but the role of carbohydrate binding by SP-A in physiological and pathological phenomena is not yet established. In this paper we report the binding of SP-A to pollen from Populus nigra italica (Lombardy Poplar), Poa pratensis (Kentucky blue grass),Secale cerale (cultivated rye) and Ambrosia elatior (short ragweed). Saturable and concentration dependent binding of SP-A to pollen grains was observed. Interaction of SP-A with pollen grains takes place through waterextractable components, in which the major species present, in Lombardy poplar pollen,are 57 kD and 7 kD (glyco)proteins. The binding of SP-A to pollen grains and their aqueous extracts was calcium ion dependent and was inhibited by mannose, and is therefore mediated by the lectin domain. Binding of SP-A to pollen grains was found to mediate adhesion of pollen grains to A549 cells. The results suggest that pollen grains or other carbohydrate-bearing particles (e. g. microorganisms) could potentially interact with different cell types via the collectin receptor (C1q Receptor) in the presence of SP-A.     

Granzyme A and thrombin differentially promote the release of interleukin-8 from alveolar epithelial A549 cells

Some of extracellular serine proteases with trypsin-like specificity of cleavage have been known to increase the release of inflammatory mediators from various cell types. For instance, two well-known trypsin-like serine proteases circulating in blood, granzyme A (GrA) and thrombin, have been found to promote interleukin (IL)-8 release from an alveolar epithelial A549 cell line. However, the mechanisms by which the proteases promote IL-8 release from the cells are not fully understood. In the present study, using A549 cells we found that (1) thrombin promoted IL-8 release from the cells via a mechanism partially involving activation of protease-activated receptor-1, a G-protein coupled receptor, whereas a recombinant form of GrA (rGrA) did it via a mechanism that does not involve the receptor activation; that (2) unlike rGrA, thrombin did not cause detachment and microtubule disruption of the cells; and that (3) the release of IL-8 induced by rGrA was inhibited in the presence of taxol, a microtubule-stabilizing reagent, whereas that induced by thrombin was not. These findings suggest that rGrA and thrombin promote the release of IL-8 from A549 cells through distinct mechanisms.     

Carrageenan inhibits granzyme A-induced detachment of and interleukin-8 release from alveolar epithelial A549 cells

Granzyme A (GrA) is a lymphocyte serine protease that is believed to enter virus-infected cells and growing tumors and induce apoptosis. We found recently that recombinant rat GrA (rGrA) promotes detachment of and interleukin (IL)-8 release from alveolar epithelial A549 cells and suggested that this protease is involved in the pathogenesis of certain inflammatory lung diseases. In the present study, we found that λ-carrageenan (a sulfated oligosaccharide constituting the cell walls of seaweeds) potently inhibits rGrA-induced detachment and IL-8 release of A549 cells. This sulfated oligosaccharide might be useful for suppressing the development of inflammatory lung diseases in which GrA is thought to be involved.     

Diallyl trisulfide induces apoptosis and inhibits proliferation of A549 cells in vitro and in vivo

Lung cancer is the leading cause of cancer-related mortality all over the world. In recent years, pulmonary adenocarcinoma has surpassed squamous cell carcinoma in frequency and is the predominant form of lung cancer in many countries. Epidemiological investigations have shown an inverse relationship between garlic (Allium sativum) consumption and death rate from many cancers. Diallyl trisulfide (DATS) is one of the garlic-derived compounds (also known as: organosulfer compounds, OSC). DATS can induce apoptosis and inhibit the growth of many cancer cell lines. Our study demonstrated that the apoptotic incidents induced by DATS were a mitochondria-dependent caspase cascade through a significant decrease of the anti-apoptotic Bcl-2 that resulted in up-regulation of the ratio of Bax/Bcl-2 and the activity of caspase-3, -8, and -9. Eventually, DATS induced the apoptosis and inhibited the proliferation in a concentration- and time-dependent manner. Furthermore, by establishing an animal model of female BALB/c nude mice with A549 xenografts, we found that oral gavage of DATS significantly retarded growth of A549 xenografts in nude mice without causing weight loss or any other side effects compared with the control group. All the evidence both in vitro and in vivo suggested that DATS could be an ideal anti-cancer drug.     

Effects of pulmonary surfactant and surfactant protein A on phagocytosis of fractionated alveolar macrophages: relationship to starvation.

Pulmonary surfactant isolated from bronchoalveolar lavage fluid of rat lung contained a high content of surfactant protein A (SP-A) in starved for 2 days compared to fed controls, but this phenomena returned to baseline following more than 4 days starvation. As determined by immunoperoxidase staining of lung sections using SP-A antibody, SP-A could be consistently observed in nonciliated bronchiolar (Clara) cells, alveolar type II cells and some alveolar macrophages (AM). Fc receptor-mediated phagocytosis of AM was enhanced by SP-A, which was dependent on the dosis and reached a maximum at 10 micrograms of SP-A/ml. Antibody to SP-A completely inhibited the enhanced response of phagocytosis. When exposed AM subpopulations, separated into four fractions (I, II, III and IV) by discontinuous Percoll gradient, to SP-A or pulmonary surfactant prepared from rats fed and starved for 2 days enhanced their phagocytic activity in high dense cells (III and IV), particularly to SP-A and pulmonary surfactant from rats starved for 2 days. Whereas little change in lower dense fractions (I and II) were seen in all exposures except for SP-A that enhanced the cells of fraction II. These results supported the concept that pulmonary surfactant and its apoprotein, SP-A, are a factor to regulate lung defense system including activation of AM that undergo different processes following starvation.     

Bim mediates mitochondria-regulated particulate matter-induced apoptosis in alveolar epithelial cells

We studied the role of Bim, a pro-apoptotic BCL-2 family member in Airborne particulate matter (PM 2.5 microm)-induced apoptosis in alveolar epithelial cells (AEC). PM induced AEC apoptosis by causing significant reduction of mitochondrial membrane potential and increase in caspase-9, caspase-3 and PARP-1 activation. PM upregulated pro-apoptotic protein Bim and enhanced translocation of Bim to the mitochondria. ShRNABim blocked PM-induced apoptosis by preventing activation of the mitochondrial death pathway suggesting a role of Bim in the regulation of mitochondrial pathway in AEC. Accordingly, we provide the evidence that Bim mediates PM-induced apoptosis via mitochondrial pathway.     

Effects of 7-hydroxy-3-methoxycadalene on cell cycle, apoptosis and protein translation in A549 lung cancer cells

Previously we reported that cadalene extracted from Zelkova serrata inhibited lung tumorigenesis in mice. However, the precise mechanism has not yet investigated. Here, we examined the effects of cadalene on signal pathways important for apoptosis, cell cycle, and protein translation in lung cancer cells. Our results showed that cadalene suppressed the expression of Akt and its phosphor-forms through controlling PI3K and PTEN. Cadalene also induced apoptosis through facilitating pro-apoptotic protein expression. In addition, cadalene caused cell cycle arrest and decreased mTOR-mediated protein translation. Taken together, cadalene may be developed as a lung cancer therapeutic agent in the future.     

Amiodarone induces apoptosis of human and rat alveolar epithelial cells in vitro

The antiarrhythmic amiodarone (AM) and its metabolite desethylamiodarone (Des) are known to cause AM-induced pulmonary toxicity, but the mechanisms underlying this disorder remain unclear. We hypothesized that AM might cause AM-induced pulmonary toxicity in part through the induction of apoptosis or necrosis in alveolar epithelial cells (AECs). Two models of type II pneumocytes, the human AEC-derived A549 cell line and primary AECs isolated from adult Wistar rats, were incubated with AM or Des for 20 h. Apoptotic cells were determined by morphological assessment of nuclear fragmentation with propidium iodide on ethanol-fixed cells. Necrotic cells were quantitated by loss of dye exclusion. Both AM and Des caused dose-dependent necrosis starting at 2.5 and 0.1 microg/ml, respectively, in primary rat AECs and at 10 and 5 microg/ml in subconfluent A549 cells (P < 0.05 and P < 0.01, respectively). AM and Des also induced dose-dependent apoptosis beginning at 2.5 microg/ml in the primary AECs (P < 0.05 for both compounds) and at 10 and 5 microg/ml, respectively, in the A549 cell line (P < 0.01). The two compounds also caused significant net cell loss (up to 80% over 20 h of incubation) by either cell type at drug concentrations near or below the therapeutic serum concentration for AM. The cell loss was not due to detachment but was blocked by the broad-spectrum caspase inhibitor Z-Val-Ala-Asp-fluoromethylketone. Furthermore, the angiotensin-converting enzyme inhibitor captopril (500 ng/ml) and the angiotensin-receptor antagonist saralasin (50 microg/ml) significantly inhibited both the induction of apoptosis and net cell loss in response to AM. These results are consistent with recent work from this laboratory demonstrating potent inhibition of apoptosis in human AECs by captopril (Uhal BD, Gidea C, Bargout R, Bifero A, Ibarra-Sunga O, Papp M, Flynn K, and Filippatos G. Am J Physiol Lung Cell Mol Physiol 275: L1013-L1017, 1998). They also suggested that the accumulation of AM and/or its primary metabolite Des in lung tissue may induce cytotoxicity of AECs that might be inhibitable by angiotensin-converting enzyme inhibitors or other antagonists of the renin-angiotensin system.