Isoform-specific roles of prolyl hydroxylases in the regulation of pancreatic β-cell function

Pancreatic β-cells can secrete insulin via two pathways characterized as KATP channel-dependent and independent. The KATP channel-independent pathway is characterized by a rise in several potential metabolic signaling molecules, including the NADPH/NADP + ratio and α-ketoglutarate (αKG). Prolyl hydroxylases (PHDs), which belong to the αKG-dependent dioxygenase superfamily, are known to regulate the stability of hypoxia-inducible factor α (HIFα). In the current study, we assess the role of PHDs in vivo using the pharmacological inhibitor dimethyloxalylglycine (DMOG) and generated β-cell specific knockout (KO) mice for all three isoforms of PHD (β-PHD1 KO, β-PHD2 KO, and β-PHD3 KO mice). DMOG inhibited in vivo insulin secretion in response to glucose challenge and inhibited the 1 st phase of insulin secretion but enhanced the second-phase of insulin secretion in isolated islets. None of the β-PHD KO mice showed any significant in vivo defects associated with glucose tolerance and insulin resistance except for β-PHD2 KO mice which had significantly increased plasma insulin during a glucose challenge. Islets from both β-PHD1 KO and β-PHD3 KO had elevated β-cell apoptosis and reduced β-cell mass. Isolated islets from β-PHD1 KO and β-PHD3 KO had impaired glucose-stimulated insulin secretion and glucose-stimulated increases in the ATP/ADP and NADPH/NADP + ratio. All three PHD isoforms are expressed in β-cells, with PHD3 showing the most unique expression pattern. The lack of each PHD protein did not significantly impair in vivo glucose homeostasis. However, β-PHD1 KO and β-PHD3 KO mice had defective β-cell mass and islet insulin secretion, suggesting that these mice may be predisposed to developing diabetes.

Deficiency of myeloid prolyl hydroxylase domain proteins aggravates atherogenesis via macrophage apoptosis and paracrine fibrotic signaling

Background Atherosclerotic plaque hypoxia is detrimental for macrophage function. Prolyl hydroxylases (PHDs) initiate cellular hypoxic responses, possibly influencing macrophage function in plaque hypoxia. Thus, we aimed to elucidate the role of myeloid PHDs in atherosclerosis. Methods Myeloid specific PHD knockout (PHDko) mice were fed high cholesterol diet for 6–12 weeks to induce atherosclerosis. Plaque parameters, e.g. plaque size and macrophage content, were analyzed. Bulk and single cell RNA sequencing was performed on PHD2 BMDMs and plaque macrophages, respectively. Results Aortic root plaque size was augmented 2.6fold in PHD2cko, and 1.4-fold in PHD3ko, but not in PHD1ko mice compared to controls. Macrophage apoptosis was promoted in PHD2cko and PHD3ko mice in vitro and in vivo, via the HIF1α/BNIP3 axis. Bulk and single cell RNA data of PHD2cko bone-marrow-derived macrophages (BMDM) and plaque macrophages, respectively, confirmed these findings and were validated by siRNA silencing. Human plaque BNIP3 mRNA associated with plaque necrotic core, suggesting similar adverse effects. Further, PHD2cko plaques displayed enhanced fibrosis, independent of macrophage MMP activity, collagen secretion or proliferation and of SMC collagen production, or proliferation. Rather, PHD2cko BMDMs enhanced fibroblast collagen secretion in a paracrine manner. Nichenet in silico analysis of macrophage-fibroblast communication predicted SPP1 signaling as regulator, in line with enhanced plaque SPP1 protein content, and SPP1 mRNA in TREM2-foamy plaque macrophages, but not in neutrophils. Conclusion Myeloid PHD2cko and PHD3ko enhanced plaque growth, macrophage apoptosis, and PHD2cko activated paracrine collagen secretion by fibroblasts. FUNDunding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): NWO, Leducq

Abstract P466: Sqstm1/p62 Regulates Hypoxia Inducible Factor 1 Alpha Stabilization And Protects The Heart From Hypoxic Injury

Ischemic heart disease (IHD) is characterized by cardiac tissue hypoxia, dysregulatedmetabolism, and cell death. Hypoxia inducible factor 1a (HIF1α) is a major component of thehypoxia pathway that regulates metabolic and angiogenic genes. Under normoxia, HIF1α getshydroxylated by the prolyl hydroxylases (PHDs), followed by its ubiquitination, and degradationby the Ubiquitin Proteasome System (UPS). Given the short half-life of HIF1α (>5mins), PHDinhibitors are employed to stabilize HIF1α and improve cardiac function in animal models ofischemia/infarction. Because PHD inhibitors exert off-target effects, alternative strategies tostabilize HIF1α are needed. In cancer cells, p62 stabilizes HIF1α by binding PHD3. Wehypothesized that p62 would stabilize HIF1α and provides cardioprotection from hypoxia. Wegenerated mice with tamoxifen-inducible cardiomyocyte-specific p62 deletion (cip62KO mice)and exposed them to 7% O2 for 6h. Compared to wild-type (WT) littermates, cip62KO micedisplayed increased oxidative stress and enhanced cell death. Hypoxia caused contractiledysfunction in cip62KO vs. WT mice. To gain mechanistic insights as to how lack of p62exacerbated hypoxic injury, cultured H9c2 cardiac myoblasts were exposed to 21% (normoxia)or 1% (hypoxia) O2 for 24h. Hypoxia increased HIF1α and p62 protein expression in H9c2 cells(p<0.05). To determine whether hypoxia-induced p62 accumulation is required for HIF1αstabilization, H9c2 cells were transfected with p62 or scrambled (ctrl) siRNA for 48h, andexposed to hypoxia. Compared to ctrl siRNA cells, hypoxia-induced HIF1α protein accumulationwas reduced (p<0.05) after p62 knockdown. Additionally, hypoxia increased expression ofHIF1α downstream targets: Egln1 , Vegfa , Bnip3 and Hmox1 mRNA, in ctrl siRNA cells, but theresponse was blunted (p<0.05) after p62 knockdown. These data indicate that p62 contributes tohypoxia-induced HIF1α stabilization and transcriptional activation. Defining how p62contributes to HIF1α stabilization and hypoxia tolerance is relevant clinically and could identifyp62 as a therapeutic target for treating IHD.

Breakthrough Science: Hypoxia-Inducible Factors, Oxygen Sensing, and Disorders of Hematopoiesis

Hypoxia-inducible factors (HIF) were discovered as activators of erythropoietin gene transcription in response to reduced O2 availability. O2-dependent hydroxylation of HIFs on proline and asparagine residues regulates protein stability and transcription activity, respectively. Mutations in genes encoding components of the oxygen sensing pathway cause familial erythrocytosis. Several small molecule inhibitors of HIF prolyl hydroxylases are currently in clinical trials as erythropoiesis stimulating agents. HIFs are overexpressed in bone marrow neoplasms, and the development of HIF inhibitors may improve outcome in these disorders.

Stabilization of Hypoxia-Inducible Factor Promotes Antimicrobial Activity of Human Macrophages Against Mycobacterium tuberculosis

Hypoxia-inducible factor (HIF) is a key oxygen sensor that controls gene expression patterns to adapt cellular metabolism to hypoxia. Pharmacological inhibition of prolyl-hydroxylases stabilizes HIFs and mimics hypoxia, leading to increased expression of more than 300 genes. Whether the genetic program initialized by HIFs affects immune responses against microbial pathogens, is not well studied. Recently we showed that hypoxia enhances antimicrobial activity against Mycobacterium tuberculosis (Mtb) in human macrophages. The objective of this study was to evaluate whether the oxygen sensor HIF is involved in hypoxia-mediated antimycobacterial activity. Treatment of Mtb-infected macrophages with the prolyl-hydroxylase inhibitor Molidustat reduced the release of TNFα and IL-10, two key cytokines involved in the immune response in tuberculosis. Molidustat also interferes with the p38 MAP kinase pathway. HIF-stabilization by Molidustat also induced the upregulation of the Vitamin D receptor and human β defensin 2, which define an antimicrobial effector pathway in human macrophages. Consequently, these immunological effects resulted in reduced proliferation of virulent Mtb in human macrophages. Therefore, HIFs may be attractive new candidates for host-directed therapies against infectious diseases caused by intracellular bacteria, including tuberculosis.

MO541HEMATOLOGIC EFFICACY OF VADADUSTAT FOR ANEMIA IN PATIENTS WITH NON--DIALYSIS-DEPENDENT CHRONIC KIDNEY DISEASE

Background and Aims Vadadustat is a small-molecule inhibitor of hypoxia-inducible factor prolyl hydroxylases under development to treat anemia associated with chronic kidney disease (CKD). The vadadustat phase 3 program includes four efficacy and cardiovascular safety outcome trials of vadadustat versus the erythropoiesis-stimulating agent (ESA) darbepoetin alfa. Here we describe detailed results on hematologic efficacy in two phase 3, randomized trials (the PRO2TECT trials) in adult patients with non–dialysis-dependent (NDD) CKD and anemia, in which vadadustat met prespecified noninferiority criteria compared to darbepoetin alfa, with respect to hematologic efficacy (correction/maintenance of hemoglobin [Hb] target concentrations). Method The mean screening Hb level for the ESA-untreated NDD-CKD trial (NCT02648347) had to be &lt;10.0 g/dL, and for the ESA-treated NDD-CKD trial (NCT02680574), the range had to be from 8.0-11.0 g/dL in the United States (US) and from 9.0-12.0 g/dL non-US. In the ESA-untreated trial, patients received no ESA within 8 weeks before randomization; in the ESA-treated trial, patients were maintained on ESA therapy, with ≥1 dose received within 6 weeks prior to or during screening. The vadadustat starting dose was 300 mg/day for all patients, whereas the initial darbepoetin alfa dose depended on each patient’s prior dose or the product label. Both vadadustat and darbepoetin alfa doses were titrated according to prespecified dosing algorithms to achieve target Hb concentrations (US: 10-11 g/dL; non-US: 10-12 g/dL) both during the primary (PEP; weeks 24-36) and secondary (SEP; weeks 40-52) evaluation periods. Herein, we present topline results from the PEP and SEP endpoints, in addition to more detailed erythrocyte parameters. Results A total of 3,476 patients (1751 ESA-untreated and 1725 ESA-treated) were randomized 1:1 to vadadustat or darbepoetin alfa. In both trials, vadadustat was noninferior to darbepoetin alfa with regard to the difference of mean change in Hb concentrations between baseline and PEP, as well as between baseline and SEP. The respective proportions of patients (vadadustat vs. darbepoetin alfa) with an average Hb value within the geography-specific target range in the PEP and SEP were 50.4% versus 50.2% and 43.1% versus 43.5% in the ESA-untreated trial and 60.1% versus 60.7% and 50.7% versus 49.0% in the ESA-treated trial. The proportion of patients (vadadustat vs darbepoetin alfa) who achieved an Hb increase &gt;1.0 g/dL from baseline to week 52 was assessed only for the ESA-untreated trial and was 87.7% (95% CI: 85.4%, 89.8%) for vadadustat versus 88.0% (95% CI: 85.6%, 90.0%) for darbepoetin alfa. Hematologic parameters at time points within the PEP and SEP are presented in Table 1. In both the ESA-untreated and ESA-treated trials, the reticulocyte count trended up from baseline through week 52 for vadadustat and trended down from baseline for darbepoetin alfa. Trends in erythrocyte mean corpuscular volume and erythrocyte mean corpuscular Hb were largely unremarkable by week 52 in both treatment groups. Conclusion Vadadustat demonstrated similar profiles across erythrocyte parameters compared with darbepoetin alfa in the treatment of adults with anemia in CKD not on dialysis, whether ESA-untreated or ESA-treated at study entry.

HIF-α Prolyl Hydroxylase Inhibitors and Their Implications for Biomedicine: A Comprehensive Review

Oxygen is essential for the maintenance of the body. Living organisms have evolved systems to secure an oxygen environment to be proper. Hypoxia-inducible factor (HIF) plays an essential role in this process; it is a transcription factor that mediates erythropoietin (EPO) induction at the transcriptional level under hypoxic environment. After successful cDNA cloning in 1995, a line of studies were conducted for elucidating the molecular mechanism of HIF activation in response to hypoxia. In 2001, cDNA cloning of dioxygenases acting on prolines and asparagine residues, which play essential roles in this process, was reported. HIF-prolyl hydroxylases (PHs) are molecules that constitute the core molecular mechanism of detecting a decrease in the partial pressure of oxygen, or hypoxia, in the cells; they can be called oxygen sensors. In this review, I discuss the process of molecular cloning of HIF and HIF-PH, which explains hypoxia-induced EPO expression; the development of HIF-PH inhibitors that artificially or exogenously activate HIF by inhibiting HIF-PH; and the significance and implications of medical intervention using HIF-PH inhibitors.