Role Of Hypoxia-inducible Factor Research Articles

L-mimosine and hypoxia can increase angiogenin production in dental pulp-derived cells

BackgroundAngiogenin is a key molecule in the healing process which has been successfully applied in the field of regenerative medicine. The role of angiogenin in dental pulp regeneration is unclear. Here we aimed to reveal the impact of the hypoxia mimetic agent L-mimosine (L-MIM) and hypoxia on angiogenin in the dental pulp.MethodsHuman dental pulp-derived cells (DPC) were cultured in monolayer and spheroid cultures and treated with L-MIM or hypoxia. In addition, tooth slice organ cultures were applied to mimic the pulp-dentin complex. We measured angiogenin mRNA and protein levels using qPCR and ELISA, respectively. Inhibitor studies with echinomycin were performed to reveal the role of hypoxia-inducible factor (HIF)-1 signaling.ResultsBoth, L-MIM and hypoxia increased the production of angiogenin at the protein level in monolayer cultures of DPC, while the increase at the mRNA level did not reach the level of significance. The increase of angiogenin in response to treatment with L-MIM or hypoxia was reduced by echinomycin. In spheroid cultures, L-MIM increased angiogenin at protein levels while the effect of hypoxia was not significant. Angiogenin was also expressed and released in tooth slice organ cultures under normoxic and hypoxic conditions and in the presence of L-MIM.ConclusionsL-MIM and hypoxia modulate production of angiogenin via HIF-1 differentially and the response depends on the culture model. Given the role of angiogenin in regeneration the here presented results are of high relevance for pre-conditioning approaches for cell therapy and tissue engineering in the field of regenerative endodontics.

Role of hypoxia-inducible factors (HIF) in the maintenance of stemness and malignancy of colorectal cancer

Hypoxia is a condition of insufficient tissue oxygenation, which is observed during normal development as well as tumorigenesis and its response at the cellular level is primarily mediated through hypoxia inducible factors (HIFs). HIFs have a significant role in the maintenance of stemness in both stem cells as well as in cancer stem cells (CSC) by acting as transcription factors. The CSCs are proposed to be the driving force of colon tumorigenesis and malignancy. These HIFs play a significant role in a wide range of diseases including colon cancer. HIF's signaling functions with stemness, and maintaining Wnt/β-catenin signaling pathways. Due to HIFs functional significance in stemness maintenance in malignancy, targeting HIFs might provide a new approach for development of new therapy for colon cancer. In this review, we will be briefing on the colon and its stem cells, various molecular signaling pathways involved in stemness preservation, and the role hypoxia and its HIFs in the maintenance of stemness in colon stem cells and colon cancer stem cells.

The Role of Hypoxia-Inducible Factors in Cancer Resistance

Diminished oxygen availability (hypoxia) is a hallmark of the tumor microenvironment. A major regulator of cellular adaptation to hypoxia is the hypoxia-inducible factor (HIF) family of transcription factors, which play key roles in many crucial aspects of cancer biology including angiogenesis, stem cell maintenance, metabolic reprogramming, resistance to apoptosis, autocrine growth factor signaling, the EMT program, invasion and metastasis. Resistance to chemotherapy/radiotherapy is the primary cause for treatment failure in clinical oncology. Hypoxia and accumulation of hypoxia-inducible factors (HIFs) in solid tumors have been associated with resistance to treatment and poor prognosis. HIFs causes autophagy establishment to promote survival of cancer cells, and is also associated with the promotion and maintenance of cancer stem cells, a minority subpopulation within the tumor responsible for tumor recurrence and resistance to chemotherapy. In this review, we provide a concise comparative description of the structure, regulation, transcribed genes and roles played by each HIFα subunit in coordinating the transcriptional responses to hypoxia and on the roles they play in the promotion of resistance to anti-cancer therapy.

Role of hypoxia-inducible factor in diabetic myocardial hypertrophy

Purpose: This study was carried out to investigate the role of hypoxia-inducible factor (HIF) in diabetic cardiomyopathy in vitro . Methods: Hypoxia was induced chemically in H9C2 cells (cardiac hypertrophy model), and the cells were treated with phenylephrine (PE), deferoxamine (DFO), PE + DFO, and HIF-1α siRNA under conditions of high and normal glucose. Western blot was used to analyze the expression of some glycolytic proteins, including Glut-1, hexokinase (HXK-2), and enolase, while apoptosis of H9C2 was determined by flow cytometry. Results: PE caused hypertrophy in H9C2, which was ameliorated by HIF-1α. Compared to normal, under prolonged high glucose, the low expression of HIF-1α led to low expressions of Glut-1, HXK-2 and enolase. However, expression of HIF-1α decreased, while those of Bax and Caspase 3 increased, and Bcl-2 expression decreased. Furthermore, under short time high glucose, HIF-1α caused apoptosis of hypertrophic cardiomyocytes. Conclusion: HIF-1 mediates diabetic myocardial hypertrophy, probably as a function of the degree of high glucose exposure and hypoxia. Keywords: H9C2 cardiomyocyte, Hypoxia-inducible factor, Myocardial hypertrophy, Diabetic cardiomyopathy

Altered Blood Gene Transcription in Chuvash Polycythemia and Its Cell Lineage Specificity

Chuvash polycythemia (CP) is a monogenic disorder characterized by an upregulated hypoxic response at normoxia. Homozygosity for the VHLR200W mutation leads to decreased degradation of the a subunits of hypoxia inducible factor (HIF)-1 and HIF-2 by the hypomorphic variant of VHL, the principal negative regulator of HIFs. An array of HIF-regulated genes, including the principal regulators of erythropoiesis and iron metabolism, have altered expression. Previous studies in CP using peripheral blood mononuclear cells (PBMCs), a heterogeneous mixture of cells, identified significant gene expression differences from wild type controls, but the cell linage specificity of these hypoxia-regulated genes remains unknown. In this study, we systematically analyzed gene expression by unbiased deep RNA sequencing in purified reticulocytes, granulocytes and platelets of CP and control individuals living at the same altitude of ~200 meters.Thirty-one samples passed quality control: reticulocytes from 10 individuals (5 VHLR200W homozygotes and 5 wild type controls), platelets from 7 individuals (3 VHL homozygotes and 4 controls) and granulocytes from 14 individuals (5 VHL homozygotes, 1 heterozygote and 8 controls). The samples were analyzed for expression differences (VHL homozygote/heterozygote versus wild type) in each cell type. We found abundant gene expression differences in these three cell types. The differential genes detected in the three cell types showed no more overlap than expected by random (Binomial test P=1 for all pairings of the three cell types), suggesting cell lineage specificity of hypoxic gene expression in CP.At 5% false discovery rate (FDR, i.e., <5 false positives in 100 detected genes), 737 of 7238 analyzed genes (10%) were altered in the reticulocytes of VHLR200W homozygotes, 271 up-regulated and 466 down-regulated. The up-regulated genes were enriched in pathways of "Telomere maintenance", "Oxidative phosphorylation", "Parkinson’s disease", "Ribosome", "Systemic lupus erythematosis", "Apoptosis", "Influenza Infection", "Metabolism of proteins", "Huntington’s disease", and "Integration of energy metabolism". The down-regulated genes were enriched in pathways of "Cell cycle" and "Ubiquitin mediated proteolysis".At 5% FDR, 3646 of 12,334 analyzed genes (30%) were differentially expressed in the platelets of VHLR200W homozygotes, 1830 up-regulated and 1816 down-regulated. The up-regulated genes were enriched in pathways of "Lysosome" and "Signaling in immune system". The down-regulated genes were enriched in pathways of "Hemostasis" and "Opioid signaling".At 5% FDR, 3423 of 11,274 analyzed genes (30%) were differentially expressed in the granulocytes of VHLR200W homozygotes, 1490 up-regulated and 1933 down-regulated. The up-regulated genes were enriched in pathways of "Gene Expression", "Metabolism of nucleotides", "Metabolism of proteins", and "Aminoacyl-tRNA biosynthesis". The down-regulated genes were highly enriched in immune pathways including "Chemokine signaling pathway", "Fc gamma R-mediated phagocytosis", "Endocytosis", "Neurotrophin signaling pathway", "B cell receptor signaling pathway", "Fc epsilon RI signaling pathway", as well as several cancer-related pathways.The relative abundance of alternative transcript isoforms differed in VHLR200W homozygotes relative to wild type controls for many genes in these three blood lineages indicating a role for HIFs in regulation of mRNA processing. At 1% FDR, 3121 of 12,514 analyzed genes (25%) in platelets, 233 of 7342 analyzed genes (3%) in reticulocytes, and 224 of 11,306 analyzed genes (2%) in granulocytes contained alternative exon(s) in VHLR200W homozygotes compared to wild type controls.In conclusion, we report marked gene expression variation in three blood cell lineages from individuals with CP, the first described disorder of congenital augmentation of hypoxia sensing. Dysregulated expression of genes not known to be transcriptionally regulated by HIFs may be due to the well-known but poorly defined effects of HIFs on epigenetic regulation of transcription. Our results demonstrate extensive cell lineage specificity in blood gene expression variations induced by augmented signaling of HIFs caused by the VHLR200W mutation. This provides novel insights to our understanding of clinical complications in CP and more broadly of hypoxic gene regulation. DisclosuresNo relevant conflicts of interest to declare.

The Role of Hypoxia-Inducible Factor 1 in Mild Cognitive Impairment.

Neuroinflammation and reactive oxygen species are thought to mediate the pathogenesis of Alzheimer's disease (AD), suggesting that mild cognitive impairment (MCI), a prodromal stage of AD, may be driven by similar insults. Several studies document that hypoxia-inducible factor 1 (HIF-1) is neuroprotective in the setting of neuronal insults, since this transcription factor drives the expression of critical genes that diminish neuronal cell death. HIF-1 facilitates glycolysis and glucose metabolism, thus helping to generate reductive equivalents of NADH/NADPH that counter oxidative stress. HIF-1 also improves cerebral blood flow which opposes the toxicity of hypoxia. Increased HIF-1 activity and/or expression of HIF-1 target genes, such as those involved in glycolysis or vascular flow, may be an early adaptation to the oxidative stressors that characterize MCI pathology. The molecular events that constitute this early adaptation are likely neuroprotective, and might mitigate cognitive decline or the onset of full-blown AD. On the other hand, prolonged or overwhelming stressors can convert HIF-1 into an activator of cell death through agents such as Bnip3, an event that is more likely to occur in late MCI or advanced Alzheimer's dementia.

Abstract 59: Antimir Therapy Ameliorates HIF1-mediated Loss of SERCA2 and Cardiac Contractility

Ischemic heart disease is a major precipitant of heart failure in the developed world. Hypoxia inducible factor 1 (HIF1) is a highly oxygen-sensitive protein and the principal regulator of hypoxia-related signaling including changes in metabolism and angiogenesis. To more fully understand the role of HIF1 in the heart, our lab has developed a transgenic mouse model in which a stable form of HIF-1alpha can be inducibly expressed under normoxic conditions in cardiomyocytes. Upon HIF1 induction, genes associated with calcium signaling are downregulated and metabolism is shifted toward glycolysis. Strikingly, these mice also show a rapid decrease in cardiac contractility and concurrent loss of SERCA2 protein expression (up to 90%) within one week. SERCA2a (sarcoplasmic/endoplasmic reticulum calcium ATPase 2) is the ATP-dependent calcium pump required for calcium re-uptake during excitation-contraction coupling in the heart and its dysfunction has been implicated in heart failure. To extend the physiological significance of these findings, we examined ischemic heart tissue from WT mice subjected to ligation of the left anterior descending (LAD) artery to mimic myocardial infarction. At one day and 3 days post-ligation, we observed an increase in transcripts needed for glycolysis and decreased abundance of transcripts from the genes for proteins that regulate excitation-contraction including the ryanodine receptor, phospholamban and SERCA2. We also observed that while SERCA2 transcript decreased in HIF1 transgenic hearts, it did not fully account for the observed protein loss. Microarray analysis found miR-29c to be substantially upregulated and a potential regulator of SERCA2 expression. Subsequent in vitro analysis confirmed miR-29c reduced SERCA2 expression by 20-30% and could be modulated with an anti-sense inhibitor. In vivo administration of the antimir to miR-29c also improved cardiac contractility and SERCA2 expression in HIF1 transgenic mice. These results suggest that a HIF1 regulated microRNA, miR-29c, contributes to loss of SERCA2 in hypoxia and can be therapeutically targeted to improve cardiac function.

Role of Hypoxia Inducible Factor 1 in Hyperglycemia-Exacerbated Blood-Brain Barrier Disruption in Ischemic Stroke

Diabetes is a major stroke risk factor and is associated with poor functional recovery after stroke. Accumulating evidence indicates that the worsened outcomes may be due to hyperglycemia-induced cerebral vascular complications, especially disruption of the blood-brain barrier (BBB). The present study tested a hypothesis that the activation of hypoxia inducible factor-1 (HIF-1) was involved in hyperglycemia-aggravated BBB disruption in an ischemic stroke model. Non-diabetic control and Streptozotocin-induced type I diabetic mice were subjected to 90min transient middle cerebral artery occlusion (MCAO) followed by reperfusion. Our results demonstrated that hyperglycemia induced higher expression of HIF-1α and vascular endothelial growth factor (VEGF) in brain microvessels after MCAO/reperfusion. Diabetic mice showed exacerbated BBB damage and tight junction disruption, increased infarct volume as well as worsened neurological deficits. Furthermore, suppressing HIF-1 activity by specific knock-out endothelial HIF-1α ameliorated BBB leakage and brain infarction in diabetic animals. Moreover, glycemic control by insulin abolished HIF-1α up-regulation in diabetic animals and reduced BBB permeability and brain infarction. These findings strongly indicate that HIF-1 plays an important role in hyperglycemia-induced exacerbation of BBB disruption in ischemic stroke. Endothelial HIF-1 inhibition warrants further investigation as a therapeutic target for the treatment of stroke patients with diabetes.

Abstract 1628: MAFF, a new hypoxia target gene involving tumor invasion and metastasis

Abstract Activation of hypoxia-inducible factor (HIF) under hypoxia is significantly correlated with tumor progression and treatment resistance by regulating target genes involved in invasion, metastasis, angiogenesis, apoptosis, and metabolism. This important role of HIF and hypoxia in promoting tumor progression provides a strong rationale to develop therapies that disrupt or target critical effectors of these pathways. V-Musculoaponeurotic Fibrosarcoma homolog F (MAFF) is a basic leucine zipper (bZIP) transcription factor that regulates antioxidative responses, hematopoiesis, and inflammation. However, its role in tumor hypoxia has not been well studied. In this study, we demonstrated that hypoxia specifically regulated MAFF in various tumor cell types and it was specifically induced by HIF-1 but not HIF-2. To determine the role of MAFF in tumor progression, we investigated tumor cells by both gain and loss of function studies. Knocking down MAFF in metastatic cancer cell lines, MDAMB-231 and OVCAR-8, resulted in reduced tumor cell invasion and metastasis both in vitro and in vivo. In contrast, overexpressing MAFF in non-metastatic cell lines, MCF7 and A549 increased tumor cell invasion. To elucidate the underlying mechanisms of MAFF-mediated tumor progression, we performed RNA sequencing in MDAMB-231 cells, which were exposed to either 20% O2 or 0.5% O2 with or without genetic inhibition of MAFF using siRNA. After performing a functional annotation using DAVID (Database for Annotation, Visualization, and Integrated Discovery) analysis, we observed genes involved in “blood vessel development” and “cell motility” were dysregulated in the absence of MAFF expression. In addition, genes relevant to “apoptosis”, “transcription”, and “inflammatory response” were altered when MAFF was inhibited. These observations suggest novel aspects of MAFF-mediated gene regulation. To further determine genes which were directly regulated by MAFF, we performed ChIP-sequencing with cells treated under normoxia or hypoxia. By combining profiles from RNA-sequencing and ChIP-sequencing, we highlighted 45 genes under normoxia and 44 genes under hypoxia. Among these, eight genes were regulated both under normoxia and hypoxia. Future studies will determine the set of genes that are responsible for tumor invasion and metastasis as well as other aspects of tumor progression. In conclusion, our study identifies MAFF as a novel HIF-1 target gene and also demonstrates its role in cell invasion and metastasis, which has not previously been described in cancer. This work may point towards a new therapeutic strategy to target tumor progression. Citation Format: Eui Jung Moon, Stephano S. Mello, Jen-Tsan Chi, Adam J. Krieg, Amato Giaccia. MAFF, a new hypoxia target gene involving tumor invasion and metastasis. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1628.

Amelioration of ER stress by 4-phenylbutyric acid reduces chronic hypoxia induced cardiac damage and improves hypoxic tolerance through upregulation of HIF-1α

While endoplasmic reticulum (ER) stress has been observed in several human diseases, few studies have reported the involvement of ER stress in chronic hypoxia (CH) induced cardiac damage. Hypoxia, such as that prevalent at high altitude (HA), forms the underlying cause of several maladies including cardiovascular diseases. While the role of hypoxia inducible factor-1 (HIF-1α) in the adaptive responses to hypoxia is known, the role of the unfolded protein response (UPR) is only recently being explored in the HA pathophysiologies. The present study investigates the effect of ER stress modulation on CH mediated injury and the cardioprotective action of 4-phenylbutyric acid (PBA) in enhancing survival response under hypoxia. Here, we observed that exposure of rats, for 1, 7 and 14days CH to a simulated altitude of 7620m, led to cardiac hypertrophy and significant protein oxidation. This induced the activation of UPR signaling mechanisms, mediated by PERK, IRE1α and ATF6. By 14days, there was a marked upregulation of apoptosis, evident in increased CHOP and caspase-3/9 activity. PBA reduced CH induced right ventricular enlargement and apoptosis. Further, in contrast to tunicamycin, PBA considerably enhanced hypoxic tolerance. An elevation in the level of antioxidant enzymes, HIF-1α and its regulated proteins (HO-1, GLUT-1) was observed in the PBA administered animals, along with a concomitant suppression of UPR markers. Our study thus emphasizes upon the attenuation of ER stress by PBA as a mechanism to diminish CH induced cardiac injury and boost hypoxic survival, providing an insight into the novel relationship between the HIF-1α and UPR under hypoxia.

622 Pathological Acid Reflux Stimulates Esophageal Epithelial Production of Hypoxia Inducible Factor (HIF)-2a and Its Target Pro-Inflammatory Molecules: A Role for HIFs in GERD Pathogenesis

622 Pathological Acid Reflux Stimulates Esophageal Epithelial Production of Hypoxia Inducible Factor (HIF)-2a and Its Target Pro-Inflammatory Molecules: A Role for HIFs in GERD Pathogenesis

Abstract PR03: Hypoxia preconditioning affects metastatic seeding and proliferation through vascular and immune function, in a process regulated by lung endothelial HIFa

Abstract Metastases are the main cause of mortality in solid cancers. Current evidence shows that tumor cells are present in circulation from early stages of malignancy, and this initial dissemination implies that early diagnosis alone is not a consistent or reliable predictor of outcome. A deeper understanding of factors affecting extravasation and secondary proliferation is crucial to develop strategies to prevent and/or treat metastatic disease. Our research and that of others have shown that tumor dissemination rely on properties of the tumor cells, but also on the individual cell types in the microenvironments, as well as on the physiological context in the host organ. Tumor progression is, consequently, a function of the individual cell types in the microenvironments of the primary tumor and metastatic sites. Activation of hypoxia-inducible factors (HIFs) in most solid tumor types is directly correlated with poor prognosis. However, we have shown that when in host organ cells, and specifically endothelial cells, the role of HIF is isoform-specific: mice lacking endothelial HIF-1α develop less lung metastases, whereas those lacking endothelial HIF-2α have more. This is a very intriguing phenomenon that revealed a decisive role for the endothelium in allowing or preventing metastatic success, shown to be, in part, dependent on an imbalanced production of nitric oxide (NO), with clear NO-driven effects on vessel function. Upon exposure of mice to low atmospheric oxygen levels, we found that there is a time-dependent activation of HIF-1α and HIF-2α in lung tissue, which results in differential gene expression and inflammatory response/inflammatory cell recruitment, affecting both extravasation and secondary proliferation of circulating tumor cells. In this study, mice were hypoxia-conditioned prior to intravenous injection of cancer cells, to investigate the effect of hypoxia-driven vascular changes on pulmonary metastases. Mice exposed to either short or prolonged hypoxia show increased number of lung monocytes when compared to normoxic controls. However, acute hypoxia (24h), which correlates with higher HIF-1α levels, results in increased numbers of lung tumors, whereas chronic hypoxia exposure (10d, higher HIF-2α activation) results in fewer and less proliferative tumors. Using a variety of approaches, including cytokine and transcriptional screens, imaging and cell-specific HIF-deletion models, both in vivo and ex-vivo, we are investigating which parameters affecting metastatic disease are changed during acute and prolonged hypoxia, resulting in differential metastatic incidence. We found that both endothelial permeability and endothelial-derived signaling is affected differently upon acute or chronic hypoxia stimulus, with also different impact on extravasation and immune cell recruitment and activation. We aim to identify what molecular mechanism(s) underlying hypoxia, HIF and vascular endothelium responses condition the establishment of secondary tumors. Citation Format: Cristina Branco, Helene Rundqvist, Renato Colaco, Randall S. Johnson. Hypoxia preconditioning affects metastatic seeding and proliferation through vascular and immune function, in a process regulated by lung endothelial HIFa. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Metastasis; 2015 Nov 30-Dec 3; Austin, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(7 Suppl):Abstract nr PR03.

Role of Hypoxia‐Inducible Factor (HIF) on colonic goblet cell differentiation and function during mucosal healing

Secreted mucus is essential for intestinal homeostasis, separating luminal bacteria from the colonic epithelium and preventing inappropriate inflammation. Depletion of the mucus‐secreting goblet cells has been shown in mice to lead to development of spontaneous colitis. Treatment of mice with a prolyl hydroxylase inhibitor (AKB‐4924) leads to stabilization of hypoxia inducible factor‐1 alpha (HIF‐1α) and ultimately regulation of hypoxia‐responsive genes that promote mucosal protection. Interestingly, goblet cell hyperplasia was observed following in vivo administration of AKB‐4924, which may account for the mucosal protection observed. Thus, we hypothesized epithelial stabilization of HIF‐1α leads to goblet cell differentiation and hyperplasia within the GI tract.Utilizing a three‐dimensional murine intestinal stem cell spheroid culture system, we were able to analyze the influence of HIF‐1α stabilization on epithelial differentiation in a physiologically relevant context. Spheroids were treated with a goblet cell promoting factor (a gamma secretase inhibitor, DBZ), AKB‐4924 or hypoxia and analyzed by qPCR for changes in differentiation markers for secretory goblet cells (i.e. Atoh1, Muc2, and SPDEF) or absorptive enterocytes (i.e. Hes1, ALPI, and Fabp2). Additionally, DBZ and AKB‐4924 treated organoids were visualized using immunofluorescence for changes in the expression of Muc2, the predominant colonic mucin. Contrary to our hypothesis, neither AKB‐4924 nor hypoxia resulted in the induction of goblet cell differentiation factors. Furthermore, mice lacking HIF‐1α in the intestinal epithelium (HIF‐1αfl/fl‐VillinCRE) exhibited normal colonic goblet cell numbers. However, goblet cells in mice lacking epithelial HIF‐1α appeared less organized with mucin granules less uniform in size and more randomly dispersed, and have a significantly altered expression profile of both secreted and adherent mucins, indicating that epithelial HIF‐1α is involved in goblet cell function.Therefore, we conclude that AKB‐4924 does not act directly on the epithelium to promote epithelial goblet cell differentiation, however, epithelial HIF‐1α does alter goblet cell function, potentially regulating intestinal mucus layer, which may contribute to the mucosal‐protective role for stabilized HIF.Support or Funding InformationR01 DK103639 from NIDDK, CDA from Crohn's and Colitis Foundation of America to E.L.C.

Hypoxia-inducible factor-1α inhibits interleukin-6 and -8 production in gingival epithelial cells during hypoxia.

Hypoxia has been widely studied in inflammatory diseases as it can modulate the inflammatory response, mainly via the hypoxia-inducible factor (HIF). However, little is known about the effects of hypoxia and the role of HIF in the inflammatory responses to periodontitis. In this study, we focused on the gingival epithelium that is exposed to relatively low levels of oxygen. We investigated whether hypoxic conditions have an impact on inflammatory responses in human gingival epithelial cells (HGECs). Pimonidazole HCl, which accumulates in hypoxic cells, was administered intraperitoneally to C57BL/6 mice with or without Porphyromonas gingivalis infection. Immunohistochemistry was then performed to detect the hypoxic cells in periodontal tissue. Immortalized HGECs were cultured under hypoxic conditions with or without interleukin (IL)-1β, and the expression levels of IL-6 and IL-8 were measured by real-time reverse transcription-polymerase chain reaction and enzyme-linked immunosorbent assay. HIF-1α expression was detected by western blotting. The DNA-binding activity of HIF-1α was determined by a DNA-binding enzyme-linked immunosorbent assay. The involvement of HIF-1α in the hypoxic response was examined by transfection with HIF-1α siRNA. Immunohistochemistry revealed pimonidazole HCl accumulation in the gingival epithelium of both normal and P. gingivalis-infected mice, with a slightly stronger signal in the P. gingivalis-infected mice than in the normal mice. The IL-1β-induced IL-6 and IL-8 production by HGECs was suppressed under hypoxic conditions. HIF-1α accumulated during hypoxia, and this accumulation was further enhanced by IL-1β treatment. The hypoxia-dependent suppression of IL-6 and IL-8 expression was reversed by treating the cells with HIF-1α siRNA. Our results suggest that the gingival epithelium is exposed to low oxygen tension in periodontal tissue and that this hypoxic condition modulates the local inflammatory response of gingival epithelial cells in an HIF-1α-dependent manner.

Expanding roles of the hypoxia-response network in chronic kidney disease.

Studies over the last two decades have established tubulointerstitial hypoxia as a final common pathway leading to end-stage kidney disease (ESKD). Chronic kidney disease (CKD) is frequently associated with various degrees of hypoxic injury in distinct tubular segments, depending on the etiology and pathological stages, which constitutes an intricate link among inflammation, oxidative stress and fibrosis. Resident cells in the kidney are equipped with mechanisms through which they cope with hypoxia. Here, transcription of genes by hypoxia-inducible factors (HIFs) plays a central role. In the ischemic kidney, HIF-1 is expressed in tubular and glomerular epithelial cells and in papillary interstitial cells, whereas HIF-2 is expressed in endothelial cells and interstitial fibroblasts. There is ample evidence that HIF protects the kidney from acute ischemic damage. In CKD, studies suggest that the function of HIF may be suppressed because of factors, such as oxidative stress and uremia, which may underlie the pathogenesis of both CKD and co-existing problems, such as renal anemia. Based on these observations, efforts are in progress to test whether restoration and activation of HIF might protect the kidney from CKD. Initial studies using non-specific or supraphysiological HIF activation suggested that the role of HIF may be multifactorial and depend on pathological context. On the other hand, specific HIF stabilizers, such as prolyl hydroxylase (PHD) inhibitors, are being developed for the treatment of renal anemia. Application of these compounds in experimental CKD may override those previous findings and provide deeper insight into the roles of hypoxia and oxygen-sensing pathways.

Role of Hypoxia Inducible Factors in Obesity Pathogenesis

The prevalence of obesity, metabolic syndrome and diabetes has been increasing rapidly worldwide. These are a group of metabolic disorders characterized by a chronic hyperglycaemic condition resulting from defects in insulin secretion, insulin action or both. The control of body weight and blood glucose concentrations depends on the exquisite coordination of the function of several cells, organs and tissues. Underlying mechanisms of obesity and insulin resistance remain uncertain. Adipose tissue is composed of heterogeneous cell types. Immune cells within adipose tissue also likely contribute to systemic metabolic processes. Increased production of local and systemic adipokines and cytokines, polarization of macrophages, T helper subtype changes could contribute to pathologies linking obesity to diabetes, both by decreasing insulin sensitivity, by compromising β-cell function and disturbing adipose tissue metabolism and distribution. Tissue oxygen (O 2) levels, hypoxia inducible factor (s) (HIFs) secretion differences regulate the plasticity of macrophages and the polarization of macrophages controls functionally divergent processes in

"A small leak will sink a great ship": hypoxia-inducible factor and group III pulmonary hypertension.

Pulmonary hypertension complicating idiopathic pulmonary fibrosis, also known as secondary pulmonary hypertension, represents a major source of morbidity and mortality in affected patients. While the study of primary pulmonary arterial hypertension has yielded several therapies, the same is not true for the treatment of pulmonary hypertension secondary to pulmonary fibrosis. Recent studies have indicated an important role of hypoxia-inducible factor (HIF) – a regulatory protein that is vital in adaptation to hypoxic conditions – in the development of secondary pulmonary hypertension. HIF influences development of hypoxia-induced pulmonary hypertension through alteration in voltage-gated potassium channels and homeostatic calcium regulation, resulting in disruption of endothelial cell-cell communication, and eventual vascular remodeling. This article summarizes salient literature related to HIF and secondary pulmonary hypertension, in addition to proposing a final common pathway in known mechanistic pathways that result in endothelial barrier integrity loss – vascular “leak” – primarily through a shared endothelial-epithelial signaling protein family, CCN.

Effects of the hypoxia-inducible factor-1 inhibitor echinomycin on vascular endothelial growth factor production and apoptosis in human ectopic endometriotic stromal cells

Recent evidence points to a possible role for hypoxia-inducible factor (HIF)-1 in the pathogenesis and development of endometriosis. The objectives of this study were to investigate the critical role of HIF-1 in endometriosis and the effect of the HIF-1 inhibitor echinomycin on human ectopic endometriotic stromal cells (eESCs). Ectopic endometriotic tissues were obtained from 20 patients, who received an operation for ovarian endometriomas. We examined vascular endothelial growth factor (VEGF) and stromal cell-derived factor-1 (SDF-1) production, HIF-1 expression, cell proliferation and apoptosis of eESCs. Cobalt chloride (CoCl2) significantly induced expression of HIF-1α protein and VEGF production in a time-dependent manner in eESCs, but reduced SDF-1 production. VEGF production was significantly suppressed by treatment of 100 nM echinomycin without causing cell toxicity, but 0.1–10 nM echinomycin or 100 nM progestin had no significant effect. SDF-1 production was not affected by echinomycin treatment at any dose. Echinomycin inhibited cell proliferation and induced apoptotic cell death of the eESCs, and significantly inhibited expression of the anti-apoptotic proteins Bcl-2 and Bcl-xL. Echinomycin inhibits VEGF production and induces apoptosis of eESCs by suppression of Bcl-2 and Bcl-xL. These findings suggest the unique therapeutic potential for echinomycin as an inhibitor of HIF-1 activation for endometriosis treatment.

Crucial role of hypoxia inducible factor in embryo implantation

Objective: Endometriosis is a gynecological condition of which the primary symptoms are infertility and pelvic pain. Especially for infertility women, a new therapy which possesses reproductive expectation is needed. Hence, it is essential to explore inflammatory substances which have strong impact on the disease. Bradykinin (BK) is one of the most algogenic substances, also known as an inflammatory mediator in various diseases. Therefore, BK attracts much attention as a therapeutic target. As a first step toward understanding a possible role of bradykinin in endometriosis, we investigated the presence and the regulation of BK receptors in human endometriotic tissues. Material and method: This study protocol was approved by the institutional review boards. The expression of BK receptor (type I and II) in human endometriotic lesions was investigated by immunohistochemistory. Endometriotic stromal cells (ESC) were cultured with IL-1 (10ng/ml), followed by quantitative-PCR for BK receptors. In some experiments, after priming with IL-1 for 24h, ESC were stimulated with BK (1 M) for 8h, followed by quantitative-PCR for IL-6, COX-2 and endothelin-1. Result: Immunohistochemisty showed that BK receptors were present in epithelial and stromal cells in endometriotic lesions. IL-1 induced 10-fold increase in BK receptors mRNA in ESC (p<0.01). IL-1 induced IL-6 and COX-2 significantly (p<0.01) but not endothelin-1 mRNA. On addition of BK, IL-6 and COX-2 mRNA expressions were further enhanced to the levels of approximately 2-fold compared to IL-1 only (p<0.01). Endothelin-1 was increased 2-fold in ESC treated with IL-1 and BK, compared with control. These increases of mRNA expressions by BK were completely abrogated by 1 M HOE-140, an antagonist of BK receptor (p<0.01). Conclusion: In endometriotic lesions, BK receptors were present and served as chemical mediator. We showed that a strategy for blockingBK receptorsmight be anew therapy for endometriosis from the point of suppressing inflammation and pelvic pain.

HIF-2α-induced chemokines stimulate motility of fibroblast-like synoviocytes and chondrocytes into the cartilage-pannus interface in experimental rheumatoid arthritis mouse models.

IntroductionPannus formation and resulting cartilage destruction during rheumatoid arthritis (RA) depends on the migration of synoviocytes to cartilage tissue. Here, we focused on the role of hypoxia-inducible factor (HIF)-2α-induced chemokines by chondrocytes in the regulation of fibroblast-like synoviocyte (FLS) migration into the cartilage-pannus interface and cartilage erosion.MethodsCollagen-induced arthritis (CIA), K/BxN serum transfer, and tumor necrosis factor-α transgenic mice were used as experimental RA models. Expression patterns of HIF-2α and chemokines were determined via immunostaining, Western blotting and RT-PCR. FLS motility was evaluated using transwell migration and invasion assays. The specific role of HIF-2α was determined via local deletion of HIF-2α in joint tissues or using conditional knockout (KO) mice. Cartilage destruction, synovitis and pannus formation were assessed via histological analysis.ResultsHIF-2α and various chemokines were markedly upregulated in degenerating cartilage and pannus of RA joints. HIF-2α induced chemokine expression by chondrocytes in both primary culture and cartilage tissue. HIF-2α -induced chemokines by chondrocytes regulated the migration and invasion of FLS. Local deletion of HIF-2α in joint tissues inhibited pannus formation adjacent to cartilage tissue and cartilage destruction caused by K/BxN serum transfer. Furthermore, conditional knockout of HIF-2α in cartilage blocked pannus formation in adjacent cartilage but not bone tissue, along with inhibition of cartilage erosion caused by K/BxN serum transfer.ConclusionOur findings suggest that chemokines induced by IL-1β or HIF-2α in chondrocytes regulate pannus expansion by stimulating FLS migration and invasion, leading to cartilage erosion during RA pathogenesis.