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Hypoxia-inducible factors (HIFs) play a pivotal role as master regulators of tumor survival and growth, controlling a wide array of cellular processes in response to hypoxic stress. Clinical data correlates upregulated HIF-1 and HIF-2 levels with an aggressive tumor phenotype and poor patient outcome. Despite extensive validation as a target in cancer, pharmaceutical targeting of HIFs, particularly the interaction between α and βsubunits that forms the active transcription factor, has proved challenging. Nonetheless, many indirect inhibitors of HIFs have been identified, targeting diverse parts of this pathway. Significant strides have also been made in the development of direct inhibitors of HIF-2, exemplified by the FDA approval of Belzutifan for the treatment of metastatic clear cell renal carcinoma. While efforts to target HIF-1 using various therapeutic modalities have shown promise, no clinical candidates have yet emerged. This review aims to provide insights into the intricate and extensive role played by HIFs in cancer, and the ongoing efforts to develop therapeutic agents against this target. [ABSTRACT FROM AUTHOR]

Hypoxia-inducible factors (HIFs) are essential to the homeostasis of hypoxic tissues. Although HIF-2α, is expressed in nucleus pulposus (NP) cells, consequences of elevated HIF-2 activity on disc health remains unknown. We expressed HIF-2α with proline to alanine substitutions (P405A; P531A) in the Oxygen-dependent degradation domain (HIF-2αdPA) in the NP tissue using an inducible, nucleus pulposus-specific K19CreERT allele to study HIF-2α function in the adult intervertebral disc. Expression of HIF-2α in NP impacted disc morphology, as evident from small but significantly higher scores of degeneration in NP of 24-month-old K19CreERT; HIF-2αdPA (K19-dPA) mice. Noteworthy, comparisons of grades within each genotype between 14 months and 24 months indicated that HIF-2α overexpression contributed to more pronounced changes than aging alone. The annulus fibrosus (AF) compartment in the 14-month-old K19-dPA mice exhibited lower collagen turnover and Fourier transform-infrared (FTIR) spectroscopic imaging analyses showed changes in the biochemical composition of the 14- and 24-month-old K19-dPA mice. Moreover, there were changes in aggrecan, chondroitin sulfate, and COMP abundance without alterations in NP phenotypic marker CA3, suggesting the overexpression of HIF-2α had some impact on matrix composition but not the cell phenotype. Mechanistically, the global transcriptomic analysis showed enrichment of differentially expressed genes in themes closely related to NP cell function such as cilia, SLIT/ROBO pathway, and HIF/Hypoxia signaling at both 14- and 24-month. Together, these findings underscore the role of HIF-2α in the pathogenesis of disc degeneration in the aged spine. [ABSTRACT FROM AUTHOR]

Solid tumors are characterized by hypoxic areas, which are prone for macrophage infiltration. Once infiltrated, macrophages polarize to tumor associated macrophages (TAM) to support tumor progression. Therefore, the crosstalk between TAMs and tumor cells is of current interest for the development of novel therapeutic strategies. These may comprise induction of an iron- and lipid peroxidation-dependent form of cell death, known as ferroptosis. To study the macrophage - tumor cell crosstalk we polarized primary human macrophages towards a TAM-like phenotype, co-cultured them with HT1080 fibrosarcoma cells, and analyzed the tumor cell response to ferroptosis induction. In TAMs the expression of ceruloplasmin mRNA increased, which was driven by hypoxia inducible factor 2 and signal transducer and activator of transcription 1. Subsequently, ceruloplasmin mRNA was transferred from TAMs to HT1080 cells via extracellular vesicles. In tumor cells, mRNA was translated into protein to protect HT1080 cells from RSL3-induced ferroptosis. Mechanistically this was based on reduced iron abundance and lipid peroxidation. Interestingly, in naïve macrophages also hypoxia induced ceruloplasmin under hypoxia and a co-culture of HT1080 cells with hypoxic macrophages recapitulated the protective effect observed in TAM co-cultures. In conclusion, TAMs provoke tumor cells to release iron and thereby protect them from lipid peroxidation/ferroptosis.

Abstract Chemotherapy, radiotherapy, targeted therapy, and immunotherapy are established cancer treatment modalities that are widely used due to their demonstrated efficacy against tumors and favorable safety profiles or tolerability. Nevertheless, treatment resistance continues to be one of the most pressing unsolved conundrums in cancer treatment. Hypoxia-inducible factors (HIFs) are a family of transcription factors that regulate cellular responses to hypoxia by activating genes involved in various adaptations, including erythropoiesis, glucose metabolism, angiogenesis, cell proliferation, and apoptosis. Despite this critical function, overexpression of HIFs has been observed in numerous cancers, leading to resistance to therapy and disease progression. In recent years, much effort has been poured into developing innovative cancer treatments that target the HIF pathway. Combining HIF inhibitors with current cancer therapies to increase anti-tumor activity and diminish treatment resistance is one strategy for combating therapeutic resistance. This review focuses on how HIF inhibitors could be applied in conjunction with current cancer treatments, including those now being evaluated in clinical trials, to usher in a new era of cancer therapy.

Hypoxia-inducible factors (HIFs) are essential to the homeostasis of hypoxic tissues. Although HIF-2α, is expressed in nucleus pulposus (NP) cells, consequences of elevated HIF-2 activity on disc health remains unknown. We expressed HIF-2α with proline to alanine substitutions (P405A; P531A) in the Oxygen-dependent degradation domain (HIF-2αdPA) in the NP tissue using an inducible, nucleus pulposus-specific K19CreERT allele to study HIF-2α function in the adult intervertebral disc. Expression of HIF-2α in NP impacted disc morphology, as evident from small but significantly higher scores of degeneration in NP of 24-month-old K19CreERT; HIF-2αdPA (K19-dPA) mice. Noteworthy, comparisons of grades within each genotype between 14 months and 24 months indicated that HIF-2α overexpression contributed to more pronounced changes than aging alone. The annulus fibrosus (AF) compartment in the 14-month-old K19-dPA mice exhibited lower collagen turnover and Fourier transform-infrared (FTIR) spectroscopic imaging analyses showed changes in the biochemical composition of the 14- and 24-month-old K19-dPA mice. Moreover, there were changes in aggrecan, chondroitin sulfate, and COMP abundance without alterations in NP phenotypic marker CA3, suggesting the overexpression of HIF-2α had some impact on matrix composition but not the cell phenotype. Mechanistically, the global transcriptomic analysis showed enrichment of differentially expressed genes in themes closely related to NP cell function such as cilia, SLIT/ROBO pathway, and HIF/Hypoxia signaling at both 14- and 24-month. Together, these findings underscore the role of HIF-2α in the pathogenesis of disc degeneration in the aged spine.

Kaposi sarcoma‐associated herpesvirus (KSHV) is the causative agent of Kaposi sarcoma and several other tumors and hyperproliferative diseases seen predominantly in human immunodeficiency virus‐infected and other immunocompromised persons. There is an increasing body of evidence showing that hypoxia and hypoxia‐inducible factors (HIFs) play important roles in the biology of KSHV and in the pathogenesis of KSHV‐induced diseases. Hypoxia and HIFs can induce lytic activation of KSHV and KSHV can in turn lead to a hypoxic‐like state in infected cells. In this review, we describe the complex interactions between KSHV biology, the cellular responses to hypoxia, and the pathogenesis of KSHV‐induced diseases. We also describe how interference with HIFs can lead to decreased tumor growth and/or death of infected cells and KSHV‐induced tumors. Finally, we show how these observations may lead to novel strategies for the treatment of KSHV‐induced diseases. [ABSTRACT FROM AUTHOR]

Chemotherapy, radiotherapy, targeted therapy, and immunotherapy are established cancer treatment modalities that are widely used due to their demonstrated efficacy against tumors and favorable safety profiles or tolerability. Nevertheless, treatment resistance continues to be one of the most pressing unsolved conundrums in cancer treatment. Hypoxia-inducible factors (HIFs) are a family of transcription factors that regulate cellular responses to hypoxia by activating genes involved in various adaptations, including erythropoiesis, glucose metabolism, angiogenesis, cell proliferation, and apoptosis. Despite this critical function, overexpression of HIFs has been observed in numerous cancers, leading to resistance to therapy and disease progression. In recent years, much effort has been poured into developing innovative cancer treatments that target the HIF pathway. Combining HIF inhibitors with current cancer therapies to increase anti-tumor activity and diminish treatment resistance is one strategy for combating therapeutic resistance. This review focuses on how HIF inhibitors could be applied in conjunction with current cancer treatments, including those now being evaluated in clinical trials, to usher in a new era of cancer therapy. [ABSTRACT FROM AUTHOR]

The ability of cells to communicate with their surrounding is a prerequisite for essential processes such as proliferation, apoptosis, migration, and differentiation. To this purpose, primary cilia serve as antennae-like structures on the surface of most mammalian cell types. Cilia allow signaling via hedgehog, Wnt or TGF-beta pathways. Their length, in part controlled by the activity of intraflagellar transport (IFT), is a parameter for adequate function of primary cilia. Here we show, in murine neuronal cells, that intraflagellar transport protein 88 homolog (IFT88) directly interacts with the hypoxia-inducible factor-2α (HIF-2α), hitherto known as an oxygen-regulated transcription factor. Furthermore, HIF-2α accumulates in the ciliary axoneme and promotes ciliary elongation under hypoxia. Loss of HIF-2α affected ciliary signaling in neuronal cells by decreasing transcription of Mek1/2 and Erk1/2. Targets of the MEK/ERK signaling pathway, such as Fos and Jun, were significantly decreased. Our results suggest that HIF-2α influences ciliary signaling by interacting with IFT88 under hypoxic conditions. This implies an unexpected and far more extensive function of HIF-2α than described before. [ABSTRACT FROM AUTHOR]

BACKGROUND: Hypoxia-inducible factor is a key gene expression regulator in the bone hypoxia microenvironment. It is of great significance to understand the mechanism of hypoxia-inducible factors in bone formation and remodeling, and to analyze its effect on bone homeostasis imbalance diseases for exploring new ways to prevent and treat bone homeostasis imbalance diseases. OBJECTIVE: To consult relevant medical articles at home and abroad to review and analyze the mechanism of hypoxia-inducible factors in the process of bone formation and bone remodeling, as well as the research progress of the relationship between hypoxia-inducible factors and bone homeostasis disorders. METHODS: In April 2021, we searched CNKI, Wanfang, VIP, NCBI, and PubMed for articles published from 2000 to 2021, with the Chinese and English key words of “hypoxia inducible factor, bone formation, bone remodeling, bone homeostasis disorders, regulatory mechanism”. Through the sorting and analysis of relevant data, the mechanisms of hypoxia-inducible factors on bone formation and bone remodeling were reviewed and the effect of hypoxia-inducible factors on bone homeostasis disorders was analyzed. RESULTS AND CONCLUSION: (1) Hypoxia-inducible factor-1α overexpression in osteoblasts under the hypoxic stress promotes bone formation and increases long bone volume, while the significant upregulation of hypoxia-inducible factor-2α expression in osteoblasts promotes osteoclast maturation and differentiation and causes progressive bone loss. (2) Inhibition of hypoxia-inducible factor-1α expression in osteoblasts impairs osteoclast activity, reduces bone resorption, and improves bone remodeling. Hypoxia-inducible factor-2α mediates the regulation between osteoblasts and osteoclasts to achieve bone remodeling. (3) The mechanism of hypoxia-inducible factor-3 in bone formation and bone remodeling has not been studied yet. (4) In osteoporosis, the decrease in osteoblast number, osteogenic function and angiogenic capacity inhibits the expression of hypoxia-inducible factor-α, causing a decrease in bone mass and bone density, which increases the risk of fracture. (5) In osteoarthritis, hypoxia-inducible factor-2α is significantly overexpressed, which stimulates chondrocyte apoptosis mediated by regulating the expression of inflammatory factors, matrix catabolic enzymes and other catabolic genes, and accelerates cartilage component. (6) In chondrosarcoma, neoangiogenesis is associated with hypoxia-inducible factor-1α overexpression, and hypoxia-inducible factor-1α can be used as a malignant marker associated with tumor neoangiogenesis in chondrosarcoma, and hypoxia-inducible factor-1 inhibition therapy can interfere with angiogenesis in chondrosarcoma. (7) Hypoxia-inducible factor plays an important role in regulating the angiogenesis process and the proliferation and activity of osteoblasts, osteoclasts, and chondrocytes in bone homeostasis imbalance diseases. The development of hypoxia-inducible factor-related activating or inhibiting drugs is very positive for repairing bone defects, accelerating fracture healing, improving bone joint inflammation and other bone homeostasis imbalance lesions. [ABSTRACT FROM AUTHOR]

Bortezomib is the first proteasomal inhibitor (PI) to be used therapeutically for treating relapse cases of multiple myeloma and mantle cell lymphoma. A proposed mechanism for its action is that it prevents the proteasomal degradation of proapoptotic proteins, leading to enhanced apoptosis. Although the α subunit of hypoxia-inducible factor (HIF)-1 is not degraded with bortezomib treatment, the heterodimeric HIF-1 fails to transactivate target genes. HIF-1 and HIF-2 are related hypoxia-inducible transcription factors that are important for the survival of hypoxic tumor cells. The majority of reports have focused on the effects of bortezomib on the transcriptional activities of HIF-1, but not HIF-2. The present study investigated the effects of bortezomib on HIF-2 activity in cancer cells with different levels of HIF-1α and HIF-2α subunits. HIF-α subunit levels were detected using specific antibodies, while HIF transcriptional activities were evaluated using immunodetection, reverse transcription-polymerase chain reaction and luciferase reporter assay. Bortezomib treatment was found to suppress the transcription and expression of CA9, a HIF-1-specific target gene; however, it had minimal effects on EPO and GLUT-1, which are target genes of both HIF-1 and HIF-2. These data suggest that bortezomib attenuates the transcriptional activity only of HIF-1, and not HIF-2. This novel finding on the lack of an inhibitory effect of bortezomib on HIF-2 transcriptional activity has implications for the improvement of design and treatment modalities of bortezomib and other PI drugs.

HIF-2 represents a tissue-specific isoform of the hypoxia-inducible factors (HIFs) which regulate oxygen homeostasis in the cell. In acute oxygen deficiency, HIF transcription factors ensure the timely restoration of adequate oxygen supply. Particularly in medical conditions such as stroke, which have a high mortality risk due to ischaemic brain damage, rapid recovery of oxygen supply is of extraordinary importance. Nevertheless, the endogenous mechanisms are often not sufficient to respond to severe hypoxic stress with restoring oxygenation and fail to protect the tissue. Herein, we analysed murine neurospheres without functioning HIF-2α and found that special importance in the differentiation of neurons can be attributed to HIF-2 in the brain. Other processes, such as cell migration and signal transduction of different signalling pathways, appear to be mediated to some extent via HIF-2 and illustrate the function of HIF-2 in brain remodelling. Without hypoxic stress, HIF-2 in the brain presumably focuses on the fine-tuning of the neural network. However, a therapeutically increase of HIF-2 has the potential to regenerate or replace destroyed brain tissue and help minimize the consequences of an ischaemic stroke. [ABSTRACT FROM AUTHOR]

Solid tumors are characterized by hypoxic areas, which are prone for macrophage infiltration. Once infiltrated, macrophages polarize to tumor associated macrophages (TAM) to support tumor progression. Therefore, the crosstalk between TAMs and tumor cells is of current interest for the development of novel therapeutic strategies. These may comprise induction of an iron- and lipid peroxidation-dependent form of cell death, known as ferroptosis. To study the macrophage - tumor cell crosstalk we polarized primary human macrophages towards a TAM-like phenotype, co-cultured them with HT1080 fibrosarcoma cells, and analyzed the tumor cell response to ferroptosis induction. In TAMs the expression of ceruloplasmin mRNA increased, which was driven by hypoxia inducible factor 2 and signal transducer and activator of transcription 1. Subsequently, ceruloplasmin mRNA was transferred from TAMs to HT1080 cells via extracellular vesicles. In tumor cells, mRNA was translated into protein to protect HT1080 cells from RSL3-induced ferroptosis. Mechanistically this was based on reduced iron abundance and lipid peroxidation. Interestingly, in naïve macrophages also hypoxia induced ceruloplasmin under hypoxia and a co-culture of HT1080 cells with hypoxic macrophages recapitulated the protective effect observed in TAM co-cultures. In conclusion, TAMs provoke tumor cells to release iron and thereby protect them from lipid peroxidation/ferroptosis., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Abstract Hypoxia-inducible factors (HIF) are master regulators of the response to hypoxia. Although several kinases are known to modify their oxygen sensitive HIF-α subunits or affect indirectly their function, little is known about the role of phosphatases in HIF control. To address this issue, a library containing siRNAs for the 25 known catalytic subunits of human phosphatases was used to screen for their effect on HIF transcriptional activity in HeLa cells. Serine-threonine phosphatase PPP3CA (calcineurin A, isoform a) was identified as the strongest candidate for a negative regulator of HIF activity. Indeed, independent silencing of PPP3CA expression stimulated HIF transcriptional activity under hypoxia, without increasing the protein levels of HIF-1α or HIF-2α. Overexpression of a constitutively active PPP3CA form, but not its catalytically inactive counterpart, inhibited HIF activity and expression of HIF target genes but did not affect HIF-1α or HIF-2α expression. These results were phenocopied by treatment with the ionophore ionomycin, that activates endogenous PPP3CA. The effect of ionomycin was mediated by PPP3CA as it was largely abolished by PPP3CA silencing. Furthermore, ionomycin enhanced the down-regulation of HIF activity by wild-type PPP3CA overexpression. Overall, our results suggest the involvement of PPP3CA in fine-tuning the HIF-dependent transcriptional response to hypoxia. [ABSTRACT FROM AUTHOR]

Hypoxia-inducible transcription factors (HIFs) drive angiogenesis and cancer cell growth, contributing to an aggressive tumor phenotype. HIF-α protein levels and activity are controlled at the post-translational level by HIF hydroxylases. Hydroxylated HIF-α is recognized by the von Hippel Lindau (VHL) tumor suppressor and targeted for degradation. The HIF hydroxylases are members of the iron and 2-oxoglutarate-dependent dioxygenases, which require ascorbate as cofactor for activity. Clear cell renal cell carcinomas (ccRCC) harbor mutations in the VHL gene, whereas papillary RCC (pRCC) have a functional VHL. These natural occurring VHL variants in RCC enable the testing, in clinical samples, of the hypothesis that ascorbate modulates HIF-α levels through its role as a cofactor for the HIF hydroxylases. We measured ascorbate, HIF-1α, and HIF-2α protein and HIF downstream targets BNIP3, CA9, cyclin D1, GLUT1, and VEGF (combined to generate the HIF pathway score) in VHL-defective ccRCC (n = 73) and VHL-proficient pRCC human tumor tissue (n = 41). HIF and ascorbate levels were increased in ccRCC and pRCC tumors compared to matched renal cortex. HIF-1 and total HIF pathway activation scores were decreased with higher ascorbate in pRCC tumors (Spearman r = −0.38, p < 0.05 and r = −0.35, p < 0.05). This was not evident for ccRCC tumors. In mechanistic studies in vitro, ascorbate influenced HIF-1 activity in VHL-proficient, but not VHL-defective ccRCC cells. Our results indicate that ccRCC, which lacks a functional VHL, does not respond to ascorbate-mediated modulation of the HIF response. This contrasts with the demonstrated association between ascorbate content and the HIF pathway observed in pRCC and other tumors with a functional VHL. The results support a role for ascorbate as a modulator of HIF activity and tumor aggression in cancer types with a functional hypoxic response.

Hypoxia-inducible transcription factors (HIFs) drive angiogenesis and cancer cell growth, contributing to an aggressive tumor phenotype. HIF-α protein levels and activity are controlled at the post-translational level by HIF hydroxylases. Hydroxylated HIF-α is recognized by the von Hippel Lindau (VHL) tumor suppressor and targeted for degradation. The HIF hydroxylases are members of the iron and 2-oxoglutarate-dependent dioxygenases, which require ascorbate as cofactor for activity. Clear cell renal cell carcinomas (ccRCC) harbor mutations in the VHL gene, whereas papillary RCC (pRCC) have a functional VHL. These natural occurring VHL variants in RCC enable the testing, in clinical samples, of the hypothesis that ascorbate modulates HIF-α levels through its role as a cofactor for the HIF hydroxylases. We measured ascorbate, HIF-1α, and HIF-2α protein and HIF downstream targets BNIP3, CA9, cyclin D1, GLUT1, and VEGF (combined to generate the HIF pathway score) in VHL-defective ccRCC (n = 73) and VHL-proficient pRCC human tumor tissue (n = 41). HIF and ascorbate levels were increased in ccRCC and pRCC tumors compared to matched renal cortex. HIF-1 and total HIF pathway activation scores were decreased with higher ascorbate in pRCC tumors (Spearman r = −0.38, p < 0.05 and r = −0.35, p < 0.05). This was not evident for ccRCC tumors. In mechanistic studies in vitro , ascorbate influenced HIF-1 activity in VHL-proficient, but not VHL-defective ccRCC cells. Our results indicate that ccRCC, which lacks a functional VHL, does not respond to ascorbate-mediated modulation of the HIF response. This contrasts with the demonstrated association between ascorbate content and the HIF pathway observed in pRCC and other tumors with a functional VHL. The results support a role for ascorbate as a modulator of HIF activity and tumor aggression in cancer types with a functional hypoxic response. [ABSTRACT FROM AUTHOR]

Abstract: Targeting transcription factors HIF‐2 is currently considered to be the most direct way for the therapy of clear cell renal cell carcinoma. The preclinical inhibitor PT2399 and artificial inhibitor 0X3 have been identified as promising on‐target inhibitors to inhibit the heterodimerization of HIF‐2. However, the inhibition mechanism of PT2399 and 0X3 on HIF‐2 remains unclear. To this end, molecular dynamics (MD) simulations and molecular docking were applied to investigate the effects of 2 inhibitors on structural motifs and heterodimerization of HIF‐2. Our simulation results reveal that the binding of inhibitors disrupts the crucial hydrogen bond and hydrophobic interactions of interdomain of HIF‐2 heterodimer due to the local conformational changes of binding interface, confirming the hypothesis that the perturbation of few residues is sufficient to disrupt the heterodimerization of HIF‐2. In addition, it can be found that PT2399 with dominant substituents (cyano, fluorine, sulfuryl, and hydroxyl) is more preferred than 0X3 as HIF‐2 inhibitor and these substituents play a crucial role in involving more hydrogen bond interactions with residues of interface and then cause the larger structural change of protein. This study may provide a deeper atomic‐level insight into the effect of on‐target inhibitors on HIF‐2 heterodimer, which is expected to contribute to further rational design of effective clear cell renal cell carcinoma drugs. [ABSTRACT FROM AUTHOR]

Abstract: Hypoxia‐inducible factors mediate adaptive responses to reduced O2 availability. In patients with obstructive sleep apnoea, repeated episodes of hypoxaemia and reoxygenation (intermittent hypoxia) are sensed by the carotid body (CB). The ensuing CB chemosensory reflex activates the sympathetic nervous system and increased secretion of catecholamines by the adrenal medulla, resulting in hypertension and breathing abnormalities. In the CB, intermittent hypoxia induces the formation of reactive oxygen species (ROS) and increased intracellular Ca2+ levels, which drive increased expression of hypoxia‐inducible factor (HIF) 1α and a decrease in the levels of HIF‐2α.  Intermittent hypoxia increases HIF‐1α‐dependent expression of Nox2, encoding the pro‐oxidant enzyme NADPH oxidase 2, and decreased HIF‐2α‐dependent expression of Sod2, encoding the anti‐oxidant enzyme superoxide dismutase 2. These changes in gene expression drive persistently elevated ROS levels in the CB, brainstem, and adrenal medulla that are required for the development of hypertension and breathing abnormalities. The ROS generated by dysregulated HIF activity in the CB results in oxidation and inhibition of haem oxygenase 2, and the resulting reduction in the levels of carbon monoxide leads to increased hydrogen sulfide production, triggering glomus cell depolarization. Thus, the pathophysiology of obstructive sleep apnoea involves the dysregulation of O2‐regulated transcription factors, gasotransmitters, and sympathetic outflow that affects blood pressure and breathing. [ABSTRACT FROM AUTHOR]

The role of microRNAs in controlling angiogenesis is recognized as a promising therapeutic target in both cancer and cardiovascular disorders. However, understanding a miRNA’s pleiotropic effects on angiogenesis is a limiting factor for these types of therapeutic approaches. Using genome-wide next-generation sequencing, we examined the role of an antiangiogenic miRNA, miR-200b, in primary human endothelial cells. The results indicate that miR-200b has complex effects on hypoxia-induced angiogenesis in human endothelia and importantly, that many of the reported miR-200b effects using miRNA overexpression may not be representative of the physiological role of this miRNA. We also identified the antiangiogenic KLF2 gene as a novel target of miR-200b. Our studies indicate that the physiological changes in miR-200b levels during acute hypoxia may actually have a proangiogenic effect through Klf2 downregulation and subsequent stabilization of HIF-1 signaling. Moreover, we provide a viable approach for differentiating direct from indirect miRNA effects in order to untangle the complexity of individual miRNA networks. [ABSTRACT FROM AUTHOR]

Hypoxia-inducible factor-2α ( HIF-2α) plays an important role in increasing cancer progression and distant metastasis in a variety of tumour types. We aimed to investigate its biological function and clinical significance in human pancreatic ductal adenocarcinoma ( PDAC). A total of 283 paired PDAC tissues and adjacent normal tissues were collected from patients who underwent surgery or biopsy at Sun Yat-sen Memorial Hospital between February 2004 and October 2016. In this study, we noted that HIF-2α expression was significantly up-regulated in PDAC, positively associated with disease stage, lymph-node metastasis and patient survival, and identified as an independent prognostic factor of PDAC patients. We demonstrated that HIF-2α silencing could reduce proliferation, migration and invasion of PDAC cells in vitro. The similar effect on growth was demonstrated in vivo. Furthermore, we noted that knock-down of HIF-2α significantly decreased the expression of glutamate oxaloacetate transaminase 1 ( GOT1). Importantly, we confirmed that the PI3K/ mTORC2 pathway promoted GOT1 expression by targeting HIF-2α. Our study validated HIF-2α was an important factor in PDAC progression and poor prognosis and may promote non-canonical glutamine metabolism via activation of PI3K/ mTORC2 pathway. Targeting HIF-2α represents a novel prognostic biomarker and therapeutic target for patients with PDAC. [ABSTRACT FROM AUTHOR]

Here, we have investigated the participation of nuclear factors NF-kB, HIF-1 and HIF-2, VEGF, VEGFR2, and carboanhydrase IX in clear-cell renal cancer. We have determined the expression and protein level of transcription factors, VEGF, VEGFR2, and carboanhydrase IX in tumor and normal tissues of 30 patients with kidney cancer. The Real-Time PCR and ELISA were used in the study. The low levels of HIF-1 mRNA expression associated with high levels of HIF-1 protein were also associated with metastasis. The expression levels of VEGF, VEGFR2, and their protein levels are increased in primary tumors of patients with disseminated kidney cancer compared to nonmetastatic cancer. No correlation was revealed between the content of mRNA and encoded proteins in the kidney cancer tissues. The changes in the ratios of mRNA levels and the respective proteins (HIF-1α, HIF-2, NF-kB, VEGF, VEGFR2, and carboanhydrase IX) may contribute to kidney- cancer metastasis. [ABSTRACT FROM AUTHOR]

[Display omitted] Benzisothiazole dioxide compound was reported to agonize HIF-2 stabilization and improve EPO production, thus conceiving a potential strategy to treat disease with chronic hypoxia exemplified by renal anemia. Herein, on the bases of multiple molecular and cellular assays, a series of benzisothiazole derivatives have been synthesized and their structure–activity relationship was evaluated. The SAR and molecular docking studies have revealed the structural insights on the rational design of HIF-2 agonist and discovered a more potential 5-bromine substituted analogue, which showed 2–4 times improvement of HIF-2 downstream gene transcriptions, including EPO production. The present results suggest the therapeutic potential of the compounds for diseases related to EPO insufficiency. [ABSTRACT FROM AUTHOR]

Hypoxia inducible factor 2 (HIF-2) is a transcriptional activator implicated in the cellular response to hypoxia. Regulation of its inducible subunit, HIF-2α (also known as EPAS1), involves posttranslational modifications. Here, we demonstrate that casein kinase 1δ (CK1δ; also known as CSNK1D) phosphorylates HIF-2α at Ser383 and Thr528 in vitro. We found that disruption of these phosphorylation sites, and silencing or chemical inhibition of CK1δ, reduced the expression of HIF-2 target genes and the secretion of erythropoietin (EPO) in two hepatic cancer cell lines, Huh7 and HepG2, without affecting the levels of HIF-2α protein expression. Furthermore, when CK1δ-dependent phosphorylation of HIF-2α was inhibited, we observed substantial cytoplasmic mislocalization of HIF-2α, which was reversed upon the addition of the nuclear protein export inhibitor leptomycin B. Taken together, these data suggest that CK1δ enhances EPO secretion from liver cancer cells under hypoxia by modifying HIF-2α and promoting its nuclear accumulation. This modification represents a new mechanism of HIF-2 regulation that might allow HIF isoforms to undertake differing functions. [ABSTRACT FROM AUTHOR]

PDGFR-β-expressing cells of the kidneys are considered as a relevant site of erythropoietin (EPO) production. The origin of these cells, their contribution to renal EPO production, and if PDGFR-β-positive cells in other organs are also capable to express EPO are less clear. We addressed these questions in mice, in which hypoxia-inducible transcription factors were stabilized in PDGFR-β cells by inducible deletion of the von Hippel-Lindau (Vhl) protein. Vhl deletion led to a 600-fold increase of plasma EPO concentration, 170-fold increase of renal EPO messenger RNA (mRNA) levels, and an increase of hematocrit values up to 70 %. Intrarenal localization of EPO-expressing cells coincided with the zonal heterogeneity and distribution of cells expressing PDGFR-β. Amongst a variety of extrarenal organs only adrenal glands showed significant EPO mRNA expression after Vhl deletion in PDGFR-β cells. EPO mRNA, plasma EPO, and hematocrit fell to subnormal values if HIF-2α, but not HIF-1α, was deleted either alone or in combination with Vhl in PDGFR-β cells. Treatment of mice with a prolyl-hydroxylase inhibitor caused an increase of EPO mRNA abundance and plasma EPO concentrations in wild-type mice and in mice lacking HIF-1α in PDGFR-β cells but exerted no effect in mice lacking HIF-2α in PDGFR-β cells. These findings suggest that PDGFR-β cells are the only relevant site of EPO expression in the kidney and that HIF-2 is the essential transcription factor triggering EPO expression therein. Moreover, our findings suggest that PDGFR-β cells elaborating EPO might arise from the metanephric mesenchyme, rather than from the neural crest. [ABSTRACT FROM AUTHOR]

Aims Intervertebral disc (IVD) degeneration was believed to contribute to lower back pain. The aim of the study was to investigate the pathogenesis and regulatory mechanism of puncture-induced IVD degeneration. Main methods We established a rat-tail puncture model using Kirschner wire and a homemade stopper. The progress of disc degeneration was evaluated by histological examination and the quantitative measurement of type I, type II collagen and other factors expression at 0.5, 1, 2, 6, and 12 weeks after puncture and was compared with control rats of the same age. Key findings Histological examination and Safranin-O staining revealed progressive degeneration of the punctured disc. Matrix metalloproteinase 13 (MMP13) was increased at 1 week after puncture but did not change in the control group. The interleukin-1 beta (IL-1β) mRNA expression level was elevated at the acute stage after puncture compared with the control group. The hypoxia inducible factor 2 (HIF-2) increased expression in punctured groups. Additionally, compare to adjacent non-punctured segments, HIF-2 α expression level transiently increased and then decreased in the nucleus pulposus immediately following puncture, and it then increased 12 weeks after puncture. Significance The degenerative changes observed in this rat-tail puncture model are similar to human disc degeneration and that this model may be valuable for elucidating the molecular mechanisms and pathways underlying disc degeneration. [ABSTRACT FROM AUTHOR]

Mitochondrial dysfunction plays an important role in obesity-induced cardiac impairment. SIRT3 is a mitochondrial protein associated with increased human life span and metabolism. This study investigated the functional role of SIRT3 in obesity-induced cardiac dysfunction. Wild-type ( WT) and SIRT3 knockout ( KO) mice were fed a normal diet ( ND) or high-fat diet ( HFD) for 16 weeks. Body weight, fasting glucose levels, reactive oxygen species ( ROS) levels, myocardial capillary density, cardiac function and expression of hypoxia-inducible factor ( HIF)-1α/-2α were assessed. HFD resulted in a significant reduction in SIRT3 expression in the heart. Both HFD and SIRT3 KO mice showed increased ROS formation, impaired HIF signalling and reduced capillary density in the heart. HFD induced cardiac hypertrophy and impaired cardiac function. SIRT3 KO mice fed HFD showed greater ROS production and a further reduction in cardiac function compared to SIRT3 KO mice on ND. Thus, the adverse effects of HFD on cardiac function were not attributable to SIRT3 loss alone. However, HFD did not further reduce capillary density in SIRT3 KO hearts, implicating SIRT3 loss in HFD-induced capillary rarefaction. Our study demonstrates the importance of SIRT3 in preserving heart function and capillary density in the setting of obesity. Thus, SIRT3 may be a potential therapeutic target for obesity-induced heart failure. [ABSTRACT FROM AUTHOR]

Sphingosine kinase 1 (SK1), the enzyme responsible for sphingosine 1-phosphate (S1P) production, is overexpressed in many human solid tumors. However, its role in clear cell renal cell carcinoma (ccRCC) has not been described previously. ccRCC cases are usually associated with mutations in von Hippel-Lindau (VHL) and subsequent normoxic stabilization of hypoxia-inducible factor (HIF). We previously showed that HIF-2α up-regulates SK1 expression during hypoxia in glioma cells. Therefore, we hypothesized that the stabilized HIF in ccRCC cells will be associated with increased SK1 expression. Here, we demonstrate that SK1 is overexpressed in 786-0 renal carcinoma cells lacking functional VHL, with concomitant high S1P levels that appear to be HIF-2α mediated. Moreover, examining the TCGA RNA seq database shows that SK1 expression was ∼2.7-fold higher in solid tumor tissue from ccRCC patients, and this was associated with less survival. Knockdown of SK1 in 786-0 ccRCC cells had no effect on cell proliferation. On the other hand, this knockdown resulted in an ∼3.5-fold decrease in invasion, less phosphorylation of focal adhesion kinase (FAK), and an ∼2-fold decrease in angiogenesis. Moreover, S1P treatment of SK1 knockdown cells resulted in phosphorylation of FAK and invasion, and this was mediated by S1P receptor 2. These results suggest that higher SK1 and S1P levels in VHL-defective ccRCC could induce invasion in an autocrine manner and angiogenesis in a paracrine manner. Accordingly, targeting SK1 could reduce both the invasion and angiogenesis of ccRCC and therefore improve the survival rate of patients. [ABSTRACT FROM AUTHOR]

ABSTRACT We investigated the feasibility of the intra-articular injection of resveratrol for preventing the progression of existing cartilage degeneration in a mouse model of osteoarthritis (OA). The effects of resveratrol on the expression of silent information regulator 2 type 1 (SIRT1), hypoxia-inducible factor-2α (HIF-2α) and catabolic factors in OA cartilage was explored. OA was induced in the mouse knee via destabilization of the medial meniscus (DMM). Resveratrol was injected weekly into the operated knee beginning 4 weeks after surgery. The OA phenotype was evaluated via histological and immunohistochemical analyses at 8 weeks after DMM. Western blot analysis was performed to identify whether resveratrol modulated the interleukin (IL)-1β-induced expression of HIF-2α in human chondrocytes. Histologically, resveratrol treatment preserved the structural homeostasis of the articular cartilage and the subchondral bone. Following resveratrol injection, the expression of collagen type II was retained, but the expression of inducible nitric oxide synthase and matrix metalloproteinase-13 was reduced in OA cartilage. Moreover, the administration of resveratrol significantly induced the activation of SIRT1 and the inhibition of HIF-2α expression in mouse OA cartilage and in IL-1β-treated human chondrocytes. These findings indicate that the intra-articular injection of resveratrol significantly prevents the destruction of OA cartilage by activating SIRT1 and thereby suppressing the expression of HIF-2α and catabolic factors. © 2015 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 33:1061-1070, 2015. [ABSTRACT FROM AUTHOR]

The hypoxic condition is a common feature that negatively impacts the efficacy of radio- and chemotherapy in solid tumors. Hypoxia-inducible factors (HIF-1, 2, 3) predominantly regulate the adaptation to hypoxia at the cellular or organismal level. HIF-2 is one of the three known alpha subunits of HIF transcription factors. Previous studies have shown that HIF-1 is associated with chemotherapy failure. Accumulating evidence in recent years suggests that HIF-2 also contributes to chemo/radioresistance in solid tumors. Despite sharing similar structures, HIF-1α and HIF-2α had highly divergent and even opposing roles in solid tumors under hypoxic conditions. Recent studies have also implied that HIF-2α had a role in chemo/radioresistance through different mechanisms, at least partly, compared to HIF-1α. The present paper summarizes the function of HIF-2 in chemo/radioresistance in solid tumors as well as some of its novel mechanisms that contributed to this pathological process. [ABSTRACT FROM AUTHOR]

Hypoxia-inducible factors (HIFs) 1 and 2 are dimeric α/β transcription factors that regulate cellular responses to low oxygen. HIF-1 is induced first, whereas HIF-2 is associated with chronic hypoxia. To determine how HIF1AmRNA, the inducible subunit of HIF-1, is regulated during hypoxia, we followed HIF1A mRNA levels in primary HUVECs over 24 hours using quantitative PCR. HIF1A and VEGF A (VEGFA) mRNA, a transcriptional target of HIF-1, increased ~2.5- and 8-fold at 2-4 hours, respectively. To determine how the mRNAs were regulated, we identified a microRNA (miRNA), miR-429, that destabilized HIF1A message and decreased VEGFA mRNA by inhibiting HIF1A Target protector analysis, which interferes with miRNA-mRNA complex formation, confirmed that miR-429 targeted HIF1A message. Desferox-amine treatment, which inhibits the hydroxylases that promote HIF-1 a protein degradation, stabilized HIF-1 activity during normoxic conditions and elevated miR-429 levels, demonstrating that HIF-1 promotes miR-429 expression. RNA-sequencing-based transcriptome analysis indicated that inhibition of miRNA-429 in HUVECs up-regulated 209 mRNAs, a number of which regulate angio-genesis. The results demonstrate that HIF-1 is in a negative regulatory loop with miR-429, that miR-429 attenuates HIF-1 activity by decreasing HIF1A message during the early stages of hypoxia before HIF-2 is activated, and this regulatory network helps explain the HIF-1 transition to HIF-2 during chronic hypoxia in endothelial cells. [ABSTRACT FROM AUTHOR]

Under obese conditions, adipose tissue can become oxygen-deficient or hypoxic. Extensive work has been done using various diet-induced obesity models to demonstrate an important role of hypoxia-induced signaling in adipose tissue and its impact on adipose functions related to adipogenesis, insulin sensitivity, and inflammation.We have recently identified a new mechanism connecting activation of the hypoxia-sensing pathway manifested by hypoxia-inducible factor (HIF) 2α to adipose tissue inflammation and hypertrophic cardiomyopathy. Interestingly, this observation is consistent with the clinical evidence showing that obesity is often associated with ventricular hypertrophy and dysfunction as well as congestive heart failure independent of other well-established risk factors including diabetes, hypertension, and coronary artery disease. This brief review will discuss the currently published genetic mouse models to determine the role of the HIF pathway in adipose tissue-associated diseases with a focus on the newly identified role of adipocyte HIF-2 in the development of hypertrophic cardiomyopathy. [ABSTRACT FROM AUTHOR]

An introduction is presented in which the editors discuss various reports within the issue on the relationship between obesity and hypoxia.

Glioblastoma multiforme (GBM) is the deadliest form of primary brain cancer. Several reports have indicated aberrant levels of βIII-tubulin (βIII-t) in human GBM. βIII-t overexpression was linked to increasing malignancy in glial tumors and described to determine the onset of resistance to chemotherapy. Furthermore, a linkage was suggested between the induction of βIII-t expression and hypoxia, a hallmark of GBM. We investigated the role of hypoxia-inducible factor (HIF)-1α and HIF-2α in the regulation of the βIII-t gene ( TUBB3) in GBM cells cultured in either normoxia or hypoxia. We report for the first time that HIF-2α, but not HIF-1α, is involved in hypoxia-induced βIII-t expression in GBM cells. By gene-reporter experiments and site-directed mutagenesis, we found that two overlapping hypoxia response elements located in the 3′ UTR of the gene were involved in the activation of TUBB3. This occurred through an enhanced binding of HIF-2α to the 3′ region, as revealed by an electrophoretic mobility shift assay. Conversely, the promoter of TUBB3 was shown to be inactive. In addition, we observed that HIF-1α exhibits a repressive effect on βIII-t expression in cells cultured in normoxia. These results show that both HIF-α isoforms have opposing effects on βIII-t expression in GBM cells. Finally, we observed that hypoxia-induced βIII-t expression is well correlated with the kinetics of HIF-2α protein stabilization. The evidence for a direct linkage between HIF-2α and increased expression of βIII-t by hypoxia suggests that an anti-HIF-2α strategy (i.e. by downregulating βIII-t) could be of potential interest for improving the treatment of GBM. [ABSTRACT FROM AUTHOR]

Background Cardiovascular patients suffer from reduced blood flow leading to ischaemia and impaired tissue metabolism. Unfortunately, an increasing group of elderly patients cannot be treated with current revascularization methods. Thus, new treatment strategies are urgently needed. Hypoxia-inducible factors ( HIFs) upregulate the expression of angiogenic mediators together with genes involved in energy metabolism and recovery of ischaemic tissues. Especially, HIF-2α is a novel factor, and only limited information is available about its therapeutic potential. Methods Gene transfers with adenoviral HIF-1α and HIF-2α were performed into the mouse heart and rabbit ischaemic hindlimbs. Angiogenesis was evaluated by histology. Left ventricle function was analysed with echocardiography. Perfusion in rabbit skeletal muscles and energy recovery after electrical stimulation-induced exercise were measured with ultrasound and 31 P-magnetic resonance spectroscopy (31 P- MRS), respectively. Results HIF-1α and HIF-2α gene transfers increased capillary size up to fivefold in myocardium and ischaemic skeletal muscles. Perfusion in skeletal muscles was increased by fourfold without oedema. Especially, Ad HIF-1α enhanced the recovery of ischaemic muscles from electrical stimulation-induced energy depletion. Special characteristic of HIF-2α gene transfer was a strong capillary growth in muscle connective tissue and that HIF-2α gene transfer maintained left ventricle function. Conclusions We conclude that both Ad HIF-1α and Ad HIF-2α gene transfers induced beneficial angiogenesis in vivo. Transient moderate increases in angiogenesis improved energy recovery after exercise in ischaemic muscles. This study shows for the first time that a moderate increase in angiogenesis is enough to improve tissue energy metabolism, which is potentially a very useful feature for cardiovascular gene therapy. [ABSTRACT FROM AUTHOR]

Adult hematopoiesis depends on rare multipotent hematopoietic stem cells (HSCs) that self-renew and give rise to progenitor cells, which differentiate to all blood lineages. The strict regulation of the fine balance between self-renewal and differentiation is essential for normal hematopoiesis and suppression of leukemia development. HSCs and progenitor cells are commonly assumed to reside within the hypoxic BM microenvironment, however, there is no direct evidence supporting this notion. Nevertheless, HSCs and progenitors do exhibit a hypoxic profile and strongly express Hif-1α. Although hypoxia signaling pathways are thought to play important roles in adult HSC maintenance and leukemogenesis, the precise function of Hif-dependent signaling in HSCs remains to be uncovered. Here we discuss recent gain-of-function and loss-of-function studies that shed light on the complex roles of hypoxia-signaling pathways in HSCs and their niches in normal and malignant hematopoiesis. Importantly, we comment on the current and often contrasting interpretations of the role of Hif-dependent signaling in stem cell functions. S tem C ells 2014;32:1390-1397 [ABSTRACT FROM AUTHOR]

Abstract: Background: Pre-clinical studies have implicated hypoxia inducible factor (HIF)-2α as an important oncogene for clear cell renal cell carcinoma (ccRCC). Generally considered to act as a nuclear transcription factor, a recent study has also implicated HIF-2α as a protein translational initiation complex function within the cytoplasm (Uniacke et al., 2012). We hypothesised that both the absolute expression as well as the sub-cellular localisation of HIF-2α would predict clinicopathological features and cancer specific survival (CSS) in ccRCC. Methods: A tissue microarray (TMA) study was conducted on three hundred and eight ccRCC patients. Survival differences were investigated with the log rank test and associations with CSS with uni- and multivariate Cox regression analyses. Recursive partition tree analysis was used to identify relevant cutoff values. Results: High HIF-2α nuclear (N) (cutoff >32%) expression was associated with smaller tumour sizes (p =0.002) and lower Fuhrman grades (p =0.044), whereas tumours with high cytoplasmic (C) HIF-2α (>0%) more often had positive lymph nodes (p =0.004), distant metastases (p =0.021) and higher Fuhrman grades (p <0.0001). After adjustment for TNM stage, Eastern Cooperative Oncology Group performance status (ECOG PS), and Fuhrman grade, both continuous (p <0.0001) and dichotomised (p <0.0001) HIF-2α C variables remained significant predictors of CSS, while neither HIF-2α N variable was retained. Conclusion: Our investigation supports that HIF-2α may have a unique tumour promoter role in the cytoplasm. This preliminary finding justifies further experimental and mechanistic studies that examine the biological functions of HIF-2α when located in the cytoplasm. [Copyright &y& Elsevier]

Abstract: Hypoxia inducible factor-1 (HIF-1), a dimeric transcription factor of the bHLH-PAS family, is comprised of HIF-1α, which is inducible by hypoxia and ARNT or HIF-1β, which is constitutively expressed. HIF-1 is involved in cellular homeostasis under hypoxia, in development and in several diseases affected by oxygen availability, particularly cancer. Since its expression is positively correlated with poor outcome prognosis for cancer patients, HIF-1 is a target for pharmaceutical therapy. We have previously shown that male germ cell Rac GTPase activating protein (MgcRacGAP), a regulator of Rho proteins which are principally involved in cytoskeletal organization, binds to HIF-1α and inhibits its transcriptional activity. In this work, we have explored the mechanism of the MgcRacGAP-mediated HIF-1 inactivation. We show that the Myo domain of MgcRacGAP, which is both necessary and sufficient for HIF-1 repression, binds to the PAS-B domain of HIF-1α. Furthermore MgcRacGAP competes with ARNT for binding to the HIF-1α PAS-B domain, as shown by in vitro binding pull down assays. In mammalian cells, ARNT overexpression can overcome the MgcRacGAP-mediated inhibition and MgcRacGAP binding to HIF-1α in vivo inhibits its dimerization with ARNT. We additionally present results indicating that MgcRacGAP binding to HIF-1α is specific, since it does not affect the transcriptional activity of HIF-2, a close evolutionary relative of HIF-1 also involved in hypoxia regulation and cancer. Our results reveal a new mechanism for HIF-1 transcriptional activity regulation, suggest a novel hypoxia-cytoskeleton link and provide new tools for selective HIF-1 inhibition. [Copyright &y& Elsevier]

Abstract: Background: Along with other regulators of cell metabolism, hypoxia-inducible factors HIF-1 and HIF-2 differentially regulate cell adaptation to hypoxia. Switches in HIF-1/HIF-2 signaling in chronic hypoxia have not been fully investigated. Methods: Proliferation, viability, apoptosis, neuronal and bioenergetic markers, mitochondrial function, respiration, glycolysis, HIF signalling, responses to O2 and glucose deprivation (OGD) were examined using tumor PC12 and SH-SY5Y cells continuously grown at 3% O2. Results: Hypoxic PC12 cells (H-cells) exhibit reduced proliferation and histone H4 acetylation, NGF-independent differentiation, activation of AMPK, inhibition of Akt, altered mitochondria and response to NGF. Cellular cytochrome c is increased with no effect on apoptosis. Reduction in respiration has minor effect on cellular ATP which is maintained through activated uptake (GLUT1) and utilization (HK2, PFK2) of glucose. H-cells exhibit resistance to OGD linked to increased glycogen stores. HIF-2alpha protein is decreased without changes in mRNA. Unlike HIF-1alpha, HIF-2alpha is not stabilized pharmacologically or by O2 deprivation. Capacity for HIF-2alpha stabilization is partly restored when H-cells are cultured at normoxia. In low-respiring SH-SY5Y cells cultured under the same conditions HIF-2alpha stabilization and energy budget are not affected. Conclusions: In chronically hypoxic PC12 cells glycolytic energy budget, increased energy preservation and low susceptibility to OGD are observed. HIF-2alpha no longer orchestrates adaptive responses to anoxia. General significance: Demonstrated switch in HIF-1/HIF-2 signaling upon chronic hypoxia can facilitate cell survival in energy crisis, by regulating balance between energy saving and decrease in proliferation, on one hand and active cell growth and tumor expansion, on the other. [Copyright &y& Elsevier]

The author of this lecture has been especially honored to be selected to deliver the Ernest Beutler Memorial Lecture at the Acute Leukemia Forum 2012 and to write this overview. Ernest Beutler was the pivotal influence in my introduction to academic life, and his contribution to hematology in the last 5 decades was unsurpassed. Taking a cue from Ernie''s example, I have elected in the keynote speech and this brief treatise, to start with an unconventional introduction and to expand on some discoveries made in my laboratory. Then I will extend these findings to the focus of the Acute Leukemia Forum to address potentially new approaches to therapies of acute leukemias. Somatic and germline mutations of acute leukemias are unfortunately caused by arrays of somatic and germline mutations. Simultaneous targeting of so many mutations makes it not possible to efficiently target all for cure. Albeit we should be aware that we should not in the near future ignore targeted therapy of those functionally important genetic and epigenetic events that are either initiating or contributing to aggressivity of acute leukemia, as these may be ameliorated by targeted intervention against one, or even a few together, of these defined molecular lesions. Yet, leukemic cells, like other cancer cells, have the unique metabolic feature to generate energy, referred as the Warburg effect, which can potentially be targeted to suppress or even eradicate cancer. [Copyright &y& Elsevier]

During hypoxia, cells undergo transcriptional changes to adjust to metabolic stress, to promote cell survival, and to induce pro-angiogenic factors. Hypoxia-induced factors (HIFs) regulate these transcriptional alterations. Failure to restore oxygen levels results in cell death by necrosis. IL-1α is one of the most important mediators of sterile inflammation following hypoxia-mediated necrosis. During hypoxia, IL-1α is up-regulated and released from necrotic cells, promoting the initiation of sterile inflammation. This study examined the role of IL-1α transcription in initiation of hypoxic stress and the correlation between IL-1α transcription and HIFα factors. In an epithelial cell line cultured under hypoxic conditions, IL-1α transcription was up-regulated in a process mediated and promoted by HIFα factors. IL-1α transcription was also up-regulated in hypoxia in a fibroblast cell line, however, in these cells, HIFα factors inhibited the elevation of transcription. These data suggest that HIFα factors play a significant role in initiating sterile inflammation by controlling IL-1α transcription during hypoxia in a differential manner, depending on the cell type. [ABSTRACT FROM AUTHOR]

Abstract: Recent studies have shown that acute intermittent hypoxia (IH) induces sensory plasticity of the carotid body manifested as sensory long-term facilitation (LTF), which requires prior conditioning with chronic IH and is mediated by reactive oxygen species (ROS). The purpose of this article is to provide a brief review of chronic IH-induced sensory LTF of the carotid body, sources of ROS, mechanisms underlying sensory LTF and its functional significance. Development of sensory LTF requires conditioning with several days of chronic IH. It is completely reversible following re-oxygenation, does not depend on the severity of hypoxia used for IH conditioning, not species specific and is selectively evoked by acute repetitive hypoxia but not by repetitive hypercapnia. Sensory LTF is not associated morphological changes in the carotid body and is expressed in chronic IH treated adult but not in neonatal rat carotid bodies. Chronic IH increases ROS levels in the carotid body involving 5-HT mediated activation of NADPH oxidase-2 (NOX2) and subsequent inhibition of the mitochondrial complex I. Sensory LTF can be prevented by inhibitors of 5-HT receptors, NOX inhibitors as well as by anti-oxidants. The signaling pathways mediating the sensory LTF are summarized in the second figure. It is suggested that sensory LTF contributes to the persistent sympathetic excitation under chronic IH. [Copyright &y& Elsevier]

Hypoxic signaling plays an essential role in maintaining oxygen homeostasis and cell survival. Hypoxia-inducible transcription factors HIF-1 and HIF-2 are central mediators of the cellular response to hypoxia by regulating the expression of genes controlling metabolic adaptation, oxygen delivery, and survival in response to oxygen deprivation. Recent studies have identified an important role for HIF-1 and HIF-2 in the regulation of skeletal development, bone formation, and regeneration, as well as joint formation and homeostasis. In addition, overexpression of HIF-1 and HIF-2 is clinically associated with osteosarcoma and osteoarthritis. Together, these findings implicate hypoxic signaling as a central regulator of bone biology and disease. [ABSTRACT FROM AUTHOR]

Abstract: Hypoxia inducible factor (HIF)-1 and HIF-2 are transcription factors that mediate the cellular response to hypoxia. Although HIF-1 and HIF-2 share the same target genes, both proteins activate a distinct subset of genes. To identify the target genes preferentially activated by HIF-2 in endothelial cells, DNA microarray analysis was performed to human umbilical vein endothelial cells (HUVECs) with forced expression of either HIF-1α or HIF-2α. In the present study, which is the first comparative study of target genes induced by either HIF-1 or HIF-2 in HUVECs, HIF-1 (and not HIF-2) stimulated mainly glycolytic, hexose metabolic and alcohol metabolic gene expression. However, HIF-2 (but not HIF-1) induced developmental gene expressions such as Fms-like tyrosine kinase 1 (Flt-1) and angiopoietin 2 (Angpt2). Furthermore, CD82 was up-regulated by HIF-2, but not by HIF-1, in response to hypoxia. HIF-2 regulated CD82 gene expression by binding to its HRE consensus sequence located within its first intron. Assessing the function of CD82 in HUVECs forced its expression. This result revealed that CD82 negatively regulates the HUVECs cell migration. The induction of CD82 gene expression in endothelial cells provided new insights into a specific function of HIF-2. [Copyright &y& Elsevier]

Endosialin is a transmembrane glycoprotein selectively expressed in blood vessels and stromal fibroblasts of various human tumours. It has been functionally implicated in angiogenesis, but the factors that control its expression have remained unclear. As insufficient delivery of oxygen is a driving force of angiogenesis in growing tumours, we investigated whether hypoxia regulates endosialin expression. Here, we demonstrate that endosialin gene transcription is induced by hypoxia predominantly through a mechanism involving hypoxia-inducible factor-2 (HIF-2) cooperating with the Ets-1 transcription factor. We show that HIF-2 activates the endosialin promoter both directly, through binding to a hypoxia-response element adjacent to an Ets-binding site in the distal part of the upstream regulatory region, and indirectly, through Ets-1 and its two cognate elements in the proximal promoter. Our data also suggest that the SP1 transcription factor mediates responsiveness of the endosialin promoter to high cell density. These findings elucidate important aspects of endosialin gene regulation and provide a rational frame for future investigations towards better understanding of its biological significance.British Journal of Cancer (2008) 99, 1348–1356. doi:10.1038/sj.bjc.6604685 www.bjcancer.com Published online 23 September 2008 [ABSTRACT FROM AUTHOR]

Abstract: Changes in oxygen concentrations affect many of the innate characteristics of stem and progenitor cells. Human mesenchymal stem cells (hMSCs) were maintained under hypoxic atmospheres (2% O2) for up to seven in vitro passages. This resulted in approximately 30-fold higher hMSC expansion over 6 weeks without loss of multi-lineage differentiation capabilities. Under hypoxia, hMSCs maintained their growth-rates even after reaching confluence, resulting in the formation of multiple cell layers. Hypoxic hMSCs also displayed differences in the cell and nuclear morphologies as well as enhanced ECM formation and organization. These changes in cellular characteristics were accompanied by higher mRNA levels of Oct-4 and HIF-2α, as well as increased expression levels of connexin-43, a protein used in gap junction formation. The results from this study demonstrated that oxygen concentrations affected many aspects of stem-cell physiology, including growth and in vitro development, and may be a critical parameter during expansion and differentiation. [Copyright &y& Elsevier]