thrombospondin-1 inhibits vegf levels in the ovary directly by binding and internalization via the...

JOURNAL OF CELLULAR PHYSIOLOGY 210:807–818 (2007)

Thrombospondin-1 Inhibits VEGF Levels in theOvary Directly by Binding and Internalization

Via the Low Density LipoproteinReceptor-Related Protein-1 (LRP-1)

JAMES GREENAWAY,1 JACK LAWLER,2 ROGER MOOREHEAD,1 PAUL BORNSTEIN,3

JONATHAN LAMARRE,1 AND JIM PETRIK1*1Department of Biomedical Sciences, University of Guelph, Guelph, Ontario

2Beth Israel Deaconess Medical Center and Harvard Medical School,Boston, Massachusetts

3Departments of Biochemistry and Medicine, University of Washington,Seattle, Washington

VEGF is a potent pro-angiogenic factorwhose effects are opposed by a host of anti-angiogenic proteins, including thrombospondin-1 (TSP-1). We have previously shown that VEGF has important extravascular roles in the ovary and that VEGF and TSP-1 areinversely expressed throughout the ovarian cycle. To date, however, a causal interaction between TSP-1 and VEGF has not beenidentified.Here, we show that TSP-1 has a direct inhibitory effect on VEGF by binding the growth factor and internalizing it via LRP-1. Mice lacking TSP-1 are subfertile and exhibited ovarian hypervascularization and altered ovarian morphology. Treatment ofovarian cellswith TSP-1 decreasedVEGF levels and rendered the cellsmore susceptible to TNFa-induced apoptosis. KnockdownofTSP-1, through RNA interference, resulted in overexpression of VEGF and reduced cytokine-induced apoptosis. In conclusion, wedemonstrate adirect inhibitory effect of TSP-1onVEGF in theovary. TSP-1’s regulation ofVEGFappears to be an importantmediatorof ovarian angiogenesis and follicle development. J. Cell. Physiol. 210: 807–818, 2007. � 2006 Wiley-Liss, Inc.

The growth of normal tissues and pathologicalstructures such as tumors are dependent upon theformation of blood vessels for nutrient delivery andwaste removal (Folkman, 1992). Growth of new vascu-lature is regulated by a balance between pro- and anti-angiogenic factors. A potent pro-angiogenic factor isvascular endothelial growth factor (VEGF), which is aheparin-binding glycoprotein secreted as a homodimerthat stimulates endothelial cell proliferation and migra-tion (Bernatchez et al., 2002; Castellon et al., 2002),promotes new vessel formation, increases vascularpermeability (Ferrara, 2004), and acts as a survivalfactor for endothelial cells in vitro and in vivo (Gerberet al., 1998; Jia et al., 2004). In addition to its effects onendothelial cells, we recently reported that VEGFprotects ovarian cells from apoptosis by signalingthrough VEGFR-2 expressed by these cells (Greenawayet al., 2004).

The effects of pro-angiogenic factors are balanced byanti-angiogenic factors such as members of the throm-bospondin (TSP) family, which consists of five proteins(TSP-1–5), of which TSP-1 and -2 share structural andfunctional homology (Bornstein, 1992; Adams andLawler, 2004). TSP-1 is a secreted glycoprotein locatedin the extracellular matrix that has been shown to be apotent inhibitor of angiogenesis (Lawler, 2002; Arm-strong and Bornstein, 2003; Wang et al., 2003; Cursiefenet al., 2004; Lawler and Detmar, 2004). The mechanismsby which the TSPs inhibit angiogenesis are not yet fullyunderstood, although recent work has suggested somepossibilities. One mechanism may involve specificinteractions with pro-angiogenic factors to inhibit theirexpression and function. TSP-1 has been shown tomaintain an inverse relationship in expression with thepotent pro-angiogenic factor, VEGF, in a number ofphysiological and pathological systems (Detmar, 2000).

In addition, a direct interaction of VEGF with TSP-1 andwith a TSP-1 type I repeat domain of connective tissuegrowth factor has been reported (Gupta et al., 1999;Inoki et al., 2002). TSP-1 is also known to bind ligandsand interact with the low-density lipoprotein receptor-related protein (LRP-1), resulting in internalization anddegradation of the protein (Mikhailenko et al., 1995;Emonard et al., 2004; Wang et al., 2004). LRP-1 is amember of the low-density lipoprotein receptor genefamily, which also includes the LDL, VLDL, andapolipoprotein E receptor-2 (Nykjaer and Willnow,2002; Strickland et al., 2002). These family membersare endocytic receptors that regulate the concentrationof extracellular ligand by transporting ligand throughclathrin-coated pits into intracellular vesicles. LRP-1 isexpressed in a host of cell types, including endothelialcells (Yamamoto et al., 1997) and granulosa cells in theovary (25Moestrup et al., 1992). Since TSP-1 binds toboth LRP-1 and matrix metalloproteinases, it serves tomediate the clearance of MMPs from the extracellularspace (Bein and Simons, 2000; Yang et al., 2001). Ashared feature of the members of the LDL receptorfamily is the inhibition of ligand interaction by a 39-kDa

� 2006 WILEY-LISS, INC.

Contract grant sponsor: Natural Sciences and EngineeringResearch Council of Canada; Contract grant sponsor: OntarioCancer Research Network; Contract grant number: 047142;Contract grant sponsor: NIH; Contract grant number: HL68003.

*Correspondence to: Dr. Jim Petrik, University of Guelph,Guelph, Ontario, Canada N1G 2W1. E-mail: [email protected]

Received 21 July 2006; Accepted 7 September 2006

DOI: 10.1002/jcp.20904

receptor-associated protein (RAP). RAP acts as amolecular chaperone and is involved in the folding oflipoprotein receptors, resulting in a conformationalchange that can prevent ligand binding (Neels et al.,1999; Bu and Marzolo, 2000).

In addition to ligand-binding to pro-angiogenic fac-tors, TSP-1 may also affect vascular formation byinducing endothelial cell apoptosis and inhibitingendothelial cell proliferation in part through its inter-action with CD36 (Dawson et al., 1997; Guo et al., 1997;Armstrong and Bornstein, 2003). The signaling path-way that leads to apoptosis includes recruitment of theSrc-family tyrosine kinase, p59fyn, and the activation ofp38 stress kinase and caspases (Jimenez et al., 2000;Yano et al., 2002; Sund et al., 2005). While stimulatingendothelial cell apoptosis, TSP-1 simultaneously inhi-bits proliferative and migratory influences of othermolecules such as estrogen (Sengupta et al., 2004).

The ovary provides a unique opportunity to studyangiogenic processes, as it is one of the few tissues in theadult that undergoes repetitive blood vessel formation,maturation, and regression (Tsafriri, 1995). We haveshown previously that TSP-1 and VEGF are inverselyexpressed, with VEGF expression levels increasingthroughout ovarian follicle development, and TSP-1levels decreasing as follicle progression occurs (Petriket al., 2002; Greenaway et al., 2004). This inverserelationship has been seen in other normal systems andalso in pathological conditions, such as in tumorformation, where VEGF levels are elevated and TSP-1expression is reduced (Doll et al., 2001; Kwak et al.,2002). In experimental tumors in which TSP-1 expres-sion is reduced, VEGF increases to a level where it canoverride the inhibitory effect of TSP-1 on angiogenesis(Gonzalez et al., 2004; Fontana et al., 2005). TSP-1 hasalso been implicated as a functional mediator of the anti-angiogenic effect of chronic low-dose metronomic che-motherapeutic approaches (Bocci et al., 2003; Kerbeland Kamen, 2004). Although an inverse relationshiphas been observed in a number of physiological systems,a characterization of the interaction between TSP-1 andVEGF has not been performed.

The generation of TSP-1-null mice has furtherilluminated the importance of this protein in inhibitingangiogenesis. TSP-1-null mice demonstrate signifi-cantly increased retinal vascularization (Wang et al.,2003) and display two to threefold increase in tumorgrowth rates (Lawler et al., 2001; Sund et al., 2005).Interestingly, these mice also have reduced litter size,suggesting reproductive dysfunction (Lawler et al.,1998).

This study characterizes the ovarian morphology ofTSP-1 null mice and identifies mechanisms by whichTSP-1 might directly influence VEGF levels in theovary.

EXPERIMENTAL PROCEDURESCell lines and tissue collection

Spontaneously immortalized rat granulosa cells(SIGC) were a generous gift from Dr. Robert Burghardt(Texas A&M University, College Station, TX). LRP1þ

and LRP1� Chinese hamster ovary (CHO) cells werekindly donated by Dr. David Fitzgerald (NCI, BethesdaMD). SIGC were cultured in DMEM/F12 supplementedwith 10% FBS and 2% penicillin/streptomycin (LifeTechnologies, Grand Island, NY) and treated asdescribed below. CHO cells were maintained in DMEMsupplemented with 10% FBS and 2% penicillin/strepto-mycin (Life Technologies) and treated as described

below. Murine microvascular endothelial cells (ATCCNumber CRL-2586) were purchased from the AmericanType Culture Collection (ATCC, Manassus, VA) andmaintained in DMEM supplemented with 10% FBS and2% penicillin/streptomycin (Life Technologies).

TSP-1-null mice were generated as described pre-viously and backcrossed eight times to the C57Bl-6background (Lawler et al., 1998). Ovaries were removedvia laparotomy, with one ovary being immersed imme-diately in liquid nitrogen and stored at�808C for proteinisolation, and the other ovary fixed overnight in 10%(vol/vol) neutral-buffered formalin at 48C for immuno-histochemistry/immunofluorescence experiments.

Small interfering RNA and transfection reagents

StealthTM siRNA targeting TSP-1 (BLOCK-ITTM

RNAi Express; 1299003) and negative control siRNAswere purchased from Invitrogen. Negative controls in-cluded in this study were vehicle control and StealthTM

RNAi Negative Control with medium GC content(Invitrogen). Cells were transfected with siRNAs usinglipofectamine 2000 (Invitrogen) in Opti-MEM.

Transfection of small interfering RNAs

SIGC were plated in DMEM-F12 at a density of2� 105 cells/5 ml in 60-mm dishes or 1� 105 cells/2 mlDMEM-F12 on glass coverslips in six-well plates. Atapproximately 50% confluence, cells were transfectedwith 200 nmol/L siRNA in lipofectamine 2000 reagentaccording to the manufacturer’s instructions. Briefly,20ml lipofectamine 2000 was diluted in 500ml Opti-MEMand incubated for 5 min at room temperature. In aseparate tube, 10 ml of 50 mmol/L siRNA was dilutedin 450 ml of Opti-MEM. Diluted oligofectamine (250 ml)was added to the diluted siRNA and the complex wasincubated for 20 min at room temperature. Cells werewashed in Opti-MEM and 1.5 ml Opti-MEM was addedto each 60-mm dish and 500 ml Opti-MEM was added toeach well of the 6-well plates. siRNAþOpti-MEMcomplexes (500 ml) were added to each dish/well. Thefinal concentration of the siRNA was 200 nmol/L. After6 h, 2 ml of DMEM-F12 with 10% FBS was addedwithout removing the transfection media. Transfectionswere allowed to continue for 48 h. Transfection controlsincluded medium controls (cells in Opti-MEM), vehiclecontrols (20 ml lipofectamine 2000 in 500 ml Opti-MEM)and transfection with negative control siRNA (StealthTM

RNAi Negative Control scrambled sequence withmedium GC content) as described.

Immunohistochemistry/immunofluorescence

Five micrometer-thick paraffin embedded ovariantissue sections from TSP-1-null and wild-type (C57BL-6) mice were incubated for 24 h at 48C in a humidifiedchamber with mouse anti-CD31 (1:500 dilution;Pharmingen, Mississauga, ON), rabbit polyclonal anti-VEGF (1:600 dilution; Santa Cruz Biotechnology, CA),or rabbit polyclonal anti-VEGFR-2 (1:500 dilution,Sigma, Oakville, ON). Antisera were diluted in0.01 PBS (pH 7.5) containing 2% (wt/vol) BSA and0.01% (wt/vol) sodium azide (100 ml/slide). All subse-quent incubations were at room temperature. Biotiny-lated anti-mouse or anti-rabbit IgGs (both 1:100dilution; Vector Laboratories, Burlingame, CA) werediluted in the same buffer and applied for 2 h. The slideswere then washed in PBS and incubated with avidin andbiotinylated horseradish peroxidase (1:30 dilution)(Extravidin, Sigma). Peptide immunoreactivity was

808 GREENAWAY ET AL.

Journal of Cellular Physiology DOI 10.1002/jcp

localized by incubation in fresh diaminobenzidinetetrahydrochloride (DAB tablets, 10 mg, Sigma) with0.03% (vol/vol) hydrogen peroxide for 2 min. Tissuesections were counterstained with Carazzi’s Hematox-ylin for 1 min. The percentage of immunopositive versusimmunonegative follicular cells was calculated withintegrated morphometry software (Metamorph, Uni-versal Imaging Inc., California).

For immunofluorescence experiments, SIGC werecultured to approximately 80% confluence on glasscoverslips housed in 6-well dishes. SIGC were serum-starved and treated with 50 ng/ml VEGF, or 100 ng/mlTSP-1, or a combination of VEGF and TSP-1, overnight.After treatment, cells were fixed in 10% neutral bufferedformalin and permeabilized in 0.1% Triton X-100(Sigma) for 15 min at room temperature. Cells wereblocked in PBS containing 5% (wt/vol) bovine serumalbumin (BSA) and 0.01% (wt/vol) sodium azide andthen primary antibodies (VEGF, 1:600 dilution, SantaCruz; VEGFR2, 1:500 dilution, Santa Cruz; proliferat-ing cell nuclear antigen (PCNA), 1:600 dilution, Sigma;Bcl-2, 1:500 dilution, Santa Cruz; Fas, 1:600 dilution,Pharmingen) were added to slides overnight at 48C.Secondary antibodies, conjugated to Alexa Fluor 488 orAlexa Fluor 594 dyes (Molecular Probes, Invitrogen,Burlington, ON), were added for 2 h at room tempera-ture, followed with application of antifade (ProLongGold, Molecular Probes). Cells were imaged with aninverted Olympus microscope using phase contrast todemonstrate cell morphology. Fluorescence stainingwas captured, and overlay of immunofluorescence andphase contrast images was performed using integratedmorphometry software (Metamorph).

To establish specificity of staining in histology andfluorescence experiments, the primary antiserum wassubstituted with non-immune serum and secondaryantibody was omitted. In each case, staining wasabolished.

Detection of apoptosis

To determine the effect of TSP-1 on granulosa cellapoptosis, SIGC were cultured to 90% confluence oncoverslips, serum starved overnight and then culturedin the presence or absence of 100 ng/ml TNFa (which wehave previously shown to be an effective inducer ofapoptosis in these cells (7)), 50 ng/ml VEGF andincreasing concentrations of purified TSP-1 (10, 50,100, 1,000 ng/ml) for 6 h. After treatment, cells werewashed and fixed in 10% (vol/vol) neutral bufferedformalin. To evaluate the effect of TSP-1 knockdown onSIGC apoptosis, TSP-1 RNAi experiments were con-ducted as described above. Cells either remaineduntreated in serum-free DMEM-F12, or were subjectedto cytokine challenge (100 ng/ml TNFa) in the presenceor absence of 50 ng/ml VEGF for 6 h. After treatment,cells were fixed in 10% (vol/vol) neutral bufferedformalin overnight at 48C. Detection of immunofluores-cence in apoptotic cells was performed using a TUNELassay according to manufacturer’s instructions (RocheApplied Science, Laval, Quebec). Briefly, after fixationcells were permeabilized in 0.1% (vol/vol) Triton X-100(Sigma), washed in PBS and incubated with the FITC-conjugated TUNEL enzyme for 60 min to detect DNAfragmentation. Nuclei were counterstained with DAPIand imaged with an Olympus BX-61 microscope andintegrated morphometry software (Metamorph). Foranalysis, 10 fields of view at magnification 250� werequantified in each experiment (n¼3). Apoptosis wasquantified as the percentage of TUNEL-positive cells.

Western blotting

Twenty microgram of total protein extracted fromTSP-1-null and wild-type ovarian lysates and from cellssubjected to TSP-1 RNAi was run on an 8% SDS–PAGEseparating gel. Commercially available VEGF proteinstandard (Santa Cruz Biotechnology) was used aspositive control for blots probing for VEGF. Theseparated proteins were electro-transferred to PVDFmembrane (Millipore, Billerica, MA). Membranes wereblocked overnight with 5% (wt/vol) skim milk at 48C andthen were incubated for 2 h at room temperature withrabbit polyclonal anti-VEGF (1:600 dilution, SantaCruz) or mouse monoclonal anti-TSP-1 (1:500 dilution,Abcam, Cambridge, MA) on a rocking platform. Afterwashing with TTBS (TBS, 1% (vol/vol) Tween 20) and re-blocking, blots were incubated with peroxidase-conju-gated anti-rabbit secondary antibody (1:1,000 dilution;Vector Laboratories) or anti-mouse IgG (1:1,000 dilu-tion, Vector Laboratories) for 1 h at room temperatureon a rocking platform. Reactive protein was detectedwith ECL chemiluminescence (Boehringer Manheim,MD) and Konica medical X-ray film (Wayne, NJ). AfterECL detection, membranes were probed with a-tubulinantibody (1:1,000 dilution, Santa Cruz) and finallystained with Coomassie Blue to ensure equal proteinload.

Cell binding and internalization assays

Purified human VEGF (R&D Systems, MN) andhuman TSP-1 (Sigma) were fluorescently-labeled withAlexa Fluor 488 Protein Labeling Kit (Molecular Probes)according to the manufacturer’s instructions. Afterconjugation of the proteins, SIGC (approximately 90%confluent) were cultured in the presence of labeledVEGF (50 ng/ml) or TSP-1 (100 ng/ml) overnight inserum-free DMEM/F12. For labeled VEGF, unlabeledpurified TSP-1 (100 ng/ml) was also added to media, andfor labeled TSP-1, unlabeled VEGF (50 ng/ml) wasadded. Following treatment, SIGC were fixed with 10%(vol/vol) neutral buffered formalin at room temperaturefor 1 h, followed by PBS washes. Immunofluorescenceexperiments were performed as described above withVEGF-treated SIGC incubated overnight with a goatpolyclonal anti-TSP-1 antibody (1:500 dilution; SantaCruz) and TSP-1-treated SIGC incubated with a rabbitpolyclonal anti-VEGF antibody (1:600 dilution, SantaCruz). Protein and antibody localization and co-localiza-tion was determined with laser scanning confocalmicroscopy (Olympus Fluoview 500 scanning confocalmicroscope) and integrated morphometric analysis(Image Pro Plus, MediaCybernetics, SanDiego,CA).

To determine whether TSP-1-mediated internaliza-tion of VEGF occurred through the LRP-1, initialexperiments were performed on LRP-1-positive andLRP-1-negative CHO cells. To evaluate the role of LRP-1in TSP-1-mediated VEGF internalization in granulosacells, the above protein labeling and internalizationexperiment was repeated on SIGC and murine micro-vascular endothelial cells (mEC) that either remainedunconditioned or were pre-treated with 500 nM RAP.RAP is a known antagonist of LRP-1 and prevents ligandbinding to the receptor (Bu and Marzolo, 2000; Lazicet al., 2003). Human RAP was expressed in bacteria as afusion protein with glutathione S-transferase. The RAP-GST fusion protein was prepared and purified asdescribed previously (Laithwaite et al., 2001).

SIGC, mEC, and LRP-positive and -negative CHOcells were seeded into 12-well dishes (Corning, NY) at

TSP-1 BINDS AND INTERNALIZES VEGF IN THE OVARY 809


1�105 cells/well 24 h prior to the assay in DMEM/F12 at378C, and 5% CO2 in air. After incubation, cells werecultured with 1 nM 125I VEGF (Amersham Life Sciences,Oakville, ON) for the times indicated (15, 30, 60, 90, 120,150, 300 min) in the absence or presence of 500 nM RAP.After incubation, cells were washed in PBS and releasedwith trypsin (50 mg/ml) and proteinase K (50 mg/ml) inbuffer (containing 5 mM EDTA). Internalized isotopewas measured with a gamma counter and corrected bysubtracting the amount of degradation products presentin the absence of cells.

Statistical analysis

Results are reported as mean� standard error of themean (SEM). For densitometric analysis of immuno-blots, statistical differences between groups were mea-sured with 2-way analysis of variance (ANOVA),followed by Tukey’s post-hoc tests for individual differ-ences when indicated by ANOVA (a¼0.05). For in vitroexperiments, data were collected from three indepen-dent cultures for each treatment group, each in tripli-cate. For morphometric analysis of histologic images, sixseparate follicles from TSP-1-null and wild-type micewere used and the entire follicle was included in theanalysis. One-way ANOVA, followed by Bonferroni’stest for differences between means was used to assessstatistical differences between the percentage of immu-nopositive follicular and cultured cells. Data wereanalyzed using SAS statistical analysis software (SASInstitute, Cary, NC). The level of significance was set atP<0.05.

RESULTSFollicle and vascular morphology is

altered in TSP-1-null mice

Using integrated morphometric image analysis soft-ware (MetaMorph), ovarian follicle number and sizewere quantified from paraffin-embedded ovaries fromwild-type and TSP-1-null mice. TSP-1-null mice exhib-ited a significant (P<0.05) increase in the number ofboth pre-antral and antral follicles (Table 1). Along withthe increase in follicle number, the mean antral folliclesize was reduced in TSP-1 null mice (36�4 mm)compared to wild-type controls (44�6 mm) (Table 1).To determine changes in ovarian vasculature, ovariesfrom wild-type and TSP-1-null mice were immunos-tained with an anti-mouse CD31 antibody, whichspecifically stains endothelial cells (Fig. 1A). Ovarianvessel density was calculated as the area of CD31-positive blood vessels divided by the total tissue area.TSP-1-null mice demonstrated a threefold (P< 0.05)increase in ovarian blood vessel density, compared tocontrols (Fig. 1C). In addition to increased vesseldensity, there was a significant increase in CD31 proteinin whole ovary homogenates in TSP-1-null mice,compared to controls (Fig. 1B).

Ovaries of TSP-1-null mice expresselevated levels of VEGF

Ovaries were collected from TSP-1 null and wild-typemice and subjected to immunohistochemistry andWestern blot analysis. TSP-1 null ovaries exhibitedan increase in VEGF staining, which was primarilylocalized to endothelial, granulosa, and theca cells(Fig. 2A). Western blot analysis demonstrated a sig-nificant (P<0.05) increase in VEGF protein in ovariesfrom TSP-1 null mice compared to wild-type controls(Fig. 2B).

TSP-1 reduces VEGF levels in granulosacells and induces apoptosis

To evaluate whether TSP-1 has a direct inhibitoryeffect on VEGF levels, SIGC were cultured in theabsence or presence of purified TSP-1 at varyingconcentrations (10, 50, 100, 1,000 ng/ml). Treatment at50 and 100 ng/ml significantly (P<0.05) reduced VEGFprotein levels, compared to untreated cells (Fig. 3A).As our previous studies show that VEGF is a potentcytoprotective factor for granulosa cells (Greenawayet al., 2004), we wanted to evaluate whether the TSP-1-induced reduction in VEGF levels would make the SIGCmore vulnerable to TNFa challenge. Serum-starved,VEGF-treated SIGC were more vulnerable (P<0.05) to100 ng/ml TNFa in the presence of 100 or 1,000 ng/mlTSP-1 (Fig. 3B).

TSP-1 alters the expression of proliferativeand apoptotic proteins in vitro

Immunofluorescence (Fig. 4A,B) and immunoblot(Fig. 4C) experiments were performed on untreatedSIGC, and those treated either with 100 ng/ml TSP-1alone or with 100 ng/ml TSP-1 and 50 ng/ml VEGF. TSP-1 100 ng/ml significantly reduced the levels of VEGF andits main signaling receptor, VEGFR2 (Fig. 4). Concomi-tant treatment of 100 ng/ml TSP-1 and 50 ng/ml VEGFresulted in levels of VEGF and VEGF R2 similar to thatof controls. The rate of granulosa cell proliferation, asdetermined by PCNA expression, was unchanged byTSP-1 or combination treatment (Fig. 4). Expression ofcytoprotective proto-oncogene bcl-2 was reduced aftertreatment with TSP-1, while concurrent treatment withboth TSP-1 and VEGF resulted in bcl-2 levels similar tocontrols (Fig. 4). Conversely, exposing SIGC to 100 ng/ml TSP-1 significantly (P<0.05) increased expression ofpro-apoptotic Fas (Fig. 4), and this increase wasreversed when cells were cultured in the presence ofboth TSP-1 and VEGF.

TSP-1 binds VEGF resulting ininternalization by LRP-1

The above experiments demonstrate that TSP-1 hasan inhibitory influence on VEGF levels in SIGC. TSP-1has been shown to bind VEGF (Gupta et al., 1999) andwe wanted to determine whether a mechanism by whichTSP-1 inhibited VEGF levels was through binding andinternalization via LRP-1. To evaluate whether TSP-1acted as a molecular linker that formed a complex withVEGF, which was in turn internalized by LRP-1, weperformed reciprocal fluorochrome labeling of TSP-1and VEGF. Internalization was followed in the presenceor absence of the LRP-1 antagonist, RAP, and in CHOcells known to possess or be deficient in LRP-1. In LRP-1-positive CHO cells, TSP-1 and VEGF co-localizedintracellularly following co-incubation, whereas inter-nalization did not occur in LRP-negative CHO cells (data

TABLE 1. Follicle morphology of wild-type and TSP-1-null mice

Follicle number Follicle diameter (mm)

Pre-antral Antral Pre-antral Antral

Wild-type 21�4 10� 3 31�6 44�6TSP-1-null 28�5* 17� 5* 28�5 36�4*

Total follicle number and follicle diameters were calculated from a minimum of 10ovaries at the pre-antral and antral stages of development from wild-type andTSP-1-null mice.* denotes statistically significant difference (P< 0.05) between TSP-1-null andwild-type group.



not shown). In SIGC, labeled TSP-1 co-localized intra-cellularly with VEGF (Fig. 5A). Reciprocal experimentsshowed that labeled VEGF co-localized with TSP-1 incultured granulosa cells (Fig. 5A). This internalizationwas abolished in the presence of 500 nM RAP (Fig. 5A).In another labeling experiment, labeled TSP-1 co-localized with VEGF in LRP-1-positive CHO cells,whereas no internalization was visible in LRP-deficientCHO cells (data not shown). Using 125I labeled VEGF,SIGC, mEC and LRP-1þ CHO cells accumulatedmaximal isotope by approximately 90 min in culture,at which time radioactivity began to diminish (Fig. 5B).In LRP-1-deficient CHO cells or in the presence of500 nM RAP, however, 125I VEGF was not internalizedthroughout the culture period (Fig. 5B). RNA inter-ference of TSP-1 also inhibited 125I-VEGF internaliza-tion (Fig. 5B), as described in more detail below.

TSP-1 knockdown increases VEGF levels andinhibits VEGF internalization

SIGC were transfected with siRNA directed againstTSP-1 to specifically address whether TSP-1 directlyaffected VEGF levels in granulosa cells and to evaluatethe role of TSP-1 in VEGF internalization. Figure 6Aand B show immunofluorescence and Western blotanalyses of SIGC after 48 h transfection with vehiclecontrol, RNAi control, and TSP-1 RNAi constructs. TSP-1 RNA interference resulted in approximately an 80%reduction in protein expression by SIGC, as determinedby immunofluorescence staining and integrated mor-phometry analysis (data not shown). TSP-1 proteinlevels in cells transfected with vehicle or RNAi controlwere similar to TSP-1 expression in non-transfectedcells. In cells transfected with TSP-1 siRNA, there was a

Fig. 1. Expression of CD31 and quantification of vessel density inovaries from wild-type and TSP-1-null mice. A: Ovaries from wild-typeand TSP-1 null mice were immunostained for CD31 (brown stain).Ovaries of wild type mice demonstrated peri-follicular, thecal, andluteal immunopositive blood vessels. TSP-1-null mice exhibitedincreased expression of CD31, with the presence of large CD31-positive blood vessels. B: Western blot; there was a significant

increase in CD31 protein in ovaries from TSP-1-null versus wild-typemice. C: Ovarian vessel density was calculated as the total area ofCD31-positive blood vessels in wild-type and TSP-1- null mice. TSP-1null ovaries had a significantly elevated blood vessel density,compared to wild-type controls. For the Western blot graph, * denotesa statistical difference in CD31 protein compared to wild-type controls(P< 0.05).



significant increase in VEGF, as identified by immuno-fluorescence (Fig. 6A) and by Western blot analysis(Fig. 6B). TSP-1 knockdown resulted in a significantincrease in VEGF expression. As we know that VEGF iscytoprotective for granulosa cells, we wanted to deter-mine the effect of TSP-1 inhibition on SIGC’s response tocytokine-induced apoptosis. Serum starvation resultedin a basal apoptotic rate of approximately 16�5%(Fig. 6C). Exposure to 100 ng/ml TNFa caused asignificant (P<0.05) increase in apoptosis, with62�14% of the cells dying. When VEGF (50 ng/ml)was added, together with vehicle, and in RNAi controltransfections, the incidence of apoptosis decreased to15�7% and 14�5%, respectively. There was a furthersignificant (P<0.05) reduction in apoptosis (5� 3%) inSIGC transfected with TSP-1 siRNA (Fig. 6C). We have

shown that TSP-1 binds and internalizes VEGF (Fig. 5),possibly contributing to the reduced VEGF expressionthat occurs following exposure to TSP-1 (Fig. 3). Weperformed labeling and internalization experiments onSIGC that had TSP-1 knocked down through RNAinterference to determine whether VEGF internaliza-tion occurred through TSP-1 specifically. In SIGCsubjected to TSP-1 RNAi, there was a reduction inVEGF internalization of approximately 80%, comparedto untreated SIGC (Fig. 5B), which corresponded to theapproximate 80% reduction in TSP-1 expression due toRNAi.

DISCUSSION

Regulation of angiogenesis requires a complexinteraction between pro- and anti-angiogenic factors.

Fig. 2. Expression of VEGF in ovaries from wild-type and TSP-1-null mice. A: Ovaries from TSP-1-nullmice exhibited an increase in VEGF-positive cells (brown stain) that were localized to the corpus luteum,granulosa/theca cells of the follicle, and endothelial cells. B: There was a significant increase VEGFprotein in TSP-1-null ovaries compared to wild-type ovaries, as measured by Western blotting. For theWestern blot graph, * denotes a statistically significant difference in VEGF protein compared to wild-typecontrols (P< 0.05).



Disruption of this balance can result in vasculardisorders that contribute to pathologies such as tumorformation (Scharovsky et al., 2004; Nyberg et al., 2005).In the ovary, follicular development and function areinherently dependent upon vascularization, whichoccurs in a tightly regulated fashion. Avascular primor-dial follicles rely on nutrient delivery from the stroma(Fraser and Wulff, 2001). After recruitment and initialdevelopment of a primary follicle, a distinct thecal celllayer is formed and endothelial cells are recruited fromneighboring blood vessels in the stroma (Barboni et al.,

2000). As the follicle progresses to the antral phase,blood vessel formation continues in the thecal layer andangiogenic growth into the avascular granulosa celllayer is prevented by its basement membrane. As thefollicle continues to grow, approximately 25%–30% ofproliferating cells are endothelial cells that are extend-ing the vascular network within the thecal layer (Fraserand Wulff, 2001). It is thought that the level ofvascularization may be a decisive factor in selectionof the dominant follicle by increasing the supply ofnutrients, growth factors, and hormones, and thereforesupporting the growth of the dominant follicle (Berishaet al., 2000; Zimmermann et al., 2001). After ovulationoccurs, the follicle wall collapses, the granulosa celllayer involutes, and there is a disintegration of thebasement membrane (Tsukada et al., 1996). These earlyluteal events are accompanied by differentiation of thethecal and granulosa cells into steroidogenically activeluteal cells and an invasion of new blood vessels thatdevelop from pre-existing vasculature. This explosiveincrease in angiogenesis continues so that by the mid-luteal phase, virtually every differentiated luteal cell isin contact with the endothelium. As a result, the maturecorpus luteum is reported to have the highest blood flowof any tissue in the body (Wiltbank et al., 1988).

In addition to the known angiogenic effects of VEGFin the ovary, we have previously demonstrated thatovarian granulosa cells express the VEGF kinasereceptor VEGFR-2 (Flk-1) (Greenaway et al., 2004).Results from the current study show that the loss of theinhibitory effect of TSP-1 in TSP-1-null mice results inincreased levels of follicular VEGF. VEGF exerts acytoprotective effect on granulosa cells during growthand development of the follicle and the elevated VEGFpresent may elicit a stronger cytoprotective effect on thegranulosa cells, inhibiting granulosa cell apoptosiswhich is a necessary component of follicle atresia (Wulffet al., 2002; Manabe et al., 2004; Valdez et al., 2005).Reduced follicle atresia may contribute to the increase infollicle number seen in the TSP-1 null mice in thepresent study. In the polycystic ovarian syndrome(PCOS) there are elevated ovarian and serum VEGFlevels, and this overexpression is associated with anincrease in the number of ovarian follicles and reducedfollicle atresia (Jonard et al., 2005). The expressionlevels of TSP-1 in PCOS and the potential role of VEGFas a cytoprotective agent in this disease have not yetbeen determined.

TSP-1 is a scavenger for fibroblast growth factor(FGF)-2 (Margosio et al., 2003) and is known to bindVEGF (Gupta et al., 1999). Our study demonstrates thatTSP-1 is internalized via LRP. These results agree withothers that have shown that TSP-1 is not degraded incells deficient in LRP (Godyna et al., 1995; Mikhailenkoet al., 1997) and that catabolism of TSP-1 is inhibited byRAP (Margosio et al., 2003). We determined in this studythat TSP-1 bound VEGF and internalized it via LRP inour granulosa and endothelial cells, demonstrating thatLRP is an essential mediator of the catabolism of thecomplex. These results suggest that in the ovary, TSP-1may have a bimodal effect. TSP-1 may exert a classicalanti-angiogenic effect by binding and internalizingVEGF in endothelial cells, but may have an importantextravascular role as well. As VEGF is a potent mitogen(Li et al., 2005) and survival factor (Pauli et al., 2005)for endothelial cells, the reduced bioavailability of thefactor would significantly reduce the pro-angiogenicstimulus. As we have shown previously (Greenawayet al., 2004, 2005), VEGF has a very important

Fig. 3. TSP-1 treatment of SIGC inhibits VEGF protein expressionand induces apoptosis. A: Western blot analysis of VEGF protein fromSIGC treated for 24 h with increasing amounts of purified TSP-1.Graph represents densitometric analysis of VEGF protein from threedifferent in vitro experiments (mean�SEM). B: Percent apoptosis ingranulosa cells subjected to TNFa challenge in the absence or presenceof variable concentrations of TSP-1. All cultures were performed inserum-free media. Lane 1: No treatment; Lane 2: 100 ng/ml TNFa;Lane 3: 100 ng/ml TNFaþ 50 ng/ml VEGFþ 10 ng/ml TSP-1; Lane 4:100 ng/ml TNFaþ 50 ng/ml VEGFþ50 ng/ml TSP-1; Lane 5: 100 ng/ml TNFaþ 50 ng/ml VEGFþ 100 ng/ml TSP-1; Lane 5: 100 ng/mlTNFaþ 50 ng/ml VEGFþ1 mg/ml TSP-1. * denotes statisticallydifferent from serum free control (Lane 1) (P<0.05) (n¼ 3 experi-ments).



Fig. 4. Expression of proteins relevant to proliferation and apoptosisin response to VEGF, TSP-1, or combination treatment. A: Reliefcontrast overlay immunofluorescence images of untreated granulosacells or after treatment with 50 ng/ml VEGF, 100 ng/ml TSP-1 or acombination of 50 ng/ml VEGF and 100 ng/ml TSP-1. B: Quantifica-tion of the percentage of immunopositive in vitro cells from (A).Graphs represent four replicate experiments (mean�SEM).

C: Representative (of four replicate) western blots of proteins isolatedfrom granulosa cells treated with VEGF, TSP-1, or both. A decrease incytoprotective VEGF, VEGR2, and bcl-2 is observed after treatmentwith 100 ng/ml TSP-1 compared to treatment with 50 ng/ml VEGF.Conversely, an increase in pro-apoptotic Fas occurs following treat-ment with TSP-1. * denotes statistically different from serum freecontrols (P>0.05).



extravascular cytoprotective role in granulosa cells. Theanti-VEGF effect of TSP-1 in ovarian cells may havea significant influence on ovarian morphometry andfunction. Our TSP-1 null mice demonstrate elevated

ovarian VEGF levels, likely due to loss of inhibitoryinfluence of TSP-1. Concurrent with the overexpressionof VEGF, the ovaries of TSP-1 null mice had an increasein follicle number, which is consistent with our previous

Fig. 5. Binding and internalization of TSP-1/VEGF by the LRPreceptor. A: Immunofluorescent co-localization of TSP-1 and VEGF inthe absence or presence of the LRP-1 antagonist RAP. Cells weretreated for 6 h with purified VEGF or TSP-1 labeled with AlexaFluor488 dye (green stain). Co-staining for TSP-1 or VEGF was performedwith an AlexaFluor 594 conjugated antibody (red stain). Reciprocalexperiments are shown. A sample containing only AlexaFluor dye(no protein) is shown as negative control. B: 125I labeled VEGF isinternalized by LRP-1-expressing Chinese hamster ovary (CHO) cells

and native murine endothelial (mEC) and granulosa cells (SIGC), butnot LRP-1-deficient CHO cells or by SIGC and mEC treated with500 nM RAP, or by SIGC treated with TSP-1 siRNA. CHO, SIGC, andmEC were plated into 12-well dishes 24 h prior to the assay. 1 nM 125I-Labeled VEGF was added to LRP-positive and -negative CHO cells, toSIGC and mEC in the presence or absence of 500 nM RAP, and toSIGC treated with TSP-1 siRNA. At the indicated times, the amount ofinternalized radioactivity was determined. Each data point representsthe average of duplicate assays (n¼ 3).



findings that VEGF inhibits granulosa cell death andfollicle atresia (Greenaway et al., 2004, 2005). Reducedatresia would be an important factor contributing to thedevelopment of a greater than normal number of antralfollicles, as is seen in PCOS. Along with its inhibitoryinfluence on VEGF, TSP-1 also inhibited expression of

VEGFR-2 in cultured granulosa cells. Although themechanism for this inhibition is not yet known, thereduced VEGFR-2 expression may be secondary tothe lower levels of VEGF, as VEGF is known to regulatethe expression of VEGFR-2 in an autocrine and para-crine fashion (Vacca et al., 2003).

Fig. 6. TSP-1 knockdown results in increased VEGF expression andreduced apoptosis. A: Immunofluorescence for TSP-1 (green) andVEGF (red) SIGC treated with scrambled sequence (RNAi control) orTSP-1 siRNA. Images are acquired with identical acquisition settings.B: Western blot analysis of TSP-1 and VEGF in control and TSP-1siRNA treated SIGC. C: Percent apoptosis in untreated, and cyokine-challenged SIGC in the presence or absence of controls or TSP-1

siRNA. All cultures were performed in serum free media. Lane 1: Notreatment; Lane 2: 100 ng/ml TNFa; Lane 3: 100 ng/ml TNFaþ 50 ng/ml VEGFþ vehicle control; Lane 4: 100 ng/ml TNFaþ 50 ng/mlVEGFþRNAi control; Lane 5: 100 ng/ml TNFaþ 50 ng/mlVEGFþTSP-1 RNAi. * denotes statistically different from serumfree control (Lane 1) (P< 0.05) (n¼ 3 experiments).



Knockdown of TSP-1 in vitro and in vivo resulted in anincrease in VEGF levels in the current study. Theincreased VEGF in the ovaries of TSP-1 null mice maybe responsible for the dramatic ovarian hypervascular-ization, which is similar to that seen in differenttumors associated with elevated VEGF expression. Thisincreased blood vessel density mirrors the hypervascu-larization seen in ovarian tumors that are associatedwith poor clinical outcome and disease progression(Shen et al., 2000; Amis et al., 2005).

In conclusion, the present study demonstrates asignificant increase in ovarian VEGF levels, hypervas-cularization, and altered ovarian morphology in micelacking the TSP-1 gene. TSP-1 directly inhibits VEGFexpression at least partly through binding to the growthfactor and cellular internalization through the LRPreceptor. Other potential inhibitory mechanisms remainto be investigated. The results from this study provideinsight into regulatory mechanisms that may beimportant in mediating ovarian vasculature and ovar-ian cell survival.

ACKNOWLEDGMENTS

We thank Michelle Ross and Katrina Watson forexcellent technical assistance.

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