Expression Of Hypoxia-inducible Transcription Factors Research Articles

The Role of Hypoxia-Inducible Factor in the Mechanisms of Aging.

Aging is accompanied by a reduction in the oxygen delivery to all organs and tissues and decrease in the oxygen partial pressure in them, resulting in the development of hypoxia. The lack of oxygen activates cell signaling pathway mediated by the hypoxia-inducible transcription factor (HIF), which exists in three isoforms - HIF-1, HIF-2, and HIF-3. HIF regulates expression of several thousand genes and is a potential target for the development of new drugs for the treatment of many diseases, including those associated with age. Human organism and organisms of laboratory animals differ in their tolerance to hypoxia and expression of HIF and HIF-dependent genes, which may contribute to the development of inflammatory, tumor, and cardiovascular diseases. Currently, the data on changes in the HIF expression with age are contradictory, which is mostly due to the fact that such studies are conducted in different age groups, cell types, and model organisms, as well as under different hypoxic conditions and mainly in vitro. Furthermore, the observed discrepancies can be due to the individual tolerance of the studied organisms to hypoxia, which is typically not taken into account. Therefore, the purpose of this review was to analyze the published data on the connection between the mechanisms of aging, basal tolerance to hypoxia, and changes in the level of HIF expression with age. Here, we summarized the data on the age-related changes in the hypoxia tolerance, HIF expression and the role of HIF in aging, which is associated with its involvement in the molecular pathways mediated by insulin and IGF-1 (IIS), sirtuins (SIRTs), and mTOR. HIF-1 interacts with many components of the IIS pathway, in particular with FOXO, the activation of which reduces production of reactive oxygen species (ROS) and increases hypoxia tolerance. Under hypoxic conditions, FOXO is activated via both HIF-dependent and HIF-independent pathways, which contributes to a decrease in the ROS levels. The activity of HIF-1 is regulated by all members of the sirtuin family, except SIRT5, while the mechanisms of SIRT interaction with HIF-2 and HIF-3 are poorly understood. The connection between HIF and mTOR and its inhibitor, AMPK, has been identified, but its exact mechanism has yet to be studied. Understanding the role of HIF and hypoxia in aging and pathogenesis of age-associated diseases is essential for the development of new approaches to the personalized therapy of these diseases, and requires further research.

Anisodine hydrobromide alleviates hypoxia/reoxygenation (H/R)‐induced brain microvascular endothelial cell injury via muscarinic acetylcholine receptor 4

Anisodine hydrobromide (Ah) could delivery to the brain across the blood-brain barrier, exerting its protective effect in cerebral ischemia/reperfusion injury. Metabolic alterations under hypoxic conditions lead to endothelial dysfunction. In the present study, we tested that whether Ah regulates the production of nitric oxide and reactive oxygen species (ROS) and the glycolytic pathway via muscarinic receptors under hypoxia/reoxygenation (H/R) conditions. Under H/R conditions, brain microvascular endothelial cells were treated with Ah. Then, the expression of muscarinic acetylcholine receptors was detected by western blot. The production of nitric oxide was detected by Greiss reaction. ROS was measured by DCFH-DA probes. The expression of hypoxia-inducible transcription factor 1 (HIF-1α) was detected. Specific inhibitor of M2 and M4 muscarinic acetylcholine receptors were used to assess the role of muscarinic receptors in Ah protects against endothelial cell dysfunction. Results showed that H/R induced the expression of M2- and M4-AchRs, but not M1-, M3-, and M5-AchRs. Ah significantly suppressed the H/R-upregulated M2- and M4-AchRs. H/R induced production of NO and ROS. Ah and M4-AchRs inhibitor significantly inhibited the H/R-induced production of NO and ROS. HIF-1α was significantly induced by H/R, whereas it was significantly suppressed by Ah. Therefore, Ah protects against H/R injury in brain microvascular endothelial cells via activating HIF-1α, producing NO and ROS, which is dependent on M4-AchR.

Preeclampsia for the Nephrologist: Current Understanding in Diagnosis, Management, and Long-term Outcomes.

Preeclampsia is a multisystem progressive disorder of pregnancy that can be potentially catastrophic for the mother and the fetus. It involves complex perturbations of the kidney and systemic physiology, along with long-term effects on vascular and kidney health. Thus, the nephrologist plays a key role in the peripartum and long-term management of preeclampsia. Recent translational research has improved our understanding of its pathophysiology, and there is hope for novel therapies. In this review, we discuss the evolution of diagnostic criteria and dilemmas in the diagnosis of hypertensive disorders in pregnancy. We summarize the advances in the pathogenesis and prediction of preeclampsia. We describe the management and prevention of preeclampsia focusing specially on the forthcoming strategies from the nephrologist's perspective. We address the evidence regarding long-term outcomes for the mother and the child. We end with exploring areas warranting future research.

The role of hypoxia-induced modulation of alveolar epithelial Na+- transport in hypoxemia at high altitude.

Reabsorption of excess alveolar fluid is driven by vectorial Na+-transport across alveolar epithelium, which protects from alveolar flooding and facilitates gas exchange. Hypoxia inhibits Na+-reabsorption in cultured cells and in-vivo by decreasing activity of epithelial Na+-channels (ENaC), which impairs alveolar fluid clearance. Inhibition also occurs during in-vivo hypoxia in humans and laboratory animals. Signaling mechanisms that inhibit alveolar reabsorption are poorly understood. Because cellular adaptation to hypoxia is regulated by hypoxia-inducible transcription factors (HIF), we tested whether HIFs are involved in decreasing Na+-transport in hypoxic alveolar epithelium. Expression of HIFs was suppressed in cultured rat primary alveolar epithelial cells (AEC) with shRNAs. Hypoxia (1.5% O2, 24 h) decreased amiloride-sensitive transepithelial Na+-transport, decreased the mRNA expression of α-, β-, and γ-ENaC subunits, and reduced the amount of αβγ-ENaC subunits in the apical plasma membrane. Silencing HIF-2α partially prevented impaired fluid reabsorption in hypoxic rats and prevented the hypoxia-induced decrease in α- but not the βγ-subunits of ENaC protein expression resulting in a less active form of ENaC in hypoxic AEC. Inhibition of alveolar reabsorption also caused pulmonary vasoconstriction in ventilated rats. These results indicate that a HIF-2α-dependent decrease in Na+-transport in hypoxic alveolar epithelium decreases alveolar reabsorption. Because susceptibles to high-altitude pulmonary edema (HAPE) have decreased Na+-transport even in normoxia, inhibition of alveolar reabsorption by hypoxia at high altitude might further impair alveolar gas exchange. Thus, aggravated hypoxemia might further enhance hypoxic pulmonary vasoconstriction and might subsequently cause HAPE.

Hypoxic Regulation of Neutrophils in Cancer.

Neutrophils have been well-characterized for their role in the host anti-microbial response. However, it is now appreciated that neutrophils have a critical role in tumorigenesis and tumor progression in the majority of solid tumors. Recent studies have indicated a critical role for hypoxia in regulating neutrophil function in tumors. Furthermore, neutrophil-specific expression of hypoxia-inducible transcription factors may represent a novel therapeutic target for human cancer. In this review, we highlight the function of neutrophils in cancer and the role of the neutrophil hypoxic response in regulating the neoplastic progression of cancer.

Pilocarpine-Induced Status Epilepticus Is Associated with P-Glycoprotein Induction in Cardiomyocytes, Electrocardiographic Changes, and Sudden Death.

Sudden unexpected death in epilepsy (SUDEP) is the major cause of death in those patients suffering from refractory epilepsy (RE), with a 24-fold higher risk relative to the normal population. SUDEP risk increases with seizure frequency and/or seizure-duration as in RE and Status Epilepticus (SE). P-glycoprotein (P-gp), the product of the multidrug resistant ABCB1-MDR-1 gene, is a detoxifying pump that extrudes drugs out of the cells and can confer pharmacoresistance to the expressing cells. Neurons and cardiomyocytes normally do not express P-gp, however, it is overexpressed in the brain of patients or in experimental models of RE and SE. P-gp was also detected after brain or cardiac hypoxia. We have previously demonstrated that repetitive pentylenetetrazole (PTZ)-induced seizures increase P-gp expression in the brain, which is associated with membrane depolarization in the hippocampus, and in the heart, which is associated with fatal SE. SE can produce hypoxic-ischemic altered cardiac rhythm (HIACR) and severe arrhythmias, and both are related with SUDEP. Here, we investigate whether SE induces the expression of hypoxia-inducible transcription factor (HIF)-1α and P-gp in cardiomyocytes, which is associated with altered heart rhythm, and if these changes are related with the spontaneous death rate. SE was induced in Wistar rats once a week for 3 weeks, by lithium-pilocarpine-paradigm. Electrocardiograms, HIF-1α, and P-gp expression in cardiomyocytes, were evaluated in basal conditions and 72 h after SE. All spontaneous deaths occurred 48 h after each SE was registered. We observed that repeated SE induced HIF-1α and P-gp expression in cardiomyocytes, electrocardiographic (ECG) changes, and a high rate of spontaneous death. Our results suggest that the highly accumulated burden of convulsive stress results in a hypoxic heart insult, where P-gp expression may play a depolarizing role in cardiomyocyte membranes and in the development of the ECG changes, such as QT interval prolongation, that could be related with SUDEP. We postulate that this mechanism could explain, in part, the higher SUDEP risk in patients with RE or SE.

HIF is not essential for suppression of experimental tumor growth by mTOR inhibition.

The Hypoxia Inducible Transcription Factor (HIF) is the master regulator of cellular response to hypoxic adaptation. Solid tumors inevitably harbour hypoxic regions with subsequent stabilization and activation of HIF and HIF target genes due to poor vascularization and rapid growth. The mammalian target of rapamycin (mTOR) is a global regulator of cellular growth and proliferation, which can also regulate HIF expression independantly of hypoxia via specific activation of cellular translation and transcription. An effective blockade of mTOR results in attenuation of HIF under hypoxic conditions in vitro. This mechanism could enable a simultaneous inhibition of both the mTOR- and the HIF-pathway, resulting in an effective tool for cancer targeting. We set out to analyze the effect of mTOR inhibition and the involvement of mTOR regulation on HIF in vivo in a subcutaneous xenograft model in nude mice. Our results demonstrate that mTOR inhibition in our model leads to a clear reduction in tumor growth of various cellular origins, most likely due to inhibition of cellular proliferation. Moreover, these effects can also be achieved independently of the HIF status of the tumor cells. The HIF levels per se seem to remain unaffected by mTOR inhibition, probably due to the profound hypoxic environment in these threedimensional structures, consequently leading to a strong HIF stabillization. Therefore, treatment of these experimental tumors with mTOR inhibitors is an effective tool to achieve size regression. The involvement of and the effect on HIF in this in vivo setting is nevertheless negligible.

Notch2 Signaling Regulates the Proliferation of Murine Bone Marrow-Derived Mesenchymal Stem/Stromal Cells via c-Myc Expression.

Mesenchymal stem/stromal cells (MSCs) reside in the bone marrow and maintain their stemness under hypoxic conditions. However, the mechanism underlying the effects of hypoxia on MSCs remains to be elucidated. This study attempted to uncover the signaling pathway of MSC proliferation. Under low-oxygen culture conditions, MSCs maintained their proliferation and differentiation abilities for a long term. The Notch2 receptor was up-regulated in MSCs under hypoxic conditions. Notch2-knockdown (Notch2-KD) MSCs lost their cellular proliferation ability and showed reduced gene expression of hypoxia-inducible transcription factor (HIF)-1α, HIF-2α, and c-Myc. Overexpression of the c-Myc gene in Notch2-KD MSCs allowed the cells to regain their proliferation capacity. These results suggested that Notch2 signaling is linked to c-Myc expression and plays a key role in the regulation of MSC proliferation. Our findings provide important knowledge for elucidating the self-replication competence of MSCs in the bone marrow microenvironment.

Inhibition of the hypoxia-inducible factor pathway by a G-quadruplex binding small molecule

The hypoxia-inducible transcription factor (HIF) co-ordinates the response of tumours to low oxygen by stimulating genes involved in metabolism and angiogenesis. HIF pathway activation is associated with decreased progression-free survival and increased mortality; compounds that target this pathway are potential agents for the treatment of a range of solid tumour malignancies. Renal cancers are likely to be particularly sensitive to inhibition of the HIF pathway since ~80% show constitutive activation of HIF. We have previously described the di-substituted naphthalene derivative, CL67, which binds to a G-quadruplex higher-order structure in the HIF promoter sequence in vitro. We show here that CL67 blocks HIF expression leading to inhibition of HIF-transactivation and down-regulation of downstream target genes and proteins in renal carcinoma cell lines and in a mouse xenograft model of renal cancer. This inhibition is independent of pathways that control HIF abundance through oxygen-dependant degradation and oxygen dependant HIF sub-unit expression.

Prolyl hydroxylase domain enzyme 2 is the major player in regulating hypoxic responses in rheumatoid arthritis

Rheumatoid arthritis (RA) is characterized by hypoxia and the expression of hypoxia-inducible transcription factors (HIFs), which coordinate cellular responses to hypoxia. The objective of this study was to analyze the expression and regulation of prolyl hydroxylase domain (PHD) enzymes and factor-inhibiting HIF-1α (FIH-1), which regulate cellular HIF levels, and to study the roles of these enzymes in RA fibroblast-like synoviocytes (RA FLS). The expression of PHD and FIH and downstream target genes was assessed by quantitative polymerase chain reaction and Western blotting. A small interfering RNA (siRNA) approach and an in vitro endothelial cell angiogenesis assay were used to analyze the roles of HIF hydroxylases. In human RA FLS, knockdown of PHD-2, but not knockdown of PHD-1 or FIH-1, dramatically augmented HIF-1α expression, modestly increased HIF-2α protein expression under normoxic conditions, and up-regulated HIF-dependent gene expression. In contrast, silencing of PHD-3 up-regulated HIF-2α but reduced HIF-1α, thereby decreasing the expression of HIF-regulated genes. A similar effect of PHD-2 knockdown was observed in osteoarthritis FLS (OA FLS) but not in nondiseased primary human dermal fibroblasts. These findings correlated with the induction of in vitro angiogenesis by supernatants from RA FLS and OA FLS transfected with siPHD-2 but not by supernatants from nondiseased fibroblasts or from siPHD-3-transfected cells. Our data suggest that PHD-2 is the major hydroxylase regulating HIF levels and the expression of angiogenic genes in arthritic cells. PHD-2 appears to regulate responses relevant to arthritis via HIF-α, highlighting the major importance of this enzyme in hypoxia- and angiogenesis-dependent inflammatory diseases such as RA.

Mutual Regulation of Hypoxia-Inducible Factor and Mammalian Target of Rapamycin as a Function of Oxygen Availability

The mammalian target of rapamycin (mTOR) regulates cellular growth and proliferation, mainly by controlling cellular translation. Most tumors show constitutive activation of the mTOR pathway. In hypoxia, mTOR is inactivated, which is believed to be part of the program of the cell to maintain energy homeostasis. However, certain proteins are believed to be preferentially translated during hypoxia via 5' terminal oligopyrimidine tract mechanisms with controversial discussion about the involvement of the mTOR-dependent ribosomal protein S6 (rpS6). The hypoxia-inducible transcription factor (HIF) is the master regulator of hypoxic adaptation and itself strongly implicated in tumor growth. HIF is translationally regulated by mTOR. The regulatory features and the involvement of molecular oxygen itself in this regulation of HIF by mTOR are poorly understood. mTOR inhibition leads to profound attenuation of HIFalpha protein in the majority of primary and cancer cells studied. Under severe hypoxia, no influence of mTOR inhibitors was observed; thus, stimulation of HIFalpha by mTOR may only be relevant under mild hypoxia or even normoxia. HIF expression and phosphorylated rpS6 negatively correlate in experimental tumors. In cell culture, prolonged hypoxia abolishes rpS6 phosphorylation, which seems to be partly independent of the upstream p70S6 kinase. We show that hypoxic repression of rpS6 is largely dependent on HIF, implicating a negative feedback loop, which may influence cellular translational rates and metabolic homeostasis. These data implicate that the hypoxic microenvironment renders tumor cells resistant to mTOR inhibition, at least concerning hypoxic gene activation, which would add to the difficulties of other established therapeutic strategies in hypoxic cancer tissues.

Hypoxia, Hypoxia-Inducible Transcription Factor, and Macrophages in Human Atherosclerotic Plaques Are Correlated With Intraplaque Angiogenesis

We sought to examine the presence of hypoxia in human carotid atherosclerosis and its association with hypoxia-inducible transcription factor (HIF) and intraplaque angiogenesis. Atherosclerotic plaques develop intraplaque angiogenesis, which is a typical feature of hypoxic tissue and expression of HIF. To examine the presence of hypoxia in atherosclerotic plaques, the hypoxia marker pimonidazole was infused before carotid endarterectomy in 7 symptomatic patients. Also, the messenger ribonucleic acid (mRNA) and protein expression of HIF1 alpha, HIF2 alpha, HIF-responsive genes (vascular endothelial growth factor [VEGF], glucose transporter [GLUT]1, GLUT3, hexokinase [HK]1, and HK2), and microvessel density were determined in a larger series of nondiseased and atherosclerotic carotid arteries with microarray, quantitative reverse transcription polymerase chain reaction, in situ hybridization, and immunohistochemistry. Pimonidazole immunohistochemistry demonstrated the presence of hypoxia, especially within the macrophage-rich center of the lesions. Hypoxia correlated with the presence of a thrombus, angiogenesis, and expression of CD68, HIF, and VEGF. The mRNA and protein expression of HIF, its target genes, and microvessel density increased from early to stable lesions, but no changes were observed between stable and ruptured lesions. This is the first study directly demonstrating hypoxia in advanced human atherosclerosis and its correlation with the presence of macrophages and the expression of HIF and VEGF. Also, the HIF pathway was associated with lesion progression and angiogenesis, suggesting its involvement in the response to hypoxia and the regulation of human intraplaque angiogenesis.

Hypoxic conditions increase hypoxia-inducible transcription factor 2α and enhance chondrogenesis in stem cells from the infrapatellar fat pad of osteoarthritis patients

Stem cells derived from the infrapatellar fat pad (IPFP) are a potential source of stem cells for the repair of articular cartilage defects. Hypoxia has been shown to improve chondrogenesis in adult stem cells. In this study we investigated the effects of hypoxia on gene expression changes and chondrogenesis in stem cells from the IPFP removed from elderly patients with osteoarthritis at total knee replacement. Adherent colony-forming cells were isolated and cultured from the IPFP from total knee replacement. The cells at passage 2 were characterised for stem cell surface epitopes, and then cultured for 14 days as cell aggregates in chondrogenic medium under normoxic (20% oxygen) or hypoxic (5% oxygen) conditions. Gene expression analysis, DNA and glycosoaminoglycan assays and immunohistochemical staining were determined to assess chondrogenesis. IPFP-derived adherent colony-forming cells stained strongly for markers of adult mesenchymal stem cells, including CD44, CD90 and CD105, and they were negative for the haematopoietic cell marker CD34 and for the neural and myogenic cell marker CD56. Cell aggregates of IPFP cells showed a chondrogenic response. In hypoxic conditions there was increased matrix accumulation of proteoglycan but less cell proliferation, which resulted in 3.5-fold more glycosoaminoglycan per DNA after 14 days of culture. In hypoxia there was increased expression of hypoxia-inducible transcription factor (HIF)2α and not HIF1α, and the expression of key transcription factors SOX5, SOX6 and SOX9, and that of aggrecan, versican and collagens II, IX, X and XI, was also increased. These results show that cells with stem cell characteristics were isolated from the IPFP of elderly patients with osteoarthritis and that their response to chondrogenic culture was enhanced by lowered oxygen tension, which upregulated HIF2α and increased the synthesis and assembly of matrix during chondrogenesis. This has important implications for tissue engineering applications of cells derived from the IPFP.

Expression of hypoxia-inducible transcription factors in developing human and rat kidneys

Early kidney development is associated with the coordinated branching of the renal tubular and vascular system and hypoxia has been proposed to be a major regulatory factor in this process. Under low oxygen levels, the hypoxia-inducible transcription factor (HIF) regulates the expression of genes involved in angiogenesis, erythropoiesis and glycolysis. To investigate the role of HIF in kidney development, we analyzed the temporal and spatial expression of the oxygen regulated HIF-1alpha and -2alpha subunits at different stages of rat and human kidney development. Using double-staining procedures, localization of the HIF target geneproducts vascular endothelial growth factor (VEGF) and endoglin was studied in relation to HIFalpha. In both species, we found marked nuclear expression of HIF-1alpha in medullary and cortical collecting ducts and in glomerular cells. In contrast, HIF-2alpha was expressed in interstitial and peritubular cells podocytes of the more mature glomeruli. After completion of glomerulogenesis and nephrogenesis, HIF-1alpha and -2alpha were no longer detectable. The HIF-target gene VEGF colocalized with HIF-1alpha protein in glomeruli and medullary collecting ducts. HIF-2alpha colocalized with the endothelium-associated angiogenic factor, endoglin. Both HIFalpha isoforms are activated in the developing kidney in a cell-specific and temporally controlled manner, indicating a regulatory role of oxygen tension in nephrogenesis. HIF-1alpha seems to be primarily involved in tubulogenesis and HIF-2alpha in renal vasculogenesis. Both isoforms are found in glomerulogenesis, potentially having synergistic effects.