Effects of hypoxia and hypercapnia on surfactant protein expression proliferation and apoptosis in A549 alveolar epithelial cells

During lung injury alveolar epithelial cells are directly exposed to changes in PO(2) and PCO(2). Integrity of alveolar epithelial type II cells (AECII) is critical in lung injury but the effect of hypoxia and hypercapnia on AECII function, viability and proliferation has not been clearly investigated. Aim of the present work was to determine the direct effect of hypoxia and hypercapnia on surfactant protein expression, proliferation and apoptosis of lung epithelial cells in vitro. A549 alveolar epithelia cells were subjected to hypoxia (1%O(2)-5% CO(2)) or hypercapnia (21% O(2-) 15% CO(2)) and expression of surfactant protein C was measured and compared to normal conditions (21% O(2)- 5% CO(2)). Cell cycle progression and apoptosis were measured by flow cytometric analysis. RESULTS: A549 alveolar epithelial cells produce surfactant proteins, including surfactant protein C, when cultured under normal conditions, which is reduced under hypoxic conditions. Specifically, pro-SpC expression is moderately decreased after 8 h of culture in hypoxia, and is completely attenuated after 48 h. Hypercapnia decreases pro-SpC expression only after 48 h of exposure. Stimulation with TNF-alpha partly reverses pSPC decrease observed under hypoxic and hypercapnic conditions. Hypoxic culture of A549 cells results in progressive arrest of cells in the G1 phase of the cell cycle and increased apoptosis first observed 4 h following exposure and peaking at 24 h. In contrast hypercapnia has no significant effect on alveolar epithelial cell proliferation or apoptosis. CONCLUSIONS: Taken together we can conclude that hypoxia rapidly and severely affects AECII function and viability while hypercapnia has an inhibitory effect on pro-SpC production only after prolonged exposure.     

Inhibition of proliferation and induction of apoptosis by abrogation of heat-shock protein (HSP) 70 expression in tumor cells

Tumor cells often express elevated levels of heat-shock protein (HSP) 70. The present study was designed to invesitgate the role of HSP70 in the proliferation and survival of tumor cells in the human system. When Molt-4 and other tumor cells were treated in vitro with HSP70 antisense oligomer, they displayed propidiumiodide-stained condensed nuclei (intact or fragmented). A ladder-like pattern of DNA fragments was observed with HSP70 antisense-oligomer-treated tumor cells in agrose gel electrophoresis, which was consistent with internucleosomal DNA fragmentation. Flow cytometry analysis revealed the hypodiploid DNA peak of propidium-iodide-stained nuclei in the antisense-oligomer-treated cells. The apoptosis induced by HSP antisense oligomer was dose- and time-dependent. The antisense oligomer induced apoptosis mainly in tumor cells at G1 and S phase, resulting in an inhibition of cell proliferation. HSP70 antisense oligomer caused DNA-sequence-specific inhibition of HSP70 expression, which preceded apparent apoptosis. These results indicate that HSP70 antisense treatment inhibits the expression of HSP70, which in turn inhibits cell proliferation and induces apoptosis in tumor cells and suggest that HSP70 is required for tumor cells to proliferate and survive under normal condition.     

Lamellar bodies in the epithelial bronchiolar cells in the mouse

Summary Lamellar bodies are described in the non-ciliated epithelial bronchiolar cells of the normal mouse lung. They are constituted of smooth concentric membranes, with a cytoplasmic center. They are related to mitochondria. They seem to belong to smooth endoplasmic reticulum. An origin from Golgi elements is discussed. Acknowledgment. This work was supported by Grant No 69088 of Conseil de la Recherche Médicale du Québec.     

Heat shock proteins, HSP25 and HSP70, and apoptosis in developing endocardial cushion of the mouse heart

Formation of the atrioventricular channels and valves from the endocardial cushion occurs through growth and remodeling of the initial endocardial cushion. This process requires balanced coordination of proliferation and apoptosis by still unknown factors. To detect a possible role for the heat shock proteins 25 and 70 (HSP25 and HSP70) as apoptosis-associated proteins and differentiation factors in the development of the endocardial cushion, we analyzed their temporal and regional occurrence during cell proliferation and apoptosis in E11-E17 embryos. The distribution and timing of these events and factors were consistent with the hypothesis that HSP25 is related to myocardial development whereas HSP70 is related to differentiation of the endocardial cushion by cell proliferation and apoptosis.     

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。     

急性冷应激对牦牛乳腺上皮细胞 HSP70 mRNA 表达的影响

本文主要研究了急性冷应激对牦牛乳腺上皮细胞热休克蛋白７０ （Heat stress protein,ＨＳＰ７０） 表达量的影响。采用实时荧光定量ＰＣＲ 技术,以急性冷刺激１０℃ 为典型研究环境,分析了ＨＳＰ７０ ｍＲＮＡ 表达的变化规律。结果显示,乳腺上皮细胞在１０℃分别冷处理２ ｈ、４ ｈ、６ ｈ 和８ ｈ,其ＨＳＰ７０ ｍＲＮＡ 的表达量变化均不显著（Ｐ ＞０. ０５）;分别在１０℃冷处理２ ｈ、４ ｈ、６ ｈ 和８ ｈ,再复温培养４ ｈ,ＨＳＰ７０ ｍＲＮＡ 的表达量均极显著增加（Ｐ ＜０. ０１）,于６ ｈ 达到峰值;在１０℃先冷处理４ ｈ,然后分别复温２ ｈ、４ ｈ、６ ｈ 和８ ｈ,ＨＳＰ７０ ｍＲＮＡ 的表达量亦均显著增加（Ｐ ＜０. ０１）,并于４ ｈ 达到峰值。结论：急性冷应激诱导牦牛乳腺上皮细胞ＨＳＰ７０ 表达量的增加不是发生在冷处理过程中,而是发生在复温过程中,并且在一定范围内随冷处理时间的增长表达量增高。     

Coelomocyte numbers and expression of HSP70 in wounded sea stars during hypoxia

Hypoxia, mainly caused by eutrophication, is a common and growing problem on marine soft bottoms. Echinoderms are known for their ability to regenerate tissue after wounding but hypoxia has a negative influence on regeneration and also on reproduction in echinoderms. We have investigated the cellular and molecular responses to wounding stress and hypoxia in the sea star Asterias rubens by using the total coelomocyte count (TCC) and the expression of heat shock proteins (HSPs). As early as 1 h after wounding, sea stars under hypoxic conditions show significantly increased TCC and, after 6 h, cell numbers increase approximately two-fold. After a 3-h hypoxia exposure of wounded animals, Western blot analysis reveals highly elevated coelomocyte cytoplasmic HSP70 expression. Non-wounded sea stars exposed to hypoxia and wounded animals kept in normoxia show enhanced HSP70 expression only after 24 h. Immunocytochemical analysis has not demonstrated any translocation of HSP70 from the cytoplasm to the nucleus. We conclude that both wounding and hypoxia elicit a stress response in sea stars and that the combined stress produces synergistic effects that may inhibit the initial processes of wound healing and regeneration.     

Mycobacterial and mouse HSP70 have immuno-modulatory effects on dendritic cells

Previously, it has been shown that heat shock protein 70 (HSP70) can prevent inflammatory damage in experimental autoimmune disease models. Various possible underlying working mechanisms have been proposed. One possibility is that HSP70 induces a tolerogenic phenotype in dendritic cells (DCs) as a result of the direct interaction of the antigen with the DC. Tolerogenic DCs can induce antigen-specific regulatory T cells and dampen pathogenic T cell responses. We show that treatment of murine DCs with either mycobacterial (Mt) or mouse HSP70 and pulsed with the disease-inducing antigen induced suppression of proteoglycan-induced arthritis (PGIA), although mouse HSP70-treated DCs could ameliorate PGIA to a greater extent. In addition, while murine DCs treated with Mt- or mouse HSP70 had no significantly altered phenotype as compared to untreated DCs, HSP70-treated DCs pulsed with pOVA (ovalbumin peptide 323–339) induced a significantly increased production of IL-10 in pOVA-specific T cells. IL-10-producing T cells were earlier shown to be involved in Mt HSP70-induced suppression of PGIA. In conclusion, this study indicates that Mt- and mouse HSP70-treated BMDC can suppress PGIA via an IL-10-producing T cell-dependent manner.     

Contrasting effects of NO and peroxynitrites on HSP70 expression and apoptosis in human monocytes

The free radicals nitric oxide(·NO) and superoxide (O₂·) react to formperoxynitrite (ONOO), a highly toxic oxidant species. Inthis study we investigated the respective effects of NO andONOO in monocytes from healthy human donors. Purifiedmonocytes were incubated for 6 or 16 h with a pure NO donor(S-nitroso-N-acetyl-DL-penicillamine, 0-2 mM), an ·NO/ONOO donor(3-morpholinosydnonimine chlorhydrate, 0-2 mM) with and withoutsuperoxide dismutase (200 IU/ml), or pure ONOO. Weprovide evidence that 3-morpholinosydnonimine chlorhydrate alonerepresents a strong stress to human monocytes leading to adose-dependent increase in heat shock protein-70 (HSP70) expression, mitochondrial membrane depolarization, and cell death by apoptosis andnecrosis. These phenomena were abolished by superoxide dismutase, suggesting that ONOO, but not ·NO, was responsible forthe observed effects. This observation was further strengthened by theabsence of a stress response in cells exposed toS-nitroso-N-acetyl-DL-penicillamine. Conversely, exposure of cells to ONOO alone also inducedmitochondrial membrane depolarization and cell death by apoptosis andnecrosis. Thus ONOO formation may well explain the toxiceffect generally attributed to ·NO.

     

低氧对大鼠肺动脉平滑肌细胞pHi及细胞增殖、凋亡的影响

目的: 探讨低氧肺血管结构重建时是否伴有PASMCs凋亡.方法: 体外低氧培养大鼠PASMCs,BCECF法测定细胞内pH值、光镜及电镜观察细胞形态、流式细胞仪测细胞周期、原位细胞凋亡(TUNEL法)观察细胞凋亡.结果: PASMCs在低氧早期即出现细胞内碱化,低氧6 h即出现G0/G1期细胞比例减少,G2/M期细胞比例增加,至24 h丝裂活动最强,但细胞凋亡率无显著变化.结论: 低氧PASMCs在细胞内碱化、细胞增殖的过程中不伴有细胞凋亡的改变.     

Characterisation of lectin binding patterns of mouse bronchiolar and rat alveolar epithelial cells in culture

Lung epithelial cell differentiation pathways remain unclear. This is due in part to the plasticity of these cells and the lack of markers which accurately reflect their differentiation status. The aim of this study was to determine if lectin binding properties are useful determinants of functional differentiation status in vitro. Mouse Clara cells were cultured for 5 days. During this time, no alteration in differentiation was evident by electron microscopy. No significant alteration in binding reactivity of Bauhinia purpurea (BPA), Maclura pomifera (MPA), Concanavalin A, Wheat germ or Helix pomatia lectins occurred in cultures compared with Clara cells in mouse lung tissue. In contrast, nitrotetrazolium blue reductase activity and CC10 expression declined in culture. Rat type II cells were cultured for 8 days. Between days 0 and 4, the number of type II cells identified by electron microscopy was constant at 70–80%, decreasing to 8% by day 6. In contrast, by day 4, only 42% cells retained alkaline phosphatase activity. BPA and MPA reactivity was altered at day 0 and day 4 respectively, compared with cells in situ. Therefore, the reactivity of lectins analysed here does not reflect functional differentiation status of cultured mouse Clara cells. However, BPA and MPA reactivity may be a sensitive indicator of alterations in rat type II cell differentiation in vitro.     

HSP70 desensitizes osteosarcoma cells to baicalein and protects cells from undergoing apoptosis

Baicalein is a new drug that has shown promising anti-cancer effects against a broad spectrum of tumors. However, the potential effect on osteosarcoma cells and the mechanisms involved are still largely unknown. Resistance to chemotherapy remains a major obstacle in cancer therapy. Therefore, the aim of the present study was to investigate the anti-tumor effect of baicalein on human osteosarcoma cancer cells and the molecular mechanism involved, as well as identify possible mechanisms of drug resistance. Our results revealed that baicalein-induced apoptosis in osteosarcoma cells was via a mitochondrial pathway involving both caspase-dependent and independent mechanisms. Notably, baicalein treatment upregulated the expression of HSP70, which partially prevented human osteosarcoma cells from undergoing apoptosis. Moreover, it was revealed that HSP70 expression decreased the sensitivity of osteosarcoma cells to baicalein via activation of PI3K/AKT and MAPK/ERK pathways. These results suggest that targeting HSP70-mediated drug resistance, in combination with chemotherapy drugs, may provide novel therapeutic opportunities.     

Cul5 mediates taurine-stimulated mTOR mRNA expression and proliferation of mouse mammary epithelial cells

Cullin5 (Cul5) protein can regulate multiple signaling pathways; however, it is still largely unknown the role and molecule mechanism of Cul5 in regulation of the mTOR signaling. In this study, we determined the effect of Cul5 on the proliferation of HC11 cells, a mouse mammary epithelial cell line, and explored the corresponding molecular mechanism. We found that Cul5 was highly expressed in mammary gland tissues in the lactation stage compared with that in puberty and involution. Using gene knockdown and activation methods, we showed that Cul5 promoted proliferation of HC11 cells, mRNA expression and protein phosphorylation of mTOR. Taurine (Tau) affected Cul5 mRNA and protein levels in a dose-dependent manner. Cul5 localized to the nucleus and knockdown of Cul5 almost totally blocked the stimulation of Tau on mTOR mRNA expression and protein phosphorylation. PI3K inhibition almost totally abolished the stimulation of Tau on Cul5 expression. In summary, our data uncover that Cul5 is a positive regulator of proliferation of HC11 cells, and mediates the stimulation of Tau on mRNA expression and subsequent protein phosphorylation of mTOR. Our data lay a new theoretical foundation for regulating mammary cell proliferation and promoting milk yield.

     

缺氧培养对大鼠神经干细胞体外增殖影响及其机制的研究

目的：研究应用缺氧对体外培养的大鼠神经干细胞增殖的影响。方法：将细胞分为4小时缺氧组、12小时缺氧组、促红细胞生成素（EPO）中和抗体组、IgG组以及对照组．测定大鼠神经干细胞经缺氧培养后的各组细胞克隆形成率以及EPO的表达变化。结果：单细胞培养条件下,与对照组相比,4小时缺氧组和IgG组克隆形成率明显增高;中和抗体组无明显变化;12小时组克隆形成率降低。但无统计学意义。缺氧4小时后,EPO蛋白在预处理后即刻出现表迭,4h达高峰,8h仍有部分表达。结论：短时间缺氧可以促进神经干细胞增殖．长时间缺氧则作用相反。缺氧对NSCs增殖作用的影响可能是由EPO介导产生。     

Preinduction of HSP70 promotes hypoxic tolerance and facilitates acclimatization to acute hypobaric hypoxia in mouse brain

It has been shown that induction of HSP70 by administration of geranylgeranylacetone (GGA) leads to protection against ischemia/reperfusion injury. The present study was performed to determine the effect of GGA on the survival of mice and on brain damage under acute hypobaric hypoxia. The data showed that the mice injected with GGA survived significantly longer than control animals (survival time of 9.55 ± 3.12 min, n = 16 vs. controls at 4.28 ± 4.29 min, n = 15, P < 0.005). Accordingly, the cellular necrosis or degeneration of the hippocampus and the cortex induced by sublethal hypoxia for 6 h could be attenuated by preinjection with GGA, especially in the CA2 and CA3 regions of the hippocampus. In addition, the activity of nitric oxide synthase (NOS) of the hippocampus and the cortex was increased after exposure to sublethal hypoxia for 6 h but could be inhibited by the preinjection of GGA. Furthermore, the expression of HSP70 was significantly increased at 1 h after GGA injection. These results suggest that administration of GGA improved survival rate and prevented acute hypoxic damage to the brain and that the underlying mechanism involved induction of HSP70 and inhibition of NOS activity.     

Nerve growth factor stimulates proliferation, adhesion and thymopoietic cytokine expression in mouse thymic epithelial cells in vitro

Thymic epithelial cells, which constitute a major component of the thymic microenvironment, provide a crucial signal for intrathymic T cell development and selection. Neuroimmune networks in the thymic microenvironment are thought to be involved in the regulation of T cell development. NGF is increasingly recognized as a potent immunomodulator, promoting “cross-talk” between various types of immune system cells. The present study clearly shows that NGF stimulates mouse thymic epithelial cell activities in vitro including cell proliferation, thymocyte adhesion to thymic epithelial cells, and the expression of cell adhesion molecules such as ICAM-1 and VCAM-1, and thymopoietic factors including IL-7, GM-CSF, SDF-1, TARC and TECK. Thus, our data are of considerable clinical importance showing that trophic NGF activity could be used to enhance the thymus regeneration and develop methods to improve host immunity when the immune function is depressed due to thymic involution.     

Long-term proliferation of mouse thymic epithelial cells in culture

Summary Epithelial cells from the normal mouse thymus were successfully cultivated on tissue culture plastic when plated with lethally irradiated support cells of the LA7 rat mammary tumor line. As the irradiated LA7 cells slowly decreased in number the thymus cells proliferated concomitantly to form a confluent monolayer. The cells now in culture have been subcultured 8 times, have doubled in number at least 30 times, and are still proliferating vigorously. The culture technique also supported clonal growth from a single cell, and nine clones have been isolated. The colony-forming efficiency of thymic cells plated at low concentrations was about 8%. These cultures were never overgrown by fibroblasts. The thymus cells were characterized as epithelial by the presence of cytoplasmic keratin and numerous desmosomes and tonofilaments. They were shown to be mouse cells by immunocytochemistry with species specific antibodies, by isoenzyme analysis, and by karyology. The cells stained when reacted with antibodies to tubulin, vimentin, and actin, but not with antibodies to Thy-1.2, Lyt-1, Lyt-2, Ia, or H-2 proteins. More than 85% of the cells had a normal mouse diploid chromosome number of 40. This culture technique opens the way for future studies of T-cell education with homogeneous thymic epithelial cell populations both in vitro and after reimplantation into genetically defined strains of mice. This work was supported by the Veterans Administration, Washington, D.C.     

Temperature-dependent enhancement of cell proliferation and mRNA expression for type I collagen and HSP70 in primary cultured goldfish cells

Goldfish (Carasius auratus) primary culture cells derived from caudal fin were incubated over a temperature range of 20-35 degrees C. The population doubling time of cells cultured at 20, 25, 30 and 35 degrees C were 34, 29, 17 and 14 h, respectively. Interestingly, cDNA-representational difference analysis revealed type I collagen alpha chain (colalpha(I)) as a candidate for a warm temperature-specific gene. mRNA levels of colalpha(I) increased with an increase of incubation temperature and days of culture. Furthermore, the cell growth rate and colalpha(I) mRNA levels were rapidly changed following temperature shifts. To examine the effects of culture temperature shift on the cellular physiological states, mRNA levels of HSP70 were additionally investigated. HSP70 mRNA levels in the cells cultured at 30 and 35 degrees C were again 2-3 times higher than those at 20 and 25 degrees C. When the culture temperature was shifted from 20 to 35 degrees C, HSP70 mRNA levels were rapidly increased within 1 h. Subsequently, mRNA levels of the 35 degrees C-treated cells decreased, but remained doubled compared with those of the 20 degrees C-treated cells, even 4 h following the temperature shift. When the culture temperature was lowered from 35 to 20 degrees C, HSP70 mRNA levels decreased to about 70% of the original levels in 4 h. These results indicate that goldfish cells cultured at different temperatures easily develop temperature-associated steady physiological states within 4 h of temperature shifts.     

Effect of nitric oxide on expression of apoptotic genes and HSP70 in drosophila

Data on involvement of nitric oxide in apoptosis are contradictory. The balance between anti- and proapoptotic activities of nitric oxide depends on many factors, including its concentration in a tissue and interactions with other regulators of apoptosis. This paper describes the results of a series of experiments on the effect of nitric oxide donors and inhibitors as well as dNOS1 and dNOS4 transgenes on the apoptosis on drosophila Lobe ^RSV mutant strain and wild-type strain Oregon R. It has been shown that a high nitric oxide content in cells is able to inhibit antiapoptotic effect of HSP70 and stimulate apoptosis, possibly, via the grim-mediated apoptotic pathway. Moreover, long-term action of a high nitric oxide concentration during the entire development more efficiently stimulates the proapoptotic genes as compared with short-term action of this agent.