Induction Of Hypoxia-inducible Factor Research Articles

Hypoxia-inducible factor 1 signaling drives placental aging and can provoke preterm labor.

Most cases of preterm labor have unknown cause, and the burden of preterm birth is immense. Placental aging has been proposed to promote labor onset, but specific mechanisms remain elusive. We report findings stemming from unbiased transcriptomic analysis of mouse placenta, which revealed that hypoxia-inducible factor 1 (HIF-1) stabilization is a hallmark of advanced gestational timepoints, accompanied by mitochondrial dysregulation and cellular senescence; we detected similar effects in aging human placenta. In parallel in primary mouse trophoblasts and human choriocarcinoma cells, we modeled HIF-1 induction and demonstrated resultant mitochondrial dysfunction and cellular senescence. Transcriptomic analysis revealed that HIF-1 stabilization recapitulated gene signatures observed in aged placenta. Further, conditioned media from trophoblasts following HIF-1 induction promoted contractility in immortalized uterine myocytes, suggesting a mechanism by which the aging placenta may drive the transition from uterine quiescence to contractility at the onset of labor. Finally, pharmacological induction of HIF-1 via intraperitoneal administration of dimethyloxalyl glycine (DMOG) to pregnant mice caused preterm labor. These results provide clear evidence for placental aging in normal pregnancy, and demonstrate how HIF-1 signaling in late gestation may be a causal determinant of the mitochondrial dysfunction and senescence observed within the trophoblast as well as a trigger for uterine contraction.

Hypoxia-inducible factor 1 signaling drives placental aging and can provoke preterm labor

Most cases of preterm labor have unknown cause, and the burden of preterm birth is immense. Placental aging has been proposed to promote labor onset, but specific mechanisms remain elusive. We report findings stemming from unbiased transcriptomic analysis of mouse placenta, which revealed that hypoxia-inducible factor 1 (HIF-1) stabilization is a hallmark of advanced gestational timepoints, accompanied by mitochondrial dysregulation and cellular senescence; we detected similar effects in aging human placenta. In parallel in primary mouse trophoblasts and human choriocarcinoma cells, we modeled HIF-1 induction and demonstrated resultant mitochondrial dysfunction and cellular senescence. Transcriptomic analysis revealed that HIF-1 stabilization recapitulated gene signatures observed in aged placenta. Further, conditioned media from trophoblasts following HIF-1 induction promoted contractility in immortalized uterine myocytes, suggesting a mechanism by which the aging placenta may drive the transition from uterine quiescence to contractility at the onset of labor. Finally, pharmacological induction of HIF-1 via intraperitoneal administration of dimethyloxalyl glycine (DMOG) to pregnant mice caused preterm labor. These results provide clear evidence for placental aging in normal pregnancy, and demonstrate how HIF-1 signaling in late gestation may be a causal determinant of the mitochondrial dysfunction and senescence observed within the trophoblast as well as a trigger for uterine contraction.

Abstract 677: Bi-directional crosstalk between the HIF and AHR transcription factors in clear cell kidney cancer

Abstract Hypoxia is a key feature of the solid tumour microenvironment, in which it is associated with poor prognosis and resistance to therapy. Hypoxia-inducible factor (HIF) is the central mediator of cellular responses to hypoxia and drives a transcriptional program with central roles in tumorigenesis. Clear cell renal cell cancers (ccRCC) exhibit the most direct and profound upregulation of the HIF pathway - through bi-allelic inactivation of the VHL tumour suppressor, a ubiquitin ligase that targets HIF-alpha subunits for degradation under normal oxygenated conditions. HIF-alpha proteins are members of the bHLH-PAS family and interact with HIF-1beta (also known as ARNT) to form functional heterodimers binding chromatin. However, the aryl hydrocarbon receptor (AHR), another bHLH-PAS protein that dimerises with HIF-1beta, is also up-regulated in ccRCC, raising the possibility of crosstalk between these two transcriptional pathways. Previous studies support an antagonistic relationship between HIF and AHR, with evidence of a context-dependent and gene-specific crosstalk. Here, we investigate the pan-genomic and precise relationship between the HIF and AHR transcription factors in ccRCC. Using immunoprecipitation, we show direct competition for HIF-1beta binding between the HIF-alpha and AHR proteins in renal cancer cell lines. Pan-genomic analysis of HIF-1alpha, HIF-2alpha, HIF-1beta and AHR chromatin binding by ChIP-seq analysis reveals a widespread competitive interaction between HIF and AHR. In RCC4 cells, this interaction is bidirectional, with induction of HIF leading to a reduction in AHR binding, while induction of AHR reduces binding of both HIF-1alpha and HIF-2alpha to chromatin. However, this competition is diminished at sites bound by both HIF and AHR, indicating a cooperative interaction between the two transcription factors in cis. Thus, while the HIF-alpha and AHR proteins compete for the common binding partner HIF-1beta, they appear to form a cooperative interaction when bound to neighbouring sites on chromatin. Linking these findings to changes in gene expression by RNA-seq analysis, we show the global downstream effects of the competition between HIF and AHR, but also reveal a degree of overlap and cooperativity between their transcriptional programs. Furthermore, we provide evidence of this antagonistic relationship in ccRCC tumours (TCGA database), in which we observe a negative correlation between HIF and AHR target gene expression. These findings shed light on the complex crosstalk between the HIF and AHR transcriptional pathways, which encompasses both a widespread inhibitory interaction and specific cooperative events and highlight a role for AHR in modifying the HIF transcriptional output in ccRCC. Citation Format: Véronique N. Lafleur, Silvia Halim, Peter J. Ratcliffe, David R. Mole. Bi-directional crosstalk between the HIF and AHR transcription factors in clear cell kidney cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 677.

Pericyte, but not astrocyte, hypoxia inducible factor-1 (HIF-1) drives hypoxia-induced vascular permeability in vivo

BackgroundWays to prevent disease-induced vascular modifications that accelerate brain damage remain largely elusive. Improved understanding of perivascular cell signalling could provide unparalleled insight as these cells impact vascular stability and functionality of the neurovascular unit as a whole. Identifying key drivers of astrocyte and pericyte responses that modify cell–cell interactions and crosstalk during injury is key. At the cellular level, injury-induced outcomes are closely entwined with activation of the hypoxia-inducible factor-1 (HIF-1) pathway. Studies clearly suggest that endothelial HIF-1 signalling increases blood–brain barrier permeability but the influence of perivascular HIF-1 induction on outcome is unknown. Using novel mouse lines with astrocyte and pericyte targeted HIF-1 loss of function, we herein show that vascular stability in vivo is differentially impacted by perivascular hypoxia-induced HIF-1 stabilization.MethodsTo facilitate HIF-1 deletion in adult mice without developmental complications, novel Cre-inducible astrocyte-targeted (GFAP-CreERT2; HIF-1αfl/fl and GLAST-CreERT2; HIF-1αfl/fl) and pericyte-targeted (SMMHC-CreERT2; HIF-1αfl/fl) transgenic animals were generated. Mice in their home cages were exposed to either normoxia (21% O2) or hypoxia (8% O2) for 96 h in an oxygen-controlled humidified glove box. All lines were similarly responsive to hypoxic challenge and post-Cre activation showed significantly reduced HIF-1 target gene levels in the individual cells as predicted.ResultsUnexpectedly, hypoxia-induced vascular remodelling was unaffected by HIF-1 loss of function in the two astrocyte lines but effectively blocked in the pericyte line. In correlation, hypoxia-induced barrier permeability and water accumulation were abrogated only in pericyte targeted HIF-1 loss of function mice. In contrast to expectation, brain and serum levels of hypoxia-induced VEGF, TGF-β and MMPs (genes known to mediate vascular remodelling) were unaffected by HIF-1 deletion in all lines. However, in agreement with the permeability data, immunofluorescence and electron microscopy showed clear prevention of hypoxia-induced tight junction disruption in the pericyte loss of function line.ConclusionThis study shows that pericyte but not astrocyte HIF-1 stabilization modulates endothelial tight junction functionality and thereby plays a pivotal role in hypoxia-induced vascular dysfunction. Whether the cells respond similarly or differentially to other injury stimuli will be of significant relevance.

The Impact of Hypoxia in Early Pregnancy on Placental Cells.

Oxygen levels in the placental microenvironment throughout gestation are not constant, with severe hypoxic conditions present during the first trimester. This hypoxic phase overlaps with the most critical stages of placental development, i.e., blastocyst implantation, cytotrophoblast invasion, and spiral artery remodeling initiation. Dysregulation of any of these steps in early gestation can result in pregnancy loss and/or adverse pregnancy outcomes. Hypoxia has been shown to regulate not only the self-renewal, proliferation, and differentiation of trophoblast stem cells and progenitor cells, but also the recruitment, phenotype, and function of maternal immune cells. In this review, we will summarize how oxygen levels in early placental development determine the survival, fate, and function of several important cell types, e.g., trophoblast stem cells, extravillous trophoblasts, syncytiotrophoblasts, uterine natural killer cells, Hofbauer cells, and decidual macrophages. We will also discuss the cellular mechanisms used to cope with low oxygen tensions, such as the induction of hypoxia-inducible factor (HIF) or mammalian target of rapamycin (mTOR) signals, regulation of the metabolic pathway, and adaptation to autophagy. Understanding the beneficial roles of hypoxia in early placental development will provide insights into the root cause(s) of some pregnancy disorders, such as spontaneous abortion, preeclampsia, and intrauterine growth restriction.

Targeting hypoxia-inducible factor 1 (HIF-1) signaling with natural products toward cancer chemotherapy.

Tumor cells are often exposed to hypoxia because of the lower oxygen supply deep inside the tumor tissues. However, tumor cells survive in these severe conditions by adapting to hypoxic stress through the induction of hypoxia-inducible factor 1 (HIF-1) signaling. HIF-1 activation is responsible for the expression of numerous HIF-1 target genes, which are related to cell survival, proliferation, angiogenesis, invasion, metastasis, cancer stemness, and metabolic reprogramming. Therefore, HIF-1 is expected to be a potential pharmacological target for cancer therapy. Small molecules derived from natural products (microbial origin, plant-derived, or marine organisms) have been shown to have unique chemical structures and biological activities, including HIF-1 inhibition. Several studies identified HIF-1 inhibitors from natural products. In this review, we summarize the current HIF-1 signaling inhibitors originating from natural products with a variety of modes of action, mainly focusing on microbial metabolites.

Inhibition of the HIF‐1α/BNIP3 pathway has a retinal neuroprotective effect

Retinal ischemia is a leading cause of irreversible blindness worldwide. Inner retinal dysfunction including loss of retinal ganglion cells is encountered in a number of retinal ischemic disorders. We previously reported administration of two different hypoxia-inducible factor (HIF) inhibitors exerted neuroprotective effects in a murine model of retinal ischemia/reperfusion (I/R) which mimics these disorders, as inner retinal degeneration could be involved in pathological HIF induction. However, this notion needs further investigation. Therefore, in this study, we attempted to use retina-specific Hif-1α conditional knockout (cKO) mice to uncover this notion more clearly under the same condition. Hif-1α cKO mice showed inner retinal neurodegeneration to a lesser extent than control mice. Hif-1α depletion in a murine 661W retinal cell line reduced cell death under pseudohypoxic and hypoxic conditions. Among hypoxia-related genes, the expression of BCL2 19kDa protein-interacting protein 3 (Bnip3) was substantially upregulated in the inner retinal layer after retinal I/R. In this regard, we further examined Bnip3 depletion in retinal neurons in vitro and in vivo and found the similar neuroprotective effects. Our results support the notion that the HIF-1α/BNIP3 pathway may have a critical role in inner retinal neurodegeneration, which can be linked with the development of new promising therapeutics for inner retinal ischemic disorders.

Hypoxia-inducible miR-196a modulates glioblastoma cell proliferation and migration through complex regulation of NRAS.

Glioblastoma (GBM) is the most common and aggressive malignant brain tumor in humans. Hypoxia has been correlated with the aggressive form of glial tumors, poor prognosis, recurrence and resistance to various therapies. MicroRNAs (miRNAs) have emerged as critical mediators of hypoxic responses and have shown great potential for cancer diagnostics and therapeutics. Here, we focus on the regulatory and functional characterization of miR-196a, a hypoxia-inducible miRNA, in GBM. Hypoxia/HIF regulation of miR-196a was assessed by RT-qPCR, promoter-luciferase and ChIP assays in GBM cell lines. miR-196a levels were analyzed inThe Cancer Genome Atlas (TCGA)-GBM, Chinese Glioma Genome Atlas (CGGA) and Indian GBM patient cohorts. miR-target interactions were studied using RNA/protein quantification and 3'UTR luciferase assays. The effect of miR-196a overexpression/inhibition wasassessed on cellular viability, migration and apoptosis under hypoxia and normoxia. Microarray-based gene expression profiling studies wereperformrd to study the effect of miR-196a ontheGBM cellular transcriptome under hypoxia. Weidentified miR-196a as a hypoxia-inducible and hypoxia-inducible factor (HIF)-regulated miRNA that plays an oncogenic role in GBM. miR-196a was found to be significantly up-regulated in TCGA-GBM, CGGA glioma as well as Indian GBM patient cohorts. miR-196a overexpression was found to induce cellular proliferation, migration, spheroid formation and colony formation and to inhibit apoptosis, while miR-196a inhibition using anti-miR-196ayielded opposite results, suggesting an oncogenic role of miR-196a in GBM. We further unveiled NRAS, AJAP1, TAOK1 and COL24A1 as direct targets of miR-196a. We also report a complex competitive regulation of oncogenic NRAS by miR-196a, miR-146a and let-7 in GBM. Analysis of microarray-based gene expression data obtained by miR-196a inhibition under hypoxia revealed a role of miR-196a inHIF, calcium adhesion, Wnt and cell adhesion pathways. Interestingly, miR-196a was found to positively regulate the expression of various genes involved in the induction or stabilization of HIFs andin maintenance of hypoxic conditions, thereby suggesting the existence of an indirect miR-196a/HIF positive feedback loop under hypoxia. Overall, our work identifies a novel association between hypoxia/HIF signalling and miR-196a in GBM and suggests its therapeutic significance.

Hypoxia-mediated drug resistance in breast cancers.

Tissue hypoxia in solid tumors is caused by several pathological changes associated with tumor growth, including altered microvasculature structure, increased diffusional distances, and tumor-associated anemia. As the oxygen tension decreases, tumor cells adapt to the limited oxygen supply. Previous studies have shown that such adaptation leads to an aggressive phenotype that is resistant to many anti-cancer therapies. Induction of hypoxia inducible factors (HIFs) mediates many proteomic and genomic changes associated with tumor hypoxia. In breast cancers, HIFs not only predict poor prognosis, but also promote metastasis and drug resistance. Several studies have proposed HIF-1α as a druggable target in drug-resistant breast cancers, leading to the synthesis and development of small molecule inhibitors. Disappointingly, however, none of these small molecule inhibitors have progressed to clinical use. In this review, we briefly discuss the role of HIF-1α in breast cancer drug resistance and summarize the current and future approaches to targeting this transcription factor in breast cancer treatment.

Metronomic chemotherapy offsets HIFα induction upon maximum-tolerated dose in metastatic cancers.

Conventional maximum‐tolerated dose (MTD) chemotherapy relies on periodic, massive cancer cell ablation events followed by treatment‐free intermissions, stereotypically resulting in resistance, relapse, and mortality. Furthermore, MTD chemotherapy can promote metastatic dissemination via activation of a transcriptional program dependent on hypoxia‐inducible factor (HIF)‐1α and (HIF)‐2α (hereafter referred to as HIFα). Instead, frequent low‐dose metronomic (LDM) chemotherapy displays less adverse effects while preserving significant pre‐clinical anticancer activity. Consequently, we hereby compared the effect of MTD or LDM chemotherapy upon HIFα in models of advanced, metastatic colon and breast cancer. Our results revealed that LDM chemotherapy could offset paralog‐specific, MTD‐dependent HIFα induction in colon cancers disseminating to the liver and lungs, while limiting HIFα and hypoxia in breast cancer lung metastases. Moreover, we assessed the translational significance of HIFα activity in colorectal and breast TCGA/microarray data, by developing two compact, 11‐gene transcriptomic signatures allowing the stratification/identification of patients likely to benefit from LDM and/or HIFα‐targeting therapies. Altogether, these results suggest LDM chemotherapy as a potential maintenance strategy to stave off HIFα induction within the intra‐metastatic tumor microenvironment.

Silencing of HIF-1α/CD73 axis by siRNA-loaded TAT-chitosan-spion nanoparticles robustly blocks cancer cell progression

Induction of Hypoxia Inducible Factor (HIF) as a direct consequence of oxygen deficiency in tumor tissues is a potent stimulus of CD73 (ecto-5′-nucleotidase) expression. Hypoxic environment and CD73 overexpression are associated with altered metabolism, elevated cancer cell proliferation, and tumor vascularization. Herein, a delivery system was developed for silencing CD73 and HIF-1α gene using siRNA-loaded Superparamagnetic iron oxide (SPION) nanocarriers for cancer treatment. SPIONs were encapsulated with thiolated chitosan (TC) and trimethyl chitosan (TMC) for improving their stabilization and functionalization. The nanoparticles (NPs) were about 133 nm in size, spherical, and non-toxic, and the addition of TAT peptide (derived from HIV-1 TAT protein) to TMC-TC-SPIONs significantly increased their cellular uptake by cancer cells. The produced NPs could efficiently accumulate in the tumor site, indicating their stability and targeting ability in reaching the tumor region. TAT-conjugated TMC-TC-SPIONs containing siRNAs could significantly reduce the HIF-1α and CD73 expression levels in cancer cells. Following transfection, cancer cells showed a significant reduction in migration and proliferation. Moreover, siRNA-loaded NPs could effectively reduce tumor growth and angiogenesis, as investigated by the chick chorioallantoic membrane (CAM) assay. This study suggested that TAT-TMC-TC-SPIONs can be potential nanocarrier for gene transfection in cancer therapy. Moreover, the co-silencing of CD73 and HIF-1α can be assumed as a novel anti-cancer treatment strategy with high tumor suppression potential.

Hard Metal Lung Disease: Update in Diagnosis and Management

Hard metal lung disease is a rare entity due to cobalt and tungsten carbide exposure. This review summarizes the literature focusing on pathogenesis, clinical features, diagnosis, and management. Theories for pathogenesis include immunogenic activation, oxidant injury, and induction of hypoxia-inducible factor in macrophages. Multinucleated giant cells with bizarre cannibalistic features are the most common pathological finding; however, it is not pathognomonic. Corticosteroids remain the mainstay of treatment based on data from small studies. Hard metal lung disease exposure can lead to an occupational asthma or parenchymal lung disease, potentially leading to respiratory failure. A history of exposure and clinical findings along with appropriate radiographic and pathological findings are essential to establish a diagnosis. Treatment options include exposure cessation, corticosteroids, immunosuppressive therapies, and lung transplantation.

Meloxicam, a Selective COX-2 Inhibitor, Mediates Hypoxia-Inducible Factor- (HIF-) 1α Signaling in Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) is regarded as a leading cause of cancer-related deaths, and its progression is associated with hypoxia and the induction of hypoxia-inducible factor (HIF). Meloxicam, a selective cyclooxygenase-2 (COX-2) inhibitor, induces cell death in various malignancies. However, the underlying mechanism remains to be elucidated in HCC, especially under hypoxic conditions. The alteration of COX-2 and HIF-1α oncogenicity was evaluated in HCC specimens by tissue microarray. Cell viability, angiogenesis assays, and xenografted nude mice were used to evaluate the effects of meloxicam, along with flow cytometry to detect the cell cycle, apoptosis, and mitochondrial membrane potential (ΔΨm) of HCC. qRT-PCR, Western blotting, immunofluorescence, immunohistochemistry, luciferase assay, and RNAi were carried out to determine the HIF-1α signaling affected by meloxicam. In this study, we showed that meloxicam exerts antiproliferative and antiangiogenesis efficacy in vitro and in vivo and causes disruption of mitochondrial membrane potential (ΔΨm), thus leading to caspase-dependent apoptosis under hypoxic environments. Exposure to meloxicam significantly reduced HIF-1α transcriptional activation and expression through sequestering it in the cytoplasm and accelerating degradation via increasing the von Hippel-Lindau tumor suppressor protein (pVHL) in HCC. These data demonstrated that inhibition of HIF-1α by meloxicam could suppress angiogenesis and enhance apoptosis of HCC cells. This discovery highlights that COX-2 specific inhibitors may be a promising therapy in the treatment of HCC.

Hypoxic stabilization of mRNA is HIF-independent but requires mtROS

BackgroundTissue ischemia can arise in response to numerous physiologic and pathologic conditions. The cellular response to decreased perfusion, most notably a decrease in glucose and oxygen, is important for cellular survival. In response to oxygen deprivation or hypoxia, one of the key response elements is hypoxia inducible factor (HIF) and a key protein induced by hypoxia is vascular endothelial growth factor (VEGF). Under hypoxia, we and others have reported an increase in the half-life of VEGF and other hypoxia related mRNAs including MYC and CYR61; however, the mediator of this response has yet to be identified. For this study, we sought to determine if HIF-mediated transcriptional activity is involved in the mRNA stabilization induced by hypoxia.MethodsHEK293T or C6 cells were cultured in either normoxic or hypoxic (1% oxygen) conditions in the presence of 1 g/L glucose for all experiments. Pharmacological treatments were used to mimic hypoxia (desferroxamine, dimethyloxaloglutamate, CoCl2), inhibit mitochondrial respiration (rotenone, myxothiazol), scavenge reactive oxygen species (ROS; ebselen), or generate mitochondrial ROS (antimycin A). siRNAs were used to knock down components of the HIF transcriptional apparatus. mRNA half-life was determined via actinomycin D decay and real time PCR and western blotting was used to determine mRNA and protein levels respectively.ResultsTreatment of HEK293T or C6 cells with hypoxic mimetics, desferroxamine, dimethyloxaloglutamate, or CoCl2 showed similar induction of HIF compared to hypoxia treatment, however, in contrast to hypoxia, the mimetics caused no significant increase in VEGF, MYC or CYR61 mRNA half-life. Knockdown of HIF-alpha or ARNT via siRNA also had no effect on hypoxic mRNA stabilization. Interestingly, treatment of HEK293T cells with the mitochondrial inhibitors rotenone and myxothiazol, or the glutathione peroxidase mimetic ebselen did prevent the hypoxic stabilization of VEGF, MYC, and CYR61, suggesting a role for mtROS in the process. Additionally, treatment with antimycin A, which has been shown to generate mtROS, was able to drive the normoxic stabilization of these mRNAs.ConclusionOverall these data suggest that hypoxic mRNA stabilization is independent of HIF transcriptional activity but requires mtROS.

PKM2 and HIF-1α regulation in prostate cancer cell lines.

Prostate cancer (PCA) is one of the most common cancer types in men, with cancer progression being linked to hypoxia and the induction of hypoxia-inducible factor (HIF).We investigated the expression of pyruvate kinase M2 (PKM2), its regulation by HIF isoforms 1α and 2α, and its role in HIF stabilization. We additionally examined cell survival in the prostate cancer cell lines PC3 and LNCaP under severe hypoxic (0.1% O2) and normoxic (20% O2) conditions. qRT-PCR showed higher up-regulation of PKM2 mRNA expression in LNCaP cells than in PC3 cells, while western blotting showed that PKM2 protein levels were up-regulated only in LNCaP cells. Inhibition of HIF-1α and HIF-2α by small interfering RNA (si-RNA) demonstrated HIF-1α dependent up-regulation of PKM2 at the mRNA and protein levels in LNCaP cells. PKM2 inhibition by si-RNA significantly decreased hypoxia-response element (HRE) activation in a gene reporter assay and down-regulated HIF-1α target vascular endothelial growth factor (VEGF) mRNA expression in PC3 cells, whereas HIF-1α protein levels were not significantly reduced. Additionally, PKM2 inhibition significantly reduced clonogenic survival in both cell lines in a colony formation assay. Prolyl hydroxylase 3 (PHD3) mRNA expression was up-regulated in both cell lines. It has been shown that PKM2 expression is regulated by HIF-1α and that PKM2 favors HIF-1α transactivation under mild (1% O2) but not severe (0.1% O2) hypoxic conditions, and some of our findings are consistent with these previous results. However, this mechanism was not fully observed in our studied cell lines, as PKM2 regulation and HIF-1α stabilization at the transactivation level occurred under severe hypoxic conditions. This discrepancy suggests that tumor tissue origin and cell type influence this model. Our findings expand the current knowledge of the mechanisms of PCA regulation, and would be important in developing novel therapeutic strategies.

HIF-1α promotes cyst progression in a mouse model of autosomal dominant polycystic kidney disease

Autosomal dominant polycystic kidney disease (ADPKD) is mainly caused by mutations of the PKD1 gene and characterized by growth of bilateral renal cysts. Cyst growth is accompanied by regional hypoxia and induction of hypoxia-inducible factor (HIF)-1α in cyst-lining epithelial cells. To determine the relevance of HIF-1α for cyst growth invivo we used an inducible kidney epithelium-specific knockout mouse to delete Pkd1 at postnatal day 20 or 35 to induce polycystic kidney disease of different severity and analyzed the effects of Hif-1α co-deletion and HIF-1α stabilization using a prolyl-hydroxylase inhibitor. HIF-1α expression was enhanced in kidneys with progressive cyst growth induced by early Pkd1 deletion, but unchanged in the milder phenotype induced by later Pkd1 deletion. Hif-1α co-deletion significantly attenuated cyst growth in the severe, but not in the mild, phenotype. Application of a prolyl-hydroxylase inhibitor resulted in severe aggravation of the mild phenotype with rapid loss of renal function. HIF-1α expression was associated with induction of genes that mediate calcium-activated chloride secretion. Thus, HIF-1α does not seem to play a role in early cyst formation, but accelerates cyst growth during progressive polycystic kidney disease. This novel mechanism of cyst growth may qualify as a therapeutic target.

Prolyl Hydroxylase Inhibitors: A Breakthrough in the Therapy of Anemia Associated with Chronic Diseases.

Chronic kidney disease, cancer, chronic inflammatory disorders, nutritional, and genetic deficiency can cause anemia. Hypoxia causes induction of hypoxia-inducible factor (HIF), which stimulates erythropoietin (EPO) synthesis. Prolyl hydroxylase domain (PHD) enzyme inhibition can stabilize hypoxia-inducible factor (HIF). HIF stabilization also decreases hepcidin, a hormone of hepatic origin, which regulates iron homeostasis. PHD inhibitors represent a novel pharmacological treatment of anemia associated with chronic diseases. Many orally active PHD inhibitors like roxadustat, molidustat, vadadustat, and desidustat are in late phase clinical trials. This review discusses the role of PHD inhibitors in the treatment of anemia associated with chronic diseases.

Donor Treatment With a Hypoxia-Inducible Factor-1 Agonist Prevents Donation After Cardiac Death Liver Graft Injury in a Rat Isolated Perfusion Model.

The protective role of hypoxia-inducible factor-1 (HIF-1) against liver ischemia-reperfusion injury has been well proved. However its role in liver donation and preservation from donation after cardiac death (DCD) is still unknown. The objective of this study was to test the hypothesis that pharmaceutical stabilization of HIF-1 in DCD donors would result in a better graft liver condition. Male SD rats (6 animals per group) were randomly given the synthetic prolyl hydroxylase domain inhibitor FG-4592 (Selleck, 6 mg/kg of body weight) or its vehicle (dimethylsulfoxide). Six hours later, cardiac arrest was induced by bilateral pneumothorax. Rat livers were retrieved 30 min after cardiac arrest, and subsequently cold stored in University of Wisconsin solution for 24 h. They were reperfused for 60 min with Krebs-Henseleit bicarbonate buffer in an isolated perfused liver model, after which the perfusate and liver tissues were investigated. Pretreatment with FG-4592 in DCD donors significantly improved graft function with increased bile production and synthesis of adenosine triphosphate, decreased perfusate liver enzyme release, histology injury scores and oxidative stress-induced cell injury and apoptosis after reperfusion with the isolated perfused liver model. The beneficial effects of FG-4592 is attributed in part to the accumulation of HIF-1 and ultimately increased PDK1 production. Pretreatment with FG-4592 in DCD donors resulted in activation of the HIF-1 pathway and subsequently protected liver grafts from warm ischemia and cold-stored injury. These data suggest that the pharmacological HIF-1 induction may provide a clinically applicable therapeutic intervention to prevent injury to DCD allografts.

Effect of erythropoietin production induced by hypoxia on autophagy in HepG2 cells

The induction of hypoxia inducible factor (HIF) and autophagy are important cellular responses to hypoxia. The production of erythropoietin (EPO) regulated by HIF is increased by hypoxia and participates in cell protection in various organs and tissues. Signal Transducers and Activator of Transcription 3 (STAT3) is a regulatory factor that is common to autophagy induction and EPO-EPO receptor signaling. In this study, we analyzed the promotion of EPO production and autophagy, and the participation of STAT3, in HepG2 cells under hypoxia. Treatment with EPO siRNA (si-EPO) significantly increased autophagy induction by hypoxia, while treatment with recombinant EPO inhibited the effect of si-EPO. NSC74859, an inhibitor of the phosphorylation of STAT3, increased autophagy induction to the same extent as si-EPO treatment. Even when 3-Methyladenine, an inhibitor of autophagy, was added, the increase of EPO mRNA expression due to hypoxia was not affected. Hypoxia-induced EPO restrained autophagy induction through the EPO receptor and phosphorylation of STAT3. Because cell viability with treatment of si-EPO under hypoxia did not increase over the control, our results suggested that EPO produced by hypoxia prevented excess autophagy induction.

Ischemic Postconditioning Protects Against Intestinal Ischemia/Reperfusion Injury via the HIF-1\u03b1/miR-21 Axis

Intestinal ischemia/reperfusion (I/R) can lead to tissue damage associated with inflammation and mucosal apoptosis. Ischemic postconditioning (IPostC), a series of repeated, brief, intermittent periods of ischemia and reperfusion, has beneficial effects against I/R-induced injury in the heart and intestine, although the underlying mechanisms for these effects remain unclear. We evaluated the involvement of microRNA-21 (miR-21) in the protective effects of IPostC in a rat model of I/R induced by superior mesenteric artery occlusion and reopening. IPostC decreased I/R injury and suppressed apoptosis in the intestinal tissues concomitant with the induction of hypoxia inducible factor 1 alpha (HIF-1α) and the upregulation of miR-21. In vitro experiments in the intestinal epithelial cell line IEC-6 showed that hypoxia induced miR-21 and this effect was abolished by silencing HIF1-α, confirming the induction of miR-21 by HIF1-α, HIF1-α or miR-21 inhibition exacerbated I/R induced apoptosis, and programmed cell death 4 (PDCD4) and Fas-L was involved in miR-21 mediated anti-apoptotic effects on intestinal epithelial cells. Knockdown of miR-21 or inhibition of HIF1-α abolished the IPostC-mediated attenuation of intestinal injury and apoptosis and the downregulation of PDCD4 and Fas-L. A potential mechanism underlying the protective effect of IPostC may therefore involve the induction of miR-21 by HIF1-α and the attenuation of apoptosis via the downregulation of PDCD4 and Fas-L.