HIF-1α Negatively Regulates Irisin Expression Which Involves in Muscle Atrophy Induced by Hypoxia

Exposure to high altitude environment leads to skeletal muscle atrophy. As a hormone secreted by skeletal muscles after exercise, irisin contributes to promoting muscle regeneration and ameliorating skeletal muscle atrophy, but its role in hypoxia-induced skeletal muscle atrophy is still unclear. Our results showed that 4 w of hypoxia exposure significantly reduced body weight and gastrocnemius muscle mass of mice, as well as grip strength and the duration time of treadmill exercise. Hypoxic treatment increased HIF-1α expression and decreased both the circulation level of irisin and its precursor protein FNDC5 expression in skeletal muscle. In in vitro, CoCl2-induced chemical hypoxia and 1% O2 ambient hypoxia both reduced FNDC5, along with the increase in HIF-1α. Moreover, the decline in the area and diameter of myotubes caused by hypoxia were rescued by inhibiting HIF-1α via YC-1. Collectively, our research indicated that FNDC5/irisin was negatively regulated by HIF-1α and could participate in the regulation of muscle atrophy caused by hypoxia.

Large-scale mapping of positional changes of hypoxia-responsive genes upon activation

Chromosome structure and nuclear organization are important factors in the regulation of gene expression. Transcription of a gene is influenced by local and global chromosome features such as condensation status and histone modifications. The relationship between the position of a gene in the cell nucleus and its activity is less clear. Here, we used high-throughput imaging to perform a large-scale analysis of the spatial location of a set of nearly 100 hypoxia-inducible genes to determine whether their location within the nucleus is correlated with their activity state upon stimulation. Radial distance analysis demonstrated that the majority of HIF- and CREB-inducible hypoxia responsive genes are located in the intermediate region of the nucleus. Radial position of numerous responsive genes changed upon hypoxic treatment. Analysis of the relative distances amongst a subset of HIF target gene groups revealed that some gene pairs also altered their relative location to each other upon hypoxic treatment, suggesting higher order chromatin rearrangements. While these changes in location occurred in response to hypoxic activation of the target genes, they did not correlate with the extent of their activation. These results suggest that induction of the hypoxia-responsive gene expression program is accompanied by spatial alterations of the genome, but that radial and relative gene positions are not directly related to gene activity.

HFD and HFD-provoked hepatic hypoxia act as reciprocal causation for NAFLD via HIF-independent signaling

Background The occurrence of non-alcoholic fatty liver disease (NAFLD) is found to be higher in patients with obstructive sleep apnea (OSA), which is characterized by intermittent hypoxia. Activation of hypoxia-inducible factors has been shown in the development and progression of NAFLD, implying a cause and effects relationship between NAFLD and hypoxia. The present study was designed to investigate the interaction of lipotoxicity and hypoxia in the pathogenesis of NAFLD using mice model with high-fat diet (HFD) feeding or hypoxic treatment. Methods NAFLD model was induced in mice by HFD feeding, and in cultured primary hepatocytes by administration of palmitate acid. Mouse hypoxic model was produced by placing the mice in a Animal incubator with oxygen concentration at 75% followed by a 21% oxygen supplement. Hypoxic condition was mimicked by treating the hepatocytes with cobalt chloride (CoCl2) or 1% oxygen supply. Pimonidazole assay was conducted to evaluate hypoxia. Lipid metabolic genes were measured by real-time polymerase-chain reaction. HIF-1α and HIF-2α genes were silenced by siRNA. Results HFD feeding and palmitate acid treatment provoked severe hepatic hypoxia along with TG accumulation in mice and in cultured primary hepatocytes respectively. Conversely, hypoxia induced hepatic TG accumulation in mice and in cultured primary hepatocytes. Hypoxic treatment inhibited the expression of lipolytic genes, while increased the expression of lipogenicgenes in mice. Although both lipotoxicity and hypoxia could activate hepatic hypoxia-induced factor 1α and 2α, while neither lipotoxicity- nor hypoxia- induced hepatic steatosis was affected when HIF was knocked down. Conclusions HFD resulted in hepatic TG accumulation and concomitant hypoxia. Conversely, hypoxia induced hepatic TG accumulation in mice and in cultured heptocytes. Thus lipotoxicity and hypoxia might work as reciprocal causation and orchestrate to promote the development of NAFLD.

Cyclic Hypoxia Exposure Accelerates the Progression of Amoebic Gill Disease

Amoebic gill disease (AGD), caused by the amoeba Neoparamoeba perurans, has led to considerable economic losses in every major Atlantic salmon producing country, and is increasing in frequency. The most serious infections occur during summer and autumn, when temperatures are high and poor dissolved oxygen (DO) conditions are most common. Here, we tested if exposure to cyclic hypoxia at DO saturations of 40–60% altered the course of infection with N. perurans compared to normoxic controls maintained at ≥90% DO saturation. Although hypoxia exposure did not increase initial susceptibility to N. perurans, it accelerated progression of the disease. By 7 days post-inoculation, amoeba counts estimated from qPCR analysis were 1.7 times higher in the hypoxic treatment than in normoxic controls, and cumulative mortalities were twice as high (16 ± 4% and 8 ± 2%), respectively. At 10 days post-inoculation, however, there were no differences between amoeba counts in the hypoxic and normoxic treatments, nor in the percentage of filaments with AGD lesions (control = 74 ± 2.8%, hypoxic = 69 ± 3.3%), or number of lamellae per lesion (control = 30 ± 0.9%, hypoxic = 27.9 ± 0.9%) as determined by histological examination. Cumulative mortalities at the termination of the experiment were similarly high in both treatments (hypoxic = 60 ± 2%, normoxic = 53 ± 11%). These results reveal that exposure to cyclic hypoxia in a diel pattern, equivalent to what salmon are exposed to in marine aquaculture cages, accelerated the progression of AGD in post-smolts.

IRE-1α regulates expression of ubiquitin specific peptidases during hypoxic response in U87 glioma cells

IRE-1α (inositol requiring enzyme-1α), the most evolutionarily conserved of the endoplasmic reticulum stress signaling pathways, is highly implicated in sustaining the proliferation of glioma cells and subsequent tumor growth, which is decreased by the inhibition of IRE-1α. To explore the IRE-1α mediated regulation of ubiquitin system in glioma cells, the expression of a subset of ubiquitin specific peptidases (USP) and of ubiquitin activating enzyme E1-like protein/autophagy related 7 (GSA7/ATG7) genes was studied, during hypoxic stress in wild type and U87 glioma cells with inhibited IRE-1α. Hypoxic treatment of wild type glioma cells leads to the up-regulation of USP25 and the concomitant downregulation of USP1, USP10, USP14, and GSA7 genes. USP4 and USP22 genes expression did not significantly change with hypoxic treatment. Inhibition of IRE-1α activity led to up-regulation of USP1, USP4, USP10, USP22, and USP25, while USP14 and GSA7 genes were down-regulated. Therefore, IRE-1α activity modifies substrate-targeting specificity to proteasome during hypoxic stress, which in turn can affect cell survival. Inhibition of IRE-1α correlates directly with deregulation of ubiquitin specific peptidases and GSA7 in a fashion that ultimately slows tumor growth.

Abstract 16186: Hypoxic Pre-conditioning on Human CD34+ Stem Cells Enhances Exosome Therapeutics of Ischemic Tissue Repair Through ETS-1-Regulated Pathway

Previous studies in our lab have revealed a novel mechanism that therapeutically important human CD34+ stem cells secrete exosomes (Exo) to extracellular milieu that induce angiogenic activity independent of the cells. Further studies demonstrated that CD34+ exosomes (CD34Exo) carry and transfer of proangiogenic miRNAs, such as miR-126, which affect the therapeutic function of the exosomes. Herein we hypothesized that hypoxic treatment of CD34+ stem cells can modulate the miRNA content and regenerative efficacy of CD34Exo. Methods and Results: Exosomes from human CD34+ cells cultured under hypoxia (H-Exo) were more proliferative, anti-apoptotic and angiogenic in vitro, as compared to exosomes from cells under normoxia (N-Exo). In a mouse model of hind limb ischemia, H-Exo treatment significantly enhanced perfusion (ratio: 0.93±0.05 v 0.77±0.02), increased capillary density (1.6±0.2 v 1.1±0.1/HPF) and prevented ischemic limb amputation (0% v 37.5%) as compared to N-Exo (p<0.05; n=7-8). Flow cytometry and confocal microscopy indicated that H-Exo was uptaken by endothelial cells in the ischemic limb. Interestingly, hypoxic treatment did not alter the size and quantity of Exo or expression of major proteins of CD34Exo compared with normoxic treatment. However, hypoxic treatment altered the proangiogenic miRNA expression in H-Exo including miR-210 and miR-126. ETS-1, a transcription factor induced by hypoxia-inducible fator-1 (HIF-1) is known to regulate expression of miR-126. We have examined the protein and RNA expression of HIF-1 and ETS-1 in Sca-1 positive mouse hematopoietic stem cells, and propose that HIF-1/ETS-1 regulatory mechanisms affect the expression of exosomal miR-126 under hypoxia. These results are being confirmed in human CD34+ cells using siRNA silencing and HIF hydroxylase inhibitor dimethyloxalylglycine. Conclusion: Hypoxia induced miR-126 expression in CD34 cell-derived exosomes stimulating exosomes-mediated angiogenesis and therapeutic recovery via ETS-transcriptional pathway. Our work has important clinical implications for therapeutic angiogenesis, especially in diabetic and cardiovascular patients who have stem cells with diminished angiogenic potential.

Morphological root responses of soybean to rhizosphere hypoxia reflect waterlogging tolerance

Jitsuyama, Y. 2015. Morphological root responses of soybean to rhizosphere hypoxia reflect waterlogging tolerance. Can. J. Plant Sci. 95: 999–1005. Excess soil moisture induces hypoxia, causing waterlogging injury in soybeans [Glycine max (L.) Merr.]. Twelve Japanese soybean cultivars with varying hypoxia tolerance were used. Of these, 11 (all but Hayahikari) were evaluated for waterlogging tolerance using a scaled index with data from previous studies. To investigate hypoxic responses, cultivars were grown under hydroponic conditions for 2 wk a year for 2 yr, with aerobic or hypoxic oxygen concentrations artificially maintained in the rhizosphere. Hypoxic responses (measured as plant dry weight and root morphology) were assessed at the early vegetative stage. The effects of hypoxic treatment on root dry weight were significant, and the effect of year on soybean dry weight was not significant. The change in root dry weight, and particularly, in coarse root length, was significantly correlated with waterlogging tolerance index at the 0.001 probability level. This study showed that root responses to rhizosphere hypoxia might reflect waterlogging tolerance in soybeans.