Stabilization Of Hypoxia-inducible Factor Research Articles

HIF-2α-dependent induction of miR-29a restrains TH1 activity during T cell dependent colitis

Metabolic imbalance leading to inflammatory hypoxia and stabilization of hypoxia-inducible transcription factors (HIFs) is a hallmark of inflammatory bowel diseases. We hypothesize that HIF could be stabilized in CD4+ T cells during intestinal inflammation and alter the functional responses of T cells via regulation of microRNAs. Our assays reveal markedly increased T cell-intrinsic hypoxia and stabilization of HIF protein during experimental colitis. microRNA screen in primary CD4+ T cells points us towards miR-29a and our subsequent studies identify a selective role for HIF-2α in CD4-cell-intrinsic induction of miR-29a during hypoxia. Mice with T cell-intrinsic HIF-2α deletion display elevated T-bet (target of miR-29a) levels and exacerbated intestinal inflammation. Mice with miR-29a deficiency in T cells show enhanced intestinal inflammation. T cell-intrinsic overexpression of HIF-2α or delivery of miR-29a mimetic dampen TH1-driven colitis. In this work, we show a previously unrecognized function for hypoxia-dependent induction of miR-29a in attenuating TH1-mediated inflammation.

Immunotherapeutic Innovations in Clear Cell Renal Cell Carcinoma: Current Strategies and Future Directions.

Approximatively 80% of kidney cancers globally are clear cell kidney cancers (ccRCCs), with 80% of these malignancies featuring an inactivating mutation of the Von Hippel-Lindau gene. This genetic alteration leads to the stabilization of hypoxia inducible factors 1 and 2 alpha (HIF 1 and 2α), resulting in the over-expression of target genes such as vascular endothelial growth factor (VEGF), which is crucial for angiogenesis. As a result, ccRCCs are highly vascularized and serve as models for anti-angiogenic treatments (AAT). Current AAT therapies comprise antibodies targeting VEGFs, tyrosine kinase inhibitors (TKi) (Sunitinib) that target neo-angiogenesis receptors, and competitive inhibitor receptors (Aflibercept) that trap VEGFA and PlGF. The over-expression of VEGF and related members such as VEGFC significantly influences angiogenesis, lymph-angiogenesis, and immune tolerance. This has resulted in the approval of various immune checkpoint inhibitors (known as anti-PD-1, anti-PD-L1, and anti-CTLA-4) as viable treatment options for kidney cancer. Despite these advances, ccRCC remains challenging to treat adequately. Thus, future research is imperative to better understand the biology and pathophysiology of RCC, the tumor microenvironment, and mechanisms of resistance, with the aim of developing new therapies.

Genetic modifications of EGLN1 reactivate HbF production in β0-thalassemia/HbE

Reactivation of fetal hemoglobin (HbF, α2γ2) potentially alleviates clinical presentation in β-thalassemia. Prolyl hydroxylase domain enzymes (PHDs) play roles in the canonical oxygen-sensing pathway and maintain the stability of cellular hypoxia-inducible factor α (HIF-α) in response to low oxygen levels or hypoxia. Pharmacological inhibition of PHDs has been shown to increase HbF production in erythroid progenitors derived from healthy donors. Here, we demonstrated the relationship between PHD2, the main PHD isoform, and clinical phenotypes in β0-thalassemia/HbE disease. Although the targeted sequencing annotated several common variants within EGLN1, the gene encoding PHD2, none of these variants were located in the functional domains of PHD2 and were irrelevant to the clinical phenotypes. CRISPR-mediated EGLN1 modifications at the functional regions; however, led to significantly reduce PHD2 expression and increase HbF expression levels in severe β-thalassemia erythroblasts. Moreover, these beneficial phenotypes were independent to the two well-known HbF regulators including BCL11A and GATA1. Our findings introduce an additional mechanism for HbF regulation in β-thalassemia and propose that targeting the canonical oxygen-sensing pathway, particularly PHD2 functional domains, might offer a promising therapeutic strategy to β-thalassemia diseases.

Pharmacologic HIF stabilization activates costimulatory receptor expression to increase antitumor efficacy of adoptive T cell therapy.

Adoptive cell transfer (ACT) is a therapeutic strategy to augment antitumor immunity. Here, we report that ex vivo treatment of mouse CD8+ T cells with dimethyloxalylglycine (DMOG), a stabilizer of hypoxia-inducible factors (HIFs), induced HIF binding to the genes encoding the costimulatory receptors CD81, GITR, OX40, and 4-1BB, leading to increased expression. DMOG treatment increased T cell killing of melanoma cells, which was further augmented by agonist antibodies targeting each costimulatory receptor. In tumor-bearing mice, ACT using T cells treated ex vivo with DMOG and agonist antibodies resulted in decreased tumor growth compared to ACT using control T cells and increased intratumoral markers of CD8+ T cells (CD7, CD8A, and CD8B1), natural killer cells (NCR1 and KLRK1), and cytolytic activity (perforin-1 and tumor necrosis factor-α). Costimulatory receptor gene expression was also induced when CD8+ T cells were treated with three highly selective HIF stabilizers that are currently in clinical use.

Hydrogen sulfide upregulates hypoxia inducible factors and erythropoietin production in chronic kidney disease induced by 5/6 nephrectomized rats.

In end stage renal disease )ESRD(, reduced EPO production resulted in decreased oxygen diffusion that cause Hypoxia-inducible factors (HIFs) stabilization. The mechanism of beneficial effects of H2S in chronic kidney disease (CKD) is the aim of the present study to examine the effects of the H2S donor sodium hydrosulfide (NaHS) on renal function parameters, oxidative stress indices and expression levels of HIF-2α gene and erythropoietin protein in 5/6 nephrectomy-induced chronic renal failure in rats. Male rats were assigned into 3 groups (n = 8): Sham, CKD and NaHS groups. In the CKD group, 5/6 nephrectomy was performed. In the sham group, rats were anesthetized but 5/6 nephrectomy was not induced. In the NaHS group, 30 µmol/L of NaHS in drinking water for 8 weeks was adminstrated 4 weeks after 5/6 nephrectomy induction. At the end of the 12 week, blood and renal tissues were taken to evaluate renal function parameters, oxidative stress indices and expression levels of HIF-2α gene and erythropoietin protein. The induction of 5/6 nephrectomy significantly caused renal dysfunction, oxidative stress, increased HIF-2α gene expression and decreased erythropoietin levels in renal tissue samples. NaHS administration resulted in a marked improvement in renal function and oxidative stress indicators, a marked reduction in HIF-2α gene expression as well as an increase in erythropoietin protein levels in comparison with the CKD group. In this study, regional hypoxia and oxidative stress in CKD, may cause the stabilization of the HIFs complexes, although erythropoietin synthesis was not increased due to destructive effects of CKD on the kidney tissues. Administration of NaHS caused up-regulating HIF-erythropoietin signaling pathway.

Protective Effects of Velvet Antler Methanol Extracts on Hypoxia-Induced Damage in Caenorhabditis elegans through HIF-1 and ECH-8 Mediated Lipid Accumulation.

Velvet antler, a traditional tonic widely used in East Asia for its health benefits, is explored in this study for its protective effects against hypoxia-induced damage using Caenorhabditis elegans (C. elegans) as a model. Hypoxia, characterized by low oxygen availability, induces significant physiological stress and potential tissue damage. Our research demonstrates that methanol extracts from velvet antler (MEs) enhance the survival of C. elegans under hypoxic conditions. This enhancement is achieved through the stabilization of hypoxia-inducible factor-1 (HIF-1) and the promotion of lipid accumulation, both of which are crucial for mitigating cellular damage. Specifically, MEs improve mitochondrial function, increase ATP production, and aid in the recovery of physical activity in C. elegans post-hypoxia or following hypoxia-reoxygenation (HR). The pivotal role of HIF-1 is underscored by the loss of these protective effects when HIF-1 function is inhibited. Additionally, our findings reveal that the gene related to lipid metabolism, ech-8, significantly contributes to the lipid accumulation that enhances resilience to hypoxia in C. elegans treated with MEs. These results not only highlight the therapeutic potential of velvet antler in modern medical applications, particularly for conditions involving hypoxic stress, but also provide insights into the molecular mechanisms by which MEs confer protection against hypoxic damage.

Simultaneous detection of three hypoxia-inducible factor stabilizers-molidustat, roxadustat, and vadadustat-in multiple keratinized matrices and its application in a doping context.

In a doping case, a top athlete challenged an anti-doping rule violation, involving molidustat. Molidustat is a stabilizing agent of the hypoxia-inducible factor (HIF) recently developed. It is currently undergoing clinical trials for anemia associated with chronic kidney disease. HIF stabilizers are banned at all times by the World Anti-Doping Agency (class S2). Because of their pharmacological proprieties, these new drugs can enhance athletic performance. The athlete's defense wanted to analyze multiple keratinized matrices as they allow long-term investigations. Requests concerning HIF stabilizers are constantly growing. We have therefore developed a liquid chromatography coupled with tandem mass spectrometry method to identify and quantify three molecules of this class: molidustat, vadadustat, and roxadustat. Thirty milligrams of keratinized matrices were incubated in 1mL of pH8.4 diammonium hydrogen phosphate buffer for 16 h at 40°C with 1ng of testosterone-D3, used as internal standard. After extraction with ethyl acetate/diethyl ether (80/20), the organic phase was evaporated, and the dry residue was reconstituted in 30 μL of initial phase. The method was linear from 5 to 1000 pg/mg for the three analytes. Limits of quantification were 2, 0.5, and 5pg/mg for molidustat, roxadustat, and vadadustat, respectively. The analysis of the athlete's head hair (collected 1 month after the urine test) showed a concentration of molidustat of 135 pg/mg, and his beard hair and his fingernails clippings contained 55 and 40 pg/mg, respectively.

HIF Stabilizer Desidustat Protects against Complement-Mediated Diseases.

Complement cascade is a defence mechanism useful for eliminating pathogenic microorganisms and damaged cells. However, activation of alternative complement system can also cause inflammation and promote kidney and retinal disease progression. Inflammation causes tissue hypoxia, which induces hypoxia-inducible factor (HIF) and HIF helps the body to adapt to inflammation. In this study, we investigated the effect of HIF stabilizer desidustat in complement-mediated diseases. Oral administration of desidustat (15 mg/kg) was effective to reduce the kidney injury in mice that was induced by either lipopolysaccharide (LPS), doxorubicin or bovine serum albumin (BSA)-overload. Complement activation-induced membrane attack complex (MAC) formation and factor B activity were also reduced by desidustat treatment. In addition, desidustat was effective against membranous nephropathy caused by cationic BSA and retinal degeneration induced by sodium iodate in mice. C3-deposition, proteinuria, malondialdehyde, and interleukin-1ß were decreased and superoxide dismutase was increased by desidustat treatment in cBSA-induced membranous nephropathy. Desidustat specifically inhibited alternative complement system, without affecting the lectin-, or classical complement pathway. This effect appears to be mediated by inhibition of factor B. These data demonstrate the potential therapeutic value of HIF stabilization by desidustat in treatment of complement-mediated diseases.

Revisiting reactive oxygen species production in hypoxia.

Cellular responses to hypoxia are crucial in various physiological and pathophysiological contexts and have thus been extensively studied. This has led to a comprehensive understanding of the transcriptional response to hypoxia, which is regulated by hypoxia-inducible factors (HIFs). However, the detailed molecular mechanisms of HIF regulation in hypoxia remain incompletely understood. In particular, there is controversy surrounding the production of mitochondrial reactive oxygen species (ROS) in hypoxia and how this affects the stabilization and activity of HIFs. This review examines this controversy and attempts to shed light on its origin. We discuss the role of physioxia versus normoxia as baseline conditions that can affect the subsequent cellular response to hypoxia and highlight the paucity of data on pericellular oxygen levels in most experiments, leading to variable levels of hypoxia that might progress to anoxia over time. We analyze the different outcomes reported in isolated mitochondria, versus intact cells or whole organisms, and evaluate the reliability of various ROS-detectingtools. Finally, we examine the cell-type and context specificity of oxygen's various effects. We conclude that while recent evidence suggests that the effect of hypoxia on ROS production is highly dependent on the cell type and the duration of exposure, efforts should be made to conduct experiments under carefully controlled, physiological microenvironmental conditions in order to rule out potential artifacts and improve reproducibility in research.

Placental apoptosis increased by hypoxia inducible factor-1 stabilization is counteracted by leptin†.

During pregnancy, apoptosis is a physiological event critical in the remodeling and aging of the placenta. Increasing evidence has pointed toward the relevance of hypoxia as modulator of trophoblast cell death. Previous reports have shown that leptin, a placental cytokine, promotes cell survival in both cell culture and placental explant models. The aim of this work is to establish the role of leptin in apoptosis under hypoxic condition in trophoblast cells. In this study, we evaluated the effect of cobalt chloride, a hypoxia mimicking agent that stabilizes the expression of hypoxia-inducible factor-1 alpha, on Swan-71 and human placental explants. Hypoxia chamber was also used to generate 2% oxygen. Apoptosis was determined by the presence of apoptotic nucleus, fragmentation of DNA and Caspase-3 and PARP-1 cleavage. The pro-apoptotic proteins BAX, BID, BAD, and BAK and the anti-apoptotic effectors BCL-2, B-cell lymphoma-extra-large, and myeloid cell leukemia-1 were also analyzed. We found that hypoxia-inducible factor-1 alpha stabilization increased the appearance of apoptotic nucleus, fragmentation of DNA, and Caspase-3 and PARP-1 cleavage. Hypoxia mimicking conditions enhanced the expression of pro-apoptotic effectors BAX, BID, BAD, and BAK. Hypoxia-inducible factor-1 alpha stabilization also downregulated the level of BCL-2, B-cell lymphoma-extra-large, and myeloid cell leukemia-1. All these apoptotic parameters changes were reversed with leptin treatment. Moreover, we showed that leptin action on apoptosis modulation involves PI3K and MAPK signaling pathways. Obtained data demonstrate that hypoxia-inducible factor-1 alpha stabilization induces apoptosis in human placenta and leptin counteracts this effect, reinforcing its role as a survival cytokine.

Hypoxia inducible factor 2α promotes tolerogenic macrophage development during cardiac transplantation through transcriptional regulation of colony stimulating factor 1 receptor

Solid organ transplantation mobilizes myeloid cells, including monocytes and macrophages, which are central protagonists of allograft rejection. However, myeloid cells can also be functionally reprogrammed by perioperative costimulatory blockade to promote a state of transplantation tolerance. Transplantation tolerance holds promise to reduce complications from chronic immunosuppression and promote long-term survival in transplant recipients. We sought to identify different mediators of transplantation tolerance by performing single-cell RNA sequencing of acute rejecting or tolerized cardiac allografts. This led to the unbiased identification of the transcription factor, hypoxia inducible factor (HIF)-2α, in a subset of tolerogenic monocytes. Using flow cytometric analyses and mice with conditional loss or gain of function, we uncovered that myeloid cell expression of HIF-2α was required for costimulatory blockade-induced transplantation tolerance. While HIF-2α was dispensable for mobilization of tolerogenic monocytes, which were sourced in part from the spleen, it promoted the expression of colony stimulating factor 1 receptor (CSF1R). CSF1R mediates monocyte differentiation into tolerogenic macrophages and was found to be a direct transcriptional target of HIF-2α in splenic monocytes. Administration of the HIF stabilizer, roxadustat, within micelles to target myeloid cells, increased HIF-2α in splenic monocytes, which was associated with increased CSF1R expression and enhanced cardiac allograft survival. These data support further exploration of HIF-2α activation in myeloid cells as a therapeutic strategy for transplantation tolerance.

Prolonged Hypoxic Exposure Impairs Endothelial Functions: Possible Mechanism of HIF-1α Signaling

PURPOSE: Hypoxic training enhances oxygen availability by elevating hemoglobin, red blood cells, and capillaries. hypoxia-inducible factor (HIF) stabilization under hypoxic conditions triggers glycolysis, erythropoiesis, and angiogenesis. However, prolonged hypoxic exposure causes endothelial cell dysfunction, contributing to cardiovascular diseases. Furthermore, endothelial mitochondria play a crucial role in maintaining endothelial cell homeostasis through the processes of biogenesis, signaling cellular response, mitochondrial dynamics, and calcium homeostasis. However, the molecular response of endothelial mitochondria has not been fully elucidated. Therefore, this study aimed to investigate the endothelial mitochondria functions and morphological change in response to hypoxia.METHODS: Cobalt chloride and 2% hypoxic condition were applied to HUVECs. The endothelial cell functions were assessed using BrdU-based proliferation assay and scratch wound healing assays. Mitochondrial functions were evaluated using Mitosox and MitoTracker staining for mitochondrial reactive oxygen species (mtROS) and mitochondrial morphology analysis, respectively.RESULTS: The hypoxic condition (2% of O2) significantly decreased endothelial proliferation/migration and mitochondrial function. Furthermore, CoCl2 treatment increased the mtROS levels and mitochondrial fragmentation in HUVECs. Additionally, hypoxic conditions were found to regulate the phosphorylation of eNOS, mitochondrial biogenesis, mitophagy, and cell cycle-related protein expression.CONCLUSIONS: These results suggest that the HIF-α pathway leads to endothelial dysfunction via mitochondrial dysfunction. Further mechanistic studies will be needed to elucidate the cellular and molecular mechanisms by which hypoxic-response pathways influence the health and homeostasis of endothelial cells. The findings reported herein underscore the importance of strategies for hypoxic training to prevent cardiovascular diseases.

Ischemia does not provoke the full immune training repertoire in human cardiac fibroblasts.

Trained immunity of monocytes, endothelial, and smooth muscle cells augments the cytokine response to secondary stimuli. Immune training is characterized by stabilization of hypoxia-inducible factor (HIF)-1α, mTOR activation, and aerobic glycolysis. Cardiac fibroblast (CF)-myofibroblast transition upon myocardial ischemia/reperfusion (I/R) features epigenetic and metabolic adaptations reminiscent of trained immunity. We assessed the impact of I/R on characteristics of immune training in human CF and mouse myocardium. I/R was simulated in vitro with transient metabolic inhibition. CF primed with simulated I/R or control buffer were 5days later re-stimulated with Pam3CSK for 24h. Mice underwent transient left anterior descending artery occlusion or sham operation with reperfusion for up to 5days. HIF-regulated metabolic targets and cytokines were assessed by qPCR, immunoblot, and ELISA and glucose consumption, lactate release, and lactate dehydrogenase (LDH) by chromogenic assay. Simulated I/R increased HIF-1α stabilization, mTOR phosphorylation, glucose consumption, lactate production, and transcription of PFKB3 and F2RL3, a HIF-regulated target gene, in human CF. PGK1 and LDH mRNAs were suppressed. Intracellular LDH transiently increased after simulated I/R, and extracellular LDH showed sustained elevation. I/R priming increased abundance of pro-caspase-1, auto-cleaved active caspase-1, and the expression and secretion of interleukin (IL)-1β, but did not augment Pam3CSK-stimulated cytokine transcription or secretion. Myocardial I/R in vivo increased abundance of HIF-1 and the precursor and cleaved forms of caspase-1, caspase-11, and caspase-8, but not of LDH-A or phospho-mTOR. I/R partially reproduces features of immune training in human CF, specifically HIF-1α stabilization, aerobic glycolysis, mTOR phosphorylation, and PFKB3 transcription. I/R does not augment PGK1 or LDH expression or the cytokine response to Pam3CSK. Regulation of PAR4 and inflammasome caspases likely occurs independently of an immune training repertoire.

Iron Metabolism and Inflammatory Mediators in Patients with Renal Dysfunction.

Chronic kidney disease (CKD) affects around 850 million people worldwide, posing significant challenges in healthcare due to complications like renal anemia, end-stage kidney disease, and cardiovascular diseases. This review focuses on the intricate interplay between iron metabolism, inflammation, and renal dysfunction in CKD. Renal anemia, prevalent in CKD, arises primarily from diminished erythropoietin (EPO) production and iron dysregulation, which worsens with disease progression. Functional and absolute iron deficiencies due to impaired absorption and chronic inflammation are key factors exacerbating erythropoiesis. A notable aspect of CKD is the accumulation of uremic toxins, such as indoxyl sulfate (IS), which hinder iron metabolism and worsen anemia. These toxins directly affect renal EPO synthesis and contribute to renal hypoxia, thus playing a critical role in the pathophysiology of renal anemia. Inflammatory cytokines, especially TNF-α and IL-6, further exacerbate CKD progression and disrupt iron homeostasis, thereby influencing anemia severity. Treatment approaches have evolved to address both iron and EPO deficiencies, with emerging therapies targeting hepcidin and employing hypoxia-inducible factor (HIF) stabilizers showing potential. This review underscores the importance of integrated treatment strategies in CKD, focusing on the complex relationship between iron metabolism, inflammation, and renal dysfunction to improve patient outcomes.

Lactate in breast cancer cells is associated with evasion of hypoxia-induced cell cycle arrest and adverse patient outcome.

Tumor hypoxia is a common microenvironmental factor in breast cancers, resulting in stabilization of Hypoxia-Inducible Factor 1 (HIF-1), the master regulator of hypoxic response in cells. Metabolic adaptation by HIF-1 results in inhibition of citric acid cycle, causing accumulation of lactate in large concentrations in hypoxic cancers. Lactate can therefore serve as a secondary microenvironmental factor influencing cellular response to hypoxia. Presence of lactate can alter the hypoxic response of breast cancers in many ways, sometimes in opposite manners. Lactate stabilizes HIF-1 in oxidative condition, as well as destabilizes HIF-1 in hypoxia, increases cellular acidification, and mitigates HIF-1-driven inhibition of cellular respiration. We therefore tested the effect of lactate in MDA-MB-231 under hypoxia, finding that lactate can activate pathways associated with DNA replication, and cell cycling, as well as tissue morphogenesis associated with invasive processes. Using a bioengineered nano-patterned stromal invasion assay, we also confirmed that high lactate and induced HIF-1α gene overexpression can synergistically promote MDA-MB-231 dissemination and stromal trespass. Furthermore, using The Cancer Genome Atlas, we also surprisingly found that lactate in hypoxia promotes gene expression signatures prognosticating low survival in breast cancer patients. Our work documents that lactate accumulation contributes to increased heterogeneity in breast cancer gene expression promoting cancer growth and reducing patient survival.

The Prolyl Hydroxylase Domain 2 (PHD2) Deficiency Enhances Maximal Exercise Capacity

PURPOSE: Hypoxic training is beneficial for improving endurance performance. Hypoxia-inducible factor (HIF) is a key transcription factor that regulates the hypoxic-responsive pathway. Stabilization of HIF by inactivating prolyl hydroxylase domain protein 2 (PHD2) under low oxygen tension, triggers angiogenesis, erythropoiesis, and glycolysis. Glycolytic capacity is essential to perform anaerobic exercise. This study aims to verify whether the upregulation of glycolytic metabolism under the hypoxic-responsive pathway enhances exercise capacity.METHODS: We employed tamoxifen-inducible conditional PHD2 knock-out (KO) mice that exhibited stabilized HIF-alpha. To evaluate anaerobic exercise capacity, a rodent treadmill exercise test was performed. RNA sequencing (RNA-seq) analysis was conducted using C2C12 myoblasts after PHD2 siRNA transfection.RESULTS: PHD2KO mice exhibited increased red blood cell counts, hemoglobin, and hematocrit values, along with enhanced maximal exercise performance compared to littermate wild-type (WT) mice. PHD2 deficiency promoted maximal exercise capacity and resulted in lower blood lactate levels. RNAseq analysis revealed that PHD2-deficient myoblasts increased the expression of genes related to glycolysis, gluconeogenesis, and muscle contraction.CONCLUSIONS: These findings suggest that PHD2 deficiency-induced activation of the hypoxic-responsive pathway enhances maximal exercise capacity.

Prolyl hydroxylase domain inhibitor is an effective pre-hospital pharmaceutical intervention for trauma and hemorrhagic shock

Pre-hospital potentially preventable trauma related deaths are mainly due to hypoperfusion-induced tissue hypoxia leading to irreversible organ dysfunction at or near the point of injury or during transportation prior to receiving definitive therapy. The prolyl hydroxylase domain (PHD) is an oxygen sensor that regulates tissue adaptation to hypoxia by stabilizing hypoxia inducible factor (HIF). The benefit of PHD inhibitors (PHDi) in the treatment of anemia and lactatemia arises from HIF stabilization, which stimulates endogenous production of erythropoietin and activates lactate recycling through gluconeogenesis. The results of this study provide insight into the therapeutic roles of MK-8617, a pan-inhibitor of PHD-1, 2, and 3, in the mitigation of lactatemia in anesthetized rats with polytrauma and hemorrhagic shock. Additionally, in an anesthetized rat model of lethal decompensated hemorrhagic shock, acute administration of MK-8617 significantly improves one-hour survival and maintains survival at least until 4 h following limited resuscitation with whole blood (20% EBV) at one hour after hemorrhage. This study suggests that pharmaceutical interventions to inhibit prolyl hydroxylase activity can be used as a potential pre-hospital countermeasure for trauma and hemorrhage at or near the point of injury.

Hypoxia-inducible factor induces cysteine dioxygenase and promotes cysteine homeostasis in Caenorhabditis elegans

Dedicated genetic pathways regulate cysteine homeostasis. For example, high levels of cysteine activate cysteine dioxygenase, a key enzyme in cysteine catabolism in most animal and many fungal species. The mechanism by which cysteine dioxygenase is regulated is largely unknown. In an unbiased genetic screen for mutations that activate cysteine dioxygenase (cdo-1) in the nematode Caenorhabditis elegans, we isolated loss-of-function mutations in rhy-1 and egl-9, which encode proteins that negatively regulate the stability or activity of the oxygen-sensing hypoxia inducible transcription factor (hif-1). EGL-9 and HIF-1 are core members of the conserved eukaryotic hypoxia response. However, we demonstrate that the mechanism of HIF-1-mediated induction of cdo-1 is largely independent of EGL-9 prolyl hydroxylase activity and the von Hippel-Lindau E3 ubiquitin ligase, the classical hypoxia signaling pathway components. We demonstrate that C. elegans cdo-1 is transcriptionally activated by high levels of cysteine and hif-1. hif-1-dependent activation of cdo-1 occurs downstream of an H2S-sensing pathway that includes rhy-1, cysl-1, and egl-9. cdo-1 transcription is primarily activated in the hypodermis where it is also sufficient to drive sulfur amino acid metabolism. Thus, the regulation of cdo-1 by hif-1 reveals a negative feedback loop that maintains cysteine homeostasis. High levels of cysteine stimulate the production of an H2S signal. H2S then acts through the rhy-1/cysl-1/egl-9 signaling pathway to increase HIF-1-mediated transcription of cdo-1, promoting degradation of cysteine via CDO-1.

Hypoxia-inducible factor induces cysteine dioxygenase and promotes cysteine homeostasis in Caenorhabditis elegans.

Dedicated genetic pathways regulate cysteine homeostasis. For example, high levels of cysteine activate cysteine dioxygenase, a key enzyme in cysteine catabolism in most animal and many fungal species. The mechanism by which cysteine dioxygenase is regulated is largely unknown. In an unbiased genetic screen for mutations that activate cysteine dioxygenase (cdo-1) in the nematode Caenorhabditis elegans, we isolated loss-of-function mutations in rhy-1 and egl-9, which encode proteins that negatively regulate the stability or activity of the oxygen-sensing hypoxia inducible transcription factor (hif-1). EGL-9 and HIF-1 are core members of the conserved eukaryotic hypoxia response. However, we demonstrate that the mechanism of HIF-1-mediated induction of cdo-1 is largely independent of EGL-9 prolyl hydroxylase activity and the von Hippel-Lindau E3 ubiquitin ligase, the classical hypoxia signaling pathway components. We demonstrate that C. elegans cdo-1 is transcriptionally activated by high levels of cysteine and hif-1. hif-1-dependent activation of cdo-1 occurs downstream of an H2S-sensing pathway that includes rhy-1, cysl-1, and egl-9. cdo-1 transcription is primarily activated in the hypodermis where it is also sufficient to drive sulfur amino acid metabolism. Thus, the regulation of cdo-1 by hif-1 reveals a negative feedback loop that maintains cysteine homeostasis. High levels of cysteine stimulate the production of an H2S signal. H2S then acts through the rhy-1/cysl-1/egl-9 signaling pathway to increase HIF-1-mediated transcription of cdo-1, promoting degradation of cysteine via CDO-1.

Discovery of Novel and Potent Prolyl Hydroxylase Domain-Containing Protein (PHD) Inhibitors for The Treatment of Anemia.

Stabilization of hypoxia-inducible factor (HIF) by inhibiting prolyl hydroxylase domain enzymes (PHDs) represents a breakthrough in treating anemia associated with chronic kidney disease. Here, we identified a novel scaffold for noncarboxylic PHD inhibitors by utilizing structure-based drug design (SBDD) and generative models. Iterative optimization of potency and solubility resulted in compound 15 which potently inhibits PHD thus stabilizing HIF-α in vitro. X-ray cocrystal structure confirmed the binding model was distinct from previously reported carboxylic acid PHD inhibitors by pushing away the R383 and Y303 residues resulting in a larger inner subpocket. Furthermore, compound 15 demonstrated a favorable in vitro/in vivo absorption, distribution, metabolism, and excretion (ADME) profile, low drug-drug interaction risk, and clean early safety profiling. Functionally, oral administration of compound 15 at 10 mg/kg every day (QD) mitigated anemia in a 5/6 nephrectomy rat disease model.