Inhibition Of Hypoxia-inducible Factor Research Articles

Oxidative Stress Induces a VEGF Autocrine Loop in the Retina: Relevance for Diabetic Retinopathy.

Background: Oxidative stress (OS) plays a central role in diabetic retinopathy (DR), triggering expression and release of vascular endothelial growth factor (VEGF), the increase of which leads to deleterious vascular changes. We tested the hypothesis that OS-stimulated VEGF induces its own expression with an autocrine mechanism. Methods: MIO-M1 cells and ex vivo mouse retinal explants were treated with OS, with exogenous VEGF or with conditioned media (CM) from OS-stressed cultures. Results: Both in MIO-M1 cells and in retinal explants, OS or exogenous VEGF induced a significant increase of VEGF mRNA, which was abolished by VEGF receptor 2 (VEGFR-2) inhibition. OS also caused VEGF release. In MIO-M1 cells, CM induced VEGF expression, which was abolished by a VEGFR-2 inhibitor. Moreover, the OS-induced increase of VEGF mRNA was abolished by a nuclear factor erythroid 2-related factor 2 (Nrf2) blocker, while the effect of exo-VEGF resulted Nrf2-independent. Finally, both the exo-VEGF- and the OS-induced increase of VEGF expression were blocked by a hypoxia-inducible factor-1 inhibitor. Conclusions: These results are consistent with the existence of a retinal VEGF autocrine loop triggered by OS. This mechanism may significantly contribute to the maintenance of elevated VEGF levels and therefore it may be of central importance for the onset and development of DR.

Overexpression of bHLH domain of HIF-1 failed to inhibit the HIF-1 transcriptional activity in hypoxia

BackgroundHypoxia inducible factor-1 (HIF-1) is considered as the most activated transcriptional factor in response to low oxygen level or hypoxia. HIF-1 binds the hypoxia response element (HRE) sequence in the promoter of different genes, mainly through the bHLH domain and activates the transcription of genes, especially those involved in angiogenesis and EMT. Considering the critical role of bHLH in binding HIF-1 to the HRE sequence, we hypothesized that bHLH could be a promising candidate to be targeted in hypoxia condition.MethodsWe inserted an inhibitory bHLH (ibHLH) domain in a pIRES2-EGFP vector and transfected HEK293T cells with either the control vector or the designed construct. The ibHLH domain consisted of bHLH domains of both HIF-1a and Arnt, capable of competing with HIF-1 in binding to HRE sequences. The transfected cells were then treated with 200 µM of cobalt chloride (CoCl2) for 48 h to induce hypoxia. Real-time PCR and western blot were performed to evaluate the effect of ibHLH on the genes and proteins involved in angiogenesis and EMT.ResultsHypoxia was successfully induced in the HEK293T cell line as the gene expression of VEGF, vimentin, and β-catenin were significantly increased after treatment of untransfected HEK293T cells with 200 µM CoCl2. The gene expression of VEGF, vimentin, and β-catenin and protein level of β-catenin were significantly decreased in the cells transfected with either control or ibHLH vectors in hypoxia. However, ibHLH failed to be effective on these genes and the protein level of β-catenin, when compared to the control vector. We also observed that overexpression of ibHLH had more inhibitory effect on gene and protein expression of N-cadherin compared to the control vector. However, it was not statistically significant.ConclusionbHLH has been reported to be an important domain involved in the DNA binding activity of HIF. However, we found that targeting this domain is not sufficient to inhibit the endogenous HIF-1 transcriptional activity. Further studies about the function of critical domains of HIF-1 are necessary for developing a specific HIF-1 inhibitor.

Bone Marrow Mesenchymal Stem Cell-Derived Exosomes Attenuate LPS-Induced ARDS by Modulating Macrophage Polarization Through Inhibiting Glycolysis in Macrophages.

Macrophages play a key role in the development of sepsis-induced acute respiratory distress syndrome (ARDS). Recent evidence has proved that glycolysis plays an important role in regulating macrophage polarization through metabolic reprogramming. Bone marrow mesenchymal stem cells (BMSCs) can alleviate sepsis-induced lung injury and possess potent immunomodulatory and immunosuppressive properties via secreting exosomes. However, it is unknown whether BMSCs-derived exosomes exert their therapeutic effect against sepsis-induced lung injury by inhibiting glycolysis in macrophages. Therefore, the present study aimed to evaluate the anti-inflammatory effects of exosomes released from BMSCs on acute lung injury induced by lipopolysaccharide (LPS) in mice and explored the possible underlying mechanisms in vitro and in vivo. We found that BMSCs inhibited M1 polarization and promoted M2 polarization in MH-S cells (a murine alveolar macrophage cell line) by releasing exosomes. Further experiments showed that exosomes secreted by BMSCs modulated LPS-treated MH-S cells polarization by inhibiting cellular glycolysis. Moreover, our results showed that BMSCs-derived exosomes down-regulated the expression of several essential proteins of glycolysis via inhibition of hypoxia-inducible factor 1 (HIF-1)α. Finally, a model of LPS-induced ARDS in mice was established, we found that BMSCs-derived exosomes ameliorated the LPS-induced inflammation and lung pathological damage. Meanwhile, we found that intratracheal delivery of BMSCs-derived exosomes effectively down-regulated LPS-induced glycolysis in mice lung tissue. These findings reveal new mechanisms of BMSCs-derived exosomes in regulating macrophage polarization which may provide novel strategies for the prevention and treatment of LPS-induced ARDS.

Vitamin C decreases VEGF expression levels via hypoxia‑inducible factor‑1α dependent and independent pathways in lens epithelial cells.

Posterior capsular opacification (PCO) is the main complication following cataract surgery. The proliferation of the residual lens epithelial cells (LECs) serves an important role in PCO formation. The authors' previous study revealed that vitamin C inhibited the proliferation of human LECs by increasing the rapid degradation of hypoxia-inducible factor-1 (HIF-1α), and hence inhibited the expression of vascular endothelial growth factor (VEGF). The present study aimed to further investigate the mechanisms underlying the effects of vitamin C on the expression levels of VEGF. The present study demonstrated that the HIF-1 inhibitor BAY 87–2243 significantly inhibited the cell proliferation and the expression levels of VEGF in LECs through the use of colony formation, western blotting and ELISA assays. Moreover, it was revealed that vitamin C could further inhibit the cell proliferation and the expression levels of VEGF in LECs following the cotreatment with the HIF-1 inhibitor. The proline hydroxylation of HIF-1α by prolyl hydroxylases (PHDs) was previously discovered to be responsible for the rapid degradation of HIF-1α. Thus, the present study subsequently used three PHD inhibitors to investigate their effects on the expression levels of VEGF; it was found that the PHD2 specific inhibitor increased the expression levels of VEGF to the greatest extent. Moreover, the genetic knockdown of PHD2 by lentiviral transfection also significantly increased the expression levels of VEGF, whereas the PHD2 specific inhibitor did not alter the expression levels of VEGF in the PHD2 knockdown LECs. AKT kinase activity is an important mediator known to upregulate VEGF expression. Using an immunoprecipitation assay to isolate endogenous AKT, it was demonstrated that AKT was prolyl hydroxylated by PHD2, which inhibited its activity. It was also revealed that vitamin C enhanced the proline-hydroxylation and inhibited the activity of AKT. Furthermore, an AKT inhibitor increased the effects of vitamin C on the expression levels of VEGF. However, the AKT inhibitor did not affect the expression levels of glucose transporter 1, which is a HIF-1α target gene. In conclusion, the findings of the present study suggested that vitamin C may inhibit the expression levels of VEGF via HIF-1α-dependent and AKT-dependent pathways in LECs.

Hypoxia-Inducible Factor Inhibitors Derived from Marine Products Suppress a Murine Model of Neovascular Retinopathy.

Neovascular retinal degenerative diseases are the leading causes of blindness in developed countries. Anti-vascular endothelial growth factor (VEGF) therapy is commonly used to treat these diseases currently. However, recent reports indicate that long term suppression of VEGF in the eye is associated with chorioretinal atrophy. Therefore, a physiological amount of VEGF is required for retinal homeostasis. Hypoxia-inducible factor (HIF) is a transcriptional factor upstream of VEGF. We previously reported that HIF regulated pathological angiogenesis in the retina of murine models of oxygen-induced retinopathy and laser-induced choroidal neovascularization. Most of the known HIF inhibitors are anti-cancer agents which may have systemic adverse effects in for clinical use; thus, there is a need for safer and less invasive HIF inhibitors. In this study, we screened marine products, especially fish ingredients, and found that six species of fish had HIF inhibitory effects. Among them, administration of Decapterus tabl ingredients significantly suppressed retinal neovascular tufts by inhibiting HIF expression in a murine oxygen-induced retinopathy model. These results indicate that particular fish ingredients can act as anti-angiogenic agents in retinal neovascularization diseases.

Antidepressant-like action of hypoxic postconditioning is accompanied by the up-regulation of hippocampal HIF-1α and erythropoietin

Objective. The aim of the study was to reveal the role of HIF-1 in the effects of hypoxic postconditioning in the rat experimental model of depression learned helplessness.
 Materials and methods. The studies were performed in the learned helplessness paradigm which represents a reliable experimental model of depression in rats. The development of the pathology was evaluated in the behavioral open field test and by the baseline level of plasma corticosterone. Correction of behavioral deficit was performed by three episodes of hypoxic postconditioning (360 mmHg, 2 h). Changes in the immunopositivity of HIF-1 and erythropoietin in the hippocampus of rats were evaluated. An inhibitor of HIF-1 subunit translation topotecan (1 mg/kg, i.p., Santa Cruz, USA) was used on the 4th day after the footshock stress. On the 9th day, animals were tested in the open field test to assess the level of depressive-like behavior.
 Results. It was shown that postconditioning by three episodes of mild hypobaric hypoxia resulted in the correction of behavioral deficit produced by the learned helplessness aversive stress, and the levels of corticosterone did not differ from the baseline in these animals. These behavioral and hormonal effects were accompanied by the increased level of immunopositive HIF-1 and its transcriptional downstream target erythropoietin in the dorsal and ventral hippocampus. Using of HIF-1 inhibitor topotecan dramatically worsen the severity of the depressive-like symptoms.
 Conclusion. The findings suggest that HIF-1 appears to have the antidepressant-like activities and that hypoxic postconditioning-induced stimulation of HIF-1 and erythropoietin level might contribute to the endogenous mechanisms which compensate for the pathogenic effects of stressors, particularly for the development of stress-induced depression.

Hypoxia Inducible Factor-1α Inhibition in Von Hippel Lindau-mutant Malignant Pleural Mesothelioma Cells.

Molecular targeted agents have been successfully developed against solid tumors and their use is also being investigated for the treatment of malignant pleural mesothelioma (MPM). We have previously reported von Hippel Lindau (VHL) mutations detected by massive parallel sequencing technology in samples of patients with MPM. Here, we conducted an in vitro study to investigate the therapeutic approaches in VHL-mutant MPM. Three MPM cell lines with or without a VHL mutation were used and the effects of molecular-targeted agents on growth inhibition were evaluated. Based on the characteristics of the molecular targeted agents that exhibited growth inhibitory effect, the effects of knockdown by siRNA were also evaluated. NCI-H28 MPM cells harboring the VHL L89H mutation were sensitive to YC-1, known as an inhibitor of hypoxia inducible factor (HIF)-1α, and YC-1treatment induced massive apoptosis in a dose-and-time-dependent manner. Knockdown of HIF-1α by siRNA partially inhibited the growth of NCI-H28 cells, suggesting that an additional blockade may be required to completely inhibit growth signaling. The VHL mutation may predict tumor responses to YC-1, a HIF-1α inhibitor.

Design, Synthesis, and Conformation-Activity Study of Unnatural Bridged Bicyclic Depsipeptides as Highly Potent Hypoxia Inducible Factor-1 Inhibitors and Antitumor Agents.

By carrying out structural modifications based on the bicyclic peptide structure of echinomycin, we successfully synthesized various powerful antitumor derivatives. The ring conformation in the obtained compounds was restricted by cross-linking with an unnatural bond. The prepared derivatives were demonstrated to strongly suppress the hypoxia inducible factor (HIF)-1 transcriptional activation and hypoxia induction of HIF-1 protein expression. Particularly, alkene-bridged derivative 12 exhibited remarkably potent cytotoxicity (IC50 = 0.22 nM on the MCF-7 cell line) and HIF-1 inhibition (IC50 = 0.09 nM), which considerably exceeded those of echinomycin. Conformational analyses and molecular modeling studies revealed that the biological activities were enhanced following restriction of the conformation by cross-linking through a metabolically stable and rigid bridge bond. In addition, we proposed a new globular conformation stabilized by intramolecular π stacking that can contribute to the biological effects of bicyclic depsipeptides. The developments presented in the current study serve as a useful guide to expand the chemical space of peptides in drug discovery.

Marked and rapid effects of pharmacological HIF-2α antagonism on hypoxic ventilatory control.

Hypoxia-inducible factor (HIF) is strikingly upregulated in many types of cancer, and there is great interest in applying inhibitors of HIF as anticancer therapeutics. The most advanced of these are small molecules that target the HIF-2 isoform through binding the PAS-B domain of HIF-2α. These molecules are undergoing clinical trials with promising results in renal and other cancers where HIF-2 is considered to be driving growth. Nevertheless, a central question remains as to whether such inhibitors affect physiological responses to hypoxia at relevant doses. Here, we show that pharmacological HIF-2α inhibition with PT2385, at doses similar to those reported to inhibit tumor growth, rapidly impaired ventilatory responses to hypoxia, abrogating both ventilatory acclimatization and carotid body cell proliferative responses to sustained hypoxia. Mice carrying a HIF-2α PAS-B S305M mutation that disrupts PT2385 binding, but not dimerization with HIF-1β, did not respond to PT2385, indicating that these effects are on-target. Furthermore, the finding of a hypomorphic ventilatory phenotype in untreated HIF-2α S305M mutant mice suggests a function for the HIF-2α PAS-B domain beyond heterodimerization with HIF-1β. Although PT2385 was well tolerated, the findings indicate the need for caution in patients who are dependent on hypoxic ventilatory drive.

Hypoxia-Inducible Factor 1α and 2α Have Beneficial Effects in Remote Ischemic Preconditioning Against Stroke by Modulating Inflammatory Responses in Aged Rats

Limb remote ischemic preconditioning (RIPC) has been proven to alleviate stroke injury in young rats, but its protective effect and its mechanism in aged rats are still unclear. Hypoxia-inducible factor (HIF) is one of the important markers of stroke, and its high expression plays an important role in the pathogenesis of stroke. In this study, we tested the hypothesis that RIPC could regulate the expression of HIF, leading to reduced inflammatory responses in aged rats. Stroke was induced by transient middle cerebral artery occlusion (MCAo) in aged rats, and RIPC was conducted in both hind limbs. The HIF-1α and HIF-2α mRNA and protein were examined by real-time RT-PCR and western blotting (WB). Inflammatory cytokines in the peripheral blood and brain were measured using AimPlex multiplex immunoassays. The protein levels of p-Akt, Akt, p-ERK, and ERK were examined by WB. We investigated that RIPC reduced the infarct size, improved neurological functions, and decreased the expression of HIF-1α and HIF-2α in the ischemic brain. RIPC reduced the levels of IL-1β, IL-6 and IFN-γ in the peripheral blood and the levels of IL-1β and IFN-γ in the ischemic brain 48 h post-stroke. Moreover, intraperitoneal injection of the HIF inhibitor, acriflavine hydrochloride (ACF), abolished the protection of RIPC with respect to infarct size and neurological functions and neutralized the downregulation of pro-inflammatory IL-1β, IL-6 and IFN-γ. ACF also reversed the activation of the Akt signaling pathway induced by RIPC following stroke. HIF may play a key role in RIPC, which was likely mediated by the Akt signaling pathway and systemic modulation of the inflammatory response in aged rats.

Lactoferrin Has a Therapeutic Effect via HIF Inhibition in a Murine Model of Choroidal Neovascularization.

BackgroundLactoferrin, a type of glycoprotein, is contained in exocrine fluids such as tears, breast milk, sweat, and saliva, and is known to have anti-microbial, antioxidant, and anti-cancer effects. In the ophthalmological field, topical administration of lactoferrin has been reported to have a therapeutic effect in a murine dry eye model. Hypoxia-inducible factor (HIF) regulates various gene expressions under hypoxia, including vascular endothelial growth factor (VEGF), and is considered as an alternative target for neovascular ocular diseases such as age-related macular degeneration (AMD). We previously screened natural products and identified lactoferrin as a novel HIF inhibitor. In this study, we confirmed that lactoferrin has an HIF inhibitory effect and a therapeutic effect in a murine model of neovascular AMD.MethodsHIF inhibitory effects of lactoferrin were evaluated using a luciferase assay and western blotting in vitro. The quantified volume of choroidal neovascularization (CNV) induced by laser irradiation was compared with oral lactoferrin administration or conditional tissue specific Hif1a knockout mice.ResultsLactoferrin administration showed a significant HIF inhibitory effect in the retinal neuronal cells. Oral administration of lactoferrin or conditional Hif1a gene deletion significantly reduced CNV volume compared to controls.ConclusionsLactoferrin has a therapeutic effect in a laser CNV model by suppressing the retinal HIF activity.

Downregulation of Hypoxia-Inducible Factor-1α by RNA Interference Alleviates the Development of Collagen-Induced Arthritis in Rats

Rheumatoid arthritis (RA) is the most common type of autoimmune arthritis. Hypoxia-inducible factor-1α (HIF-1α) as a transcription factor in response to hypoxia suggests that it could be a potential therapeutic target for the treatment of RA. In this study, we assessed whether the HIF pathway blockade attenuates the manifestations of RA in the collagen-induced arthritis (CIA) rat model. We constructed a short hairpin RNA (shRNA) lentiviral expression vector targeting HIF-1α (pLVX-shRNA-HIF-1α) and to achieve HIF-1α RNA interference. Quantitative RT-PCR, immunofluorescence staining, and western blot were used to detect the expressions of HIF-1α, vascular endothelial growth factor (VEGF), phsopho (p)-p65, and p-IКBɑ mRNA and protein, respectively. Micro-computed tomography was used to investigate joint morphology at different time points after CIA induction. Moreover, enzyme-linked immunosorbent assay (ELISA) was used to monitor the expression of inflammatory cytokines. In vitro analyses revealed that pLVX-shRNA-HIF-1α effectively inhibited the expression of HIF-1α and VEGF and led to the activation of p-65 and p-IКBɑ, as well as decreased proinflammatory cytokine expression in cell culture. Inhibition of HIF-1α in rats decreased signs of a systemic inflammatory condition, together with decreased pathological changes of RA. Moreover, downregulation of HIF-1α expression markedly reduced the synovitis and angiogenesis. In conclusion, we have shown that pharmacological inhibition of HIF-1 may improve the clinical manifestations of RA.

The HIF1αn gene and its association with hypoxia tolerance in the Asian seabass

Hypoxia is one of the major challenges in aquaculture industry. Breeding of fish tolerant to hypoxia is an important task in genetic improvement of aquaculture species. The Asian seabass, Lates calcarifer, is an important foodfish species. We identified and characterized the hypoxia-inducible factor inhibitor (HIF1αn) gene in the Asian seabass. The full-length cDNA sequence of the HIF1αn was 3425 bp, with an ORF of 1065 bp, encoding 354 amino acids. The genomic sequence of the gene was 8667 bp in length, and contained eight exons and seven introns. Phylogenetic analysis of the gene in fish and tetrapods revealed that the HIF1αn in the Asian seabass was closely related to that of tilapia (Oreochromis niloticus). The HIF1αn was highly up-regulated in the gill, spleen and heart after 3.5-hours hypoxia treatment. We identified three SNPs in the third and fourth introns of the HIF1αn gene. The SNP (i.e. SNP 9332241 (C/T)) in the fourth intron was significantly (P < 0.01) associated with hypoxia tolerance. This SNP might be useful in selecting Asian seabass for improved tolerance to hypoxia. Our data also provide useful information for further detailed analysis of the function of the HIF1αn gene in hypoxia tolerance.

Pharmacological HIF1 Inhibition Eliminates Downregulation of the Pentose Phosphate Pathway and Prevents Neuronal Apoptosis in Rat Hippocampus Caused by Severe Hypoxia.

The pentose phosphate pathway (PPP) of glucose metabolism in the brain serves as a primary source of NADPH which in turn plays a crucial role in multiple cellular processes, including maintenance of redox homeostasis and antioxidant defense. In our model of protective mild hypobaric hypoxia in rats (3MHH), an inverse correlation between hypoxia-inducible factor-1 (HIF1) activity and mRNA levels of glucose-6-phosphate dehydrogenase (G6PD), the key enzyme of PPP, was observed. In the present study, it was demonstrated that severe hypobaric hypoxia (SH) induced short-term upregulation of HIF1 alpha-subunit (HIF1α) in the hippocampal CA1 subfield and decreased the activity of G6PD. The levels of NADPH were also reduced, promoting oxidative stress, triggering apoptosis, and neuronal loss. Injection of a HIF1 inhibitor (HIF1i), topotecan hydrochloride (5mg/kg, i.p.), before SH prevented the upregulation of HIF1α and normalized G6PD activity. In addition, HIF1i injection caused an increase in NADPH levels, normalization of total glutathione levels and of the cellular redox status as well as suppression of free-radical and apoptotic processes. These results demonstrate a new molecular mechanism of post-hypoxic cerebral pathology development which involves HIF1-dependent PPP depletion and support a recently suggested injurious role of HIF1 activation in the acute phase of cerebral hypoxia/ischemia. Application of PPP stimulators in early post-hypoxic/ischemic period might represent a promising neuroprotective strategy. Graphical abstract HIF1-dependent down-regulation of the pentose phosphate pathway contributes to the hypoxia-induced oxidative stress and neuronal apoptosis in the rat hippocampus.

Possible Contribution of Inflammation-Associated Hypoxia to Increased K2P5.1 K+ Channel Expression in CD4+ T cells of the Mouse Model for Inflammatory Bowel Disease.

Previous studies have reported the up-regulation of the two-pore domain K+ channel K2P5.1 in the CD4+ T cells of patients with multiple sclerosis (MS) and rheumatoid arthritis (RA), as well as in a mouse model of inflammatory bowel disease (IBD). However, the mechanisms underlying this up-regulation remain unclear. Inflammation-associated hypoxia is involved in the pathogenesis of autoimmune diseases, such as IBD, MS, and RA, and T cells are exposed to a hypoxic environment during their recruitment from inflamed tissues to secondary lymphoid tissues. We herein investigated whether inflammation-associated hypoxia is attributable to the increased expression and activity of K2P5.1 in the splenic CD4+ T cells of chemically-induced IBD model mice. Significant increases in hypoxia-inducible factor (HIF)-1α transcripts and proteins were found in the splenic CD4+ T cells of the IBD model. In the activated splenic CD4+ T cells, hypoxia (1.5% O2) increased K2P5.1 expression and activity, whereas a treatment with the HIF inhibitor FM19G11 but not the selective HIF-2 inhibitor exerted the opposite effect. Hypoxia-exposed K2P5.1 up-regulation was also detected in stimulated thymocytes and the mouse T-cell line. The class III histone deacetylase sirtuin-1 (SIRT1) is a downstream molecule of HIF-1α signaling. We examined the effects of the SIRT1 inhibitor NCO-01 on K2P5.1 transcription in activated CD4+ T cells, and we found no significant effects on the K2P5.1 transcription. No acute compensatory responses of K2P3.1–K2P5.1 up-regulation were found in the CD4+ T cells of the IBD model and the hypoxia-exposed T cells. Collectively, these results suggest a mechanism for K2P5.1 up-regulation via HIF-1 in the CD4+ T cells of the IBD model.

EPO could be regulated by HIF-1 and promote osteogenesis and accelerate bone repair

Bone defects caused by many factors prompt further study of pathological process and restoration methods. This study was aimed to clarify the effect of erythropoietin on the repair of bone defect. We added the designated concentration of rhEPO to endothelial progenitor cells and marrow stromal cells, then detected its osteogenic and angiogenesis effects. The results showed that rhEPO promoted the proliferation of EPC and ST2 by promoting the mitosis without affecting cell apoptosis. The protein and mRNA levels of angiogenesis and osteogenic related factors exhibited higher expressions. Additionally, rhEPO encapsulated in PLGA scaffolds accelerated the new bone formation in rat calvaria bone defect model. Since the centre of bone defect was hypoxia environment, we cultured EPC and ST2 under hypoxia. SiRNA and an inhibitor of HIF-1 were used to interfere HIF-1, then the following changes of VEGF and EPO were detected. The results showed that all the factors were upregulated under the hypoxia environment. The expression of VEGF at protein and mRNA level decreased as HIF-1 was inhibited or interfered from 6 h, while the mRNA expression of EPO from 6 h and changed significantly at protein level from 12 h. Therefore, EPO is a promising factor for further studies.

Evidence for the Capability of Roxadustat (FG-4592), an Oral HIF Prolyl-Hydroxylase Inhibitor, to Perturb Membrane Ionic Currents: An Unidentified yet Important Action.

Roxadustat (FG-4592), an analog of 2-oxoglutarate, is an orally-administered, heterocyclic small molecule known to be an inhibitor of hypoxia inducible factor (HIF) prolyl hydroxylase. However, none of the studies have thus far thoroughly investigated its possible perturbations on membrane ion currents in endocrine or heart cells. In our studies, the whole-cell current recordings of the patch-clamp technique showed that the presence of roxadustat effectively and differentially suppressed the peak and late components of IK(DR) amplitude in response to membrane depolarization in pituitary tumor (GH3) cells with an IC50 value of 5.71 and 1.32 μM, respectively. The current inactivation of IK(DR) elicited by 10-sec membrane depolarization became raised in the presence of roxadustatt. When cells were exposed to either CoCl2 or deferoxamine (DFO), the IK(DR) elicited by membrane depolarization was not modified; however, nonactin, a K+-selective ionophore, in continued presence of roxadustat, attenuated roxadustat-mediated inhibition of the amplitude. The steady-state inactivation of IK(DR) could be constructed in the presence of roxadustat. Recovery of IK(DR) block by roxadustat (3 and 10 μM) could be fitted by a single exponential with 382 and 523 msec, respectively. The roxadustat addition slightly suppressed erg-mediated K+ or hyperpolarization-activated cation currents. This drug also decreased the peak amplitude of voltage-gated Na+ current with a slowing in inactivation rate of the current. Likewise, in H9c2 heart-derived cells, the addition of roxadustat suppressed IK(DR) amplitude in combination with the shortening in inactivation time course of the current. In high glucose-treated H9c2 cells, roxadustat-mediated inhibition of IK(DR) remained unchanged. Collectively, despite its suppression of HIF prolyl hydroxylase, inhibitory actions of roxadustat on different types of ionic currents possibly in a non-genomic fashion might provide another yet unidentified mechanism through which cellular functions are seriously perturbed, if similar findings occur in vivo.

Abstract TMIM-081: THE HYPOXIC TUMOR-MESOTHELIAL NICHE PROMOTES OVARIAN CANCER METASTASIS THROUGH COLLAGEN REMODELING

Abstract High grade serous ovarian cancer (HGSOC) is a leading cause of cancer related deaths among women. The primary cause of morbidity and mortality in HGSOC patients is metastasis to organs within the peritoneal cavity. Mesothelial cells line the surface of peritoneal organs and are a key stromal cell in HGSOC metastatic niche. However, the mechanisms by which mesothelial cells promote ovarian cancer peritoneal metastasis remain largely unknown. Here we demonstrate that tumor associated mesothelial cells promote tumor invasion by increasing collagen deposition and remodeling. Mechanistically, we demonstrate the tumor-mesothelial niche is hypoxic where both tumor cells and mesothelial cells express the hypoxia inducible factors HIF-1 and HIF-2 and their downstream target gene lysyl oxidase (LOX). LOX is an enzyme that crosslinks collagen fibrils to promote collagen remodeling. In tumor-mesothelial co-culture experiments, we demonstrate that hypoxia enhances extracellular fibrillar collagen deposition by mesothelial cells. Conditioned media from hypoxic tumor-mesothelial co-cultures promotes collagen remodeling and HGSOC tumor cell invasion. Genetic inactivation of either HIF-1 and HIF-2 or LOX reduces the hypoxic induction of collagen remodeling and tumor cell invasion. Importantly, pharmacologic inhibition of HIF-1 and HIF-2 with digoxin reduces the colonization of disseminated HGSOC cells to the omentum, the preferential migration site of peritoneal cancer metastasis. Moreover, pharmacologic inhibition of LOX with BAPN (beta-aminopropionitrile) is sufficient to inhibit metastatic tumor burden and collagen remodeling at metastatic sites in preclinical models of HGSOC metastasis. These data reveal a novel role for mesothelial cells in the production of type I collagen and collagen remodeling in the HGSOC metastatic microenvironment. Furthermore, these studies demonstrate a role for the HIF/LOX signaling axis in the HGSOC tumor-mesothelial niche that can be therapeutically targeted to inhibit collagen remodeling and ovarian cancer metastatic progression. Citation Format: Suchitra Natarajan, Katie Foreman, Michaela Soriano, Hussein Shehade, Daniel Fregoso, Joshua Eggold, Ninna S. Rosen, Sarah Heilshorn, Adam J. Krieg, Venkatesh Krishnan, Oliver Dorigo, Subarna Sinha, Katherine C. Fuh, Erinn B. Rankin. THE HYPOXIC TUMOR-MESOTHELIAL NICHE PROMOTES OVARIAN CANCER METASTASIS THROUGH COLLAGEN REMODELING [abstract]. In: Proceedings of the 12th Biennial Ovarian Cancer Research Symposium; Sep 13-15, 2018; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2019;25(22 Suppl):Abstract nr TMIM-081.

Hypoxia Inducible Factors' Transcriptional Activity in Polycythemia Vera

Polycythemia vera (PV), a myeloproliferative neoplasms (MPN), is characterized by high red cell mass due to excessive erythrocytosis and by clonal proliferation of myeloid cells. The major PV complication, increased risk of thrombosis, is also seen in Chuvash erythrocytosis (CE), a disorder of upregulated hypoxia sensing. Hypoxia inducible factor (HIF)-1 and HIF-2 are the principle regulators of the hypoxic response by regulating the transcription of hypoxia responsive genes. We previously showed that MPN patients have upregulation of some HIF target genes in granulocytes and platelets along with increased glucose uptake (Sana, et al., Blood 2013 122:1604). Further, stem cell factor and thrombopoietin signaling increase HIF-1 activity (Kirito et al, Blood 2005); however, the role of HIFs in PV is unknown. We evaluated HIF transcriptional activity in PV by unbiased RNA sequencing of JAK2V617F positive PV granulocytes (22 patients and 10 controls) and platelets (24 patients and 4 controls). Differential gene expression was determined by DEseq2 and data stratified by p&lt;0.05 and Log2fold change&gt;1. BaseSpace Correlation Engine (Illumina Inc.) was used to identify genes regulated by HIF. Fifty-seven HIF target genes were dysregulated in PV granulocytes. There were 44 upregulated genes involved in apoptosis, metabolism, cellular proliferation, intracellular signal transduction, and ion transport. Thirteen genes were downregulated in PV, all associated with Ras and Rap1 signaling. Thirty-five genes were correlated with JAK2V617F allele burden; 27 genes were positively correlated while 8 genes were inversely correlated. SOCS2, FLT1, PDLIM1, ALDH1A2, NPR3, and BATF3 were regulated by both STAT5 and HIF suggesting that HIFs and the JAK-STAT pathway may cross-talk. CAV1, PLOD2, NRIP3, NGFRAP1, and UBXN10 inversely correlated with the white blood cell counts. Eighty-seven HIF target genes were differentially expressed in PV platelets. Seventy-two genes were upregulated and 15 genes were downregulated. Upregulated genes were associated with apoptosis, proliferation, and metabolism. Unlike in PV granulocytes, some of these upregulated genes were associated with immune responses including the TNF and NF-kB signaling pathways; NF-kB and HIF-1 co-stimulate each other. Twenty genes positively correlated with JAK2V617F allele burden but BCL2 and PNOC inversely correlated. There were 13 genes regulated by both STAT5 and HIF. DOT1L, GYG1, HOXA1, PRELID1, and RPIA transcript levels inversely correlated with the platelet counts. In both granulocytes and platelets, 17 genes were dysregulated. CAV1, CHAD, and SOCS2 are associated with negative regulation of receptor signaling via JAK-STAT. CHAD was downregulated while CAV1 and SOCS2 were upregulated in both granulocytes and platelets. ALDH1A2, CAV1, KCNMA1, and MMP9 genes were upregulated and associated with apoptosis. We also observed an increased transcript of inflammatory genes in PV. PV granulocytes had 82 upregulated and 11 downregulated inflammatory genes. Among these upregulated genes, MGLL, PROK2, and MAP2K6 were also HIF target genes. In platelets, 34 genes were dysregulated (31 genes were upregulated and 3 genes were downregulated). It is of interest that 2 prothrombotic genes are reported to be regulated by HIFs, tissue factor encoded by F3 (Giannarelli, et al., Circulation 2014) and protein S (encoded by PROS1). We found F3 transcript to be upregulated also in PV and CE granulocytes. PROS1 was reported to be negatively regulated by HIF-1 as detected by decreased protein S in mouse plasma and by decreased PROS1mouse liver transcript and human cell line HepG2 (Pilli, et al., Blood 2018). We found PROS1 to be also expressed in human granulocytes and platelets; however, it was overexpressed in PV and CP granulocytes but not in platelets. We demonstrate here high HIF transcriptional activity in granulocytes and platelets of PV; some of these HIF-regulated genes are also associated with immune response genes. Dysregulated HIF target genes were involved in apoptosis and cell proliferation pathways. Most HIF target gene transcription correlated with JAK2V617F allele burden, platelet counts, and white blood cell counts. These results suggest that HIF might be a therapeutic target of PV. Of drugs in clinical use, digoxin is a potent HIF-1 inhibitor (Zhang, et al., PNAS 2008); its potential role in PV and CE is being explored. Disclosures Gordeuk: Inctye: Research Funding; CSL Behring: Consultancy, Honoraria, Research Funding; Ironwood: Research Funding; Imara: Research Funding; Pfizer: Research Funding; Modus Therapeutics: Consultancy, Honoraria; Novartis: Consultancy, Honoraria, Research Funding; Emmaus: Consultancy, Honoraria; Global Blood Therapeutics: Consultancy, Honoraria, Research Funding.

Vitexin, an inhibitor of hypoxia-inducible factor-1α, enhances the radiotherapy sensitization of hyperbaric oxygen on glioma.

Vitexin, an inhibitor of hypoxia-inducible factor (HIF)-1α, has anti-tumor effect. However, whether it can enhance the radiotherapy sensitization of hyperbaric oxygen (HBO) on glioma is unclear. This study aimed to investigate the effect of vitexin. The nude mice with paw-transplanted glioma were divided into four groups: control group, HBO + radiation group, HBO + vitexin group, and HBO + vitexin + radiation group. The mice of last two groups were daily given vitexin 75mg/kg by intraperitoneal injection. 30min after administration of vitexin, the HBO-treated mice were daily placed in HBO chamber for 60min. The radiation-treated mice were given local tumor irradiation once every week during the HBO treatment, and the dose of irradiation was 10Gy/time. The experimental treatment lasted for 21days. Compared with the HBO + radiation group, the tumor volume, tumor weight, and tumor weight coefficient in the HBO + vitexin + radiation group were lower (p < 0.05). Importantly, the contents of reduced glutathione and glutathione peroxidase as well as expressions of HIF-1α, vascular endothelial growth factor, glucose transporter (GLUT)-1, and GLUT-3 proteins in tumor tissues were also lower in the HBO + vitexin + radiation group than in the HBO + radiation group (p < 0.01). Vitexin can cooperate with HBO to sensitize the glioma radiotherapy, and its mechanisms may be correlated to the inhibition of HIF-1α protein expression and subsequent decrements of its downstream protein expressions, which finally cause the reduction of antioxidant capacity.