Upregulation Of Hypoxia-inducible Factor Research Articles

MicroRNA-210 Regulates Mitochondrial Free Radical Response to Hypoxia and Krebs Cycle in Cancer Cells by Targeting Iron Sulfur Cluster Protein ISCU

BackgroundHypoxia in cancers results in the upregulation of hypoxia inducible factor 1 (HIF-1) and a microRNA, hsa-miR-210 (miR-210) which is associated with a poor prognosis.Methods and FindingsIn human cancer cell lines and tumours, we found that miR-210 targets the mitochondrial iron sulfur scaffold protein ISCU, required for assembly of iron-sulfur clusters, cofactors for key enzymes involved in the Krebs cycle, electron transport, and iron metabolism. Down regulation of ISCU was the major cause of induction of reactive oxygen species (ROS) in hypoxia. ISCU suppression reduced mitochondrial complex 1 activity and aconitase activity, caused a shift to glycolysis in normoxia and enhanced cell survival. Cancers with low ISCU had a worse prognosis.ConclusionsInduction of these major hallmarks of cancer show that a single microRNA, miR-210, mediates a new mechanism of adaptation to hypoxia, by regulating mitochondrial function via iron-sulfur cluster metabolism and free radical generation.

Abstract LB-376: Hypoxia-inducible factor-1 alpha is an unfavorable determinant of relapse in gastric cancer patients who underwent curative surgery followed by adjuvant 5-fluorouracil chemotherapy

Abstract Background: Among several chemotherapeutic agents, 5-Fluorouracil (5-FU) has been widely used as a key drug in adjuvant chemotherapy for gastric cancer. However, no reliable marker, which predicts the response to 5-FU in an adjuvant setting, has been identified. Hypoxia-induced drug resistance, via up-regulation of hypoxia-inducible factor (HIF) -1α, is a major obstacle in the development of effective cancer therapy. Despite the numerous investigations, few clinical studies have so far assessed the relationship between the HIF-1α expression and the chemo-resistance of gastric cancer patients in an adjuvant setting. Objective: To determine whether the expression of HIF-1α predicts the relapse of gastric cancer patients who underwent curative surgery followed by adjuvant 5-FU chemotherapy. Materials and Methods: Two HIF-1α knockdown gastric cancer cell lines were established in order to clarify the role of HIF-1α in chemo-resistance against 5-FU. The sensitivity to 5-FU was evaluated by MTT assay, and compared between HIF-1α knockdown cells and control scrambled cells. 5-FU-induced apoptosis was assessed by flow cytometry and the DNA ladder. Furthermore, HIF-1α knockdown xenograft models of nude mice were established to confirm the role of HIF-1α in 5-FU chemo-resistance in vivo. A total of 91 patients with locally advanced gastric cancer who underwent a curative resection were enrolled. Sixty-four of 91 patients received 5-FU based adjuvant chemotherapy after curative surgery. The expression of HIF-1α was immunohistochemically assessed, and correlated with the patient outcome. Results: The inhibition of HIF-1α improved the sensitivity to 5-FU in gastric cancer cell lines, and promoted 5-FU-induced apoptosis in vitro and in vivo. The effect of HIF-1α knockdown on 5-FU-induced apoptosis was more distinctly observed in xenograft tumor than in cell culture. In immunohistochemical analysis, HIF-1α expression was associated with the significantly shorter relapse-free survival and disease-specific survival in all gastric cancer patients (p = 0.045, 0.025), and in the 64 patients in the adjuvant group (p = 0.026, 0.014), but not in the 27 in the surgery group. Multivariate analysis showed that HIF-1α was an independent risk factor for relapse in 64 patients in the adjuvant group (p = 0.029). Conclusions: The current study demonstrated, for the first time, that HIF-1α expression is an independent risk factor for relapse in advanced gastric cancer patients who underwent curative surgery followed by adjuvant 5-FU chemotherapy. A favorable effect of 5-FU might therefore be expected in patients that do not express HIF-1α, whereas, other types of chemotherapy or additional treatments, such as HIF-1α inhibitors, should be considered in patients that do express HIF-1α. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-376.

Abstract 459: Caveolin-1 mediated signaling and proliferation is regulated through the oxygen sensing pathway

Abstract Caveolin-1 (Cav-1) is a multifunctional scaffolding protein with multiple binding partners that is associated with cell surface caveolae and the regulation of lipid raft domains. Cav-1 regulates multiple cancer-associated processes including cellular transformation, tumour growth, cell death and survival, and multidrug resistance. A high expression of Cav-1 is found in various cancer , including clear cell renal cell carcinoma (CCRCC), correlating with large, high grade tumors and poor prognosis. However, the role of Cav-1 in the oncogenesis of CCRCC remains unclear, and its role as either an oncogene or tumour suppressor is still under debate. Here, we investigated the mechanism of Cav-1 in controlling cell proliferation using in vitro and in vivo assays. We report that Cav-1 is highly expressed in tumour compared to normal tissue in patients with CCRCC, thus Cav-1 likely functions as an oncogene. Upon knock-down of Cav-1, tumour cells (786-VHL-/-) showed a decrease in proliferation. We showed that Cav-1 is associating with EGFR, a receptor tyrosine kinase, to activate the Ras-RAF-MEK-ERK kinase pathway and induce cell proliferation. We found that Cav-1 binds to EGFR in immunoprecipitation. The Ras pathway is upregulated in cells overexpressing Cav-1 after EGF stimulation. Upon knockdown of Cav-1, the level of p-ERK, p-MEK, and p-c-Raf decreased, as well as the rate of cell proliferation. Upstream of the Ras pathway, we report that Cav-1 appears to be under the control of hypoxia-inducible factor (HIF). Under hypoxia, VHL (the principal negative regulator of HIF) is downregulated, thus leading to upregulation of HIF. We showed that under hypoxia, Cav-1 increased in 786-VHL, Hela and A431 cell lines. Similarly, cells with VHL loss (786-VHL-/-), thus high HIF level, had high levels of Cav-1. We are currently examining the role of Cav-1 in tumour growth by injecting SCID mice with either 786-VHL-/-, or Cav-1 knock-down 786-VHL-/- tumour cells. Since Cav-1 level seems to be under the control of HIF in renal, cervical and squamous carcinoma cell lines, it suggests that the oxygen sensing pathway plays a general role in controlling cell proliferation through upregulating Cav-1. Cav-1 should be considered as a target for drug design as the knock down of Cav-1 decreased cell proliferation and could potentially decrease tumour growth. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 459.

Up-Regulation of Hypoxia-Inducible Factor (HIF)-1α and HIF-Target Genes in Cortical Neurons by the Novel Multifunctional Iron Chelator Anti-Alzheimer Drug, M30

Up-Regulation of Hypoxia-Inducible Factor (HIF)-1α and HIF-Target Genes in Cortical Neurons by the Novel Multifunctional Iron Chelator Anti-Alzheimer Drug, M30

Hypoxia Inducible Factor-1 Protects Against Nitrate Tolerance and Stunning in Rabbit Cardiac Myocytes

We tested whether upregulation of hypoxia inducible factor-1 (HIF-1) would restore the blunted effects of natriuretic peptides and nitric oxide caused by chronic nitrate exposure and stunning in cardiac myocytes. HIF-1alpha was increased with deferoxamine (150 mg/kg for 2 days). Nitrate tolerance was induced by a chronic nitroglycerin patch (0.3 mg/h for 5 days). We used freshly isolated rabbit ventricular myocytes. Half the myocytes were subjected to simulated ischemia [15 min 95% N(2)-5% CO(2)] and reperfusion [reoxygenation] to produce stunning. Cell function was measured utilizing a video-edge detector. Shortening was examined at baseline and after brain natriuretic peptide (BNP, 10(-8), 10(-7) M) or S-nitroso-N-acetyl-penicillamine (SNAP, 10(-6), 10(-5) M) followed by KT5823 (cyclic GMP protein kinase inhibitor, 10(-6) M). We also measured cyclic GMP protein kinase protein levels and kinase activity. In control, BNP (-29%) reduced percent shortening, while KT5823 partially restored function. Deferoxamine treated control myocytes responded similarly. In patched nonstunned myocytes, BNP (-12%) reduced shortening less and KT5823 did not increase function. However, deferoxamine restored the blunted effects of BNP (-21%) and KT5823. In stunned myocytes, BNP (-11%) reduced shortening less and KT5823 did not affect function. Deferoxamine increased the effects of BNP (-27%) and KT5823 in stunning. Patch combined with stunning also similarly blunted the effects of BNP (-12%) and KT5823. Deferoxamine improved the effects of BNP (-22%) and KT5823. Similar results were observed after SNAP. Stunning reduced cyclic GMP protein kinase activity and deferoxamine restored activity. Deferoxamine had no effect on kinase activity in nitrate tolerance. We found that upregulation of HIF-1 could protect isolated cardiac myocytes against nitrate tolerance through a cyclic GMP protein kinase-independent mechanism and through a kinase-dependent mechanism in stunning.

Prolonged Mechanical Stretch is Associated with Upregulation of Hypoxia-inducible Factors and Reduced Contraction in Rat Inferior Vena Cava

Prolonged Mechanical Stretch is Associated with Upregulation of Hypoxia-inducible Factors and Reduced Contraction in Rat Inferior Vena Cava

Intermittent hypoxia during sleep induces reactive gliosis and limited neuronal death in rats: implications for sleep apnea

Sleep apnea (SA) can be effectively managed in humans but it is recognized that when left untreated, SA causes long-lasting changes in neuronal circuitry in the brain. Recent neuroimaging studies gave suggested that these neuronal changes are also present even in patients successfully treated for the acute effects of SA. The cellular mechanisms that account for these changes are not certain but animal models of intermittent hypoxia (IH) during sleep have shown neuronal death and impairment in learning and memory. Reactive gliosis has a drastic effect on neuronal survival and circuitry and in this study we examined the neuro-glial response in brain areas affected by SA. Glial and neuronal alterations were analyzed after 1, 3, 5 and 10 days of exposure to IH (8 h/day during the sleep phase, cycles of 6 min each, 10-21% O2) and observed significant astroglial hyperplasia and hypertrophy in parietal brain cortex and hippocampus by studying gliofibrillary acidic protein, Vimentin, S100B and proliferating cell nuclear antigen expression. In addition, altered morphology, reduced dendrite branching and caspase activation were observed in the CA-1 hippocampal and cortical (layers IV-V) pyramidal neurons at short exposure times (1-3 days). Surprisingly, longer exposure to IH reduced the neuronal death rate and increased neuronal branching in the presence of persistent reactive gliosis. Up-regulation of hypoxia inducible factor 1 alpha (HIF-1alpha) and mdr-1, a HIF-1alpha target gene, were observed and increased expression of receptor for advanced end glycated products and its binding partner S100B were also noted. Our results show that a low number of hypoxic cycles induce reactive gliosis and neuronal death whereas continuous exposure to IH cycles reduced the rate of neuronal death and induced neuronal branching on surviving neurons. We hypothesize that HIF-1alpha and S100B glial factor may improve neuronal survival under hypoxic conditions and propose that the death/survival/re-growth process observed here may underlie brain circuitry changes in humans with SA.

HYPOXIA‐INDUCIBLE FACTOR‐1 IMPROVES THE ACTIONS OF POSITIVE INOTROPIC AGENTS IN STUNNED CARDIAC MYOCYTES

1. In the present study, we tested hypothesis that upregulation of hypoxia-inducible factor-1 (HIF-1) would improve the actions of positive inotropic agents in cardiac myocytes after simulated ischaemia-reperfusion (I/R). 2. Hypoxia-inducible factor-1α was upregulated with deferoxamine (150 mg/kg per day for 2 days). Rabbit cardiac myocytes were subjected to simulated ischaemia (15 min, 95% N(2)-5% CO2) and reperfusion (re-oxygenation) and compared with control myocytes. Cell contraction and calcium transients were measured at baseline and after forskolin (10(-7) and 10(-6) mol/L) or ouabain (10(-5) and 10(-4) mol/L). 3. Under control conditions, high-dose forskolin and ouabain increased percentage shortening by 20 and 18%, respectively. Deferoxamine-treated control myocytes responded similarly. In stunned myocytes, forskolin and ouabain did not significantly increase shortening (increases of 8% and 9%, respectively). Deferoxamine restored the effects of forskolin (+26%) and ouabain (+28%) in stunning. The results for maximum shortening and relaxation rates were similar. The increased calcium transients caused by forskolin and ouabain were also depressed in stunned myocytes, but were maintained by HIF-1 upregulation. 4. These results suggest that simulated I/R impaired the functional and calcium transient effects of positive inotropic agents. Upregulation of HIF-1 protects cardiac myocyte function after I/R by maintaining calcium release.

Neuroprotective and neuritogenic activities of novel multimodal iron‐chelating drugs in motor‐neuron‐like NSC‐34 cells and transgenic mouse model of amyotrophic lateral sclerosis

Novel therapeutic approaches for the treatment of neurodegenerative disorders comprise drug candidates designed specifically to act on multiple central nervous system targets. We have recently synthesized multifunctional, nontoxic, brain-permeable iron-chelating drugs, M30 and HLA20, possessing the N-propargylamine neuroprotective moiety of rasagiline (Azilect) and the iron-chelating moiety of VK28. The present study demonstrates that M30 and HLA20 possess a wide range of pharmacological activities in mouse NSC-34 motor neuron cells, including neuroprotective effects against hydrogen peroxide- and 3-morpholinosydnonimine-induced neurotoxicity, induction of differentiation, and up-regulation of hypoxia-inducible factor (HIF)-1alpha and HIF-target genes (enolase1 and vascular endothelial growth factor). Both compounds induced NSC-34 neuritogenesis, accompanied by a marked increase in the expression of brain-derived neurotrophic factor and growth-associated protein-43, which was inhibited by PD98059 and GF109203X, indicating the involvement of mitogen-activated protein kinase and protein kinase C pathways. A major finding was the ability of M30 to significantly extend the survival of G93A-SOD1 amyotrophic lateral sclerosis mice and delay the onset of the disease. These properties of the novel multimodal iron-chelating drugs possessing neuroprotective/neuritogenic activities may offer future therapeutic possibilities for motor neurodegenerative diseases.

A phase I trial of liposomal doxorubicin (D), bevacizumab (A), and temsirolimus (T) in advanced malignancy

e13508 Background: Liposomal doxorubicin (D) and bevacizumab (A) are active drugs in multiple malignancies. Unfortunately, resistance mechanisms have been described for each that limit their efficacy following repeated exposure. These include aberrant activation of alternate signaling pathways, such as PI3K/AKT/mTOR and upregulation of hypoxia inducible factor (HIF-1α). We therefore added temsirolimus (T), an agent known to inhibit mTOR and to downregulate HIF-1α, to the combination of D and A. The objectives of this trial were to assess the safety of this combination, obtain preliminary efficacy data and to identify biologic correlates for treatment response. Methods: D, A and T were administered intravenously on a 21 day cycle. D, A and T were given day 1, and T was administered alone weeks 2 and 3. A standard 3+3 trial design was used for dose escalation; 6 dose levels are planned. Patient serum and biopsies were also collected pre- and post-treatment for batched analysis to assess biologic response correlates. Results: 6/12 treated patients are evaluable (remaining patients have not yet completed 1st restaging). 5 patients are male; median age (all patients) 53, range 12 to 59. Median number of prior treatments = 5, range 2 - 8. At first restaging, 2/2 patients with endometrial cancer were found to have ≥ 25% decrease in tumor size (RECIST), and one patient with colorectal carcinoma had a 15% decrease. 1 patient had stable disease. One of the patients with endometrial cancer and extensive intra-abdominal disease who showed rapid tumor regression (≥ 25% decrease at 2 months) developed an entero-colonic fistula, and chose hospice care. Grade 2 stomatitis and fatigue (CTCAEv3)have also been seen. The current doses being used are 20mg/m2 of D and 5mg/m2 of A every 21 days, and 25mg of T every 7 days (dose level 3). Conclusions: Preliminary data indicate that the combination of D, A and T has anti-tumor activity and is well tolerated at the doses used. Patients with intra-abdominal disease who experience rapid tumor regression may be at risk for fistula formation. Biomarker analysis for modulation of the mTOR signal (via PI3K mutations or other aberrations) and the HIF-1α pathway is ongoing. No significant financial relationships to disclose.

Involvement of Erythropoietin in Retinal Ischemic Preconditioning

The purpose of this study was to examine the role of erythropoietin in retinal ischemic preconditioning (IPC). Rats were subjected to retinal ischemia after IPC. Electroretinography assessed functional recovery after ischemia; retinal sections were examined to determine loss of retinal ganglion cells, and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling was used to assess apoptosis. Levels of downstream mediators were measured in retinal homogenates by Western blotting. To assess the involvement of erythropoietin in IPC, Western blotting was used to measure levels of erythropoietin and its receptor (EPO-R) in retinal homogenates after IPC. To examine erythropoietin's role in IPC, the impact of blocking erythropoietin via intravitreal injection of soluble EPO-R (sEPO-R) before IPC was studied. Erythropoietin levels did not change after IPC, but EPO-R increased. Intravitreal injection of sEPO-R significantly attenuated both the functional and histologic neuroprotection produced by IPC in comparison to control injection of denatured sEPO-R. Apoptotic damage after ischemia was enhanced in the sEPO-R-treated retinas as indicated by fluorescent terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling. Phosphorylated extracellular-signal-regulated kinase and heat shock protein 27, but not protein kinase B, upregulated in denatured sEPO-R-treated retinae, were attenuated in eyes injected with sEPO-R. These results indicate that EPO-R upregulation is a critical component of the functional, histologic, and antiapoptotic protective effect of IPC on ischemia in the retina and that several downstream effectors may be involved in the neuroprotective actions of erythropoietin.

Hypoxia-induced CCR7 expression via HIF-1α and HIF-2α correlates with migration and invasion in lung cancer cells

Hypoxia, a common phenomenon in human solid tumors, is associated with invasion and metastasis in various tumors. Hypoxia inducible factors (HIFs) are key molecules in the hypoxic response, and regulate the activation of specific genes, which mediate many of the adaptations to hypoxia. CC chemokine receptor 7 (CCR7) has been shown to play a critical role in cell chemotaxis and homing, which are key steps in cancer metastasis. A study has demonstrated that hypoxia could up-regulate CCR7 in breast cancer cells. The CCR7 gene presents hypoxia response element (HRE; A/GCGTG). We presumed that hypoxia induced up-regulation of HIFs promoted the expression of CCR7 facilitating tumor cells invasion and metastasis. In this study, we firstly examined the relationship of CCR7 and HIF-1α, HIF-2α in 94 cases non-small cell lung cancer (NSCLC) tissues by Immunohistochemistry. The results showed that CCR7 expression correlated positively with HIF-1α and HIF-2α, all of them correlated with clinical stage and lymph node metastasis. Then, we investigated whether hypoxia induce the expression of CCR7 through HIF-1α and/or HIF-2α and observed the effects of up-regulated CCR7 on the migration and invasion of lung cancer cells. We found that hypoxia induced HIF-1α and HIF-2α expression, which up-regulated CCR7 expression; inhibiting HIF-1α or HIF-2α expression in BE1 and A549 cells by RNAi led to the decrease of CCR7 expression, inhibition of migratory and invasive abilities, and the effects of HIF-1α were more significant. Moreover, the migration and invasion of BE1 cells were increased as well as the expression of p-ERK1/2 after CCR7 transfection, but the cells invasive ability was inhibited after blocking p-ERK1/2 with PD98059 and CCR7 with specific antibody. In summary, our study demonstrated that hypoxia-HIF-1α, 2α-CCR7-ERK1/2 pathway could regulate the migration and invasion of lung cancer cells under hypoxic conditions, and promote metastasis of lung cancer.

Sulfonylurea Receptor 1 in the Germinal Matrix of Premature Infants

Germinal matrix (GM) hemorrhage (GMH) is a major cause of mortality and of life-long morbidity from cerebral palsy. GMH is typically preceded by hypoxic/ischemic events and is believed to arise from rupture of weakened veins in the GM. In the CNS, hypoxia/ischemia up-regulate sulfonylurea receptor 1 (SUR1)-regulated NCCa-ATP channels in microvascular endothelium, with channel activation by depletion of ATP being responsible for progressive secondary hemorrhage. We hypothesized that this channel might be up-regulated in the GM of preterm infants at risk for GMH. Here, we studied expression of the regulatory subunit of the channel, SUR1, and its transcriptional antecedent, hypoxia inducible factor 1 (HIF1), in postmortem tissues of premature infants who either were at risk for or who sustained GMH. We found regionally specific up-regulation of HIF1 and of SUR1 protein and mRNA in GM tissues, compared with remote cortical tissues. Up-regulation was prominent in most progenitor cells, whereas in veins, SUR1 was found predominantly in infants who had sustained GMH compared with those without hemorrhage. Our data suggest that the SUR1-regulated NCCa-ATP channel may be associated with GMH, and that pharmacological block of these channels could potentially reduce the incidence of this devastating complication of prematurity.

In Vivo Bioluminescence Imaging Monitoring of Hypoxia-Inducible Factor 1α, a Promoter That Protects Cells, in Response to Chemotherapy

Bioluminescence imaging is a powerful technique that has shown that hypoxia-inducible factor 1 (HIF-1), a transcription factor that protects tumor cells from hypoxia, is up-regulated in tumors after radiation therapy. We tested the hypothesis that bioluminescence imaging would successfully and noninvasively depict an increase in HIF-1 in the novel therapeutic environment of chemotherapy and that, as in radiation therapy, the underlying mechanism involves inducible nitric oxide synthase originating in macrophages. Active HIF-1 consists of alpha and beta subunits that bind to promoter sequences in many genes, including those that protect endothelial cells, promote angiogenesis, and alter metastasis and tumor cell metabolism. We grew 4T1 murine breast carcinoma cells with an HIF-1alpha luciferase reporter construct to 7 mm in the right rear flanks of 18 Balb-C mice. The mice were evenly randomized to receive one of the following single intraperitoneal doses: maximum tolerated dose cyclophosphamide (231.5 mg/kg), maximum tolerated dose paclitaxel (10 mg/kg), or control saline solution. Immunohistochemical analysis of tumor sections from the cyclophosphamide and control groups was performed 10 days after treatment to assess the intensity and distribution of HIF-1alpha expression, hypoxia, macrophage infiltration, and expression of macrophage-derived inducible nitric oxide synthase in tumor tissues treated with maximum tolerated dose cyclophosphamide compared with control tumors. Cyclophosphamide, but not paclitaxel, significantly inhibited tumor growth and caused a significant increase in HIF-1alpha protein levels, which peaked at a 10-fold increase from baseline on day 10 after administration. In contrast, paclitaxel did not have an antitumor effect in this model and did not cause a significant increase in HIF-1alpha. Immunohistochemical analysis showed increased and more evenly dispersed levels of HIF-1alpha protein, macrophage infiltration, and expression of inducible nitric oxide synthase originating in macrophages after cyclophosphamide treatment. We successfully monitored increased expression of a tumor protective protein in a noninvasive manner. Such monitoring may be a means of detection of resistance to therapy, and it may be possible to use the monitoring findings to alter treatment strategies in real time. The tumor microenvironment seen at immunohistochemical analysis supports the hypothesized mechanism that the cytotoxic effects of radiation therapy that attract macrophages, causing the release of macrophage-derived inducible nitric oxide synthase and production of HIF-1alpha under aerobic conditions, also underlie chemotherapy. Such noninvasive imaging may be a means to development of therapeutic strategies that prevent HIF-1 up-regulation after chemotherapy treatments.

The impact of N‐nitrosomelatonin as nitric oxide donor in cell culture experiments

N-nitrosomelatonin (NOMela) is well-known for its capabilities of transnitrosating nucleophiles such as thiols and ascorbate, thereby generating nitric oxide (NO)-releasing compounds. It is unknown, however, whether NOMela can be successfully applied as a precursor of NO in a complex biological environment like a cell culture system. NO donors may be useful to induce the transcription factor hypoxia inducible factor 1 (HIF-1), which coordinates the protection of cells and tissues from the lack of oxygen (hypoxia). In this study, the effects of NOMela in an in vitro cell-free assay [NO-release, inhibition of prolylhydroxylase1 (PHD1)] and in living cells (upregulation of HIF-1, reduction of HIF-1 hydroxylation, upregulation of the HIF-1-target gene PHD2) were compared with those of the frequently applied NO donor S-nitrosoglutathione (GSNO) under normoxic and hypoxic conditions. In contrast to GSNO, NOMela released NO in a predictable manner and this release in vitro was found to be independent of the composition of the buffer system. The NOMela-mediated effects in oxygenated cells were in all cases comparable to the hypoxic response, whereas unphysiological strong effects were observed with GSNO. Probably, because of the antioxidative power of the NOMela-dependent formation of melatonin, cells were completely protected against the attack of reactive nitrogen oxygen species, which are generated by autoxidation of NO. In conclusion, NOMela had to be an excellent NO precursor for cells in culture and potentially tissues.

Neuroprotection in Diet-Induced Ketotic Rat Brain after Focal Ischemia

Neuroprotective properties of ketosis may be related to the upregulation of hypoxia inducible factor (HIF)-1alpha, a primary constituent associated with hypoxic angiogenesis and a regulator of neuroprotective responses. The rationale that the utilization of ketones by the brain results in elevation of intracellular succinate, a known inhibitor of prolyl hydroxylase (the enzyme responsible for the degradation of HIF-1alpha) was deemed as a potential mechanism of ketosis on the upregulation of HIF-1alpha. The neuroprotective effect of diet-induced ketosis (3 weeks of feeding a ketogenic diet), as pretreatment, on infarct volume, after reversible middle cerebral artery occlusion (MCAO), and the upregulation of HIF-1alpha were investigated. The effect of beta-hydroxybutyrate (BHB), as a pretreatment, via intraventricular infusion (4 days of infusion before stroke) was also investigated following MCAO. Levels of HIF-1alpha and Bcl-2 (anti-apoptotic protein) proteins and succinate content were measured. A 55% or 70% reduction in infarct volume was observed with BHB infusion or diet-induced ketosis, respectively. The levels of HIF-1alpha and Bcl-2 proteins increased threefold with diet-induced ketosis; BHB infusions also resulted in increases in these proteins. As hypothesized, succinate content increased by 55% with diet-induced ketosis and fourfold with BHB infusion. In conclusion, the biochemical link between ketosis and the stabilization of HIF-1alpha is through the elevation of succinate, and both HIF-1alpha stabilization and Bcl-2 upregulation play a role in ketone-induced neuroprotection in the brain.

Preclinical rationale for combination targeted therapy in advanced clear cell renal cell carcinoma (RCC): Abrogation of rapamycin-mediated induction of AKT phosphorylation by perifosine

16083 Background: RCCs are highly vascularized tumors characterized by VHL abnormalities resulting in the upregulation of hypoxia inducible factor (HIF). Inhibition of mTOR, an upstream modulator of HIF, is an established therapeutic strategy in advanced RCC. One proposed resistance mechanism for mTOR inhibition is the compensatory upregulation of AKT, a promoter of cell growth and proliferation. Perifosine, an orally bioavailable AKT inhibitor, has clinical activity against RCC. We sought to determine whether treatment with perifosine and an mTOR-inhibitor (rapamycin) would synergistically inhibit RCC in preclinical models. Methods: Four clear cell RCC cell lines were studied: A498, CAKI-1, 769-P, and 786-O. Dose curves of single-agent and combination perifosine + rapamycin were performed by MTT. Combination studies were designed for median-effect. Status of pAKT (S473), pmTOR (S2481) and HIF-2alpha was assessed by Western blot. Cell lines had low basal levels of pAKT and VHL, moderate levels of pmTOR and HIF-2alpha, and no detectable HIF-1alpha. Results: CAKI-1, 769-P, and 786-O were sensitive to perifosine with an IC50 between 5–10 uM, while the IC50 of A498 was > 20 uM. Growth inhibition by rapamycin plateaued by 0.5–1 nM with none of the lines achieving an IC50. Combination treatments were essentially additive at lower doses of perifosine, becoming sub-additive or antagonistic at higher doses. Perifosine blocked phosphorylation of AKT induced by rapamycin. As a single agent and in combination, perifosine inhibited HIF-2alpha expression in the 769-P and CAKI-1 cell lines. Conclusions: Perifosine has high single-agent activity in select RCC lines and can abrogate the induction of AKT phosphorylation mediated by mTOR inhibition. Perifosine + rapamycin was at best additive, suggesting that pAKT is not the sole mechanism of resistance. Studies to explore the roles of VHL and HIF in response to this combination are ongoing. A Southwest Oncology Group trial in RCC patients testing the rational combination of perifosine and the mTOR inhibitor temsirolimus is now under development. No significant financial relationships to disclose.

Isoflurane Preconditioning Decreases Myocardial Infarction in Rabbits via Up-regulation of Hypoxia Inducible Factor 1 That Is Mediated by Mammalian Target of Rapamycin

Volatile anesthetics are known to protect the heart against ischemia-reperfusion injury. The authors tested whether anesthetic preconditioning with isoflurane is mediated via activation of the transcription factor hypoxia inducible factor 1 (HIF-1) and evaluated the role of mammalian target of rapamycin signaling in this process. New Zealand White rabbits subjected to 40 min of regional myocardial ischemia, followed by 180 min of reperfusion, were assigned to the following groups: ischemia and reperfusion (I/R) only, isoflurane (1 minimal alveolar concentration) preconditioning, and isoflurane preconditioning in the presence of the mammalian target of rapamycin inhibitor rapamycin (0.25 mg/kg). Sham-operated, isoflurane + sham, rapamycin + sham, rapamycin + I/R, and dimethyl sulfoxide + I/R groups were also included. Creatine kinase-MB levels were assessed as an indicator of myocardial damage, and infarct size was evaluated by triphenyl tetrazolium chloride staining. HIF-1alpha expression and DNA binding were assessed by Western blotting and electrophoretic mobility shift analysis, respectively. Isoflurane preconditioning reduced infarct size compared with the I/R group: 26 +/- 4% versus 44 +/- 6% (P < 0.05). Creatine kinase-MB concentrations in the preconditioned animals (103 +/- 8% above baseline) were lower than in the I/R group (243 +/- 12% above baseline; P < 0.05). Rapamycin inhibited the cardioprotective effect of isoflurane: myocardial infarction increased to 44 +/- 4% and creatine kinase-MB level increased to 254 +/- 9% above baseline. HIF-1alpha protein expression and DNA binding activity increased after isoflurane preconditioning compared with the ischemia group. These effects were also inhibited by rapamycin. The current results indicate that isoflurane-induced myocardial protection involves activation of the HIF-1 pathway that is mediated by the mammalian target of rapamycin.

A new mouse model of Peyronie's disease: An increased expression of hypoxia-inducible factor-1 target genes during the development of penile changes

Peyronie's disease (PD) is characterized by an inflammatory response beneath the tunica albuginea with fibroblast proliferation forming a thickened fibrous plaque that may cause pain, penile curvature and erectile dysfunction. The progression of the PD plaque may eventually lead to calcification or ossification. Current therapeutic success is often unsatisfactory because of limited insight into disease mechanisms. Research has been hampered by the lack of a universally accepted animal model. We describe an animal model of spontaneous PD in tight skin ( Tsk) mice , a C57Bl/6J subline that reproduces with age important features of the human disease (fibrous plaque formation, penile bending and areas of chondroid metaplasia with heterotopic ossification). Histological analysis demonstrated an evident structural disorganization of the tunica albuginea with excessive accumulation of type I collagen. At 12 months of age, fibrous plaques with areas of chondroid metaplasia and heterotopic ossification characterized Tsk penises. The up-regulation of hypoxia-inducible factor-1 (HIF-1) leads to an increased downstream expression of HIF-1 target genes, such as TGFβ and iNOS. These factors, together with some PDGF family members, can cause collagen deposition in Tsk penises. They can also influence chondrocyte differentiation and heterotopic bone formation. In conclusion, hypoxia, HIF-1 and HIF-1 target genes appear to play an important role in the pathogenesis of PD in Tsk mice. This mouse model that is the first example of naturally occurring model of PD in laboratory animals may aid in the identification of signalling pathways crucial for PD and should facilitate the designing and testing of new therapeutic interventions.

Renal Parenchymal Hypoxia, Hypoxia Adaptation, and the Pathogenesis of Radiocontrast Nephropathy

Renal parenchymal Po(2) declines after the administration of iodinated radiocontrast agents, reaching critically low levels of approximately 10 mmHg in medullary structures. In this review, the causes of renal parenchymal hypoxia and its potential role in the pathogenesis of contrast nephropathy are appraised. Commonly associated predisposing factors are associated with a propensity to enhance renal hypoxia. Indeed, animal models of radiocontrast nephropathy require the induction of such predisposing factors, mimicking clinical scenarios that lead to contrast nephropathy in high-risk individuals. In these models, in association with medullary hypoxic damage, a transient local cellular hypoxia response is noted, initiated at least in part by hypoxia-inducible factors. Some predisposing conditions that are distinguished by chronically aggravated medullary hypoxia, such as tubulointerstitial disease and diabetes, are characterized by a priori upregulation of hypoxia-inducible factors, which seems to confer tolerance against radiocontrast-related hypoxic tubular damage. Renal dysfunction under such circumstances likely reflects to some extent altered intrarenal hemodynamics, rather than acute tubular injury. Real-time, noninvasive novel methods may help to differentiate between evolving tubular damage and altered hemodynamics and in the design of appropriate preventive interventions.