B R A I N R E S E A R C H 1 2 8 0 ( 2 0 0 9 ) 4 3 – 5 1

ava i l ab l e a t www.sc i enced i rec t . com

www.e l sev i e r. com/ l oca te /b ra in res

Research Report

Short-term effects of pharmacologic HIF stabilization onvasoactive and cytotrophic factors in developing mouse brain

Christina Schneidera, Gudrun Krischkea, Stephan Kellerb, Gail Walkinshawc,Michael Arendc, Wolfgang Raschera, Max Gassmannb, Regina Trollmanna,⁎aDepartment of Pediatrics, Friedrich-Alexander-University of Erlangen-Nuremberg, Loschgestrasse 15, 91054 Erlangen, GermanybInstitute of Veterinary Physiology, and Zurich Center for Integrative Human Physiology (ZIHP), University of Zurich, SwitzerlandcFibroGen Inc., San Francisco, California, USA

A R T I C L E I N F O

⁎ Corresponding author. Fax: +49 9131 853 338E-mail address: regina.trollmann@uk-erlaAbbreviations: ADM, adrenomedullin; EPO

porphobilinogen deaminase; PHI, prolyl hydr

0006-8993/$ – see front matter © 2009 Elsevidoi:10.1016/j.brainres.2009.05.023

A B S T R A C T

Article history:Accepted 11 May 2009Available online 18 May 2009

Hypoxia-inducible transcription factors (HIFs) are crucially involved in brain developmentand cellular adaptation to hypoxia and ischemia. Degradation of HIF is regulated undernormoxia by oxygen-dependent hydroxylation of specific prolyl residues on the labile α-subunit by HIF prolyl hydroxylases (PHD). Prolyl-4-hydroxylase inhibitors (PHI) have shownprotective effects in vitro and in vivo in adult kidney and brain. The aim of the present studywas to investigate in vivo short-term effects of a novel lowmolecular weight PHI, FG-4497, onHIF-regulated cytotrophic and vasoactive factors in developing mouse brain. Neonatal (P7,n =26) C57/BL6 mice were treated with PHI FG-4497 (30–100 mg/kg, i.p., duration 6 h). Geneexpression was analyzed by TaqMan RT-PCR in kidney and developing brain in comparisonto controls (NaCl 0.9% and non-treated animals). HIF-1α protein was quantified by Westernblot analysis. Dose–response studies revealed prominent effects of FG-4497 at a dose of100 mg/kg as assessed by significant up-regulation of mRNA in both kidney and brain of thefollowing HIF-dependent genes: vascular endothelial growth factor, adrenomedullin anderythropoietin. Organ-specific transcriptional regulation was evident from analysis ofhexokinase 2, inducible NO synthase and PHD3 mRNA concentrations. In the brain, HIF-1αand HIF-2α protein markedly accumulated in response to FG-4497. Besides vasoactivefactors, PHI significantly increased cerebral chemokine receptor CXCR-4 mRNA levels. Inconclusion, the novel PHI FG-4497 activates HIFs at an early stage of brain maturation andmodulates neurotrophic processes known to be crucially involved in brain development andhypoxia-induced brain pathology.

© 2009 Elsevier B.V. All rights reserved.

Keywords:Hypoxia-inducibletranscription factorVascular endothelial growth factorChemokine receptor CXCR-4ErythropoietinAdrenomedullinDeveloping mouse brain

1. Introduction

Endogenous hypoxia-inducible mechanisms are cruciallyinvolved in early physiologic brain development (Iyer et al.,

9.ngen.de (R. Trollmann)., erythropoietin; HIF, hypoxylase inhibitor; VEGF, v

er B.V. All rights reserved

1998, Lee et al., 2001, Tomita et al., 2003) and protectivemodulation of hypoxia-induced brain pathology during earlystages of brain maturation (Bernaudin et al., 2002). Amongthese adaptive systems hypoxia-inducible transcription fac-

oxia-inducible factor; iNOS, inducible nitric oxide synthase; PBGD,ascular endothelial growth factor

.

44 B R A I N R E S E A R C H 1 2 8 0 ( 2 0 0 9 ) 4 3 – 5 1

tors (HIFs) are characterized as the most important mediatorsof cellular and molecular responses to hypoxia and ischemiaof many organs including the brain (Fandrey et al., 2006). HIFsare heterodimeric transcription factors, consisting of the O2-regulated α- and the constitutively expressed β-subunit. Theα-subunit is degraded rapidly by the ubiquitin-proteasomepathway mediated by specific prolyl residues that are hydro-xylated by an enzyme family of HIF prolyl hydroxylases (prolylhydroxylation domain protein [PHD]) that require di-oxygenand 2-oxoglutarate as co-substrates (Fandrey et al., 2006).Reduced activity of the PHDs under hypoxia results instabilization of the HIF-α protein, heterodimerization (α/β),activation of nuclear translocation and binding to hypoxiaresponse elements of promoters of specific responsive genes.HIF targets such as erythropoietin (EPO), glycolytic enzymesincluding lactate dehydrogenase and hexokinase (HK2), vas-cular endothelial growth factor (VEGF), adrenomedullin (ADM)and inducible NO synthase (iNOS), are involved in themodulation of oxygen and energy supply by activation ofangiogenesis, erythropoiesis, glycolysis and cell survival(Fandrey et al., 2006). Interestingly, specific hypoxia responseelements have been identified in the promoter region of PHD2and PHD3 preventing overstimulation of the HIF systemduring persisting hypoxia and reoxygenation (D'Angelo et al.,2003, Pescador et al., 2005).

During early stages of brain maturation, HIF target genesmodulate essential developmental processes including vas-culogenesis (e.g. VEGF) and neuronal and glial survival anddifferentiation (e.g. VEGF, EPO) (Lee et al., 2001, Ogunshola etal., 2002, Sun et al., 2003). Moreover, cell migration in hypoxicbrain regions is modified by HIF. Chemokine receptor CXCR-4and its physiologic ligand stromal-derived factor-1 (SDF-1)are up-regulated by HIF-1 in different cell types (Ceradini etal., 2004, Schioppa et al., 2004, Staller et al., 2003) includinghuman neural stem cells (Chu et al., 2008) and mouse corticaland hippocampal neurons (Stumm et al., 2002). As demon-strated by knockout experiments, SDF-1 and CXCR-4 arecrucial factors for physiological cerebellar (Ma et al., 1998)and cortical development (Paredes et al., 2006). Beyondembryogenesis, SDF-1/CXCR-4 signaling is essential forsurvival and directed migration of neural and oligodendro-cyte precursors during postnatal brain maturation (Dziem-bowska et al., 2005).

This broad spectrum of HIF effects is of special interestin terms of neuroprotection upon hypoxic and ischemicinjury of the neonatal brain considering possible synergisticeffects of HIF target genes. Indeed, pharmacologic HIFstabilization by desferioxamine (DFX) or cobalt chloridethat inhibit the activity of iron-dependent HIF prolyl-4-hydroxylases revealed neuroprotective effects in vitro (Chuet al., 2008) and decreased ischemic brain damage in vivo inadult (Bergeron et al., 1999) and neonatal rats (Bergeron etal., 2000). Novel low molecular weight inhibitors of HIFprolyl hydroxylases (PHI) which did not show toxic effectsupon systemic administration (Bernhardt et al., 2006,Robinson et al. 2008) have been shown to protect theadult rat kidney (Bernhardt et al., 2006, Rosenberger et al.2008), murine bowel (Robinson et al. 2008) and brain (Siddiq etal., 2005) from ischemic and oxidative stress-induced injury.Assessed in vitro by embryonic cortical neurons (Siddiq et al.,

2005) and a hippocampal cell line (Aminova et al. 2005,2008), oxidative neuronal damage decreased in response toPHI implicating future clinical perspectives. However, dataon neonatal brain in vivo is not available. Therefore, weaimed to use a novel PHI, FG-4497, to analyze short-term invivo effects of pharmacological HIF stabilization on expres-sion of specific HIF-regulated target genes in normoxicdeveloping mouse brain at postnatal day 7 (P7). Significantprotective effects of FG-4497 have been demonstrated instudies on hypoxic kidney injury in adult rats (Rosenbergeret al., 2008) and inflammatory bowel disease in adult mice(Robinson et al., 2008). Here we extend the present knowl-edge about in vivo effects of FG-4497 by demonstrating firsttime its property to stabilize HIF and up-regulate vasoactiveand cytotrophic target genes in the brain at early stage ofdevelopment.

2. Results

2.1. Dose–response studies

To determine organ-specific activation of HIF-regulated sys-tems by PHI, we analyzed mRNA levels of specific HIF targetgenes in developing brain and kidney of neonatal mice atpostnatal day 7 (P7). FG-4497 treatment at a dose of 30 and60 mg/kg did not seem to have consistently significant effectsin neonatal mice during normoxia assessed by changes ofmRNA concentrations of HIF target genes in developing brainand kidney. At this dose range, mean mRNA levels of VEGF,iNOS and EPO in both brain and kidney of the treated micewere not significantly different from those of controls (datanot shown). Increasing the dose to 100 mg/kg was efficient toup-regulate specific HIF-regulated genes encoding vasoactiveand cytotrophic factors in the kidney (Fig. 1) and developingbrain (Fig. 2). As expected HIF-1α mRNA levels did not changeupon FG-4497 treatment (100mg/kg) in either tissue comparedto controls (mean±SEM, normalized to PBGD mRNA levels;kidney: FG-4497, 0.75±0.12; vs NaCl 0.9%, 0.99±0.11, non-treated, 0.84±0.06; brain: FG-4497, 2.66±1.79; vs NaCl 0.9%,1.46±0.26, non-treated, 2.35±0.81, ns).

2.2. Effects of pharmacologic HIF stabilization on HIFtarget gene expression in the kidney

In neonatal kidney we observed significant changes invasoactive and cytotrophic HIF target genes in response toFG-4497 (100 mg/kg) (Fig. 1). Among vasoactive factors, wefound a 2-fold increase in VEGF (Fig. 1A, p <0.05) and 3-foldincrease in ADM mRNA levels (Fig. 1B, p <0.01) in FG-4497-treated mice compared to controls. In contrast, iNOS mRNAlevels tended to increase, but were not significantly changedby FG-4497 treatment (mean±SEM, normalized to β-actinmRNA levels; FG-4497, 1.42±0.32; vs NaCl 0.9%, 0.36±0.06,non-treated, 0.72±0.08, ns). In terms of metabolic andcytotrophic factors we showed a significant increase oferythropoietin (EPO) (Fig. 1C, p <0.01) and hexokinase 2 (HK2)(Fig. 1D, p <0.05) in response to FG-4497 compared to controls.Moreover, PHD3 mRNA levels (Fig. 1E, p <0.01) were up-regulated by FG-4497 compared to controls.

Fig. 1 – HIF target gene expression (mRNA levels normalized to β-actin, mean±SEM) in mouse kidney at P7 in response to PHIFG-4497 (100 mg/kg, n =5) compared to controls (NaCl 0.9%, n =5; non-treated, n =5). *p <0.05, **p <0.01.

45B R A I N R E S E A R C H 1 2 8 0 ( 2 0 0 9 ) 4 3 – 5 1

2.3. Stabilization of HIF-α proteins in developing mousebrain in response to PHI

Western blot analysis revealed that HIF-1α protein wasdetectable in developing mouse brains under normoxicconditions (Fig. 2A). FG-4497 strongly induced HIF-1α proteinaccumulation in developing brain compared to controls (Figs.2A, B, p <0.05). Similarly, HIF-2α protein levels also markedlyincreased upon FG-4497 treatment compared with almostundetectable levels in non-treated normoxic tissues (Figs. 2C,D, p <0.05).

2.4. Differential up-regulation of HIF target genes indeveloping mouse brain in response to PHI

To analyze cerebral functional activity of the HIF system inresponse to PHI treatment specific vasoactive and cytotrophictarget genes were investigated. FG-4497 significantlyincreased mean VEGF and ADM mRNA levels (Figs. 3A, B,

p <0.05) in developing brain compared to controls but did notincrease iNOS mRNA levels upon 6 h (Table 1). Among themetabolic and cytotrophic factors investigated in this study,FG-4497 treatment up-regulated cerebral EPO mRNA levels(Fig. 3C, p <0.05), but did not change HK2 mRNA expression(Table 1) compared to controls. In addition, cerebral PHD3mRNA levels were not altered by FG-4497 treatment (Table 1).This was in contrast to our observations in the kidney (Fig. 1).Interestingly, chemokine receptor CXCR-4 mRNA concentra-tions were significantly increased in FG-4497-treated brainscompared to controls (Fig. 3D, p <0.05) whereas mRNA levelsof its physiological ligand, SDF-1, were unchanged at the timepoint analyzed (Table 1).

3. Discussion

HIFs play a crucial role in cerebral angiogenesis, neurogenesis,metabolic adaptation, cell migration and differentiation

Fig. 2 – Western blot analysis of HIF-1α (A) and HIF-2α protein (C) in developing mouse brain (P7) in response to PHIFG-4497 (100mg/kg) compared to controls. NT, non-treated control. B, D. Densitometric quantification of HIF-1α (B) and HIF-2αprotein accumulation (D) (n =3 per group; normalized to β-actin). *p <0.05.

46 B R A I N R E S E A R C H 1 2 8 0 ( 2 0 0 9 ) 4 3 – 5 1

during early brain development (Lee et al., 2001, Iyer et al.,1998). HIF-1α protein detectable in normoxic developingmouse brain, as shown in this study, reflects availability ofHIF-1α in the regulation of physiological oxygen demands (Lee

Fig. 3 – HIF target gene expression (mRNA levels, normalized toresponse to PHI FG-4497 (100 mg/kg, n =5) compared to controls

et al., 2001) allowing immediate adaptation to fluctuations ofcellular oxygen tension in the developing brain. Underpathological conditions of hypoxia and ischemia, stabilizationof HIFs by novel low molecular weight PHI has been shown to

b-actin; mean±SEM) in developing mouse brain at P7 in(NaCl 0.9%, n =5; non-treated, n =5). *p <0.05.

Table 1 – HIF target gene expression in developing mousebrain in response to PHI FG-4497 (100 mg/kg) compared tocontrols.

mRNA ratio a

(mean±SEM)PHI, 100

mg/kg n =5NaCl 0.9%

n =5Non-treated

n =5

PHD3/β-actin 0.86±0.09 0.52±0.03 0.62±0.02 nsHK2/β-actin 0.95±0.23 0.79±0.05 0.71±0.08 nsiNOS/β-actin 0.65±0.05 0.60±0.08 0.59±0.06 nsSDF-1/β-actin 0.26±0.05 0.54±0.05 0.31±0.04 ns

a Specific mRNA normalized to β-actin mRNA. Similar results wereobtained in relation to PBGD.

47B R A I N R E S E A R C H 1 2 8 0 ( 2 0 0 9 ) 4 3 – 5 1

protect against subsequent renal (Bernhardt et al., 2006,Rosenberger et al. 2008) and cerebral ischemia in adult rats(Siddiq et al., 2005) without toxic effects. Of importance,developmental age- and organ-specific effects of PHI treat-ment have to be assumed from studies on hypoxia-inducedHIF stabilization in rodents (Bernaudin et al., 2002, Stroka etal., 2001). This study demonstrates for the first time markedshort-term in vivo changes in endogenous, developmentallyregulated vasoactive and cytoprotective HIF-dependent fac-tors at an early stage of brain development in response to thenovel PHI FG-4497. To determine the functional response toHIF stabilization, selected HIF target genes known to beinvolved in early responses to hypoxia- and DFX-inducedHIF accumulation in developing mouse brain from P0 to P7(Bernaudin et al., 2002, Trollmann et al., 2008a,b) wereinvestigated. The major findings of the present short-termstudy were first, that HIF stabilization by FG-4497 inducedorgan-specific responses at the time point analyzed, andsecond, that FG-4497 treatment produced differential HIFtarget gene activation in developing mouse brain suggestingeffective penetration of FG-4497 through immature blood-brain-barrier under normoxia.

3.1. Organ-specific regulation of the HIF system

In response to systemic hypoxia, organ-specific regulation ofthe HIF system has been described in adult (Stroka et al., 2001,Wiesener et al., 2003) and neonatal rodents (Bernaudin et al.,2002, Trollmann et al., 2008b). Notably, more severe hypoxiawas required in adult mouse kidney than in brain to induceHIF-1α protein accumulation (Stroka et al., 2001). A compar-ison of HIF-2α protein accumulation in multiple organs ofadult rats exposed to CO (0.1%) revealed protein stabilizationat higher oxygen concentrations compared to HIF-1α, andstronger and earlier protein accumulation in the kidney thanin the brain (Wiesener et al., 2003). In neonatal mice exposedto 8% O2 for 3 h (Bernaudin et al., 2002) and to 6% O2 for 6 h(Trollmann et al., 2008b), ADM and VEGF were immediatelyup-regulated in the brain but not GLUT-1 and EPOwhich were,however, strongly induced in the kidney (Bernaudin et al.,2002). Beyond degree of hypoxia, age- and cell type-dependentregulation of HIF activity and target gene activation (Chavez etal., 2006; Hu et al., 2003, Stroka et al., 2001, Trollmann et al.,2008b, Wiesener et al., 2003) as well as tissue-specificsensitivity (Rosenberger et al., 2002, Wiesener et al., 2003)might explain organ-specific responses to hypoxia. Theseobservations confirm the functional significance of the HIF

system reflecting a readily available and highly flexible systemto activate adaptive mechanisms under hypoxic conditions atthe cellular level (Fandrey et al., 2006, Rosenberger et al., 2002).Supporting this view, activity and distribution of the oxygen-sensing prolyl hydroxylases (PHD) have been shown to varybetween different cell types (Bruick and McKnight, 2001,Willam et al., 2006) indicating organ-specific HIF regulationat the level of PHDs. Here, we show differences in HIF-α targetgene up-regulation between kidney and developing brain inresponse to HIF stabilization by FG-4497 at P7. In neonatalkidneys, FG-4497 increased mRNA levels of vasoactive (VEGF,ADM), cytotrophic and metabolic (EPO, HK2) as well astranscriptional activity modifying (PHD3) HIF target genes. Inthe brain, VEGF, ADM and EPO mRNA levels were similarlyincreased in response to FG-4497. However, there were nochanges in HK2 and PHD3 mRNA expression compared tocontrols. This difference may not be attributable to differenteffects of FG-4497 on HIF-α isoforms as HK2 is thought to beregulated primarily by HIF-1α (Hu et al., 2003, Mense et al.,2006), whereas PHD3 has been shown to be mainly under thecontrol of HIF-2α (Appelhoff et al., 2004). We speculate thatthis differential regulation of HIF target genes due to PHItreatment, similar to hypoxia- and iron chelators-inducedactivation of the HIF system (Bernaudin et al., 2002), maydepend on cell type and developmental stage. Moreover,evidence arises that prolyl-4-hydroxylase inhibition mayalter expression of hypoxia-induced regulatory factors otherthan HIF such as iron regulatory protein-2 (Hanson et al., 2003)and RNA polymerase II (Lee et al., 2002), as well as NFkB(Cummins et al., 2006) that might influence target geneexpression. Of note, we are fully aware that our results mayreflect only a selected spectrumof HIF-α target genesmodifiedby short-term PHI treatment. However, it confirms the ideathat pharmacologic HIF stabilization reveals organ-specificeffects, shown here at the level of target gene activation, thatare particularly relevant at early stage of development, e.g. interms of consequences for physiological maturational pro-cesses. Of note, besides protective effects, age-specific con-sequences of HIF stabilization on various developmentalprocesses at early stage of brain maturation (Curristin et al.,2002, Dziembowska et al., 2005) need special attention andremain to be determined by future studies.

Strong cerebral HIF-1α and HIF-2α protein accumulation aswell as transcriptional up-regulation of specific target genes inresponse to FG-4497 may suggest effective penetration of thisnovel PHI through the blood-brain barrier of developingmousebrain under normoxia. A direct comparison of blood-brainbarrier penetration of FG-4497 between adult and neonatalanimals is not available from literature. Other PHI have beenreported to be active in adult rat brain upon oral andintraperitoneal application (Siddiq et al. 2005, Witten et al.2009), but the use of different compounds, the stage of brainmaturation, and differences in experimental setting limit anycomparisons with these studies.

3.2. Cerebral HIF target gene up-regulation by PHI

HIF target genes modify basic mechanisms of cellular proli-feration and survival under normoxic and hypoxic conditionsin the brain. Todemonstrate thatHIF is transcriptionally active

48 B R A I N R E S E A R C H 1 2 8 0 ( 2 0 0 9 ) 4 3 – 5 1

in developing mouse brain after PHI treatment, we analyzedvasoactive and cytotrophic target genes that have been shownto be differentially up-regulated by systemic hypoxia indeveloping mouse brain at P0 and P7 (Bernaudin et al., 2002,Trollmann et al., 2008a,b). Present short-term observationsdemonstrate that VEGF and ADM, genes shown to be up-regulated by cerebral hypoxia (Bernaudin et al., 2002, Troll-mann et al., 2008a,b), are also up-regulated by FG-4497 indeveloping mouse brain. Our data might suggest the ability ofPHI to allow vascular and neuronal adaptation under condi-tions of low oxygen partial pressure. VEGF is involved in basicprocesses of vascular development (Ogunshola et al., 2002),neuronal and glial differentiation and survival via its Flk-1receptor and phosphorylation of downstream signaling mole-cules including MAPK, p90RSK, and STAT family members(Ogunshola et al., 2002). ADM secreted in the brain by cerebralendothelial and glial cells modifies cerebral blood flow,neuronal and glial survival, blood-brain barrier functions andvascular regeneration (Kis et al., 2001, Ladoux and Frelin, 2000,Xia et al., 2004).

Protective effects of the HIF system are attributed to manycellular trophic factors as well. Among them, EPO diminishesneuronal apoptosis by activation of anti-apoptotic proteinsbcl-2 and bcl-XL and inhibition of pro-apoptotic caspases,protects neurons from inflammatory and excitotoxic injuryand activates endogenous protective factors such as BDNF (Yuet al., 2002, Noguchi et al., 2007). In contrast to EPO mRNAregulation under conditions of systemic hypoxia (8% O2, 6 h) inneonatal mice at P7 (Trollmann et al., 2008a), FG-4497treatment led to a significant increase of cerebral EPO mRNAlevels upon 6 h. This may indicate more pronounced short-term effects of FG-4497 than systemic hypoxia (8% O2, 6 h) onHIF stabilization in developing mouse brain. Moreover, thisobservation suggests effective HIF-2α protein activation by FG-4497, since transcription of EPO is mainly regulated by HIF-2α(Chavez et al., 2006).

Extending the present knowledge on CXCR-4 signaling inthe neonatal brain, present short-term data demonstrates up-regulation of cerebral CXCR-4 expression in vivo in response topharmacologic HIF stabilization at early stage of postnatalbrain maturation in mice. This supports observations byothers on cerebral CXCR-4 expression under conditions ofhypoxia and ischemia in vitro (Frøyland et al., 2008) and in vivo(Stumm et al., 2002). CXCR-4 increased significantly in humanneuronal NT2-N cells during acidotic hypoxia and reoxygena-tion (Frøyland et al., 2008) and in human neural stem cellsafter treatment with DFX (Chu et al., 2008). In vivo, transientup-regulation of CXCR-4 was present in adult mouse corticalneurons upon focal ischemia (Stumm et al., 2002). In thiscontext, our data implicates that PHI could promote migrationand cell survival (Dziembowska et al., 2005) at early stage ofpostnatal brain maturation.

Finally, it is important to mention that the role of other HIFtarget genes that are potentially activated by PHI in the brain,including those encoding for pro-apoptotic proteins (Helton etal., 2005, Aminova et al., 2005, 2008), as well as possibleregional-specific HIF target gene activation need furtherinvestigation. However, the present data provides importantbasic information about short-term effects of a novel PHI, FG-4497, in neonatal mice to support future studies on pharma-

cologic stabilization of HIFs as a promising option forneuroprotection in hypoxic neonatal brain injury.

4. Experimental procedures

4.1. Animal experiments

All animal experiments were reviewed and performed underthe approval of the national care committee (RegierungMittelfranken, Germany) according to national and Europeanlaw. The prolyl hydroxylase inhibitor (PHI), FG-4497, wasprovided by FibroGen Co (San Francisco; USA). Tolerance ofFG-4497 at a dose of 6–100mg/kg and significant up-regulationof serum EPO levels (within 4–6 h) by FG-4497 have beenshown in adult rats and mice upon systemic (i.v., i.p.)application (Rosenberger et al. 2008, Robinson et al. 2008).The specific ability of FG-4497 to induce HIF activity has beendemonstrated in HeLa cells (Robinson et al., 2008). In ourstudy, a 6-hour treatment interval was decided according tothese published observations (Robinson et al., 2008) and, inaddition, to studies on protective effects of other PHI than FG-4497 in vivo in adult rats (Siddiq et al., 2005, Bernhardt et al.,2006). A freshly prepared solution of FG-4497 (100 mg FG-4497to 9.675 ml of 5% Dextrose to 325 μl of 1 N NaOH; according tomanufacturer's instructions) was administered intraperitone-ally (injection volume 0.1 ml). Pilot experiments on adultC57BL/6 wild-type mice under normoxia (n =3 per dose, vscontrols, n =3) treated with FG-4497 i.p. showed a dose-dependent and significant increase of serum EPO and VEGFprotein levels (10- and 2-fold increase, respectively) at a doseof 60 mg/kg compared to controls. In the brain, at this doseonly a two-fold increase of VEGF but not EPOmRNA levels waspresent. Thus, considering inconsistent cerebral target geneup-regulation, PHI dose was adjusted up to 100 mg/kg.

4.1.1. Neonatal mouse experimentsSeven-day old neonatal C57BL/6 wild-type mice (P7) weretreated with FG-4497 (n =26) at a single dose of 30 (n =8), 60(n =8) and 100 mg/kg (n =10). Controls were treated i.p. withNaCl 0.9% (n =20) or remained non-treated (n =20). At the ageof P7 stage of brain development corresponds to that ofhumans at near-term (Dobbing and Sands, 1979). Aftertreatment period of 6 h kidneys and brains were immedi-ately dissected, frozen in liquid nitrogen and stored at−80 °C until mRNA (n =5 per group) and protein extraction(n =3 per group). During treatment period adult mice wereprovided with food and water ad libitum, and pups werekept together with the dam to provide normal temperatureand nutrition.

4.2. RNA isolation and RT-PCR

Total RNA was extracted from whole brains and kidneysusing Trizol isolation method (Invitrogen, Germany) accord-ing to the manufacturer's instructions. RT-PCR was per-formed as described previously (Trollmann et al., 2008a,b).Commercial reagents (TaqMan PCR Reagent Kit, Eurogentec,Germany) and conditions have been used according to themanufacturer's protocol. The PCR reaction was performed in

49B R A I N R E S E A R C H 1 2 8 0 ( 2 0 0 9 ) 4 3 – 5 1

an ABI 7500 real-time PCR thermocycler (Applied Biosys-tems). All reactions were performed in duplicate using β-actin and porphobilinogen deaminase (PBGD) as endogenouscontrols. The following primers and TaqMan probes based onpublished reports were used:

β-actin, forward: 5′-ATGCTCCCCGGGCTGTAT-3′;reverse: 5′-TCACCCACATAGGAGTCCTTCTG-3′;TaqMan probe: 5′(FAM)-ATCACACCCTGGTGCCTAGGGCG-(TAMRA)-3′;ADM, forward: 5′-TGGACGAGCAGAACACAACTG-3′reverse: 5′-CTGGCGGTAGCGTTTGACA-3′;TaqMan probe: 5′(FAM)-CCCTACAAGCCAGCAATCAGA-GCGAA-(TAMRA)-3′CXCR-4, forward: 5′-GCTGGCTGAAAAGGCAGTCTA-3′reverse: 5′-CGTCGGCAAAGATGAAGTCA-3′;TaqMan probe: 5′(FAM)-TCTGGATCCCAGCCCTCCTCCTG-(TAMRA)-3′EPO, forward: 5′-AAGGTCCCAGACTGAGTGAAAATATTAC-3′reverse: 5′-GGACAGGCCTTGCCAAACT-3′;TaqMan probe: 5′(FAM)-TCTATGGCCTGTTCTTCCACC-TCCATTCT-(TAMRA)-3′HIF-1α, forward: 5′-AGACAGACAAAGCTCATCCAAGG-3′;reverse: 5′-GCGAAGCTATTGTCTTTGGGTTTAA-3′;TaqMan probe: 5′(FAM)-CTGCCACTTTGAATCAAAGAAAT-ACTGTTCCTGAG-(TAMRA)-3′;HK2, forward: 5′-CAACATCCTGATCGATTTCACAA-3′reverse: 5′-GCAGTCACTCTCGATCTGAGACA-3′;TaqMan probe: 5′(FAM)-CGCATCTCAGAGCGCCTCAA-GACAA-(TAMRA)-3′iNOS, forward: 5′-CAGCTGGGCTGTACAAACCTT-3′;reverse: 5′-CATTGGAAGTGAAGCGTTTCG-3′;TaqMan probe: 5′(FAM)-CGGGCAGCCTGTGAGACCTTTGA-(TAMRA)-3′.PBGD, forward: 5′-ACAAGATTCTTGATACTGCACTCTCTAAG-3′;reverse: 5′-CCTTCAGGGAGTGAACAACCA-3′;TaqMan probe: 5′(FAM)-TCTAGCTCCTTGGTAAACAGGC-TCTTCTCTCCA-(TAMRA)-3′;PHD3, forward: 5′-AGCCCATTTTTGACAGACTTCTG-3′reverse: 5′-AGCGTACCTGGTGGCATAGG-3′;TaqMan probe: 5′(FAM)-TCTGGTCAGACCGCAGGAATC-CACAT-(TAMRA)-3′SDF-1 gamma. forward: 5′-GAGCCAACGTCAAGCATCTG-3′reverse: 5′-TCTTCAGCCGTGCAACAATC-3′;TaqMan probe: 5′(FAM)-AAATCCTCAACACTCCAAACTGTG-CCCTTC-(TAMRA)-3′VEGF; forward: 5′-GCACTGGACCCTGGCTTTACT-3′;reverse: 5′-ACTTGATCACTTCATGGGACTTCTG-3′;TaqMan probe: 5′(FAM)-CCATGCCCAGTGGTCCCAGGCTG-(TAMRA)-3′.

4.3. Protein extraction and quantification

Whole brains were homogenized in lysis buffer A (buffer A:10mMHEPES, 10mMKCl, 1.5mMMgCl2, 0,5mMdithiothreitol,5 mM sodium flourid, 1% NP-40 and protease inhibitors indistilledwater). Samples were centrifuged at 4 °C for 5min, thesupernatant (cytosolic extracts) was separated and stored at−80 °C. The resulting pellet was resuspended in buffer B(buffer B: 10 mM HEPES, 10 mM KCl, 1.5 mM MgCl2, 0.5 mM

dithiothreitol, 5 mM sodium flourid, 420 mM NaCl, 25%glycerol and protease inhibitors in distilled water). Afterincubation on ice for 20 min samples were centrifuged for 5min at 4 °C. Supernatants (nuclear extracts) were kept at−80 °C. Nuclear and cytosolic protein contents were analyzedby commercial protein assay (cytosolic extracts: BCA proteinassay, Pierce Biological, Rockford, USA; nuclear extracts: Bio-Rad protein assay, Bio-Rad, Munich, Germany).

4.4. Western blot analysis

Nuclear protein (30 μg)was runon7.5%-SDS-polyacrylamidegeland transferred to a nitrocellulosemembrane. After blocking in5% nonfat milk-TBS for 90 min at room temperature mem-branes were incubated overnight at 4 °C with the polyclonalrabbit anti-HIF-1α antibody (Novus Biologicals, Littleton, CO;1:1000), or the polyclonal rabbit anti-HIF-2α antibody (NovusBiologicals, Littleton, CO; 1:500). After washing in TBS contain-ing 0.05% Tween blots were incubated with a horseradishperoxidase-conjugated secondary antibody (Cell Signaling,Danvers, USA, 1:2000) for 1 h at room temperature, followedby chemiluminescent detection. Three samples per groupwereanalyzed and blots were quantified densitometrically using theAida Image program (Version 2.0; Raytest, Straubenhardt,Germany). β-actin was used as a loading control.

4.5. ELISA

SerumVEGFandEPOconcentrations inadultmiceweremeasuredby specific ELISA (mouse VEFG Immunoassay, mouse Erythro-poietin Immunoassay; R and D Systems Europe, Abingdon, U.K.)according to the manufacturer's recommendations. The meandetection limits for VEGF and EPO were 7.8 up to 1000 pg/ml. Allsampleswere assayed induplicate. Proteinwasdeterminedby theBCA protein assay reagent (Pierce Biological, Rockford, USA).

4.6. Statistical analysis

Results are shown as mean±SEM. Statistical significance wasdetermined by One-way ANOVA (p value<0.05).

Acknowledgments

This work was supported by a grant of Deutsche Forschungs-gemeinschaft (TR 726/2-1).

R E F E R E N C E S

Aminova, L.R., Chavez, J.C., Lee, J., Ryu, H., Kung, A., Lamanna, J.C.,Ratan, R.R., 2005. Prosurvival and prodeath effects of hypoxia-inducible factor-1alpha stabilization in a murine hippocampalcell line. J. Biol. Chem. 280, 3996–4003.

Aminova, L.R., Siddiq, A., Ratan, R.R., 2008. Antioxidants, HIFprolyl hydroxylase inhibitors or short interfering RNAs toBNIP3 or PUMA, can prevent prodeath effects of thetranscriptional activator, HIF-1alpha, in a mouse hippocampalneuronal line. Antioxid. Redox Signal. 10, 1989–1998.

A ppelhoff, R.J., Tian, Y.M., Raval, R.R., Turley, H., Harris, A.L., Pugh,C.W., Ratcliffe, P.J., Gleadle, J.M., 2004. Differential function ofthe prolyl hydroxylases PHD1, PHD2, and PHD3 in the

50 B R A I N R E S E A R C H 1 2 8 0 ( 2 0 0 9 ) 4 3 – 5 1

regulation of hypoxia-inducible factor. J. Biol. Chem. 279,38458–38465.

Bergeron, M., Yu, A.Y., Solway, K.E., Semenza, G.L., Sharp, F.R.,1999. Induction of hypoxia-inducible factor-1 (HIF-1) and itstarget genes following focal ischaemia in rat brain. Eur. J.Neurosci. 11, 4159–4170.

Bergeron, M., Gidday, J.M., Yu, A.Y., Semenza, G.L., Ferriero, D.M.,Sharp, F.R., 2000. Role of hypoxia-inducible factor-1 inhypoxia-induced ischemic tolerance in neonatal rat brain.Ann. Neurol. 48, 285–296.

Bernaudin, M., Tang, Y., Reilly, M., Petit, E., Sharp, F.R., 2002. Braingenomic response following hypoxia and re-oxygenation in theneonatal rat. Identification of genes that might contribute tohypoxia-induced ischemic tolerance. J. Biol. Chem. 277,39728–39738.

Bernhardt, W.M., Campean, V., Kany, S., Juergensen, J.S.,Weidemann, A., Warnecke, C., Arend, M., Klaus, S., Guenzler,V., Amann, K., Willam, C., Wiesener, M.S., Eckardt, K.U., 2006.Preconditional activation of hypoxia-inducible factorsameliorates ischemic acute renal failure. J. Am. Soc. Nephrol.17, 1970–1978.

Bruick, R.K., McKnight, S.L., 2001. A conserved family ofprolyl-4-hydroxylases that modify HIF. Science 294, 1337–1340.

Ceradini, D.J., Kulkarni, A.R., Callaghan, M.J., Tepper, O.M.,Bastidas, N., Kleinman, M.E., Capla, J.M., Galiano, R.D., Levine,J.P., Gurtner, G.C., 2004. Progenitor cell trafficking is regulatedby hypoxic gradients through HIF-1 induction of SDF-1. Nat.Med. 10, 858–864.

Chavez, J.C., Baranova, O., Lin, J., Pichiule, P., 2006. Thetranscriptional activator hypoxia inducible factor 2(HIF-2/EPAS-1) regulates the oxygen-dependent expression oferythropoietin in cortical astrocytes. J. Neurosci. 26, 9471–9481.

C hu, K., Jung, K.H., Kim, S.J., Lee, S.T., Kim, J., Park, H.K., Song, E.C.,Kim, S.U., Kim, M., Lee, S.K., Roh, J.K., 2008. Transplantation ofhuman neural stem cells protect against ischemia in apreventive mode via hypoxia-inducible factor-1alphastabilization in the host brain. Brain Res. 1207, 182–192.

Cummins, E.P., Berra, E., Comerford, K.M., Ginouves, A., Fitzgerald,K.T., Seeballuck, F., Godson, C., Nielsen, J.E., Moynagh, P.,Pouyssegur, J., Taylor, C.T., 2006. Prolyl hydroxylase-1 nega-tively regulates IkB kinase-beta, giving insight into hypoxia-induced NFkB activity. Proc. Natl. Acad. Sci. USA 103,18154–18159.

Curristin, S.M., Cao, A., Stewart, W.B., Zhang, H., Madri, J.A.,Morrow, J.S., Ment, L.R., 2002. Disrupted synaptic developmentin the hypoxic newborn brain. Proc. Natl. Acad. Sci. USA 99,15729–15734.

D'Angelo, G., Duplan, E., Boyer, N., Vigne, P., Frelin, C., 2003.Hypoxia up-regulates prolyl hydroxylase activity: a feedbackmechanism that limits HIF-1 responses during reoxygenation.J. Biol. Chem. 278, 38183–38187.

Dobbing, J., Sands, J., 1979. Comparative aspects of the braingrowth spurt. Early Hum. Dev. 3, 79–83.

Dziembowska, M., Tham, T.N., Lau, P., Vitry, S., Lazarini, F.,Dubois-Dalcq, M., 2005. A role for CXCR4 signaling in survivaland migration of neural and oligodendrocyte precursors. Glia50, 258–269.

Fandrey, J., Gorr, T.A., Gassmann, M., 2006. Regulating cellularoxygen sensing by hydroxylation. Cardiovasc. Res. 71, 642–651.

Frøyland, E., Skjaeret, C., Wright, M.S., Dalen, M.L., Cvancarova, M.,Kasi, C., Rootwelt, T., 2008. Inflammatory receptors andpathways in human NT2-N neurons during hypoxia andreoxygenation. Impact of acidosis. Brain Res. 1217, 37–49.

Hanson, E.S., Rawlins, M.L., Leibold, E.A., 2003. Oxygen and ironregulation of iron regulatory protein 2. J. Biol. Chem. 278,40337–40342.

Helton, R., Cui, J., Scheel, J.R., Ellison, J.A., Ames, C., Gibson, C.,Blouw, B., Ouyang, L., Dragatsis, I., Zeitlin, S., Johnson, R.S.,Lipton, S.A., Barlow, C., 2005. Brain-specific knock-out of

hypoxia-inducible factor-1alpha reduces rather than increaseshypoxic–ischemic damage. J. Neurosci. 25, 4099–4107.

Hu, C.J., Wang, L.Y., Chodosh, L.A., Keith, B., Simon, M.C., 2003.Differential roles of hypoxia-inducible factor 1alpha(HIF-1alpha) and HIF-2alpha in hypoxic gene regulation. Mol.Cell. Biol. 23, 9361–9374.

Iyer, N.V., Kotch, L.E., Agani, F., Leung, S.W., Laughner, E., Wenger,R.H., Gassmann, M., Gearhart, J.D., Lawler, A.M., Yu, A.Y.,Semenza, G.L., 1998. Cellular and developmental control of O2

homeostasis by hypoxia inducible factor 1α. Genes Dev. 12,149–162.

Kis, B., Abraham, C.S., Deli, M.A., Kobayashi, H., Wada, A., Niwa,M., Yamashita, H., Ueta, Y., 2001. Adrenomedullin in thecerebral circulation. Peptides 22, 1825–1834.

Ladoux, A., Frelin, C., 2000. Coordinated up-regulation by hypoxiaof adrenomedullin and one of its putative receptors (RDC-1) incells of the rat blood-brain barrier. J. Biol. Chem. 275,39914–39919.

Lee, K.B., Wang, D., Lippard, S.J., Sharp, P.A., 2002. Transcription-coupled and DNA damage-dependent ubiquitination of RNApolymerase II in vitro. Proc. Natl. Acad. Sci. USA 99, 4239–4244.

Lee, Y.M., Jeong, C.H., Koo, S.Y., Son, M.J., Song, H.S., Bae, S.K.,Raleigh, J.A., Chung, H.Y., Yoo, M.A., Kim, K.W., 2001.Determination of hypoxic region by hypoxia marker indeveloping mouse embryos in vivo: a possible signal for vesseldevelopment. Dev. Dyn. 220, 175–186.

Ma, Q., Jones, D., Borghesani, P.R., Segal, R.A., Nagasawa, T.,Kishimoto, T., Bronson, R.T., Springer, T.A., 1998. ImpairedB-lymphopoiesis, myelopoiesis, and derailed cerebellar neuronmigration in CXCR4- and SDF-1-deficient mice. Proc. Natl.Acad. Sci. USA 95, 9448–9453.

Mense, S.M., Sengupta, A., Zhou, M., Lan, C., Bentsman, G., Volsky,D.J., Zhang, L., 2006. Gene expression profiling reveals theprofound upregulation of hypoxia-responsive genes in primaryhuman astrocytes. Physiol. Genomics 25, 435–449.

Noguchi, C.T., Asavaritikrai, P., Teng, R., Jia, Y., 2007. Role oferythropoietin in the brain. Crit. Rev. Oncol. Hematol. 64,159–171.

Ogunshola, O.O., Antic, A., Donoghue, M.J., Fan, S.Y., Kim, H.,Stewart, W.B., Madri, J.A., Ment, L.R., 2002. Paracrine andautocrine functions of neuronal vascular endothelial growthfactor (VEGF) in the central nervous system. J. Biol. Chem. 277,11410–11415.

Paredes, M.F., Li, G., Berger, O., Baraban, S.C., Pleasure, S.J., 2006.Stromal-derived factor-1 (CXCL12) regulates laminar positionof Cajal–Retzius cells in normal and dysplastic brains.J. Neurosci. 26, 9404–9412.

Pescador, N., Cuevas, Y., Naranjo, S., Alcaide, M., Villar, D.,Landazuri, M.O., Del Peso, L., 2005. Identification of a functionalhypoxia-responsive element that regulates the expression ofthe egl nine homologue 3 (egln3/phd3) gene. Biochem. J. 390,189–197.

Robinson, A., Keely, S., Karhausen, J., Gerich, M.E., Furuta, G.T.,Colgan, S.P., 2008. Mucosal protection by hypoxia-induciblefactor prolyl hydroxylase inhibition. Gastroenterology 134,145–155.

Rosenberger, C., Mandriota, S., Jurgensen, J.S., Wiesener, M.S.,Horstrup, J.H., Frei, U., Ratcliffe, P.J., Maxwell, P.H., Bachmann,S., Eckardt, K.U., 2002. Expression of hypoxia-inducible factor-1alpha and -2alpha in hypoxic and ischemic rat kidneys. J. Am.Soc. Nephrol. 13, 1721–1732.

Rosenberger, C., Rosen, S., Shina, A., Frei, U., Eckardt, K.U., Flippin,L.A., Arend, M., Klaus, S.J., Heyman, S.N., 2008. Activation ofhypoxia-inducible factors ameliorates hypoxic distal tubularinjury in the isolated perfused rat kidney. Nephrol. Dial.Transplant. 23, 3472–3478.

Schioppa, T., Uranchimeg, B., Saccani, A., Biswas, S.K., Doni, A.,Rapisarda, A., Bernasconi, S., Saccani, S., Nebuloni, M., Vago, L.,Mantovani, A., Melillo, G., Sica, A., 2003. Regulation of the

51B R A I N R E S E A R C H 1 2 8 0 ( 2 0 0 9 ) 4 3 – 5 1

chemokine receptor CXCR4 by hypoxia. J. Exp. Med. 198,1391–1402.

Siddiq, A., Ayoub, I.A., Chavez, J.C., Aminova, L., Shah, S.,LaManna, J.C., Patton, S.M., Connor, J.R., Cherny, R.A., Volitakis,I., Bush, A.I., Langsetmo, I., Seeley, T., Gunzler, V., Ratan, R.R.,2005. Hypoxia-inducible factor prolyl 4-hydroxylase inhibition.A target for neuroprotection in the central nervous system.J. Biol. Chem. 280, 41732–41743.

Staller, P., Sulitkova, J., Lisztwan, J., Moch, H., Oakeley, E.J., Krek,W., 2003. Chemokine receptor CXCR4 downregulated by vonHippel–Lindau tumour suppressor pVHL. Nature 425, 307–311.

Stroka, D.M., Burkhardt, T., Desbaillets, I., Wenger, R.H., Neil,D.A.H., Bauer, C., Gassmann, M., Candinas, D., 2001. HIF-1 isexpressed in normoxic tissues and displays an organ-specificregulation under systemic hypoxia. FASEB J. 15, 2445–2453.

Stumm, R.K., Rummel, J., Junker, V., Culmsee, C., Pfeiffer, M.,Krieglstein, J., Hollt, V., Schulz, S., 2002. A dual role for theSDF-1/CXCR4 chemokine receptor system in adult brain:isoform-selective regulation of SDF-1 expression modulatesCXCR4-dependent neuronal plasticity and cerebral leukocyterecruitment after focal ischemia. J. Neurosci. 22, 5865–5878.

Sun, Y., Jin, K., Xie, L., Childs, J., Mao, X.O., Logvinova, A.,Greenberg, D.A., 2003. VEGF-induced neuroprotection,neurogenesis, and angiogenesis after focal cerebral ischemia.J. Clin. Invest. 111, 1843–1851.

Tomita, S., Ueono, M., Sakamotoa, M., Kitahama, Y., Ueki, M.,Maekawa, N., Sakamoto, H., Gassmann, M., Kageyama, R.,Ueda, N., Gonzalez, F.J., Takahama, Y., 2003. Defective braindevelopment in mice lacking the Hif-1α gene in neural cells.Mol. Cell. Biol. 23, 6739–6749.

Trollmann, R., Schneider, J., Keller, S., Strasser, K., Wenzel, D.,Rascher, W., Ogunshola, O.O., Gassmann, M., 2008a.

HIF-1-regulated vasoactive systems are differentially involvedin acute hypoxic stress responses of the developing brain ofnewbornmice and are not affected by levetiracetam. Brain Res.1199, 27–36.

Trollmann, R., Strasser, K., Keller, S., Antoniou, X., Grenacher, B.,Ogunshola, O.O., Dötsch, J., Rascher, W., Gassmann, M., 2008b.PlacentalHIFsasmarkersof cerebralhypoxicdistress in fetalmice.Am. J. Physiol. Regul. Integr. Comp. Physiol. 295, R1973–R1981.

Wiesener, M.S., Jürgensen, J.S., Rosenberger, C., Scholze, C.K.,Hörstrup, J.H., Warnecke, C., Mandriota, S., Bechmann, I., Frei,U.A., Pugh, C.W., Ratcliffe, P.J., Bachmann, S., Maxwell, P.H.,Eckardt, K.U., 2003. Widespread hypoxia-inducible expressionof HIF-2alpha in distinct cell populations of different organs.FASEB J. 17, 271–273.

Willam, C., Maxwell, P.H., Nichols, L., Lygate, C., Tian, Y.M.,Bernhardt, W., Wiesener, M., Ratcliffe, P.J., Eckardt, K.U., Pugh,C.W., 2006. HIF prolyl hydroxylases in the rat; organdistribution and changes in expression following hypoxia andcoronary artery ligation. J. Mol. Cell Cardiol. 41, 68–77.

Witten, L., Sager, T., Thirstrup, K., Johansen, J.L., Larsen, D.B.,Montezinho, L.P., Mørk, A., 2009. HIF prolyl hydroxylaseinhibition augments dopamine release in the rat brain in vivo.J. Neurosci. Res. 87, 1686–1694.

Xia, C.F., Yin, H., Borlongan, C.V., Chao, J., Chao, L., 2004.Adrenomedullin gene delivery protects against cerebralischemic injury by promoting astrocytemigration and survival.Hum. Gene Ther. 15, 1243–1254.

Yu, X., Shacka, J.J., Eells, J.B., Suarez-Quian, C., Przygodzki, R.M.,Beleslin-Cokic, B., Lin, C.S., Nikodem, V.M., Hempstead, B.,Flanders, K.C., Costantini, F., Noguchi, C.T., 2002.Erythropoietin receptor signalling is required for normal braindevelopment. Development 129, 505–516.