HIF-1alpha Protein Levels Research Articles

Prostaglandin E1 reduces apoptosis and improves the homing of mesenchymal stem cells in pulmonary arterial hypertension by regulating hypoxia-inducible factor 1 alpha

BackgroundPulmonary arterial hypertension (PAH) is associated with oxidative stress and affects the survival and homing of transplanted mesenchymal stem cells (MSCs) as well as cytokine secretion by the MSCs, thereby altering their therapeutic potential. In this study, we preconditioned the MSCs with prostaglandin E1 (PGE1) and performed in vitro and in vivo cell experiments to evaluate the therapeutic effects of MSCs in rats with PAH.MethodsWe studied the relationship between PGE1 and vascular endothelial growth factor (VEGF) secretion, B-cell lymphoma 2 (Bcl-2) expression, and C-X-C chemokine receptor 4 (CXCR4) expression in MSCs and MSC apoptosis as well as migration through the hypoxia-inducible factor (HIF) pathway in vitro. The experimental rats were randomly divided into five groups: (I) control group, (II) monocrotaline (MCT) group, (III) MCT + non-preconditioned (Non-PC) MSC group, (IV) MCT + PGE1-preconditioned (PGE1-PC) MSC group, and (V) MCT+PGE1+YC-1-PCMSC group. We studied methane dicarboxylic aldehyde (MDA) levels, MSC homing to rat lungs, mean pulmonary artery pressure, pulmonary artery systolic pressure, right ventricular hypertrophy index, wall thickness index (%WT), and relative wall area index (%WA) of rat pulmonary arterioles.ResultsPreconditioning with PGE1 increased the protein levels of HIF-1 alpha (HIF-1α) in MSCs, which can reduce MSC apoptosis and increase the protein levels of CXCR4, MSC migration, and vascular endothelial growth factor secretion. Upon injection with PGE1-PCMSCs, the pulmonary artery systolic pressure, mean pulmonary artery pressure, right ventricular hypertrophy index, %WT, and %WA decreased in rats with PAH. PGE1-PCMSCs exhibited better therapeutic effects than non-PCMSCs. Interestingly, lificiguat (YC-1), an inhibitor of the HIF pathway, blocked the effects of PGE1 preconditioning.ConclusionsOur findings indicate that PGE1 modulates the properties of MSCs by regulating the HIF pathway, providing insights into the mechanism by which PGE1 preconditioning can be used to improve the therapeutic potential of MSCs in PAH.

MP39-18 AN AUTOCHTHONOUS MOUSE MODEL OF KIDNEY CANCER WITH T CELL INFILTRATION

You have accessJournal of UrologyKidney Cancer: Basic Research & Pathophysiology I (MP39)1 Sep 2021MP39-18 AN AUTOCHTHONOUS MOUSE MODEL OF KIDNEY CANCER WITH T CELL INFILTRATION Nitika Sharma, Bo Xu, Jacky Chow, Jason Muhitch, and Eric Kauffman Nitika SharmaNitika Sharma More articles by this author , Bo XuBo Xu More articles by this author , Jacky ChowJacky Chow More articles by this author , Jason MuhitchJason Muhitch More articles by this author , and Eric KauffmanEric Kauffman More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000002054.18AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Immunocompetent animal models that recapitulate human renal cell carcinoma (RCC) and its tumor-immune microenvironment are critical for the preclinical study of novel immunotherapeutic strategies. Abundant CD8+ T cell lymphocyte infiltration is a defining feature of human clear cell RCC but is lacking from many mouse models. Iron is a known carcinogen with increased cellular levels in human RCC. In the FeNTA mouse model, chronic systemic administration of iron (Fe) with a chelator (nitrilotriacetic acid, NTA) induces renal carcinogenesis, however the tumor biology is largely uncharacterized. We studied whether FeNTA mouse model cancers have similar biology as human RCC, including an abundance of tumor-infiltrating lymphocytes. METHODS: 60 male mice (AJ strain) were treated for 12 weeks (5X/week) with intraperitoneal FeNTA injection to induce renal tumorigenesis. Renal tumors were harvested at 12-15 months post-treatment and underwent H&E histologic evaluation by a clinical genitourinary pathologist. Tumor lysates were analyzed for PAX8 and HIF2-alpha protein levels by Western Blot, while tumor single-cell suspensions were analyzed for immune cell antigens (CD45, CD3, CD4, CD8) by imaging flow cytometry to quantify tumor-infiltrating T cell lymphocyte populations. RESULTS: Of 60 FeNTA-treated mice, 14 (23.3%) developed renal tumors as confirmed by magnetic resonance imaging and histology. Mean tumor size was 1.5 cm (interquartile range 1.1-1.8 cm). Tumor histology was most similar to clinical clear cell RCC with eosinophilic cytoplasm. The clinical RCC biomarker, PAX8, was expressed in murine tumors, as was the HIF2-alpha protein that typifies human clear cell RCC. Infiltrating immune cells (CD45+) comprised on average 33.2% (+/- standard error, 4.6%) of all tumor cells, including 11.9% (+/- 3.9%) of tumor cells that were specifically T cell lymphocytes (CD45+/CD3+). Most tumor-infiltrating T cells were CD8+/CD4- (74.3+/- 3.7%), while the remainder were CD4+/CD8- (25.7+/- 3.7%). CONCLUSIONS: The FeNTA autochthonous immunocompetent mouse model recapitulates key clinical RCC biology, including a tumor-infiltrating T cell profile similar to that of the human disease. This model may provide a valuable resource for preclinical evaluation of novel immunotherapeutic strategies for RCC. Source of Funding: American Cancer Society © 2021 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 206Issue Supplement 3September 2021Page: e711-e712 Advertisement Copyright & Permissions© 2021 by American Urological Association Education and Research, Inc.MetricsAuthor Information Nitika Sharma More articles by this author Bo Xu More articles by this author Jacky Chow More articles by this author Jason Muhitch More articles by this author Eric Kauffman More articles by this author Expand All Advertisement Loading ...

Endogenous reactive oxygen species and nitric oxide have opposite roles in regulating HIF-1alpha expression in hypoxic astrocytes.

Ischemic stroke results in cerebral tissue hypoxia and increased expression of hypoxia-inducible factor (HIF), which is critically implicated in ischemic brain injury. Understanding the mechanisms of HIF-1alpha regulation in the ischemic brain has been an important research focus. The generation of both nitric oxide (NO) and reactive oxygen species (ROS) is increased under hypoxic/ischemic conditions and each of them has been independently shown to regulate HIF-1alpha expression. In this study, we investigated the cross-effects of NO and ROS on the expression of HIF-1alpha in hypoxic astrocytes. Exposure of astrocytes to 2 h-hypoxia remarkably increased HIF-1alpha protein levels, which was accompanied by increased NO and ROS production. Decreasing ROS with NAC, NADPH oxidase inhibitor DPI, or SOD mimetic MnTMPyP decreased hypoxia-induced HIF-1alpha protein accumulation and increased NO level in hypoxic astrocytes. The NO synthase (NOS) inhibitor L-NAME inhibited ROS generation, which led to a reduction in hypoxia-induced HIF-1alpha protein expression. Although NOS inhibitor or ROS scavengers alone reduced HIF-1alpha protein levels, the reduction was reversed when NOS inhibitor and ROS scavenger present together. The NO scavenger PTIO increased hypoxia-induced HIF-1alpha protein expression and ROS production, while HIF-1alpha protein level was decreased in the presence of NO scavenger and ROS scavenger together. These results suggest that ROS, NO, and their interaction critically contribute to the regulation of hypoxia-induced HIF-1alpha protein accumulation under hypoxic condition. Furthermore, our results indicate that hypoxia-induced NO generation may represent an endogenous mechanism for balancing ROS-mediated hypoxic stress, as reflected by inhibiting hypoxia-induced HIF-1alpha protein accumulation.

Vandetanib (ZD6474) induces antiangiogenesis through mTOR-HIF-1 alpha-VEGF signaling axis in breast cancer cells.

ObjectiveVandetanib, also known as ZD6474, has recently been proved to be a clinical drug for cancer by targeting vascular endothelial growth factor receptor 2 (VEGFR2), EGFR, and RET tyrosine kinases. We hypothesized that vandetanib will be a drug candidate for breast cancer treatment by targeting angiogenesis.Materials and methodsVandetanib was used to treat different breast cancer cell lines, and its effect on growth, apoptosis, and cell cycle was studied by MTT assay and flow cytometry. VEGF level in culture medium was measured by ELISA. Gene expression of mechanistic target of rapamycin (mTOR), hypoxia-inducible factor (HIF)-1 alpha, and VEGF at mRNA and protein level were analyzed by quantitative real-time-PCR and Western blot. The cellular behavior variations were investigated by using wound healing assay, transwell invasion assay, and tubular formation assay as well as experiments in vivo.ResultWe found that vandetanib can inhibit breast cancer cell line growth via apoptosis and cell cycle regulation. VEGF secretion decreases upon treatment. Vandetanib can reduce both mRNA and protein level of mTOR, HIF-1 alpha, and VEGF. Angiogenesis assays showed that vandetanib can inhibit wound healing, invasion, and tubular formation in culture. Furthermore, vandetanib inhibited the growth of breast tumor in vivo.ConclusionIn short, our study showed that vandetanib can control angiogenesis of breast cancer in culture via mTOR, HIF-1 alpha, and VEGF signaling pathway.

Bisphenol A and Its Derivatives Induce Degradation of HIF-1alpha via the Lysosomal Pathway in Human Hepatocarcinoma Cell Line, Hep3B.

Bisphenol A (BPA, 2,2-bis(4-hydroxyphenyl)propane), one of the phenolic compounds widely used in the manufacture of plastic and epoxy resins, is known as an endocrine disruptor. In a previous study, we found that BPA induced hypoxia inducible factor-1alpha (HIF-1alpha) degradation by dissociation from heat shock protein 90 (Hsp90). In this study, to investigate the structural requirements for degradation of HIF-1alpha, we estimated the effect of BPA derivatives (BPE, BPF, BPB, Dimethyl butylidene diphenol (DMBDP), Ethyl hexylidene diphenol (EHDP), Bishydroxyphenyl cyclohexane (BHCH), and Methyl benzylidene bisphenol (MBBP)) on HIF-1alpha protein degradation, using human hepatocarcinoma cell line, Hep3B. BPB, DMBDP, BHCH, and MBBP decreased HIF-1alpha protein levels more efficiently than BPA, but BPE, BPF, and EHDP did not affect HIF-1alpha protein levels. BPA degraded HIF-1alpha even in the presence of MG132, a proteasome inhibitor. In this study, we found that ammonium chloride (NH4Cl), a lysosomal enzyme inhibitor, efficiently restored the decrease in HIF-1alpha protein levels by BPA. Recent studies indicated that HIF-1alpha is degraded by the lysosomal pathway as well as the proteasomal pathway. Therefore, we investigated the levels of heat shock cognate 70 kDa protein (HSC70) protein after treatment with BPA. We found that BPA induced HSC70 protein and overexpression of HSC70 enhanced HIF-1alpha degradation in Hep3B cells. These results suggested that BPA causes the degradation of HIF-1alpha by induction of HSC70, leading lysosomal degradation of HIF-1alpha.

A hypoxia response element in the Vegfa promoter is required for basal Vegfa expression in skin and for optimal granulation tissue formation during wound healing in mice.

Hypoxia in skin wounds is thought to contribute to healing through the induction of hypoxia inducible factor-1 (HIF-1). Although HIF-1 can regulate the expression of vascular endothelial growth factor A (Vegfa), whether hypoxia and HIF-1 are required to induce Vegfa expression in the context of wound healing is unknown. To test this hypothesis, we evaluated Vegfa expression and wound healing in mutant mice that lack a functional HIF-1 binding site in the Vegfa promoter. Full-thickness excisional wounds were made using a biopsy punch, left to heal by second intention, and granulation tissue isolated on a time course during healing. mRNA levels of Vegfa and its target genes platelet-derived growth factors B (Pdgfb) and stromal cell-derived factor-1 (Sdf1) were measured by RT-qPCR, and HIF-1alpha and VEGFA protein levels measured by immunoblotting. Lower levels of Vegfa, Pdgf1 and Sdf1 mRNA were found in intact skin of mutant mice relative to wild-type controls (n = 6 mice/genotype), whereas levels in granulation tissue during wound healing were unaltered. VEGFA protein levels were also lower in intact skin of the mutant versus the wild-type mice. Decreased Vegfa mRNA levels in skin of mutant mice could not be attributed to decreased HIF-1alpha protein expression, and were therefore a consequence of the loss of HIF-1 responsiveness of the Vegfa promoter. Comparative histologic analyses of healing wounds in mutant and wild-type mice (n = 8 mice/genotype) revealed significant defects in granulation tissue in the mutant mice, both in terms of quantity and capillary density, although epithelialization and healing rates were unaltered. We conclude that HIF-1 is not a major regulator of Vegfa expression during wound healing; rather, it serves to maintain basal levels of expression of Vegfa and its target genes in intact skin, which are required for optimal granulation tissue formation in response to wounding.

Abstract 891: Annexin A2 (ANXA2) promotes migration and invasion of esophageal cancer cells via stabilizing c-Myc and promoting HIF-1α transcription

Abstract Purpose: We previously revealed that the expression of Annexin A2 (ANXA2) was increased in ESCC by two-dimensional electrophoresis and MALDI-TOF and LC-ESI-IT MS. This study aims to investigated the implication of Annexin A2 in ESCC. Annexin A2 (ANXA2) promotes migration and invasion of esophageal cancer cells via stabilizing c-Myc and promoting HIF-1α transcription. Experimental Design: Immunohistochemistry was performed to analyze the expression of ANXA2 in esophageal squamous cell carcinomas (ESCC). siRNA knockdown, transwell assay and wound-healing assays were used to investigate the function of ANXA2 in cells. Quantitative reverse-transcription polymerase chain reaction, Western blot analysis were used to seek the potential effector molecule of ANXA2. Chromatin immunoprecipitation, and reporter gene assays were used to confirm the relationship between c-Myc and HIF-1 alpha. Co-immunoprecipitation assay was performed to demonstrate the relationship between ANXA2 and c-Myc. Immunofluorescence was used to reveal the location of ANXA2. Results: Knockdown of Annexin A2 significantly inhibited the migration and invasion of ESCC cells. At the molecular level, VEGF, MMP-2, MMP-9, p-MEK and p-ERK were down-regulated in ANXA2-siRNA cells. The HIF-1 alpha mRNA and protein levels were significantly lowered after knocked down the Annexin A2. HIF-1 alpha is the effector molecule of ANXA2 in ESCC cells, and that ANXA2 affects the expression of HIF-1 alpha at the transcription level because when knocking down HIF-1 alpha, the level of VEGF, MMP-2, MMP-9, p-MEK and p-ERK reduced as well. c-Myc is a transcription factor for HIF-1 alpha. ANXA2 has a direct protein interaction with c-Myc and knockdown of ANXA2 down-regulated the expression of c-Myc. The Tyr 24 phosphorylation form of Annexin A2 (Y24D-ANXA2) was located in the nucleus, and the non-phosphorylation form (Y24A-ANXA2), in the cytoplasm and cell membrane. Transfection of pcDNA3.1-ANXA2-Y24D enhanced the migration and invasion as compared with that of pcDNA3.1-ANXA2-Y24A in esophageal cancer cells. A treatment with the Annexin A2 phosphorylation inhibitor, Dasatinib, changed the location of Annexin A2 and lowered the expression of c-myc and HIF-1 alpha. Conclusions: These results suggest that in esophageal cancer cells, phosphorylated Annexin A2 could enter into nucleus, which stabilizes c-myc and promotes the transcription of HIF-1 alpha, and the activates MAPK pathway, increases matrix metalloproteinase (MMP) activity and vascular endothelial growth factor (VEGF) expression, which results in the enhancement of cell invasion and migration. Citation Format: Sai Ma, Yan-Yi Jiang, Li-Fei Wu, Jia-Jie Hao, Yu Zhang, Xin Xu, Yan Cai, Ming-Rong Wang. Annexin A2 (ANXA2) promotes migration and invasion of esophageal cancer cells via stabilizing c-Myc and promoting HIF-1α transcription [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 891. doi:10.1158/1538-7445.AM2017-891

The stabilization of hypoxia inducible factor modulates differentiation status and inhibits the proliferation of mouse embryonic stem cells

Hypoxic conditions are suggested to affect the differentiation status of stem cells (SC), including embryonic stem cells (ESC). Hypoxia inducible factor (HIF) is one of the main intracellular molecules responsible for the cellular response to hypoxia. Hypoxia stabilizes HIF by inhibiting the activity of HIF prolyl-hydroxylases (PHD), which are responsible for targeting HIF-alpha subunits for proteosomal degradation. To address the impact of HIF stabilization on the maintenance of the stemness signature of mouse ESC (mESC), we tested the influence of the inhibition of PHDs and hypoxia (1% O2 and 5% O2) on spontaneous ESC differentiation triggered by leukemia inhibitory factor withdrawal for 24 and 48 h. The widely used panhydroxylase inhibitor dimethyloxaloylglycine (DMOG) and PHD inhibitor JNJ-42041935 (JNJ) with suggested higher specificity towards PHDs were employed. Both inhibitors and both levels of hypoxia significantly increased HIF-1alpha and HIF-2alpha protein levels and HIF transcriptional activity in spontaneously differentiating mESC. This was accompanied by significant downregulation of cell proliferation manifested by the complete inhibition of DNA synthesis and partial arrest in the S phase after 48 h. Further, HIF stabilization enhanced downregulation of the expressions of some pluripotency markers (OCT-4, NANOG, ZFP-42, TNAP) in spontaneously differentiating mESC. However, at the same time, there was also a significant decrease in the expression of some genes selected as markers of cell differentiation (e.g. SOX1, BRACH T, ELF5). In conclusion, the short term stabilization of HIF mediated by the PHD inhibitors JNJ and DMOG and hypoxia did not prevent the spontaneous loss of pluripotency markers in mESC. However, it significantly downregulated the proliferation of these cells.

Maternal omega-3 fatty acid supplementation to a vitamin B12 deficient diet normalizes angiogenic markers in the pup brain at birth

Vitamin B12 and omega-3 fatty acids are critical for normal brain development and function and their deficiencies during pregnancy could have adverse effects on cognitive performance in children. Our earlier studies indicate that both maternal vitamin B12 and omega-3 fatty acids influence brain development by regulating the levels of neurotrophins. Literature suggests that there exists a cross talk between neurotrophins like nerve growth factor (NGF) and angiogenic factors like vascular endothelial growth factor (VEGF). It remains to be established whether maternal nutrients like vitamin B12 and omega-3 fatty acids influence the levels of angiogenic markers like VEGF and NGF in the brain of the offspring. Therefore the present study examines the effect of maternal vitamin B12 and omega-3 fatty acids on protein and mRNA levels of VEGF, HIF-1 alpha (hypoxia inducible factor alpha) and NGF in the pup brain at birth. Pregnant Wistar rats were divided into five dietary groups (n=8 each): control, vitamin B12 deficient, vitamin B12 deficient+omega-3 fatty acid, vitamin B12 supplemented, vitamin B12 supplemented+omega-3 fatty acid. At birth the pups were dissected to collect the brain tissue. Maternal vitamin B12 deficiency showed lower (p<0.05) pup brain mRNA and protein levels (p<0.01) of VEGF, higher (p<0.01) HIF-1 alpha protein levels, lower (p<0.05) NGF protein levels while NGF mRNA levels were not altered. Omega-3 fatty acid supplementation to a vitamin B12 deficient group normalized the VEGF mRNA levels, NGF protein levels and HIF-1 alpha protein levels. Vitamin B12 supplementation showed similar protein and mRNA levels of VEGF and NGF as well as HIF-1 alpha protein levels as compared to control. Omega-3 fatty acid supplementation to the vitamin B12 supplemented group showed higher (p<0.01) protein and mRNA levels of NGF but the protein and mRNA levels of VEGF were comparable to control. In conclusion maternal vitamin B12 and omega-3 fatty acids both influence the levels and expression of neurotrophins and angiogenic factors in the offspring brain suggesting a possible benefit of combined maternal supplementation of these vital nutrients.

Abstract 727: Does gene transfer of gulono-lactone oxidase into human hepatocellular carcinoma cells restore ascorbate biosynthesis

Abstract Humans are unable to synthesise ascorbate (vitamin C) due to a mutation in the gene for gulono-lactone oxidase (Gulo), the terminal enzyme in the synthesis pathway. Ascorbate is a vital micronutrient required for many biological functions, including its action as a vital cofactor for Cu- and Fe-containing enzymes. As enzyme cofactor for HIF-hydroxylases, it is important for the regulation of hypoxia-inducible factor-1 (HIF-1), which governs cancer growth and spread. A lack of ascorbate increases both the level and activity of HIF-1 in cell culture. This study aimed to restore ascorbate synthesis to human cells and to determine the effect of intracellular ascorbate biosynthesis on HIF-1 levels. HepG2 cells were gene modified using Lipofectamine LTX with a plasmid encoding the mouse Gulo cDNA and a control plasmid encoding the green fluorescent protein (GFP). Modified HepG2 cells were tested for genomic incorporation by PCR, for Gulo enzyme production by Western blotting, and ascorbate synthesis by high performance liquid chromatography with electrochemical detection (HPLC-ECD). Protein levels of HIF-1alpha in hypoxic cells were measured by Western blot analysis. Parental HepG2 cells accumulated a mean of 120 nmol ascorbate per 1 million cells when loaded for 24h with 0.5 mM ascorbate. Gene-modified HepG2Gulo cells demonstrated a change in morphology and an increase in adherence, compared to HepG2GFP and parental HepG2 cells. A PCR-positive stable clone was able to synthesise ascorbate but only when the Gulo substrate, L-gulono-1,4-lactone, was supplied. Intracellular ascorbate levels then reached 5% of saturation levels. Hypoxic HIF-1 accumulation was reduced by addition of gulonolactone to this clone. However, gulonolactone also reduced HIF-1 levels in parental HepG2 cells, suggesting that it may similarly act as cofactor for the HIF-hydroxlases. This study indicated that the ascorbate biosynthesis pathway in human cells is significantly modified and may contain numerous non-functional members besides gulono-lactone oxidase. Future biochemical studies will determine which other enzymes are non-functional. Citation Format: Teresa Flett, Elizabeth Campbell, Elisabeth Phillips, Margreet CM Vissers, Gabi U. Dachs. Does gene transfer of gulono-lactone oxidase into human hepatocellular carcinoma cells restore ascorbate biosynthesis. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 727. doi:10.1158/1538-7445.AM2014-727

IDH1 p.R132 mutations may not be actively involved in the carcinogenesis of hepatocellular carcinoma

BackgroundRecent studies have identified prevalent isocitrate dehydrogenase 1 (IDH1) codon 132 mutations (p.R132) in gliomas and acute myeloid leukemia (AML). The IDH1 mutations lead to a loss of its normal enzymatic activity and acquisition of neomorphic activity in production of α-ketoglutarate (α-KG) and 2-hydroxyglutarate (2-HG), which finally cause alterations of multiple gene expression of tumorigenesis-associated α-KG-dependent enzymes. The aim of this study was to determine whether IDH1 p.R132 mutations are involved in the carcinogenesis of hepatocellular carcinoma.Material/MethodsA total of 87 Han Chinese patients with primary hepatocellular carcinoma (HCC) were analyzed by direct DNA sequencing for IDH1 p.R132 mutations. The expression levels of multiple α-KG-dependent enzymes and associated genes were quantified in HepG2 cells overexpressing IDH1 p.R132 mutants by Western blotting and real-time PCR.ResultsNone of 87 Han Chinese patients with HCC harbored any IDH1 p.R132 mutations. The protein levels of HIF-1α and histone methylation marker (H3K4me3 and H3K79me2) were determined in HepG2 cells overexpressing IDH1 p.R132 mutants, but we discerned no difference. Measurement of mRNA expression levels of VEGF, GLUT1, and HOXA genes also showed no significant difference between cells overexpressing IDH1 wild-type and p.R132 mutants.ConclusionsOur negative results, together with some previous reports of the absence of IDH1 p.R132 mutations in HCC tissues, suggests that IDH1 p.R132 mutations are not actively involved in the development of HCC.

Abstract 545: PLacental eXpanded (PLX) Cell Treatment Ameliorates Preeclampsia Induced by TLR3 or TLR7 Activation in Mice

PLacental eXpanded (PLX) cells (Pluristem Therapeutics Inc.) are human placenta-derived, mesenchymal-like adherent stromal cells that release proteins in response to the environment of the host. PLX cells are non-immunogenic and have been shown to decrease inflammation and increase angiogenesis in inflammatory and ischemic conditions. Therefore, we tested whether PLX cell treatment could attenuate symptoms of preeclampsia (PE) in mice. We hypothesized that one-time PLX cell treatment would decrease the pregnancy-dependent hypertension, proteinuria, endothelial dysfunction, splenomegaly, inflammation, and placental injury induced by Toll-like receptor (TLR) activation during pregnancy. Pregnant C57BL/6 mice were given ip injections of saline vehicle (P), the TLR3 agonist poly I:C (PPIC), or the TLR7 agonist R837 (PR) on days 13, 15, and 17 of gestation. P, PPIC, and PR mice were also given either plasmalyte A (PLA, vehicle) or PLX cells (1 million) by im injection in the right leg on gestational day 14 (n=8 in each group). PLX cell treatment progressively decreased SBP over 3 days in PPIC and PR mice and had no effect in P control mice (day 17 SBP in mmHg: P+PLA = 100±4, P+PLX = 96±4, PPIC+PLA = 144±3, PPIC+PLX = 111±1, PR+PLA = 145±2, PR+PLX = 106±3; PPIC+PLA and PR+PLA p&lt;0.05 vs. P+PLA). PLX cell treatment also normalized the urinary protein/creatinine ratio and aortic endothelium-dependent relaxation responses in PPIC and PR mice to that of P mice while having no significant effects on the number of fetuses or incidence of fetal demise per litter. Inflammation plays a central role in the development of TLR-induced PE and PLX cell treatment reduced spleen weight/body weight ratios, normalized splenic levels of gamma-delta T cells, decreased plasma IL-6 levels, and restored plasma IL-4 levels in PPIC and PR mice. Additionally, PLX cell treatment reduced fibrin deposition in the placental vasculature and significantly reduced placental HIF-1alpha protein levels. These data demonstrate that one-time PLX cell treatment after PE is induced was able to decrease inflammation, proteinuric hypertension, endothelial dysfunction, and placental injury in mice and may be beneficial in women with PE.

Development of Lymphoproliferative Diseases by Hypoxia Inducible Factor-1alpha Is Associated with Prolonged Lymphocyte Survival

Hypoxia-inducible factor-1alpha (HIF-1 alpha) plays an essential role in the regulation of various genes associated with low oxygen consumption. Elevated expression of HIF-1alpha has been reported to be associated with tumor progression, invasion and metastasis in many cancers. To investigate the role of HIF-1alpha in tumor development and metastasis, we established transgenic mice constitutively expressing HIF1A gene under regulation of the cytomegalovirus gene promoter. Although HIF-1alpha protein levels varied among organs, expression of HIF1A mRNA in most organs gradually increased in an age-dependent manner. The transgenic mice showed no gross morphological abnormality up to 8 weeks after birth, although they subsequently developed tumors in the lymphoid, lung, and breast; the most prominent tumor was lymphoma appearing in the intestinal mucosa and intra-mesenchymal tissues. The prevalence of tumors reached 80% in 13 months after birth. The constitution of lymphocyte populations in the transgenic mice did not differ from that in wild-type mice. However, lymphocytes of the transgenic mice revealed prolonged survival under long-term culture conditions and revealed increased resistance to cytotoxic etoposide. These results suggest that HIF-1alpha itself is not oncogenic but it may play an important role in lymphomagenesis mediated through the prolonged survival of lymphocytes in this transgenic mouse model.

Glycerol accumulation in edema formation following diffuse traumatic brain injury

Traumatic brain injury (TBI) induces brain edema via water and glycerol transport channels, called aquaporins (AQPs). The passage of glycerol across brain cellular compartments has been shown during edema. Using a modified impact/head acceleration rodent model of diffuse TBI, we assessed the role of hypoxia inducible factor (HIF)-1alpha in regulating AQP9 expression and glycerol accumulation during the edema formation. Adult (400–425 g) male Sprague–Dawley rats received a closed head injury with a weight drop (450 g, 2-m height) and were allowed to survive up to 48 hours. Some rat groups were administered 2-methoxyestradiol (2ME2, a HIF-1alpha inhibitor) 30 minutes after injury and were euthanized at 4 and 24 hours after injury. Brain edema was measured directly by water content, and glycerol concentration was determined by the Cayman Glycerol Assay. HIF-1alpha and AQP9 protein levels were assessed by Western immunoblotting. This study demonstrated a significant (P<0·05) increase in brain water content at 4–48 hours following impact. Cerebral glycerol was significantly (P<0·05) up-regulated at as early as 1 hour and remained at high levels for up to 48 hours. Similarly, significant (P<0·05) increases in HIF-1alpha and AQP9 protein levels were found at 1 hour and up to 48 hours after injury. Compared to untreated but injured rats, inhibition of HIF-1alpha by 2ME2 significantly (P<0·05) reduced the TBI-induced AQP9 up-regulation. This reduction was temporally associated with significant (P<0·05) decreases in both edema and glycerol accumulation. The data suggested an associated induction of HIF-1alpha, AQP9, and extracellular glycerol accumulation in edema formation following diffuse TBI. The implication of HIF-1alpha and AQP9 underlying TBI-induced edema formation offers possibilities for novel TBI therapies.

Differential Distribution of miR-20a and miR-20b may Underly Metastatic Heterogeneity of Breast Cancers

The discovery that microRNA (miRNA) regulates metastasis provide a principal molecular basis for tumor heterogeneity. A characteristic of solid tumors is their heterogenous distribution of blood vessels, with significant hypoxia occurring in regions (centers of tumor) of low blood flow. It is necessary to discover the mechanism of breast cancer metastasis in relation to the fact that there is a differential distribution of crucial microRNA in tumors from centers to edges. Breast tissues from 48 patients (32 patients with breast cancer) were classified into the high invasive and metastatic group (HIMG), low invasive and metastatic group (LIMG), and normal group. Samples were collected from both the centers and edges of all tumors. The first six specimens were detected by microRNA array, and the second ten specimens were detected by real-time qRT- PCR and Western blot analyses. Correlation analysis was performed between the miRNAs and target proteins. The relative content of miR-20a and miR-20b was lower in the center of the tumor than at the edge in the LIMG, lower at the edge of the tumor than in the center in the HIMG, and lower in breast cancer tissues than in normal tissues. VEGF-A and HIF-1alpha mRNA levels were higher in the HIMG than in the LIMG, and levels were higher in both groups than in the normal group; there was no difference in mRNA levels between the edge and center of the tumor. VEGF-A and HIF-1alpha protein levels were higher in the HIMG than in the LIMG, and protein levels in both groups were higher than in the normal group; there was a significant difference in protein expression between the edge and center of the tumor. Correlation analysis showed that the key miRNAs (miR-20a and miR-20b) negatively correlated with the target proteins (VEGF-A and HIF-1alpha). Our data suggest that miR-20a and miR-20b are differentially distributed in breast cancer, while VEGF-A and HIF-1alpha mRNA had coincident distributions, and VEGF-A and HIF-1alpha proteins had uneven and opposing distributions to the miRNAs. It appears that one of the most important facets underlying metastatic heterogeneity is the differential distribution of miR-20a and miR-20b and their regulation of target proteins.

Combined Preconditioning and Postconditioning Provides Synergistic Protection against Liver Ischemic Reperfusion Injury

Hepatic Ischemia and Reperfusion Injury (IRI) is a major cause of liver damage during liver surgery and transplantation. Ischemic preconditioning and postconditioning are strategies that can reduce IRI. In this study, different combined types of pre- and postconditioning procedures were tested in a murine warm hepatic IRI model to evaluate their protective effects. Proanthocyanidins derived from grape seed was used before ischemia process as pharmacological preconditioning to combine with technical preconditioning and postconditioning. Three pathways related to IRI, including reactive oxygen species (ROS) generation, pro-inflammatory cytokines release and hypoxia responses were examined in hepatic IRI model. Individual and combined pre- and postconditioning protocols significantly reduce liver injury by decreasing the liver ROS and cytokine levels, as well as enhancing the hypoxia tolerance response. Our data also suggested that in addition to individual preconditioning or postconditioning, the combination of these two treatments could reduce liver ischemia/reperfusion injury more effectively by increasing the activity of ROS scavengers and antioxidants. The utilization of grape seed proanthocyanidins (GSP) could improve the oxidation resistance in combined pre- and postconditioning groups. The combined protocol also further increased the liver HIF-1 alpha protein level, but had no effect on pro-inflammatory cytokines release compared to solo treatment.

Methylglyoxal‐induced imbalance in the ratio of vascular endothelial growth factor to angiopoietin 2 secreted by retinal pigment epithelial cells leads to endothelial dysfunction

Progressive microvascular complications are a main feature of diabetes and are associated with impairment of the angiogenic response. Methylglyoxal (MGO) has been implicated in the molecular events that lead to endothelial dysfunction in diabetes. In this study, we hypothesize that increased levels of MGO disrupt the ratio of vascular endothelial growth factor (VEGF) to angiopoietin 2 (Ang 2) secreted by retinal pigment epithelial (RPE) cells, which provides a key destabilizing signal that leads to apoptosis and decreased proliferation of retinal endothelial cells. Indeed, we show that MGO increases the levels of Ang 2 and dramatically decreases the levels of VEGF secreted by RPE cells in response to hypoxia. Downregulation of VEGF is likely to be related to decreased hypoxia-inducible factor-1alpha (HIF-1alpha) protein levels and HIF-1 transcriptional activity. Data further show that MGO-induced imbalance in the VEGF/Ang II ratio significantly changes the levels of BAX and Bcl-2 in endothelial cells. Moreover, this imbalance is accompanied by an increase in the activity of caspase-3 and decreased proliferation of endothelial cells. Data obtained in cell culture systems are consistent with observations in retinas of diabetic animals, where increased availability of MGO is associated with changes in distribution and levels of HIF-1alpha, VEGF and Ang 2 and increased microvascular permeability. In conclusion, the MGO-induced imbalance in the VEGF/Ang 2 ratio secreted by retinal epithelial cells activates apoptosis and decreases proliferation of retinal endothelial cells, which are likely to contribute to endothelial dysfunction in diabetic retinopathy.

FOXO4 Induces Human Plasminogen Activator Inhibitor-1 Gene Expressionviaan Indirect Mechanism by Modulating HIF-1α and CREB Levels

The plasminogen activator inhibitor-1 (PAI-1) expression can be enhanced by hypoxia and various stimuli associated with oxidative stress. Among the FOXO transcription factors, FOXO4 appears to be crucial in the response against oxidative stress. Therefore, it was the aim of this study to investigate the role of peroxide-induced oxidative stress and FOXO4 on PAI-1 expression under normoxia and hypoxia. Treatment of cells with hydrogen peroxide increased PAI-1 mRNA, protein, and promoter activity, and knocking down FOXO4 abolished the peroxide-dependent PAI-1 induction. PAI-1 promoter reporter gene assays revealed that the peroxide and FOXO4-dependent induction was mediated through the HIF-1 and CREB-binding HRE within the PAI-1 promoter. Western blot analyses then indicated that peroxide and FOXO4 downregulated HIF-1alpha levels, whereas CREB levels were increased. Chromatin immunoprecipitations showed that FOXO4 did not bind the PAI-1 promoter, whereas CREB binding was enhanced on FOXO4 overexpression. In addition, knockdown of CREB abolished the FOXO4-mediated PAI-1 induction. Together, these findings provide the first evidence that oxidative stress and FOXO4 induce PAI-1 expression through an indirect mechanism involving modulation of HIF-1alpha and CREB protein levels and that enhanced CREB binding to the PAI-1 promoter is critical for the PAI-1 induction under oxidative stress.

Cytoprotective effects of the volatile anesthetic sevoflurane are highly dependent on timing and duration of sevoflurane conditioning: Findings from a human, in-vitro hypoxia model

Using animal models, volatile anesthetics have been recognized for their neuroprotective effects. Nevertheless, there is still disagreement about the optimal duration and timing of conditioning with the volatile anesthetic sevoflurane in the human system. In the study presented, we employed a human neuronal cell culture model to investigate the effects of hypoxia and to evaluate potential cytoprotective properties of different sevoflurane conditioning strategies. Sevoflurane was applied to human IMR-32 cells in which hypoxic conditions were induced for 2 h using our recently described two-enzyme model (Zitta et al., Eur. J. Pharmacol., 2010). Cellular effects of hypoxia and sevoflurane conditioning were evaluated by lactate dehydrogenase (LDH) measurements, brightfield microscopy, ELISAs, cytometric bead arrays, Westernblotting and RT-PCR. Hypoxia increased the release of LDH into the culture medium after 24 h (normoxia: 0.15 ± 0.02 a.u; hypoxia: 0.69 ± 0.08 a.u, P < 0.001) and expression of hypoxia associated genes HIF-1alpha, VEGF, catalase. Cytoprotective effects were observed in cultures that received sevoflurane for 30 min before hypoxia (preconditioning: 0.41 ± 0.07 a.u., P < 0.01) and for 30 min during the hypoxic period (intraconditioning: 0.20 ± 0.01 a.u., P < 0.001). Application of sevoflurane after the hypoxic insult did not lead to cytoprotection (postconditioning: 0.73 ± 0.12 a.u., P > 0.05). Conditioning with sevoflurane for a total of 3 h before, during and after hypoxia, however, resulted in an enhanced release of LDH (periconditioning: 0.97 ± 0.10 a.u., P < 0.01) and additional cell damage. Hypoxia and sevoflurane intraconditioning were associated with changes in erk1/2 phosphorylation (T202/Y204) and HIF-1alpha protein levels, whereas phosphorylation of akt (S473) was not significantly altered. Our results suggest short pre- and intraconditioning with sevoflurane as most potent strategies to reduce hypoxia induced neuronal cell damage.

Muscle‐specific expression of hypoxia‐inducible factor in human skeletal muscle

Skeletal muscle is well known to exhibit a high degree of plasticity depending on environmental changes, such as various oxygen concentrations. Studies of the oxygen-sensitive subunit alpha of hypoxia-inducible factor-1 (HIF-1) are difficult owing to the large variety of functionally diverse muscle fibres that possess unique patterns of protein and gene expression, producing different capillarization and energy metabolism systems. In this work, we analysed HIF-1alpha mRNA and protein expression related to the fibre-type composition in untrained human skeletal muscle by obtaining muscle biopsies from triceps brachii (characterized by a high proportion of type II fibres), from soleus (characterized by a high proportion of type I fibres) and from vastus lateralis (characterized by an equal proportion of type I and II fibres). The hypothesis was that type I muscle fibres would have lower HIF-1alpha mRNA and protein owing to their higher oxidative capacity. We have shown, in normoxic conditions, a higher HIF-1alpha protein expression in predominantly oxidative muscles than in predominantly glycolytic muscles. However, the HIF-1alpha mRNA expression pattern was not in agreement with the HIF-1alpha protein level. Interestingly, none of the HIF-1alpha target genes, like the most studied angiogenic factor involved in muscle angiogenesis, vascular endothelial growth factor (VEGF), exhibited a muscle fibre-specific-related mRNA expression at rest in normoxia. However, soleus presented a significantly higher VEGF protein content than vastus lateralis and triceps muscle. In conclusion, we have shown that there are muscle-specific differences in HIF-1alpha and VEGF expression within human skeletal muscle at rest in normoxic conditions. Recent results, when combined with the findings described here, support a key role for HIF-1alpha for maintaining muscle homeostasis in non-hypoxic conditions.