drug-induced regeneration in adult mice -...

R E S EARCH ART I C L E

WOUND HEAL ING

Drug-induced regeneration in adult miceYong Zhang,1*† Iossif Strehin,2*‡ Khamilia Bedelbaeva,1† Dmitri Gourevitch,1 Lise Clark,1

John Leferovich,1§ Phillip B. Messersmith,2¶ Ellen Heber-Katz1†ǁ

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Whereas amphibians regenerate lost appendages spontaneously, mammals generally form scars over the injury sitethrough the process of wound repair. The MRL mouse strain is an exception among mammals because it shows aspontaneous regenerative healing trait and so can be used to investigate proregenerative interventions in mam-mals. We report that hypoxia-inducible factor 1a (HIF-1a) is a central molecule in the process of regeneration inadult MRL mice. The degradation of HIF-1a protein, which occurs under normoxic conditions, is mediated by prolylhydroxylases (PHDs). We used the drug 1,4-dihydrophenonthrolin-4-one-3-carboxylic acid (1,4-DPCA), a PHD inhib-itor, to stabilize constitutive expression of HIF-1a protein. A locally injectable hydrogel containing 1,4-DPCA wasdesigned to achieve controlled delivery of the drug over 4 to 10 days. Subcutaneous injection of the 1,4-DPCA/hydrogel into Swiss Webster mice that do not show a regenerative phenotype increased stable expression of HIF-1a protein over 5 days, providing a functional measure of drug release in vivo. Multiple peripheral subcutaneousinjections of the 1,4-DPCA/hydrogel over a 10-day period led to regenerative wound healing in Swiss Webster miceafter ear hole punch injury. Increased expression of the HIF-1a protein may provide a starting point for futurestudies on regeneration in mammals.

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INTRODUCTION
Wound repair and regeneration are two separate biological processesby which organisms heal wounds (1). There are several models of re-generation in mammals including ear hole closure in rabbits (2, 3), inthe inbred mouse strains MRL/MpJ and LG/J (4, 5), and in other mu-tant mouse strains (6–10). These models show similarities to limb re-generation in amphibians (11, 12) including the replacement of cartilage(4, 13, 14) and the lack of scarring (4, 15, 16). Other less well-knownclassical regenerative phenotypes, not seen during wound repair, havebeen observed in the ear injury model of the MRL (Murphy Roths Large)mouse. The MRL mouse is an inbred strain with a genetic backgroundcomprising 75% LG (Large) and a 25% mixture of AKR, C3H, andC57BL/6 (4, 5). Regeneration in the MRL mouse includes rapidre-epithelialization, enhanced tissue remodeling, basement membranebreakdown, and blastema growth and redifferentiation (4, 15). Inflam-mation is now considered a key factor in regenerative processes (17–22).The role of inflammation in ear hole closure has been demonstrated inmice with an acute inflammatory reactivity (AIR) phenotype, whichhave the ability to close holes made in their ear pinnae (23). A furthercharacteristic of adult MRL mice is their ability to use aerobic glycol-ysis for normal metabolism, which may contribute to the regenerativeresponse (24). This metabolic state contributes to inflammation, withglycolysis playing an important role in the migration and activity ofinflammatory cells (25, 26). The protein hypoxia-inducible factor-1a (HIF-1a) is a central node in all of these processes and could beimportant in ear hole closure in MRL mice.

1Molecular and Cellular Oncogenesis Program, The Wistar Institute, Philadelphia, PA19104, USA. 2Department of Biomedical Engineering, Northwestern University, Evanston,IL 60208, USA.*These authors are first co-authors.†Present address: Laboratory of Regenerative Medicine, Lankenau Institute for MedicalResearch, Wynnewood, PA 19096, USA.‡Present address: Allergan, Santa Barbara, CA 93111, USA.§Present address: Department of Pathology and Laboratory Medicine, University ofPennsylvania, Philadelphia, PA 19104, USA.¶Present address: Bioengineering, Materials Science and Engineering, University ofCalifornia, Berkeley, Berkeley, CA 94720, USA.ǁCorresponding author. E-mail: [email protected]

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HIF-1a is an oxygen-regulated protein that functions as part of aheterodimeric complex, which it forms with HIF-1b in the nucleus. Thiscomplex binds to DNA at specific promoter or enhancer sites [hypoxiaresponse elements (HREs)], resulting in transcriptional regulation ofmore than 100 gene products (27, 28). These include molecules of in-terest in regenerative processes such as angiogenesis, which is induced byvascular endothelial growth factor (VEGF), VEGF receptor-1 (VEGFR-1),platelet-derived growth factor (PDGF), and erythropoietin (EPO). Oth-er processes involved in regeneration include tissue remodeling, whichis induced by urokinase-type plasminogen activator receptor (uPAR),matrix metalloproteinase 2 (MMP2), MMP9, and tissue inhibitors ofmetalloproteinase (TIMPs), and glycolytic metabolism induced by lac-tate dehydrogenase (LDH), which converts pyruvate into lactate andpyruvate dehydrogenase kinase (PDK), which blocks the entry of pyr-uvate into the tricarboxylic acid (TCA) cycle. HIF-1a protein is gen-erally short-lived in the cytoplasm because, under normoxic conditions,it is continually being hydroxylated by prolyl hydroxylases (PHDs)and then is bound by the von Hippel-Lindau tumor suppressor pro-tein (pVHL) and the recently identified SAG/ROC/RBX2 E3 ubiquitinligase complex, which targets HIF-1a protein for proteolysis (29). Un-der low-oxygen conditions, hydroxylation is inhibited, and HIF-1a pro-tein survives and is translocated to the nucleus where it binds to HIF-1b andacts as a transcription factor, binding to the appropriate DNA elementsor HREs (30).

The stabilization of HIF-1a protein can be accomplished throughinhibition of PHDs, molecules that are also actively involved in colla-gen secretion and cross-linking. PHDs control collagen deposition infibrosis, the response to ischemia, and wound repair (31–36). Consideringthe negative impact of scar formation on regeneration, inhibition ofPHDs could have a two-fold effect: stabilization of HIF-1a and down-regulation of scarring. In a chronic diabetic wound repair mouse model,the use of compounds that inhibit PHDs, when applied locally to awound, can accelerate wound repair in the presence of increased vas-cularity and granulation tissue that is rich in collagen and other extra-cellular matrix components (37, 38).

Here, we show that there is increased HIF-1a protein expression inMRL mice after wounding compared to C57BL/6 (B6) mice that do

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not show a regenerative phenotype. A small interfering RNA (siRNA)against HIF-1a (siHif1a) blocked regeneration inMRLmice after wound-ing. In another nonregenerative Swiss Webster mouse strain, administeringthe PHD inhibitor 1,4-DPCA (1,4-dihydrophenonthrolin-4-one-3-carboxylic acid), which stabilizes HIF-1a protein (39, 40), induced regen-erative wound healing in these animals.

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RESULTS

HIF-1a protein overexpression in MRL miceAfter wounding of the ear pinnae of MRL and B6 mice (Fig. 1A), HIF-1a protein expression was measured by immunohistochemistry andWestern blot analysis (Fig. 1, B toD and table S3). HigherHIF-1a expression

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Fig. 1. HIF-1a protein is required for the regenerative response of theMRL mouse. (A) Schematic shows ear punch injury, and images show re-

tector, measured photon number in steradians (a unit of measure based ona solid angle) on days 0 and 7 in the healing ear and whole MRL cross

sulting histological sections and processed tissue samples. (B to F) HIF-1a protein expression in MRL and B6 mice before and after ear wounding.(B) Pooled ear hole donuts (n = 4) were processed for HIF-1a Western blotanalysis with Coomassie blue–stained protein (seen as red with the Odys-sey Classic Infrared Imaging System) as a loading control; N = 2 experi-ments. (C) Ear tissue was used for HIF-1a immunostaining (green; arrowsshow epidermal HIF-1a expression and arrowheads show dermal HIF-1a ex-pression). Scale bar, 0.1 mm. (D) Multiple ear tissue samples for each timepoint after injury (n = 3 to 7 samples, N = 2 experiments) were quantita-tively analyzed for MRL and B6 mice on day 7 (*P < 0.05) and on days 3 and10 (**P < 0.01). (E) Further confirmation of HIF-1a expression was carriedout in MRL and B6 mice backcrossed (BC4) to transgenic HIF-1a peptide–luciferase reporter mice. MRL.HIF-luc and B6.HIF-luc mice showed luciferaseactivity as measured by bioluminescence. This is seen as red for the highestbioluminescent activity and blue for the lowest activity. Arrowheads indi-cate ear bioluminescence; asterisks indicate body bioluminescence. (F) IVIS(in vivo imaging system, Perkin Elmer), a small animal bioluminescence de-

mouse (red) and in the wounded ear and whole B6 cross mouse (blue); dataare expressed as photons per second per square centimeter per steradian(p−1 s−1 cm−2 sr−1). Panel (E) shows one representative mouse per group asan example of a set of multiple experiments with n = 5 mice per group. In (F),the total number of photons detected in each of the mice from (E) is shown.(G and H) HIF-1a expression is required during MRL ear hole closure. (G)Treatment with Hif1a siRNA (siHif1a) blocked Hif1a mRNA expression in vitroin MRL mouse ear fibroblasts (n = 3 replicates); Gapdh is the loading control(N = 2 experiments). (H) Treatment of MRL.HIF-luc mice after ear punchingwith siHif1a_3 in vivo. Mice were injected subcutaneously with either JETPEI-siHif1a_3 mixture at days 0 to 20 after injury (15 mg per mouse; green) orphosphate-buffered saline (PBS; red). (a) Ear hole closure was blocked onday 28; **P < 0.001, Student’s t test (n = 4 ears per group, N = 2 experiments).(b) HIF-1a was determined by bioluminescence (dorsal and ventral) in reportermice treated with siHif1a_3. (c) Number of photons detected on day 14 in thewhole mouse (n = 2) or injured ear; *P < 0.05, Student’s t test (n = 4 earsamples per group) for siHif1a_3 compared to control (N = 2 experiments).




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was observed in MRL mouse tissue after injury with peak expressionat day 7. For longitudinal studies, HIF-1a reporter mice were createdby backcrossing MRL and B6 mice to a transgenic HIF-1a peptide–luciferase reporter mouse [FVB.129S6-Gt(ROSA)26S]; the transgeneconstruct used was made by fusing luciferase to the domain of HIF-1a that binds to pVHL in an oxygen-dependent way (41). The MRL.HIF-luc mice showed higher luciferase activity, which was a correlateof HIF-1a protein expression, compared to B6.HIF-luc mice both be-fore injury in the liver and after injury in the ear (Fig. 1, E and F).

Inhibition of ear hole closure in MRL mice by blocking HIF-1aThe requirement for increased HIF-1a protein expression in ear holeclosure was demonstrated using Hif1a siRNA. Treatment of ear-derived fibroblasts in vitro with a panel of Hif1a siRNAs showed thatone of four tested siRNAs (siHif1a_3) could completely inhibit con-stitutive Hif1a mRNA expression in MRL fibroblasts (Fig. 1G) and infibroblasts from B6 and Swiss Webster mice (fig. S1). Next, siHif1a_3was tested in vivo in MRL.HIF-luc backcross mice for its effect onHIF-1a expression and ear hole closure. Previous studies determinedthat a hole size of 0 to 0.4 mm in diameter 30 days after injury repre-sented a regenerative response in MRL mice; a 30-day hole size of 1.2to 1.6 mm in diameter represented wound repair with scar tissue for-mation and lack of regeneration in B6 mice (4, 42). Ear hole closure(Fig. 1H and table S3) was blocked by treatment with siHif1a_3 in the

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MRL.HIF-luc backcross mice (Fig. 1Ha). HIF-1a expression deter-mined by bioluminescence was reduced in MRL reporter mice bothin the ear and throughout the body (Fig.1H, b and c).

Stabilization of HIF-1a protein in vitro with a hydrogelloaded with 1,4-DPCAThe compound 1,4-DPCA has been reported to be a potent inhibitorof PHDs and factors inhibiting HIF both in vitro and in vivo. Ratsgiven this compound showed inhibition of collagen hydroxylationand a reduction in collagen deposition (33). By blocking PHDs, 1,4-DPCA stabilizes HIF-1a protein (40) (Fig. 2A). As a delivery systemfor 1,4-DPCA, we created a polymer hydrogel composed of a cross-linked network of polyethylene glycol molecules that was capable ofrapid in situ gel formation from a liquid precursor. This hydrogel sys-tem was chosen as the delivery vehicle because of its rapid gelationunder physiological conditions, its biocompatibility, and other favor-able properties for in vivo use (39). Drug-loaded hydrogels wereformed by suspending polymer-stabilized 1,4-DPCA microcrystals inan aqueous mixture of the hydrogel precursors P8Cys and P8NHS(Fig. 2B), which solidified rapidly to entrap the drug microcrystalswithin the hydrogel (Fig. 2C). In vitro drug release studies demon-strated the delivery of 1,4-DPCA from the hydrogel over several days(Fig. 2, D and E). Because 1,4-DPCA is a poorly soluble drug, crystalswere surface-modified with F127 polymer to aid in homogeneous


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Fig. 2. The drug 1,4-DPCA, an inhibitor of PHDs, is encapsulated in an in-jectable polymer hydrogel and released over several days. (A) Diagramshowing that blockade of PHDs by 1,4-DPCA slows degradation of HIF-1a. (B)Chemical structures of the drug delivery components include the drug1,4-DPCA;Pluronic F127, which coats drug crystals; and P8NHS and P8Cys, which react toform the polymer hydrogel that contains the drug. (C) The presence of drug mi-crocrystals does not interfere with the gelation kinetics of the hydrogel. (D) Thedrug was encapsulated in the hydrogel, yielding a white, opaque cylindrical
hydrogel (left), which when incubated with PBS released the drug, leaving
behind a clear hydrogel (right). (E) In vitro experiments showed that drug release occurred over several days for hydrogels containing 119 to 477 mgof the 1,4-DPCA drug. Colored lines represent total drug loading for each formulation. Panels (C) and (E) show data from replicate samples (n = 3).





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distribution throughout the hydrogel (fig.S2, A and B). Furthermore, entrapmentof drug within the hydrogel helped toavoid cytotoxicity observed upon directcontact between cells and drug crystals(fig. S2C).

Fibroblasts from B6mice cultured withthis drug/hydrogel combination (Gd) for24 hours showed increased expression ofHIF-1a, but not HIF-2a, protein in boththe cytoplasm and nucleus as determinedby immunohistochemistry (Fig. 3A) andWestern blot analysis (Fig. 3B) when com-pared to cells incubated with either gel alone(G0) or no drug/no gel (Nor.). To determineactivation of HIF-1a target gene transcrip-tion, we performed reverse transcriptionpolymerase chain reaction (RT-PCR) analysisof mRNA from treated B6 cells. Analysisrevealed that the expression of multipletarget genes was specifically increasedby 1,4-DPCA including the proangiogenictarget genes Vegfa and Hmox1 and pro-glycolytic targets Ldh-a, Pgk1, Pdk1, andGlut1 (Fig. 3C). Furthermore, the expres-sion of all of these genes was blocked bytreatment with siHif1a_3 (fig. S3).

The effect of the 1,4-DPCAdrug/hydrogel in vivo in SwissWebster miceTo test the drug/hydrogel combinationin vivo, we used Swiss Webster mice thatdo not show a regenerative phenotype. Aninitial attempt to directly apply gel tothe ear hole injury site in these mice failedbecause it could not be maintained on thewound. Thus, the drug/hydrogel was in-jected subcutaneously at the back of theneck, distal to the wound to attempt toachieve a pharmacological effect. The ki-netics of effectiveness of the drug/hydrogelin the injured ear were examined to de-termine how often reinjection would benecessary. After ear hole punching, a sin-gle injection (100 ml) of hydrogel contain-ing drug at either 2 mg/ml (Gd) or 0 mg/ml(gel alone, G0) was given, and these SwissWebster mouse groups were comparedto an uninjected group. Ear tissue washarvested daily for 5 days (Fig. 3D). Theeffect of 1,4-DPCA on HIF-1a proteinexpression in ear tissue was determinedby Western blot analysis (Fig. 3E) and byimmunohistochemistry (Fig. 3, F and Gand table S4). HIF-1a expression increasedon day 1, becoming maximal on days 3 to4 after injection.

Fig. 3. 1,4-DPCA drug/gel stabilizes HIF-1a, but not HIF-2a, in vitro and in vivo. (A) Ear fibroblastsfrom B6 mice were cultured with normal medium (Nor.), gel alone (G ), or drug (2 mg/ml)/gel (G ) in 100-ml
0 d
total volume, which formed a solid disc in 24-well plates. Addition of 1,4-DPCA drug/gel (Gd) induced HIF-1a protein expression as determined by immunostaining (green, panel c). Scale bar, 50 mm. (B) Cell lysates(n = 3 per lane) from (A) were used for Western blot analysis for HIF-1a protein (green) and HIF-2a (red)compared to control protein a-tubulin (red) (N = 4). (C) Activation of HIF-1a target gene transcription. RT-PCR was used to analyze mRNA extracted from drug-treated mouse B6 cells (n = 3) from (A). The expres-sion of multiple genes was increased by the 1,4 DPCA/gel treatment, including proangiogenic targetgenes Vegf and Hmox1, and proglycolytic targets Ldh-a, Pgk, Pdk1, and Glut1. Gapdh and 18S ribosomalRNA were used as internal controls for all RT-PCR reactions (N = 3). (D to G) Swiss Webster mice weretreated with a single injection of drug/gel on day 0, the day of injury, and ear tissue was harvestedeveryday for 5 days and was tested for HIF-1a up-regulation. (D) Schematic illustrates the in vivo treat-ment schedule. Swiss Webster mice were ear-punched and injected several hours later in the back of theneck with either gel alone (G0) or drug/gel (Gd). Ear donut tissue was collected for protein and immuno-histochemical (IHC) analysis on days 1 to 5. In (E), hole donuts (n = 6) were processed, and Western blotanalysis was carried out using antibody to HIF-1a (green) and Coomassie-stained samples as loading controls(red) (N = 3). (F) Immunostaining of ear tissue with anti–HIF-1a antibody (green) and 4′,6-diamidino-2-phenylindole (DAPI) counterstain (blue). (G) Immunohistochemical quantitation for G0 (blue) and Gd (red)tissue showed nonoverlapping differences at all time points (n = 2 per treatment group, N = 2experiments).

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Induction of a regenerative response in SwissWebster mice by multiple injections of drug/hydrogelDay 7 after injury was the approximate peak of HIF-1a pro-tein expression in MRL mice, and HIF-1a protein was stillelevated in MRL mice on day 14 (Fig. 1D). Therefore, mul-tiple injections of drug/hydrogel were administered to SwissWebster mice to achieve MRL-like HIF-1a protein expres-sion kinetics. Because a single injection of drug/hydrogelhad an effect on HIF-1a expression through day 5, drug/gelor gel alone was injected subcutaneously on days 0, 5, and10 into three adjacent sites in the back of the neck (Fig. 4A).Complete ear hole closure (Fig. 4, B and C and table S5)
was achieved with multiple injections of drug (2 mg/ml)/gel (Gd), withongoing ear hole closure occurring up to day 35. Furthermore, West-ern blot analysis (Fig. 4D) showed that although HIF-1a expressionwas slightly increased in Swiss Webster mice treated with gel alone onday 7 after injury, it was markedly increased in mice treated with drug(2 mg/ml)/gel, similar to what was seen in MRL mice (Fig. 1B). Incontrast, HIF-2a expression was barely affected by 1,4-DPCA treat-ment in the Swiss Webster mice (Fig. 4D), consistent with in vitroexperiments (fig. S4). Next, we tested the effects of drug/gel treatmenton more distal sites. Mice were injected subcutaneously with drug/gel(on days 0, 5, and 10 after injury) into the left and right rear flankregions (fig. S5A). Only partial ear hole closure (fig. S5B) was achievedat 2 mg/ml, and no closure was observed with drug (1 mg/ml)/gel. We
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also examined the long-term effects of treatment with drug/gel or gelalone; no histopathological effects or weight changes were seen up to 3months after treatment (fig. S5C). Finally, Swiss Webster mice wereinjected three times with drug (2 mg/ml)/gel at the back of the neckand were simultaneously injected with siHif1a_3 every other day for20 days beginning on day 0 after injury. Ear hole closure induced by1,4-DPCA/gel on days 14 and 21 was blocked by siHif1a (P < 0.0001),indicating that HIF-1a expression induced by the drug/gel contributedto the healing effect (Fig. 4E and table S5).

A potential role for HIF-1a in the regenerative responseWe previously showed that the stem cell markers NANOG and SOX2were up-regulated in regeneration-competent adult MRL mice (24)

Fig. 4. Sequential injections of 1,4-DPCA drug/gel intomice at 5-day intervals promote ear hole closure. (A) In-
jection scheme for Swiss Webster mice ear-punched on day 0and injected with drug/gel on days 0, 5, and 10 into separatelocations at the base of the neck (one site every 5 days, indi-cated by arrows). (B) Mice were injected with either gel alone[G0; drug (0 mg/ml)/gel, blue line] or drug/gel [Gd; drug (2 mg/ml)/gel, red line] and were followed for 35 days. Results of woundhealing with three injections of drug (2 mg/ml)/gel were com-pared to G0 on day 35 (**P < 0.0001; Student’s t test; n = 10 ears,N = 4). Images on the left are representative of day 35 ear pinnae(arrows point to ear holes) (a, 0 mg drug/gel; b and c, 2 mg drug/gel). (C) Histological analysis of Alcian blue–stained day 35 eartissue treated with 1,4-DPCA/gel (a and b) with the 2-mm areaof the original hole indicated; (c and d) higher magnification(n = 2). Areas of cartilaginous condensation are shown (black ar-rows); blue staining indicates the presence of proteoglycans.Panel (c) shows a less complete closure where epithelial cells(red arrow) still persist in the new bridge region. In panel (d),ear hole closure is more complete with the bridge filled withmesenchymal cells. (D) Hole donuts (n = 6) from punched earstreated with gel alone (G0) or drug/gel (Gd) were processed, andWestern blot analysis (using the Odyssey Classic Infrared ImagingSystem) was carried out (N = 3) with anti–HIF-1a (green) or anti–HIF-2a (red) antibodies; Coomassie protein staining is theloading control (red). (E) Treatment of Swiss Webster mice withdrug/gel and siHif1a showed inhibition of ear hole closure atday 21 after injury (green line) compared to mice treated withdrug/gel only (red line) (**P < 0.001). Drug/gel + siHif1a treat-ment (green line) compared to gel only (G0) (blue line) showeddifferences in inhibition of ear hole closure on day 14 (*P < 0.05)but not on day 21 (P = 0.051). Comparing ear hole closure in G0-treated (blue line) and Gd-treated (red line) Swiss Webster miceshowed differences at day 21 (*P < 0.05; n = 4 to 7, N = 2). Anal-ysis of variance (ANOVA) statistical analysis was used.



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and that ear-derived fibroblasts from MRL mice in culture displayedmultiple stem cell markers not expressed by ear-derived fibroblastsfrom B6 mice. Fibroblasts from MRL mice (Fig. 5A, first row) stainedpositive for HIF-1a expression and for a suite of immature cell markersincluding NANOG. Ear fibroblasts from B6 mice (Fig. 5A, secondrow) stained negative for HIF-1a expression and immature cell markers,although rare positive staining was seen in dividing cells. However, B6mouse ear fibroblasts treated in vitro with 1,4-DPCA/gel for 24 hours(Fig. 5A, third row) stained positive for HIF-1a expression as well asfor the immature cell markers, displaying both cytoplasmic and nuclearstaining that was indistinguishable from that for MRL mouse ear fi-broblasts. These results were confirmed by quantitative PCR (qPCR),which showed significant differences between B6 mouse ear fibroblaststreated with drug/gel versus no gel (Fig. 5B and table S6) (PNANOG =0.0004; POCT3/4 = 0.0071). To address the effects of blocking HIF-1a expression on stem cell marker expression, we treated NANOG-positive MRL mouse ear-derived fibroblasts with siHif1a (Fig. 5C); thefibroblasts showed almost complete suppression of NANOG. Similarly,after siHif1a treatment, B6 mouse ear-derived fibroblasts that hadbeen treated with 1,4-DPCA in vitro to induce marker expressionshowed suppression of these markers (fig. S3).

Very early and rapid re-epithelialization is a feature that distin-guishes regeneration from wound repair (43). In the amphibian, re-epithelialization is complete within the first 12 hours after injury.Re-epithelialization after ear hole injury in MRL mice occurred be-tween 1 and 2 days after injury, but not until as late as 5 to 10 daysafter injury in the nonregenerating B6 and other mouse strains (4, 16).Swiss Webster mice treated with drug/gel showed re-epithelializationby day 2 after injury, in contrast to mice treated with gel only (Fig. 6A,

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a and b). HIF-1a expression in Swiss Webster mouse ear epidermiswas seen on day 1, followed by WNT5a expression on day 2 (Fig. 6A,c to f), similar to what was observed in drug-treated ear-derived fibro-blasts in vitro (Fig. 6A, g to i). Paralleling the response in vitro (Fig. 5A),cellular dedifferentiation was also seen in vivo after a single injectionof 1,4-DPCA drug/gel into Swiss Webster mice (Fig. 6B, a and b). Ex-pression of the stem cell markers NESTIN, OCT3/4, neurofilament,and PAX7 was detected by immunohistochemistry (Fig. 6B, c to f) andqPCR (Fig. 6Bg) and peaked at days 4 to 5 after injury in Swiss Webstermice receiving the drug/gel.

Expression of laminin, a major component of the basement mem-brane, was reduced in SwissWebster mouse ear tissue after 1,4-DPCA/geltreatment (Fig. 6C, a and b), and laminin expression was absent fromthe epithelial-mesenchymal border. Expression of MMP9, a major pro-tease involved in laminin breakdown, increased after drug treatment(Fig. 6Cc), along with markers for inflammatory cells that have beenassociated with tissue remodeling, such as the neutrophil markersmyeloperoxidase (MPO) and Ly6G (Fig. 6C, d and e) and mast cellmarkers (fig. S6). Next, we examined scar formation after drug/geltreatment of Swiss Webster mice using staining with picrosirius red(a marker of collagen cross-linking). Scar formation was reduced afterdrug/gel treatment but was restored after treatment of the mice withsiHif1a (Fig. 6D, a to d and table S7). In addition to reduced PHDfunction after 1,4-DPCA/gel treatment, the expression of lysyl oxidase–like 4 (Loxl4) and connective tissue growth factor (Ctgf), elements of scartissue formation, was also reduced by drug/gel treatment as shown byqPCR in ear tissue from Swiss Webster mice (Fig. 6De).

Finally, blastema growth and redifferentiation including chondro-genesis and hair follicle growth, which are generally considered later


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Fig. 5. HIF-1a stabilization by 1,4-DPCA drug/gel induces stem andprogenitor cell marker expression in vitro. (A) Stem and progenitor

(B) Quantitative PCR analysis. Results of qPCR were consistent with im-munohistochemistry showing marked changes in mRNA expression for

cell marker expression. Cultured MRL mouse fibroblasts (top row) andB6 mouse fibroblasts (middle row) were grown on coverslips and immu-nostained with antibodies against HIF-1a, NANOG, OCT3/4, CD133,PAX7, PREF1 (DLK1), NESTIN, von Willebrand factor (vWF), and CD34.B6 mouse fibroblasts were cultured with drug (2 mg/ml)/gel (B6 +Gd,; bottom row) for 24 hours (n = 5 to 10 fields per coverslip, N = 3).

all stem cell markers in the drug-treated cells compared to untreatedcells (n = 3, N = 3, see table S6; *P < 0.05, **P < 0.01, Student’s t test). (C)NANOG expression. MRL mouse fibroblasts (n = 3 coverslips per group)were treated with either siRNA control (left) or siHif1a (right) for 48 hoursand were immunostained with anti-NANOG antibody (N = 2). All scalebars, 50 mm.





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Ly6G protein in Gd-treated versus G0-trea(A to C) Blue is DAPI counterstain; redimmunostaining. (D) Picrosirius red stainlagen cross-linking in ear tissue fromwith gel only G0 (a), drug/gel Gd (b), opolarized light analysis (d) shows nonovG0-treated and Gd-treated ear tissue andsiHif1a–treated ear tissue (n = 2). (e) qPC

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Fig. 6. Major steps in tis-sue regeneration. (A) Re-
epithelialization of punchedear tissue (n = 4 per group)was seen on day 2 after punchhole injury of Swiss Webstermice. Ear tissue from micetreated with gel only (G0) ordrug/gel (Gd) was stained withhematoxylin and eosin (H&E);black arrows show incom-plete (a) versus complete ep-idermis (b). Immunostainingof ear tissue was done forHIF-1a expression (c and d)and WNT5a expression (e andf) after G0 (c and e) or Gd (dand f) treatment; white ar-rows indicate epidermis. Im-munohistochemistry of WNT5aprotein expression in vitro with-out (g) or with (h) 1,4-DPCAdrug/gel treatment; (i) West-ern blot analysis of pooledtissue (n = 3, N = 2). (B) H&E-immunostained sections ofmouse ear tissue (day 4 afterinjury) show dedifferentiationin the area of the Gd-treatedwound site (b, dashed blacklines). Gd- and G0-treated eartissues were immunostainedfor NESTIN, OCT3/4, neurofila-ment (NF), and PAX7 (c to f )(n = 4). G0 controls are shownin insets. Results of qPCR forthese same genes are seenin (g) (n = 3, N = 3; *P < 0.05,**P < 0.01, Student’s t test).(C) Ear tissue from Gd-treatedversus G0-treatedmicewas im-munostained to show lamininexpression andbasementmem-brane breakdown (white ar-rows, a and b, inset). (c to e)Expression of MMP9, MPO, and
ted (insets) mice (n = 4, N = 3).or green color shows specificing analysis shows day 14 col-Swiss Webster mice treated

r Gd + siHif1a (c). Quantitatederlapping differences betweenbetween Gd-treated and Gd +R results show early (days 2 to

3 after injury) Loxl4 and Ctgf expression in Gd- and G0-treated ear tissue samples (n = 3 per group, N = 3; **P < 0.01, Student’s t test). (E) Blastema growthand redifferentiation into cartilage and hair follicles was seen in Swiss Webster ear tissue after Gd treatment. (a) Low-magnification histological image ofear hole tissue stained with Alcian blue shows chondrogenesis in Gd-treated but not in G0-treated ear tissue. Solid vertical lines indicate ends of cartilage;broken lines show soft tissue borders. (b) Quantitation of soft tissue ear hole diameter at day 35 after injury. (c) A magnified image of Gd-treated mouseear tissue indicates two new areas of chondrogenesis (g and h) and new hair follicles (k). Results of qPCR show up-regulated chondrogenesis-associated(d) and hair follicle–associated (i) genes on day 21 after injury in Gd-treated but not in G0-treated mouse ear tissue (*P < 0.05, **P < 0.01, Student’s t test;n = 3, N = 3). Cartilage hole diameter (e) and cartilage area (f) (a histomorphometric measurement of Alcian blue staining in new growth area) weresignificantly different between Gd-treated and G0-treated mouse ear tissue (**P < 0.01, Student’s t test; n = 4 to 6 ears). (j) Number of keratin 14–positivehair follicles in Gd-treated mouse ear tissue compared to Go-treated and normal mouse ear tissue. **P < 0.0001, ANOVA (n = 4 ears). Scale bars, 0.1 mm forall panels except for panel (Ah) (50 mm).

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events in the regenerative process, were also affected by 1,4-DPCA/geltreatment. A major difference in ear hole size with accompanyingchondrogenesis and appearance of hair follicles (Fig. 6E, a to h andtable S7) was apparent by day 35 after drug/gel treatment of SwissWebster mice. Differences in inward tissue growth were seen (Fig.6Ec) as well as changes at the end of the cartilage at the site of theoriginal punch margin (Fig. 6E, e to h and table S7), which showednew chondrogenesis. Furthermore, expression of the chondrogenesis-associated genes Mgp, Itm2a, Matn3, Mia2, Col11a1, Prg4, Fmod, andChad was up-regulated on day 21 after injury in Swiss Webster micetreated with drug/gel (Fig. 6Ed and table S7). In addition, hair folliclesexpressing keratin 14 were found in ear tissue from drug/gel-treatedbut not in ear tissue from gel only–treated mice, with keratin 14 expres-sion similar to that found for normal ear tissue (Fig. 6E, j and k and tableS7). Expression of the genes S100a4, Wif1, Dkk3, Dcn, and Tgfb2 in thegrowing hair follicle bulge region was also increased in drug/gel-treatedbut not in gel-treated Swiss Webster mice (Fig. 6Ei and table S7).


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DISCUSSION

Following its discovery (27), there has been a growing recognition thatHIF-1a is a master regulator of cellular processes including regulationof oxygen concentrations, aerobic glycolysis, cell migration, and in-flammation. Our study suggests that another role for HIF-1a is inmammalian tissue regeneration. Given the role of HIF-1a in cellularprocesses that distinguish tissue regeneration from scar formation, weexplored the role of HIF-1a in regenerative wound healing in theMRL mouse. The MRLmouse strain uses aerobic glycolysis as its basalmetabolic state (24) and is able to regenerate many tissue types afterinjury. Furthermore, a recent genetic mapping study shows thatRNF7, an E3 ligase necessary for HIF-1a ubiquitination, is associatedwith ear hole regeneration in LG/J mice. Expression of Rnf7 is down-regulated in both MRL and LG/J mice (an MRL mouse parental straincontributing 75% of the MRL genome), resulting in a predicted in-crease in HIF-1a expression (44). Here, we show that HIF-1a is up-regulated in unwounded MRL mice compared to B6 and SwissWebster mice. Expression of HIF-1a was further increased in MRLmice after wounding (ear hole punching). Treatment of MRL miceafter injury with siHif1a, which blocked HIF-1a expression, inhibitedear hole closure. To determine the effects of increased HIF-1a expres-sion in Swiss Webster mice that do not have a regenerative phenotype,we subcutaneously injected these animals with the HIF-1a–stabilizingdrug 1,4-DPCA in a hydrogel (39). Administering the drug/gel sub-cutaneously both proximal and distal to the ear wound sites resultedin accelerated ear hole closure. As in MRL mice, siHif1a treatmentblocked this drug-induced regenerative response, supporting the no-tion that up-regulation of HIF-1a in mice is sufficient to lead to aregenerative response in ear pinnae after wounding.

Formulating the 1,4-DPCA drug in a hydrogel enabled slow deliv-ery of the drug over 4 to 10 days, depending on the gel formulationand drug dose. Treatment of mice with the drug/gel increased HIF-1a protein expression for up to 5 days. Trapping microcrystals of thedrug within a hydrogel avoided potential cytotoxicity associated withdirect uptake of drug crystals by cells. In terms of specificity, 1,4-DPCA interacted with and blocked PHD function and increasedHIF-1a expression. We showed that the drug did not affect the ex-pression of HIF-2a (also regulated by PHDs) in either fibroblasts or

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endothelial cells (fig. S4) and that HIF-2a expression was not affectedin ear tissue after drug/gel treatment.

Central to limb regeneration in amphibians is the blastema. Theblastema is a tissue structure seen in the developing embryo and inregenerating tissue that is made up of a mass of undifferentiated plu-ripotent cells that can proliferate and then produce a copy of the loststructure (12, 45). The process starts with rapid coverage of the woundby epithelial cells (re-epithelialization), which occurs in the absence ofa basement membrane potentially due to increased MMP protease ac-tivity (46, 47). Reforming the basement membrane in regeneratingamphibian limbs after retinoic acid treatment leads to scarring andlack of blastema growth (48, 49). In the next step of blastema growth,nondividing mesenchymal cells migrate under the new epidermis asthe accumulation blastema and then divide, resulting in tissue elonga-tion and expansion; the cells finally redifferentiate into the lost ap-pendage (50).

HIF-1a–regulated ear hole closure in MRL mice and drug-treatedSwiss Webster mice appears to go through a process that has some ofthe hallmarks of the blastema and its formation. HIF-1a is most high-ly expressed in the early phase of the regenerative response in whichcells are migratory, nonproliferative, and dedifferentiated. This isconsistent with up-regulation of molecules such as WNT5a, involvedin cell migration (51–53), and NANOG and OCT3/4, which are markersof pluripotent stem cells, oocytes, and the early embryo (54, 55). De-velopment of the embryo occurs under low-oxygen conditions, result-ing in increased expression of HIF-1a, morphogenesis (56), andincreased stem cell marker expression (57, 58).

MRL mouse ear tissue showed unusual expression of a range ofdiverse stem cell markers both in vitro and in vivo, including NANOG,SOX2, OCT3/4, CD34, and CD133. NESTIN is a neuronal stem andprogenitor cell marker, PAX7 is a satellite muscle-associated stem cellmarker, WNT5a is an early marker involved in migration, and PREF1or DLK1 is a preadipocyte/hepatocyte stem cell marker. Expression ofthese molecules was not observed in ear tissue of B6 or Swiss Webstermice. HIF-1a stabilization by the drug 1,4-DPCA led to an increase inexpression of all of these differentiation markers in Swiss Webster andB6 mouse fibroblasts, although only transiently. Treatment with siHif1ablocked NANOG expression in MRL mouse ear fibroblasts, suggestingthat the expression of this marker is due to increased HIF-1a. In additionto a hypoxic environment and elevated HIF-1a, pluripotent embryonicstem cells maintain a glycolytic metabolic state (59). Adult quiescentmesenchymal and hematopoietic stem cells also may utilize glycolyticmetabolism (59, 60) like in adult MRL mice (24), other animal modelsof regeneration (61, 62), and some examples of surgical wounds (63).Surprisingly, HIF-2a expression, reported to modulate expression ofNANOG and OCT3/4 (64), does not increase during ear hole closure;however, a recent study shows that other factors such as miR-302 canalso regulate expression of OCT3/4 (65).

HIF-1a regulates MMPs, which in turn degrade extracellular matrixincluding laminin and basement membrane remodeling proteins. LikeMRL mice (15, 16), Swiss Webster mice treated with the drug 1,4-DPCA (a PHD inhibitor) show increased MMP9 expression and avanishing basement membrane (Fig. 6C, a and b). A second regeneration-promoting effect of 1,4-DPCA is collagen hydroxylation, leading toreduced scarring and increased collagen degradation (33, 40, 66, 67).MRL mice and 1,4-DPCA–treated Swiss Webster mice also show re-duced expression of scar-associated markers Loxl4 and Ctgf (17, 68);in contrast, hypoxia-induced remodeling molecules are increased,




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including lysyl oxidase (69) and collagen prolyl (P4HA1,2) and lysyl(PLOD2) hydroxylases. These molecules are also regulated by HIF-1a andresult in increased extracellular matrix stiffness and alignment (70),indicating that hypoxia may mediate tissue component structure be-yond a specific HIF-1a effect (see fig. S4).

In MRL mice, elastic and articular cartilage formation begins atabout 1 month after injury and can be fully regenerated by 3 to 4months after injury (13, 14). With 1,4-DPCA treatment of SwissWebster mice after ear hole punch injury, the new growth area showschondrogenesis by day 35, with up-regulation of multiple chondro-genesis markers including those found in chondrogenic precursor cells(71–74) and in cartilage extracellular matrix (75, 76). Hair follicle for-mation was also found in the new growth area at a level seen in nor-mal tissue, and multiple markers of bulge-derived keratinocyte stemcells (77) and epithelial sac cells involved in regeneration were alsoexpressed in drug-treated Swiss Webster mice (78). Together, our datademonstrate that spontaneous regeneration in MRL mice with earhole punch injury can be mimicked in nonregenerating Swiss Webstermice by treatment with the drug 1,4-DPCA/hydrogel, which stabilizesHIF-1a, indicating that HIF-1a is a regulator of mammalian regenera-tion in vivo. However, a key limitation of this study is that it remainsuntested whether modulating HIF-1a expression in other mammalsand in humans will induce a regenerative healing response to wounds.Finally, whereas the ear hole punch model has proven to be a reliableproxy for wound healing studies in multiple tissues in regeneratingand nonregenerating mice, future HIF-1a modulation studies willbe required to generalize these findings to other tissue injury sites.


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MATERIALS AND METHODS

Study designWe used inbred mouse strains to study the effect of a small-moleculeinhibitor of PHDs on the in vitro and in vivo expression of HIF-1a andthe impact on quantitative regenerative ear hole closure (4, 42). Inanimal studies, 2.1-mm ear hole punch wounds were created in earpinnae, and a 1,4-DPCA–containing hydrogel was subcutaneously im-planted in the back of the neck of the wounded mice at multiple timepoints. Healing was monitored by measuring hole diameters. Endpoints of the study were previously determined to be 30+ days afterinjury (4, 15, 24, 42) and included key indices of tissue regenerationsuch as blastema formation and epithelial, dermal, and cartilaginouswound closure with hair follicle replacement plus expression ofmultiple molecular markers of cellular dedifferentiation, redifferentia-tion, and stem cell state. These parameters were determined by phys-ical measurements of wound closure, standard tissue histology andhistomorphometry, and gene expression using quantitative immuno-histochemistry, Western blot analysis, and qPCR. The experimentalgroups were coded, and different laboratory personnel were involvedin doing ear wounding, injections, phenotyping, and data analysis.

Animals and in vivo proceduresMRL/MpJ and HIF-1a::ODD-luciferase reporter [FVB.129S6-Gt(RO-SA)26Sortm2(HIF1A/luc)Kael/J] mice were obtained from The Jack-son Laboratory; C57BL/6 mice were from Taconic Laboratories; SwissWebster mice were from Charles River. Mice were used at about 8 to10 weeks of age in all experiments under standard conditions at theWistar Institute Animal Facility, and the protocols were in accordance

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with the National Institutes of Health (NIH) Guide for the Care andUse of Laboratory Animals. Through-and-through ear hole puncheswere carried out as previously described (4), and each ear hole diam-eter was measured in the direction of the ear pinna long axis and thenthe perpendicular axis.

IVIS luciferase scanningTo detect luciferase expression in vivo, mice were given a single intra-peritoneal injection of D-luciferin (37.5 mg/kg, Gold BiotechnologyInc.) in sterile water. Fifteen minutes later, mice were anesthetizedusing isoflurane and placed in a light-tight chamber equipped witha charge-coupled device IVIS imaging camera (Xenogen). Photons werecollected for a period of 1 to 5 min, and images were obtained by usingLiving Image software (Xenogen) and IGOR image analysis software(WaveMetrics). HIF-1a ODD luc expression after ear punching was de-termined in MRL and B6 mice backcrossed to the transgenic HIF-1apeptide–luciferase reporter mouse FVB.129S6-Gt(ROSA)26S, made byfusing luciferase to the domain of HIF-1a that binds to pVHL in an oxygen-dependent way (ODD peptide) and selected for luciferase positivity.

Hif1a siRNA transfection in vitro and in vivoB6, Swiss Webster, and MRL mouse ear fibroblast-like cells at 70% ofconfluence were transfected with 100 nM of four different Hif1a siRNAs(SI00193025, SI00193032, Sl00193011, and SI00193018; purchased fromQiagen) and scramble siRNA (sc-37007, Santa Cruz Biotechnology),using Lipofectamine 2000 according to the manufacturer’s protocol.Transfected cells were examined for knockdown efficiency after 48 hoursof transfection. siRNA Mm_Hif1a_3 (SI00193025) was selected forthe in vitro experiments because of its high efficiency. In vivo, siRNAMm_Hif1a_3 was used for HIF-1a inhibition. SiHif at 75 mg/kg wasmixed with Jetpei (Polyplus, Genycell) following the manufacturer’sinstructions and was then injected into animals subcutaneously every48 hours.

In vivo hydrogel injectionSwiss Webster mice were injected subcutaneously at the base of theneck with 100 ml of 10% (w/v) 1:1 (w/w) ratio of P8Cys (with or with-out 1,4-DPCA drug crystal) to P8NHS hydrogel prepared in PBS (Fig.2B and Supplementary Materials). Each component was kept cold andmixed just before injection. At different time points, mice were eutha-nized and tissues were removed for protein and RNA analysis.

Statistical analysisAll experiments were repeated multiple times (N). The data representpooled samples for Western blot analysis and qPCR, and individualsamples in healing studies and tissue analysis (n) as indicated in thefigure legends. All experiments used inbred mouse strains reducingindividual-to-individual variation. Student’s t test was carried out to com-pare differences of means from independent samples between twogroups. The ANOVA test was performed to determine if there weresignificant differences among the means of more than two groups. Ifthe P value from ANOVA analysis was significant, then the post hocTukey test was applied to compare the mean between each group. Pvalues ≤0.05 were considered significant, and P values ≤0.01 wereconsidered highly significant. All error bars shown on the graphs rep-resent SEs, except in Fig. 2 where SD is used. The software used forthe ANOVA analysis and the post hoc Tukey test is R version 2.14.1.All other statistical analyses were done using Microsoft Excel 2010.




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SUPPLEMENTARY MATERIALS

www.sciencetranslationalmedicine.org/cgi/content/full/7/290/290ra92/DC1Materials and MethodsFig. S1. Knockdown of Hif1a in B6 and Swiss Webster mouse ear fibroblasts.Fig. S2. Effects of Pluronic F127 on microcrystal growth and stability.Fig. S3. HIF-1a target genes and dedifferentiation marker genes up-regulated by 1,4-DPCA/gelwere blocked by knockdown of Hif1a.Fig. S4. HIF-2a is not affected by the 1,4-DPCA drug/gel construct in vitro.Fig. S5. Distal and long-term effects and survival of drug/gel-treated Swiss Webster mice.Fig. S6. Inflammatory responses are affected by 1,4-DPCA treatment.Fig. S7. Synthesis of 1,4-DPCA.Table S1. Primer sequences used for RT-PCR.Table S2. Antibodies used for immunostaining.Table S3. Raw data and statistical significance testing for Fig. 1.Table S4. Raw data and statistical significance testing for Fig. 3G.Table S5. Raw data and statistical significance testing for Fig. 4.Table S6. Raw data and statistical significance testing for Fig. 5.Table S7. Raw data and statistical significance testing for Fig. 6.Table S8. Raw data and statistical significance testing for fig. S5.References (79–82)


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Acknowledgments: We thank X. Yin, Q. Liu, R. Davulari, Y. Bi, and A. Weeraratna for usefuldiscussions and statistical advice, and M. O’Connell and A. Weeraratna for providing reagents.Funding: Supported, in whole or in part, by NIH grants from the National Institute of Dental andCraniofacial Research, RO1 DE021215 and DE021104, with other funding from the F. M. KirbyFoundation and the G. Harold and Leila Y. Mathers Foundation. These studies used the WistarInstitute Animal Facilities, Histotechnology Facility, and Research Supplies Facility, which aresupported by a Wistar Cancer Center grant from the National Cancer Institute, NIH (P30CA010815). Author contributions: E.H.-K. and P.B.M. designed the studies, analyzed and in-terpreted results, and wrote the paper. Y.Z. carried out most of the animal experiments andthe Western and RT-PCR experiments. I.S. analyzed and carried out drug synthesis, drug crystalsynthesis, hydrogel polymer synthesis, drug cytotoxicity studies, and drug release studies. K.B.carried out immunohistochemistry and cell culture studies. D.G. carried out histology and dataanalysis and prepared the figures. L.C. carried out mouse genetics and phenotyping. J.L. carriedout original bioluminescence studies. Competing interests: A U.S. patent application “Epimorphicregeneration and related hydrogel delivery systems” has been filed in connection with this work(P.B.M., I.S., and E.H.-K.). The other authors declare that they have no competing interests.

Submitted 1 August 2014Accepted 15 May 2015Published 3 June 201510.1126/scitranslmed.3010228

Citation: Y. Zhang, I. Strehin, K. Bedelbaeva, D. Gourevitch, L. Clark, J. Leferovich,P. B. Messersmith, E. Heber-Katz, Drug-induced regeneration in adult mice. Sci. Transl. Med.7, 290ra92 (2015).

nceTranslationalMedicine.org 3 June 2015 Vol 7 Issue 290 290ra92 11


Drug-induced regeneration in adult mice

and Ellen Heber-KatzYong Zhang, Iossif Strehin, Khamilia Bedelbaeva, Dmitri Gourevitch, Lise Clark, John Leferovich, Phillip B. Messersmith

DOI: 10.1126/scitranslmed.3010228, 290ra92290ra92.7Sci Transl Med

driver of regeneration in mice. is a centralαregeneration in both untreated MRL mice and drug-treated Swiss Webster mice, showing that HIF-1 blockedHif1aobserved in MRL mice during regeneration in response to injury. A small interfering RNA against

authors observed ear hole healing with closure and a pattern of expression of cellular markers similar to thatexpression after ear hole punch injury in adult Swiss Webster mice that do not normally show regeneration. The

proteinα used a drug in a slow-release hydrogel formulation to up-regulate HIF-1et al.this hypothesis, Zhang testthe MRL mouse could account for some of the cellular, molecular, and metabolic hallmarks of regeneration. To

) found both before and after injury inα (HIF-1αThe markedly increased expression of hypoxia-inducible factor-1The MRL mouse is capable of regenerating injured tissues, making it a rare exception among mammals.

Driving regeneration in mice

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