The American Journal of Pathology, Vol. 181, No. 2, August 2012

Copyright © 2012 American Society for Investigative Pathology.

Published by Elsevier Inc. All rights reserved.

http://dx.doi.org/10.1016/j.ajpath.2012.05.010

Metabolic, Endocrine, and Genitourinary Pathobiology

Thrombospondin-1 Mimetic Peptide ABT-898 AffectsNeovascularization and Survival of Human Endometriotic

Lesions in a Mouse Model

Diane S. Nakamura,* Andrew K. Edwards,*Sophia Virani,* Richard Thomas,† andChandrakant Tayade*†

From the Departments of Biomedical and Molecular Sciences*

and Obstetrics and Gynecology,† Queen’s University, Kingston,

Ontario, Canada

Endometriosis is a common cause of pelvic pain andinfertility in women, and a common indication for hys-terectomy, yet the disease remains poorly diagnosedand ineffectively treated. Because endometriotic lesionsrequire new blood supply for survival, inhibiting angio-genesis could provide a novel therapeutic strategy. ABT-898 mimics the antiangiogenic properties of thrombos-pondin-1, so we hypothesized that ABT-898 will preventneovascularization of human endometriotic lesions andthat ABT-898 treatment will not affect reproductive out-comes in a mouse model. Endometriosis was induced inBALB/c-Rag2�/�Il2rg�/� mice by surgical implantationof human endometrial fragments in the peritoneal cav-ity. Mice received daily injections of ABT-898 for 21days. Flow cytometry was performed to measure circu-lating endothelial progenitor cells in peripheral blood.Cytokines were measured in plasma samples. Half ofthe ABT-898-treated and control mice were euthanizedto assess neovascularization of endometriotic lesions,using CD31� immunofluorescence. The remaining micewere mated and euthanized at gestation day 12. Endo-metriotic lesions increased circulating endothelial progen-itor cells 13 days after engraftment, relative to baseline.Endometriotic lesions from ABT-898-treated mice exhib-ited reduced neovascularization, compared with controls,and lesions had fewer CD31� microvessels. Chronic treat-ment with ABT-898 did not lead to any fetal anomalies oraffect litter size at gestation day 12, compared with con-trols. Our results suggest that ABT-898 inhibits neovas-cularization of human endometriotic lesions withoutaffecting mouse fecundity. (Am J Pathol 2012, 181:570–582; http://dx.doi.org/10.1016/j.ajpath.2012.05.010)

Endometriosis is characterized by the growth of endome-

trial tissue (normal uterine lining) outside of the uterus. It is

570

estimated that 176 million women of reproductive age areaffected by this disease worldwide, including 8.5 millioncases in North America.1–5 In the United States in 2002, theestimated costs of diagnosis and treatment of endometrio-sis was $22 billion (USD).2 In Canada, annual estimatedcost of endometriosis is $1.8 billion (CAD), with a mean of$1164 per patient in direct health care costs and $5206 intotal, including indirect costs due to loss in productivity.6

Endometriosis is responsible for 50% of infertility cases and60% of pelvic pain cases in women.1 Even though it is oneof the most prevalent global causes of infertility and pelvicpain, and a major indication for hysterectomy, endometrio-sis remains poorly diagnosed and ineffectively treated. Cur-rent treatment options include hormone therapy, pain killers,or surgery to remove recurrent endometriotic lesions or theuterus itself, but none offer long-term relief and all can carryserious adverse effects.1,7 Gonadotropin-releasing hor-mone (GnRH) agonists are used to treat pelvic pain asso-ciated with endometriosis and function by inhibiting theproduction of endogenous GnRH resulting in hypoestro-genism and degeneration of endometrium.1,7,8 Althoughthis treatment is effective in reducing pelvic pain, womenare unable to become pregnant during treatment becauseof the anovulatory state created. For this reason, we havefocused on developing a noninvasive therapeutic optionthat will reduce symptoms of endometriosis without interfer-ing with fertility.

The establishment of a new blood supply is fundamentalfor the support, growth, and survival of endometriotic le-sions. Indeed, dense vascularization is a characteristic fea-ture of endometriotic lesions.9–14 This has led to the ideathat antiangiogenic therapy might be a successful thera-peutic approach for endometriosis. Potential effectiveness of

Supported by the Canadian Institute of Health Research, Natural Sci-ences and Engineering Research Council, and the Principal’s Develop-ment Fund–Queen’s University, Kingston, Ontario, Canada.

Accepted for publication May 2, 2012.

Disclosure: ABT-898 was provided by Abbott Laboratories.

D.S.N. and A.K.E. contributed equally to this work.

Address reprint requests to Chandrakant Tayade, D.V.M., Ph.D., De-partment of Biomedical and Molecular Sciences, Queen’s University, 18

Stuart St., Kingston, ON K7L 3N6, Canada. E-mail: tayadec@queensu.ca.

ABT-898 Reduces Endometriosis Vascularity 571AJP August 2012, Vol. 181, No. 2

antiangiogenic therapies has been assessed in limited animalstudies, but to date there have been no clinical trials.14–16

ABT-898 is an octapeptide that mimics the heptapeptidesequence of TSR-1, within the protein thrombospondin-1(TSP-1), responsible for its antiangiogenic activity.17 TSP-1is a large homotrimeric protein that mediates its antiangio-genic effects via the CD36 pathway by inhibiting VEGFreceptor-2, resulting in the inability of endothelial cells torespond to the proangiogenic factor VEGF.18 TSP-1 alsoinduces endothelial cell apoptosis and inhibition of cell cy-cle progression through the CD36 pathway19 and bindsand sequesters VEGF, inhibiting its biological activity.20

ABT-898 is capable of inducing tumor regression in a mousemodel of ovarian cancer by inhibiting angiogenesis.21

ABT-898 may be an effective treatment in that it hasdual functionality, inhibiting VEGF while simultaneouslycausing apoptosis and inhibiting cell cycle progres-sion.17 A recent study in marmosets demonstrated thatABT-898 does not inhibit ovulation or folliculogenesis, aprocess that requires physiological angiogenesis.17 ABT-898 could inhibit the pathological angiogenesis requiredfor endometriotic lesion growth without affecting thephysiological angiogenesis required during the ovariancycle and pregnancy. In the present study, we set out todetermine the effects of ABT-898 on neoangiogenesisand on growth and survival of human endometriotic le-sions in an alymphoid mouse model. We further evalu-ated whether chronic treatment with ABT-898 affects re-productive outcomes in treated mice.

Materials and Methods

HUVEC Proliferation Assay

The effect of ABT-898 (Abbott Laboratories, North Chicago,IL) on proliferation of human umbilical vein endothelial cells(HUVECs) was assessed using a WST-1 proliferation assay(Roche Diagnostics, Indianapolis, IN) according to themanufacturer’s instructions. WST-1 is a nonradioactive sub-strate that measures metabolic activity of living cells. Briefly,HUVECs obtained from Cell Applications (San Diego, CA)and grown in endothelial cell growth medium (Cell Applica-tions) at 37°C and 5% CO2 were harvested by trypsin di-gestion. Cells were stained with Trypan Blue and countedusing a hemocytometer (Hausser Scientific, Horsham, PA).HUVECs (5 � 103 in 100 �L of endothelial cell growthmedium) were added to each well in a 96-well plate andwere incubated in RPMI 1640 medium (HyClone; ThermoFisher Scientific, Logan, UT) for 24 hours at 37°C and 5%CO2. HUVECs were treated with varying concentrations ofABT-898 reconstituted in 5% dextrose (0.1, 1, 10, 100, or1000 ng/mL) or 5% dextrose control (n � 3 wells per treat-ment group). After incubation for 24 hours, 10 �L of WST-1was added to each well. Absorption at 450 nm was mea-sured 4 hours later, to quantify HUVEC proliferation.

Matrigel Angiogenic Tube Formation Assay

To investigate the effects of ABT-898 on endothelial celltube formation, a Cell Biolabs (San Diego, CA) endothe-

lial tube formation assay was performed according to the

manufacturer’s instructions. In brief, HUVECs (1 � 104 in150 �L of endothelial cell growth medium per well) wereadded onto 50 �L of solidified extracellular matrix gel(Cell Biolabs) and were incubated for 18 hours with dif-ferent concentrations of ABT-898 (0.01, 0.1, 1.0, or 100ng/mL) or PBS alone at 37°C and 5% CO2. The growthmedium was removed, cells were washed with 100 �L ofPBS, and 50 �L of a staining solution containing fluoresceinisothiocyanate was added. After 30 minutes of incubation at37°C, cells were washed with PBS and then visualized un-der bright-field and fluorescent confocal microscopy (LeicaTCS SP2 Multi Photon; Leica Microsystems, Concord, ON,Canada). Using ImageJ Pro Plus software version 6.0 (NIH,Bethesda, MD), the distance between branching pointswas measured in arbitrary units to assess tube length. Sim-ilarly, tube thickness was measured to quantify branchwidth.

Mouse Model of Endometriosis

Breeding pairs of Rag2�/�Il2rg�/� double-knockout mice(alymphoid) on a BALB/c background were kindly pro-vided by Dr. M. Ito (Central Institute for ExperimentalAnimals, Kawasaki, Japan). These mice were bred in-house at Queen’s University (Kingston, ON, Canada) un-der barrier husbandry. All experiments were performedunder protocols approved by the Queen’s University In-stitutional Animal Care Committee. To allow for the growthof estrogen-dependent endometriotic lesions, 5- to7-week-old female Rag2�/�Il2rg�/� mice (n � 12) wereimplanted with 60-day slow-release �-estradiol 17-ace-tate pellets (15 mg/pellet; Innovative Research of Amer-ica, Sarasota, FL) on day 0. On day 5, endometriosis wasinduced intraperitoneally, using nonpathological eutopichuman endometrial tissue collected from hysterectomiesperformed at Kingston General Hospital. Uteri were bi-sected with a scalpel and endometrium was scraped off theuterine wall. Endometrium was divided into 24 equal sec-tions (mean weight � 0.0260 g) in a Petri dish containingRPMI 1640 medium (Gibco; Invitrogen-Life Technologies,Grand Island, NY). Endometrial sections were washed inPBS, and kept on ice until surgically implanted in mice.Small incisions were made in the abdomen of each mouse,and sections of human endometrium were dropped into theleft side of the peritoneal cavity. Mice were monitored for 4to 8 weeks, depending on the therapeutic regimen.

Treatment with ABT-898

Beginning on day 12 (7 days after endometriosis induc-tion), experimental mice were given daily intraperitonealinjections of ABT-898 (100 mg/kg in 100 �L of 5% dex-trose; n � 6) or 100 �L of 5% dextrose (n � 6; BaxterCorporation, Mississauga, ON, Canada) for 21 consecu-tive days. The decision to administer daily injections atthe concentration used was based on pharmacokinetic

and half-life studies performed by Abbott Laboratories.

572 Nakamura et alAJP August 2012, Vol. 181, No. 2

Detection of Circulating Endothelial ProgenitorCells in Peripheral Blood of Mice

Peripheral blood was collected from the submandibularvein of each mouse, to quantify levels of SCA-1�/c-Kit�/CD31�/CD45� circulating endothelial progenitor cells(CEPCs) by four-color flow cytometry. Plasma sampleswere stored in �80°C and used subsequently for 23-plexcytokine assay (Bio-Rad Laboratories, Mississauga, ON,Canada). In summary, 100 �L of peripheral blood inheparinized tubes (100 �L heparin 500 USP/mL; Sigma-Aldrich, St. Louis, MO) was collected per mouse on day7 (after endometriosis induction, but before ABT-898treatment), day 18 (7 days after starting ABT-898 treat-ment), day 32 (the final day of 21-day ABT-898 treat-ment), and day 39 (7 days after the end of ABT-898treatment). Blood was centrifuged at 1000 � g for 5minutes; plasma was collected and stored at �80°C.Erythrocytes were lysed using 2 mL of deionized water for2 minutes. To restore isotonic balance, 600 �L of 0.6mol/L KCl was added. Fluorochrome-conjugated [phyco-erythrin-cyanine 5 (PEC5) for CD45; phycoerythrin (PE)for CD31; phycoerythrin-cyanine 7 (PEC7) for Sca-1, andfluorescein isothiocyanate (FITC) for c-Kit, each at 0.4�g/mL] anti-mouse antibodies (BD Biosciences, Missis-sauga, ON, Canada) were used to identify Sca-1�/c-Kit�/CD31�/CD45� cells by four-channel flow cytometry.

Cytokine Profiles in Mouse Peripheral Blood

Plasma cytokine levels in peripheral blood were analyzedusing a Bio-Plex Pro mouse cytokine 23-plex assay on aBio-Plex 200 suspension array system (both from Bio-RadLaboratories), according to the manufacturer’s protocol. Inthis assay, 15 �L of plasma per sample was used. Sampleswere diluted fourfold, and 100 �L of assay buffer and 50 �Lof beads were added to the assay plate. After two washingswith 100 �L of wash buffer, 50 �L of sample was added toeach well. After 1 hour of incubation (in the dark, with shak-ing at 300 rpm), wells were washed three times with 100 �Lof wash buffer before addition of 25 �L of detection anti-body. Samples were incubated for 30 minutes, thenwashed three times. Streptavidin-phycoerythrin (50 �L) wasadded to each well and was incubated for 10 minutes. Afterthree washings, beads were resuspended in 125 �L ofassay buffer, with shaking at 1100 rpm for 30 seconds. Theplate was read under the Bio-Plex 200 suspension arraysystem (Bio-Rad Laboratories).

Ultrasound Image Acquisition of EndometrioticLesions in Vivo

Ultrasound image acquisition was used to further assessvascularization of endometriotic lesions of ABT-898-treated mice and 5% dextrose controls. Mice anesthe-tized with isoflurane were restrained on a heated platformin the supine position with electrocardiogram electrodesand heart rate display. Fur on the abdomen was chemi-cally removed (Nair hair removal lotion; Church & Dwight

Canada, Mississauga, ON, Canada). Ultrasound images

were acquired with a Vevo 770 high-resolution microimag-ing system (VisualSonics, Toronto, ON, Canada) equippedwith a real-time microvisualization VisualSonics 704 scan-head (center frequency, 40 MHz; focal depth, 6 mm). Theregion of interest was fully scanned, and two-dimensionalimages were taken in uniform steps of 50 �m. Ultrasoundexamination was conducted by a trained technician. Im-ages for blood flow were analyzed using a software pro-gram provided with the Vevo 770 imaging system. Ultra-sound images were acquired at three time points: week 1,week 2, and week 3 of treatment. Blood flow to the endo-metriotic lesions was quantified with Image-Pro Plus 6.0software (Media Cybernetics, Silver Spring, MD).

Necropsy and Lesion Assessment in a MouseModel

On day 39 (7 days after completion of treatment), one halfof the mice were sacrificed from the ABT-898 treatmentgroup (n � 3) and from the 5% dextrose control group(n � 3). The abdominal cavity was exposed for grossobservation of the growth and vascularity of endometri-otic lesions. The endometriotic lesions were then excised,snap-frozen in Cryomatrix optimal cutting temperature

0

0.5

1

1.5

2

2.5*

HU

VE

C p

rolif

erat

ion

(450

nm-6

90nm

)

ABT-89

80.1

ng/m

L5 %

dext

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cont

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Figure 1. ABT-898 inhibits HUVEC proliferation in vitro. The proliferation ofHUVECs after ABT-898 or dextrose control treatment was quantified bycalculating the optical density of each treatment group (ie, subtracting theabsorbance at 690 nm from the absorbance at 450 nm). All ABT-898 treatmentgroups exhibited a significant reduction in HUVEC proliferation, comparedwith control. Data were analyzed using one-way analysis of variance with a

Tukey post-test. Data are expressed as means � SD and are representative oftwo independent experiments. *P � 0.05.

ABT-898 Reduces Endometriosis Vascularity 573AJP August 2012, Vol. 181, No. 2

compound (Thermo Scientific, Kalamazoo, MI), andstored at �80°C for future analysis.

Endometriotic Lesion Histology and CD31Immunofluorescence

Endometriotic lesions snap-frozen in Cryomatrix optimalcutting temperature compound from ABT-898-treatedand 5% dextrose control mice were cut into 6-�m serialsections. Sections were transferred onto Superfrost Plusslides (Fisher Scientific, Ottawa, ON, Canada) andstained with H&E or were colocalized with CD31 andDAPI immunofluorescence. For H&E staining, sectionswere air-dried for 30 seconds at room temperature, fixedin 70% ethanol for 2 minutes, and then rinsed in deion-ized water. After staining with Harris’ hematoxylin (FisherScientific) for 60 seconds, sections were rinsed with wa-

A

F

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G

FITC

Brightfield

PBScontrol

ABT-8980.01ng/mL

Arb

itrar

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K

020406080

100120140

TT

***

***

Figure 2. ABT-898 prevents three-dimensional endothelial tube formation in H0.1, 1.0, or 100 ng/mL) or PBS control on a solidified extracellular matrix gel for 1and branching (arrows). ABT-898 caused a reduction in tube formation at 0.01tubes were visualized under fluorescent (A–E) and bright-field (F–J) confocal m

(L) were significantly reduced at all concentrations of ABT-898, compared with PBS cona Tukey post-test. Data are expressed as means � SD and are representative of two ind

ter and dipped in 1% eosin (Fisher Scientific) for 15seconds. Tissues were then dehydrated in increasingconcentrations of ethanol (70%, 95%, and 100%) for 2minutes and xylene for 5 minutes. Slides were then cov-ered with coverslips, using Permount mounting medium(Electron Microscopy Sciences, Hatfield, PA).

CD31 is an endothelial cell-specific marker, and itslocalization was used to qualitatively assess blood vesseldensity in endometriotic lesions. Sections were air-driedfor 30 seconds at room temperature, fixed in 70% ethanolfor 2 minutes, and then rinsed in deionized water. Sec-tions were blocked in 5% bovine serum albumin in PBSfor 60 minutes and then rinsed in PBS for 30 seconds.Sections were then incubated for 120 minutes with phy-coerythrin mouse anti-human CD31 antibody (BD Biosci-ences) at a concentration of 33.3 �g/mL. Sections werethen rinsed in PBS, dehydrated in increasing concentra-

D

I

E

J

BT-898.1ng/mL

ABT-8981ng/mL

ABT-898100ng/mL

L

engthidth

n vitro. HUVECs were incubated with different concentrations of ABT-898 (0.01,Endothelial tube formation was qualitatively assessed by examining tube lengthnd completely abrogated tube formation at 0.1, 1.0, and 100 ng/mL. Endothelialy. L and K: Endothelial cell tube length (yellow lines) and width (blue lines)

C

H

A0

ube lube w

UVECs i8 hours.ng/mL, aicroscop

trol (K). Data were analyzed using repeated measures analysis of variance withependent experiments. ***P � 0.001. Original magnification, �150.

574 Nakamura et alAJP August 2012, Vol. 181, No. 2

tions of ethanol and xylene as described above, andcovered with coverslips with ProLong Gold antifade re-agent with DAPI (Invitrogen-Life Technologies, Burling-ton, ON, Canada). Image-Pro Plus 6.0 software (MediaCybernetics) was used to quantify CD31� staining ofendometriotic lesions, using integrated optical density.

Effects of ABT-898 on Reproductive Outcomesin Mice

Mice that had been chronically treated for 21 days withABT-898 (n � 3) or with 5% dextrose control (n � 3) and thephysiological controls (n � 3) were put in breeding pairs onday 70 to assess the effects of ABT-898 treatment on femalereproduction. Day 70 was chosen to allow these mice tofade out the effects of human estrogen pellets and restorethe estrous cycle. A second pregnancy trial was conductedin which a group of mice treated with ABT-898 alone (no

Day 0:Subcutaneous human β-estradiol implant(15mg/pellet) 6- to 8-week-old femalealymphoid Rag2-/-Ilrg-/- mice (n=12)

Day 5: Induction of endometriosis with human endometrial tissue (n=12)

Day 11-32:Daily intraperitoneal injections:-100 μL ABT-898 (100mg/kg) in 5% dextrose (n=6)

-100 μL 5% dextrose (n=6)

Day 33: End of treatment

Day 39:Sacrifice ABT-898 treated (n=3) and 5 % dextrose control subjects (n=3) to observe endometriotic lesion and vasculature development

Day 60:Release of human estrogen is stopped

Day 70:Set up breeding pairs

Identify co

Collect implantation sites at

estradiol pellet or endometriosis) was included. To ensurethat mice were undergoing normal estrous cycling, vaginalcytology was performed for 4 consecutive days. In brief, thevaginal canal was aspirated with 50 �L of PBS and theaspirate was smeared onto Superfrost Plus slides (FisherScientific). Vaginal cells were air-dried for 5 minutes, fixed inchilled methanol for 60 seconds, and then stained with H&Eas described above. Gestation day 0 was indicated by thepresence of a copulation plug, and animals were sacrificedon gestation day 12. Implantation sites and ovaries werecollected, fixed in 4% paraformaldehyde, dehydrated inincreasing concentrations of ethanol and xylene, and em-bedded in paraffin wax.

Implantation Site Histology and Isolectin Staining

Paraffin-embedded implantation sites cut into 5-�m sec-tions were stained according to a standard H&E protocol.

n plug.

Day 7: Flow cytometry

Day 18: Flow cytometry

Day 32: Flow cytometry

Day 39: Flow cytometry

Figure 3. Experimental outline of investigationinto effects of ABT-898 on endometriotic lesiongrowth, vasculature development, and fecundity.To allow for the growth of human endometrium,60-day, slow-release human estradiol pellets wereplaced subcutaneously in female Rag2�/�Il2rg�/�

(alymphoid) mice (n � 12) on day 0. Endometri-osis was induced on day 5 by surgically placinghuman eutopic endometrium into the peritonealcavity of the human estradiol-primed mice. Fromday 11 to day 32, mice were given daily intraper-itoneal injections of ABT-898 (100 mg/kg in 100 �Lof 5% dextrose, n � 6) or 5% dextrose (100 �L,n � 6). Peripheral blood was collected on days 7,18, 32, and 39 for the detection of SCA-1�/c-Kit�/CD31�/CD45� CEPCs by flow cytometry, and forplasma cytokine profiling. On day 39, mice fromthe ABT-898-treated (n � 3) and 5% dextrose con-trol (n � 3) groups were sacrificed, and endo-metriotic lesions were retrieved for histology andimmunohistology studies. The remaining micewere placed in breeding pairs on day 70 and weresacrificed on gestation day 12, to assess implanta-tion site morphology and litter size outcomes.

pulatio

gestation day 12.

ABT-898 Reduces Endometriosis Vascularity 575AJP August 2012, Vol. 181, No. 2

Briefly, sections were deparaffinized, left in xylene for6 minutes, and then hydrated in decreasing concentra-tions of ethanol (100%, 90%, and 70%) for 4 minuteseach step. Slides were rinsed with running tap water for 3minutes and deionized water for 1 minute. Sections werestained with Harris’ hematoxylin (Fisher Scientific) for 1minute, rinsed with water for 5 minutes, and stained with1% eosin (Fisher Scientific) for 60 seconds. After dehy-dration in increasing concentrations of ethanol (70%,90%, and 100% for 4 minutes each step), sections werekept in xylene for 4 minutes. Slides were then coveredwith coverslips, using Permount mounting medium (Elec-tron Microscopy Sciences). Paraffin-embedded implan-tation sites were cut into 5-�m sections and deparaf-finized as described above. Sections were then incubatedwith isolectin-IB4 conjugated with Alexa Fluor 488 at a con-centration of 1:300 in PBS (Invitrogen-Life Technologies) for120 minutes in the dark. The glycoprotein isolectin, ex-tracted from the seeds of the African legume Griffonia sim-plicifolia, is well known to bind endothelial cells. Sectionswere washed in PBS and stored after putting coverslips withProLong Gold antifade reagent with DAPI (Invitrogen-LifeTechnologies).

Statistical Analysis

All data were analyzed with repeated-measures analysisof variance with a Tukey post-test using SigmaStat 3.0software (Systat Software, San Jose, CA). All data passednormal distribution and variance tests. A P value of �0.05was considered significant.

Results

Effects of ABT-898 on Human Umbilical VeinEndothelial Cell Proliferation and TubeFormation in Vitro

To assess the direct effects of the TSP-1 mimetic peptideABT-898 on endothelial cell proliferation and in vitro an-giogenesis, assays were conducted using HUVECs. Pre-vious studies have demonstrated antiangiogenic and an-tiproliferative effects of ABT-898 on endothelial cells inmouse, rat, and marmoset models.17,21 Here, we dem-onstrate similar effects on a human endothelial cell line.Serial dilutions of ABT-898 (0.1, 1, 10, or 100 ng/mL) and5% dextrose control were incubated with 5 � 103

HUVECs for 24 hours. Even the lowest concentration ofABT-898 (0.1 ng/mL) caused a significant reduction inHUVEC proliferation, compared with control (P � 0.05)(Figure 1). To understand the effects of ABT-898 on en-dothelial tube formation in vitro, HUVECs were plated ona three-dimensional extracellular matrix gel and treatedwith serial dilutions of ABT-898 and PBS control. After 18hours of incubation, HUVECs formed tubes with severalbranching points in the PBS control (Figure 2, A and F).ABT-898 caused a reduction in both tube formation andbranching at 0.01 ng/mL (Figure 2, B and G) and com-pletely abrogated tube formation at 0.1 ng/mL (Figure 2,

C and H), 1 ng/mL (Figure 2, D and I), and 100 ng/mL

(Figure 2, E and J). Tube dimensions (Figure 2L) whenquantified revealed a significant decrease in branchlength and width at all concentrations of ABT-898, com-pared with PBS control (P � 0.001) (Figure 2K).

Characterization of Circulating EndothelialProgenitor Cells in Peripheral Blood ofExperimental and Control Mice

CEPCs are bone marrow-derived progenitor cells re-cruited to the sites of angiogenesis and incorporated intoneovessels. Recent studies have demonstrated recruit-ment and incorporation of CEPCs into neovasculature ofdeveloping endometriotic lesions in mouse models.11,22

Here, we used CEPC levels in peripheral blood of exper-imental mice as an indicator of neoangiogenesis or re-generative inflammatory response to the implanted hu-man endometriotic lesions at four time points (days 7, 18,32, and 39; Figure 3). Using four-color flow cytometry,CEPCs were characterized as SCA-1�/c-Kit�/CD31�/CD45� and detected in peripheral blood of experimentaland control mice on day 7 (after induction of endometri-osis, but before treatment), day 18 (7 days of treatmentwith ABT-898 or 5% dextrose control), day 32 (21 days oftreatment with ABT-898 or 5% dextrose control), and day39 (7 days after completion of treatment). Day 7 levels didnot differ significantly from physiological control baselinelevels, but by day 18 CEPC levels had elevated signifi-cantly in ABT-898-treated mice (P � 0.05) (Figure 4). Byday 32, CEPC levels returned to baseline in ABT-898-treated and 5% dextrose control groups, and remainedthere on day 39 (P � 0.05) (Figure 4). This indicates thatsignificant neovascularization of endometriotic lesions hadnot been initiated by day 7 (2 days after induction of endo-metriosis), but had been by day 18 (13 days after endome-triosis induction), and was complete by day 32 (27 daysafter endometriosis induction) in our mouse model.

Figure 4. Human endometriotic lesions induce elevated CEPC levels in periph-eral blood of mice. Flow cytometry was used to quantify levels of SCA-1�/c-Kit�/CD31�/CD45� CEPCs in peripheral blood. CEPC levels were elevated onday 18 in both ABT-898 and 5% dextrose, compared with day 7 (pretreatment)and physiological control groups. By day 32, however, CEPC levels had droppedbelow day 7 (pretreatment) levels in both the ABT-898-treated and the 5%dextrose control groups, and remained that way on day 39. Data were analyzed

using a repeated-measures analysis of variance. Data are expressed as means �SD and are representative of two independent experiments. *P � 0.05.

18 (1 wetment) i

576 Nakamura et alAJP August 2012, Vol. 181, No. 2

Plasma Cytokine Profile in Experimental andControl Mice

Peripheral blood plasma cytokines were analyzed at fourtime points (days 7, 18, 32, and 39), using a Bio-RadBio-Plex Pro mouse cytokine 23-plex assay on a Bio-Plex200 suspension array system (Figure 5, A–D). Of the 23cytokines analyzed in this multiplex mouse cytokine as-say (Table 1), 16 were present in levels above the detec-tion limit. ABT-898 caused a decrease in the proinflam-matory cytokine G-CSF on day 18 (1 week aftertreatment). Control mice had elevated G-CSF in periph-eral blood but ABT-898 had decreased levels, comparedwith pretreatment. On day 32, ABT-898 G-CSF levelsremained lower, but 5% dextrose controls returned topretreatment levels (data not shown).

Effects of ABT-898 on Neovascularization ofHuman Endometriotic Lesions in a MouseModel

After induction of human endometriotic lesions, micewere treated with ABT-898 (n � 6) or 5% dextrose (n � 6)

Figure 5. 23-Plex cytokine analysis of mouse plasma on days 7 (A), 18 (B), 32 (200 suspension array system. Peripheral blood was collected from experimentadifferent cytokines. Sixteen analytes were found at levels greater than the detectiG-CSF plasma levels had decreased from pretreatment values (day 7) by dayremained lower at day 32 (3 weeks of treatment) and day 39 (1 week after trea

for 21 days (days 11 to 32). At 7 days after the treatment

ended (day 39), half of the ABT-898 mice (n � 3) and halfof the 5% dextrose control mice (n � 3) were eutha-nized. Human endometriotic lesions from mice treatedwith ABT-898 (Figure 6A) had decreased vasculature

39 (D). Plasma cytokines from peripheral blood were analyzed using a Bio-Plexntrol mice on days 7, 18, 32, and 39 and plasma samples were analyzed for 23or the assay. There was no significant difference between groups or time points.ek of treatment) in both ABT-898-treated and 5% dextrose control mice, andn both groups (data not shown). Data are expressed as means � SD.

Table 1. Cytokines in 23-Plex Cytokine Assay

Analyte Limit of detection (pg/mL) Dynamic range (pg/mL)

IL-1� 2 1.79–29,248IL-1� 7 1.95–31,872IL-2 3 3.22–52,736IL-3 2 1.99–32,640IL-4 3 4.33–70,912IL-5 2 1.94–31,744IL-6 2 1.21–19,776IL-9 15 1.12–18,304IL-10 2 2.29–37,586IL-12p40 2 1.02–16,768IL-12p70 4 1.88–30,720IL-13 9 2.20–25,968IL-17A 1 1.71–28,096Eotaxin 148 1.46–23,920G-CSF 1 1.94–31,808GM-CSF 7 2.14–35,008IFN-� 6 1.55–25,472KC 3 1.91–31,360MCP-1 14 2.02–33,152MIP-1� 24 1.97–32,320MIP-1� 2 1.68–27,456RANTES 5 1.43–23,424

C), andl and coon limit f

TNF-� 6 2.28–37,312

ABT-898 Reduces Endometriosis Vascularity 577AJP August 2012, Vol. 181, No. 2

(gross observation) supplying the lesion, compared with5% dextrose controls (Figure 6B). Harvested tissues hadseveral characteristics of endometriotic lesions, includingglandular cysts and endometrial glands, in both the ABT-898 and the 5% dextrose control groups (data not shown).

In another set of experiments, the size of and bloodflow to the endometriotic lesions in ABT-898-treated and

B 5% dextrose controlA ABT-898 treated

DC

2450

2500

2550

2600

2650

2700

2750

2800

ABT-898treated

5 % dextrose

AAre

a (a

rbitr

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)

E

Figure 6. ABT-898 affects neoangiogenesis of endometriotic lesions. Humanendometrium introduced into the abdominal cavity of human estradiol-primed Rag2�/�Il2rg�/� mice adhered to the peritoneum and induced neo-vascularization. A and B: Based on the gross observations, mice treated withABT-898 (n � 3) had reduced peritoneal vasculature supplying the endo-metriotic lesions (A), compared with 5% dextrose controls (n � 3) (B).Circles indicate areas of vasculature; arrows indicate endometriotic lesions.C and D: Doppler imaging of endometriotic lesions was performed at weeks1, 2, and 3 of the ABT-898 treatment. Reduced blood flow to the lesions(dotted outlines) was revealed only at week 3 in ABT-898-treated mice (C),compared with 5% dextrose controls (D). E: Blood flow to the endometrioticlesions was assessed by quantifying the total area representative of vascula-ture in Doppler images, revealing a decrease in blood flow in ABT-898-treated mice, compared with 5% dextrose controls.

control mice were evaluated at time points week 1, week

2, and week 3 of treatment using a high-resolution micro-imaging system and real-time microvisualization. Al-though no significant differences were observed for le-sion size using ultrasound three-dimensional imaging,reduced blood flow to the endometriotic lesion (Figure 6,C–E) was observed in ABT-898-treated mice at week 3 oftreatment with ABT-898 (Figure 6C), compared with con-trol (Figure 6D). Immunofluorescence for CD31, a markerof endothelial cells, was used to visualize microvessels inendometriotic lesions. Human endometriotic lesions fromABT-898-treated mice had reduced levels of CD31� cells(Figure 7, A–C), compared with lesions from the 5% dex-trose control mice (Figure 7, D–F). Intensity of CD31staining (quantified using Image-Pro Plus software pro-gram) was lower in the endometriotic lesions from ABT-898-treated mice (Figure 7G), compared with 5% dex-trose controls. The reduced peritoneal vasculature, alongwith fewer CD31� microvessels, provides evidence thatABT-898 is affecting neovascularization of human endo-metriotic lesions.

Effects of ABT-898 on Reproductive Outcomesin Mice

Current medical and surgical therapies for endometrio-sis, such as the use of GnRH agonists are not compatiblewith pregnancy.1 Here, we assessed the effects of theantiangiogenic peptide ABT-898 on mouse fecundity.Mice not sacrificed on day 39 from the ABT-898 (n � 3)and 5% dextrose control groups (n � 3) were placed inbreeding pairs on day 70. Mice were sacrificed 12 days

PE-CD31 DAPI Merge

ABT-898treated

5% dextrose control

D E

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ensit

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G

Figure 7. ABT-898 causes a reduction in CD31� endothelial cells in endometri-otic lesions. Human endometriotic lesions were stained with anti-human CD31primary antibody conjugated to phycoerythrin, then covered with a coverslipand mounting medium containing DAPI. Images were merged. Lesions fromRag2�/�Il2rg�/� mice treated with ABT-898 (n � 3) (A–C) exhibited reducednumbers of CD31� endothelial cells, compared with 5% dextrose control (n � 3)(D–F). G: Quantification of CD31� staining confirmed reduced vasculariza-

tion in the endometriotic lesions from ABT-898-treated mice, compared withcontrols. Original magnification, �50. Scale bar � 100 �m.

pitheliaOriginal

578 Nakamura et alAJP August 2012, Vol. 181, No. 2

after identification of a copulation plug. Although all micefrom both the ABT-898-treated and the 5% dextrose con-trol groups successfully mated, none were pregnant 12days after identifying the copulation plug. Ovarian histol-ogy revealed that no corpus luteum had formed in eitherABT-898-treated or 5% dextrose control groups (data notshown). We concluded that estrogen from the implanted�-estradiol pellet might have impaired normal folliculo-genesis.

A second mouse experiment was conducted with a sim-ilar experimental outline as described above. Group 1 re-ceived daily injections of 5% dextrose (n � 5), group 2received daily injections of ABT-898 (n � 5), and group 3received daily injections of ABT-898 and implantation of a�-estradiol pellet (n � 5). Treatment lasted for 21 days.Instead of setting up breeding pairs on day 70, we waiteduntil day 90, to ensure sufficient time for fading out effectsof implanted estrogen effects and for the return of mouseestrous cyclicity. To confirm that mice were undergoingnormal estrous cycling, vaginal cytology was analyzedfor 4 consecutive days. Physiological control (Figure 8, A,E, I, and M), 5% dextrose control (Figure 8, B, F, J, and

5% dextrosecontrol

Physiological control

Day 1

Day 2

Day 3

Day 4

A

E

I

M

B

F

J

N

Figure 8. ABT-898 does not affect mouse estrous cycle. All experimental grABT-898 with estradiol (D–P)]. All experimental groups (physiological contrvaginal cell ratio over 4 days of the estrous cycle. Beginning on day 80, vagicycle from ABT-898-treated, ABT-898 with estradiol, physiological controlproportion of leukocytes (blue arrow), cornified cells (black arrow), and efour stages of the estrous cycle (diestrus, proestrus, estrus, and metestrus).

N), ABT-898 treatment (Figure 8, C, G, K, and O), and

ABT-898 treatment plus estrogen (Figure 8, D, H, L,and P) groups all had changes in vaginal cytologyindicative of a normal estrous cycle. Mice were placedin breeding pairs, and sacrificed on gestation day 12. Allmice were pregnant at gestation day 12, and no signifi-cant differences were seen in implantation site numbersbetween control and treatment groups (Figure 9, A–E).Histological evaluation of implantation sites revealed nogross abnormalities of implantation site structure in eithertreatment or control groups (Figure 10, A–H). Fluoro-chrome-tagged isolectin staining exhibited normal vas-cularization of the maternal-fetal interface in the implan-tation sites of all groups (Figure 10, I, J, M, N, Q, R, U, andV), as well as typical maternal vessel structure (Figure 10,K, L, O, P, S, T, W, and X).

Discussion

In the present study, we have elucidated the effects ofantiangiogenic ABT-898 on the neovascularization,growth, and survival of human endometriotic lesions in a

ABT-898treated

ABT-898 withestrogen

C

G

K

O

D

H

L

P

ysiological control (A–M), 5% dextrose control (B–N), ABT-898 (C–O), andextrose control, ABT-898, and ABT-898 with estradiol) exhibited changes insamples were acquired on 4 consecutive days to analyze the mouse estrous% dextrose control mice. Determination of cycle stage was based on thel cells (white arrow) present. Results demonstrate progression through themagnification, �100. Scale bar � 150 �m.

oups [phol, 5% dnal cell, and 5

murine model. Other studies have demonstrated the in-

ABT-898 Reduces Endometriosis Vascularity 579AJP August 2012, Vol. 181, No. 2

hibitory effects of ABT-898 and of the first-generationthrombospondin-1 (TSP-1) mimetic ABT-510 on thegrowth of tumors.23–26 The utilization of ABT-898 as apotential treatment for endometriosis, however, is a novelarea of study. Unlike other antiangiogenic therapiestested in animal models, which target either endothelialcells or proangiogenic factors alone,14,27,28 TSP-1 (mim-icked by ABT-898) uniquely sequesters VEGF while si-multaneously inhibiting proliferation and migration of en-dothelial cells. Furthermore, we have evaluated theeffects of ABT-898 on reproductive success in chroni-cally treated alymphoid mice using a xenograft model ofendometriosis.

ABT-898 mimics the antiangiogenic activity of TSP-1, apotent regulator of neovascularization. At the cellularlevel, TSP-1 induces apoptosis19 and inhibits prolifera-tion29 and migration30 of endothelial cells via the CD36pathway. Angiogenesis is also inhibited by CD36 signal-ing through inhibiting VEGFR-2 up-regulation, whichplays a key role in early stages of vascular develop-ment.31 TSP-1 directly binds and sequesters VEGF,32

and can internalize it through an LRP-1 mechanism.20 Inphysiological angiogenesis involved in the regrowth ofendometrium, endothelial cells sprout from pre-existingvessels, creating tube-like structures.33 Guided byproangiogenic factors, these new branches will anasto-mose with each other to form a solid blood vessel. Duringvessel maturation, a process regulated by the Notch sig-naling pathway,33 protective pericytes are recruited tosurround the basement membrane.33,34 In comparison,endothelial cells lining the newly formed blood vessels ofthe pathological lesions are not stabilized by pericytesand are therefore susceptible to the apoptotic effects of

controlcontrolB 5% dextroseA Physiological

E

Ave

rage

Num

ber

0

2

4

6

8

10

12

14

Physiological control

5 % dextrose control

ABT-898treated

ABT-898with estrogen

Implantation sites

Resorptions

ABT-898.34,35

To understand the antiangiogenic effects of ABT-898 invitro, proliferation and endothelial tube formation assayswere performed, using HUVECs. At concentrations greaterthan 0.01 ng/mL, ABT-898 inhibited proliferation of HUVECsand completely blocked branching of endothelial tubes.Our in vitro findings confirm earlier reports on ABT-510, afirst-generation antiangiogenic peptide, and its effects onapoptosis and proliferation of HUVECs.36,37 Another studyon ABT-898 also confirmed its apoptotic effects on granu-losa cells in vitro.17 To assess the effects of ABT-898 on thegrowth and survival of human endometriotic lesions in vivo,we induced endometriosis in Rag2�/�Il2rg�/� miceprimed with human estradiol. Unlike nude mice com-monly used in endometriosis research, these alymphoidmice lack all mature lymphocytes (T�B�NK�). Becausein our mouse model the NK cells that can be activated innude mice are lacking, our model more widely acceptshuman tissue grafts. Importantly, alymphoid mice do notdemonstrate age-related compensatory immunity or thy-momas, as seen in SCID mice, permitting xenographicstudies of much longer duration.38

Recent evidence has shown that endometriotic lesionsmust recruit bone marrow-derived endothelial progenitorcells (EPCs) into the peripheral blood for the formation ofnew blood vessels at the lesion.11,22,39,40 Indeed, themobilization and recruitment of EPCs from bone marrowto endometriotic lesions has been demonstrated inmouse models of endometriosis in which GFP-positivebone marrow-derived EPCs were found within lesions 7days after induction of endometriosis.11,22,40 Similarly,cancer studies have shown the importance of endothelialprogenitor cell recruitment in the pathological neovascu-

-898 with estrogen

D ABTABT-898 treated

re 9. ABT-898 does not affect implantation sites or litter size. Experimentalwere sacrificed on gestation day 12. Compared with physiological controlnd 5% dextrose control (B), mice treated with ABT-898 (C) or with ABT-898estradiol (D) exhibited no significant differences in implantation site num-(E). Resorption sites were sporadically found, but were evenly distributedng the experimental groups (E). Scale bar � 1 cm.

C

Figumice(A) aandbersamo

larization of tumors.41–43

tation sl structu

580 Nakamura et alAJP August 2012, Vol. 181, No. 2

To understand the correlation of CEPCs with thegrowth of endometriotic lesions in our mouse model,CEPCs were quantified from the peripheral blood of ex-perimental mice on a weekly basis, using four-color flowcytometry. At 1 week after induction of endometriosis, a

Physiological control

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40x

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I J

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U V

DAPIsolectin

2.5x

Figure 10. ABT-898 does not affect implantation site structure or vascularizaA–H: H&E staining of implantation sites from all groups revealed normal stdecidua basalis (DB), and placenta (P). I–X: Isolectin staining of implanexperimental groups (bottom two rows), as well as normal maternal vesse

significant increase in circulating EPCs was observed in

the treated group, which we hypothesized to indicaterecruitment of EPCs from the bone marrow to the endo-metriotic lesions for neovascularization.39 A significantdecrease in EPC levels was observed at 3 weeks afterinduction of endometriosis in the ABT-898-treated group.

ABT-898treated

ABT-898 with estrogen

C D

G H

P

DB

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MLAp

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O P

S T

W X

DAPIIsolectin

Maternal vessel (40x)

plantation sites were harvested from experimental mice on gestation day 12.organization of the mesometrial lymphoid aggregate of pregnancy (MLAp),ites indicated normal vascularization of the maternal-fetal interface in allre (top two rows). Scale bar � 75 �m.

I

tion. Imructural

We presume that extensive recruitment of bone marrow-

ABT-898 Reduces Endometriosis Vascularity 581AJP August 2012, Vol. 181, No. 2

derived EPCs is required during initial neovascularizationof the lesion; in accord, CEPCs were increased in bothABT-898 and control groups at day 18. CEPCs are notrequired in later stages of development, when endometri-otic lesions have fully established their vasculature.42 Nodifferences between EPC levels in treated and control micewere seen for the remainder of the treatment. In a relatedstudy, decreased numbers of EPCs were incorporated intolung carcinoma tumors in mice after treatment with recom-binant VEGI, an inhibitor of endothelial cell proliferation.39

Nonetheless, there is lively debate in the literature regardingthe role of EPCS in neovascularization.

Several recent reports confirm that circulating EPCs donot play a role in the neovascularization of tumors orduring wound repair.44–46 We conducted 23-plex cyto-kine analysis to assess whether ABT-898 modulates theoverall inflammatory response to the human endometri-otic lesions. Plasma from ABT-898-treated mice hadlower levels of G-CSF, compared with controls; however,no other significant differences were found betweentreated and control groups. The alymphoid mice used inthe present study lack immune cells, which normally playa major role in cytokine production, and therefore ourmodel does not allow us to completely assess the effectsof ABT-898 on the inflammatory profiles of treated mice.

Neovascularization of endometriotic lesions is a pro-cess absolutely crucial for the progression and develop-ment of endometriosis, and therefore limiting the bloodsupply to endometriotic lesions could potentially sup-press their growth.47 The inhibitory effects of ABT-898 onendothelial cells may prove to be an effective method oflimiting angiogenesis.17,21 In fact, ABT-898 reduced tu-mor growth and prolonged survival in a mouse model ofepithelial ovarian cancer.21 In support of previous studiessuggesting the antiangiogenic activity of ABT-898 invivo,17,21 we found that gross vascularization of endo-metriotic lesions was reduced in mice treated with ABT-898, compared with control; these findings were furthersupported by results of Doppler ultrasound imaging. Ad-ditionally, the microvessel density of these lesions, visu-alized using CD31� cells, indicated fewer CD31� mi-crovessels in the treated group, compared with controls.This provides strong evidence that ABT-898 not only af-fects blood supply to the endometriotic lesion but alsoaffects vascularization within the lesion itself. Thus, wecan surmise that treatment with ABT-898 limits the bloodsupply of the endometriotic lesions and potentially inhib-its their survival and growth.

Currently, short-term relief from symptoms of endome-triosis is achieved through hormone therapy (includingGnRH agonists, progestins, and oral contraceptives) andsurgery; however, adverse effects and the recurrence ofendometriosis limit use of these approaches.1,48 Mostsignificantly, these therapies can often preclude preg-nancy.1 The majority of the women diagnosed with endo-metriosis are of fertile age, and thus it is of paramountimportance to find a viable treatment option that relievessymptoms while maintaining reproductive status.

In our original pregnancy trial, using mice chronicallytreated with ABT-898, vaginal cytology revealed that

mice given subcutaneous 60-day slow-release estradiol

pellets remained in estrus, compared with physiologicalcontrols, which cycled normally. We postulated that theestradiol pellet kept these mice in estrus, resulting in adepletion of fully developed ova throughout the 60-dayperiod. Ovaries isolated from physiological controls con-tained primary, secondary, and mature follicles, whereasmice with estradiol implants lacked secondary and ma-ture follicles. Previous report indicates that ABT-898 doesnot interfere with ovulation,17 so we can infer that it isperhaps the estradiol pellet that interfered with reproduc-tive success. To overcome this difficulty, we establishedbreeding pairs of ABT-898-treated mice 10 days after therelease of estradiol had ended. To assess effects of thedrug alone on pregnancy, a group treated only with ABT-898 alone (no estradiol pellet and no endometriosis) wasadded. As predicted, mice in all groups yielded normalnumbers of implantation sites by gestation day 12, withfew or no resorptions. Histological analysis confirmed novariation in structural organization of the implantationsites. This suggests that ABT-898 does not interfere withthe reproductive outcomes in chronically treated mice.Moreover, these findings suggest that ABT-898 specifi-cally targets pathological angiogenesis without affectingthe physiological angiogenesis required during preg-nancy or wound healing.

Here, for the first time, we provide evidence that ABT-898can inhibit blood vessel development of human endometri-otic lesions in a mouse model of endometriosis withoutaffecting fecundity. We have shown that ABT-898 inhibitsthe proliferation of endothelial cells in vitro and have shownthat it consequently reduces vascularization of endo-metriotic lesions in vivo. Importantly, despite chronictreatment with ABT-898, mice successfully yielded nor-mal numbers of implantation sites, suggesting that thisdrug does not interfere with fertility. Further research isneeded to elucidate the effects of ABT-898 on the off-spring of treated mice, on ovarian and endometrial phys-iological angiogenesis, and on reproductive outcomes ofmice treated during pregnancy.

Acknowledgments

We thank Abbott Laboratories for providing ABT-898, Dr.Jack Henkin (Northwestern University; formerly at AbbottLaboratories) for his advice, and Queen’s University An-imal Care staff for their excellent support.

References

1. Giudice LC: Endometriosis. N Engl J Med 2010, 362:2389–23982. Bulun SE: Endometriosis. N Engl J Med 2009, 360:268–2793. Adamson GD, Kennedy SH, Hummelshoj L: Creating solutions in

endometriosis: global collaboration through the World EndometriosisResearch Foundation. J Endometriosis 2010, 2:3–6

4. Asante A, Taylor RN: Endometriosis: the role of neuroangiogenesis.Annu Rev Physiol 2011, 73:163–182

5. Halme J, Hammond MG, Hulka JF, Raj SG, Talbert LM: Retrogrademenstruation in healthy women and in patients with endometriosis.Obstet Gynecol 1984, 64:151–154

6. Levy AR, Osenenko KM, Lozano-Ortega G, Sambrook R, Jeddi M,

Belisle S, Reid RL: Economic burden of surgically confirmed endo-metriosis in Canada. J Obstet Gynaecol Can 2011, 33:830–837

582 Nakamura et alAJP August 2012, Vol. 181, No. 2

7. Jacobson TZ, Duffy JM, Barlow D, Koninckx PR, Garry R: Laparo-scopic surgery for pelvic pain associated with endometriosis. Co-chrane Database Syst Rev 2009, (4):CD001300

8. Petraglia F, Hornung D, Seitz C, Faustmann T, Gerlinger C, Luisi S,Lazzeri S, Strowitzki T: Reduced pelvic pain in women withendometriosis: efficacy of long-term dienogest treatment. Arch Gyne-col Obstet 2012, 285:167–173

9. D’Hooghe TM, Debrock S: Endometriosis, retrograde menstruationand peritoneal inflammation in women and in baboons. Hum ReprodUpdate 2002, 8:84–88

10. Risau W: Mechanisms of angiogenesis. Nature 1997, 386:671–67411. Becker CM, Beaudry P, Funakoshi T, Benny O, Zaslavsky A, Zura-

kowski D, Folkman J, D’Amato R, Ryeom S: Circulating endothelialprogenitor cells are up-regulated in a mouse model of endometriosis.Am J Pathol 2011, 178:1782–1791

12. Asahara T, Masuda H, Takahashi T, Kalka C, Pastore C, Silver M,Kearne M, Magner M, Isner JM: Bone marrow origin of endothelialprogenitor cells responsible for postnatal vasculogenesis in physiologi-cal and pathological neovascularization. Circ Res 1999, 85:221–228

13. Becker CM, Sampson DA, Short SM, Javaherian K, Folkman J,D’Amato RJ: Short synthetic endostatin peptides inhibit endothelialmigration in vitro and endometriosis in a mouse model. Fertil Steril2006, 85:71–77

14. Ricci AG, Olivares CN, Bilotas MA, Meresman GF, Barañao RI: Effectof vascular endothelial growth factor inhibition on endometrial implantdevelopment in a murine model of endometriosis. Reprod Sci 2011,18:614–622

15. Ferrara N, Hillan KJ, Gerber HP, Novotny W: Discovery and develop-ment of bevacizumab, an anti-VEGF antibody for treating cancer. NatRev Drug Discov 2004, 3:391–400

16. Laschke M, Elitzsch A, Vollmar B, Vajkoczy P, Menger M: Combinedinhibition of vascular endothelial growth factor (VEGF), fibroblastgrowth factor and platelet-derived growth factor, but not inhibition ofVEGF alone, effectively suppresses angiogenesis and vessel matu-ration in endometriotic lesions. Hum Reprod 2006, 21:262–268

17. Garside SA, Henkin J, Morris KD, Norvell SM, Thomas FH, Fraser HM:A thrombospondin-mimetic peptide, ABT-898, suppresses angiogen-esis and promotes follicular atresia in pre- and early-antral follicles invivo. Endocrinology 2010, 151:5905–5915

18. Yang JL, Zhang CP, Li L, Huang L, Ji SY, Lu CL, Fan CH, Cai H, RenY, Hu ZY, Gao F, Liu YX: Testosterone induces redistribution offorkhead box-3a and down-regulation of growth and differentiationfactor 9 messenger ribonucleic acid expression at early stage ofmouse folliculogenesis. Endocrinology 2010, 151:774–782

19. Volpert OV, Zaichuk T, Zhou W, Reiher F, Ferguson TA, Stuart PM,Amin M, Bouck NP: Inducer-stimulated fas targets activated endo-thelium for destruction by anti-angiogenic thrombospondin-1 andpigment epithelium-derived factor. Nat Med 2002, 8:349–357

20. Greenaway J, Lawler J, Moorehead R, Bornstein P, LaMarre J, PetrikJ: Thrombospondin-1 inhibits VEGF levels in the ovary directly bybinding and internalization via the low density lipoprotein receptor-related protein-1 (LRP-1). J Cell Physiol 2007, 210:807–818

21. Campbell N, Greenaway J, Henkin J, Petrik J: ABT-898 induces tumorregression and prolongs survival in a mouse model of epithelialovarian cancer. Mol Cancer Ther 2011, 10:1876–1885

22. Laschke MW, Giebels C, Nickels RM, Scheuer C, Menger MD: En-dothelial progenitor cells contribute to the vascularization of endo-metriotic lesions. Am J Pathol 2011, 178:442–450

23. Hoekstra R, de Vos FYFL, Eskens FALM, Gietema JA, van der GaastA, Groen HJM, Knight RA, Carr RA, Humerickhouse RA, Verwij J, deVries EG: Phase I safety, pharmacokinetic, and pharmacodynamicstudy of the thrombospondin-1-mimetic angiogenesis inhibitor ABT-510in patients with advanced cancer. J Clin Oncol 2005, 23:5188–5197

24. Yang Q, Tian Y, Liu S, Zeine R, Chlenski A, Salwen HR, Henkin J,Cohn SL: Thrombospondin-1 peptide ABT-510 combined with val-proic acid is an effective antiangiogenesis strategy in neuroblastoma.Cancer Res 2007, 67:1716–1724

25. Yap R, Veliceasa D, Emmenegger U, Kerbel RS, McKay LM, HenkinJ, Volpert OV: Metronomic low-dose chemotherapy boosts CD95-dependent antiangiogenic effect of the thrombospondin peptideABT-510: a complementation antiangiogenic strategy. Clin CancerRes 2005, 11:6678–6685

26. Greenaway J, Henkin J, Lawler J, Moorehead R, Petrik J: ABT-510induces tumor cell apoptosis and inhibits ovarian tumor growth in an

orthotopic, syngeneic model of epithelial ovarian cancer. Mol CancerTher 2009, 8:64–74

27. Becker CM, D’Amato RJ: Angiogenesis and antiangiogenic therapy inendometriosis. Microvasc Res 2007, 74:121–130

28. Becker CM, Sampson DA, Rupnick MA, Rohan RM, Efstathiou JA,Short SM, Taylor GA, Folkman J, D’Amato RJ: Endostatin inhibits thegrowth of endometriotic lesions but does not affect fertility. Fertil Steril2005, 84 Suppl 2:1144–1155

29. Armstrong LC, Björkblom B, Hankenson KD, Siadak AW, Stiles CE,Bornstein P: Thrombospondin 2 inhibits microvascular endothelialcell proliferation by a caspase-independent mechanism. Mol Biol Cell2002, 13:1893–1905

30. Short SM, Derrien A, Narsimhan RP, Lawler J, Ingber DE, Zetter BR:Inhibition of endothelial cell migration by thrombospondin-1 type-1repeats is mediated by beta1 integrins [Erratum appeared in J CellBiol 2005, 169:541]. J Cell Biol 2005, 168:643–653

31. Scappaticci FA: Mechanisms and future directions for angiogenesis-based cancer therapies. J Clin Oncol 2002, 20:3906–3927

32. Gupta K, Gupta P, Wild R, Ramakrishnan S, Hebbel RP: Binding anddisplacement of vascular endothelial growth factor (VEGF) bythrombospondin: effect on human microvascular endothelial cell pro-liferation and angiogenesis. Angiogenesis 1999, 3:147–158

33. Carmeliet P, Jain RK: Molecular mechanisms and clinical applica-tions of angiogenesis. Nature 2011, 473:298–307

34. Laschke MW, Menger MD: In vitro and in vivo approaches to studyangiogenesis in the pathophysiology and therapy of endometriosis.Hum Reprod Update 2007, 13:331–342

35. Bergers G, Song S: The role of pericytes in blood-vessel formationand maintenance. Neuro Oncol 2005, 7:452–464

36. Isenberg JS, Jia Y, Fukuyama J, Switzer CH, Wink DA, Roberts DD:Thrombospondin-1 inhibits nitric oxide signaling via CD36 by inhib-iting myristic acid uptake. J Biol Chem 2007, 282:15404–15415

37. Isenberg JS, Yu C, Roberts DD: Differential effects of ABT-510 and aCD36-binding peptide derived from the type 1 repeats of thrombos-pondin-1 on fatty acid uptake, nitric oxide signaling, and caspaseactivation in vascular cells. Biochem Pharmacol 2008, 75:875–882

38. Bruner-Tran KL, Carvalho-Macedo AC, Duleba AJ, Crispens MA,Osteen KG: Experimental endometriosis in immunocompromisedmice after adoptive transfer of human leukocytes. Fertil Steril 2010,93:2519–2524

39. Liang PH, Tian F, Lu Y, Duan B, Stolz DB, Li LY: Vascular endothelialgrowth inhibitor (VEGI; TNFSF15) inhibits bone marrow-derived en-dothelial progenitor cell incorporation into Lewis lung carcinoma tu-mors. Angiogenesis 2011, 14:61–68

40. Laschke M, Giebels C, Menger M: Vasculogenesis: a new piece ofthe endometriosis puzzle. Hum Reprod Update 2011, 17:628–636

41. Asahara T, Kawamoto A, Masuda H: Concise review: circulatingendothelial progenitor cells for vascular medicine. Stem Cells 2011,29:1650–1655

42. Ahn GO, Brown JM: Role of endothelial progenitors and other bonemarrow-derived cells in the development of the tumor vasculature.Angiogenesis 2009, 12:159–164

43. Lyden D, Hattori K, Dias S, Costa C, Blaikie P, Butros L, Chadburn A,Heissig B, Marks W, Witte L, Wu Y, Hicklin D, Zhu Z, Hackett NR,Crystal RG, Moore MA, Hajjar KA, Manova K, Benezra R, Rafii S:Impaired recruitment of bone-marrow-derived endothelial and hema-topoietic precursor cells blocks tumor angiogenesis and growth. NatMed 2001, 7:1194–1201

44. Purhonen S, Palm J, Rossi D, Kaskenpää N, Rajantie I, Ylä-HerttualaS, Alitalo K, Weissman IL, Salven P: Bone marrow-derived circulatingendothelial precursors do not contribute to vascular endothelium andare not needed for tumor growth. Proc Natl Acad Sci USA 2008,105:6620–6625

45. Wickersheim A, Kerber M, de Miguel LS, Plate KH, Machein MR:Endothelial progenitor cells do not contribute to tumor endothelium inprimary and metastatic tumors. Int J Cancer 2009, 125:1771–1777

46. Bluff JE, Ferguson MWJ, O’Kane S, Ireland G: Bone marrow-derivedendothelial progenitor cells do not contribute significantly to new vesselsduring incisional wound healing. Exp Hematol 2007, 35:500–506

47. Carmeliet P: Angiogenesis in life, disease and medicine. Nature2005, 438:932–936

48. Dodin S, Lemay A, Maheux R, Dumont M, Turcot-Lemay L: Bone

mass in endometriosis patients treated with GnRH agonist implant ordanazol. Obstet Gynecol 1991, 77:410–415