The Prostate 68:893 ^ 901 (2008)

Endothelial Cells Supportthe GrowthofProstateTissue InVivo

Michael Bates, Bruce Kovalenko, E. Lynette Wilson, and David Moscatelli*

Departmentof Cell Biology,KaplanCancer Center,NewYorkUniversity SchoolofMedicine,NewYork,NewYork

INTRODUCTION. The contribution of vascular endothelial cells to prostate growth has notbeen investigated. We examined whether endothelial cells support growth of prostate tissuewhen co-inoculated with prostate epithelial cells under the renal capsule.METHODS. Vascular endothelial cells were isolated from mice and co-inoculated under therenal capsule with a prostate luminal or basal epithelial cell line. After 60 days, kidneys wereexamined for growth of prostate tissue. Prostatic tissues were examined by immuno-histochemistry for expression of cytokeratins 5 and 8, and vascular density was determined.To determine if increased expression of VEGF-A would increase prostatic growth, transfectedendothelial cells overexpressing VEGF-A were co-inoculated with the prostate luminal or basalepithelial lines.RESULTS. Co-inoculation of endothelial cells and prostate luminal or basal epithelial cellsresulted in significant growth of prostatic tissue, whereas inoculation of any of the cell linesalone resulted in little growth. The growths from co-inoculation of endothelial cells and luminalepithelial cells contained duct-like structures that stained with antibodies to cytokeratin 8,whereas those from co-inoculation of endothelial cells and basal epithelial cells contained cordsof cells that stained with antibodies to cytokeratin 5. Overexpression of VEGF-A had no effect ongrowth of the prostatic tissues.CONCLUSION. Endothelial cells contribute to the growth of prostatic epithelial cells. Prostate68: 893–901, 2008. # 2008 Wiley-Liss, Inc.

KEY WORDS: vascular endothelial growth factor; flt-1; endothelial cells

INTRODUCTION

The development of the murine prostate depends onreciprocal signals between the stroma and epithelium[1,2]. The importance of stromal signals has beenshown in prostate reconstitution experiments in whichcombinations of dissociated prostate epithelial cellsand undifferentiated urogenital sinus mesenchymeare inoculated under the renal capsule where theyform a prostate-like organ [3–5]. The urogenitalsinus mesenchyme is required for formation ofprostate-like structures. The stromal cells presumablyprovide appropriate signals that stimulate the epithe-lial cells to grow and organize into functional prostaticstructures. In addition, the prostate stroma appears toregulate differentiation of prostate epithelium. WhenUGM is implanted under the renal capsule togetherwith bladder or urethral epithelium, it directs theseepithelia to develop into prostate tissue [6,7]. These

differentiative and organizing signals also appear to bepresent to some extent in mature prostate stroma [8].Prostate smooth muscle cells, fibroblasts, and evenadipocytes support the development of prostate-liketissues when co-inoculated under the renal capsulewith prostate epithelial cells [9–11]. Another potentialstromal source of signals to the prostatic epithelium isthe endothelium of the blood vessels, but the ability of

Grant sponsor: National Institutes of Health; Grant numbers:DK52644, CA90593, CA132641.

*Correspondence to: Dr. David Moscatelli, Department of CellBiology, New York University School of Medicine, 550 First Ave.,New York, NY 10016. E-mail: moscad01@med.nyu.eduReceived 12 October 2007; Accepted 13 February 2008DOI 10.1002/pros.20762Published online 24 March 2008 in Wiley InterScience(www.interscience.wiley.com).

) 2008 Wiley-Liss, Inc.

vascular endothelium to support prostatic epithelialgrowth has not been investigated.

There is ample evidence that there is a link betweengrowth or regression of prostatic epithelium andprostatic blood vessels [12]. During the involution ofthe prostate after castration of rats, blood vessels andepithelium regressed in parallel [13]. Apoptosis ofthe epithelial cells correlated with apoptosis of theendothelial cells [14,15]. Similarly, in castrated animalsthat were administered testosterone, increased DNAsynthesis was observed in both prostate epithelial cellsand prostate capillary endothelial cells after a 1 day lag[13,14]. The labeling of the epithelial cells followedthe same kinetics as the labeling of endothelial cells.Moreover, inhibition of vascular growth in castratedanimals administered testosterone prevented theregeneration of the prostate and inhibited prostateepithelial proliferation [16,17]. Together, these obser-vations suggest that in the prostate epithelial growth istightly coupled to vascular growth.

Considering the close relation between prostaticvascular growth and prostatic epithelial growth, it islikely that prostatic endothelial cells also contribute toprostatic epithelial growth. However, possible contri-bution of signals from the endothelium have notbeen investigated. Here we show that endothelialcells contribute to prostate epithelial growth anddevelopment.

MATERIALS ANDMETHODS

Isolation of Endothelial Cells

Endothelial cell cultures were established by amodification of the methods of Folkman et al. [18]and Voyta et al. [19]. Major airways were cut away fromthe lungs of Balb/c or FVB/N-Tg(TIE2-lacZ)182Sato/Jmice, and the remaining lungs were minced into smallfragments, washed in phosphate-buffered saline, andincubated in 0.15% (w/v) collagenase (Worthington) inalpha modified Eagle’s medium (aMEM) for 20 minat 378C. The tissue suspension was pipetted againstthe side of the tube to break it into smaller piecesand centrifuged at 200g for 5 min. The pellet wasresuspended in aMEM containing 10% fetal bovineserum and 5 ng/ml FGF-2 and plated onto 60 mmtissue culture dishes. After 4 hr, unattached cells werewashed away.

Cells from FVB/N-Tg(TIE2-lacZ)182Sato/J micewere plated at low density, and, after 2 days of culture,groups of cells with an endothelial cell morphologywere identified and their position marked on the dish.Surrounding cells were dislodged from the dish usinga drawn-out Pasteur pipet, and the remaining cellswere grown in aMEM with 10% fetal bovine serum and

5 ng/ml FGF-2 until they formed a colony ofapproximately 200 cells. These colonies were trypsiniz-ed using cloning rings and transferred to wells of a24-well plate. Cells from confluent wells were clonedby limiting dilution. Confluent cultures of thecloned cells were analyzed for b-galactosidase activity.The cells were fixed for 15 min with 2% formaldehydeand 0.2% glutaraldehyde in phosphate-bufferedsaline containing 2 mM MgCl2. Cultures were stainedwith 1 mg/ml X-gal (Sigma), 5 mM potassium fer-ricyanide, 5 mM potassium ferrocyanide, 2 mM MgCl2,0.01% sodium deoxycholate, and 0.02% Nonidet P-40 inphosphate-buffered saline overnight at 378C.

Endothelial cells from Balb/c mice were isolatedbased on their ability to take up acetylated low densitylipoprotein [19]. Cells harvested from the lungs wereplated at high density on 60 mm dishes and incubatedfor 1 week in aMEM containing 10% fetal bovineserum and 5 ng/ml FGF-2. The cultures were incubatedfor 4 hr in medium containing 10 mg/ml acetylated lowdensity lipoprotein labeled with 1,10-dioctadecyl-3,3,30,30-tetramethyl-indocarbocyanine perchlorate (Bio-medical Technologies, Inc., Stoughton, MA). Cells werewashed with phosphate-buffered saline, trypsinized,and sorted on a FACScalibur fluorescence activated cellsorter (Becton Dickinson) as described [19]. Cells in theupper 5% of fluorescence intensity were collectedand plated at 5� 103 cells/well in a 24-well plate. Afterexpansion, the cells were cloned by limiting dilutionand verified as endothelial by staining with antibodiesto von Willebrand factor.

Western Blots

Endothelial cells were extracted in a buffer contain-ing 0.5% Triton X-100, 150 mM NaCl, 10 mM Tris–HCl,30 mg/ml of aprotinin and 10 mM EDTA. In otherexperiments, confluent cultures of endothelial cellstransfected with an expression vector for VEGF-A orwith the vector alone were incubated for 24 hr in serum-free aMEM, and the conditioned medium was col-lected. EDTA was added to the conditioned mediumto a final concentration of 10 mM, and the mediumwas concentrated 20-fold with Centricon-YM10 centri-fugal filter devices (Millipore). Equal amounts of cellprotein or concentrated conditioned medium wereseparated by electrophoresis on an 8% SDS–polyacry-lamide gel. Proteins were transferred to nitrocellulosemembrane (Schleicher & Schuell Protran, BioSciences)by electroblotting (Bio-Rad Laboratories, Hercules,CA). The membrane was blocked with 5% (wt/vol)fat-free milk powder, 0.5% (vol/vol) Tween-20 inphosphate-buffered saline overnight at 48C and in-cubated with rabbit anti-human von Willebrandfactor antibody (Dako), rabbit anti-Tie-2 (Santa Cruz

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Biotechnology, Santa Cruz, CA), or rabbit anti-humanVEGF-A (Santa Cruz Biotechnology) at a dilutionof 1:300 for 2 hr at room temperature. After washingin TBS, blots were incubated at room temperature for1 hr with a 1:2,000 dilution of horseradish peroxidase-conjugated anti-rabbit IgG secondary antibodies (SantaCruz Biotechnology). Blots were washed three timeswith TBS. Immunocomplexes were developed usingWestern Lightning Chemiluminescence Reagent Plus(PerkinElmer Life Science, Inc., Boston, MA). Themembrane was exposed to an X-ray film for imaging.Proteins were sized with Rainbow markers (AmershamPharmacia Biotech).

RT-PCR

Confluent cultures of PE-L-1 cells or MLE-bgal cellswere extracted with TriZol reagent (GIBCO-BRL), andtotal RNA was isolated according to the manufacturer’sinstructions. The RNA was quantitated from its opticaldensity, and its integrity assessed by 1.2% formalde-hyde gel electrophoresis. Total RNA (2.5 mg) from eachsample was reverse transcribed for 90 min at 428C toobtain first-strand cDNAs using random hexamerprimers and Maloney murine leukemia virus reversetranscriptase (Roche Diagnostics Corporation, Indian-apolis, IN). The cDNA products in one-twentieth of theRT reaction mix were amplified by PCR using Thermusaquaticus DNA polymerase (Roche Diagnostics Corpo-ration). The primer pair used for PCR amplification ofmouse VEGF-A cDNA was 50-TGAGACCCTGGTG-GACATCT-30 and 50-CACCGCCTTGGCTTGTCAC-30.These primers amplify the region of the VEGF-Amessage that is alternatively spliced and yield bandsof 483, 411, 351, and 279 bp, representing messages forthe 188, 164, 144, and 120 amino acid isoforms of VEGF-A. The primer pair used for amplification of mouse flk-1cDNA was 50-GTCATGGATCCAGATGAATTGC-30

and 50-CGAAGTCACAGATCTTAACCAC-30 whichyielded a band of 728 bp. For amplification of mouseflt-1 the primers 50-GCACCCAGCATGTCATGCAAG-30 and 50-TCAATCCGCTGCCTTATAGATG-30 yieldeda band of 738 bp. As a control, the cDNA of b-actin wasamplified using the primers 50-ATCTGGCACCA-CACCTTCTACAATGAGCTGCG-30 and 50-CGTCA-TACTCCTGCTTGCTGATCCACATCTGC-30 The PCRprofile was as follows: 10 min at 958C, followed by30 cycles of 1 min at 948C, 2 min at 588C, and 3 min at748C. cDNA was isolated from the agarose gels usingthe GFX PCR DNA and gel band purification kit(Amersham Biosciences) and sequenced to verify itsidentity.

Transfection of Cell Lines

The cDNA for the 164 amino acid form of mouseVEGF-A (a gift from Genentech, Inc., South San

Francisco, CA) was inserted between the BamH1 andHinD III sites of the pZeo SV vector (Invitrogen).Correct orientation of the insert was determinedby restriction digestion, and identity of the insertwas confirmed by DNA sequencing. MLE-bgal cellsat 40% confluence were transfected with pZeo SVvector containing the VEGF-A insert or with theempty vector using Lipofectamine (Invitrogen).Two days after transfection, the cells were trypsinizedand replated at a 1:3 dilution in aMEM containing 10%fetal bovine serum, 5 ng/ml FGF-2, and 250 mg/mlZeocin (Invitrogen). Colonies that survived Zeocinselection were analyzed by RT-PCR for VEGF-Aexpression. Clones that expressed 5–10 times moreVEGF-A than non-transfected MLE-bgal cells wereselected for further study. Western blot analysisconfirmed that MLE-bgal cells transfected with theVEGF-A expression vector secreted 5–10 times moreVEGF-A protein than MLE-bgal cells transfectedwith the empty vector. There was no difference ingrowth rate between MLE-bgal cells transfected withthe VEGF-A expression vector and MLE cells trans-fected with the empty vector when plated in aMEMwith 5%, 1%, or 0.2% serum.

Growthof ProstaticTissues Under the Renal Capsule

Prostatic luminal (PE-L-1) and basal (PE-B-1) epithe-lial cell lines established from p53 null C57BL/6 micewere described previously [20,21]. Cells were harvestedby trypsin treatment when approximately 80% con-fluent, and 2.5� 105 PE-L-1 or PE-B-1 cells were mixedwith 5� 105 MLE-bgal endothelial cells. Cells werepelleted and resuspended in 20 ml of type I collagen(Vitrogen-100 collagen from Collagen Corporation, PaloAlto, CA), and the collagen was allowed to gel at 378C for15 min. Collagen gels were also prepared containing7.5� 105 PE-L-1, PE-B-1, or MLE-bgal cells alone or1.5� 106 MLE-bgal cells alone. The gels were graftedbeneath the renal capsule of 6-week-old male athymicmice (National Cancer Institute, Frederick, MD) asdescribed [9]. Each cell combination was implantedunder the renal capsule into at least five kidneys foreach experiment. Mice were sacrificed 6 weeks after gelimplantation and the dimensions of each graft weremeasured.

Immunocytochemistry

At the time of sacrifice, kidneys were removedand cut with a scalpel through the highest part ofthe tissue growths under the renal capsule. Half ofeach kidney was embedded in OCT, and snap-frozen.The remainder of the kidney was fixed in formalin.Vascular density was determined in frozen sectionsusing antibodies to PECAM-1, and differentiation

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of the implanted epithelial cells was determinedusing antibodies to cytokeratins 5 and 8. Eight-micrometer-thick-frozen sections were post-fixed inacetone for 10 min, treated with 0.3% hydrogenperoxide/PBS for 15 min, blocked in 5% normal rabbitserum or goat serum for 30 min, and biotin/avidinblocking reagents for 15 min each (Vector Laboratories,Inc., Burlingame, CA). The sections were then incu-bated with rat anti-mouse PECAM-1 antibodies (diluted1:200; Pharmingen) for 1 hr at room temperature. Theantibody–antigen complexes were visualized bythe avidin/biotin method (Vectastain Elite ABC Kit,Vector Laboratories, Inc.) according to the manufac-turer’s instructions using diaminobenzidine as asubstrate (Vector Laboratories, Inc.). The sections werecounterstained with Mayer’s hematoxylin (Sigma),dehydrated in graded concentrations of alcohol,and coverslipped with resin (Permount, FisherScientific Co.). Control sections were incubatedwith non-immune rabbit immunoglobulins usingthe same working dilution as the primary antibody.Vessels in the growths were counted in three non-adjacent sections per kidney using a Leica DMLBmicroscope equipped with an ocular grid.

The tissue fixed in formalin was embedded inparaffin, sectioned (5 mm), and stained with hematox-ylin and eosin. Formalin-fixed sections were alsostained with antibodies to cytokeratin 8 or cytokeratin5 and counter-stained with Mayer’s hematoxylin asdescribed above.

RESULTS

Establishmentof Mouse Endothelial Cell Cultures

Endothelial cells were cultured from lungs of FVB/N-Tg(TIE2-lacZ)182Sato/J mice. These mice expressb-galactosidase under control of the endothelial cell-specific tie-2 promoter. Primary digests of the tissueswere plated at low density, and clones of endothelial

cells were selected based on their morphology [18] andgrown to confluence in medium containing 10% fetalcalf serum and 5 ng/ml FGF-2. Confluent cultures ofthese cells (MLE-bgal) were verified as endothelialby their expression of b-galactosidase (Fig. 1B) andthe endothelial cell-specific markers von Willebrandfactor, Tie-2 (Fig. 1A), and PECAM-1 (data not shown).Cells with similar morphology were isolated from thelungs of wild-type Balb/c mice (MLE cells) by sortingfor cells that took up fluorescent labeled acetylated lowdensity lipoprotein, a characteristic of endothelial cells[19]. These cells also expressed von Willebrand factor,Tie-2 (Fig. 1A), and PECAM-1 (data not shown) atsimilar levels. Both lines have maintained expression ofthese markers for over 50 passages. Neither line istumorigenic as no growths were detected 60 days after5� 106 cells are implanted subcutaneously in athymicmice.

Endothelial Cells Support Growth ofProstateTissue InVivo

To determine if endothelial cells support the growthof prostate epithelial cells in vivo, we adapted the invivo co-culture system in which combinations ofprostatic cells inoculated under the renal capsule formprostatic tissues [9]. In these experiments, 2.5� 105

MLE-bgal endothelial cells were co-inoculated with5� 105 PE-L-1 prostate luminal epithelial cells underthe renal capsule. When inoculated alone, 7.5� 105 or1.5� 106 MLE-bgal cells did not grow, and 7.5� 105 PE-L-1 cells grew to a minor extent (Fig. 2A). However, co-inoculation of PE-L-1 cells with endothelial cells led tosignificant growth of prostatic tissue (Fig. 2A). Growthswere not as large as those obtained when smoothmuscle cells were co-inoculated with PE-L-1 cells [9].The growths contained large, epithelial-lined cavitiesreminiscent of prostatic ducts (Fig. 3A) that oftencontained secretory products (not shown).

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Fig. 1. Characterizationofendothelialcell lines.A:Expressionof vonWillebrand factor andTie-2bymouseendothelialcells.Proteinswereextracted from confluent cultures of lung endothelial cells fromwild-typemice (MLE 0.5 ^1) or frommice expressing b-galactosidase undercontrolof the tie-2promoter (MLE-bgal).As acontrol,proteinswereextractedfromculturesofB16melanomacells.Equalamountsofproteinwereseparatedbyelectrophoresisonan8%polyacrylamide^SDSgelandblottedontonitrocellulose.Blotswereprobedwithantibodies tovonWillebrandfactor(vWF)orTie-2.B:ConfluentculturesofMLE-bgalcellswerelightly fixedwith0.2%glutaraldehydeandstainedforb-galacto-sidase activityusing theX-gal substrate. [Color figurecanbeviewedin theonlineissue,whichis availableatwww.interscience.wiley.com.]

896 Bates et al.

The effect of co-inoculation of endothelial cells withPE-L-1 cells on the vascularity of the prostatic growthswas also investigated. Vascular density in the growthsresulting from co-inoculation of MLE-bgal cells and PE-L-1 cells was approximately double that in the smallgrowths that occurred after inoculation of either celltype alone (Fig. 4). Using RT-PCR the mRNA forbacterial b-galactosidase was detected in the prostaticgrowths, indicating that the implanted MLE-bgal cellssurvived and incorporated into the growths. However,the MLE-bgal cells could not be localized in the vesselsof the growths by staining with the X-gal substrate forb-galactosidase or immunostaining with antibodies tob-galactosidase. Failure to detect the MLE-bgal cellswas probably due to the low level of expression ofb-galactosidase in these cells.

We also examined if endothelial cells would supportthe growth of prostate basal epithelial cells in vivo.When MLE-bgal cells were co-inoculated with thePE-B-1 basal epithelial cell line under the renal capsule,significant growths of prostate-like tissue occurred(Fig. 2B). These growths contained small nests andcords of epitheloid cells (Fig. 3D) that did not haveeasily identifiable lumens. No growth was observed ifMLE-bgal cells or PE-B-1 cells were inoculated alone(Fig. 2B).

Endothelial Cells Support Differentiation ofProstatic Cells

To determine if endothelial cells also support thedifferentiation of the prostate cells, we determinedif the cells in the implants expressed luminal or basalcell-specific cytokeratins. In prostatic growths resultingfrom the implant of MLE-bgal cells with PE-L-1 cells, allepithelial cells lining the duct-like structures stainedpositive for cytokeratin 8, a prostate luminal cell-specific cytokeratin (Fig. 3B). No expression of the basalcell-specific cytokeratin, cytokeratin 5, was observed(Fig. 3C). Similarly, in prostatic growths resulting fromthe implant of MLE-bgal cells and PE-B-1 cells, all cellsin the cord-like structures stained positive for basalcell-specific cytokeratin 5 (Fig. 3F), and expression ofcytokeratin 8 was not observed (Fig. 3E). Thus, co-inoculation with endothelial cells supported continuedexpression of differentiated markers by the inoculatedluminal and basal epithelial cells but did not promotetransdifferentiation from one phenotype to the other.

Prostate Epithelial Cell Growth IsNot Stimulatedby Overproduction of VEGF-A

As it has been reported that prostate tumor cellsexpress receptors for VEGF [22–28], the expression of

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Fig. 2. Growth of prostatic cells under the renal capsule.MLE-bgalmouse endothelial cells (2.5�105 cells) transfectedwith an expressionvectorencodingVEGF-A(MLE-VEGF)orwith thevector alone (MLE)were co-inoculatedunder therenalcapsulewith5�105PE-L-1prostateluminal epithelial cells (A) or PE-B-1 prostate basal epithelial cells (B). As a control 7.5�105 MLE, MLE-VEGF, PE-L-1, or PE-B-1 cells wereimplantedalone.After 60days, themicewere sacrificed, and the size of theprostatic growthmeasured. *P< 0.01compared toMLE alone andP< 0.05comparedtoPE-L-1alone;**P< 0.01comparedtoMLE-VEGFaloneorPE-L-1alone;***P< 0.01comparedtoMLEaloneorPE-B-1alone;****P< 0.01comparedtoMLE-VEGF aloneorPE-B-1alone.

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VEGF receptors by the prostate luminal epithelial cellline, PE-L-1, was investigated. RT-PCR revealed thatthe PE-L-1 cells expressed mRNA for VEGF receptor-1(flt-1) but at a lower level than in the culturedendothelial cells (Fig. 5A). There was no detectablemessage for VEGF receptor-2 (flk-1; data not shown).

We hypothesized that increased expression ofVEGF might lead to increased growth of prostatic cellimplants either by direct stimulation of PE-L-1 cellgrowth or by increased recruitment of endothelial cells.To examine if altered VEGF-A expression modifiedprostate epithelial growth in vivo, the MLE-bgal cellswere transfected with a pZeo SV VEGF-A expressionvector and clones that expressed approximately10 times more VEGF-A than cells transfected with thevector alone were chosen (Fig. 5B,C). PE-L-1 cells wereimplanted under the renal capsule with endothelialcells overexpressing VEGF-A or endothelial cells trans-fected with vector alone. Overexpression of VEGF-Adid not alter the growth of MLE-bgal cells in cultureor when inoculated alone in vivo. Co-inoculation ofPE-L-1 cells with the MLE-bgal cells overexpressingVEGF-A under the renal capsule resulted in the same

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Fig. 3. Histological and immunohistochemical analysis of growth under the renal capsule. Growths arising from the implantation of2�105MLE-bgal cells and5�105 PE-L-1cells (A^C) or PE-B-1cells (D^F)wereharvestedafter 60days, and5mmsectionswere stainedwithhematoxylin and eosin (A and D). Sections were also immunostainedwith antibodies to cytokeratin 8 (B and E) or cytokeratin 5 (C and F).ArrowsinDpointoutcordsofepitheloidcells.Bar inAequals50mm.Allpanelshave the samemagnification. [Color figurecanbeviewedin theonlineissue,whichis availableatwww.interscience.wiley.com.]

Fig. 4. Vascularity of prostatic growths under the renal capsule.Sections fromtheprostaticgrowthwere stainedwithantibodies toPECAM-1 and vessel segments were counted in the prostaticgrowths. *P< 0.05comparedtoMLEaloneorPE-L-1alone.

898 Bates et al.

amount of prostatic growth as obtained when PE-L-1cells were co-inoculated with MLE-bgal cells trans-fected with the vector alone (Fig. 2A). Similarly, co-inoculation of PE-B-1 cells with MLE cells overexpress-ing VEGF-A resulted in the same amount of prostaticgrowth as when PE-B-1 cells were co-inoculatedwith MLE-bgal cells transfected with the vectoralone (Fig. 2B). Thus, increased VEGF-A expressionby endothelial cells had no effect on growth of prostaticcells under the renal capsule. Increased expression ofVEGF-A by the implanted endothelial cells also did notalter the number of blood vessels surrounding theprostatic growths (data not shown).

DISCUSSION

We have found that vascular endothelial cellsare able to support the growth and maintenance ofdifferentiation of prostatic epithelium in vivo. Sub-stantial evidence supports a close relation betweenendothelial growth or regression and epithelial growthor regression in the prostate. This close relationshipmay partly be the result of secretion of angiogenicfactors by prostatic epithelium. Prostatic epithelial cellsproduce several angiogenic factors, including vascularendothelial growth factor (VEGF), fibroblast growthfactors, and the angiopoietins [29–34]. Synthesis ofVEGF is regulated by androgens in prostatic luminal

epithelial cells [31,35–38]. Thus, castration maydecrease the balance of pro-angiogenic factors pro-duced by the epithelium, resulting in regression of thevasculature. Vascular regression would starve theepithelium of essential nutrients, resulting in epithelialregression. Similarly, restoration of androgen tocastrated animals would stimulate VEGF productionby prostate epithelial cells leading to expansion of thevasculature. The growing vasculature would supplynutrients to support the expansion of the epithelialcompartment.

VEGF seems to play an essential role in theregulation of vascular growth in response to androgensas blocking VEGF signals with soluble VEGF receptorsreduced vascular growth in the prostates of castratedmice administered testosterone [17,39] and inhibitedregeneration of the prostate [16,17]. However, in theexperiment presented here, increased expression ofVEGF did not alter the ability of endothelial cellsto potentiate prostate tissue growth. VEGF is highlyexpressed in the kidney [40]. Thus, it is likely that thekidney already produced adequate amounts of VEGFto support endothelial cell expansion in the implants,and increased amounts were superfluous.

The relationship between prostatic epithelial celland blood vessel health has been used to suppressprostate tumor growth. As in other tumors, prostatetumors regulate their vascularization by the release ofangiogenesis factors that stimulate the surroundingvasculature to sprout new capillaries that grow intothe tumor. Factors that target angiogenic factors orendothelial cells themselves block the recruitmentof new blood vessels and thereby inhibit prostatetumor growth [41–47]. These anti-vascular therapiesare believed to act by starving the prostatic tumors ofnutrients supplied by the blood vessels.

However, in the experiments presented here, endo-thelial cells themselves, without being organized intovessels, were able to support the establishment andgrowth of prostatic tissue in vivo. These results may berelated to the observation that the vascular endothe-lium appears to have a direct role in supporting growthof some epithelial tissues. In mice that do not developblood vessels because of the knockout of the endothe-lial cell specific receptor, VEGF receptor-2, liver doesnot develop [48]. Similarly, the islets of Langerhansdo not develop in Xenopus embryos in which theearly dorsal aorta has been ablated, thereby blockingfurther vascular development [49]. The developmentof these tissues is blocked at a stage before bloodbegins to flow in the embryo, suggesting that there isa direct requirement for endothelium rather than thenutrients delivered through the blood. Developmentof these tissues can be rescued in vitro by addition ofendothelial cells. Thus, development of some epithelia

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Fig. 5. Characterizationofcell lines.A:Flt-1expressioninPE-L-1cells.RNAisolated fromPE-L-1cells orMLE-bgal cellswas analyzedby RT-PCR with primers specific for flt-1. As a control, thesame samples were analyzed by RT-PCRwith primers specific forb-actin. B: VEGF expression in transfected MLE-bgal cells. RNAisolated fromMLE-bgal cells transfectedwith anexpressionvectorfor VEGF-A (MLE-VEGF) or with the vector alone (MLE) was ana-lyzedbyRT-PCRwithprimers specific forVEGF orb-actin.C:VEGFprotein expression in transfected MLE-bgal cells. Conditionedmedium from confluent cultures of MLE-bgal cells transfectedwith an expression vector for VEGF-A (MLE-VEGF) or with thevector alone (MLE) was concentrated 20-fold, run on an 8% poly-acrylamide gel, andblotted onto nitrocellulose.Blots were probedwithantibodies toVEGF-A.

Endotheliumand Prostate Growth 899

requires factors directly supplied by endothelial cells.Even in adult animals, liver responds to factors secretedby the endothelium. Administration of VEGF to micestimulates liver growth by stimulating growth ofsinusoidal endothelial cells [50]. Growth of the endo-thelium stimulates the growth of the liver epithelium.Epithelial growth is partly due to hepatocyte growthfactor (HGF) produced by the endothelial cells [50]. Arecent study reported that in mice deficient in theangiogenesis inhibitor, pigment epithelium-derivedfactor, there is overgrowth of blood vessels and hy-perplasia of prostatic epithelium, suggesting thatprostatic epithelium may also respond to direct signalsfrom endothelial cells [51].

ACKNOWLEDGMENTS

The authors thank Gilbert Kim and Xiao-li Liu fortheir expert technical assistance.

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