Hypoxic Activation Research Articles

Erythropoietin and its antagonist regulate hypoxic fictive breathing in newborn mice

Clinical use of erythropoietin in adult and newborn patients has revealed its involvement in neuroprotection, neurogenesis, and angiogenesis. More recently, we showed in adult mouse, that brain erythropoietin interacts with the major brainstem centers associated with respiration to enhance the ventilatory response to acute and chronic conditions of physiological hypoxia (e.g., as occurring at high altitude). However, whether brain erythropoietin is involved in breathing regulation in newborns remains unknown. In this study, en bloc brainstem–spinal cord preparations were obtained from mice at postnatal day 4. After various periods (30, 60, or 90min) of incubation with 0, 25, or 250U of erythropoietin, preparations were superfused with artificial cerebrospinal fluid bubbled with normoxic or hypoxic gas mixtures. The electrophysiological fictive breathing produced by axons at the C4 ventral root was next recorded. Our results show that erythropoietin attenuates the hypoxia-mediated decrease of the central respiratory activity and improves post-hypoxic recovery. Additional analysis revealed that the soluble erythropoietin receptor (the endogenous erythropoietin antagonist) dramatically decreases neural hypoxic respiratory activity, confirming the specific erythropoietin effect on respiratory drive. These results imply that erythropoietin exerts main modulation and maintenance of respiratory motor output during hypoxic and post-hypoxic challenges in 4-days old mice.

Induction of Gastrin Expression in Gastrointestinal Cells by Hypoxia or Cobalt Is Independent of Hypoxia-Inducible Factor (HIF)

Gastrin and its precursors have been shown to promote mitogenesis and angiogenesis in gastrointestinal tumors. Hypoxia stimulates tumor growth, but its effect on gastrin gene regulation has not been examined in detail. Here we have investigated the effect of hypoxia on the transcription of the gastrin gene in human gastric cancer (AGS) cells. Gastrin mRNA was measured by real-time PCR, gastrin peptides were measured by RIA, and gastrin promoter activity was measured by dual-luciferase reporter assay. Exposure to a low oxygen concentration (1%) increased gastrin mRNA concentrations in wild-type AGS cells (AGS) and in AGS cells overexpressing the gastrin receptor (AGS-cholecystokinin receptor 2) by 2.1 ± 0.4- and 4.1 ± 0.3-fold (P < 0.05), respectively. The hypoxia mimetic, cobalt chloride (300 μM), increased gastrin promoter activity in AGS cells by 2.4 ± 0.3-fold (P < 0.05), and in AGS-cholecystokinin receptor 2 cells by 4.0 ± 0.3-fold (P < 0.05), respectively. The observations that either deletion from the gastrin promoter of the putative binding sites for the transcription factor hypoxia-inducible factor 1 (HIF-1) or knockdown of either the HIF-1α or HIF-1β subunit did not affect gastrin promoter inducibility under hypoxia indicated that the hypoxic activation of the gastrin gene is likely HIF independent. Mutational analysis of previously identified Sp1 regulatory elements in the gastrin promoter also failed to abrogate the induction of promoter activity by hypoxia. The observations that hypoxia up-regulates the gastrin gene in AGS cells by HIF-independent mechanisms, and that this effect is enhanced by the presence of gastrin receptors, provide potential targets for gastrointestinal cancer therapy.

Erythropoietin and its antagonist regulate the hypoxic fictive breathing in newborn mice

Erythropoietin (Epo) in adult mouse interacts with the major brainstem centers associated with respiration to enhance the ventilatory response to acute and chronic conditions of physiological hypoxia (e.g. as occuring at high altitude). However, whether Epo is as well implicated in the regulation of breathing in newborns remains unknown. To answer this question en bloc brainstem‐spinal cord preparations were obtained from mice at postnatal day 4 (P4). After time‐ (30, 60 or 90 min) and dose‐dependent (0, 25 or 250 U) incubation with or without Epo, en bloc preparations were superfused with artificial cerebrospinal fluid (aCSF) bubbled with normoxic or hypoxic gas mixtures. Subsequently, the electrophysiological fictive breathing produced by axons at the C4 ventral root was recorded. Our results show that upon hypoxic conditions Epo stimulates the neural hypoxic respiratory activity and improves the post hypoxic recovery. In addition, proof‐of‐concept experiments showed that soluble Epo receptor (sEpoR), the endogenous antagonist of Epo, dramatically decreases the neural hypoxic respiratory activity. These results imply that Epo and its endogenous antagonist in P4 mice are implicated in the modulation and maintenance of respiratory motor output during hypoxic and post‐hypoxic challenges. As Epo is a highly promising protective therapeutic agent for several brain diseases, these results suggest that Epo applications may be also relevant for neuronal‐related respiratory disorders in term and preterm neonates.

Crucial role of phospholipase C gamma 2 in hypoxic pulmonary vasoconstriction

Hypoxic pulmonary vasoconstriction (HPV) may ensure the physiological regional alveolar ventilation‐pulmonary perfusion matching, but can also serve as a key pathological mechanism leading to pulmonary hypertension and associated right ventricular failure. An increase in [Ca2+]i in pulmonary artery smooth muscle cells (PASMCs) is vital for HPV. However, the underlying molecular mechanisms remain incompletely understood. We have found that acute hypoxia significantly increases the activity of total phospholipase C (PLC) in isolated, endothelium‐denuded mouse resistance pulmonary arteries. The activity of PLCγ2 is largely augmented as well following hypoxic exposure. The PLC inhibitor U73122 (1 μM) greatly diminishes the hypoxia‐induced increase in [Ca2+]i in PASMCs, whereas U73433 (1 μM), an inactive analog of U73122, has no effect. 2‐aminoethoxydiphenyl borate (2‐APB) and xestospongin C to block inositol trisphosphate receptors (IP3Rs) also inhibit the hypoxic Ca2+ response. Excitingly, shRNA‐mediated PLCγ2 gene silencing diminishes the hypoxia‐induced increase in [Ca2+]i. Using myxothiazol to inhibit mitochondrial ROS production blocks the hypoxic activation of PLCγ2. In conclusion, hypoxia can specifically activate PLCγ2 and then IP3Rs to increase [Ca2+]i in PASMCs by enhancing mitochondrial ROS production, which may play an important role in HPV and hypoxic pulmonary hypertension. This study was supported by AHA EIA 0340160N and NIH R01 HL64043, HL064043‐S1, and HL075190.

Activated CD47 promotes pulmonary arterial hypertension through targeting caveolin-1

Pulmonary arterial hypertension (PAH) is a progressive lung disease characterized by pulmonary vasoconstriction and vascular remodelling, leading to increased pulmonary vascular resistance and right heart failure. Loss of nitric oxide (NO) signalling and increased endothelial nitric oxide synthase (eNOS)-derived oxidative stress are central to the pathogenesis of PAH, yet the mechanisms involved remain incompletely determined. In this study, we investigated the role activated CD47 plays in promoting PAH. We report high-level expression of thrombospondin-1 (TSP1) and CD47 in the lungs of human subjects with PAH and increased expression of TSP1 and activated CD47 in experimental models of PAH, a finding matched in hypoxic human and murine pulmonary endothelial cells. In pulmonary endothelial cells CD47 constitutively associates with caveolin-1 (Cav-1). Conversely, in hypoxic animals and cell cultures activation of CD47 by TSP1 disrupts this constitutive interaction, promoting eNOS-dependent superoxide production, oxidative stress, and PAH. Hypoxic TSP1 null mice developed less right ventricular pressure and hypertrophy and markedly less arteriole muscularization compared with wild-type animals. Further, therapeutic blockade of CD47 activation in hypoxic pulmonary artery endothelial cells upregulated Cav-1, increased Cav-1CD47 co-association, decreased eNOS-derived superoxide, and protected animals from developing PAH. Activated CD47 is upregulated in experimental and human PAH and promotes disease by limiting Cav-1 inhibition of dysregulated eNOS.

Hypoxic activation of ATR and the suppression of the initiation of DNA replication through cdc6 degradation

Many severely hypoxic cells fail to initiate DNA replication, but the mechanism underlying this observation is unknown. Specifically, while the ataxia-telangiectasia-rad3 related (ATR) kinase has been shown to be activated in hypoxic cells, several studies have not been able to document down-stream consequences of ATR activation in these cells. By clearly defining the DNA replication initiation checkpoint in hypoxic cells, we now demonstrate that ATR is responsible for activating this checkpoint. We show that the hypoxic activation of ATR leads to the phosphorylation dependent degradation of the cdc25a phosphatase. Down regulation of cdc25a protein by ATR in hypoxic cells decreases CDK2 phosphorylation and activity, which results in the degradation of cdc6 by APC/CCdh1. These events do not occur in hypoxic cells when ATR is depleted, and the initiation of DNA replication is maintained. We therefore present a novel mechanism of cdc6 regulation in which ATR can play a central role in inhibiting the initiation of DNA replication via the regulation of cdc6 by APC/CCdh1. This model provides insight into the biology and therapy of hypoxic tumors.

Hypoxic damage to pancreatic beta cells – The hidden link between sleep apnea and diabetes

Despite a large body of epidemiologic and clinical evidence suggesting that sleep disordered breathing is an independent risk factor for development of type 2 diabetes (T2DM), the underlying pathogenesis of altered glucose metabolism in sleep apnea remains to be unraveled. While previous studies have proposed some causal pathways linking sleep apnea with T2DM through increased insulin resistance and deterioration in insulin sensitivity, there has been a particular lack of research into sleep apnea-related alterations in pancreatic beta-cell function. Drawing upon our previous observation that sleep apnea is independently associated with an increased basal pancreatic beta-cell function in adults with normal glucose metabolism [1], the idea presented here suggests that sleep apnea imposes an excessive demand for insulin secretion, which may lead to progressive pancreatic beta-cell failure in high-risk individuals. Specifically, we hypothesize that in addition to diabetogenic effects of acute hypoxic activation of the sympathetic nervous system, the chronic intermittent hypoxemia represses the expression of key genes regulating biosynthesis of pancreatic proinsulin convertases with a resultant progressive decrease in their catalytic activity. The long-term hypoxic damage to pancreatic beta-cells may thus contribute to progression of glucose dysregulation in persons with untreated sleep apnea over time. Strategies to prevent and decrease the high prevalence and associated morbidity of T2DM are critically needed. The ideas and hypotheses presented here address the unexplored pathophysiological mechanisms underlying the potential causal link between sleep apnea and T2DM. Future hypotheses-testing will seek to delineate the role of sleep apnea in the development of T2DM, probe the underlying molecular mechanisms for pancreatic beta-cell dysfunction in sleep apnea, and obtain information on clinical, epidemiologic, and other factors responsible for protecting individuals from alterations in insulin-glucose homeostasis. These results could further be utilized in testing genetic susceptibilities and various therapy modalities to prevent pancreatic beta-cell dysfunction and maintain normal glucose status in persons with sleep apnea in the long term.

The kreisler mutation leads to the loss of intrinsically hypoxia-activated spots in the region of the retrotrapezoid nucleus/parafacial respiratory group

Acute hypoxia elicits a biphasic respiratory response characterized in the newborn by a transient hyperventilation followed by a severe decrease in respiratory drive known as hypoxic respiratory depression. Medullary O(2) chemosensitivity is known to contribute to respiratory depression induced by hypoxia, although precise involvement of cell populations remains to be determined. Having a thorough knowledge of these populations is of relevance because perturbations in the respiratory response to hypoxia may participate in respiratory diseases in newborns. We aimed to analyze the hypoxic response of ponto-medullary cell populations of kreisler mutant mice. These mice have defects in a gene expressed in two rhombomeres encompassing a part of the medulla oblongata implicated in hypoxic respiratory depression. Central responses to hypoxia were analyzed in newborn mice by measuring respiratory rhythm in ex vivo caudal pons-medullary-spinal cord preparations and c-fos expression in wild-type and kreisler mutants. The homozygous kreisler mutation, which eliminates most of rhombomere 5 and mis-specifies rhombomere 6, abolished (1) an early decrease in respiratory frequency within 10 min of hypoxia and (2) an intrinsic hypoxic activation, which is characterized by an increase in c-fos expression in the region of the ventral medullary surface encompassing the retrotrapezoid nucleus/parafacial respiratory group expressing Phox2b. This increase in c-fos expression persisted in wild-type Phox2b-negative and Phox2b-positive cells after blockade of synaptic transmission and rhythmogenesis by a low [Ca(2+)](0). Another central response was retained in homozygous kreisler mutant mice; it was distinguished by (1) a delayed (10-30 min) depression of respiratory frequency and (2) a downregulation of c-fos expression in the ventrolateral reticular nucleus of the medulla, the nucleus of the solitary tract, and the area of the A5 region. Thus, two types of ponto-medullary cell groups, with distinct anatomical locations, participate in central hypoxic respiratory depression in newborns.

Impaired hypoxic response in senescent mouse brain

Tissue hypoxia leads to activation of endogenous adaptive responses that involve a family of prolyl hydroxylase domain proteins (PHD1-3) with oxygen sensing properties, hypoxia inducible transcription factors (HIFs), and cytoprotective HIF target genes such as erythropoietin (EPO) and vascular endothelial growth factor (VEGF). The hypoxic induction of these genes is regulated by oxygen-dependent hydroxylation of HIFα subunits by PHDs, which signals their proteasomal degradation. In this study, mice of different age were exposed to hypoxia or subjected to cerebral ischemia after hypoxic pre-conditioning. We found an impaired hypoxic response in the brain, characterized by elevated levels and impaired downregulation of PHD1. Furthermore, an attenuated hypoxic activation of VEGF and EPO, as well as of other HIF-target genes such glucose transporter-1 and carbonic anhydrase 9 was found in senescent brain. Finally, we observed a loss of the protective effect of hypoxic pre-conditioning on subsequent cerebral ischemia with increasing age. Thus, the impaired hypoxic adaptation, resulting in compromised hypoxic activation of neuroprotective factors, could contribute to neurodegenerative processes with increasing age, and might have implications for treating age-related disorders.

The Hog1 Mitogen-Activated Protein Kinase Mediates a Hypoxic Response inSaccharomyces cerevisiae

We have studied hypoxic induction of transcription by studying the seripauperin (PAU) genes of Saccharomyces cerevisiae. Previous studies showed that PAU induction requires the depletion of heme and is dependent upon the transcription factor Upc2. We have now identified additional factors required for PAU induction during hypoxia, including Hog1, a mitogen-activated protein kinase (MAPK) whose signaling pathway originates at the membrane. Our results have led to a model in which heme and ergosterol depletion alters membrane fluidity, thereby activating Hog1 for hypoxic induction. Hypoxic activation of Hog1 is distinct from its previously characterized response to osmotic stress, as the two conditions cause different transcriptional consequences. Furthermore, Hog1-dependent hypoxic activation is independent of the S. cerevisiae general stress response. In addition to Hog1, specific components of the SAGA coactivator complex, including Spt20 and Sgf73, are also required for PAU induction. Interestingly, the mammalian ortholog of Spt20, p38IP, has been previously shown to interact with the mammalian ortholog of Hog1, p38. Taken together, our results have uncovered a previously unknown hypoxic-response pathway that may be conserved throughout eukaryotes.

A DNA-dependent stress response involving DNA-PK occurs in hypoxic cells and contributes to cellular adaptation to hypoxia

DNA-dependent protein kinase (DNA-PK) is involved in DNA double-strand break (DSB) signalling and repair. We report that DNA-PK is activated by mild hypoxia conditions (0.1-1% O₂) as shown by (1) its autophosphorylation on Ser2056, and (2) its mobilisation from a soluble nucleoplasmic compartment to a less extractable nuclear fraction. The recruitment of DNA-PK was not followed by activation and recruitment of the XRCC4-DNA-ligase-IV complex, suggesting that DSBs are not responsible for activation of DNA-PK. To unravel the mechanism of DNA-PK activation, we show that exposure of cells to trichostatin A, a histone deacetylase inhibitor, leads to DNA-PK autophosphorylation and relocalisation to DNA. Histone acetylation (mainly H3K14) is increased in hypoxic cells and treatment with anacardic acid, an inhibitor of histone acetyl transferase, prevented both histone modifications and DNA-PK activation in hypoxic conditions. Importantly, in using either silenced DNA-PK cells or cells exposed to a specific DNA-PK inhibitor (NU7026), we demonstrated that hypoxic DNA-PK activation positively regulates the key transcription factor HIF-1 and one subsequent target gene, GLUT1. Our results show that hypoxia initiates chromatin modification and consequently DNA-PK activation, which positively regulate cellular oxygen-sensing and oxygen-signalling pathways.

Abstract 344: Oxygen sensors are poor prognosticators for survival in NSCLC

Abstract Background: Most solid tumors are at least partly hypoxic. To survive hypoxia, cancer cells must adapt by hypoxic gene activation. Hypoxia-inducible factors (HIFs) have been recognized as key regulators of this transcriptional control. However, oxygen tension is sensed by three HIF prolyl hydroxylases (PHD1, PHD2 and PHD3) and one asparaginyl hydroxylase, FIH (Foctor inhibiting HIF). This study evaluates the prognostic impact of these four oxygen sensors in the biggest killer in cancer, Non-Small Cell Lung Cancer (NSCLC). Methods: 335 unselected stage I-IIIA NSCLC-patients were included. Duplicate 0.6 mm diameter cores from both the viable neoplastic cell compartment (tumor) and stroma were collected in tissue microarrays (TMAs). Immunohistochemistry (IHC) was used to semiquantitatively evaluate the expression. Results: There was only scorable expression in the tumor compartment. In univariate analyses high expression of all the oxygen sensors in tumor were significantly associated with a poor prognosis (PHD1, P = 0.023; PHD2, P = 0.013; PHD3, P = 0.018; FIH, P = 0.033). There were only weak correlations between these markers (strongest between PHD2 and FIH; r = 0.18, P = 0.001). In the multivariate analysis, entering all the significant clinicopathological variables and the oxygen sensors, we found only high expression of PHD2 (HR 2.03; CI 95% 1.20-3.42; P = 0.008) and PHD1 (HR 1.45; CI 95% 1.01-2.10; P = 0.047) of the molecular variables to be independent poor prognosticators. Conclusions: Oxygen sensors were only scorable in tumor. High expression of all the oxygen sensors was associated with a poor prognosis. High expression of PHD1 and PHD2 were also significantly and independently associated with a dismal prognosis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 344. doi:10.1158/1538-7445.AM2011-344

Abstract 4233: Protolimonoids potently suppress mitochondrial respiration and disrupt protein translation in breast tumor cells

Abstract Hypoxia (decreased oxygen tension) is a common feature of solid tumors and represents an obstacle for cancer treatment. The transcription factor hypoxia-inducible factor-1 (HIF-1) has emerged as an important molecular target for antitumor hypoxia drug discovery. A natural product chemistry-based approach was utilized to identify small-molecule HIF-1 inhibitors. Over 20,000 extracts of plants and marine organisms were evaluated for HIF-1 inhibitory activities. An active extract of the sacred medicinal Bael Tree (Aegle marmelos) trunk bark was subjected to bioassay-guided isolation and de-replication-based structure elucidation yielded two known protolimonoids skimmiarepin A (1) and C (2). Compounds 1 and 2 inhibited hypoxia-induced HIF-1 activation in a human breast tumor T47D cell-based reporter assay (IC50 values 0.063 and 0.068 μM, respectively). The hypoxic induction of HIF-1 target genes GLUT-1 and VEGF were also inhibited by 1 and 2. Mechanistic investigation revealed that 1 and 2 suppressed HIF-1 activation by blocking the accumulation of nuclear HIF-1α protein under hypoxic conditions. To test the hypothesis that these protolimonoids may interfere with hypoxic signaling by disrupting mitochondrial function, the effects of 1 and 2 on cellular respiration were examined. Both compounds inhibited T47D cell respiration at the range of concentrations that inhibited hypoxic HIF-1 activation. Compound 1 suppressed mitochondrial electron transport chain at complex I (NADH dehydrogenase). Under experimental conditions, neither 1 nor rotenone (positive control for complex I inhibitor) decreased cellular ATP content following a two-day exposure. However, rotenone and 1 suppressed cellular signaling pathways that control protein translation. While it is commonly believed that the antitumor activity of mitochondrial inhibitors is a consequence of their ability to inhibit cellular ATP production, the disruption of protein translation appears to constitute an important mechanism by which mitochondrial inhibitors obstruct tumor cell proliferation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4233. doi:10.1158/1538-7445.AM2011-4233

Abstract 1512: Hypoxic activation of FVII gene in cancer cells mediated by interaction among HIF2α, Sp1, and HDAC4

Abstract Blood coagulation factor VII (fVII) is a key enzyme of the extrinsic coagulation cascade that is produced predominantly by hepatocytes. Tissue factor/fVIIa complex formation on the cell surface initiates key pathogenic mechanisms in cancer, including cell motility, invasion, cell survival, and angiogenesis. We have shown that various cancer cells ectopically synthesize fVII, resulting in activation of cell motility and invasion [1]. We have also shown that fVII expression is ectopically induced in ovarian cancer cells under hypoxia, potentially leading to a major complication of cancer patients, thromboembolism via secretion of fVII-positive microparticles [2]. Hypoxia inducible factors HIF1α and HIF2α are known to form a heterodimeric complex with a constitutively expressed aryl hydrocarbon receptor nuclear translocator (ARNT). HIF-ARNT complex binds to hypoxia response elements (HREs) within gene regulatory regions, resulting in transcriptional activation. We previously found that fVII gene (FVII) is inducible under hypoxia by HIF2α dependent mechanism. Unexpectedly, chromatin immunoprecipitation (ChIP) analysis revealed that HIF2α but not HIF1α was found to bind to FVII promoter without HRE [1]. In this study, we further investigated this characteristic transcriptional activation mechanism. Reporter gene, ChIP, and RNA interference (RNAi) experiments revealed that interaction between Sp1 and HIF2α in the promoter region is essential for FVII induction. Histone acetylation is known as a major mechanism for transcriptional activation. Thus, we additionally investigated effect of histone acetylation within the promoter region on HIF2α-Sp1 mediated transcriptional activation. We unexpectedly found that unlike typical transcriptional activation mechanisms, acetylation of histones negatively regulates FVII gene induction. ChIP and RNAi, followed by real-time PCR analysis revealed that a class II histone deacetylase HDAC4 is recruited to FVII promoter under hypoxia and is a coactivator responsible for this transcriptional induction. Furthermore, immunohistochemical experiments with an ovarian cancer xenograft model revealed that fVII presents in tissues strongly expressing HIF2α. These results suggest that hypoxic activation of FVII gene in cancer cells involves novel deacetylation dependent mechanism mediated by HIF2α-Sp1 interaction.

Altered responses of MeCP2-deficient mouse brain stem to severe hypoxia

Rett syndrome (RTT) patients suffer from respiratory arrhythmias with frequent apneas causing intermittent hypoxia. In a RTT mouse model (methyl-CpG-binding protein 2-deficient mice; Mecp2(-/y)) we recently discovered an enhanced hippocampal susceptibility to hypoxia and hypoxia-induced spreading depression (HSD). In the present study we investigated whether this also applies to infant Mecp2(-/y) brain stem, which could become life-threatening due to failure of cardiorespiratory control. HSD most reliably occurred in the nucleus of the solitary tract (NTS) and the spinal trigeminal nucleus (Sp5). HSD susceptibility of the Mecp2(-/y) NTS and Sp5 was increased on 8 mM K(+)-mediated conditioning. 5-HT(1A) receptor stimulation with 8-hydroxy-2-(di-propylamino)tetralin (8-OH-DPAT) postponed HSD by up to 40%, mediating genotype-independent protection. The deleterious impact of HSD on in vitro respiration became obvious in rhythmically active slices, where HSD propagation into the pre-Bötzinger complex (pre-BötC) immediately arrested the respiratory rhythm. Compared with wild-type, the Mecp2(-/y) pre-BötC was invaded less frequently by HSD, but if so, HSD occurred earlier. On reoxygenation, in vitro rhythms reappeared with increased frequency, which was less pronounced in Mecp2(-/y) slices. 8-OH-DPAT increased respiratory frequency but failed to postpone HSD in the pre-BötC. Repetitive hypoxia facilitated posthypoxic recovery only if HSD occurred. In 57% of Mecp2(-/y) slices, however, HSD spared the pre-BötC. Although this occasionally promoted residual hypoxic respiratory activity ("gasping"), it also prolonged the posthypoxic recovery, and thus the absence of central inspiratory drive, which in vivo would lengthen respiratory arrest. In view of the breathing disorders in RTTs, the increased hypoxia susceptibility of MeCP2-deficient brain stem potentially contributes to life-threatening disturbances of cardiorespiratory control.

Hydrogen sulfide therapy rescues critical limb ischemia in aged diabetic animals through an eNOS/HIF‐1/VEGF dependent pathway

Objective To determine the effect and molecular mechanism of Na2S induced neovascularization in critical limb ischemia (CLI) during diabetes. Methods Permanent unilateral hind limb ischemia was induced in 42 wk old Db/Db diabetic or eNOSKO mice (n=8, each group). PBS or 0.5 mg/kg Na2S was administered twice daily by retro-orbital injection. Body weight, blood glucose, lipid panels, hind limb perfusion, angiogenic index, capillary to myofiber ratio, proliferation index and TUNEL assay were measured. Endothelial cell proliferation was also performed under hypoxic condition. Lastly, HIF -1 activity was measured using a HIF -1 reporter assay and VEGF expression was determined by ELISA. Result Blood tissue perfusion, angiogenic index, proliferation index, capillary to myofiber ratio, HIF-1 activity and VEGF expression were all significantly increased and conversely, apoptosis was prevented in ischemic hind limb of aged diabetic mice treated with Na2S compared to PBS control. Na2S therapy did not completely restore eNOS KO ischemic hind limb blood flow to pre-ligation levels and cPTIO treatment significantly prevented Na2S mediated hypoxic endothelial cell proliferation activity. Conclusion Sodium sulfide therapy restores tissue perfusion of critical limb ischemia in aged diabetic mice by increasing HIF-1 activation and expression of VEGF that involves NO synthesis and bioavailability.

Hypoxia-induced SOCS3 is limiting STAT3 phosphorylation and NF-κB activation in congenital heart disease

Suppressor of cytokine signaling 3 (SOCS3) is a critical attenuator of the JAK-STAT signaling pathway, and it is involved in mediating the intensity and duration of STAT3 activation in the process of myocardial protection. Nuclear factor-κB (NF-κB) has emerged as a decisive transcription factor in cardiac myocyte compensatory responses to stress that enhance survival. However, the expression, activation and regulation of this signaling molecule in response to hypoxic stress have not been elucidated. We investigated 40 infants with cyanotic or acyanotic cardiac defects, as well as H9c2 embryonic rat cardiomyocytes, to examine the effect of hypoxia on the expression or activation of SOCS3, STAT3 and NF-κB in vivo and in vitro. We found an increase in endogenous cardiac SOCS3, p-STAT3 and AC-RelA activation in the myocardium of infants with cyanotic cardiac defects. In hypoxic cultivated H9c2 cells, SOCS3, STAT3 and AC-RelA activity slowly increased and then reached a stable expression. We evaluated the interaction of SOCS3 with STAT3 and NF-κB by transfecting the SOCS3 plasmid to hypoxic cultured H9c2 cells. Forced expression of SOCS3suppressed tyrosine phosphorylation of STAT3 and transcription of the C-myc and interleukin-6 genes. AC-RelA activation was also suppressed by over expression of SOCS3. These findings suggest that the mechanism of a positive transactivation loop that maintains higher levels of NF-κB and p-STAT3 and the negative feedback factor SOCS3, which maintains balanced NF-κB and p-STAT3 activities, is important in the process of myocardial adaptation to chronic hypoxia. SOCS3 is a rapid hypoxia inducible gene and acts to inhibit activation of the cellular signaling pathway in a classical negative feedback loop. Upregulated SOCS3 might play an important role in cardiocytes during chronic hypoxia as SOCS3 regulates cell signaling crosstalking between NF-κB and p-STAT3 under stressful conditions.

Synthesis, hypoxia-selective cytotoxicity of new 3-amino-1,2,4-benzotriazine-1,4-dioxide derivatives

We reported the synthesis, hypoxic cytotoxic activities and selectivities of 18 new 3-(alkoxymethylamino)-1,2,4-benzotriazine 1,4-dioxides. The synthesized compounds were screened in vitro against 5 cell lines: K562, SMMC-7721, A549, PC-3 and KB in hypoxia and in normoxia. Some of them showed higher or similar cytotoxic activity when compared to tirapazamine. Physico-chemical study showed the positive correlation between hypoxic activity and lipophilicity within a certain range. Preliminary mechanism study on the potent derivatives 4b, 4l and 4m indicated that the cytotoxic activities of these compounds might be mediated by inducing apoptosis.

Regulatory Effects of microRNA-92 (miR-92) on VHL Gene Expression and the Hypoxic Activation of miR-210 in Clear Cell Renal Cell Carcinoma

In order to understand the role of miRNAs in renal tumorigenesis, we undertook a stepwise approach that included a comprehensive differential miRNA expression analysis for the most common histological subtypes of human renal neoplasms appearing in either sporadic or hereditary forms. We also aimed to test the hypothesis that microRNAs can act as an alternative mechanism of VHL gene inactivation and therefore might be correlated with tumorigenesis in ccRCC. Finally, we wanted to explore whether the well-known hypoxic activation of ccRCC is followed by a specific pattern of miRNA expression. Tumor and normal adjacent kidney parenchyma from patients with RCC were tested for microRNA expression. Twenty cases of different histologies were used for profiling by PCR miRNA arrays. For validation, a separate cohort of samples used to test specifically miR92a expression and its involvement in VHL gene mRNA silencing. Finally, miR210 as a marker of hypoxia was evaluated. Expression values were correlated with important clinicopathologic features from the patients. We identified unique miRNA expression signatures for each histologic subtype of kidney tumors. Expression values for downregulated miRNAs ranged from 0.3-fold (in VHL-clear cell RCC) up to 0.393 fold (in papillary type II (HLRCC) tumors). For the upregulated miRNAs, fold-changes ranged from 2.1 up to 290-fold. Specific patterns together with type-specific profiles were observed. Twenty-three miRNAs were found to be differentially expressed in both sporadic and VHL-dependent ccRCC. Sporadic clear cell tumors showed a unique pattern of 14-miRNA that were absent from the VHL-dependent tumors. These also showed 15 miRNAs specific to the hereditary type. Common miRNAs to both sporadic and hereditary forms included miR-92a and miR-210. For miR-92a, and a striking inverse correlation with VHL mRNA levels was found. For the hypoxia-regulated miR-210, clear cell tumors showed significantly higher expression levels when compared to tumor of non-clear cell histology (9.90-fold vs. 1.36, p<0.001). microRNA expression seems to be involved in every step of RCC pathogenesis: both as an element for tumor development as well as a consequence of or in response to the initial malignant transformation and part of tumor progression. Our data show consistent disregulation of miRNAs in human kidney cancer, some of which are potentially involved in critical gene silencing in RCC and others that are activated as part of the pathophysiological response in these tumors.

Hypoxia and high glucose activate tetrodotoxin-resistant Na(+) currents through PKA and PKC.

Voltage-gated sodium channels are critical for the initiation and propagation of action potentials and for the regulation of neuronal excitability. Hyperglycemia and hypoxia are two main changes in diabetes frequently associated with several complications. Although many studies on streptozotocin-induced diabetic rats indicate that early diabetic neuropathy is associated with increased amplitude and faster kinetics of sodium channels, the distinctive roles of high glucose and hypoxia have not been completely clarified. Here we show that hypoxic and high glucose conditions (overnight exposure) increase activation and inactivation of TTX-RINa in DRG neurons without affecting the level of expression. Hypoxia and high glucose alone were potent enough to induce similar or even greater sensitization than when both conditions were present, without any of them having a predominant effect. PKA is mainly responsible of the one condition effect, while under both hypoxia and high glucose PKC was also contributing to alter the kinetics, although not in a cumulative manner. These data indicate that TTX-RINa is significantly modulated under short-time exposure to hypoxia and high glucose, a mechanism which might be relevant for diabetes-related complications or other diseases associated with acute hypoxia.