Prolyl Hydroxylase Domain Inhibitors Research Articles

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 prolyl hydroxylase domain inhibitors may mitigate loss of skeletal muscle mass in haemodialysis patients

AbstractBackgroundChronic kidney disease patients particularly with renal anaemia hyporesponsive to erythropoiesis‐stimulating agents (ESAs) are at a greater risk of having skeletal muscle mass (SMM) loss. Hypoxia‐inducible factor prolyl hydroxylase domain inhibitor (HIF‐PHI), a novel therapeutic agent for renal anaemia, potentially promotes angiogenesis, muscle repair, and homeostasis. However, effects of HIF‐PHIs on SMM remain unknown.MethodsThis retrospective observational cohort study enrolled 292 Japanese adults receiving maintenance haemodialysis at our dialysis centre. The dataset included 11 patients who received daprodustat for 6 months or longer during 1 December 2020 through 30 June 2022. From the previously published pooled cohort, we enrolled 281 participants from 1 August 2018 to 31 July 2019 prior to the approval of HIF‐PHIs for renal anaemia. SMM was assessed using modified creatinine index (mg/kg/day) calculated by age, sex, serum creatinine, and single‐pool Kt/V. Annual changes of SMM [ΔSMM (%)] were analysed with the least squares regression model and mixed‐effects model during 6‐ to 12‐month follow‐up period.ResultsThe median age of the participants was 63 years [interquartile range (IQR), 54–71 years], and 33% were female. The median ΔSMM levels (IQR) in the least squares regression model were 4.0% (−1.7% to 9.3%) in the HIF‐PHI group, 0.20% (−2.1% to 2.1%) in the no ESA group, and −0.94% (−3.0% to 1.3%) in the high ESA group using darbepoetin equivalent to 20 μg or more per week. Those in the mixed‐effects model were −1.7% (−1.2% to 3.8%), 0.09% (−1.4% to 1.3%), and −0.74% (−2.0% to 0.8%), respectively. The multivariable linear regression models revealed that HIF‐PHI use was associated with greater ΔSMM compared with the high ESA group [coefficient, 3.737; 95% confidence interval (CI), 1.216–6.258 in the least squares regression model or coefficient, 1.635; 95% CI, 0.068–3.201 in the mixed‐effects model, respectively].ConclusionsHIF‐PHI use led to greater ΔSMM in maintenance haemodialysis patients. HIF‐PHIs may minimize loss of SMM in patients with end‐stage kidney disease and renal anaemia.

#6153 SODIUM-GLUCOSE COTRANSPORTER 2 INHIBITORS VERSUS DIPEPTIDYL PEPTIDASE 4 INHIBITORS AS STABILIZERS OF HIF PATHWAY IN DIABETIC NEPHROPATHY RATS

Abstract Background and Aims With the diabetes pandemic, chronic kidney disease is a burden, with diabetic nephropathy (DN) being the primary leading cause of end-stage renal disease. Antidiabetic drugs have shown renoprotective effects via different mechanisms, and among those are SGLT2 inhibitors and DPP-4 inhibitors. Hypoxia-inducible factor (HIF) pathway, a significant contributor to inflammation development in the kidneys, is a major pathway that regulates erythropoietin synthesis. Prolyl hydroxylase domain (PHD) decreases erythropoietin synthesis by inhibiting HIFs. PHD inhibitors are anticipated to have additional effects, such as protection against metabolic diseases, due to HIF's impact on many genes. The diabetic kidney is characterized by enhanced HIF-1α activation but decreased HIF-2α activity. These actions may significantly contribute to glomerular and renal tubular dysfunction, renal inflammation and fibrosis, and decreased erythropoietin synthesis observed in DN. This work aims to test the renoprotective effect of SGLT2 inhibitors and DPP-4 inhibitors by controlling the proinflammatory and anti-inflammatory domains of the HIF pathway. Method Type 1 DM rat model was induced in 24-hour-fasted Sprague Dawley rats by a single intraperitoneal injection of 45 mg/kg streptozotocin (STZ). DM was verified by measuring blood glucose seven days following STZ injection, and only animals with glucose levels of ≥15 mM were included (n = 24), randomly and equally distributed, then received either saline or vildagliptin (3 mg/Kg) or empagliflozin (30 mg/Kg) once daily by oral gavage for three months. An additional group was used as a non-diabetic control. At the end of the experiment, rats were placed in metabolic cages for 24-h urine collection, then sacrificed. Blood samples and kidney tissue were collected. Kidney function was assessed by measuring serum creatinine and urinary albumin: creatinine ratio. HIF1α, HIF2α, and PHD3 mRNA expression in kidney tissues were assessed using quantitative real-time PCR, and to indirectly assess HIF2α protein stability, immunohistochemical staining for PHD3 was also performed. Results Vildagliptin and empagliflozin could significantly ameliorate the diabetic disturbance of renal function, as demonstrated in serum creatinine (F = 23.518, p<0.001) with a non-significant difference between both treated groups, and in the significantly reduced albumin/creatinine ratio (F = 14.453, p<0.001), with a significantly lower ratio for empagliflozin (4.73±0.54) than vildagliptin-treated group (9.09±1.04). Regarding the HIF pathway as a potential mechanism, empagliflozin-treated groups showed a significantly higher HIF2α gene expression in kidney tissue samples (0.98±0.10) than the vildagliptin-treated group (0.58±0.05). For the expression of its regulator, PHD3, there was a significant difference between groups (F = 5.694, p = 0.005). However, the vildagliptin-treated group (0.65±0.06) didn′t differ significantly from the untreated group (0.60±0.03), in contrast to the empagliflozin-treated group (2.01±0.50), which showed a significantly higher expression than the other groups (Figure 1). Conclusion Empagliflozin significantly increased the level of HIF-2α in renal tissue. In addition, it nearly neutralizes the tubular expression of PHD3 compared to non-treated and vildagliptin-treated rats. SGLT2 inhibition is effective for correcting the proinflammatory/anti-inflammatory imbalance in DN.

Scaffold hopping strategy to derive 4‐hydroxy‐1‐alkyl‐2‐oxo‐1,2‐dihydrothieno[2,3‐b:4,5‐b′]dipyridine‐3‐carbonylglycine derivatives as a novel hypoxia‐inducible factor prolyl hydroxylase domain inhibitor for the potential treatment of chronic kidney disease anemia

AbstractDerivatives of 4‐hydroxy‐1‐alkyl‐2‐oxo‐1,2‐dihydrothieno[2,3‐b:4,5‐b′]dipyridine‐3‐carbonylglycine were developed as a novel hypoxia‐inducible factor prolyl hydroxylase domain (PHD) inhibitor. The chemical space of the tricyclic 4‐hydroxypyridinyl glycines was examined thoroughly during our optimization study. One of our most potent compounds 12ar exhibits superior enzymatic activity to the known PHD inhibitors that are in the late stage of clinical studies. The functional efficacy of our PHD inhibitors was confirmed via the increased level of erythropoietin (EPO) expression in a dose‐dependent manner in vitro.

Pleotropic effects of hypoxia-inducible factor-prolyl hydroxylase domain inhibitors: are they clinically relevant?

Anemia is common in patients with chronic kidney disease (CKD) and is mainly caused by insufficient production of erythropoietin from fibrotic kidney. Because anemia impairs quality of life and overall prognosis, recombinant human erythropoietin-related products (erythropoiesis-stimulating agents, ESAs) have been developed to increase hemoglobin level for decades. However, many safety concerns have been announced regarding the use of ESAs, including an increased occurrence of cardiovascular events, vascular access thrombosis, cancer progression, and recurrence. Hypoxia-inducible factor (HIF) is crucial to erythropoietin production, as a result, prolyl hydroxylase domain (PHD) enzyme inhibitors have been new therapeutic agents for the treatment of anemia in CKD. They can be administered orally, which is a preferred route for patients not undergoing hemodialysis. In clinical trials, PHD inhibitor could induce noninferior effect on erythropoiesis and improve functional iron deficiency compared with ESAs. Although no serious adverse events were reported, safety is still a concern because HIF stabilization induced by PHD inhibitor has pleotropic effects, such as angiogenesis, metabolic change, and cell survival, which might lead to unwanted deleterious effects, including fibrosis, inflammation, cardiovascular risk, and tumor growth. More molecular mechanisms of PHD inhibition and long-term clinical trials are needed to observe these pleotropic effects for the confirmation of safety and efficacy of PHD inhibitors.

Synthesis and biological evaluation of (4-hydroxy-2-(substitued sulfonamido)pyrimidine-5-carbonyl)glycines as oral erythropoietin secretagogues

Erythropoietin (EPO) is the predominant regulating factor in erythropoiesis. Herein we describe the identification of (4-hydroxy-2-(substitued sulfonamido)pyrimidine-5-carbonyl) glycine-based oral EPO secretagogues. Most of these compounds obviously increased the EPO level in Hep3B cells by stabilizing the hypoxia-inducible factor-α (HIF-α). Furthermore, their potential inhibitory activities against HIF prolyl hydroxylase domain (PHD) revealed their function as PHD inhibitors in PHD-HIF pathway. Compound 6i, with a biphenyl substituent on the sulfonamido group, particularly increased plasma EPO level in mice and could serve as a candidate of EPO stimulating agent for anemia treatment.

Threshold of Serum Ferritin to Discriminate against Those at Greater Risk of Platelet Increase during Treatment with Hypoxia-Inducible Factor Prolyl Hydroxylase Domain Inhibitor

Introduction: Hypoxia-inducible factor prolyl hydroxylase domain inhibitors (HIF-PHI) are a new treatment for renal anemia. HIF-PHI is believed to increase iron usage to improve availability of iron for erythropoiesis. Therefore, there is concern that HIF-PHI might be prone to iron deficiency and that thrombosis might be induced by increased platelet and transferrin levels due to this iron deficiency. Methods: Relationship of iron-related factors with platelet count (PLT) and total iron-binding capacity (TIBC; which reflects the transferrin level) were examined in 29 patients who were treated with darbepoetin alfa (DA) and then switched to roxadustat (Rox). To determine how changes in PLT and TIBC related to changes in iron-related factors, univariable and multivariable linear regression models were applied. To examine what iron-related factors on Day 0 influenced change in PLT, we used receiver operating characteristic (ROC) curves and logistic regression analysis for a rate of change in PLT ≤0% as the endpoint. Logistic regression analysis was performed with the reference group having serum ferritin (s-ft) or Transferrin saturation below the corresponding cutoff value (low vs. high). Results: Multivariable analysis showed significant positive correlations between the rate of change in PLT and the change in s-ft and red blood cells (RBC) count {β-coefficients; 0.40 [95% confidence interval (CI): 0.17–0.62], p = 0.001} (β-coefficients; 30.45 [95% CI: 10.90–50.00], p = 0.004). The rate of change in TIBC was significantly positively correlated with only the change in RBC count. The ROC showed a significant cutoff value for s-ft of 77.2 ng/mL (sensitivity 63.6%, specificity 83.3%, area under the curve 0.76, 95% CI 0.55–0.96). Multivariable logistic regression also showed that only high s-ft was significantly elevated (9.46, 95% CI 1.42–63.30, p = 0.020). Conclusions: This study showed that changes in PLT were correlated with s-ft and amount of hematopoiesis. This suggests that an increase in PLT due to iron levels is less likely when s-ft is 77.2 ng/mL or higher at the time of switching from DA to Rox. In contrast, TIBC was only related to hematopoiesis in these patients. Control of s-ft before initiation of HIF-PHI treatment and gradual hematopoiesis might reduce the risk of thrombosis when switching from erythropoiesis-stimulating agents to HIF-PHI.

Regulation of Fetal Hemoglobin Expression By the VHL-HIF1α Oxygen Sensing System

Defining the mechanisms that control the perinatal switch from γ-globin (HBG1 and HBG2) to β-globin (HBB) gene expression in human red blood cells (RBCs) has informed novel approaches to reactivate fetal hemoglobin (HbF, α2γ2) therapeutically for sickle cell disease and β-thalassemia. However, one longstanding unsolved problem is to explain how HbF becomes elevated in conditions such as blood loss, hypoxia and hemolysis. These conditions are associated with accelerated RBC production, also referred to as stress erythropoiesis, driven by activation of hypoxia-inducible factor (HIF) via a canonical O 2 sensing pathway. At high O 2 levels (“normoxia”), O 2-dependent prolyl hydroxylase domain (PHD) enzymes hydroxylate HIFα, thereby targeting it for ubiquitination by the von Hippel-Lindau (VHL) E3 ubiquitin ligase complex, followed by proteasomal degradation. At low O 2 tension (hypoxia), PHD activity is reduced, causing HIF1α to accumulate, dimerize with constitutively expressed HIF1β, and bind hypoxia response elements (HREs) to activate a broad array of genes that facilitate hypoxic adaptation. We identified VHL and HIF1α, as negative and positive regulators of HbF expression, respectively.Disruption of the VHL gene in CD34 + hematopoietic stem and progenitor cells (HSPCs) by transfection with ribonucleoprotein (RNP) consisting of Cas9 and VHL-targeting guide RNA increased HbF expression from 7.5% ±1.2% in control cells to 30.9% ± 4.8% (mean ± SD, P<0.0001) in RBC progeny generated by in vitro differentiation. Similarly, γ-globin mRNA was induced by 5 -fold after disruption of VHL in HUDEP-2 cells, an immortalized erythroid line that expresses mainly adult hemoglobin (HbA, α2/β2). Mass spectrometry and transcriptome analysis of VHL-disrupted CD34 + HSPCs revealed increased HIF1α protein expression with no change in the corresponding mRNA. Inhibition of HIF1A mRNA by RNA interference suppressed γ-globin induction in VHL-/- HUDEP-2 clones and in RBCs generated from VHL RNP-treated CD34 + cells, indicating VHL knockout induced HbF through HIF1α protein accumulation. CUT&RUN analysis of VHL-depleted erythroblasts revealed HIF1α/HIF1β heterodimer occupancy at BGLT3, a long-noncoding RNA locus approximately 2.7kb 3' of HBG1. The HIF1α-bound BGLT3 locus contains two canonical HREs (ACGTG) separated by 13 bp. Disruption of each HRE motif in VHL-/- HUDEP-2 cells by base editing caused additive reductions in BGLT3 HIF1α occupancy and γ-globin expression. Mechanistically, VHL depletion caused the accumulation of HIFα/β heterodimers at BGLT3, recruitment of the transcriptional activators GATA1 and P300, and targeted chromatin accessibility to establish the active enhancer mark H3K27ac. These changes were accompanied by altered chromosome conformation to favor long-range interactions between the γ-globin loci (HBG1 and HBG2) and the locus control region, a powerful upstream enhancer. Treatment of healthy donor CD34 + HSPCs-derived erythroblasts with the clinically approved PHD inhibitor FG-4592 (Roxadustat) caused HbF to increase from 4.76%±1.22% at baseline to 12.86 ± 2.40% (mean ± SD in 3 biological replicates, P<0.01). Treatment of the same erythroblasts with FG-4592 and hydroxyurea, a widely used SCD drug that acts partly by inducing HbF, caused HbF to increase from 4.76% ± 1.22% at baseline to 24.1% ± 5.3% (mean ± SD in 3 biological replicates, P<0.01), indicating an additive effect. Our findings link developmental globin gene regulation with O 2 sensing, provide a mechanism for HbF induction during stress erythropoiesis, and identify a novel therapeutic approach for β-hemoglobinopathies. DisclosuresBlobel: Pfizer: Consultancy; Fulcrum Therapeutics, Inc.: Consultancy. Weiss: Beam Therapeutics: Current holder of stock options in a privately-held company; Forma Therapeutics: Consultancy; Cellarity Inc.: Consultancy; Novartis: Consultancy.

Transforming growth factor-\u03b21 decreases erythropoietin production through repressing hypoxia-inducible factor 2\u03b1 in erythropoietin-producing cells

BackgroundRenal erythropoietin (EPO)-producing (REP) cells produce EPO through hypoxia-inducible factor (HIF) 2α-activated gene transcription. Insufficient EPO production leads to anemia in patients with chronic kidney disease. Although recombinant EPO is effective to improve anemia, no reliable REP cell lines limit further progress of research and development of novel treatment.MethodsWe screened Epo mRNA expression in mouse fibroblast cell lines. Small interfering RNA specific for HIF1α or HIF2α was transfected to study Epo expression in C3H10T1/2 cells. The effect of transforming growth factor-β1 (TGF-β1) on HIF-EPO axis was studied in C3H10T1/2 cells and mice.ResultsSimilar to mouse REP pericytes, C3H10T1/2 cells differentiated to α-smooth muscle actin+ myofibroblasts after exposure to TGF-β1. Specific HIF knockdown demonstrated the role of HIF2α in hypoxia-induced Epo expression. Sustained TGF-β1 exposure increased neither DNA methyltransferase nor methylation of Epas1 and Epo genes. However, TGF-β1 repressed HIF2α-encoding Epas1 promptly through activating activin receptor-like kinase-5 (ALK5), thereby decreasing Epo induction by hypoxia and prolyl hydroxylase domain inhibitor roxadustat. In mice with pro-fibrotic injury induced by ureteral obstruction, upregulation of Tgfb1 was accompanied with downregulation of Epas1 and Epo in injured kidneys and myofibroblasts, which were reversed by ALK5 inhibitor SB431542.ConclusionC3H10T1/2 cells possessed the property of HIF2α-dependent Epo expression in REP pericytes. TGF-β1 induced not only the transition to myofibroblasts but also a repressive effect on Epas1-Epo axis in C3H10T1/2 cells. The repressive effect of TGF-β1 on Epas1-Epo axis was confirmed in REP pericytes in vivo. Inhibition of TGF-β1-ALK5 signaling might provide a novel treatment to rescue EPO expression in REP pericytes of injured kidney.

Hypoxia-inducible factor prolyl hydroxylase domain inhibitor may maintain hemoglobin synthesis at lower serum ferritin and transferrin saturation levels than darbepoetin alfa.

BackgroundHypoxia-inducible factor (HIF) prolyl hydroxylase domain inhibitors, which have recently become clinically available for treating renal anemia, are attracting attention for their novel mechanisms of action.MethodsRelationships of reticulocyte hemoglobin content (CHr), which reflects recent Hb synthesis, with serum ferritin (s-ft) and transferrin saturation (TSAT) were examined in 30 patients on hemodialysis after switching from darbepoetin alfa (DA) to roxadustat (Rox). Iron deficiency was defined as CHr < 32.0 pg. Cutoff values of s-ft and TSAT were determined using receiver operating characteristic curves for the endpoint CHr ≥ 32.0 pg. Logistic analysis was performed with the reference group having s-ft or TSAT below the corresponding cutoff value (low vs high).ResultsWith the endpoint CHr ≥ 32.0 pg on Day 0, cutoff values for s-ft and TSAT were respectively 49.7 ng/mL and 21.6% on Day 0 and 35.5 ng/mL and 16.2% on Day 28. With the endpoint CHr ≥ 32.0 pg on Day 28, cutoff values for s-ft and TSAT on Day 0 were 81.6 ng/mL and 23.9%, respectively. According to multivariable logistic analysis, the odds ratios of CHr ≥ 32.0 pg on Day 0 were significantly higher for high TSAT on Day 0 [34.7 (95% CI 2.42–131.0), p<0.003] and Day 28 [24.8 (95% CI 2.75–224.0), p = 0.004]. There were no significant differences by s-ft. Odd ratios of CHr ≥ 32.0 pg on Day 28 were also significantly higher for high s-ft on Day 0 [16.0 (95% CI 1.57–163.0), p = 0.019] and high TSAT on Day 0 [13.5 (95% CI 1.24–147.0), p<0.033].ConclusionsOur results suggest Hb synthesis was maintained with lower TSAT and s-ft during Rox therapy compared with DA therapy. To avoid iron deficiency during the 4 weeks after switching DA to Rox, ideal s-ft and TSAT levels before the switch are 81.6 ng/mL and 23.9%, respectively.

Whole-Body Prolyl Hydroxylase Domain (PHD) 3 Deficiency Increased Plasma Lipids and Hematocrit Without Impacting Plaque Size in Low-Density Lipoprotein Receptor Knockout Mice.

Background and aims: Atherosclerosis is an important cause of clinical cardiovascular events. Atherosclerotic plaques are hypoxic, and reoxygenation improves plaque phenotype. Central players in hypoxia are hypoxia inducible factors (HIF) and their regulators, HIF-prolyl hydroxylase (PHD) isoforms 1, 2, and 3. PHD inhibitors, targeting all three isoforms, are used to alleviate anemia in chronic kidney disease. Likewise, whole-body PHD1 and PHD2ko ameliorate hypercholesterolemia and atherogenesis. As the effect of whole-body PHD3 is unknown, we investigated the effects of germline whole-body PHD3ko on atherosclerosis.Approach and Results: To initiate hypercholesterolemia and atherosclerosis low-density lipoprotein receptor knockout (LDLrko) and PHD3/LDLr double knockout (PHD3dko), mice were fed a high-cholesterol diet. Atherosclerosis and hypoxia marker pimonidazole were analyzed in aortic roots and brachiocephalic arteries. In contrast to earlier reports on PHD1- and PHD2-deficient mice, a small elevation in the body weight and an increase in the plasma cholesterol and triglyceride levels were observed after 10 weeks of diet. Dyslipidemia might be explained by an increase in hepatic mRNA expression of Cyp7a1 and fatty acid synthase, while lipid efflux of PHD3dko macrophages was comparable to controls. Despite dyslipidemia, plaque size, hypoxia, and phenotype were not altered in the aortic root or in the brachiocephalic artery of PHD3dko mice. Additionally, PHD3dko mice showed enhanced blood hematocrit levels, but no changes in circulating, splenic or lymphoid immune cell subsets.Conclusion: Here, we report that whole-body PHD3dko instigated an unfavorable lipid profile and increased hematocrit, in contrast to other PHD isoforms, yet without altering atherosclerotic plaque development.

ESA, Iron Therapy and New Drugs: Are There New Perspectives in the Treatment of Anaemia?

Anemia is a well-known consequence of chronic kidney disease (CKD); it is mainly due to a relative insufficiency of erythropoietin synthesis by the failing kidneys. Over the years, the combination of erythropoiesis stimulating agents (ESA) and iron has become the standard of care of anemia. All ESAs effectively increase hemoglobin (Hb) levels in a substantial percentage of patients. However, in the last decade, their use has been surrounded by safety issues in increased cardiovascular risk, especially when used at high doses in inflamed and hyporesponsive patients. This has led to the definition of a more cautious Hb target. Iron deficiency is very frequent in CKD patients, with a higher frequency in non-dialysis patients. Traditionally, iron supplementation is mostly used as supportive therapy for anemia control. However, the concept is growing that intravenous iron therapy per se could be beneficial in the presence of heart failure. A new class of drugs, prolyl hydroxylase domain (PHD) inhibitors (PHD inhibitors) is becoming available for the treatment of anemia in CKD patients. Theoretically, these agents have a number of advantages, the main ones being that of stimulating the synthesis of endogenous erythropoietin and increasing iron availability. The impact of their future use in clinical practice is still to be defined. Another possible strategy could be targeting serum hepcidin and its related pathways. This possibility is fascinating from the scientific point of view, but at present its development phase is still far from clinical application.

Targeting HIF-α for robust prevascularization of human cardiac organoids.

Prevascularized 3D microtissues have been shown to be an effective cell delivery vehicle for cardiac repair. To this end, our lab has explored the development of self-organizing, prevascularized human cardiac organoids by co-seeding human cardiomyocytes with cardiac fibroblasts, endothelial cells, and stromal cells into agarose microwells. We hypothesized that this prevascularization process is facilitated by the endogenous upregulation of hypoxia-inducible factor (HIF) pathway in the avascular 3D microtissues. In this study, we used Molidustat, a selective PHD (prolyl hydroxylase domain enzymes) inhibitor that stabilizes HIF-α, to treat human cardiac organoids, which resulted in 150 ± 61% improvement in endothelial expression (CD31) and 220 ± 20% improvement in the number of lumens per organoids. We hypothesized that the improved endothelial expression seen in Molidustat treated human cardiac organoids was dependent upon upregulation of VEGF, a well-known downstream target of HIF pathway. Through the use of immunofluorescent staining and ELISA assays, we determined that Molidustat treatment improved VEGF expression of non-endothelial cells and resulted in improved co-localization of supporting cell types and endothelial structures. We further demonstrated that Molidustat treated human cardiac organoids maintain cardiac functionality. Lastly, we showed that Molidustat treatment improves survival of cardiac organoids when exposed to both hypoxic and ischemic conditions in vitro. For the first time, we demonstrate that targeted HIF-α stabilization provides a robust strategy to improve endothelial expression and lumen formation in cardiac microtissues, which will provide a powerful framework for prevascularization of various microtissues in developing successful cell transplantation therapies.

Hypoxia-Inducible Factor and Oxygen Biology in the Kidney.

Kidney tissue hypoxia is detected in various kidney diseases and is considered to play an important role in the pathophysiology of both AKI and CKD. Because of the characteristic vascular architecture and high energy demand to drive tubular solute transport, the renal medulla is especially prone to hypoxia. Injured kidneys often present capillary rarefaction, inflammation, and fibrosis, which contribute to sustained kidney hypoxia, forming a vicious cycle promoting progressive CKD. Hypoxia-inducible factor (HIF), a transcription factor responsible for cellular adaptation to hypoxia, is generally considered to protect against AKI. On the contrary, consequences of sustained HIF activation in CKD may be either protective, neutral, or detrimental. The kidney outcomes seem to be affected by various factors, such as cell types in which HIF is activated/inhibited, disease models, balance between two HIF isoforms, and time and methods of intervention. This suggests multifaceted functions of HIF and highlights the importance of understanding its role within each specific context. Prolyl-hydroxylase domain (PHD) inhibitors, which act as HIF stabilizers, have been developed to treat anemia of CKD. Although many preclinical studies demonstrated renoprotective effects of PHD inhibitors in CKD models, there may be some situations in which they lead to deleterious effects. Further studies are needed to identify patients who would gain additional benefits from PHD inhibitors and those who may need to avoid them.

Inhibition of prolyl hydroxylases increases hepatic insulin and decreases glucagon sensitivity by an HIF-2α-dependent mechanism

ObjectiveRecent evidence indicates that inhibition of prolyl hydroxylase domain (PHD) proteins can exert beneficial effects to improve metabolic abnormalities in mice and humans. However, the underlying mechanisms are not clearly understood. This study was designed to address this question. MethodsA pan-PHD inhibitor compound was injected into WT and liver-specific hypoxia-inducible factor (HIF)-2α KO mice, after onset of obesity and glucose intolerance, and changes in glucose and glucagon tolerance were measured. Tissue-specific changes in basal glucose flux and insulin sensitivity were also measured by hyperinsulinemic euglycemic clamp studies. Molecular and cellular mechanisms were assessed in normal and type 2 diabetic human hepatocytes, as well as in mouse hepatocytes. ResultsAdministration of a PHD inhibitor compound (PHDi) after the onset of obesity and insulin resistance improved glycemic control by increasing insulin and decreasing glucagon sensitivity in mice, independent of body weight change. Hyperinsulinemic euglycemic clamp studies revealed that these effects of PHDi treatment were mainly due to decreased basal hepatic glucose output and increased liver insulin sensitivity. Hepatocyte-specific deletion of HIF-2α markedly attenuated these effects of PHDi treatment, showing PHDi effects are HIF-2α dependent. At the molecular level, HIF-2α induced increased Irs2 and cyclic AMP-specific phosphodiesterase gene expression, leading to increased and decreased insulin and glucagon signaling, respectively. These effects of PHDi treatment were conserved in human and mouse hepatocytes. ConclusionsOur results elucidate unknown mechanisms for how PHD inhibition improves glycemic control through HIF-2α-dependent regulation of hepatic insulin and glucagon sensitivity.

Pyrazolo[4,3-d]pyrimidine Derivatives as a Novel Hypoxia-Inducible Factor Prolyl Hydroxylase Domain Inhibitor for the Treatment of Anemia.

Inhibition of hypoxia-inducible factor prolyl hydroxylase domain (HIF-PHD) promotes erythropoietin (EPO) production by stabilizing the HIFα subunit. Thieno[2,3-d]pyrimidine 8 identified based on X-ray crystal structure analysis was optimized to lead to the discovery of pyrazolo[4,3-d]pyrimidine 13 as the lead compound of orally bioavailable HIF-PHD inhibitors. Conversion of the benzyl moiety in 13 gave pyrazolopyrimidine 19 with high solubility and bioavailability, which increased hemoglobin levels in anemic model rats after repeated oral administration. It was shown that pyrazolo[4,3-d]pyrimidine derivatives are promising therapeutic agents for renal anemia through the inhibition of HIF-PHD.

Mannose Binding Lectin Is Hydroxylated by Collagen Prolyl-4-hydroxylase and Inhibited by Some PHD Inhibitors.

Mannose-binding lectin (MBL) is an important component of innate immune defense. MBL undergoes oligomerization to generate high mol weight (HMW) forms which act as pattern recognition molecules to detect and opsonize various microorganisms. Several post-translational modifications including prolyl hydroxylation are known to affect the oligomerization of MBL. Yet, the enzyme(s) which hydroxylate proline in the collagen-like domain residues have not been identified and the significance of prolyl hydroxylation is incompletely understood. To investigate post-translational modifications of MBL, we stably expressed Myc-DDK tagged MBL in HEK293S cells. We used pharmacologic and genetic inhibition of 2-oxoglutarate-dependent dioxygenases (2OGDD) to identify the enzyme required for prolyl hydroxylation of MBL. We performed mass spectrometry to determine the effects of various inhibitors on MBL modifications. Secretion of HMW MBL was impaired by inhibitors of the superfamily of 2OGDD, and was dependent on prolyl-4-hydroxylase subunit α1. Roxadustat and vadadustat, but not molidustat, led to significant suppression of hydroxylation and secretion of HMW forms of MBL. These data suggest that prolyl hydroxylation in the collagen-like domain of MBL is mediated by collagen prolyl-4-hydroxylase. Reduced MBL activity is likely to be an off-target effect of some, but not all, prolyl hydroxylase domain (PHD) inhibitors. There may be advantages in selective PHD inhibitors that would not interfere with MBL production.

Prolyl hydroxylase domain 2 reduction enhances skeletal muscle tissue regeneration after soft tissue trauma in mice.

The transcription factor Hypoxia-inducible factor 1 (HIF-1) plays a pivotal role in tissue regeneration. HIF-1 is negatively controlled by O2-dependent prolyl hydroxylases with a predominant role of prolyl hydroxylase 2 isoform (Phd2). Transgenic mice, hypomorphic for this isoform, accumulate more HIF-1 under normoxic conditions. Using these mice, we investigated the influence of Phd2 and HIF-1 on the regenerative capability of skeletal muscle tissue after myotrauma. Phd2-hypomorphic and wild type mice (on C57Bl/6 background) were grouped with regeneration times from 6 to 168 hours after closed mechanic muscle trauma to the hind limb. Tissue samples were analysed by immuno-staining and real-time PCR. Bone marrow derived macrophages of wild type and Phd2-hypomorphic mice were isolated and analysed via flow cytometry and quantitative real-time PCR. Phd2 reduction led to a higher regenerative capability due to enhanced activation of myogenic factors accompanied by induction of genes responsible for glucose and lactate metabolism in Phd2-hypomorphic mice. Macrophage infiltration into the trauma areas in hypomorphic mice started earlier and was more pronounced compared to wild type mice. Phd2-hypomorphic mice also showed higher numbers of macrophages in areas with sustained trauma 72 hours after myotrauma application. In conclusion, we postulate that the HIF-1 pathway is activated secondary to a Phd2 reduction which may lead to i) higher activation of myogenic factors, ii) increased number of positive stem cell proliferation markers, and iii) accelerated macrophage recruitment to areas of trauma, resulting in faster muscle tissue regeneration after myotrauma. With the current development of prolyl hydroxylase domain inhibitors, our findings point towards a potential clinical benefit after myotrauma.

Prolyl-hydroxylase domain inhibitors in chronic kidney disease, a promising alternative for erythropoiesis-stimulating agent

•ESA are commonly used to treat anemia in chronic kidney disease but meet several limits. Iron needs are elevated and cardio-vascular mortality may increase at high-dose. •Prolyl-hydroxylase domain is involved in hypoxia physiology and its inhibition leads to higher hemoglobin and lower hepcidin levels, decreasing iron needs. Oral PHD inhibitors will be soon available to treat CKD related anemia. •Many pathways are affected by PHD inhibition and data are lacking regarding their long term safety. For these reasons, longer studies are needed before using PHDi in clinical practice.

Are all erythropoiesis-stimulating agents created equal?

Erythropoiesis-stimulating agents (ESAs) are effective drugs to correct and maintain haemoglobin (Hb) levels, however, their use at doses to reach high Hb targets has been associated with an increased risk of cardiovascular adverse events, mortality and cancer. Presently used ESAs have a common mechanism of action but different pharmacokinetic and pharmacodynamic characteristics. Accordingly, the mode of activation of the erythropoietin (EPO) receptor can exert marked differences in downstream events. It is unknown whether the various ESA molecules have different efficacy/safety profiles. The relative mortality and morbidity risks associated with the use of different types of ESAs remains poorly evaluated. Recently an observational study and a randomized clinical trial provided conflicting results regarding this matter. However, these two studies displayed several differences in patient characteristics and ESA molecules used. More importantly, by definition, randomized clinical trials avoid bias by indication and suffer less from confounding factors. Therefore they bring a higher degree of evidence. The scenario becomes even more complex when considering the new class of ESAs, called prolyl-hydroxylase domain (PHD) inhibitors. They are oral drugs that mimic exposure to hypoxia and stabilize hypoxia-inducible factor α. They profoundly differ from presently used ESAs, as they have multiple targets of action, including the stimulation of endogenous EPO synthesis, direct mobilization/absorption of iron and a higher reduction of hepcidin. Accordingly, they have the potential to be more effective in inflamed patients with functional iron deficiency, i.e. the setting of patients who are at higher risk of cardiovascular events and mortality in response to present ESA use. As for ESAs, individual PHD inhibitors differ in molecular structure and degree of selectivity for the three main PHD isoforms; their efficacy and safety profiles may therefore be different from that of presently available ESAs.