雷帕霉素对低氧下HL-60细胞的低氧调控信号分子表达的影响

摘要目的：通过小分子化合物氯（CoCt2）模拟的低氧环境,分析低氧下及其雷帕霉素（RPM）作用下人急性髓细胞白血病细胞HL．60的低氧调控信号分子表达的变化;方法：常规方法复苏、传代、培养HL-60细胞,培养细胞进入对数生长期后用于实验。低氧模拟组、低氧雷帕霉素处理组、常氧雷帕霉素处理组分别用含2001xmol／LCoCl2、2001xmol／LCOCl2／20nmol／LRPM、20nmol／LRPM的1640培养基处理生长状态良好的细胞,对照组细胞用1640培养基培养,各组置培养箱以37℃、5％CO2培养,并于处理后24h、48h、72h收集细胞用于检测;采用实时荧光定量PCR方法检测低氧诱导因子（HIF-1α）、内皮细胞生长因于（VEGF）、雷帕霉素靶蛋白（mTOR）及GAPDH在转录水平的表达;结果：①与各时段对照组相比,低氧模拟组HIF-1α表达随时间逐渐增加,72h明显上调;与常氧雷帕霉素处理组各时段比较,低氧雷帕霉素处理组HIF-1α表达早期（24h）相对下调,后期相对上调;②．与对照组比较,各处理组mTOR表达均下调,低氧雷帕霉素处理组在早期（24h）下调显著;与常氧雷帕霉素处理组比较,低氧雷帕霉素处理组mTOR各时段的表达均相对下调;③与对照组各时段相比,低氧模拟组VEGF的表达在早期显著上调,但后期呈下调;常氧雷帕霉素处理组各时段VEGF的表达下调,与其比较,低氧雷帕霉素处理组各时段均呈相对下调。结论：常氧和低氧下RPM作用HL-60细胞后VEGF、mTOR的mRNA均表达下调,RPM可在低氧环境下增强了这种下调表达作用。     

Increased Proliferation and Analysis of Differential Gene Expression in Human Wharton's Jelly-derived Mesenchymal Stromal Cells under Hypoxia

Multipotent mesenchymal stromal cells (MSCs) from Wharton''s jelly (WJ) of umbilical cord bear higher proliferation rate and self-renewal capacity than adult tissue-derived MSCs and are a primitive stromal cell population. Stem cell niche or physiological microenvironment plays a crucial role in maintenance of stem cell properties and oxygen concentration is an important component of the stem cell niche. Low oxygen tension or hypoxia is prevalent in the microenvironment of embryonic stem cells and many adult stem cells at early stages of development. Again, in vivo, MSCs are known to home specifically to hypoxic events following tissue injuries. Here we examined the effect of hypoxia on proliferation and in vitro differentiation potential of WJ-MSCs. Under hypoxia, WJ-MSCs exhibited improved proliferative potential while maintaining multi-lineage differentiation potential and surface marker expression. Hypoxic WJ-MSCs expressed higher mRNA levels of hypoxia inducible factors, notch receptors and notch downstream gene HES1. Gene expression profile of WJ-MSCs exposed to hypoxia and normoxia was compared and we identified a differential gene expression pattern where several stem cells markers and early mesodermal/endothelial genes such as DESMIN, CD34, ACTC were upregulated under hypoxia, suggesting that in vitro culturing of WJ-MSCs under hypoxic conditions leads to adoption of a mesodermal/endothelial fate. Thus, we demonstrate for the first time the effect of hypoxia on gene expression and growth kinetics of WJ-MSCs. Finally, although WJ-MSCs do not induce teratomas, under stressful and long-term culture conditions, MSCs can occasionally undergo transformation. Though there were no chromosomal abnormalities, certain transformation markers were upregulated in a few of the samples of WJ-MSCs under hypoxia.     

Lung-selective gene responses to alveolar hypoxia: potential role for the bone morphogenetic antagonist gremlin in pulmonary hypertension

Pulmonary hypoxia is a common complication of chronic lung diseases leading to the development of pulmonary hypertension. The underlying sustained increase in vascular resistance in hypoxia is a response unique to the lung. Thus we hypothesized that there are genes for which expression is altered selectively in the lung in response to alveolar hypoxia. Using a novel subtractive array strategy, we compared gene responses to hypoxia in primary human pulmonary microvascular endothelial cells (HMVEC-L) with those in cardiac microvascular endothelium and identified 90 genes (forming 9 clusters) differentially regulated in the lung endothelium. From one cluster, we confirmed that the bone morphogenetic protein (BMP) antagonist, gremlin 1, was upregulated in the hypoxic murine lung in vivo but was unchanged in five systemic organs. We also demonstrated that gremlin protein was significantly increased by hypoxia in vivo and inhibited HMVEC-L responses to BMP stimulation in vitro. Furthermore, significant upregulation of gremlin was measured in lungs of patients with pulmonary hypertensive disease. From a second cluster, we showed that CXC receptor 7, a receptor for the proangiogenic chemokine CXCL12, was selectively upregulated in the hypoxic lung in vivo, confirming that our subtractive strategy had successfully identified a second lung-selective hypoxia-responsive gene. We conclude that hypoxia, typical of that encountered in pulmonary disease, causes lung-specific alterations in gene expression. This gives new insights into the mechanisms of pulmonary hypertension and vascular loss in chronic lung disease and identifies gremlin 1 as a potentially important mediator of vascular changes in hypoxic pulmonary hypertension.     

Temporal expression of hypoxia-regulated genes is associated with early changes in redox status in irradiated lung

The development of normal lung tissue toxicity after radiation exposure results from multiple changes in cell signaling and communication initiated at the time of the ionizing event. The onset of gross pulmonary injury is preceded by tissue hypoxia and chronic oxidative stress. We have previously shown that development of debilitating lung injury can be mitigated or prevented by administration of AEOL10150, a potent catalytic antioxidant, 24h after radiation. This suggests that hypoxia-mediated signaling pathways may play a role in late radiation injury, but the exact mechanism remains unclear. The purpose of this study was to evaluate changes in the temporal expression of hypoxia-associated genes in irradiated mouse lung and determine whether AEOL10150 alters expression of these genes. A focused oligo array was used to establish a hypoxia-associated gene expression signature for lung tissue from sham-irradiated or irradiated mice treated with or without AEOL10150. Results were further verified by RT-PCR. Forty-four genes associated with metabolism, cell growth, apoptosis, inflammation, oxidative stress, and extracellular matrix synthesis were upregulated after radiation. Elevated expression of 31 of these genes was attenuated in animals treated with AEOL10150, suggesting that expression of a number of hypoxia-associated genes is regulated by early development of oxidative stress after radiation. Genes identified herein could provide insight into the role of hypoxic signaling in radiation lung injury, suggesting novel therapeutic targets, as well as clues to the mechanism by which AEOL10150 confers pulmonary radioprotection.     

Effect of bone marrow–derived mesenchymal stromal cells on hepatoma

Background aimsThe aim of the study was to evaluate the effect of mesenchymal stromal cells (MSCs) on tumor cell growth in vitro and in vivo and to elucidate the apoptotic and anti-proliferative mechanisms of MSCs on a hepatocellular carcinoma (HCC) murine model.MethodsThe growth-inhibitory effect of MSCs on the Hepa 1–6 cell line was tested by means of methyl thiazolyl diphenyl-tetrazolium assay. Eighty female mice were randomized into four groups: group 1 consisted of 20 mice that received MSCs only by intrahepatic injection; group 2 consisted of 20 HCC mice induced by inoculation of Hepa 1–6 cells into livers without MSC treatment; group 3 consisted of 20 mice that received MSCs after induction of liver cancer; group 4 consisted of 20 mice that received MSCs after induction of liver cancer on top of induced biliary cirrhosis.ResultsMSCs exhibited a growth-inhibitory effect on Hepa 1–6 murine cell line in vitro. Concerning in vivo study, decreases of serum alanine transaminase, aspartate transaminase and albumin levels after MSC transplantation in groups 2 and 3 were found. Gene expression of α-fetoprotein was significantly downregulated after MSC injection in the HCC groups. We found that gene expression of caspase 3, P21 and P53 was significantly upregulated, whereas gene expression of Bcl-2 and survivin was downregulated in the HCC groups after MSC injection. Liver specimens of the HCC groups confirmed the presence of dysplasia. The histopathological picture was improved after administration of MSCs to groups 2 and 3.ConclusionsMSCs upregulated genes that help apoptosis and downregulated genes that reduce apoptosis. Therefore, MSCs could inhibit cell division of HCC and potentiate their death.     

Prolyl-hydroxylase inhibition and HIF activation in osteoblasts promotes an adipocytic phenotype

Bone is a dynamic environment where cells sense and adapt to changes in nutrient and oxygen availability. Conditions associated with hypoxia in bone are also associated with bone loss. In vitro hypoxia (2% oxygen) alters gene expression in osteoblasts and osteocytes and induces cellular changes including the upregulation of hypoxia inducible factor (HIF) levels. Our studies show that osteoblasts respond to hypoxia (2% oxygen) by enhancing expression of genes associated with adipocyte/lipogenesis phenotype (C/EBPbeta, PPARgamma2, and aP2) and by suppressing expression of genes associated with osteoblast differentiation (alkaline phosphatase, AP). Hypoxia increased HIF protein levels, hypoxic response element (HRE) binding, and HRE-reporter activity. We also demonstrate that prolyl-hydroxylases 2 and 3 (PHD2, PHD3), one of the major factors coordinating HIF degradation under normoxic but not hypoxic conditions, are induced in osteoblasts under hypoxic conditions. To further determine the contribution of PHDs and upregulated HIF activity in modulating osteoblast phenotype, we treated osteoblasts with a PHD inhibitor, dimethyloxaloylglycine (DMOG), and maintained cells under normoxic conditions. Similar to hypoxic conditions, HRE reporter activity was increased and adipogenic gene expression was increased while osteoblastic genes were suppressed. Taken together, our findings indicate a role for PHDs and HIFs in the regulation of osteoblast phenotype.     

Pressure load: the main factor for altered gene expression in right ventricular hypertrophy in chronic hypoxic rats

Background

The present study investigated whether changes in gene expression in the right ventricle following pulmonary hypertension can be attributed to hypoxia or pressure loading.

Methodology/Principal Findings

To distinguish hypoxia from pressure-induced alterations, a group of rats underwent banding of the pulmonary trunk (PTB), sham operation, or the rats were exposed to normoxia or chronic, hypobaric hypoxia. Pressure measurements were performed and the right ventricle was analyzed by Affymetrix GeneChip, and selected genes were confirmed by quantitative PCR and immunoblotting. Right ventricular systolic blood pressure and right ventricle to body weight ratio were elevated in the PTB and the hypoxic rats. Expression of the same 172 genes was altered in the chronic hypoxic and PTB rats. Thus, gene expression of enzymes participating in fatty acid oxidation and the glycerol channel were downregulated. mRNA expression of aquaporin 7 was downregulated, but this was not the case for the protein expression. In contrast, monoamine oxidase A and tissue transglutaminase were upregulated both at gene and protein levels. 11 genes (e.g. insulin-like growth factor binding protein) were upregulated in the PTB experiment and downregulated in the hypoxic experiment, and 3 genes (e.g. c-kit tyrosine kinase) were downregulated in the PTB and upregulated in the hypoxic experiment.

Conclusion/Significance

Pressure load of the right ventricle induces a marked shift in the gene expression, which in case of the metabolic genes appears compensated at the protein level, while both expression of genes and proteins of importance for myocardial function and remodelling are altered by the increased pressure load of the right ventricle. These findings imply that treatment of pulmonary hypertension should also aim at reducing right ventricular pressure.     

Interleukin-6 induces proinflammatory signaling in Schwann cells: A high-throughput analysis

Interleukin-6 plays an important role in peripheral nerve regeneration. We recently reported that IL-6 targets Schwann cells in the peripheral nerve for its function. In this study, we analyzed genes whose expression is regulated by IL-6 in a cell line derived from Schwann cells, the peripheral glia, using the Illumina gene microarray. At measurements 3 and 12 h after IL-6 treatment, 35 genes were found to be upregulated by IL-6. Most upregulated genes were proinflammatory genes that are known to be induced in inflammatory conditions. Interestingly, the expression of immunoproteasome subunits was upregulated by IL-6 in Schwann cells. Treatment with forskolin, an agent that mimics axonal signaling, suppressed the expression of IL-6-inducible genes. Finally, we found for the first time that sciatic nerve injury induced immunoproteasome expression in vivo. These findings indicate that IL-6 is involved in peripheral nerve regeneration by regulating proinflammatory signaling in Schwann cells.     

Proliferation and differentiation of bone marrow stromal cells under hypoxic conditions

Low oxygen tension is a potent differentiation inducer of numerous cell types and an effective stimulus of many gene expressions. Here, we described that under 8% O(2), bone marrow stromal cells (MSCs) exhibited proliferative and morphologic changes. The level of differentiated antigen H-2Dd and the number of G(2)/S/M phase cells increased evidently under 8% O(2) condition. Also, the proportion of wide, flattened, and epithelial-like cells (which were alkaline phosphatase staining positive) in MSCs increased significantly. When cultured in adipogenic medium, there was a 5- to 6-fold increase in the number of lipid droplets under hypoxic conditions compared with that in normoxic culture. We also demonstrated the existence of MSC differentiation under hypoxic conditions by electron microscopy. Expression of Oct4 was inhibited under 8% O(2) condition, but after adipocyte differentiation in normoxic culture and hypoxia-mimicking agents cobalt chloride (CoCl(2)) and deferoxamine mesylate (DFX) treatments, Oct4 was still expressed in MSCs. These results indicate hypoxia accelerates MSC differentiation and hypoxia and hypoxia-mimicking agents exert different effects on MSC differentiation.