Hypoxic Stimulation Research Articles

KDM3A Senses Oxygen Availability to Regulate PGC-1α-Mediated Mitochondrial Biogenesis.

Hypoxia, which occurs during tumor growth, triggers complex adaptive responses in which peroxisome proliferator-activated receptor gamma coactivator-1 alpha (PGC-1α) plays a critical role in mitochondrial biogenesis and oxidative metabolism. However, how PGC-1α is regulated in response to oxygen availability remains unclear. We demonstrated that lysine demethylase 3A (KDM3A) binds to PGC-1α and demethylates monomethylated lysine (K) 224 of PGC-1α under normoxic conditions. Hypoxic stimulation inhibits KDM3A, which has a high KM of oxygen for its activity, and enhances PGC-1α K224 monomethylation. This modification decreases PGC-1α's activity required for NRF1- and NRF2-dependent transcriptional regulation of TFAM, TFB1M, and TFB2M, resulting in reduced mitochondrial biogenesis. Expression of PGC-1α K224R mutant significantly increases mitochondrial biogenesis, reactive oxygen species (ROS) production, and tumor cell apoptosis under hypoxia and inhibits brain tumor growth in mice. This study revealed that PGC-1α monomethylation, which is dependent on oxygen availability-regulated KDM3A, plays a critical role in the regulation of mitochondrial biogenesis.

Baicalin relieves hypoxia-aroused H9c2 cell apoptosis by activating Nrf2/HO-1-mediated HIF1α/BNIP3 pathway

Background Myocardial ischemia is the main reason for ischemic heart disease. Baicalin is a plant-derived flavonoid with cardio-protective activity. Herein, we tested the influences of baicalin on cardiomyocytes H9c2 apoptosis aroused by hypoxia stimulation. Methods Firstly, H9c2 cells were subjected to hypoxia and/or baicalin exposure. Cell viability and apoptosis, along with hypoxia-inducible factor 1α (HIF1α) and Bcl-2/adenovirus E1B 19-KDa interacting protein 3 (BNIP3) expressions were tested respectively. Then, si-HIF1α was transfected into H9c2 cells to probe whether up-regulation of HIF1α attended to the influences of baicalin on hypoxia-stimulated H9c2 cells. Finally, the regulatory effect of nuclear factor E2-related factor 2 (Nrf2)/heme oxygenase 1 (HO-1) pathway on HIF1α expression was analyzed. Results Hypoxia exposure aroused H9c2 cell viability reduction and apoptosis. Baicalin mitigated H9c2 cell viability reduction and apoptosis aroused by hypoxia. Moreover, HIF1α/BNIP3 pathway was further activated by baicalin in hypoxia-exposed H9c2 cells. Silencing HIF1α lowered the functions of baicalin on hypoxia-exposed H9c2 cells. Besides, baicalin enhanced hypoxia-caused activation of Nrf2/HO-1 pathway. Activation of Nrf2/HO-1 pathway was associated with the up-regulation of HIF1α and protective functions of baicalin on hypoxia-exposed H9c2 cells. Conclusion Baicalin relieved cardiomyocytes H9c2 apoptosis aroused by hypoxia might be achieved through activating Nrf2/HO-1-mediated HIF1α/BNIP3 pathway. Highlights Baicalin mitigates H9c2 cell viability loss and apoptosis aroused by hypoxia; Baicalin activates HIF1a/BNIP3 pathway in hypoxia-exposed H9c2 cells; Silencing HIF1α weakens the influences of baicalin on hypoxia-exposed H9c2 cells; Baicalin promotes Nrf2/HO-1 pathway in hypoxia-exposed H9c2 cells; Promotion of Nrf2/HO-1 pathway is related to the up-regulation of HIF1α.

The MicroRNA-92a/Sp1/MyoD Axis Regulates Hypoxic Stimulation of Myogenic Lineage Differentiation in Mouse Embryonic Stem Cells.

Hypoxic microenvironments exist in developing embryonic tissues and determine stem cell fate. We previously demonstrated that hypoxic priming plays roles in lineage commitment of embryonic stem cells. In the present study, we found that hypoxia-primed embryoid bodies (Hyp-EBs) efficiently differentiate into the myogenic lineage, resulting in the induction of the myogenic marker MyoD, which was not mediated by hypoxia-inducible factor 1α (HIF1α) or HIF2α, but rather by Sp1 induction and binding to the MyoD promoter. Knockdown of Sp1 in Hyp-EBs abrogated hypoxia-induced MyoD expression and myogenic differentiation. Importantly, in the cardiotoxin-muscle injury mice model, Hyp-EB transplantation facilitated muscle regeneration invivo, whereas transplantation of Sp1-knockdown Hyp-EBs failed to do. Moreover, we compared microRNA (miRNA) expression profiles between EBs under normoxia versus hypoxia and found that hypoxia-mediated Sp1 induction was mediated by the suppression of miRNA-92a, which directly targeted the 3' untranslated region (3' UTR) of Sp1. Further, the inhibitory effect of miRNA-92a on Sp1 in luciferase assay was abolished by a point mutation in specific sequence in the Sp1 3' UTR that is required for the binding of miRNA-92a. Collectively, these results suggest that hypoxic priming enhances EB commitment to the myogenic lineage through miR-92a/Sp1/MyoD regulatory axis, suggesting a new pathway that promotes myogenic-lineage differentiation.

Insulin-like growth factor-I prevents hypoxia-inducible factor-1 alpha-dependent G1/S arrest by activating cyclin E/cyclin-dependent kinase2 via the phoshatidylinositol-3 kinase/AKT/forkhead box O1/Cdkn1b pathway in porcine granulosa cells†.

As the follicle develops, the thickening of the granulosa compartment leads to progressively deficient supply of oxygen in granulosa cells (GCs) due to the growing distances from the follicular vessels. These conditions are believed to cause hypoxia in GCs during folliculogenesis. Upon hypoxic conditions, several types of mammalian cells have been reported to undergo cell cycle arrest. However, it remains unclear whether hypoxia exerts any impact on cell cycle progression of GCs. On the other hand, although the GCs may live in a hypoxic environment, their mitotic capability appears to be unaffected in growing follicles. It thus raises the question whether there are certain intraovarian factors that might overcome the inhibitory effects of hypoxia. The present study provides the first evidence suggesting that cobalt chloride (CoCl2)-mimicked hypoxia prevented G1-to-S cell cycle progression in porcine GCs. In addition, we demonstrated that the inhibitory effects of CoCl2 on GCs cell cycle are mediated through hypoxia-inducible factor-1 alpha/FOXO1/Cdkn1b pathway. Moreover, we identified insulin-like growth factor-I (IGF-I) as an intrafollicular factor required for cell cycle recovery by binding to IGF-I receptor in GCs suffering CoCl2 stimulation. Further investigations confirmed a role of IGF-I in preserving G1/S progression of CoCl2-treated GCs via activating the cyclin E/cyclin-dependent kinase2 complex through the phoshatidylinositol-3 kinase/protein kinase B (AKT)/FOXO1/Cdkn1b axis. Although the present findings were based on a hypoxia mimicking model by using CoCl2, our study might shed new light on the regulatory mechanism of GCs cell cycle upon hypoxic stimulation.

NADPH oxidase 4 mediates TGF-β1/Smad signaling pathway induced acute kidney injury in hypoxia.

Hypoxia is an important cause of acute kidney injury (AKI) in various conditions because kidneys are one of the most susceptible organs to hypoxia. In this study, we investigated whether nicotinamide adenine dinucleotide 3-phosphate (NADPH) oxidase 4 (Nox4) plays a role in hypoxia induced AKI in a cellular and animal model. Expression of Nox4 in cultured human renal proximal tubular epithelial cells (HK-2) was significantly increased by hypoxic stimulation. TGF-β1 was endogenously secreted by hypoxic HK-2 cells. SB4315432 (a TGF-β1 receptor I inhibitor) significantly inhibited Nox4 expression in HK-2 cells through the Smad-dependent cell signaling pathway. Silencing of Nox4 using Nox4 siRNA and pharmacologic inhibition with GKT137831 (a specific Nox1/4 inhibitor) reduced the production of ROS and attenuated the apoptotic pathway. In addition, knockdown of Nox4 increased cell survival in hypoxic HK-2 cells and pretreatment with GKT137831 reproduce these results. This study demonstrates that hypoxia induces HK-2 cell apoptosis through a signaling pathway involving TGF-β1 via Smad pathway induction of Nox4-dependent ROS generation. In an ischemia/reperfusion rat model, pretreatment of GKT137831 attenuated ischemia/reperfusion induced acute kidney injury as indicated by preserved kidney function, attenuated renal structural damage and reduced apoptotic cells. Therapies targeting Nox4 may be effective against hypoxia-induced AKI.

Abstract 1718: Down-regulation of p16INK4a by hYSK1 promotes cancer cell migration under hypoxic conditions

Abstract The alteration expression of p16INK4a, a well-known cyclin-dependent kinase inhibitor involved in cancer cell cycle control, is unclear, especially under hypoxic conditions. To evaluate p16INK4a regulation, we performed a protein microarray analysis. Among 1,800 proteins, we identified hYSK1 as a novel protein that interacts with the tumor suppressor p16INK4a. hYSK1, a member of the Ste20 family of serine/threonine protein kinases, promotes cell migration & tumorigenesis and is activated by oxidative stress. However, the molecular mechanisms underlying the oncogenic potential of hYSK1 remain elusive. Here, we report here that hYSK1 interacts with p16INK4a under hypoxic conditions like tumors, where it negatively regulates p16INK4a, enhancing cancer cell migration. Hypoxic stimulation of hYSK1 reduces p16INK4a accumulation through p16 promoter regulation to interact with unphosporylated SP-1 & increases matrix metalloproteinase-2 (MMP-2) expression by activating the MMP-2 promoter associated with cell migration and proliferation.Conversely, knocking down hYSK1 expression activated p16INK4a expression & suppressed MMP-2 expression.Thus, hYSK1 is necessary as a trigger for inactivating p16INK4a & activating MMP-2 during tumor migration, suggesting that hYSK1 is a specific negative regulator of the tumor suppressor p16INK4a may represent a novel molecular target for reactivation of tumor suppressor genes in humans. Citation Format: Mee-Hyun Lee, Jung-Hyun Shim, Zigang Dong, Young-Joon Surh, Bu Young Choi. Down-regulation of p16INK4a by hYSK1 promotes cancer cell migration under hypoxic conditions [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1718.

A Long-Acting Neutralizing Monoclonal ACTH Antibody Blocks Corticosterone and Adrenal Gene Responses in Neonatal Rats.

The control of steroidogenesis in the neonatal adrenal gland is of great clinical interest. We have previously demonstrated that the postnatal day (PD) 2 rat exhibits a large plasma corticosterone response to hypoxia in the absence of an increase in plasma ACTH measured by RIA, whereas the corticosterone response to exogenous ACTH is intact. By PD8, the corticosterone response to hypoxia is clearly ACTH-dependent. We hypothesized that this apparently ACTH-independent response to hypoxia in the newborn rat is due to an increase in a bioactive, nonimmunoassayable form of ACTH. To evaluate this phenomenon, we pretreated neonatal rats with a novel, specific, neutralizing anti-ACTH antibody (ALD1611) (20 mg/kg or 1 mg/kg IP) on the morning of PD1, PD7, and PD14. Twenty-four hours later, we measured hypoxia- or ACTH-stimulated plasma ACTH and corticosterone. For long-term effects, ALD1611 was given on PD1 and pups were studied on PD8 and PD15. Pretreatment with ALD1611 significantly decreased baseline corticosterone and completely blocked the corticosterone response to hypoxia and exogenous ACTH stimulation at all ages. The effect of 1 mg/kg ALD1611 on PD1 had dissipated by PD15. The decrease in corticosterone in ALD1611-treated pups was associated with decreases in baseline and hypoxia- and ACTH-stimulated adrenal Ldlr, Mrap, and Star mRNA expression at all ages. The adrenal response to hypoxia in the newborn rat is ACTH-dependent, suggesting the release of nonimmunoassayable, biologically active forms of ACTH. ALD1611 is useful as a tool to attenuate stress-induced, ACTH-dependent adrenal steroidogenesis in vivo.

Acute Effects Of Electrical Stimulation In Hypoxia On Arterial Stiffness

Acute Effects Of Electrical Stimulation In Hypoxia On Arterial Stiffness

LncRNA MALAT1 cessation antagonizes hypoxia/reoxygenation injury in hepatocytes by inhibiting apoptosis and inflammation via the HMGB1-TLR4 axis

Hepatic ischemia-reperfusion (I/R) injury frequently occurs after liver transplantation, stroke, and trauma, resulting in organ dysfunction and failure. Hepatocyte apoptosis and inflammation are identified as the hallmarks of liver I/R injury. Long non-coding RNA (lncRNA) metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) is induced following hypoxia or ischemic stimulation, and exerts the contradictory roles in various injury progression. However, its role and mechanism lying beneath hepatic I/R remains ill defined. In this study, elevation of MALAT1 expression was corroborated in human hepatocytes under hypoxia/reoxygenation (H/R)H/R condition. Of interest, depression of MALAT1 blunted H/R-inhibited cell viability, and counteracted lactate dehydrogenase (LDH) and malondialdehyde release. Additionally, MALAT1 cessation antagonized H/R-evoked cell apoptosis and caspase-3 activity. Simultaneously, the increased inflammatory reaction triggered by H/R stimulation was also abrogated following MALAT1 suppression by reducing pro-inflammatory cytokine transcripts and productions including IL-1β and TNF-α. Mechanistically, H/R exposure activated the pathway of high-mobility group box1 (HMGB1)-TLR4, which was muted after MALAT1 inhibition. More importantly, elevation of HMGB1 reversed MALAT1 down-regulation-mediated inhibition in cell injury and inflammation. Moreover, blocking the TLR4 signaling also ameliorated H/R-evoked hepatocyte apoptosis and inflammatory response. Consequently, these data suggest that MALAT1 may aggravate hepatic I/R injury by regulating the HMGB1-TLR4-triggered cell apoptosis and inflammation, implying a promising therapeutic strategy to fight liver I/R injury.

Hypoxic regulation of angiotensin-converting enzyme 2 and Mas receptor in human CD34+ cells.

CD34+ hematopoietic stem/progenitor cells (HSPCs) are vasculogenic and hypoxia is a strong stimulus for the vasoreparative functions of these cells. Angiotensin-converting enzyme 2 (ACE2)/angiotensin-(1-7)/Mas receptor (MasR) pathway stimulates vasoprotective functions of CD34+ cells. This study tested if ACE2 and MasR are involved in the hypoxic stimulation of CD34+ cells. Cells were isolated from circulating mononuclear cells derived from healthy subjects (n = 46) and were exposed to normoxia (20% O2 ) or hypoxia (1% O2 ). Luciferase reporter assays were carried out in cells transduced with lentivirus carrying ACE2- or MasR- or a scramble-3'-untranslated region gene with a firefly luciferase reporter. Expressions or activities of ACE, angiotensin receptor Type 1 (AT1R), ACE2, and MasR were determined. In vitro observations were verified in HSPCs derived from mice undergoing hindlimb ischemia (HLI). In vitro exposure to hypoxia-increased proliferation and migration of CD34+ cells in basal conditions or in response to vascular endothelial growth factor (VEGF) or stromal-derived factor 1α (SDF) compared with normoxia. Expression of ACE2 or MasR was increased relative to normoxia while ACE or AT1R expressions were unaltered. Luciferase activity was increased by hypoxia in cells transfected with the luciferase reporter plasmids coding for the ACE2- or MasR promoters relatively to the control. The effects of hypoxia were mimicked by VEGF or SDF under normoxia. Hypoxia-induced ADAM17-dependent shedding of functional ACE2 fragments. In mice undergoing HLI, increased expression/activity of ACE2 and MasR were observed in the circulating HSPCs. This study provides compelling evidence for the hypoxic upregulation of ACE2 and MasR in CD34+ cells, which likely contributes to vascular repair.

RELA promotes hypoxia-induced angiogenesis in human umbilical vascular endothelial cells via LINC01693/miR-302d/CXCL12 axis.

Hypoxia-induced angiogenesis plays a critical role in wound healing, which could be disturbed by multifactors. Upon hypoxia stimulation, CXCL12 and its receptor CXCR4 were significantly upregulated in human umbilical vascular endothelial cells (HUVECs); thus, we attempted to investigate the role and mechanism of CXCL12 in HUVEC angiogenesis under hypoxia. Via downloading and analyzing microarray profiles (GSE76743 and GSE116909), we found that LINC01693 was positively correlated with CXCL12 and upregulated by hypoxia in HUVECs, while miR-302d was downregulated by hypoxia and might target LINC01693 and CXCL12. RELA, a critical transcriptional factor for response to hypoxia, could bind to LINC01693 promoter to activate its transcription, therefore, promoting CXCL12 expression under hypoxia. LINC01693 served as a competing endogenous RNA for miR-302d to counteract miR-302d-mediated CXCL12 suppression via direct targeting. Hypoxia-induced CXCL12 upregulation and angiogenesis in HUVECs could be significantly suppressed by LINC01693 silence while enhanced by miR-302d inhibition; the effect of LINC01693 silence could be partially reversed by miR-302d inhibition. Taken together, RELA promotes the angiogenesis in HUVECs via LINC01693/miR-302d/CXCL12 axis. We provide a novel mechanism and experimental basis of CXCL12 function in hypoxia-induced HUVEC angiogenesis.

Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Induces ATP Formation and Release by Regulating Acid‐Base Transition and Mitochondrial Oxidative Phosphorylation in the Heart

ATP is released in the heart during hypoxia, ischaemia or catecholamine stimulation. Both ischaemia and hypoxia lower intracellular pH. However, little is known about the signaling pathways involved in CFTR regulated ATP formation and release in heart.Lactic acid infusion, the mimic of ischaemia, increased left ventricular wall interstitial ATP in anaesthetized rats, which was blocked by inhibitors of CFTR. The signaling mechanism was studied in cardiomyocytes isolated from adult rat heart: lactic acid increased intracellular cAMP, Ca in the near membrane area, and ATP release. Acidosis‐induced ATP release was abolished by CFTR inhibitors or CFTR siRNA. The CFTR potentiator, apigenin, or cAMP‐elevating agents, forskolin and IBMX (F&I), also increased ATP release, confirming the role of CFTR. Inhibition of either the Na+/H+ exchanger (NHE) with amiloride or the Na+/Ca2+ exchanger (NCX) with SN6 abolished acidosis‐induced ATP release. Acidosis or F&I failed to activate ATP release with either BAPTA, an intracellular calcium chelator, or calcium free incubation medium. Thus, both cAMP and calcium activated CFTR opening during acidosis.Forskolin or acidosis‐stimulated ATP release from cardiomyocytes was abolished in bicarbonate free incubation medium, suggesting that the role of CFTR is to permit bicarbonate entry. Bicarbonate increased mitochondrial cytochrome C expression and release of both ATP and cytochrome C from isolated mitochondria; these were inhibited by KH7, a soluble adenylyl cyclase (sAC)‐specific inhibitor, suggesting that mitochondrial cyclic AMP activates mitochondrial PKA, which phosphorylates mitochondrial proteins resulting in increased mitochondrial oxidative phosphorylation and accumulation of cytochrome‐C. Cyclosporin A or V5, mitochondrial permeability transition pore/Bax inhibitors, induced accumulation of cytochrome‐C in mitochondria, whereas they inhibited ATP release from cardiomyocytes, suggesting that cytochrome‐C leaves the mitochondria through mPTP.Pannexin1 inhibitors, caspase inhibitors or pannexin1 siRNA, attenuated the ATP release during acidosis or forskolin treatment, suggesting that Pannexin1, activated by caspase cleavage, functions as the ATP release channel. Immunofluorescence imaging suggested that CFTR was co‐localized with Pannexin1.Hence, we demonstrate for the first time that the CFTR channel is activated by calcium/cAMP signaling during acidosis, allowing the movement of bicarbonate into the cell. The bicarbonate activates the HCO3‐mito‐sAC‐cAMP‐PKA signaling cascade, which serves as a metabolic sensor modulating ATP generation, and subsequently modulates Pannexin1 gating (for ATP release) by activation of caspase.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Biomolecular changes at ventrolateral medulla and respiratory deficit in an experimental model of Parkinson's disease

Parkinson's disease (PD) is a neurodegenerative disorder characterized by motor and non‐motor symptoms, and caused by degeneration of dopaminergic neurons of substantia nigra pars compacta (SNpc). Prior studies showed that in animal model of PD there is a decrease in the resting and hypercapnia‐induced respiratory rate and in the number of phox2b‐expressing RTN neurons. The aim of this study was to identify the extent to which RTN pharmacological rescue restores respiratory function and determine the extent of biomolecular changes following PD‐induction. Adult male Wistar rats with bilateral injections of 6‐hydroxy‐dopamine (6‐OHDA, 24 μg/μl) or vehicle into the striatum were used. For proteomic analyzes, laser capture and pressure catapulting was used for microdissection of the brain cuts. We also performed comparative proteomic studies followed by Ingenuity Pathway Analysis (IPA®) of the RTN region in PD‐induced rats. Stainless steel canullas were implanted into RTN region and injections of N‐methyl D‐aspartate (NMDA, 50 nl) were performed to verify the pharmacological activation of these neurons. Respiratory parameters were evaluated by whole body plethysmography. The results showed that sixty days after injection of 6‐OHDA, the number of SNpc catecholaminergic and RTN phox2b neurons reduced (73.1± 2 and 55.9 ± 17%, respectively). In PD‐animals, there is a reduction in basal ventilation, hypercapnia, hypoxia and maximal chemoreflex stimulation (MCS) (basal: 357 ± 19, vs. vehicle: 917 ± 22 ml/kg/min; hypercapnia: 1064 ± 35, vs. vehicle: 1921 ± 108 ml/kg/min; hypoxia: 711 ± 6, vs. vehicle: 1851 ± 102 ml/kg/min; MCS: 1195 ± 17, vs. vehicle: 2727 ± 26 ml/kg/min, p<0.05). However, the activation of RTN by NMDA injection showed similar respiratory responses between PD and control animals (respiratory frequency: 149 ± 12, vs. control 155 ± 12 breaths/min; ventilation: 1028 ± 191, vs. vehicle: 985 ± 199 ml/kg/min; tidal volume: 6.9 ± 0.7, vs. vehicle: 6.4 ± 0.5 ml/kg, p>0.05). The proteomic analyses of the RTN showed a biomolecular profile indicative of ongoing cell remodeling in PD‐induced animals. In PD‐group, some proteins showed a decreased fold change in quantity, such as MYH9 (cytoskeleton reorganization), CAMK2D (regulation of Ca2+ homeostasis), RAB6A (protein transport), whereas Dnah5 (cellular movement) and Rnh1 (ribonuclease inhibitor) were increased. In addition, other genes were functionally inhibited, such as ADORA‐2A (g‐protein receptor), IGF‐1 (growth factor) and SRF (transcription regulation) or activated, as RICTOR (kinases mediate responses to stress), MAP4K4 (kinase) and CAMK2A (NMDAR signaling complex). Our data suggest, therefore, that despite the fact that biomolecular changes occur at the RTN level, there may be persistence of an NMDA‐specific compensatory mechanism provided by the surviving neurons.Support or Funding InformationNIH/NHLBI, FAPESP, CNPq and CAPESThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Hypoxia-induced miR-191-C/EBP\u03b2 signaling regulates cell proliferation and apoptosis of fibroblast-like synoviocytes from patients with rheumatoid arthritis

BackgroundHypoxia plays an important role in the proliferation of rheumatoid arthritis fibroblast-like synoviocytes (RA-FLS), leading to pathology of RA. This study was conducted to evaluate hypoxia-induced microRNAs (hypoxamiR) in RA-FLS and its role in the function of RA-FLS.MethodsRA-FLS were cultured under normoxia (21% O2) or hypoxia (3% O2) condition, followed by a microRNA (miRNA) array analysis. The upregulation of miR-191 by hypoxia was confirmed in RA-FLS and FLS from osteoarthritis (OA) patients by quantitative real-time polymerase chain reaction (RT-PCR). Transfection of miR-191 mimic and inhibitor was used to investigate the function of miR-191 in RA-FLS. The functional targets of miR-191 were predicted by bioinfomatics and then validated by reporter gene assay.ResultsA subset of miRNAs was identified to be induced by hypoxia including miR-191. The upregulation of miR-191 was found to be specific in hypoxic RA-FLS, compared to hypoxic OA-FLS. We observed that miR-191 in RA-FLS increased cellular proliferation via promoting G1/S transition of the cell cycle and suppressed cell apoptosis induced by cell starvation. Bioinformatical analysis and experimental assays identified CCAAT/enhancer binding protein β (C/EBPβ) as a target gene of miR-191 in RA-FLS. Enforced expression of C/EBPβ rescued the cellular phenotypes induced by miR-191. In addition, an inverse correlation between the C/EBPβ level and hypoxia stimulation was found in RA-FLS, and overexpression of C/EBPβ could partly rescue the hypoxia-induced cell proliferation.ConclusionWe demonstrated the miR-191-C/EBPβ signaling pathway mediating the hypoxia-induced cell proliferation in RA.

Caffeine Treatment Promotes Differentiation and Maturation of Hypoxic Oligodendrocytes via Counterbalancing Adenosine 1 Adenosine Receptor-Induced Calcium Overload.

BackgroundWe aimed to explore the involvement of adenosine 1 adenosine receptor (A1AR) in hypoxia-induced poor differentiation of oligodendrocytes (OLs), and the underlying mechanism of caffeine treatment in hypoxic injuries.Material/MethodsReal-time polymerase chain reaction (RT-PCR) was used to assess the alterations of AR expression in cultured hypoxic OLs with or without caffeine treatment. Then, intracellular alterations of Ca2+ concentrations ([Ca2+]) were detected by confocal Fluo-3 imaging. The subsequent changes of myelin related protein expression were determined by western blot and immunofluorescence.ResultsThree hours after hypoxia, significantly upregulated expression of A1AR was observed, accompanied with significantly decreased expression of oligodendrocyte transcription factor (Olig2). In addition, either hypoxia stimulation or 100 μM adenosine induced apparent elevation of resting [Ca2+] in cultured OLs. However, pretreatment with DPCPX (A1AR selective antagonist) or caffeine abolished the [Ca2+] increase, and the subsequent adenosine of high dose induced Ca2+ activity in developing OLs. Furthermore, caffeine or DPCPX improved the expression MBP and CNPase proteins after hypoxia stimulation, which resulted in the morphological maturation of OLs.ConclusionsCaffeine treatment exerted protective effects on neonatal hypoxia injuries. It prevented Ca2+ overload injury, kept Ca2+ homeostasis in hypoxic developing OLs, and facilitated optimal expression of myelin related proteins by inhibiting A1AR in vitro. This study also provided experimental evidence for clinical application of caffeine in early treatment of neonatal hypoxia, and highlighted the potential significance of A1AR in anti-hypoxic drug discovery.

ILK promotes survival and self-renewal of hypoxic MSCs via the activation of lncTCF7-Wnt pathway induced by IL-6/STAT3 signaling.

Mesenchymal stem cells (MSCs) have been applied in treating various diseases including myocardial infarction (MI) and achieved a bit of success; however, the decreased survival rate of MSCs after transplantation greatly limited the efficacy for cell therapy. How to improve the MSC survival rate in stem cell transplantation has undoubtedly become urgent and genetic engineering may be an ideal and feasible way. In this study, we explored the effects on MSCs survival and self-renewal by overexpression of integrin-linked kinase (ILK) in MSCs under hypoxic stimulation and aimed to reveal the molecular mechanisms from the point of paracrine function of MSCs. We first found that overexpression of ILK induced the expression and secretion of IL-6 increased significantly in MSCs under hypoxic stimulation, and the survival and self-renewal of MSCs exposed to hypoxia were enhanced after ILK overexpression. Then the activation of JAK2/STAT3 signaling was detected because of the increased IL-6, and an lncRNA, named lncTCF7, was upregulated remarkably, promoting the activation of Wnt pathway that was required for keeping cell viability and stemness of MSCs. Moreover, we further verified that inhibition of STAT3 signaling by WP1066 and silencing lncTCF7 expression eliminated the protective effects of ILK overexpression on cell survival and self-renewal of MSCs under hypoxic sitmulation. In conclusion, our results uncovered a novel function of ILK to promote MSC survival and self-renewal, suggesting more application potentials of MSC cell therapy on MI.

Long Non Coding RNA H19: A New Player in Hypoxia-Induced Multiple Myeloma Cell Dissemination.

The long non-coding RNA H19 (lncH19) is broadly transcribed in the first stage of development and silenced in most cells of an adult organism; it appears again in several tumors where, through different molecular mediators, promotes cell proliferation, motility and metastases. LncH19 has been associated with hypoxia-inducible factor 1-alpha (HIF-1α) activation and, in some tumors, it has proved to be necessary and required to sustain hypoxic responses. Here we propose to investigate a putative role for the lncH19 in hypoxia induced multiple myeloma (MM) progression. Transcriptional analysis of MM cell lines (RPMI and MM1.S) exposed to normoxia or hypoxia (1% O2) was done in order to evaluate lncH19 levels under hypoxic stimulation. Then, to investigate the role of lncH19 in hypoxia mediated MM progression, transcriptional, protein and functional assays have been performed on hypoxia stimulated MM cell lines, silenced or not for lncH19. Our data demonstrated that hypoxic stimulation in MM cell lines induced the overexpression of lncH19, which, in turn, is required for the expression of the hypoxia induced genes involved in MM dissemination, such as C-X-C Motif Chemokine Receptor 4 (CXCR4) and Snail. Moreover, adhesion assays demonstrated that lncH19 silencing abrogates the increased adhesion on stromal cells induced by the hypoxic condition. Finally, Western blot analysis indicated that lncH19 silencing impaired HIF1α nuclear translocation. The LncH19, required for the induction of hypoxic responses in MM cells, could represent a new therapeutic target for MM.

Hemolymph and transcriptome analysis to understand innate immune responses to hypoxia in Pacific abalone

Hypoxia was thought to inhibit immune responses, causing severe mortality to marine organisms. The Pacific abalone, Haliotis discus hannai, is the most widely cultured abalone species in China, but suffering from “summer mortality” in which hypoxia has been one of the main reasons. The effect of hypoxia exposure on immune responses in H. discus hannai was investigated, including cellular immune response using flow cytometry and transcriptome profiles. The influence of hypoxia treatment on the total hemocyte count (THC) of H. discus hannai was rather limited but the hemocyte survival rate of abalone increased during 48 h exposure. There was an initial rise in the production of phagocytes and reactive oxygen species (ROS) shortly (3 h) after hypoxic stimulation, but finally decreased after 48 h. This indicates that hypoxia inhibited redox activity of abalone. RNA-seq studies of gill tissues also revealed immune response mechanism in abalone after 24 h of hypoxia. Totally 954 differentially expressed genes (DEGs) were detected under different degrees of deoxygenation. Though oxidation-reduction turbulence was a result of both up- and down-regulated DEGs, cell death/apoptosis induction resulted from up-regulated DEGs, whilst DNA metabolic and immunity suppression resulted from down-regulated DEGs. In summary, our data provides evidence that deoxygenation greatly affects abalone immunity, probably making it more vulnerable. The present study also lays the foundation for further research in hypoxia-associated conditions in abalone aquaculture.

Abstract A180: HHLA2 is a novel tumor-expressed member of the B7 immune checkpoint family

Abstract The purpose of this study was to assess the function and expression-regulation of the new B7 family member, HHLA2, in the tumor microenvironment. HHLA2 is a newly identified B7 family member that has been reported to both inhibit and stimulate T-cell function in vitro. Expression of HHLA2 has been noted on a wide variety of cancers and relatively few normal tissues. The function of HHLA2 in the tumor microenvironment remains elusive and the mechanisms regulating its expression are currently unknown. Challenges to addressing these questions include the lack of a functional HHLA2 gene in mice and, consequently, the lack of in vivo tumor models in which HHLA2 can be studied. Here we assess factors that drive expression of HHLA2 in vivo and present the first humanized mouse model to study the function of HHLA2 expression in the tumor microenvironment. Human cancer cell lines derived from lung, prostate, pancreas, kidney, and colon malignancies were identified with varying levels of HHLA2 expression. HHLA2 surface expression was monitored as these cell lines were grown in vivo as tumors in NSG mice and in vitro under conditions including cytokine stimulation, 3D culture, and hypoxia. Expression was assessed by both flow cytometry and qPCR. HHLA2-knockout cancer cell lines and controls were generated using CRISPR and grown as tumors in NSG mice with human immune cells. These tumors were dissociated and the immune infiltrates were phenotyped by flow cytometry. HHLA2 was upregulated in numerous human cancer cell lines in vivo and expression was reduced or lost in vitro under standard cell culture conditions. Conditions mimicking the tumor microenvironment including 3D culture, hypoxia, and cytokine stimulation all contributed to HHLA2 upregulation. We successfully generated HHLA2-knockout human cancer cell lines and developed a humanized tumor immunotherapy model. HHLA2-expressing tumors contained significantly less infiltrating CD8 and CD4 T-cells than the HHLA2-knockout tumors. The T-cells were also phenotypically less active in the HHLA2-expressing tumors. Expression of HHLA2 in human cancers is driven by environmental conditions that are specific to the tumor microenvironment and this expression appears to have a suppressive effect on infiltrating T-cells. HHLA2 is the newest B7 family member and a promising candidate for targeted immunotherapy against a wide range of cancers. Citation Format: Jordan Manek Chinai, Hao Wang, Xudong Tang, Murali Janakiram, Haiying Cheng, Xingxing Zang. HHLA2 is a novel tumor-expressed member of the B7 immune checkpoint family [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr A180.

Orexin Receptor Blockade-Induced Sleep Preserves the Ability to Wake in the Presence of Threat in Mice.

Retention of the ability to wake from sleep in response to dangerous situations is an ideal characteristic of safe hypnotics. We studied the effects of a dual orexin receptor antagonist-22 (DORA-22) and the GABA-A receptor modulator, triazolam, on the ability to wake in response to aversive stimuli. We examined four modalities of sensory inputs, namely, auditory (ultrasonic sound), vestibular (trembling), olfactory (predator odor), and autonomic (hypoxia) stimuli. When the mice fell asleep, one of the four stimuli was applied for 30 s. In the case of auditory stimulation, latency to arousal following vehicle, DORA-22, and triazolam administration was 3.0 (2.0–3.8), 3.5 (2.0–6.5), and 161 (117–267) s (median and 25–75 percentile in the parentheses, n = 8), respectively. Latency to return to sleep after arousal was 148 (95–183), 70 (43–98), and 60 (52–69) s, respectively. Similar results were obtained for vestibular and olfactory stimulation. During the hypoxic stimulation, latencies for arousal and returning to sleep were not significantly different among the groups. The findings of this study are consistent with the distinct mechanisms of these sleep promoting therapies; GABA-A receptor activation by triazolam is thought to induce widespread central nervous system (CNS) suppression while DORA-22 more specifically targets sleep/wake pathways through orexin receptor antagonism. These data support the notion that DORA-22 preserves the ability to wake in response to aversive and consciousness-inducing sensory stimuli, regardless of modality, while remaining effective in the absence of threat. This study provides a unique and important safety evaluation of the potential for certain hypnotics.