CXCR7 Silencing Attenuates Cell Adaptive Response to Stromal Cell Derived Factor 1α after Hypoxia

Previous studies have shown that chemotactic factor stromal-cell derived factor 1α (SDF1α) promotes cell recovery from hypoxic injury via its main receptor C-X-C chemokine receptor type (CXCR) 4. However, the role of its new receptor CXCR7 on cell repair against hypoxia and cell response to SDF1α remains largely unknown. In this study, neurons induced from hippocampal progenitor cells were pre-conditioned in hypoxia for 4h and subsequently monitored to investigate the function of SDF1α on cell repair after hypoxia. Neurons were assessed for their cell morphology, actin filament polymerization and migration capability. SDF1α protein levels increased significantly 1 h after hypoxia compared to control (P<0.01), and it reached a peak at 24 h after hypoxia. Moreover, addition of SDF1α promoted neurite outgrowth and actin filament polymerization both in normoxic and hypoxic cells compared to untreated cells. Cell migration showed a time-dependent increase with SDF1α stimulation in both groups, and hypoxic cells illustrated a significant augment at 0.5 h, 1 h and 12 h after SDF1α application compared to normoxic cells (P<0.01). CXCR7 expression also increased with time dependence after hypoxia and demonstrated a two-fold upregulation compared to control at 24 h after hypoxia. With CXCR7 silencing, axon elongation and actin filament polymerization induced by SDF1α were inhibited sharply both in normoxic and hypoxic cells. CXCR7 silencing also leads to reduced hypoxic cell migration at 0.5 h, 1 h, 12 h, 24 h and 36 h after SDF1α application (P<0.01), but it failed to reduce normoxic cell migration induced by SDF1α at 0.5 h, 1 h and 12 h (P>0.05). 24 h SDF1α stimulation led to higher ERK1/2 phosphorylation compared to control, and ERK1/2 phosphorylation increased more in hypoxic cells than that in normoxic cells. This study suggested that CXCR7 plays an important role on cell repair processing induced by SDF1α, and CXCR7 silencing attenuates cell adaptive response to acute SDF1α stimulation (≤12 h) after hypoxia.     

Increased Metabolite Levels of Glycolysis and Pentose Phosphate Pathway in Rabbit Atherosclerotic Arteries and Hypoxic Macrophage

Aims

Inflammation and possibly hypoxia largely affect glucose utilization in atherosclerotic arteries, which could alter many metabolic systems. However, metabolic changes in atherosclerotic plaques remain unknown. The present study aims to identify changes in metabolic systems relative to glucose uptake and hypoxia in rabbit atherosclerotic arteries and cultured macrophages.

Methods

Macrophage-rich or smooth muscle cell (SMC)-rich neointima was created by balloon injury in the iliac-femoral arteries of rabbits fed with a 0.5% cholesterol diet or a conventional diet. THP-1 macrophages stimulated with lipopolysaccharides (LPS) and interferon-γ (INFγ) were cultured under normoxic and hypoxic conditions. We evaluated comprehensive arterial and macrophage metabolism by performing metabolomic analyses using capillary electrophoresis-time of flight mass spectrometry. We evaluated glucose uptake and its relationship to vascular hypoxia using ¹⁸F-fluorodeoxyglucose (¹⁸F-FDG) and pimonidazole, a marker of hypoxia.

Results

The levels of many metabolites increased in the iliac-femoral arteries with macrophage-rich neointima, compared with those that were not injured and those with SMC-rich neointima (glycolysis, 4 of 9; pentose phosphate pathway, 4 of 6; tricarboxylic acid cycle, 4 of 6; nucleotides, 10 of 20). The uptake of ¹⁸F-FDG in arterial walls measured by autoradiography positively correlated with macrophage- and pimonidazole-immunopositive areas (r = 0.76, and r = 0.59 respectively; n = 69 for both; p<0.0001). Pimonidazole immunoreactivity was closely localized with the nuclear translocation of hypoxia inducible factor-1α and hexokinase II expression in macrophage-rich neointima. The levels of glycolytic (8 of 8) and pentose phosphate pathway (4 of 6) metabolites increased in LPS and INFγ stimulated macrophages under hypoxic but not normoxic condition. Plasminogen activator inhibitor-1 protein levels in the supernatant were closely associated with metabolic pathways in the macrophages.

Conclusion

Infiltrative macrophages in atherosclerotic arteries might affect metabolic systems, and hypoxia but not classical activation might augment glycolytic and pentose phosphate pathways in macrophages.     

Hypoxia Enhances Proliferation of Human Adipose-Derived Stem Cells via HIF-1ɑ Activation

BackgroundAdipose tissue-derived stem cells (ASCs) have been recently isolated from human subcutaneous adipose tissue. ASCs may be useful in regenerative medicine as an alternative to bone marrow-derived stem cells. Changes in the oxygen concentration influence physiological activities, such as stem cell proliferation. However, the effects of the oxygen concentration on ASCs remain unclear. In the present study, the effects of hypoxia on ASC proliferation were examined.MethodsNormal human adipose tissue was collected from the lower abdomen, and ASCs were prepared with collagenase treatment. The ASCs were cultured in hypoxic (1%) or normoxic (20%) conditions. Cell proliferation was investigated in the presence or absence of inhibitors of various potentially important kinases. Hypoxia inducible factor (HIF)-1α expression and MAP kinase phosphorylation in the hypoxic culture were determined with western blotting. In addition, the mRNA expression of vascular endothelial growth factor (VEGF) and fibroblast growth factor (FGF)-2 in hypoxic or normoxic conditions were determined with real-time RT-PCR. The effects of these growth factors on ASC proliferation were investigated. Chromatin immunoprecipitation (ChIP) of the HIF–1α-binding hypoxia responsive element in FGF–2 was performed. HIF–1α was knocked down by siRNA, and FGF–2 expression was investigated.ResultsASC proliferation was significantly enhanced in the hypoxic culture and was inhibited by ERK and Akt inhibitors. Hypoxia for 5–15 minutes stimulated the phosphorylation of ERK1/2 among MAP kinases and induced HIF–1α expression. The levels of VEGF and FGF–2 mRNA and protein in the ASCs were significantly enhanced in hypoxia, and FGF–2 increased ASC proliferation. The ChIP assay revealed an 8-fold increase in the binding of HIF–1α to FGF–2 in hypoxia. HIF–1α knockdown by siRNA partially inhibited the FGF–2 expression of ASCs induced by hypoxia.ConclusionASC proliferation was enhanced by hypoxia. HIF–1α activation, FGF–2 production, and the ERK1/2 and Akt pathway were involved in this regulatory mechanism.     

The real face of HIF1α in the tumor process

It is well known that the hypoxia-inducible factor 1 α (HIF1α) is detectable as adaptive metabolic response to hypoxia. However, HIF1/HIF1α is detectable even under normoxic conditions, if the metabolism is altered, e.g., high proliferation index. Importantly, both hypoxic metabolism and the Warburg effect have in common a decrease of the intracellular pH value. In our interpretation, HIF1α is not directly accumulated by hypoxia, but by a process which occurs always under hypoxic conditions, a decrease of the intracellular pH value because of metabolic imbalances. We assume that HIF1α is a sensitive controller of the intracellular pH value independently of the oxygen concentration. Moreover, HIF1α has its major role in activating genes to eliminate toxic metabolic waste products (e.g., NH₃/NH₄+) generated by the tumor-specific metabolism called glutaminolysis, which occur during hypoxia, or the Warburg effect. For that reason, HIF1α appears as a potential target for tumor therapy to disturb the pH balance and to inhibit the elimination of toxic metabolic waste products in the tumor cells.     

Astrocyte-Derived Proinflammatory Cytokines Induce Hypomyelination in the Periventricular White Matter in the Hypoxic Neonatal Brain

Hypoxic exposure in the perinatal period causes periventricular white matter damage (PWMD), a condition associated with myelination abnormalities. Under hypoxic conditions, glial cells were activated and released a large number of inflammatory mediators in the PWM in neonatal brain, which may result in oligodendrocyte (OL) loss and axonal injury. This study aims to determine if astrocytes are activated and generate proinflammatory cytokines that may be coupled with the oligodendroglial loss and hypomyelination observed in hypoxic PWMD. Twenty-four 1-day-old Wistar rats were exposed to hypoxia for 2 h. The rats were then allowed to recover under normoxic conditions for 7 or 28 days before being killed. Another group of 24 rats kept outside the chamber was used as age-matched controls. Upregulated expression of TNF-α and IL-1β was observed in astrocytes in the PWM of P7 hypoxic rats by double immunofluorescence, western blotting and real time RT-PCR. This was linked to apoptosis and enhanced expression of TNF-R₁ and IL-1R₁ in APC⁺ OLs. PLP expression was decreased significantly in the PWM of P28d hypoxic rats. The proportion of myelinated axons was markedly reduced by electron microscopy (EM) and the average g-ratios were higher in P28d hypoxic rats. Upregulated expression of TNF-α and IL-1β in primary cultured astrocytes as well as their corresponding receptors in primary culture APC⁺ oligodendrocytes were detected under hypoxic conditions. Our results suggest that following a hypoxic insult, astrocytes in the PWM of neonatal rats produce inflammatory cytokines such as TNF-α and IL-1β, which induce apoptosis of OLs via their corresponding receptors associated with them. This results in hypomyelination in the PWM of hypoxic rats.     

δ-Opioid Receptor Activation Modified MicroRNA Expression in the Rat Kidney under Prolonged Hypoxia

Hypoxic/ischemic injury to kidney is a frequently encountered clinical problem with limited therapeutic options. Since microRNAs are differentially involved in hypoxic/ischemic events and δ-opioid receptor (DOR) activation is known to protect against hypoxic/ischemic injury, we speculated on the involvement of DOR activation in altering the microRNA (miRNA) expression in kidney under hypoxic condition. We selected 31 miRNAs based on microarray data for quantitative PCR analysis. Among them, 14 miRNAs were significantly altered after prolonged hypoxia, DOR activation or a combination of both. We found that 1) DOR activation alters miRNA expression profiles in normoxic conditions; 2) hypoxia differentially alters miRNA expression depending on the duration of hypoxia; and 3) DOR activation can modify hypoxia-induced changes in miRNA expression. For example, 10-day hypoxia reduced the level of miR-212 by over 70%, while DOR activation could mimic such reduction even in normoxic kidney. In contrast, the same stress increased miR-29a by >100%, which was reversed following DOR activation. These first data suggest that hypoxia comprehensively modifies the miRNA profile within the kidney, which can be mimicked or modified by DOR activation. Ascertaining the targeted pathways that regulate the diverse cellular and molecular functions of miRNA may provide new insights into potential therapies for hypoxic/ischemic injury of the kidney.     

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.     

Elevated Cell Invasion Is Induced by Hypoxia in a Human Pituitary Adenoma Cell Line

Pituitary adenoma tissues are hypovascular, and have a lower partial oxygen pressure compared with neighboring normal organs. In this study, we investigated whether hypoxia influences the cell invasiveness of the human pituitary adenoma cell line, HP-75. HP-75 cells were exposed to hypoxic (1–10% oxygen) or normoxic (21% oxygen) conditions for 24 hours. Gelatin and reverse zymogram assays were used to determine the enzyme activities of matrix metalloproteinases (MMP) and tissue inhibitor of metalloproteinases (TIMP). Cell adhesion and Matrigel cell invasion were examined with a Boiden chamber. Finally, the mRNA gene expression profiles of cells exposed to hypoxia or normoxia were examined by cDNA microarray and confirmed with real-time RT-PCR and flow cytometry. The gelatin and reverse zymograms revealed that the activities of MMP and TIMP were not significantly altered by hypoxia. Matrigel cell invasion and cell adhesion to Matrigel or collagen type IV were increased by hypoxia (3.8- and 4.8-fold, respectively). The cDNA microarray analysis revealed that laminin β2 chain mRNA was specifically up-regulated under hypoxic conditions (4.96-fold). Finally, real-time RT-PCR and flow cytometry verified the elevated expression of laminin β2 chain at the mRNA and protein levels under hypoxic conditions. RNA interference with siRNA targeting laminin β2 inhibited Matrigel invasion and adhesion to collagen type IV in a dose.dependent manner.Collectively, these results suggested that hypoxia (1% oxygen) enhanced the cell invasion properties of a pituitary adenoma cell line in association with elevated expression of laminin β2 and enhanced binding to collagen type IV.Key Words: cell invasion, hypoxia, laminin β2, pituitary adenoma, siRNA     

Radiosensitization of normoxic and hypoxic h1339 lung tumor cells by heat shock protein 90 inhibition is independent of hypoxia inducible factor-1α

Background

Ionizing irradiation is a commonly accepted treatment modality for lung cancer patients. However, the clinical outcome is hampered by normal tissue toxicity and tumor hypoxia. Since tumors often have higher levels of active heat shock protein 90 (Hsp90) than normal tissues, targeting of Hsp90 might provide a promising strategy to sensitize tumors towards irradiation. Hsp90 client proteins include oncogenic signaling proteins, cell cycle activators, growth factor receptors and hypoxia inducible factor-1α (HIF-1α). Overexpression of HIF-1α is assumed to promote malignant transformation and tumor progression and thus might reduce the accessibility to radiotherapy.

Methodology/Principal Findings

Herein, we describe the effects of the novel Hsp90 inhibitor NVP-AUY922 and 17-allylamino-17-demethoxygeldanamycin (17-AAG), as a control, on HIF-1α levels and radiosensitivity of lung carcinoma cells under normoxic and hypoxic conditions. NVP-AUY922 exhibited a similar biological activity to that of 17-AAG, but at only 1/10 of the dose. As expected, both inhibitors reduced basal and hypoxia-induced HIF-1α levels in EPLC-272H lung carcinoma cells. However, despite a down-regulation of HIF-1α upon Hsp90 inhibition, sensitivity towards irradiation remained unaltered in EPLC-272H cells under normoxic and hypoxic conditions. In contrast, treatment of H1339 lung carcinoma cells with NVP-AUY922 and 17-AAG resulted in a significant up-regulation of their initially high HIF-1α levels and a concomitant increase in radiosensitivity.

Conclusions/Significance

In summary, our data show a HIF-1α-independent radiosensitization of normoxic and hypoxic H1339 lung cancer cells by Hsp90 inhibition.     

Hypoxia Increases Gefitinib-Resistant Lung Cancer Stem Cells through the Activation of Insulin-Like Growth Factor 1 Receptor

Accumulating evidence indicates that a small population of cancer stem cells (CSCs) is involved in intrinsic resistance to cancer treatment. The hypoxic microenvironment is an important stem cell niche that promotes the persistence of CSCs in tumors. Our aim here was to elucidate the role of hypoxia and CSCs in the resistance to gefitinib in non-small cell lung cancer (NSCLC) with activating epidermal growth factor receptor (EGFR) mutation. NSCLC cell lines, PC9 and HCC827, which express the EGFR exon 19 deletion mutations, were exposed to high concentration of gefitinib under normoxic or hypoxic conditions. Seven days after gefitinib exposure, a small fraction of viable cells were detected, and these were referred to as “gefitinib-resistant persisters” (GRPs). CD133, Oct4, Sox2, Nanog, CXCR4, and ALDH1A1–all genes involved in stemness–were highly expressed in GRPs in PC9 and HCC827 cells, and PC9 GRPs exhibited a high potential for tumorigenicity in vivo. The expression of insulin-like growth factor 1 (IGF1) was also upregulated and IGF1 receptor (IGF1R) was activated on GRPs. Importantly, hypoxic exposure significantly increased sphere formation, reflecting the self-renewal capability, and the population of CD133- and Oct4-positive GRPs. Additionally, hypoxia upregulated IGF1 expression through hypoxia-inducible factor 1α (HIF1α), and markedly promoted the activation of IGF1R on GRPs. Knockdown of IGF1 expression significantly reduced phosphorylated IGF1R-expressing GRPs under hypoxic conditions. Finally, inhibition of HIF1α or IGF1R by specific inhibitors significantly decreased the population of CD133- and Oct4-positive GRPs, which were increased by hypoxia in PC9 and HCC827 cells. Collectively, these findings suggest that hypoxia increased the population of lung CSCs resistant to gefitinib in EGFR mutation-positive NSCLC by activating IGF1R. Targeting the IGF1R pathway may be a promising strategy for overcoming gefitinib resistance in EGFR mutation-positive NSCLC induced by lung CSCs and microenvironment factors such as tumor hypoxia.     

THE LOCALIZATION OF GLYCOGEN IN THE SPERMATOZOA OF VARIOUS INVERTEBRATE AND VERTEBRATE SPECIES

With the periodic acid-thiosemicarbazide-silver proteinate procedure for the detection of polysaccharides in thin sections, glycogen is localized in the cavities of centrioles and basal bodies, within the axoneme (and surrounding it), in mitochondria, and in the "packing" cytoplasm of the middle piece of spermatozoa of several invertebrate and vertebrate species. The cytochemical localization of glycogen is verified by extraction with α-amylase solution. These findings establish the existence of stored glycogen in sperm. The polysaccharide presumably serves as an endogenous source of energy in the absence of extracellular metabolites, under either aerobic or anaerobic conditions. Other hypotheses on the physiological significance of intracellular glycogen stores in sperm are discussed. Sperm that store glycogen contain some enzymes of glycogen metabolism. In the presence of glucose-1-phosphate, ATP, and Mg⁺⁺ ions, an amylophosphorylase catalyzes the in vivo synthesis of glycogen. The newly formed product resembles γ-particles, and is digestible with α-amylase.     

Pyruvate reverses metabolic effects produced by hypoxia in glioma and hepatoma cell cultures

The intervention of pyruvate in glucose metabolism was investigated during hypoxic stress in tumour cell cultures having respiratory capacities under normoxic conditions. Results obtained with nuclear magnetic resonance (NMR) spectroscopy showed that, under normoxic conditions, rat glioma C6 and human hepatoma Hep G2 cell cultures metabolised [(13)C(1)]glucose into lactate, alanine, glutamate and other less abundant metabolites, as already known from the literature. In the absence of pyruvate, during hypoxia or cyanide poisoning, both cell types dramatically decreased the label into glutamate and accumulated [(13)C(3)]glycerol-3-phosphate. The compound was further identified by 31P NMR spectroscopy. The accumulation of the label in glycerol-3-phosphate, however, did not occur when the cells were incubated in the presence of pyruvate. The fate of the latter, followed under normoxic conditions by incubating cells with [(13)C(3)]pyruvate and natural glucose, showed that the label was mainly found in alanine, lactate and glutamate. Anoxic conditions increased the label in lactate and reduced that of glutamate. The data show a metabolic effect of pyruvate during mitochondrial blockade due to severe lack of oxygen in tumour cell lines.