Zinc Supplementation Enhances the Hematopoietic Activity of Erythropoiesis-Stimulating Agents but Not Hypoxia-Inducible Factor-Prolyl Hydroxylase Inhibitors.

Since zinc is involved in many aspects of the hematopoietic process, zinc supplementation can reduce erythropoiesis-stimulating agents (ESAs) in patients undergoing hemodialysis. However, it remains unclear whether hypoxia-inducible factor-prolyl hydroxylase inhibitors (HIF-PHIs) have similar reduction effects. HIF-PHI stabilizes HIF, which promotes hematopoiesis, although HIF-1α levels are downregulated by zinc. This study aimed to investigate the effect of zinc supplementation on the hematopoietic effect of HIF-PHI in patients undergoing hemodialysis. Thirty patients undergoing maintenance hemodialysis who underwent periods of treatment with roxadustat or darbepoetin alfa during the past 3 years were retrospectively observed. Participants who underwent periods with and without zinc supplementation were selected, with nine treated with darbepoetin alfa and nine treated with roxadustat. Similarly to the ESA responsiveness index (ERI), the hematopoietic effect of zinc supplementation was determined by the HIF-PHI responsiveness index (HRI), which was calculated by dividing the HIF-PHI dose (mg/week) by the patient's dry weight (kg) and hemoglobin level (g/L). Zinc supplementation significantly increased ERI (p < 0.05), but no significant change was observed (p = 0.931) in HRI. Although zinc supplementation did not significantly affect HRI, adequate zinc supplementation is required to alleviate concerns such as vascular calcification and increased serum copper during the use of HIF-PHI.

Multimodal Imaging Reveals that Sustained Inhibition of HIF-Prolyl Hydroxylases Induces Opposing Effects on Right and Left Ventricular Function in Healthy Rats.

PURPOSE: Hypoxia-inducible factor (HIF) drives transcription of critical hypoxia response genes, increasing the production of red blood cells in low oxygen conditions. In the absence of hypoxia, HIF is degraded by prolyl hydroxylases (HIF-PHs). Pharmacological HIF-PH inhibition stabilizes HIF and is being studied as a treatment for anemia. However, like sustained hypoxia, HIF-PH inhibition may increase pulmonary arterial pressure leading to right ventricular hypertrophy. The aim of this study was to assess the cardiac effects of sustained pharmacological HIF-PH inhibition using multimodal imaging, blood analysis, and histology. METHODS: Rats were dosed daily with a pan HIF-PH inhibitor or vehicle for 4 weeks followed by a 2-week washout period and underwent longitudinal magnetic resonance imaging (MRI) and echocardiography to simultaneously assess RV and LV function. Blood samples from weeks four and six were analyzed to determine red blood cell composition. Histology was performed on the cardiac tissue from a subset of rats at weeks four and six to assess structural effects. RESULTS: Imaging revealed that RV ejection fraction was reduced in animals receiving HIF-PH inhibitor and resulted in RV hypertrophy. Interestingly, HIF-PH inhibition had the opposite effect on the left ventricle (LV), increasing contractility measured by LV ejection fraction. LV effects were reversed by week six, while RV effects (functional and structural) were sustained. CONCLUSION: These opposing cardiac effects of HIF-PH inhibition warrant further study to both understand the potential negative effects on RV structure and function and investigate the therapeutic potential of increased LV contractility for conditions like heart failure.

ARID1A loss induces P4HB to activate fibroblasts to support lung cancer cell growth, invasion, and chemoresistance.

Loss of AT-interacting domain-rich protein 1A (ARID1A) frequently occurs in human malignancies including lung cancer. The biological consequence of ARID1A mutation in lung cancer is not fully understood. This study was designed to determine the effect of ARID1A-depleted lung cancer cells on fibroblast activation. Conditioned media was collected from ARID1A-depleted lung cancer cells and employed to treat lung fibroblasts. The proliferation and migration of lung fibroblasts were investigated. The secretory genes were profiled in lung cancer cells upon ARID1A knockdown. Antibody-based neutralization was utilized to confirm their role in mediating the cross-talk between lung cancer cells and fibroblasts. NOD-SCID-IL2RgammaC-null (NSG) mice received tumor tissues from patients with ARID1A-mutated lung cancer to establish patient-derived xenograft (PDX) models. Notably, ARID1A-depleted lung cancer cells promoted the proliferation and migration of lung fibroblasts. Mechanistically, ARID1A depletion augmented the expression and secretion of prolyl 4-hydroxylase beta (P4HB) in lung cancer cells, which induced the activation of lung fibroblasts through the ß-catenin signaling pathway. P4HB-activated lung fibroblasts promoted the proliferation, invasion, and chemoresistance in lung cancer cells. Neutralizing P4HB hampered the tumor growth and increased cisplatin cytotoxic efficacy in two PDX models. Serum P4HB levels were higher in ARID1A-mutated lung cancer patients than in healthy controls. Moreover, increased serum levels of P4HB were significantly associated with lung cancer metastasis. Together, our work indicates a pivotal role for P4HB in orchestrating the cross-talk between ARID1A-mutated cancer cells and cancer-associated fibroblasts during lung cancer progression. P4HB may represent a promising target for improving lung cancer treatment.

The effects of prolyl hydroxylase inhibition during and post, hypoxia, oxygen glucose deprivation and oxidative stress, in isolated rat hippocampal slices.

The contributions of hypoxia and oxidative stress to the pathophysiology of acute ischemic stroke are well established and can lead to disruptions in synaptic signaling. Hypoxia and oxidative stress lead to the neurotoxic overproduction of reactive oxygen species (ROS) and the stabilization of hypoxia inducible factors (HIF). Compounds such as prolyl-4-hydroxylase domain enzyme inhibitors (PHDIs) have been shown to have a preconditioning and neuroprotective effect against ischemic insults such as hypoxia, anoxia, oxygen glucose deprivation (OGD) or H2O2. Therefore, this study explored the effects of two PHDIs, JNJ-42041935 (10 µM) and roxadustat (100 µM) on cell viability using organotypic hippocampal slice cultures. We also assessed the effects of these compounds on synaptic transmission during and post hypoxia, OGD and H2O2 application in isolated rat hippocampal slices using field recording electrophysiological techniques and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor subunit trafficking using immunohistochemistry. Our organotypic data demonstrated a protective role for both inhibitors, where slices had significantly less cell death post anoxia and OGD compared to controls. We also report a distinct modulatory role for both JNJ-42041935 and roxadustat on fEPSP slope post hypoxia and OGD but not H2O2. In addition, we report that application of roxadustat impaired long-term potentiation, but only when applied post-hypoxia. This inhibitory effect was not reversed with co-application of the cyclin-dependent kinase 5 (CDK-5) inhibitor, roscovitine (10 µM), suggesting a CDK-5 independent synaptic AMPAR trafficking mechanism. Both hypoxia and OGD induced a reduction in synaptic AMPA GluA2 subunits, the OGD effect being reversed by prior treatment with both JNJ-42041935 and roxadustat. These results suggest an important role for PHDs in synaptic signaling and plasticity during episodes of ischemic stress.

Astragaloside IV restrains pyroptosis and fibrotic development of pulmonary artery smooth muscle cells to ameliorate pulmonary artery hypertension through the PHD2/HIF1α signaling pathway.

BACKGROUND: Astragaloside (AS)-IV, extracted from traditional Chinese medicine Astragalus mongholicus, has been widely used in the anti-inflammatory treatment for cardiovascular disease. However, the mechanism by which AS-IV affects pulmonary artery hypertension (PAH) development remains largely unknown. METHODS: Monocrotaline (MCT)-induced PAH model rats were administered with AS-IV, and hematoxylin-eosin staining and Masson staining were performed to evaluate the histological change in pulmonary tissues of rats. Pulmonary artery smooth muscle cells (PASMCs) were treated by hypoxia and AS-IV. Pyroptosis and fibrosis were assessed by immunofluorescence, western blot and enzyme-linked immunosorbent assay. RESULTS: AS-IV treatment alleviated pulmonary artery structural remodeling and pulmonary hypertension progression induced by MCT in rats. AS-IV suppressed the expression of pyroptosis-related markers, the release of pro-inflammatory cytokine interleukin (IL)-1ß and IL-18 and fibrosis development in pulmonary tissues of PAH rats and in hypoxic PAMSCs. Interestingly, the expression of prolyl-4-hydroxylase 2 (PHD2) was restored by AS-IV administration in PAH model in vivo and in vitro, while hypoxia inducible factor 1α (HIF1α) was restrained by AS-IV. Mechanistically, silencing PHD2 reversed the inhibitory effect of AS-IV on pyroptosis, fibrosis trend and pyroptotic necrosis in hypoxia-cultured PASMCs, while the HIF1α inhibitor could prevent these PAH-like phenomena. CONCLUSION: Collectively, AS-IV elevates PHD2 expression to alleviate pyroptosis and fibrosis development during PAH through downregulating HIF1α. These findings may provide a better understanding of AS-IV preventing PAH, and the PHD2/HIF1α axis may be a potential anti-pyroptosis target during PAH.

DPP-4i versus SGLT2i as modulators of PHD3/HIF-2α pathway in the diabetic kidney.

RATIONALE: Renal hypoxia is one of the currently highlighted pathophysiologic mechanisms of diabetic nephropathy (DN). Both hypoxia-inducible factor-1α (HIF-1α) and HIF-2α are major regulators of renal adaptive responses to hypoxia. OBJECTIVES: This study aims to compare the effects of vildagliptin (a dipeptidyl peptidase-IV inhibitor, DPP-4i) and empagliflozin (a sodium-glucose cotransporter 2 inhibitor, SGLT2i) on the differential expression of renal HIF-1α/2α. Tissue expression of prolylhydroxylase 3 (PHD3), a key regulator of HIF-2α stability, was also highlighted in a diabetic nephropathy rat model. Type 1 diabetes mellitus was induced and diabetic rats were treated with either Vildagliptin or Empagliflozin (10 mg/kg/d each) for 12 weeks. Improvements in the kidney functional and histopathological parameters were addressed and correlated to changes in the renal expression of HIF-1α/2α, and PHD3. Urinary KIM-1 concentration was tested as a correlate to HIF pathway changes. FINDINGS: Both vildagliptin- and empagliflozin-treated groups exhibited significant improvement in the functional, pathological, and ultra-structural renal changes induced by chronic diabetes. Compared to the untreated group, renal gene expression of HIF-1α was decreased while that of HIF-2α was increased in both treated groups, with significantly greater effects observed with SGLT2i. Renal PHD3 immune-reactivity was also decreased by both drugs, again with better efficacy for the SGLT2i. Importantly, improvements in the diabetic kidney biochemical and structural biomarkers were significantly correlated to PHD3 reductions and HIF-2α increments. CONCLUSIONS: Both DPP-4i and SGLT2i could delay the progression of DN through their differential modulating effects on the PHD3/ HIF-2α pathway with significantly better efficacy for SGLT2i.

Effects of hypoxia-inducible factor-prolyl hydroxylase inhibitors vs. erythropoiesis-stimulating agents on iron metabolism in non-dialysis-dependent anemic patients with CKD: A network meta-analysis.

Objective: To compare the effects of five hypoxia-inducible factor-prolyl hydroxylase domain inhibitors (HIF-PHIs), two erythropoiesis-stimulating agents (ESAs), and placebo on iron metabolism in renal anemia patients with non-dialysis-dependent chronic kidney disease (NDD-CKD). Method: Five electronic databases were searched for studies. Randomized controlled clinical trials comparing HIF-PHIs, ESAs, and placebo in NDD-CKD patients were selected. The statistical program used for network meta-analysis was Stata/SE 15.1. The main outcomes were the change in hepcidin and hemoglobin (Hb) levels. The merits of intervention measures were predicted by the surface under the cumulative ranking curve method. Results: Of 1,589 original titles screened, data were extracted from 15 trials (3,228 participants). All HIF-PHIs and ESAs showed greater Hb level-raising ability than placebo. Among them, desidustat demonstrated the highest probability of increasing Hb (95.6%). Hepcidin [mean deviation (MD) = -43.42, 95%CI: -47.08 to -39.76], ferritin (MD= -48.56, 95%CI: -55.21 to -41.96), and transferrin saturation (MD = -4.73, 95%CI: -5.52 to -3.94) were decreased, while transferrin (MD = 0.09, 95%CI: 0.01 to 0.18) and total iron-binding capacity (MD = 6.34, 95%CI: 5.71 to 6.96) was increased in HIF-PHIs versus those in ESAs. In addition, this study observed heterogeneity in the ability of HIF-PHIs to decrease hepcidin. Compared with darbepoetin, only daprodustat (MD = -49.09, 95% CI: -98.13 to -0.05) could significantly reduce hepcidin levels. Meanwhile, daprodustat also showed the highest hepcidin-lowering efficacy (84.0%), while placebo was the lowest (8.2%). Conclusion: For NDD-CKD patients, HIF-PHIs could ameliorate functional iron deficiency by promoting iron transport and utilization, which may be achieved by decreasing hepcidin levels. Interestingly, HIF-PHIs had heterogeneous effects on iron metabolism. Systematic review registration: https://www.crd.york.ac.uk/prospero/display_record.php?RecordID=242777, Identifier CRD42021242777.

Prolyl 4-hydroxylase P4HA1 Mediates the Interplay Between Glucose Metabolism and Stemness in Pancreatic Cancer Cells.

INTRODUCTION: Cancer stem cells (CSCs) are profoundly implicated in tumor initiation and progression as well as drug resistance and tumor recurrence of many cancer types, especially pancreatic ductal adenocarcinoma (PDAC). Previously, we revealed that prolyl 4-hydroxylase subunit alpha 1 (P4HA1) enhances the Warburg effect and tumor growth in PDAC. However, the possible connection between P4HA1 and cancer stemness in PDAC remains obscure. In this study, P4HA1-dependent cancer stemness was studied by sphere-formation assay and detection of stemness markers. METHODS: Glycolytic capacity in cancer stem cells and their parental tumor cells was investigated by glucose uptake, lactate secretion, and expression of glycolytic genes. Glycolysis inhibitors were used to determine the link between cancer stemness and glycolysis. A subcutaneous xenograft model was generated to investigate P4HA1-induced stemness and glycolysis in vivo. RESULTS: We revealed that ectopic expression of P4HA1 increased the stemness of PDAC cells as evidenced by the increased proportion of CD133+ cells, elevated sphere-formation ability, and the upregulated levels of cancer stemness-related proteins (SOX2, OCT4, and NANOG). Blocking tumor glycolysis with 2-Deoxy-D-glucose (2-DG) or a selective inhibitor of glucose transporter 1 (STF-31) significantly reduced the stem properties of PDAC cells, suggesting that P4HA1-induced glycolysis was essential for the stem-like phenotype of PDAC cells. In addition, in vivo study reaffirmed a promotive effect of P4HA1 on tumor glycolysis and cancer stemness. CONCLUSION: Collectively, our findings suggest that P4HA1 not only affects tumor metabolic reprogramming but also facilitates cancer stemness, which might be exploited as a vulnerable target for PDAC treatment.

Role of Arginase-II in Podocyte Injury under Hypoxic Conditions.

Hypoxia plays a crucial role in acute and chronic renal injury, which is attributable to renal tubular and glomerular cell damage. Some studies provide evidence that hypoxia-dependent upregulation of the mitochondrial enzyme arginase type-II (Arg-II) in tubular cells promotes renal tubular injury. It is, however, not known whether Arg-II is also expressed in glomerular cells, particularly podocytes under hypoxic conditions, contributing to hypoxia-induced podocyte injury. The effects of hypoxia on human podocyte cells (AB8/13) in cultures and on isolated kidneys from wild-type (wt) and arg-ii gene-deficient (arg-ii-/-) mice ex vivo, as well as on mice of the two genotypes in vivo, were investigated, respectively. We found that the Arg-II levels were enhanced in cultured podocytes in a time-dependent manner over 48 h, which was dependent on the stabilization of hypoxia-inducible factor 1α (HIF1α). Moreover, a hypoxia-induced derangement of cellular actin cytoskeletal fibers, a decrease in podocin, and an increase in mitochondrial ROS (mtROS) generation-as measured by MitoSOX-were inhibited by adenoviral-mediated arg-ii gene silencing. These effects of hypoxia on podocyte injury were mimicked by the HIFα stabilizing drug DMOG, which inhibits prolyl hydroxylases (PHD), the enzymes involved in HIFα degradation. The silencing of arg-ii prevented the detrimental effects of DMOG on podocytes. Furthermore, the inhibition of mtROS generation by rotenone-the inhibitor of respiration chain complex-I-recapitulated the protective effects of arg-ii silencing on podocytes under hypoxic conditions. Moreover, the ex vivo experiments with isolated kidney tissues and the in vivo experiments with mice exposed to hypoxic conditions showed increased Arg-II levels in podocytes and decreased podocyte markers regarding synaptopodin in wt mice but not in arg-ii-/- mice. While age-associated albuminuria was reduced in the arg-ii-/- mice, the hypoxia-induced increase in albuminuria was, however, not significantly affected in the arg-ii-/-. Our study demonstrates that Arg-II in podocytes promotes cell injury. Arg-ii ablation seems insufficient to protect mice in vivo against a hypoxia-induced increase in albuminuria, but it does reduce albuminuria in aging.

Inhibition of Hypoxia-Inducible Factor Prolyl-Hydroxylase Modulates Platelet Function.

Hypoxia-inducible factor-1α (HIF-1α) involves in redox reactions. Considering the role of reactive oxygen species (ROS) in platelet function, whether it regulates platelet function remains unclear. Using an inhibitor of HIF prolyl-hydroxylase, IOX-2, we intend to investigate its effect on platelet function. Human platelets were treated with IOX-2 (0, 10, 25, and 50 µM) followed by analysis of platelet aggregation, granule secretion, receptor expression, platelet spreading, or clot retraction. Additionally, IOX-2 (10 mg/kg) was injected intraperitoneally into mice to measure tail bleeding time and arterial thrombosis. IOX-2 significantly inhibited collagen-related peptide (CRP; 0.25 µg/mL) or thrombin (0.03 U/mL)-induced platelet aggregation and ATP release dose dependently without affecting P-selectin expression and the surface levels of glycoprotein (GP)Ibα, GPVI, or αIIbß3. In addition, IOX-2-treated platelets presented significantly decreased spreading on fibrinogen or collagen and clot retraction. Moreover, IOX-2 administration into mice significantly impaired the in vivo hemostatic function of platelets and arterial thrombus formation without affecting the number of circulating platelets and coagulation factors (FVIII and FIX). Further, IOX-2 significantly upregulated HIF-1α in platelets, decreased ROS generation, and downregulated NOX1 expression. Finally, IOX-2 increased the phosphorylation level of VASP (Ser157/239), and inhibited the phosphorylation of p38 (Thr180/Tyr182), ERK1/2 (Thr202/Tyr204), AKT (Thr308/Ser473), and PKCδ (Thr505) in CRP- or thrombin-stimulated platelets. In conclusion, inhibition of HIF prolyl-hydroxylase modulates platelet function and arterial thrombus formation, possibly through upregulation of HIF-1α expression and subsequent inhibition of ROS generation, indicating that HIF-1α might be a novel target for the treatment of thrombotic disorders.

PHD2 attenuates high-glucose-induced blood retinal barrier breakdown in human retinal microvascular endothelial cells by regulating the Hif-1α/VEGF pathway.

OBJECTIVE: Diabetic macular edema (DME) is one of the most frequent causes of severe vision loss. The pathogenesis of DME is still not fully understood; however, it is hypothesized to result from breakdown of the blood-retinal barrier (BRB) due to retinal inflammation by vascular endothelial growth factor (VEGF) secretion under hyperglycemic conditions. In this investigation, we discovered that Prolyl-4-hydroxylase 2 (PHD2), an upstream regulator of hypoxia-inducible factor 1 (HIF-1) modulates VEGF expression and thus preserves BRB function in the mouse retina. MATERIALS AND METHODS: Primary human retinal microvascular endothelial cells (hRMECs) were cultured in human endothelial serum-free growth medium and exposed to hyperglycemia. Changes in cell viability were investigated by an MTT assay. BRB function in each group was revealed by a paracellular permeability assay and trans-endothelial electrical resistance (TEER). Morphological changes in the BRB were investigated by immunofluorescence staining of occludin and zonula occludens-1 (ZO-1). The mRNA and protein levels of the tight junction proteins, PHD2, HIF-1α, and VEGF were measured by reverse transcription-quantitative PCR (RT-qPCR), western blot analysis and ELISA. RESULTS: Under hyperglycemic conditions, the viability of hRMECs was decreased, and PHD2 expression was downregulated, accompanied by increased paracellular permeability and decreased trans-endothelial electrical resistance. Additionally, HIF-1α and VEGF expression levels were increased, whereas the expression levels of tight junction proteins, including occludin and ZO-1, were decreased and BRB function was compromised. The PHD2 activator R59949 (diacylglycerol kinase inhibitor II), altered these pathological changes, and the PHD2 inhibitor dimethyloxalylglycine (DMOG) resulted in the opposite effects. CONCLUSION: These results demonstrated that PHD2 inhibited HIF-1 activity by inhibiting HIF-1α expression in hRMECs under hyperglycemic conditions, which led to the downregulation of the expression of the angiogenic factor VEGF, and thus helped to maintain the functions of hRMECs. Therefore, it is reasonable to propose that PHD2 could be a potential novel target for the treatment of DME or other diseases with a similar pathogenesis.

Prolyl hydroxylases positively regulated LPS-induced inflammation in human gingival fibroblasts via TLR4/MyD88-mediated AKT/NF-κB and MAPK pathways.

OBJECTIVES: Prolyl hydroxylases (PHDs) play essential roles in oxygen-sensing system, whereas the effects of PHDs on inflammation have not been totally uncovered. Our study aimed to investigate the role of PHDs in lipopolysaccharide (LPS)-induced inflammation of human gingival fibroblasts (HGFs) and clarify the potential mechanisms. MATERIALS AND METHODS: A pan hydroxylase inhibitor, dimethyloxallyl glycine (DMOG), and RNA interference were used to explore the role of PHDs in inflammation. Cytotoxic effect of DMOG was determined by cell-counting kit-8 and flow cytometry respectively. The secretion levels of IL-6 and IL-8 were assessed by ELISA. The mRNA levels of inflammatory cytokines, Toll-like receptor (TLR) 4 and MyD88 were evaluated by quantitative real-time PCR. The activation of NF-κB, mitogen-activated protein kinase (MAPK) and PI3K/AKT pathways were detected by western blot and the nuclear translocation of NF-κB p65 was examined by immunofluorescence. Downregulation of PHD1 and PHD2 was performed with siRNA transfection. RESULTS: Dimethyloxallyl glycine inhibited LPS-induced inflammatory cytokine, TLR4 and MyD88 expression in gene level and the elevated secretion of IL-6 and IL-8 was also downregulated. Additionally, LPS-induced activation of NF-κB, MAPK and AKT pathways was abolished by DMOG treatment. Importantly, LPS-induced inflammatory cytokine expression was merely suppressed by PHD2 knockdown. CONCLUSIONS: Prolyl hydroxylases acted as a positive regulator in LPS-induced inflammation of HGFs via TLR4/MyD88-mediated NF-κB, MAPK and AKT signalling pathways and PHD2 among three isoforms was principally responsible for the effects.

L-ascorbic acid: A true substrate for HIF prolyl hydroxylase?

L-Ascorbate (L-Asc), but not D-isoascorbate (D-Asc) and N-acetylcysteine (NAC) suppress HIF1 ODD-luc reporter activation induced by various inhibitors of HIF prolyl hydroxylase (PHD). The efficiency of suppression by L-Asc was sensitive to the nature of HIF PHD inhibitor chosen for reporter activation. In particular, the inhibitors developed to compete with alpha-ketoglutarate (αKG), were less sensitive to suppression by the physiological range of L-Asc (40-100 µM) than those having a strong iron chelation motif. Challenging those HIF activators in the reporter system with D-Asc demonstrated that the D-isomer, despite exhibiting the same reducing potency with respect to ferric iron, had almost no effect compared to L-Asc. Similarly, no effect on reporter activation was observed with cell-permeable reducing agent NAC up to 1 mM. Docking of L-Asc and D-Asc acid into the HIF PHD2 crystal structure showed interference of Tyr310 with respect to D-Asc. This suggests that L-Asc is not merely a reducing agent preventing enzyme inactivation. Rather, the overall results identify L-Asc as a co-substrate of HIF PHD that may compete for the binding site of αKG in the enzyme active center. This conclusion is in agreement with the results obtained recently in cell-based systems for TET enzymes and jumonji histone demethylases, where L-Asc has been proposed to act as a co-substrate and not as a reducing agent preventing enzyme inactivation.