Loss of HIF-2α and inhibition of VEGF impair fetal lung maturation, whereas treatment with VEGF prevents fatal respiratory distress in premature mice

702 NATURE MEDICINE • VOLUME 8 • NUMBER 7 • JULY 2002

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Preterm delivery is the chief problem in obstetrics, affecting 10%of all births and accounting for more than 70% of perinatal mor-tality1. 60% of the infants born at less than 32 weeks of gestationand weighing less than 1,000 g develop respiratory distress syn-drome (RDS) with a mortality of 50% (ref. 2). RDS results frominsufficient production of surfactant by immature alveolar type2 pneumocytes. Surfactant is a mixture of phospholipids andsurfactant-associated proteins (SP-A to SP-D), which lowers sur-face tension at the air-water interface and prevents alveolar col-lapse. Surfactant phospholipids are synthesized from metabolicsubstrates, provided by glycogen stores in fetal immature pneu-mocytes3. Neonatal intensive care and treatment with oxygenand steroids have improved the survival of infants with RDS, butoften at the expense of the development of bronchopulmonarydysplasia or chronic lung disease of prematurity and other side-effects4. The pathogenesis of RDS remains incompletely under-stood.

Interactions between airways and blood vessels are critical fornormal lung development5. A major factor in lung vascular de-velopment is vascular endothelial growth factor (VEGF), whichbinds the receptors Flk-1 (also known as VEGF receptor-2) andFlt-1 (also known as VEGF receptor-1)6. There are three VEGF iso-forms: a diffusable VEGF120, a matrix-bound VEGF188 and

VEGF164, which can bind matrix and is also diffusable. VEGF isdeposited at the leading edge of branching airways, where itstimulates vascularization7. However, indirect evidence suggeststhat VEGF also affects epithelial growth and differentiation.Type 2 pneumocytes and bronchiolar epithelial cells produceVEGF and express VEGF receptors8,9. VEGF levels are also consid-erably higher in the bronchoalveolar fluid than in the blood9,suggesting that epithelial cells affect their own function by re-leasing VEGF into the airway lumen. Furthermore, VEGF levelsin tracheal aspirate were lower in infants with lung immaturitydeveloping bronchopulmonary dysplasia than in those surviv-ing without pulmonary complications in some10–12 but not inother studies13. Exogenous VEGF stimulates growth of lung epithelial cells in vitro14, but the relevance of endogenous VEGFfor lung maturation in vivo and the possible therapeutic poten-tial of VEGF in preventing RDS in preterm infants remain un-known. Hypoxia upregulates VEGF gene transcription byactivating the hypoxia-inducible transcription factors HIF-1αand HIF-2α (refs. 15–17), which bind the hypoxia-response ele-ment in the VEGF promotor. HIF-2α is expressed in fetal type 2pneumocytes16, but its relevance for RDS remains unknown. Instudying the role of HIF-2α and VEGF in fetal-lung maturation,here we reveal a potential use of VEGF for treatment of RDS.

Loss of HIF-2α and inhibition of VEGF impair fetal lungmaturation, whereas treatment with VEGF prevents fatal

respiratory distress in premature mice

VEERLE COMPERNOLLE1, KOEN BRUSSELMANS1, TILL ACKER2, PETER HOET3, MARC TJWA1, HEIKE BECK2, STÉPHANE PLAISANCE1, YUVAL DOR4, ELI KESHET4, FLOREA LUPU5,

BENOIT NEMERY3, MIEKE DEWERCHIN1, PAUL VAN VELDHOVEN6, KARL PLATE2, LIEVE MOONS1,DÉSIRÉ COLLEN1 & PETER CARMELIET1

1The Center for Transgene Technology and Gene Therapy, Flanders InteruniversityInstitute for Biotechnology, Leuven, Belgium

2Neurological Institute, JWG Frankfurt University, Frankfurt, Germany3Laboratory of Pneumology, Unit of Lung Toxicology, KU Leuven, Leuven, Belgium

4Department of Molecular Biology, Hebrew University-Hadassah Medical School, Jerusalem, Israel5Cardiovascular Biology Research Program, Oklahoma Medical Research Foundation, Oklahoma City, Oklahoma, USA

6Department of Pharmacology, KU Leuven, Leuven, BelgiumCorrespondence should be addressed to P.C.; email: [email protected]

Published online: 10 June 2002, doi:10.1038/nm721

Respiratory distress syndrome (RDS) due to insufficient production of surfactant is a commonand severe complication of preterm delivery. Here, we report that loss of the hypoxia-inducibletranscription factor-2α (HIF-2α) caused fatal RDS in neonatal mice due to insufficient surfactantproduction by alveolar type 2 cells. VEGF, a target of HIF-2α, regulates fetal lung maturation: be-cause VEGF levels in alveolar cells were reduced in HIF-2α-deficient fetuses; mice with a defi-ciency of the VEGF164 and VEGF188 isoforms or of the HIF-binding site in the VEGF promotor diedof RDS; intrauterine delivery of anti-VEGF-receptor-2 antibodies caused RDS and VEGF stimu-lated production of surfactant proteins by cultured type 2 pneumocytes. Intrauterine delivery orpostnatal intratracheal instillation of VEGF stimulated conversion of glycogen to surfactant andprotected preterm mice against RDS. The pneumotrophic effect of VEGF may have therapeuticpotential for lung maturation in preterm infants.

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NATURE MEDICINE • VOLUME 8 • NUMBER 7 • JULY 2002 703

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HIF-2α–/– neonates succumb to RDSDeficiency of HIF-2α was accomplished by deletion of the sec-ond exon of the gene, which encodes the DNA-binding do-main18. HIF-2α–/– embryos represented approximately 25% of thelittermates until embryonic day (E) 13.5, when half of the em-bryos died of cardiac failure. At birth, wild-type (WT) neonatesbreathed regularly, were well oxygenated and actively movedtheir limbs, whereas HIF-2α–/– neonates breathed irregularly withgasping and signs of retraction, had a cyanotic skin color andsuccumbed within 2 to 3 hours in severe respiratory failure dueto extensive lung collapse (Fig. 1a–c). In WT newborns, lung aer-ation (percentage of the total surface filled with air) doubledafter birth and almost achieved adult levels (68 ± 2%), but failedto increase at all in HIF-2α–/– newborns (Table 1). RDS was not at-tributable to growth retardation, respiratory muscle dysfunc-

tion, lung hypoplasia, impaired fluid clearance, hypoxic stress orother organ defects.

Defective surfactant production in HIF-2α–/– miceLoss of HIF-2α did not affect lung development during thepseudoglandular or canalicular phase. In the saccular phase (E17.5through postnatal day (P) 0), both genotypes had a comparabledensity of terminal sacs, septa per terminal sac, amount of elastinper alveolar septa and airspace. However, thinning of the alveolarsepta at birth, a prerequisite for blood-gas exchange, was impairedin HIF-2α–/– mice (Fig. 1d and e; Table 1). This was not attributableto abnormal epithelial proliferation or apoptosis, but to impaireddifferentiation. Immature epithelial cells contain abundant PAS+

glycogen stores, which they convert to surfactant phospholipids.Beyond E18.5, PAS+ cells disappeared in WT mice but persisted in

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k lFig. 1 Impaired lung maturation and RDS in HIF-2α–/– mice. a, Skin oxy-genation is normal in WT (upper) neonates but cyanotic in HIF-2α–/– (lower)littermates. b and c, Normal inflation of WT lungs (b), but lung collapse inHIF-2α–/– littermates (c). d and e, PAS+ (glycogen-rich) cells are minimal inWT neonates (d), but abundant (arrowhead in inset) in HIF-2α–/– lungs (e). f, PAS+ cells progressively disappear in WT (�), but not in mutant lungs (�).*, P < 0.05 versus WT. n = 3–5. g and h, Semi-thin lung sections (toluidineblue), revealing thinning of the alveolar septa in WT (g) but not in mutant(h) neonates. i and j, SP-D+ alveolar type 2 cells are more numerous (arrow)in WT (i) than in HIF-2α–/– (j) neonates. k and l, Transmission electromicro-graphs revealing an alveolar type 2 cell containing surfactant lamellar bod-ies (k; arrow) in WT lungs. In HIF-2α–/– lungs (l), lamellar bodies (arrow)

persist in the alveolar lumen (‘L’, lined with dashed line) and fail to formmyelin structures and a surfactant layer. Scale bars, 100 µm (b and c), 25 µm (d and e), 20 µm (g–j).

Table 1 Pulmonary maturity evidenced by the degree of aeration and thickness of alveolar septa

E18.5 Day of birth

WT HIF-2α–/– WT HIF-2α–/– VEGF120/120 VEGF164/164 VEGF188/188 VEGF∂/∂ PlGF–/–

Aerated lung 34 ± 6 32 ± 6 57 ± 1 30 ± 2* 23 ± 4* 58 ± 4 55 ± 2 45 ± 2* 50 ± 4area (% of total)

Alveolar septal 13 ± 1 15 ± 1* 8 ± 0.1 14 ± 0.4* 13 ± 0.7* 8 ± 0.5 8 ± 0.5 14 ± 0.6* 8 ± 0.8 thickness (µm)

Values represent the mean ± s.e.m. of measurements in 4–5 mice. *, P < 0.05 versus WT by t-test.

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Fig. 2 Pulmonary vascular development in HIF-2α–/– mice. a and b,Thrombomodulin staining, revealing a double layer of capillaries, alignedin the immediate vicinity of the alveolar lumen (‘L’) in WT mice (arrows ina), whereas capillaries in ∼ 30% of the alveoli lay more distant from thealveolar lumen (‘L’) in HIF-2α–/– mice (arrows in b) at E18.5. c–e, Schematicillustration of capillary remodeling in alveolar septa. At E17.5, capillaries lierandomly scattered in the thick alveolar septum (c). At E19.0, the alveolarseptum becomes thinner, and capillaries lie in a double layer aligned withand immediately juxtaposed to the alveolar lumen (d). On ventilation atbirth, further thinning of the septum results from inflation-inducedstretching so that the alveolus expands and capillaries are aligned in a sin-gle layer (e). f and g, Laminin staining, revealing normal basement mem-brane formation of microvessels in alveolar septa in WT (f) and HIF-2α–/– (g)fetuses at E18.5. Alveolar lumen is marked with L. h and i, Smooth muscle

HIF-2α–/– mice (Fig. 1d–f). Semi-thin sections confirmed the abun-dance of immature pneumocytes in HIF-2α–/– mice (Fig. 1g and h).HIF-2α–/– mice produced less surfactant phospholipids (nmol/lungphosphatidylcholine and phospholipids, 190 ± 22 and 980 ± 38 inWT versus 110 ± 1 and 680 ± 45 in HIF-2α–/– lungs, n = 5, P < 0.05;phosphatidylcholine:sphingomyelin ratio, 3.5 ± 0.2 in WT versus2.8 ± 0.2 in HIF-2α–/– lungs; n = 5, P < 0.05) HIF-2α–/– mice also pro-duced less SP-A, SP-B and SP-D (copies per 100 copies β-actin forSP-A, SP-B and SP-D: 330 ± 14, 160 ± 11 and 5 ± 0.4 in WT versus230 ± 34, 110 ± 40 and 3 ± 1 in HIF-2α–/– lungs, respectively, n = 6,P < 0.05). They also had fewer SP-B and SP-D positive type 2 pneu-mocytes (positive cells per mm alveolus for SP-B and SP-D, 15 ± 1.3and 8 ± 1.3 in WT versus 10 ± 0.75 and 3 ± 0.75 in HIF-2α–/–, n = 5,P < 0.05, Fig. 1i and j). In contrast to the presence of surfactantlamellar bodies inside WT type 2 pneumocytes, abundant alveolarsecretions of abnormal lamellar surfactant structures were oftenpresent in HIF-2α–/– mice (Fig. 1k and l), as occurs in mice lackingSP-D (ref. 19). Alveolar epithelial defects were specific, as similarnumbers of PGP9.5+ neuroepithelial cell bodies and CC10+ Claracells were present in both genotypes.

Pulmonary angiogenesis in HIF-2α–/– miceVascular development during the pseudoglandular and canalic-ular stages was normal in both genotypes, presumably becausepulmonary VEGF levels were comparable. However, one day be-

fore birth, a subtle deficit in vascularization of alveolar septa wasdetected in HIF-2α–/– mice (vessels per alveolus, 17 ± 1 in WT ver-sus 13 ± 1 in HIF-2α–/–, n = 5, P < 0.05) (Fig. 2a and b). Onset ofthese vascular defects in HIF-2α–/– mice coincided with the timewhen expression of HIF-2α and VEGF in alveolar epithelial cellswas upregulated in WT but not in HIF-2α–/– fetuses. In addition,alveolar capillaries failed to remodel properly in HIF-2α–/– micebefore birth. At E17.5, capillaries lie scattered amidst thick septa(Fig. 2c); however, during subsequent maturation at E19.0, capil-laries become aligned in two layers, juxtaposed to the alveolarlumen (Fig. 2d). Upon ventilation after birth, alveolar expansionfurther stretches the septa, so that capillaries are aligned in a sin-gle layer and gas exchange is facilitated (Fig. 2e). In WT mice,capillaries lied juxtaposed to the lumen in 95 ± 1% of terminalsacs, whereas in HIF-2α–/– neonates, capillaries were separatedfrom the lumen in 31 ± 5% of terminal sacs (n = 5, P < 0.05) (Fig.2a and b). However, alveolar vessels in HIF-2α–/– fetuses were notleaky and had normal basement membranes (Fig. 2f and g).Muscularization of peripheral vessels (Fig. 2h and i) and branch-ing of large vessels following the bronchiolar structures (Fig. 2jand k) were also normal.

Expression of HIF-2α during pulmonary maturationHIF-2α transcript levels were comparable in the heart, lungs andkidneys in WT fetuses at E16.5 (Fig. 3a). Thereafter, expression of

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α-actin staining, revealing normal muscularization of large pulmonary ves-sels in WT (h) and HIF-2α–/– (i) mice at E18.5. j and k, Angiogram, revealingnormal branching of large pulmonary vessels in WT (j) and HIF-2α–/– (k) fe-tuses at E18.5. Scale bars, 10 µm (a, b), 20 µm (f and g), 50 µm (h and i).

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Fig. 3 Pulmonary expression of HIF-2α, VEGFand its receptors. a, HIF-2α mRNA levels in-creased more than 5-fold in WT lungs and lessthan 2-fold in hearts and kidneys during thefinal stage of fetal development. E16.5, �;E17.5, �; E18.5, � and P0, . *, P < 0.05versus E16.5 b, VEGF protein levels in lung ex-tracts increased beyond E16.5 in WT mice (�)but not in HIF-2α–/– mice (�). *, P < 0.05 versusWT. Panels c-k: sections from E18.5 WT lungs;the nucleus is marked with an asterisk; ‘L’ de-notes alveolar lumen and arrowheads in c–idenote alveolar type 2 cells. c and d, HIF-2α-immunostaining (c) and nuclear DAPI staining(d) on adjacent sections, revealing nuclear lo-calization of HIF-2-α in alveolar cells. e, HIF-1α-immunostaining, revealing expression ofHIF-1α in bronchiolar (arrows) but not in alve-olar cells. f, Double-immunostaining, revealingco-expression of cytosolic alkaline phosphatase(AP; orange) and nuclear HIF-2α (green) intype 2 pneumocytes. g, Double-immunostain-ing, revealing co-expression in type 2 pneu-mocytes of AP (red) and Flk-1 (blue), resultingin a pink colorization. Flk-1 was also detectablein other cells in the alveolar septa, presumably in endothelial cells. h, Double-immunostaining, revealing that expression of HIF-1α (green) isundetectable in AP+ (red) type 2 pneumocytes. i, Immunostaining for pimonidazole, revealing hypoxic type 2 alveolar pneumocytes. j and k, VEGF expression in type 2 cells (arrow, in situ hybridization; j) and

HIF-2α increased more than five-fold in the lungs, but less thantwo-fold in the heart and kidneys (Fig. 3a). By immunostain-ing, HIF-2α was abundantly expressed in the nucleus of alveo-lar pneumocytes (Fig. 3c and d), whereas HIF-1α was detectablein bronchiolar but not in alveolar epithelium (Fig. 3e), consis-tent with previous findings16,20. Double-immunostaining forHIF and alkaline phosphatase (AP, a marker of type 2 pneumo-cytes) confirmed that type 2 alveolar pneumocytes expressedHIF-2α (Fig. 3f) but not HIF-1α (Fig. 3h). Other cells in alveolarsepta, presumably endothelial and mesenchymal cells, also ex-

pressed HIF-2α (Fig. 3c and f). Alveolar type 2 cells stained pos-itively for the hypoxia-marker pimonidazole hydrochloride(Fig. 3i), suggesting that hypoxia might have triggered activa-tion of HIF-2α in these cells (as also shown by its nuclear local-ization).

Role of VEGF in alveolar epithelial maturationBecause VEGF is a downstream target of HIF-2α and has been im-plicated in neonatal lung disease10–12, we analyzed a number ofpreviously generated VEGF mutant mouse strains. A fraction of

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abundant epithelial localization of the protein at the alveolar side (arrow,immunostaining; k). l and m, Immunostaining for Flt-1 (l) and CD31 (m)at E17.5, revealing a similar expression pattern, suggesting that Flt-1 isprimarily expressed on endothelial cells (arrow). Scale bars, 10 µm(c,d,f,g,i,j,l and m); 20 µm (e and h), 5 µm (k).

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VEGF120/120 mice (selectively expressing VEGF120)21 died of RDSand had abundant PAS+ pneumocytes (cells per mm, 2 ± 1 in WTneonates versus 51 ± 20 in VEGF120/120 littermates, n = 3–5, P <0.05) (Table 1). In contrast, pulmonary development was normalin VEGF164/164 or VEGF188/188 mice (expressing exclusively VEGF164

or VEGF188, respectively22), or in placental growth factor (PlGF)–/–

mice lacking the VEGF homolog PlGF (Table 1)23. A small frac-tion of VEGF∂/∂ neonates that died at birth and lacked the hy-poxia-responsive HIF-binding site in their VEGF promotor alsosuffered lung prematurity (Table 1)24. Thus, absence of criticalVEGF isoforms, or impaired HIF-dependent VEGF regulationcaused RDS.

Pulmonary VEGF protein levels were comparable in bothgenotypes at E16.5 (Fig. 3b). During the final stage of fetal devel-opment, VEGF levels increased approximately four-fold in WTmice, but only minimally in HIF-2α–/– mice (Fig. 3b). Comparedwith WT neonates, pulmonary transcript levels of VEGF120,VEGF164 and VEGF188 were reduced by 31%, 39% and 39%, re-spectively, in HIF-2α–/– mice. By in situ hybridization, VEGF wasdetectable in type 2 pneumocytes but not in alveolar blood ves-sels (Fig. 3j). The co-expression of HIF-2α and VEGF in pneumo-cytes, but not in blood vessels, suggests that HIF-2α regulatesVEGF expression primarily in pneumocytes. Abundant VEGFprotein was detectable on the apical surface of type 2 cells (Fig.3k) and in the broncho-alveolar lavage fluid of WT neonates(120 ± 49 pg/mg protein), indicating that VEGF was secreted inthe alveolar lumen. No genotypic differences were detected inpulmonary expression of PlGF. Double labeling revealed thatFlk-1 was present in septal microvessels, but also in AP+ type 2pneumocytes (Fig. 3g). By triple labeling, type 2 pneumocytesexpressed both Flk-1 and HIF-2α (data not shown). Flt-1 was de-tectable in capillaries and colocalized with the endothelialmarker CD31 on adjacent sections (Fig. 3l and m), whereas theVEGF165-isoform selective receptor neuropilin-1 was unde-tectable (data not shown).

Notably, freshly isolated type 2 pneumocytes also expressedFlk-1 transcripts (data not shown) and responded to VEGF by in-creasing their expression of SP-B and SP-C (copies per 100 copiesβ-actin for SP-B and SP-C, 15 ± 1 and 12 ± 1 after saline versus 27± 4 and 21 ± 4 after VEGF, n = 4–6, P < 0.05). Thus, surfactant-producing alveolar type 2 cells produce VEGF in a HIF-2α-depen-dent manner and are responsive to VEGF.

Mouse models of lung prematurity and RDSTo evaluate the role of VEGF in lung maturation in an intact an-imal in vivo, a mouse model of lung prematurity was established.After cesarean section (C-section) at E17.5, approximately 90%of 113 premature newborns exhibited severe signs of lung imma-turity and succumbed due to respiratory failure within 10 hoursafter delivery; this model was used to evaluate the effect of intra-tracheal instillation of VEGF. After C-section of preterm fetusesat E18.5, more than 90% of 30 neonates suffered RDS during thefirst hours, but generally survived thereafter. The clinical condi-tion of preterm pups was monitored within the first 20 minutesafter delivery by determining an ‘activity pulse grimace appear-ance respiration-like’ (APGAR) score on a scale from 0 to 10 (seeMethods). In general, saline-treated pups had an APGAR score of5 ± 0.3 at 5 and 10 minutes after birth (n = 10). This model wasused to evaluate the effect of intra-amniotic injection, at E17.5,of VEGF or of antibodies against VEGF receptor. These com-pounds reliably reached the airways after intra-amniotic admin-istration from E17.5 onwards.

Inhibition of Flk-1 impairs fetal lung maturationTo analyze which VEGF receptor mediated lung maturation,neutralizing anti-Flk-1 or anti-Flt-1 antibodies23,24 were intra-am-niotically injected in WT fetuses at E17.5, and pups were deliv-ered by C-section at E18.5. Anti-Flt-1 antibodies were ineffective,but anti-Flk-1 antibodies prevented the thinning of the alveolarsepta and the disappearance of PAS+ cells (Table 2). By immuno-staining, intra-amniotically injected antibodies remained re-stricted to the alveolar compartment, suggesting that theobserved effects on lung maturation were due to inhibition ofalveolar VEGF. Taken together, Flk-1, not Flt-1, mediates the ef-fect of endogenous VEGF on lung maturation in vivo.

Intra-amniotic VEGF administration prevents RDSAfter intra-amniotic VEGF delivery, the APGAR score improved to7.5 ± 0.7 and 8 ± 0.4 after 5 and 10 minutes, respectively (n = 8),which is significantly better than after saline (P < 0.005). In con-trast to the 24 saline-treated pups, of which 75% remained com-pletely immobile after 20 minutes, 60% of the 24 VEGF-treatedpups breathed spontaneously and regularly, had a pink skin colorafter 10 minutes, and actively moved their limbs after 20 minutes(P < 0.02). As a result of the improved aeration after VEGF (Table

Table 2 Treatment with VEGF improves lung maturation in preterm WT mice

Intra-amniotic injection at E17.5 → preterm delivery at E18.5 Intratracheal injectionafter delivery at E17.5

Saline VEGF PlGF IgG Anti-Flt-1 Anti-Flk-1 Saline VEGF

Aerated lung 39 ± 2 58 ± 1* 41 ± 1 42 ± 2 41 ± 1 37 ± 4 44 ± 2 52 ± 3*area (% of total)

Alveolar septal 12 ± 0.4 8 ± 0.1* 13 ± 0.1 13 ± 0.2 13 ± 0.1 15 ± 0.5* 18 ± 1 15 ± 1* thickness (µm)

PAS+ cells/ 100 38 ± 6* 78 ± 6 100 118 ± 13 208 ± 18* 100 55 ± 7*mm alveolus(% of control)

Number of blood 17 ± 0.7 17 ± 0.2 17 ± 0.9 16 ± 0.5 16 ± 1.0 16 ± 0.5 13 ± 0.4 14 ± 0.3vessel/alveolus

Values represent the mean ± s.e.m. of measurements in 5–10 mice. *, P < 0.05 versus littermates treated with saline or IgG by t-test. For intra-amniotic injection, fetuses were in-jected at E17.5 in utero, and premature pups were then delivered by C-section at E18.5. For intratracheal injection, premature E17.5 fetuses were delivered by C-section.

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2), inflated VEGF-treated lungs floated, while atelectatic controllungs sank to the bottom in a water-filled recipient (Fig. 4d). AfterVEGF treatment, alveolar septa were thinner, PAS+ glycogen storeswere mobilized (Table 2; Fig. 4a and b) and surfactant productionwas increased (phosphatidylcholine per lung, 180 ± 14 nmol incontrol versus 220 ± 10 nmol after VEGF, n = 5, P < 0.05). The ther-apeutic effect of VEGF was specific, as intra-amniotic injection ofPlGF, a specific ligand of Flt-1 but not Flk-1, was ineffective (Fig.4c; Table 2). Notably, VEGF was comparably effective with the glu-cocorticoid dexamethasone (0.8 mg/kg), administered to preg-nant mice at gestational day 15.5 and 16.5. Dexamethasoneimproved the APGAR score to 6.7 ± 0.7 and 8.4 ± 0.4 at 5 and 10minutes, respectively (n = 71; P = n.s. versus VEGF; P < 0.005 ver-sus saline for 5 and 10 min, respectively) and stimulated lung aer-

ation (57 ± 3% after dexamethasone versus 58 ± 1% after VEGF; n = 3–9; P = n.s.; as compared with 39 ± 2% after saline) (Table 2).VEGF was slightly more efficient in thinning of the septa thandexamethasone (10 ± 0.1 µm after dexamethasone versus 8 ± 0.1µm after VEGF, n = 3–9, P < 0.05). VEGF and glucocorticoids mayinteract in pulmonary maturation, as pulmonary VEGF levelswere increased by a low dose, but suppressed by a high dose ofdexamethasone (pg/mg protein at E18.5, 270 ± 12 after saline ver-sus 330 ± 9 and 210 ± 18 after 0.8 or 2.4 mg/kg dexamethasone, re-spectively, n = 5–11, P < 0.05 versus saline). Thus, intrauterineVEGF improved fetal lung maturation and prevented RDS.

VEGF administration improves lung function and survivalAfter C-section at E17.5, approximately 60% of preterm pups

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Fig. 4 VEGF treatment improves lung maturation and protects against res-piratory distress. a–c, Intra-amniotic treatment of WT E17.5 fetuses withVEGF (b), but not with saline (a) or PlGF (c), reduced the number of PAS+

pneumocytes in fetal lungs. d, Lungs from VEGF-treated fetuses were in-flated and floated (right), while lungs from saline-treated fetuses sank to thebottom (left). e and f, Thrombomodulin-staining, revealing a similar num-ber of alveolar capillaries after intra-amniotic VEGF-treatment (f) of WT atE17.5 as compared with saline-treatment (e). g, Western blotting of IgG inplasma and perfused lung extracts, illustrating that intra-amniotic VEGF

treatment did not increase extravasation of plasma IgG into the lungparenchyma. h, Premature pups survived longer after intratracheal treat-ment with VEGF (�) than with saline (�) (P < 0.05 versus saline starting from6 hours). i, Model illustrating the proposed pneumotropic effect of HIF-2αand VEGF on fetal lung maturation: PAS+ glycogen in immature pneumo-cytes is converted to glucose, and used as substrate for synthesis of surfac-tant phospholipids. HIF-2α and VEGF stimulate this conversion of glycogento surfactant and thereby improve fetal lung maturation and protect againstRDS. Scale bars, 20 µm (a–c), 10 µm (e–f).

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had an aerated lung area of less than 25% and died immediatelyafter birth (category A), whereas another approximately 10% ofpreterm pups ventilated well and were normally oxygenated(category B). The remaining approximately 30% of preterm pupshad an aerated lung area of 39 ± 2% and suffered severe RDS.These pups were able to live for at least six hours, although theyultimately succumbed to fatal exhaustion (category C). Onlymice of class C were intratracheally injected with VEGF (500ng/pup). To allocate pups to categories A, B and C, newbornswere monitored for 30 minutes after C-section, when their lungfunction and clinical condition was easily scored. IntratrachealVEGF administration prevented RDS in preterm pups of categoryC. Within 4–6 hours after VEGF administration, breathing be-came easier and more regular, skin color turned pink and pupsmoved more actively. As a result, a third of the VEGF-treatedpups (n = 22), but none of the controls (n = 14), survived for upto 20 hours when they were killed (P < 0.05) (Fig. 4h). The aver-age survival time was significantly longer after VEGF treatmentthan in control mice (8.5 ± 1.3 h after saline versus 13 ± 1.3 hafter VEGF, n = 14–22, P < 0.05). Histological analysis after sixhours revealed that VEGF-treatment improved lung aeration, ac-celerated alveolar septal thinning and stimulated conversion ofglycogen stores, as evidenced by the disappearance of PAS+ cells(Table 2). No differences were found in the number of cells ex-pressing SP-B (positive cells per mm alveolus, 2.6 ± 0.8 aftersaline versus 3.5 ± 0.9 after VEGF, n = 4, P = n.s.). Consideringthat intratracheally delivered VEGF could only reach ventilatedlung areas (∼ 50% of the lung), and taking the short duration ofVEGF exposure (6 h) and the young fetal age (E17.5) into consid-eration, the observed improvement of the clinical condition andlung maturation is remarkable.

Safety of pulmonary VEGF treatmentWhen 1 µg hVEGF was intratracheally administered (resultingin an estimated concentration of 10 µg/ml alveolar fluid), lessthan 0.1% of the hVEGF was recovered in the fetal plasma after1 hour (500 ± 60 pg/ml hVEGF as compared with 50 ± 4 pg/mlmurine VEGF in plasma of uninjected pups). After 3 and 5hours, hVEGF plasma levels were undetectable (<2 pg/ml), con-firming previous findings that VEGF remains restricted to thealveolar compartment with minor spill-over to the interstitiumand circulation9. Similar findings were obtained after intra-am-niotic injection of hVEGF. Neither intra-amniotic nor intratra-cheal VEGF stimulated angiogenesis in alveolar septa (Table 2;Fig.4e and f), vascular leakage (amount of extravasated im-munoglobulin G (IgG) in perfused lungs, analyzed by westernblotting) (Fig. 4g) or bronchial edema. There were also no mi-croscopic abnormalities, leakiness or neovascular growth in thegastrointestinal tract, placenta or fetal membrane after intra-amniotic delivery.

DiscussionHere we show that HIF-2α and its downstream target VEGF arecritical for fetal lung maturation. Loss of HIF-2α, absence of crit-ical VEGF isoforms or inhibition of VEGF in utero impaired lungmaturation and caused RDS at birth due to insufficient surfac-tant production. When administered intra-amniotically to un-born fetuses or intratracheally after birth, VEGF increasedconversion of glycogen stores to surfactant, improved lung func-tion, protected severely preterm mice against RDS and pro-longed their survival, with a comparable efficiency as prenatalsteroid treatment but without acute adverse effects.

Our findings indicate that HIF-2α has a critical role in control-ling the conversion of glycogen to surfactant. Glycogenolysis, thefirst step in this pathway, is critical for lung maturation, as defec-tive glycogen breakdown causes insufficient surfactant produc-tion in rats with a deficiency of glycogen phosphorylase-B kinase25

and in fetuses from diabetic mothers26. Moreover, glucocorticoidsstimulate surfactant production, in part by increasing glycogeno-lysis27. Hypoxic activation of HIF-2α has a role in this process,given that hypoxia is known to enhance glycogenolysis28, fetaltype 2 cells were hypoxic, upregulation of HIF-2α in type 2 pneu-mocytes coincided with the onset of surfactant production, andHIF-binding sites are present in several genes implicated in glu-cose metabolism29. HIF-2α could also upregulate the expression ofgenes encoding surfactant-associated proteins or of genes in-volved in the conversion of glycogen to surfactant phospholipids(including VEGF). Although we did not detect HIF-1α by im-munostaining in hypoxic fetal alveolar cells, the protein has beenimmunolocalized in adult alveolar cells under extreme hypoxicconditions30, raising the question whether HIF-1α might con-tribute to fetal lung maturation under more severe conditions.

We also uncovered a novel role of VEGF in lung maturationand surfactant production in vivo. Indeed, pulmonary VEGF lev-els were reduced in HIF-2α–/– neonates with RDS, loss of the longVEGF-isoforms, impaired HIF-dependent upregulation of VEGF,and intrauterine delivery of anti-Flk-1 antibodies caused lungprematurity. HIF-2α developmentally upregulated VEGF expres-sion in alveolar pneumocytes, probably by binding the hypoxia-response element in the VEGF promoter31. VEGF seems to affectalveolar type 2 pneumocytes directly, as these cells expressedFlk-1 and synthesized more SP-B and SP-C in response to VEGF,and anti-Flk-1 antibodies caused RDS without crossing the epithelial barrier. VEGF enhanced surfactant synthesis and im-proved lung function in vivo rapidly, for example within 4–6hours, suggesting that its beneficial effect may not require exten-sive epithelial differentiation but could rely on switching on themetabolic conversion of glycogen to surfactant and/or on surfac-tant release. Although the precise mechanism remains to be determined, VEGF is known to upregulate synthesis of platelet-activating factor32, a potent inducer of glycogenolysis in fetallung33 and to activate protein kinase C (ref. 6), a central regulatorof surfactant secretion34 and glycogen metabolism35.

Loss of HIF-2α was reported to cause reduced catecholam-ine levels36 and vascular remodeling defects in embryos37.Catecholamine production was indeed lower in HIF-2α–/–

neonates, but the RDS was not rescued by treatment of pregnantHIF-2α+/– females with D,L-threo-3,4-dihydrophenylserine (DOPS;a substrate that is converted to noradrenaline) (data not shown),indicating that RDS was not attributable to insufficient cate-cholamine production. We did not detect any defects in pul-monary vascular development in HIF-2α–/– fetuses until the lastphase of fetal-lung maturation, precisely when HIF-2α and VEGFexpression were upregulated in WT but not in HIF-2α–/– lungs.Thus, early pulmonary vascular development (including branch-ing and muscularization of proximal lung vessels and initial for-mation of distal alveolar capillaries) proceeded normally as longas VEGF was not upregulated by the elevated HIF-2α levels.Another reason why pulmonary vascular defects were subtle mayrelate to the finding that HIF-2α seems to be more important forupregulation of VEGF in alveolar epithelial than in vascular cells(as suggested by our co-expression studies). Pulmonary vasculardefects were more severe in VEGF120/120 neonates (data notshown), which suggests a critical role of the longer and most

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abundant VEGF164 and VEGF188 isoforms in the lung38,39. As theselonger isoforms bind to the subepithelial heparin sulfate-rich ex-tracellular matrix at the branching tips of airways in the distallung, matrix-associated VEGF may provide critical spatial guid-ance cues for growing vessels and link airway branching withblood vessel formation7. Although these vascular defects couldcontribute to impaired lung maturation in HIF-2α–/– mice, thefindings that RDS develops in the presence (VEGF120/120) or ab-sence (HIF-2α–/–) of severe vascular defects suggest that the latterare not a prerequisite for RDS. In addition, VEGF prevented RDSvia an epithelial effect without increasing alveolar angiogenesis.

Our findings have potential medical implications. First, re-duced levels of HIF-2α and VEGF may identify infants at risk forRDS. Second, intra-amniotic or intratracheal delivery of VEGFimproved surfactant production and protected preterm new-borns against RDS. The rapidity with which VEGF stimulatesconversion of preformed glycogen to surfactant phospholipids(<5 hours) makes VEGF an attractive therapeutic target. VEGFdid not cause adverse effects on vascular leakage or bleeding inthe lung, possibly because it barely crossed the alveolar epithe-lium. Third, steroids are often used to induce lung maturationbut may cause adverse effects40. Our findings indicate that dexa-methasone upregulated pulmonary VEGF expression in fetusesat low doses41, but suppressed VEGF production at a high dose.Thus, excessive amounts of glucocorticoids might counteractthe beneficial pneumotrophic effect of VEGF. Fourth, oxygenimproves oxygenation of preterm infants with RDS but, as italso suppresses VEGF expression in alveolar type 2 pneumo-cytes8, it would deprive alveolar cells from pneumotrophic ef-fects. VEGF supplements might also lower the toxicity of highoxygen concentrations in the neonate. In concert, our findingssuggest that the pneumotrophic effect of VEGF might have atherapeutic potential for lung maturation in preterm infants atrisk for RDS.

MethodsGeneration of HIF-2α–/– transgenic mice. HIF-2α–/– ES cells18 were used togenerate HIF-2α–/– mice (129/Sv × Swiss). VEGF120/120 mice21, VEGF164/164 andVEGF188/188 mice22, PlGF–/– mice23 and VEGF∂/∂ mice24 were previously gener-ated. Quantitative real-time PCR was performed as described and tran-scripts were normalized per 100 β-actin transcripts21,23. ELISA-kits (R&DSystems, Abingdon, U.K) were used to measure murine and human VEGF.Antibodies used for western blotting23: rabbit anti-human Flt-1 (clone Flt-11; Sigma, Bornem, Belgium), rabbit anti-mouse Flk1 (Santa Cruz,Sanvertech, Boechout, Belgium), rat anti-mouse endoglin (Pharmingen, BDBiosciences, Erembodegem, Belgium) and rat anti-mouse VE-Cadherin (giftfrom E. Dejana). Housing and procedures involving experimental animalswere approved by the Institutional Animal Care and Research AdvisoryCommittee of the University in Leuven.

Histologic analysis. Lungs were paraformaldehyde-fixed and 7 µm paraffinsections were stained for H&E, Hart’s elastin, PAS, thrombomodulin (giftfrom Dr. R. Jackman, Boston, USA), smooth muscle cell α-actin (Sigma,Bornem, Belgium), SP-D and CC10 (Santa Cruz, Sanvertech, Boechout,Belgium), or PGP9.5 (UltraClone, Cambridge, UK). Apoptosis was evalu-ated by TUNEL staining (Roche Diagnostics, Mannheim, Germany), whileproliferating cells were detected using an anti-BrdU antibody (Seralab Ltd,Sussex, UK) after i.p. injection of BrdU (Sigma, 50 mg/kg body weight; at 3intervals of 90 minutes prior to c-section) in pregnant females.Immunostaining for Flt-1 (rat anti-Flt-1; MF1) and Flk-1 (goat anti-Flk-1;#457-683; gift from ImClone Systems Inc, New York, USA) was performedon acetone-fixed 7 µm frozen sections, while immunofluorescent stainingfor laminin (Sigma), HIF-2α (gift from I. Flamme) and Flk-1 (rat anti-Flk-1,gift from H. Kataoka) were performed on cryofrozen lung sections. No nu-clear HIF-2α staining was observed in HIF-2α–/– lungs. In situ hybridization

and ultrastructural analysis were performed as described42. For angiograms,ink was injected into the right fetal ventricle. Subsequently the lungs wereparaformaldehyde-fixed (pH 7.4), embedded in gelatin (30%) and sec-tioned (100 µm).

Premature mouse models. For intra-amniotic injections, pregnant WTSwiss mice were anesthesized using isoflurane and, after laparatomy, 10 µlof Evans blue (final concentration 0.5%), saline, hVEGF165 (R&D Systems;0.5 µg/10 µl saline) or hPlGF-2 (Reliatech, Braunschweig, Germany; 0.5µg/10 µl saline) were injected in the amniotic cavity of E17.5 fetusesthrough the uterine wall, taking care not to injure the fetuses, placenta orfetal membranes. Pups were prematurely delivered by C-section at E18.5(one day before the end of gestation), and scored for respiration and skinoxygenation using an APGAR-like score considering the skin color (cyanosis,0; pink, 4) and lung function: no respiration (0); no spontaneous respirationbut only respiration after pain stimulus (2); spontaneous but irregular respi-ration with RDS (4); and regular breathing without RDS (6). For intratra-cheal injections, WT E17.5 fetuses were delivered by C-section, survivingpups were anesthesized on ice and intratracheally injected with hVEGF165

(0.5 µg/5 µl) or saline (5 µl). Survival of the premature pups was followedduring 20 hours, while in other pups, lungs were analyzed histologically 6hours after intratracheal injection.

Isolation and culture of type 2 pneumocytes and measurement of sur-factant phospholipids. Alveolar type 2 cells were isolated from Wistar ratlungs according to the previously described methods43. Human VEGF (200ng/ml, R&D Systems) was added to the culture medium. SP-B and SP-Ctranscripts were measured after 30 hours. Phospholipids were measured inlung homogenates (chloroform/methanol/water; 5/10/4; v/v) as de-scribed19.

AcknowledgmentsWe thank R. Verbesselt, K. Desmet, C. Van Geet and H. Devlieger for helpfuldiscussion; and A. Bouché, M. De Mol, B. Hermans, S. Jansen, L. Kieckens,W.Y. Man, A. Manderveld, K. Maris, W. Martens, M. Nijs, S. Terclavers, A.Vandenhoeck, B. Vanwetswinkel, P. Van Wezemael and S. Wyns for technicalassistance. This work was supported by an FWO-fellowship to V.C. and S.P.and by grants from the Research Fund K.U.Leuven (GOA/2001/09) and theBIOMED (#PL963380) to P.C. and D.C.

Competing interests statementThe authors declare that they have no competing financial interests.

RECEIVED 4 MARCH; ACCEPTED 17 MAY 2002

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Loss of HIF-2α and inhibition of VEGF impair fetal lung maturation, whereas treatment with VEGF prevents fatal respiratory distress in premature mice