Comparative systems pharmacology of HIFstabilization in the prevention of retinopathyof prematurityGeorge Hoppea,b, Suzy Yoona, Banu Gopalanc, Alexandria R. Savaged, Rebecca Browna, Kelsey Casea, Amit Vasanjie,E. Ricky Chanf, Randi B. Silverd, and Jonathan E. Searsa,b,1

aCole Eye Institute, Cleveland Clinic, Cleveland, OH 44195; bDepartment of Cellular and Molecular Medicine, Cleveland Clinic, Cleveland, OH 44195; cYorgCorporation, Plano, TX 75093; dDepartment of Physiology and Biophysics, Weill Cornell Medicine, New York, NY 10065; eImageIQ Inc., Cleveland, OH 44128;and fInstitute for Computational Biology, Case Western Reserve University School of Medicine, Cleveland, OH 44106

Edited by Gregg L. Semenza, Johns Hopkins University School of Medicine, Baltimore, MD, and approved March 18, 2016 (received for review November24, 2015)

Retinopathy of prematurity (ROP) causes 100,000 new cases ofchildhood blindness each year. ROP is initiated by oxygen supple-mentation necessary to prevent neonatal death. We used organsystems pharmacology to define the transcriptomes of mice thatwere cured of oxygen-induced retinopathy (OIR, ROP model) byhypoxia-inducible factor (HIF) stabilization via HIF prolyl hydroxylaseinhibition using the isoquinolone Roxadustat or the 2-oxoglutarateanalog dimethyloxalylglycine (DMOG). Although both moleculesconferred a protective phenotype, gene expression analysis byRNA sequencing found that Roxadustat can prevent OIR by twopathways: direct retinal HIF stabilization and induction of aerobicglycolysis or indirect hepatic HIF-1 stabilization and increased serumangiokines. As predicted by pathway analysis, Roxadustat rescuedthe hepatic HIF-1 knockout mouse from retinal oxygen toxicity,whereas DMOG could not. The simplicity of systemic treatmentthat targets both the liver and the eye provides a rationale forprotecting the severely premature infant from oxygen toxicity.

HIF | BPD | prolyl hydroxylase inhibition | Roxadustat | ROP

Oxygen supplementation in severely premature infants is re-quired to prevent mortality but is simultaneously toxic to

premature developing tissues such as the retina. The retinovasculargrowth attenuation and vascular obliteration created by hyperoxialead to retinopathy of prematurity (ROP), a leading cause of child-hood blindness worldwide, accounting for 100,000 new cases of infantblindness each year (1). Recent prospective clinical trials paradoxi-cally have demonstrated that targeting oxygen saturations to levelscommensurate with in utero saturations reduces ROP but in-creases mortality (2, 3). The response to oxygen tension within thecell is regulated by hypoxia-inducible factors (HIFs), which aretranscription factors that modulate adaptation to hypoxia andnormoxia by directing the expression of gene products that in-crease glycolysis, hematopoiesis, angiogenesis, and vasculogenesis(4–6). HIF activity is quickly decreased posttranslationally by HIFprolyl hydroxylase domain proteins (HIF PHDs), which hydroxyl-ate the alpha subunit of the HIF heterodimer at Pro-402 or Pro-564 within the C-terminal oxygen-dependent degradation domainto make it a substrate of the von Hippel Lindau protein, an E3ubiquitin ligase (7–11). Polyubiquitination targets HIFα to theproteasome and thereby attenuates HIF-regulated transcription. Inhumans, this down-regulation of HIF activity is further increasedby hydroxylation of an aspargine residue within the C-terminalactivation domain by factor-inhibiting HIF, which prevents bindingof transcriptional coactivators CBP and p300 (12).HIF and HIF PHDs are particularly important to the growth

and development of the fetus, which is relatively hypoxic with re-spect to the mother and in fact has lower partial pressures of ox-ygen in comparison with an adult (for a review, see ref. 13). Thisphysiologic hypoxia drives growth, and naturally the hyperoxicenvironment of premature birth therefore induces severe growth

attenuation and vasoobliteration in tissues that are not yet fullydeveloped, which are associated with decreases in VEGF expres-sion by hyperoxia-induced down-regulation of HIF protein levels(14, 15). As a strategy to overcome the negative effects of oxygen,HIF stabilization during hyperoxia might lessen the abrupt tran-scriptional change that occurs after premature birth interrupts fetaldevelopment in utero. The basic challenge to implementing thisconcept is that HIF stabilization must be administered systemicallyand not locally because, as a preventative strategy, multiple in-traocular injections of HIF stabilizers are impractical consideringthe millions of infants at risk. In addition, ROP reflects systemicdisease, and therefore, blocking ROP by inducing normal reti-novascular growth should have the potential to protect infants fromother oxygen-induced diseases such as bronchopulmonary dyspla-sia, a major cause of chronic lung disease in children (16).We have definitively demonstrated the prevention of oxygen-

induced retinopathy (OIR) in two species using HIF PHD in-hibition (HIF PHi) during hyperoxia to stabilize HIF even in veryhigh oxygen tensions that would normally induce catabolism of theHIFα protein (17, 18). We have also demonstrated that systemicadministration of small-molecule HIF stabilizers can safely targetthe liver, inducing it to act as an endocrine organ that protectsperipheral capillary beds such as in the retina (19). The finding that

Significance

In all premature births, oxygen supplementation is a necessarylife-sustaining measure, but unfortunately for these high-riskbabies, oxygen toxicity may adversely and permanently affectthe retina. Pharmacological activation of the hypoxia-induciblefactor (HIF) pathway can prevent experimental oxygen-inducedretinopathy and thus has the potential to prevent blindness in100,000 children annually. Comprehensive analysis of liver andretinal transcriptomes after HIF stabilization demonstrates thatselect small molecules, given systemically, protect the retina bytwo pathways: stimulating the liver to secrete angiogenichepatokines or locally stimulating retinal protection. Thesefindings support a low dose, intermittent, systemic approachfor preventing oxygen induced injury to premature infants.

Author contributions: G.H., S.Y., R.B.S., and J.E.S. designed research; G.H., S.Y., A.R.S., R.B.,K.C., and J.E.S. performed research; A.V. and E.R.C. contributed new reagents/analytictools; G.H., B.G., A.R.S., A.V., E.R.C., R.B.S., and J.E.S. analyzed data; and G.H. and J.E.S.wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

Data deposition: The data reported in this paper have been deposited in the Gene Ex-pression Omnibus (GEO) database, www.ncbi.nlm.nih.gov/geo (accession no. GSE74170;NCBI tracking system no. 17567121).1To whom correspondence should be addressed. Email: searsj@ccf.org.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1523005113/-/DCSupplemental.

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the liver is essential to this protection using dimethyloxalylglycine(DMOG), via hepatic HIF-1 stabilization, provides a mechanisticbasis for solving the paradox of premature birth, as it creates the

possibility of inducing the neonatal liver to secrete necessarygrowth factors to create a systemic cytoprotective phenotypeduring the relatively short window of hyperoxia.

Fig. 1. Roxadustat targets liver and kidney. (A) Both i.p. and s.c. injections create liver- and kidney-specific luminescence in the luc-ODDmouse. (B) Quantificationof luciferase activity in organ lysates demonstrates liver and kidney tropism by either i.p. or s.c. injection of Roxadustat (RXD). (C) Luminescence of organ lysates ofi.p. Roxadustat indicates specificity for liver and kidney. *P < 5 × 10−4. (D) Time duration of HIF PHi by Roxadustat gives sustained stabilization of luc-ODD over 24 h.(E) Western blot analysis of HIF-1α protein after Roxadustat. (F) Integrated optical density of immunoblot analysis in E. (G) Densitometry of HIF-1α immunoblot(Insert) over time after Roxadustat i.p. injection. (H) Epo mRNA expression in organs after Roxadustat i.p. injection. (I) Serum EPO concentration versus time afteri.p. Roxadustat. (J) Dose-dependent expression of Epo mRNA in the liver. (K) Dose–response of HIF-1α to Roxadustat in cultured Hep3B cells analyzed by Westernblotting. (L) EPO mRNA levels in cultured Hep3B cells and EPO protein content on Hep3B culture media in response to Roxadustat.

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There are multiple examples of HIF PHD antagoniststhat function as competitive inhibitors. These inhibitors can actas competitive antagonists of 2-oxoglutarate, a cofactor that ac-cepts one oxygen from molecular dioxygen to become succinateas the second oxygen forms trans-4-hydroxyproline (20). Al-though the most basic of them structurally is DMOG, hydra-zones, carboxamides, and quinolones have been established aspotent HIF stabilizers (21–23). In this study, we use a systemspharmacology approach (comparative transcriptome analysiswith and without drugs) to compare Roxadustat (24) to DMOGin preventing OIR. We find that cytoprotection is initiatedduring hyperoxia, where capillaries survive hyperoxia under theaction of systemic HIF PHi. Systemic administration of DMOGprimarily targets visceral organs such as the liver and can protectthe lung from hyperoxia-induced alveolar destruction. Analysisof the transcriptome from the liver after systemic HIF PHidemonstrates up-regulation of angiogenic coding sequences forsecreted proteins that are secondarily validated by tissue-specificRT-PCR and immunoanalysis of serum. Although the hepatictranscriptomes induced by both small molecules are nearly iden-tical, retinal transcriptomes are different and indicate that systemicRoxadustat can also target the retina, providing a very potentprotection against OIR. Pathway analysis of retinal codingsequences demonstrates a metabolic shift toward aerobic gly-colysis in Roxadustat-treated animals, whereas DMOG-treatedanimals down-regulated cell adhesion/extracellular matrix path-ways in the retina. These findings suggest that systemic small-molecule HIF stabilizers can stimulate retinovascular protectionby two mechanisms: liver-targeted secretion of proangiogenicproteins and/or direct metabolic-based protection of retinal tis-sue during hyperoxia. The apparent synergy between thesemechanisms supports the use of low-dose, intermittent HIFstabilization in severely premature children to permit oxygensupplementation while protecting against oxygen-induced injury.

ResultsHIF PHi. Detection of organ-specific HIF PHi using the luciferaseoxygen-dependent degradation domain mouse (luc-ODD) (25)correlates to organ-specific HIF-1α stabilization (Fig. 1). Theluc-ODD mouse has a transgene comprising luciferase fused tothe ODD and therefore serves as a reporter gene in vivo ofwhere hydroxylation of the ODD is inhibited, which is observedas luminescence in tissue lysates and whole animal imaging.Ventral and dorsal views (Fig. 1A) of the luc-ODD mouse showthe highest luminescence in the liver and kidney, respectively,which are targeted by either i.p. or s.c. injections of Roxadustat.Quantitation supports the specificity of this effect in tissue ly-sates and shows sixfold luciferase activity over baseline in liver(Fig. 1 B and C). A time course of HIF stabilization in the kidneyand liver revealed that a single i.p. injection of Roxadustatprovides HIF PHi for at least 24 h with a half-life of 2–3 d (Fig.1D). We found organ-specific elevation of HIF-1α protein, de-tected by Western blotting, with a 5–10-fold increase in liver andkidney and weaker but definite twofold increase in spleen, lung,brain, and retina (Fig. 1 E and F). The kinetics of HIF stabili-zation by Roxadustat (Fig. 1G) is similar to luminescence activityin the luc-ODD mouse, showing maximal increases in serumerythropoietin (EPO) at 6 h after i.p. injection (Fig. 1I). Thiscorrelates with hepatic expression of Epo mRNA (Fig. 1H) aswell as an eightfold increase in serum EPO protein concentra-tion in response to Roxadustat (Fig. 1I). The dose–response ofRoxadustat using serum EPO as a marker of HIF-1 activitydemonstrates maximal effect in the mouse of 10 mg/kg body-weight, within an order of magnitude to the dose used in humanadults to increase red blood cell density (Fig. 1J). These effectsof Roxadustat on HIF-1α stabilization and expression of EPO,both mRNA and protein, can be recapitulated in cultured hep-atoma cell Hep3B (Fig. 1 K and L).

HIF PHi Prevents OIR. A direct comparison of the HIF PHD in-hibitor DMOG in the OIR model demonstrates at least the samebenefit with a trend to superiority of Roxadustat, reducing cap-illary dropout threefold in retinal flatmounts when each is usedin its optimal dose (Fig. 2 A and B). Roxadustat requires lessdrug concentration [10 mg/kg (28.4 μmol/kg) Roxadustat vs.200 mg/kg (1.14 mmol/kg) DMOG], which comprises about 40-fold reduction in drug molarity. The effect of Roxadustat corre-lates to a reduction in ischemic hypoxic regions of retina whenanalyzed using a hypoxia-sensitive probe (Fig. 2 C and D). Thus,Roxadustat, like DMOG, decreases hyperoxic retinal vascularobliteration, known as phase 1, facilitating normal retinal growthin relative hypoxia, known as phase 2, to induce normal andsequential retinovascular repair when administered in phase 1(26). The protection by Roxadustat was then further studied todetermine whether it protected retinal blood vessels duringhyperoxia or whether it facilitated regrowth by quantifying retinalflat mounts at P8 (8 d of age), P10 (middle of hyperoxia), P12 (theend of hyperoxia), P15 (middle of hypoxia), and P17 (end of theOIR model). Analysis of the change in the central avascular re-gion, which is the hallmark of vascular disease phenotype in themouse OIR, shows statistically significant reduction of capillaryloss at P10 and P12 after Roxadustat with similar rates of re-vascularization in treated and untreated animals (Fig. 2 E and F).The identical slope of retinovascular repair indicates thatRoxadustat protects the retina from oxygen-induced vasoobliterationduring hyperoxia while inducing normal vascular growth after phase1 HIF PHi (Fig. 2G). Pathologic neovascularization is also reducedby Roxadustat (Fig. 2H), because it reduces ischemia, which isthe substrate for abnormal vessel growth. This experimentdemonstrates that Roxadustat induces the normal sequentialgrowth of retinal capillaries, thereby depriving ROP of itsavascular substrate.Our previous publication measured retinal function using

electroretinography to demonstrate preservation of function ofretina after HIF stabilization in hyperoxia (19). Preservation ofretinal cells is now confirmed by cysteine aspartic acid protease 3(caspase 3) immunohistochemistry at P10 identifying cleaved andactivated caspase 3 in apoptotic cells. Animals treated withRoxadustat show reduction of activated caspase 3 in hyperoxia(Fig. S1 A and B) and hence reduction of apoptosis by HIF PHi.The effect was greatest in the inner nuclear layer and statisticallysignificant in Roxadustat-treated animals in hyperoxia comparedwith room air- and PBS-treated animals in hyperoxia.

Systems Pharmacology of HIF PHi: Liver Versus Retina. Completeanalysis of the transcriptome comparing Roxadustat to DMOGshows high concordance of gene expression in liver after eitherDMOG or Roxadustat but little common gene expression inretina, despite the fact that both small molecules confer protectionagainst OIR (Fig. 3 A and B and Dataset S1). Further stratificationof liver transcripts to secreted gene products (Secreted ProteinDatabase, spd.cbi.pku.edu.cn) demonstrates concordance of topresponders from the liver and includes Serpine1 (plasminogenactivator inhibitor-1, PAI-1), Epo, and Orm2 (orosomucoid)(Table 1) as candidates of hepatokines that might protect theretina remotely. Secondary validation of Serpine1 and Epo byRT-PCR of liver and ELISA of serum confirms the validity ofusing serum PAI-1 or EPO as a surrogate biomarker of HIFstabilization, which is predictably found at maximal increases 6 hafter i.p. injection (Fig. 3 C and D). There is a transient small risein serum VEGF (Fig. 3D) but not as significant as other proteins;Vegfa mRNA did not increase beyond the 2.0-fold cutoff inRoxadustat-treated animals (Dataset S1). There is no increase inVegfa mRNA in the control samples because RNA was obtainedfrom animals in hyperoxia or phase 1 ROP, which down-regulatesVefga mRNA, the period in which we envision applying HIF sta-bilization to prevent ROP. Unlike with the liver, transcriptional

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Fig. 2. Preservation of retinal blood vessels at P17 after Roxadustat injection. (A) Representative flat mounts comparing sham PBS injection to Roxadustat toDMOG. Each whole retinal flatmount image was obtained by taking nine overlapping microphotographs followed by merging corresponding micropho-tographs into one as described in SI Materials and Methods. Color coding of avascular region at P17 indicates reduction of oxygen-induced vasoobliterationprotection created by Roxadustat. (B) Quantification and statistical analysis of retinal flat mounts depicting percent avascular area of total retina area. **P = 5 ×10−9, *P = 0.02. RXD, Roxadustat. (C) Isolectin and Hypoxyprobe staining indicate a decrease in ischemic, hypoxic retina at P17 after Roxadustat injection inrepresentative retinal flat mounts. (D) Quantitative image analysis of avascular and hypoxic area expressed as percent of total retinal area (y axis). Valueswithin the bars represent hypoxic area as percent of avascular area. (E) Rate of retinal vascular growth during two phases of the OIR model, after PBS orRoxadustat treatment. Shown are representative flat mounts with computer-assisted area calculation shown in yellow. Roxadustat decreases vascularobliteration in hyperoxia. (F) Quantitation of avascular area (*P < 1 × 10−5) demonstrates consistently less ischemic retina, which is the substrate of ROP andpathologic neovascularization. (G) Rate of retinal vascularization in control and treated animals throughout the OIR cycle. The area of ischemia is less afterRoxadustat treatment, yet the slopes of regrowth are identical between control and Roxadustat-treated animals, indicating that Roxadustat protects retinaduring hyperoxia but does not induce abnormal or rapid regrowth of retinal blood vessels. (H) Decreased ischemia leads to decreased pathologic neo-vascularization. **P = 0.002, *P = 0.037.

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Fig. 3. Identification and validation of gene expression similarities and differences between animals treated with DMOG versus Roxadustat (RXD). (A) Venndiagrams of differentially expressed genes in the liver and retina, PBS vs. DMOG or PBS vs. RXD; fold-change cutoff, 2.0. (B) Pie charts depicting commonly anduniquely up-regulated genes. Liver has many common genes up-regulated, whereas retina does not. The identity of the top responders (fold-change cutoff,2.0) can be found in Tables 1 and 2 and Dataset S1, where a complete list of gene products is given, as well as online at Gene Expression Omnibus (GSE74170;NCBI tracking system no. 17567121). (C) Secondary validation of top responders—secreted liver gene products by qPCR of hepatic mRNA (*P = 9 × 10−5, **P =0.003, ***P = 0.001) and by (D) ELISA of serum proteins (*P < 0.002). (E) Immunofluorescent microscopy (40× magnification) shows partial colocalization ofMCT-4 (green) elevated by Roxadustat (RXD) with vimentin (red), specific for Muller cells. (F) Higher magnification (63×) of vimentin (red) and MCT-4 (green)demonstrates overlap within Muller cells that span the width of the retina and shows characteristic morphology of specialized retinal macroglia.

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analysis of retina revealed different patterns of gene expressiondepending on whether DMOG or Roxadustat was used to treatanimals systemically. Roxadustat stimulated expression of multiplemetabolic genes associated with aerobic glycolysis, such as Slc16a3(solute carrier family 16 member 3), Grhpr (hydroxypyruvate re-ductase), Pdk1 (pyruvate dehydrogenase kinase), Pfk1 (phospho-fructokinase), and Pgk1 (phosphoglycerate kinase) (Table 2),whereas DMOG mostly induced twofold induction in histonecluster genes (Table 2). Retinal transcripts from animals treatedwith each small molecule showed up-regulation of serine proteasesPrss56 and Upk3b (uroplakin) as well. The stimulation of a retinalcytoprotective pathway using aerobic glycolysis in Roxadustat-treated animals was further explored by correlating increases inretinal mRNA for Slc16a3, a monocarboxylate transporterspecific for lactate, also known as monocarboxylate trans-

porter 4 (MCT-4), to MCT-4 protein. In Roxadustat-treatedanimals, MCT-4 is increased in multiple cell types of the retina,including photoreceptors where MCT-4 colocalizes with rho-dopsin (Fig. S2A). At least another of these cell types within theinner plexiform layer is Muller cells, where MCT-4 colocalizeswith vimentin in vivo (Fig. 3 E and F). Expression of SLC16A3(MCT4) mRNA in cultured Muller cell line MIO-M1 afterRoxadustat treatment in vitro increases twofold (Fig. S2B).Pathway analysis of Roxadustat and DMOG liver and retinatranscriptomes demonstrates a conversion to aerobic glycolysisin liver after systemic stimulus with both small molecules but in-duction of aerobic glycolysis in retina only in Roxadustat-treatedanimals (Fig. S3 and Dataset S2).

Two Pathways for Systemic HIF Stabilization. The fact that systemicRoxadustat targets the liver and the retina whereas systemicDMOG does not is further confirmed by testing both smallmolecules in the hepatic Hif1a cre/lox knockout (KO) mouse.Roxadustat rescues the KO from OIR, whereas DMOG does not(Fig. 4 A and B), demonstrating that Roxadustat can circumventhepatic HIF-1 ablation to target retinal HIF-1 directly. Thisfinding correlates to tissue-specific gene expression profiles,collectively proving that DMOG induces retinovascular protectiononly remotely by secreted hepatokines, whereas Roxadustatoverrides the ablation of hepatic HIF-1α by directly targetingretinal tissue.The advantage of systemic HIF stabilization was explored by

investigating whether Roxadustat was also able to protect lung

Table 1. Hepatic gene identities of top responders toRoxadustat or DMOG from livers that were filtered through theSecreted Protein Database (fold-change vs. PBS)

Symbol Alternative gene name Roxadustat DMOG

Serpine1 Plasminogen activator inhibitor type 1 84.8 138.0Epo Erythropoietin 21.6 82.4Orm2 Orosomucoid 2 20.4 83.4Adm2 Adrenomedullin 2 18.4 14.6Hamp Hepcidin antimicrobial peptide 16.2 27.2Apol11a Apolipoprotein L 11A 12.2 16.6Dmkn Dermokine 9.9 6.5Apol10a Apolipoprotein L 10A — 9.5Fstl3 Follistatin-like protein — 8.3Saa2 Serum amyloid A2 6.8 4.5Orm1 Orosomucoid 1 6.6 13.5Fam132b Family with sequence similarity 132 6.3 7.5A2m Alpha-2-macroglobulin 5.9 8.1Saa1 Serum amyloid A1 5.7 4.4Bmp8b Bone morphogenetic protein 8b 5.3 10.6Apol11b Apolipoprotein L 11B 5.2 6.8Wisp2 WNT1-inducible signaling pathway — 4.9A1bg Alpha-1-B glycoprotein 4.3 —

Stc2 Stanniocalcin 2 4.1 3.1Efna1 Ephrin-A1 4.0 6.0Prss57 Serine protease 57 — 3.8Stc1 Stanniocalcin 1 3.7 3.4Mup20 Major urinary protein 20 3.5 —

Igfbp1 Insulin-like growth factor bp 1 3.2 3.1Ifng IFN gamma — 3.1Gpi1 Glucose-6-phosphate isomerase 1 2.8 6.2Mup3 Major urinary protein 3 2.8 —

Grem2 Gremlin 2 2.7 —

Ccl12 Chemokine ligand 12 — 2.7Serpina3g Serine protease inhibitor 2A — 2.6Angptl3 Angiopoietin-like 3 — 2.6Osm Oncostatin M 2.6 3.7Apol10b Apolipoprotein L 10B 2.5 —

Apoc3 Apolipoprotein C-III 2.5 2.5Apln Apelin 2.4 7.3Mmp13 Matrix metallopeptidase 13 2.4 —

Tnfaip6 Tumor necrosis factor alpha-induced 6 — 2.4Ccl2 MCP-1 — 2.4Serpina7 Antitrypsin — 2.4Vegfa Vascular endothelial growth factor A — 2.3Serpina3f Antitrypsin — 2.2Serpina3i Antitrypsin 2.2 2.7Nts Neurotensin 2.2 2.3Apoa4 Apolipoprotein A-IV 2.1 —

Lcn2 Lipocalin 2 2.1 4.1

Table 2. Retinal gene identities of top responders after systemicRoxadustat or DMOG (fold-change vs. PBS)

Symbol Alternative gene name Roxadustat DMOG

S100a9 S100 calcium binding protein A9 9.1 —

Dio3 Deiodinase, iodothyronine, type III 7.4 —

Slc16a3 Solute carrier family 16 member 3 6.8 —

Ppp1r3g Protein phosphatase 1 subunit 3G 6.5 —

Ak4 Adenylate kinase 4 5.3 —

Ca9 Carbonic anhydrase IX 4.0 —

Grhpr Glyoxylate/hydroxypyruvatereductase

3.8 —

Upk3b Uroplakin 3B 3.7 2.3Csn3 Casein kappa 3.5 —

Pdk1 Pyruvate dehydrogenase kinase 2.9 —

Pfkl Phosphofructokinase, liver 2.8 —

Prss56 Serine protease 56 2.5 2.6Hist1h4b Histone cluster 1, H2b — 2.5Hist1h2an Histone cluster 1, H2an 2.4 —

Tnfsf9 Tumor necrosis factor member 9 2.3 —

Bnip3 BCL2/adenovirus E1B protein 3 2.3 —

Ascl1 Achaete-scute complex homolog 1 2.2 —

Myocd Myocardin 2.2 —

Hist1h4a Histone cluster 1, H4a — 2.2Tpi1 Triosephosphate isomerase 1 2.1 —

Stc2 Stanniocalcin 2 2.1 —

Ankrd2 Ankyrin repeat domain 2 2.1 —

Smtnl2 Smoothelin-like 2 2.1 —

Mif Macrophage migration inhibitoryfactor

2.1 —

Hist1h3d Histone cluster 1, H3d 2.1 2.1Pgk1 Phosphoglycerate kinase 1 2.0 —

H2-Ab1 Major histocompatibility complex II 2.0 —

Selenbp2 Selenium binding protein 2 2.0 —

Ripply3 Ripply3 homolog 2.0 —

Ankrd37 Ankyrin repeat domain 37 2.0 —

Hist1h2ai Histone cluster 1, H2ai — 2.0

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tissue from hyperoxia. Roxadustat (10 mg/kg) was injected s.c.every other day in a murine model of bronchopulmonary dys-plasia and compared with hyperoxia alone and room air-raisedpups. When alveolar integrity was measured by mean linear in-tercept (Lm) to quantify alveolar cord length, Roxadustat pre-vented alveolar destruction and enlargement by hyperoxia (Fig. 4C and D). This finding provides a basic example of the power ofsystemic HIF stabilization, as it may address oxygen toxicity tomultiple organs (Fig. 4E).

DiscussionThe use of small molecules to stabilize a transcriptional activatorsuch as HIF is a powerful technique in regulating gene expres-sion noninvasively. There are many small-molecule inhibitors ofHIF PHDs (21, 23), but our experience testing them in vitro andin vivo has demonstrated that a carbonylglycine structure has avery high activity in vitro, and when it is attached to a hetero-cyclic ring comprising a fused benzene ring to a pyrimidine ringin an isoquinolone configuration, this activity increases, at leastin vivo (Fig. S4). Our comparison of DMOG [chemical name, N-(2-Methoxy-2-oxaoacetyl)glycine methyl ester] to Roxadustat [chem-ical name, N-([4-hydroxy-1-methyl-7-phenoxyisoquinolin-3-yl]car-bonylglycine)] demonstrates that DMOG solely targets the liver toinduce retinal protection, whereas Roxadustat targets both theliver and the retina, despite the fact that Roxadustat is therapeuticat 40 times less molar concentration.HIF is a transcription factor that induces the sequential, co-

ordinated, and orderly growth of blood vessels because it con-

trols the expression of multiple angiokines that act in synergy tocreate robust protection. In the case of ROP, the brief window ofopportunity available to prevent retinovascular growth attenua-tion as a means of removing the substrate for pathologic neo-vascularization suggests that systemic HIF PHi offers thepossibility of protecting other organ systems in addition to theeye, such as the lung (16). The importance of this investigation isthat we demonstrate a very potent effect of a HIF stabilizer thathas passed phase 1 and 2a trials (24) and hence may be relevantfor clinical trials in neonates once approved for adults, as theconcern for safety might be less using an agent that is alreadyproven safe in adults. The dose range in our mice is close to thedose tested in humans (10 mg/kg vs. 0.1–2 mg/kg, respectively)(24), and we speculate that weekly i.v. injections would be suf-ficient to induce retinovascular protection in severely prematureinfants, beginning a week after birth and up to 30 wk of correctedgestational age. Our retinal flat mount analysis of five differenttime points within the OIR cycle demonstrates that Roxadustatcreates an orderly, sequential protection and growth of retinalvasculature. We have previously reported that HIF PHi does notalter weight gain in pups (18), does not negatively affect liver orkidney function, and preserves retinal function (19). We arehopeful that the protective effects of HIF stabilization in thelung seen in baboon lung explants by DMOG and in our murinemodel of bronchopulmonary dysplasia by Roxadustat might im-prove gas exchange and thereby decrease the need for extendedoxygen resuscitation (Fig. 4E) (16). Less oxygen supplementationshould decrease oxygen toxicity to retina, lung, and brain tissues,

Fig. 4. Comparison of Roxadustat to DMOG using the cre/lox hepatic Hif1a KO mouse. (A) Representative retinal flat mount showing capillary dropout aftersystemic treatment with DMOG or Roxadustat in the hepatic HIF-1 KO. DMOG does not rescue the KO, whereas Roxadustat does, which is shown here toprevent OIR in the KO. (B) Quantification and statistical analysis of retinal flat mounts depicting percent avascular area of total retina area. *P = 1 × 10−12.(C) Lung histology after hematoxylin and eosin staining demonstrates the destructive alveolar enlargement by hyperoxia that is reduced by s.c. Roxadustat (RXD)to a size and structure comparable to age-matched controls in room air. (D) Quantification of alveolar size using Lm demonstrates statistically significantnormalization in alveolar size by Roxadustat (RXD) treatment during hyperoxia in the wild-type CD1 mouse pups. *P = 1 × 10−4. (E) Schematic representationof the two pathways for retinovascular protection against OIR and bronchopulmonary dysplasia, targeting extraretinal HIF-1 in the liver in the case of DMOG,or both hepatic and retinal HIF-1 pathways in the case of Roxadustat (RXD).

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much as both basic science and clinical research has suggested(16, 27, 28). The spleen is also a weak target of systemic HIFstabilization, as the luc-ODD mouse has increased luminescenceafter systemic HIF PHi. This may facilitate extramedullary he-matopoiesis after HIF-2 stabilization (29). Our systems phar-macology analysis confirms that the potent effect of Roxadustatmay derive from the finding that it stimulates two pathways ofprotection, via liver and retinal HIF stabilization. In the absenceof hepatic HIF-1α, changes in retinal gene expression induced byi.p. DMOG, in comparison with Roxadustat, are not sufficient tocreate protection against OIR. These findings leave open thepossibility that a HIF-1–dependent, secreted hepatic gene productcan remotely protect the retina (19, 30).Although a natural candidate gene for this role is EPO, we

believe that HIF stabilization is able to induce multiple pathwaysto induce the coordinated sequential growth and protection ofblood vessels. EPO has also been reported to both increase anddecrease protection in the mouse and rat (30–32) and has notbeen shown to decrease ROP in humans (33). Moreover, pro-longed and constant levels of high EPO may be deleterious, asconstitutive expression of EPO in the mouse, and exogenousadministration of EPO in infants, especially into phase 2 of ROP,is reported to be deleterious (34, 35). These findings suggest thatHIF stabilization in the neonate should be intermittent and ata low dose.We have definitively demonstrated that DMOG-dependent

protection depends on hepatic HIF-1α (19), and now our systemspharmacology analysis reveals that both DMOG and Roxadustathave similar hepatic transcriptional profiles in contrast to dif-ferent retinal gene expression profiles. Multiple gene productswe find secreted by the liver have been implicated in amelio-rating blood vessel destruction, such as EPO, PAI-1, orosomu-cosoid, adrenomedulin, apelin, VEGF, and angiopoietin-likeprotein 3, implying that the power of targeting a transcriptionfactor lies in the coordinated up-regulation of multiple proteinsnecessary for organized blood vessel growth (31, 36–41). Wehave also considered that the differences in retinal RNA inducedby DMOG or Roxadustat demonstrate HIF-1α or HIF-2α de-pendence. However, there is clear evidence that DMOG stabilizesboth HIF-1α and HIF-2α as well as the asparaginyl hydroxylasefactor-inhibiting HIF (42).Our pathway analysis demonstrates a change in both the he-

patic and retinal metabolic phenotype to aerobic glycolysis aftersystemic Roxadustat stimulus. There is up-regulation of multipleenzymes for glycolysis in the transcriptomes of liver and retinatreated with Roxadustat. Conversely, DMOG increases thesesame enzymes in liver but not retina transcriptomes (Fig. S3).Aerobic glycolysis is reported to drive endothelial growth andrepair as a stimulus for DL4/Notch-dependent endothelial tipcell sprouting (43, 44). Our analysis found multiple glycolyticgenes up-regulated by Roxadustat in the retina (Fig. S3), includingphosphofructokinase, which is reported to be essential for endo-thelial tip sprouting, but also pyruvate dehydrogenase kinase, therate-limiting enzyme that inactivates pyruvate dehydrogenase,thereby preventing pyruvate from becoming decarboxylated andentering the Kreb’s cycle (45). In addition, up-regulation of MCT-4/Slc16a3 may allow for transport of excess lactate out of Mullercells for use as an energy substrate in photoreceptors (46).In summary, our data define two pathways for retinovascular

protection against OIR: targeting extraretinal HIF-1 in the liverin the case of DMOG or both hepatic and retinal HIF-1 path-ways in the case of Roxadustat. Our data make it practical toconsider the use of low-dose, intermittent HIF PHi during thenarrow window of opportunity after premature birth, beforeROP develops, as stimulation of these two pathways simulta-neously is at the least additive and possibly synergistic to pro-tecting capillary beds against oxygen toxicity. The liver can begently stimulated to become an endocrine organ for vascular

protection in neonates, much as it is used to stimulate erythro-poiesis in adults with kidney failure (24). In the case of DMOG,this is essential to preventing OIR, as transcriptome and Westernblot analysis of retina and rescue of the hepatic HIF-1 KOsuggest that penetration of systemic DMOG to the retina isweak, whereas for Roxadustat, this effect is additive to stimula-tion of the retinal HIF pathway.

Materials and MethodsAnimals.All experimental procedures involving live animals were approved bythe Cleveland Clinic Institutional Animal Care and Use Committee (protocolno. 2013-1108). Wild-type C57BL/6 mice were supplied by Harlan and JacksonLaboratory. The Luc-ODD transgenic mouse expressing HIF-1α oxygen-dependent degradation domain fused to luciferase was purchased fromJackson Laboratory (stock 6206). The conditional liver Hif1a KO mouse wasgenerated at Jackson Laboratory by crossing a Hif1a2lox/2lox mouse (stock7561) with an Albumin-Cre mouse (stock 3574) as previously described (19).

Prolyl Hydroxylase Inhibitors. DMOG from Frontier Scientific was directlydissolved in sterile PBS to a stock concentration of 20 mg/mL, then filter-sterilized again, and stored aliquoted at –80 °C. Roxadustat (FG-4592) wassupplied by Selleck Chemicals and by AdooQ BioScience; 50 mg/mL stocksolution was first prepared in DMSO, then further diluted in sterile PBS to1 mg/mL, and stored aliquoted at –80 °C. The inhibitors were administeredto newborn pups by i.p. injections via a 31-gauge needle at a typical dose of200 mg/kg (DMOG) or 10 mg/kg (Roxadustat), unless specified otherwise.

Detection of Luc-ODD Luciferase Reporter in Vivo and in Vitro. Live imaging ofluciferase activity was performed using Bruker Xtreme small-animal X-ray, afluorescence bioluminescence imaging system (25) (SI Materials and Methods).

OIR and Retinal Vasculature Analysis. OIR as an animal model of ROP used inthis study is based on a well-established protocol by Smith et al. (47). Ourtypical pharmacological treatment of OIR was previously described in pub-lished literature (17, 19) and involves up to three i.p. injections of a prolylhydroxylase inhibitor DMOG or Roxadustat at P6, P8, and P10. OIR-associ-ated vascular pathology as well as the therapeutic effect of HIF PHi wereassessed by computer-assisted quantification of retinal capillary loss andneovascular tufts in isolectin-stained, whole-retinal flat mounts using softwaredeveloped by ImageIQ and described previously (17) (SI Materials and Methods).

Retinal Tissue Hypoxia Analysis. Tissue hypoxia was detected using theHypoxyprobe Green Kit (Natural Pharmacia) as follows. One hour beforesacrifice, mouse pups received a single i.p. dose of 60mg/kg pimonidazole HCldissolved in PBS followed by retinal dissection and immunodetection ofpimonidazole adducts combinedwith isolectin staining performed essentiallyas described above. Briefly, dissected, premeabilized, and blocked retinaswere incubated overnight at 4 °C with a mixture of GS-IB4-Alexa568 andFITC-conjugated mouse monoclonal antipimonidazole adduct antibody(1:200 dilution).

Immunofluorescent Microscopy. Enucleated unfixed mouse pup eyes weredirectly embedded in Tissue-Tek OCT compound (Electron Microscopy Sci-ences), cut into 8 μm-thick frozen sections, and stored at –80 °C until use. Thefollowing primary antibodies and their dilutions were used for the immu-nohistochemical study: MCT-4 rabbit polyclonal (a gift from Nancy Philp,Thomas Jefferson University, Philadelphia) (dilution 1:1,000), rhodopsin mousemonoclonal antibody (B6–30N, a gift from Paul Hargrave, University of Florida,Gainesville, FL) (dilution 1:10,000), vimentin chick polyclonal antibody (EMDMillipore) (dilution 1:500), and active caspase 3 rabbit polyclonal antibody(BD Pharmingen) (dilution 1:200) (SI Materials and Methods).

Oxygen-Induced Lung Injury and Lung Airspace Analysis. A well-defined mu-rine model of bronchopulmonary dysplasia using neonatal mice chronicallyexposed to hyperoxia (48–50) followed by normoxia (51) was used for ourstudies. We used the direct method for the accurate estimation of the Lmlength (cord length) to assess the mean free distance in the alveoli (52) (SIMaterials and Methods).

RNA Sequencing and Bioinformatics. RNAwas harvested from animals at P8 forliver and P12 for retina. The quality control of total RNA (TRNA) samples wasexecuted using Qubit (Invitrogen) for quantification and Agilent Bioanalyzeranalysis to assess quality using a cutoff of RNA integrity number (RIN) > 7.0 to

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select specimens for further analysis. For library preparation, the IlluminaTruSeq Stranded Total RNA kit with Ribo Zero Gold for rRNA removal wasused. This protocol starts by using the Ribo-Zero kits to remove ribosomalRNA (rRNA) from 0.10–1 μg of TRNA using a hybridization/bead captureprocedure that selectively binds rRNA species using biotinylated captureprobes. The resulting purified mRNA was used as input for the IlluminaTruSeq kit in which libraries are tagged with unique adapter indexes. Finallibraries are quality control checked (QC’d) on the Agilent 2100 Bioanalyzer,quantified via quantitative PCR (qPCR), diluted, and pooled at equimolarratios. The sample pools were assayed on a 50-cycle sequencing run onIllumina MiSeq to test for evenness of individual sample distribution in thepool and optimal cluster density for the HiSEq. 2500 Rapid Runs. Optimizedlibraries were run on a HiSEq. 2500 Paired-End Rapid Run Flow Cell. Se-quences generated from the Illumina platform were checked for overallquality using FastQC (www.bioinformatics.babraham.ac.uk/projects/fastqc/).The 101-bp paired-end reads were trimmed for quality as well as adaptersequences using custom in-house scripts. The reads were aligned to themouse transcriptome using the GENCODE annotation for mm9 using TopHat(53). Cufflinks was then used to assemble the transcripts and estimate theirabundance as FPKM (fragments per kilobase of transcript per millionmapped reads) values (54). The cuffdiff program was used to determinedifferential gene expression analysis. An adjusted P value cutoff (q ≤ 0.05)was used to determine gene significance. Functional profiling of differentiallyaffected biological processes, functions, pathways, and networks for the dif-ferentially expressed genes obtained from the DMOG- and Roxadustat-treatedsamples in liver and retina was evaluated using both open source tools and

commercial pathway packages such as Metacore (lsresearch.thomsonreuters.com), Ingenuity Pathway Analysis (IPA; www.ingenuity.com), and Database forAnnotation, Visualization and Integrated Discovery (DAVID; https://david.ncifcrf.gov/). The consensus and reliable findings from these tools were used tobetter understand the role and the underlying differences in mechanisms be-hind the two drugs.

Statistical Analysis. Aside from biostatistical processing of RNA-seq data,regular statistical analyses of all other data (qPCR, image density, or pixelnumber quantification) were performed by comparingmeans using Student’st test. The two-tailed probability associated with rejecting the null hypothesisof no difference between observed groups was calculated, using an alphalevel of 0.05. Error bars in all figures represent SD.

ACKNOWLEDGMENTS. We thank Nancy Philp for the MCT-4 antibody, PaulHargrave for the rhodopsin antibody, G. Astrid Limb for MIO-M1 cells,Lediana Goduni for Hypoxyprobe immunohistochemistry (detection of tissuehypoxia), Judy Drazba for live animal imaging of luciferase activity and forassistance with frozen sectioning, Ms. Sowbarnika Kannan for assistancewith bioinformatics analysis, and Krzysztof Palczewski for helpful discussionsin regard to systems pharmacology. This work was supported by a Researchto Prevent Blindness Unrestricted Challenge Grant from the E. MatildaZiegler Foundation for the Blind (to J.E.S.), The Hartwell Foundation Collab-orative Biomedical Research Fellowship (to J.E.S. and R.B.S.), NIH GrantR01EY024972 (to J.E.S.), and the Genomics Core Facility of the Case WesternReserve University School of Medicine Genetics and Genome Sciences De-partment and Case Comprehensive Cancer Center (Grant P30CA043703).

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