Prolyl Hydroxylase Research Articles

Inhibition of Hypoxia Inducible Factor Prolyl Hydroxylase By FG-4592 (Roxadustat) Increases Erythropoietic Stimulation in a Mouse Model of Sickle Cell Disease

Sickle cell disease (SCD) is a devastating monogenic disorder caused by a mutation in the β-globin gene, leading to production of mutant hemoglobin S (HbS). Abnormal polymerization of deoxygenated HbS causes sickling of red blood cells (RBCs), resulting in painful vaso-occlusive crises and chronic hemolytic anemia. Etiologies of chronic anemia in SCD are multifactorial, including intravascular and extravascular hemolysis, inadequate erythropoietin (EPO) production, and abnormal iron homeostasis (Xu and Thein, Blood 2022). Hydroxyurea (HU) is the standard-of-care drug therapy for SCD and primarily reduces HbS polymerization through induction of fetal Hb (HbF) expression. However, it only modestly improves anemia and does not address other causes of anemia in SCD. Additionally, lower Hb levels in SCD are associated with chronic kidney disease (CKD), stroke, and pulmonary hypertension, arguing for the need for additional therapies targeting anemia in SCD. Hypoxia-inducible factor prolyl hydroxylase inhibitors (HIF-PHIs) are a class of drugs developed for treatment of anemia in CKD and end-stage renal disease. HIF-PHIs stimulate erythropoiesis by inhibiting prolyl hydroxylase and von Hippel-Lindau (VHL)-mediated degradation of HIF-α, leading to increased hypoxic signaling and promotion of HIF-induced EPO expression in the kidneys and liver. Stimulation of erythropoiesis also increases production of erythroferrone, the main regulator of hepcidin, and suppression of hepcidin levels by HIF-PHIs may improve iron mobilization. Furthermore, HIF-PHIs can induce HbF expression in erythroblasts derived from CD34+ hematopoietic stem and progenitor cells (HSPCs) (Hsieh et al.,Blood 2007, Feng et al., ASH abstract 2021), suggesting multiple potential therapeutic benefits in SCD. Therefore, we characterized the effect of the HIF-PHI FG-4592 (roxadustat), on HbF induction in CD34+ HSPCs and on hematologic markers, EPO and hepcidin expression in a mouse model of SCD. CD34+ HSPCs from 3 donors were differentiated in vitro to erythroblasts with vehicle, 20 μM of FG-4592, or 50 μM of HU. Flow cytometric analysis on Day 9 showed that FG-4592 treatment increased % F-cells by 1.47-fold (±0.45), compared to a 2.21-fold (±0.89) increase with HU (Fig. 1). We then transplanted lethally irradiated (1000 rads) male 6-8-week-old C57BL/6J mice with bone marrow from transgenic sickle cell mice (Hb SS; Townes model, Jackson Laboratory). Twelve weeks after transplantation, mice with >80% engraftment of the donor HbSS phenotype, determined by Hb electrophoresis, were used for subsequent experiments. Vehicle, 25 mg/kg FG-4592, or 50 mg/kg FG-4592 was administered three times weekly through intraperitoneal injection for 8 weeks. All mice underwent retro-orbital bleeding at baseline (48 h pretreatment), 2, 4, 6, and 8 weeks of treatment for hematological evaluation, including Hb, hematocrit, and reticulocyte % (Fig. 1). At the end of 8 weeks, livers, kidneys, and plasma were collected. Hb and hematocrit levels increased in the 50 mg/kg FG-4592 group compared to the other groups at 4 weeks but decreased by 8 weeks for all groups. Reticulocyte % also decreased in all groups at the 4- and 8-week timepoints. Red cell distribution width (RDW) and platelet count up-trended while mean corpuscular volume (MCV) and mean corpuscular Hb (MCH) down-trended over the 8 weeks, suggestive of an evolving iron deficiency contributing to worsening anemia. Quantitative PCR detected a 182% increase in liver EPO transcript and a 68% increase in kidney EPO transcript in the 50 mg/kg FG-4592 treated group, compared to vehicle. No changes in liver hepcidin transcription were observed in any group. ELISA analysis detected increased plasma EPO and decreased plasma hepcidin levels with increasing doses of FG-4592. None of these trends, except increases in RDW, were statistically significant. Our study is the first to characterize the effect of HIF-PHI drugs in a mouse model of SCD. Preliminary data confirm an effect of FG-4592 on HbF induction and suggest that FG-4592 may increase EPO expression and decrease hepcidin levels in SCD mice. These results highlight the potential of prolyl hydroxylase inhibition as a novel therapeutic approach for chronic anemia in SCD. Future directions include testing higher doses of FG-4592 to induce a stronger erythropoietic effect and monitoring additional markers of iron homeostasis in our mouse model of SCD. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Cardiac and Kidney Adverse Effects of HIF Prolyl-Hydroxylase Inhibitors for Anemia in Patients With CKD Not Receiving Dialysis: A Systematic Review and Meta-analysis

Hypoxia-inducible factor prolyl hydroxylase inhibitors (HIF-PHIs) are novel, orally administered agents for anemia management in chronic kidney disease (CKD). We evaluated the cardiac and kidney-related adverse effects of HIF-PHIs among patients with CKD and anemia. Systematic review and meta-analysis of randomized controlled trials (RCTs). Patients with anemia and CKD not receiving maintenance dialysis. RCTs comparing HIF-PHIs to placebo or an erythropoiesis-stimulating agent (ESA) with primary outcomes of cardiac and kidney-related adverse events (AEs). Two independent reviewers evaluated RCTs for eligibility and extracted relevant data. Dichotomous variables were pooled using the Mantel-Haenszel method and presented as risk ratios (RRs). Subgroup analyses evaluated different intervention times and HIF-PHIs, as well as phase 2 versus phase 3 trials. The certainty of findings was rated according to GRADE criteria. Twenty-three studies with 15,144 participants were included. No significant difference in the risk of cardiac AEs was observed between the HIF-PHIs group and the placebo (RR, 1.02 [95% CI, 0.89-1.16]; moderate certainty) or ESA (RR, 1.06 [95% CI, 0.98-1.14]; low certainty) groups. No significant difference in the risk of kidney-related AEs was observed between the HIF-PHIs group and the placebo (RR, 1.09 [95% CI, 0.98-1.20]; moderate certainty) or ESA (RR, 1.00 [95% CI, 0.94-1.06]; low certainty) groups. The occurrence of hypertension and hyperkalemia was higher in the HIF-PHIs group than in the placebo group (RRs of 1.35 [95% CI, 1.14-1.60] and 1.25 [95% CI, 1.03-1.51], respectively; both findings had high certainty). The occurrence of hypertension was lower in the HIF-PHIs group than in the ESA group (RR, 0.89 [95% CI, 0.81-0.98]; moderate certainty). The reporting criteria of cardiac and kidney-related AEs and dosage of HIF-PHIs were inconsistent across trials. The occurrence of cardiac or kidney-related AEs in the HIF-PHI groups were not different compared with placebo or ESA groups. Registered at PROSPERO with registration number CRD42021228243.

Use of Hypoxia-Inducible Factor Prolyl Hydroxylase Inhibitor Agents for Anemia in Non-Dialysis-Dependent Chronic Kidney Disease: A Systematic Review and Meta-Analysis

Background: Anemia is a common complication associated with chronic kidney disease (CKD), driven by decreased erythropoietin (EPO) production and dysregulated iron metabolism. Anemia is associated with decreased quality of life, as well as increased cardiovascular events, dialysis requirements, and mortality. Treatment classically consists of iron repletion and erythropoietin stimulating agents (ESA). However, use of these agents can be associated with hyporesponsiveness, a need for close monitoring to avoid adverse cardiovascular events, and patient inconvenience due to subcutaneous or intravenous administration. Hypoxia-inducible factor prolyl hydroxylase inhibitors (HIF-PHIs) are novel oral therapeutic agents that promote accumulation of HIF transcription factors and expression of HIF-responsive genes, leading to increased synthesis of EPO and proteins involved in iron metabolism. Multiple large randomized controlled trials (RCTs) investigating the safety and efficacy of HIF-PHIs in the treatment of anemia of non-dialysis dependent CKD (NDD-CKD) have been completed. However, to the best of our knowledge, there have been no recent comprehensive systematic reviews synthesizing this evidence. Aim: The objective of this systematic review is to assess the safety and efficacy of HIF-PHIs in the treatment of anemia caused by NDD-CKD. Methods: A comprehensive search of MEDLINE, Embase, Cochrane (CENTRAL), and the grey literature was done from inception to April 2022. Studies were screened at abstract and full-text level independently and in duplicate. English-language phase three RCTs investigating the use of HIF-PHIs versus placebo or ESAs in anemia of NDD-CKD were included. Baseline characteristics, intervention details, comparator details, and outcome measurement timing were collected. Our primary efficacy outcome measures were mean change in hemoglobin from baseline and proportion of patients achieving a hemoglobin response. Our primary safety outcome was major adverse cardiac events (MACE) defined as a composite of nonfatal myocardial infarction, nonfatal stroke, and all-cause mortality. Meta-analysis was done using DerSimonian and Laird random effects model and reported as relative risks (RR) or mean differences (MD) with 95% confidence intervals (CI). Results: 14 studies with a total of 12,681 patients were included in the analysis. HIF-PHIs were associated with increased hemoglobin response rate compared to placebo (RR 9.02, 95%CI 6.85,11.86, p<0.005; I2=56%, N=5). There was no significant difference in hemoglobin response rate for HIF-PHIs versus ESAs (RR 0.97, 95%CI 0.9,1.04, p=0.36; I2=76%, N=9). HIF-PHIs were associated with an increased change in hemoglobin from baseline compared to placebo (MD=1.57, 95%CI 1.29,1.85, p<0.05; I2=93%, N=4). There was no significant difference in mean change in hemoglobin from baseline between HIF-PHIs and ESAs (MD -0.06, 95%CI -0.19,0.07, p=0.38; I2=0, N=2). There was no statistically significant difference in MACE between HIF-PHI and placebo (RR 1.14, 95%CI 0.93,1.40, p=0.2; I2=11%, N=4) or ESA (RR 1.03, 95%CI 0.89, 1.19, p=0.70; I2=23%, N=9). Conclusion: HIF-PHIs are associated with increased mean change in hemoglobin and hemoglobin response rate compared to placebo. HIF-PHIs are non-inferior to ESAs for these outcomes. When used to current hemoglobin targets, MACE outcomes are similar to those seen with ESAs.

Effect of Biantie pretreatment on serum level of PHD2/HIF-1α and brain tissue damage in rats during acute hypobaric hypoxia exposure

To observe the effect of Biantie (bian stone plaste) pretreatment on serum level of prolyl hydroxylase domain 2 (PHD2) and hypoxia-inducible factor-1α (HIF-1α) in rats with acute hypobaric hypoxia induced-brain injury, and to explore the possible mechanism of Biantie on preventing brain injury at high altitude. Forty-five male SD rats were randomly divided into a blank group, a model group, a Biantie group, a medication group and a Biantie+inhibitor group, 9 rats in each group. The rats in the Biantie group the and the Biantie+inhibitor group were pretreated with Biantie at "Taiyuan" (LU 9), "Neiguan" (PC 6) and "Renying" (ST 9), 2 h each time, once a day; the rats in the medication group were treated with intragastric administration of rhodiola capsule solution (280 mg/kg) for 14 d; the rats in the Biantie+inhibitor group were intraperitoneally injected with the PHD inhibitor dimethyloxalyl glycine (DMOG) at a dose of 40 mg/kg 24 h before the establishment of the model. After the intervention, except for the blank group, the rats in the remaining 4 groups were placed in the oxygen chamber to simulate a high-altitude environment to establish the acute hypobaric hypoxia brain injury model. The arterial blood-gas analysis indexes [blood oxygen saturation (SaO2), lactic acid (Lac), blood sodium (Na+), blood potassium (K+)] and brain water content were detected in each group; the histomorphology of cerebral cortex was observed by HE staining; the serum levels of PHD2 and HIF-1α as well as vascular endothelial growth factor (VEGF) were detected by ELISA; the VEGF protein expression in brain tissue was detected by Western blot; the VEGF mRNA expression in brain tissue was detected by real-time fluorescent quantitative PCR. Compared with the blank group, the levels of SaO2 and Na+ in the model group were decreased (P<0.05), while the levels of Lac and K+ as well as the water content of brain tissue were increased (P<0.05). Compared with the model group, the level of SaO2 in the Biantie group and the medication group was increased (P<0.05), while the levels of Lac, K+ and the water content of brain tissue were decreased (P<0.05); the level of Na+ in the Biantie group was increased (P<0.05). Compared with the Biantie group, the level of SaO2 in the Biantie+inhibitor group was decreased (P<0.05), and the level of Lac and the water content of brain tissue were increased (P<0.05). In the model group, the cortical tissue cells were loose and disordered, the cortical blood vessels were dilated, and the cells were obviously swollen; the anoxic injury in the Biantie group and the medication group was lighter, and the anoxic injury in the Biantie+inhibitor group was more obvious than that in the Biantie group. Compared with the blank group, the serum PHD2 content in the model group was decreased and the HIF-1α content was increased (P<0.05), and the content of VEGF in serum and VEGF protein and mRNA expressions in brain were increased (P<0.05). Compared with the model group, the content of PHD2 in serum in the Biantie group and the medication group was increased (P<0.05), and the level of HIF-1α was decreased (P<0.05), and the content of VEGF in serum as well as VEGF protein and mRNA expressions in brain were decreased (P<0.05). Compared with the Biantie group, the serum PHD2 content in the Biantie+inhibitor group was decreased and HIF-1α level were increased (P<0.05), and the content of VEGF in serum as well as VEGF mRNA expression in brain were increased (P<0.05). Biantie at "Taiyuan" (LU 9), "Neiguan" (PC 6) and "Renying" (ST 9) could regulate serum PHD2/HIF-1α to down-regulate VEGF expression, reduce brain edema and enhance anti-hypoxia ability, so as to achieve the purpose of preventing brain injury at high altitude.

Abstract 15056: Cardiac P62/sqstm1 Deficiency Exacerbates Hypoxic Stress vi a Impaired Hypoxia Inducible Factor 1α Signaling

Ischemic heart disease (IHD), a major cause of heart failure, is characterized by metabolic dysfunction and myocardial cell death. Cellular hypoxia activates hypoxia inducible factor 1α (HIF1α) to initiate metabolic, angiogenic, and growth-factor related responses. Under normoxia, HIF1α is degraded by prolyl hydroxylase (PHD) domain-containing proteins via the proteasome. Due to the short half-life of HIF1α (&lt;5mins), PHD inhibitors are utilized to stabilize HIF1α and improve cardiac function in pre-clinical models of ischemia/infarction. However, evidence exists that PHD inhibitors exert off-target effects. Based on prior studies in cancer cells demonstrating that the autophagy adaptor protein p62 prevents HIF1α degradation, we hypothesized that p62 stabilizes cardiac HIF1α and provides cardioprotection from hypoxia. We generated mice with tamoxifen-inducible cardiomyocyte-specific p62 deletion (p62icKO mice; 8 week male and female) and exposed them to 7% O 2 for 6h. Hypoxia increased cardiac mRNA expression of Sqstm1 , Bnip3 , and Hmox1 in wild-type (WT) but not p62icKO mice. Underscoring functional relevance, ejection fraction and fractional shortening were less, oxidative stress was elevated, and apoptosis was greater, in p62icKO vs. WT mice exposed to hypoxia. Mechanisms were evaluated using cultured H9c2 cardiomyoblasts exposed to 21% (normoxia) or 1% (hypoxia) O 2 for 24h. Hypoxia increased both HIF1α and p62 protein expression. To determine the role of p62, H9c2 cells were transfected with p62 or scrambled (ctrl) siRNA for 48h, followed by 24 h hypoxia. Hypoxia-induced HIF1α protein accrual and expression of HIF1α downstream targets Egln1 , Vegfa , Bnip3 and Hmox1 mRNA was attenuated in H9c2 cells transfected with p62 vs. control siRNA. These data indicate p62 stabilizes cardiac HIF1α and regulates its transcriptional activation. Thus, p62 could be a novel therapeutic target for treatment of IHD.

Hypoxia-inducible factor 1α enhances RANKL-induced osteoclast differentiation by upregulating the MAPK pathway

Hypoxia (low-oxygen tension) and excessive osteoclast activation are common conditions in many bone loss diseases, such as osteoporosis, rheumatoid arthritis (RA), and pathologic fractures. Hypoxia-inducible factor 1 alpha (HIF1α) regulates cellular responses to hypoxic conditions. However, it is not yet known how HIF1α directly affects osteoclast differentiation and activation. This study sought to. explore the effects of HIF1α on osteoclast differentiation and it's molecular mechanisms. L-mimosine, a prolyl hydroxylase (PHDs) domain inhibitor, was used to stabilize HIF1α in normoxia. In the presence of receptor activator of nuclear factor-kB (NF-kB) ligand (RANKL), RAW264.7 cells were cultured and stimulated by treatment with L-mimosine at several doses to maintain various levels of intracellular HIF1α. The multi-nucleated cells were assessed by a tartrate-resistant acid phosphatase (TRAP) and F-actin ring staining assays. The osteoclast-specific genes, such as Cathepsin K, β3-Integrin, TRAP, c-Fos, nuclear factor of activated T cells, cytoplasmic 1 (NFATc1), and matrix metallo-proteinase 9 (MMP9), were analyzed by real time-polymerase chain reaction (RT-PCR). The expression of relevant proteins was analyzed by Western blot. L-mimosine increased the content of intracellular HIF1α in a dose-dependent manner, which in turn promoted RANKL-induced osteoclast formation and relevant protein expression by upregulating the mitogen-activated protein kinase (MAPK) pathways. Our findings suggest that HIF1α directly increases the osteoclast differentiation of RANKL-mediated RAW264.7 cells in vitro by upregulating the MAPK pathways.

Potential Long-Term Economic Benefits of Switching From Epoetin Alfa to Hypoxia-Inducible Factor Prolyl Hydroxylase (HIF-PH) Inhibitors for the Management of Anemia in Hemodialysis (HD) Patients

Potential Long-Term Economic Benefits of Switching From Epoetin Alfa to Hypoxia-Inducible Factor Prolyl Hydroxylase (HIF-PH) Inhibitors for the Management of Anemia in Hemodialysis (HD) Patients

Evolving Strategies in the Treatment of Anaemia in Chronic Kidney Disease: The HIF-Prolyl Hydroxylase Inhibitors.

Chronic kidney disease (CKD) affects approximately 10% of the worldwide population; anaemia is a frequent complication. Inadequate erythropoietin production and absolute or functional iron deficiency are the major causes. Accordingly, the current treatment is based on iron and erythropoiesis stimulating agents (ESAs). Available therapy has dramatically improved the management of anaemia and the quality of life. However, safety concerns were raised over ESA use, especially when aiming to reach near-to-normal haemoglobin levels with high doses. Moreover, many patients show hypo-responsiveness to ESA. Hypoxia-inducible factor (HIF) prolyl hydroxylase domain (PHD) inhibitors (HIF-PHIs) were developed for the oral treatment of anaemia in CKD to overcome these concerns. They simulate the body's exposure to moderate hypoxia, stimulating the production of endogenous erythropoietin. Some molecules are already approved for clinical use in some countries. Data from clinical trials showed non-inferiority in anaemia correction compared to ESA or superiority for placebo. Hypoxia-inducible factor-prolyl hydroxylase domain inhibitors may also have additional advantages in inflamed patients, improving iron utilisation and mobilisation and decreasing LDL-cholesterol. Overall, non-inferiority was also shown in major cardiovascular events, except for one molecule in the non-dialysis population. This was an unexpected finding, considering the lower erythropoietin levels reached using these drugs due to their peculiar mechanism of action. More data and longer follow-ups are necessary to better clarifying safety issues and further investigate the variety of pathways activated by HIF, which could have either positive or negative effects and could differentiate HIF-PHIs from ESAs.

The Prognostic Significance and Potential Mechanism of Prolyl 3-Hydroxylase 1 in Hepatocellular Carcinoma

Prolyl 3-hydroxylase 1 (P3H1) is essential for human collagen synthesis. Here, we investigated its relevance to multiple cancers, especially hepatocellular carcinoma (LIHC). We estimated the relationship of P3H1 with 33 cancers using publicly available databases. And immunohistochemistry was utilized to verify the P3H1 expression in liver, gastric, colon, pancreatic, and rectal cancer. Then, we attenuated P3H1 expression in BEL-7402 and HLF cells by lentivirus technology and assessed the effect of P3H1 on cell proliferation, migration, and invasion. Bioinformatic analysis revealed a significantly higher expression of P3H1 in almost all tumors, which was consistent with the immunohistochemical findings in the liver, gastric, colon, pancreatic, and rectal cancers. P3H1 expression was associated with overall survival, progression-free interval, disease-specific survival, and disease-free interval in most cancers, particularly in LIHC. Besides, we also found that P3H1 expression was an independent prognostic factor for LIHC. And knockdown of P3H1 significantly reduced liver cancer cell proliferation, migration, and invasion in liver cancer cells. Interestingly, P3H1 expression levels showed a significant positive connection with Th2 infiltration through multiple immune infiltration algorithms. ICI treatment was less effective in LIHC patients with high P3H1 expression. Finally, we also identified an upstream regulatory mechanism of P3H1 in LIHC, namely, AL355488.1, HCG18, and THUMPD3-AS1/hsa-miR-29c-3p-P3H1 axis. We have systematically described for the first time that P3H1 is closely related to various tumors, particularly in LIHC, and interference with P3H1 may be a therapeutic target for patients with LIHC.

Methyltransferase SMYD3 impairs hypoxia tolerance by augmenting hypoxia signaling independent of its enzymatic activity

Hypoxia-inducible factor (HIF)1α, a main transcriptional regulator of the cellular response to hypoxia, also plays important roles in oxygen homeostasis of aerobic organisms, which is regulated by multiple mechanisms. However, the full cellular response to hypoxia has not been elucidated. In this study, we found that expression of SMYD3, a methyltransferase, augments hypoxia signaling independent of its enzymatic activity. We demonstrated SMYD3 binds to and stabilizes HIF1α via co-immunoprecipitation and Western blot assays, leading to the enhancement of HIF1α transcriptional activity under hypoxia conditions. In addition, the stabilization of HIF1α by SMYD3 is independent of HIF1α hydroxylation by prolyl hydroxylases and the intactness of the von Hippel-Lindau ubiquitin ligase complex. Furthermore, we showed SMYD3 induces reactive oxygen species accumulation and promotes hypoxia-induced cell apoptosis. Consistent with these results, we found smyd3-null zebrafish exhibit higher hypoxia tolerance compared to their wildtype siblings. Together, these findings define a novel role of SMYD3 in affecting hypoxia signaling and demonstrate that SMYD3-mediated HIF1α stabilization augments hypoxia signaling, leading to the impairment of hypoxia tolerance.

Crystal structure of the collagen prolyl 4-hydroxylase (C-P4H) catalytic domain complexed with PDI: Toward a model of the C-P4H α2β2 tetramer

Collagen prolyl 4-hydroxylases (C-P4H) are α2β2 tetramers, which catalyze the prolyl 4-hydroxylation of procollagen, allowing for the formation of the stable triple-helical collagen structure in the endoplasmic reticulum. The C-P4H α-subunit provides the N-terminal dimerization domain, the middle peptide-substrate-binding (PSB) domain, and the C-terminal catalytic (CAT) domain, whereas the β-subunit is identical to the enzyme protein disulfide isomerase (PDI). The structure of the N-terminal part of the α-subunit (N-terminal region and PSB domain) is known, but the structures of the PSB-CAT linker region and the CAT domain as well as its mode of assembly with the β/PDI subunit, are unknown. Here, we report the crystal structure of the CAT domain of human C-P4H-II complexed with the intact β/PDI subunit, at 3.8Å resolution. The CAT domain interacts with the a, b', and a' domains of the β/PDI subunit, such that the CAT active site is facing bulk solvent. The structure also shows that the C-P4H-II CAT domain has a unique N-terminal extension, consisting of α-helices and a β-strand, which is the edge strand of its major antiparallel β-sheet. This extra region of the CAT domain interacts tightly with the β/PDI subunit, showing that the CAT-PDI interface includes an intersubunit disulfide bridge with the a' domain and tight hydrophobic interactions with the b' domain. Using this new information, the structure of the mature C-P4H-II α2β2 tetramer is predicted. The model suggests that the CAT active-site properties are modulated by α-helices of the N-terminal dimerization domains of both subunits of the α2-dimer.

Metformin and simvastatin synergistically suppress endothelin 1-induced hypoxia and angiogenesis in multiple cancer types.

Multiple cancers have been reported to be associated with angiogenesis and are sensitive to anti-angiogenic therapies. Vascular normalization, by restoring proper tumor perfusion and oxygenation, could limit tumor cell invasiveness and improve the effectiveness of anticancer treatments. However, the underlying anticancer mechanisms of antiangiogenic drugs are still unknown. Metformin (MET) and simvastatin (SVA), two metabolic-related drugs, have been shown to play important roles in modulating the hypoxic tumor microenvironment and angiogenesis. Whether the combination of MET and SVA could exert a more effective antitumor effect than individual treatments has not been examined. The antitumor effect of the synergism of SVA and MET was detected in mouse models, breast cancer patient-derived organoids, and multiple tumor cell lines compared with untreated, SVA, or MET alone. RNA sequencing revealed that the combination of MET and SVA (but not MET or SVA alone) inhibited the expression of endothelin 1 (ET-1), an important regulator of angiogenesis and the hypoxia-related pathway. We demonstrate that the MET and SVA combination showed synergistic effects on inhibiting tumor cell proliferation, promoting apoptosis, alleviating hypoxia, decreasing angiogenesis, and increasing vessel normalization compared with the use of a single agent alone. The MET and SVA combination suppressed ET-1-induced hypoxia-inducible factor 1α expression by increasing prolyl hydroxylase 2 (PHD2) expression. Furthermore, the MET and SVA combination showed a more potent anticancer effect compared with bosentan. Together, our findings suggest the potential application of the MET and SVA combination in antitumor therapy.

Role of prolyl hydroxylase/HIF-1 signaling in vascular calcification.

Morbidity and mortality of chronic kidney disease (CKD) patients are largely associated with vascular calcification, an actively regulated process in which vascular smooth muscle cells (VSMCs) change into cells similar to osteocytes/chondrocytes, known as trans-differentiation. Cellular and systemic response to low oxygen (hypoxia) is regulated by the prolyl hydroxylase/hypoxia-inducible factor (HIF)-1 pathway. Recent studies highlighted that hypoxia-mediated activation of HIF-1 induces trans-differentiation of VSMCs into bone-forming type through an increase in osteo-/chondrogenic genes. Inhibition of the HIF-1 pathway abolished osteochondrogenic differentiation of VSMCs. Hypoxia strongly enhanced elevated phosphate-induced VSMC osteogenic trans-differentiation and calcification. HIF-1 was shown to be essential for phosphate enhanced VSMC calcification. O2-dependent degradation HIF-1 is triggered by the prolyl hydroxylase domain proteins (PHD). Prolyl hydroxylase inhibitors, daprodustat and roxadustat, increase high phosphate-induced VC in VSMCs, stabilizing HIF-1α and activating the HIF-1 pathway in these cells. Whether the use of these PHD inhibitors to treat anemia in CKD patients will favor the development and progression of vascular calcification remains to be explored.

Precise Monitoring and Assessing Treatment Response of Sepsis-Induced Acute Lung Hypoxia with a Nitroreductase-Activated Golgi-Targetable Fluorescent Probe.

Sepsis-induced acute lung injury (ALI) is mostly attributed to an outbreak of reactive oxygen species (ROS), which makes leukocytes infiltrate into the lung and results in lung hypoxia. Nitroreductase (NTR) is significantly upregulated under hypoxia, which is commonly regarded as a potential biomarker for assessing sepsis-induced acute lung hypoxia. Increasing evidence shows that NTR in the Golgi apparatus could be induced in sepsis-induced ALI. Meanwhile, the prolyl hydroxylase (PHD) inhibitor (dimethyloxalylglycine, DMOG) attenuated sepsis-induced ALI through further increasing the level of Golgi NTR by improving hypoxia inducible factor-1α (HIF-1α) activity, but as yet, no Golgi-targetable probe has been developed for monitoring and assessing treatment response of sepsis-induced ALI. Herein, we report a Golgi-targetable probe, Gol-NTR, for monitoring and assessing treatment response of sepsis-induced ALI through mapping the generation of NTR. The probe displayed high sensitivity with a low detection limit of 54.8 ng/mL and good selectivity to NTR. In addition, due to the excellent characteristics of Golgi-targetable, Gol-NTR was successfully applied in mapping the change of Golgi NTR in cells and zebrafish caused by various stimuli. Most importantly, the production of Golgi NTR in the sepsis-induced ALI and the PHD inhibitor (DMOG) against sepsis-induced ALI were visualized and precisely assessed for the first time with the assistance of Gol-NTR. The results demonstrated the practicability of Gol-NTR for the precise monitoring and assessing of the personalized treatment response of sepsis-induced ALI.

Activation of γ-globin expression by hypoxia-inducible factor 1α.

Around birth, globin expression in human red blood cells (RBCs) shifts from γ-globin to β-globin, which results in fetal haemoglobin (HbF, α2γ2) being gradually replaced by adult haemoglobin (HbA, α2β2)1. This process has motivated the development of innovative approaches to treat sickle cell disease and β-thalassaemia by increasing HbF levels in postnatal RBCs2. Here we provide therapeutically relevant insights into globin gene switching obtained through a CRISPR-Cas9 screen for ubiquitin-proteasome components that regulate HbF expression. In RBC precursors, depletion of the von Hippel-Lindau (VHL) E3 ubiquitin ligase stabilized its ubiquitination target, hypoxia-inducible factor 1α (HIF1α)3,4, to induce γ-globin gene transcription. Mechanistically, HIF1α-HIF1β heterodimers bound cognate DNA elements in BGLT3, a long noncoding RNA gene located 2.7 kb downstream of the tandem γ-globin genes HBG1 and HBG2. This was followed by the recruitment of transcriptional activators, chromatin opening and increased long-range interactions between the γ-globin genes and their upstream enhancer. Similar induction of HbF occurred with hypoxia or with inhibition of prolyl hydroxylase domain enzymes that target HIF1α for ubiquitination by the VHL E3 ubiquitin ligase. Our findings link globin gene regulation with canonical hypoxia adaptation, provide a mechanism for HbF induction during stress erythropoiesis and suggest a new therapeutic approach for β-haemoglobinopathies.

In Situ Inhibitor Synthesis and Screening by Fluorescence Polarization: An Efficient Approach for Accelerating Drug Discovery.

Target-directed dynamic combinatorial chemistry has emerged as a useful tool for hit identification, but has not been widely used, in part due to challenges associated with analyses involving complex mixtures. We describe an operationally simple alternative: in situ inhibitor synthesis and screening (ISISS), which links high-throughput bioorthogonal synthesis with screening for target binding by fluorescence. We exemplify the ISISS method by showing how coupling screening for target binding by fluorescence polarization with the reaction of acyl-hydrazides and aldehydes led to the efficient discovery of a potent and novel acylhydrazone-based inhibitor of human prolyl hydroxylase 2 (PHD2), a target for anemia treatment, with equivalent in vivo potency to an approved medicine.

The ribosomal chaperone NACA recruits PHD2 to cotranslationally modify HIF-α.

Prolyl hydroxylase domain protein 2 (PHD2)-catalyzed modification of hypoxia-inducible factor (HIF)-α is a key event in oxygen sensing. We previously showed that the zinc finger of PHD2 binds to a Pro-Xaa-Leu-Glu (PXLE) motif. Here, we show that the zinc finger binds to this motif in the ribosomal chaperone nascent polypeptide complex-α (NACA). This recruits PHD2 to the translation machinery to cotranslationally modify HIF-α. Importantly, this cotranslational modification is enhanced by a translational pause sequence in HIF-α. Mice with a knock-in Naca gene mutation that abolishes the PXLE motif display erythrocytosis, a reflection of HIF pathway dysregulation. In addition, human erythrocytosis-associated mutations in the zinc finger of PHD2 ablate interaction with NACA. Tibetans, who have adapted to the hypoxia of high altitude, harbor a PHD2 variant that we previously showed displays a defect in zinc finger binding to p23, a PXLE-containing HSP90 cochaperone. We show here that Tibetan PHD2 maintains interaction with NACA, thereby showing differential interactions with PXLE-containing proteins and providing an explanation for why Tibetans are not predisposed to erythrocytosis.

In Situ Inhibitor Synthesis and Screening by Fluorescence Polarization: An Efficient Approach for Accelerating Drug Discovery.

Target-directed dynamic combinatorial chemistry has emerged as a useful tool for hit identification, but has not been widely used, in part due to challenges associated with analyses involving complex mixtures. We describe an operationally simple alternative: in situ inhibitor synthesis and screening (ISISS), which links high-throughput bioorthogonal synthesis with screening for target binding by fluorescence. We exemplify the ISISS method by showing how coupling screening for target binding by fluorescence polarization with the reaction of acyl-hydrazides and aldehydes led to the efficient discovery of a potent and novel acylhydrazone-based inhibitor of human prolyl hydroxylase 2 (PHD2), a target for anemia treatment, with equivalent in vivo potency to an approved medicine.

Efficient Delivery of P3H4 siRNA and Chlorin e6 by cRGDfK-Installed Polyarginine Nanoparticles for Tumor-Targeting Therapy of Bladder Cancer.

Purpose: Prolyl 3-hydroxylase family member 4 (P3H4) is a potent prognostic oncogene in bladder cancer (BC), and the inhibition of P3H4 suppresses BC tumor growth. This study aimed to evaluate the efficiency of P3H4 inhibition for BC tumor therapy via tumor-targeting nanoparticles. Methods and results: A linear polyarginine peptide (R9) was synthesized, azide-modified, and then assembled with cyclic pentapeptide cRGDfK. Chlorin e6 (ce6)-conjugated CH3-R9-RGD nanoparticles were prepared for the delivery of siP3H4 into T24 cells in vitro and BC tumors in vivo. Dynamic light scattering analysis identified that the optimum CH3-R9-RGD@siP3H4 molar ratio was 30/1. CH3-R9-RGD@ce6/siP3H4 nanocomposites decreased P3H4 expression and cell proliferation and promoted reactive oxygen species production, apoptosis, and calreticulin exposure in T24 cells in vitro. In vivo experiments showed that CH3-R9-RGD@ce6/siP3H4 nanocomposites caused pathological changes, suppressed BC tumor growth, promoted caspase 3 expression, and enhanced calreticulin exposure in tumor cells. Conclusions: The tumor-targeting CH3-R9-RGD nanocomposites encapsulating siP3H4 and ce6 might be an alternative therapeutic strategy or intravesical instillation chemotherapy for BC.

HIF-α activation by the prolyl hydroxylase inhibitor roxadustat suppresses chemoresistant glioblastoma growth by inducing ferroptosis

Patients with glioblastoma (GBM) have poor prognosis and limited treatment options, largely due to therapy resistance upon the induction of apoptosis. Ferroptosis emerges as a potential antineoplastic strategy to bypass apoptosis resistance in traditional therapeutics. Hypoxia is a fundamental hallmark of GBM and hypoxia-inducible factor (HIF) is the main regulator of hypoxia response, however, the role of HIF has not been sufficiently explored in GBM. Herein, we first discovered that amplifying HIF signals by the prolyl hydroxylase (PHD) inhibitor roxadustat significantly suppressed GBM cell growth in vitro and in vivo, especially when the cells were resistant to temozolomide (TMZ). The accumulation of lipid peroxidation and cellular iron in GBM cells following roxadustat treatment indicated that the cells underwent ferroptosis, which was also supported by morphological changes in mitochondrial ultrastructure and immunogenic signals release. Moreover, in vivo studies further confirmed the ferroptosis induction and verified that roxadustat significantly prolonged survival of the mice harboring chemoresistant GBM without visible organ toxicity. Finally, we proved that the ferroptosis induction by roxadustat is HIF-α independent, especially activation of HIF-2α upregulating lipid regulatory genes was revealed to be mainly responsible for the enhanced lipid peroxidation. Altogether, our study provided novel evidence that amplifying HIF signals induced ferroptosis in chemoresistant GBM cells and suppressed the tumor growth in vivo, highlighting that ferroptosis induction by targeting HIF-α might provide new approaches to improve GBM treatment.