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Department of Molecular and Cellular Biology [S. J. M., S. J. R., J. A. T., F. Y., T. D. D., D. R. R.] and Cell and Molecular Biology Program [M. L.], Baylor College of Medicine, Houston, Texas 77030
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INTRODUCTION |
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Our previous studies have reported that the ps20 protein is expressed by prostate stromal cells with strong expression in vessel wall smooth muscle cells (4) . Originally, we characterized ps20 as a growth inhibitor of epithelial cells in vitro and purified ps20 to homogeneity from the conditioned medium of a fetal rat urogenital sinus mesenchymal cell line (5 , 6) . Subsequently, we cloned and characterized the cDNA encoding rat ps20 (4) , human ps20 cDNA, and genomic DNA encoding the gene we termed WFDC1, as well as genomic clones containing mouse ps20 (Wfdc1; Ref. 7 ). These studies also localized WFDC1 to human chromosome 16q24 (7) . In addition, Rodriguez-Rey et al. recently cloned and submitted a chicken ps20 cDNA to the GenBank/European Molecular Biology Laboratory database (AJ438920). There is high conservation of ps20 between species, ranging from 61% to 96% identity of the mature protein.
Each ps20 protein sequence contains a functional motif identified as a WAP type-four disulfide core domain consisting of eight cysteines forming four characteristically paired disulfide bonds. The domain and protein family is named after the first characterized member, WAP (8) . The WAP domain confers a serine protease inhibition activity to several family members including elafin and SLPI (9 , 10) . Elafin is a potent inhibitor of elastase and proteinase 3, each of which are elastin-degrading proteases (11) . SLPI inhibits a broader range of serine proteases including trypsin, chymotrypsin, elastase, and cathepsin G (11) . Both elafin and SLPI are secreted proteins known to protect host tissues from the effects of excessive proteolysis and to promote appropriate tissue repair in numerous model systems (12, 13, 14) . These data suggest a role for these protease inhibitors in reactive stroma biology at numerous tissue sites. On the basis of the immunolocalization of ps20 to smooth muscle cells and the presence of a signal peptide with secretion of the native protein from cells in vitro, it is theorized that ps20 is secreted by smooth muscle cells in vivo and is a component of the ECM environment, where it may function as a serine protease inhibitor.
The purpose of the present study was to additionally evaluate ps20 expression in human prostate stroma, including vessels, and to determine the effects of ps20 on endothelial cell migration, angiogenesis, and tumorigenesis using in vitro assays, and the DRS xenograft prostate cancer model. We report that although ps20 is expressed in normal prostate stroma, expression was restricted to vessel wall smooth muscle cells in samples of human prostate cancer Gleason 3 foci. Additionally, endothelial cell motility was stimulated in the presence of ps20, whereas endothelial cell proliferation was not significantly altered. The presence of ps20 in the DRS tumor microenvironment significantly increased both angiogenesis and tumorigenesis, as determined by microvessel density, tumor wet weights, and volumes. In addition, we determined that ps20 mRNA expression was increased by TGF-ß1, a known regulator of angiogenesis in prostate cancer. Together, these data suggest that ps20 is expressed in the vessel wall in prostate cancer and can function to promote angiogenesis, possibly by stimulating endothelial cell migration or by stabilization of the vessel wall during vessel formation. These studies also suggest that ps20 may be a target for novel prostate cancer therapeutics directed toward angiogenesis in the reactive stroma compartment.
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MATERIALS AND METHODS |
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Cell Lines.
COS-1 cells (monkey kidney fibroblast-like; American Type Culture Collection, Manassas, VA) were cultured in DMEM (Invitrogen, Carlsbad, CA), containing 10% fetal bovine serum (Hyclone, Logan, UT), 100 units/ml penicillin, and 100 µg/ml streptomycin (Sigma, St. Louis, MO). COS-1 stable cell lines expressing rat ps20 were generated and maintained as reported previously (4)
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The GeneSwitch System (Invitrogen) was used to create stable cell lines with mifepristone-inducible expression of a V5-His tagged ps20 protein. GeneSwitch-CHO and GeneSwitch-3T3 cells expressing the GeneSwitch regulatory protein from the pSwitch vector were purchased from Invitrogen. GeneSwitch-CHO cells were cultured in Ham’s F-12 medium (Invitrogen) supplemented with 10% fetal bovine serum, 2 mM L-glutamine, and 100 µg/ml hygromycin-B (Invitrogen). GeneSwitch-3T3 cells were cultured in DMEM containing 10% fetal bovine serum and 50 µg/ml hygromycin-B. Rat ps20 cDNA was inserted into the pGENE/V5-His vector to generate ps20pGENE/V5-His, which was transfected in parallel with empty vector controls into both GeneSwitch-CHO and GeneSwitch-3T3 cell lines using FuGENE 6 transfection reagent (Roche, Indianapolis, IN). Stably transfected GeneSwitch-CHO cells were selected with 400 µg/ml Zeocin (Invitrogen), whereas GeneSwitch-3T3 cells were selected with 300 µg/ml Zeocin. Resistant clones from each cell line were treated with 100 pM mifepristone (Sigma) and screened for fusion protein expression by Western analysis of the conditioned medium with anti-V5 antibody (Invitrogen). All of the stably transfected cell lines were maintained in 300 µg/ml Zeocin. Cells lines used in this study represent pooled clones of stably transfected cells.
BAE cells (kindly provided by Dr. Karen Hirschi, Departments of Pediatrics and Molecular and Cellular Biology, Baylor College of Medicine, Houston, TX; Ref. 16 ) were cultured in DMEM-Low Glucose supplemented with 10% calf serum, 100 units/ml penicillin, 100 µg/ml streptomycin, and 0.29 mg/ml L-glutamine (Invitrogen). Cultures between passages 10 and 20 were used for these studies.
HUVECs (Clonetics, Walkersville, IL) were cultured in endothelial cell growth medium-2 (Clonetics). Cells between passages 3 and 8 were used in these studies.
LNCaP human prostate carcinoma cells (American Type Culture Collection) were cultured in RPMI 1640 (Invitrogen) supplemented with 10% fetal bovine serum, 100 units/ml penicillin, and 100 µg/ml streptomycin.
HPS-19B human prostate stromal cells were established using our protocols reported previously (6
, 17)
. Briefly, prostate tissue from a 19-year-old organ donor was obtained from the Baylor College of Medicine Prostate Specialized Program of Research Excellence Pathology and Tissue Microarray Core. A tissue core was isolated from peripheral zone prostate graded as normal by histopathological criteria. The core was diced into 1-mm cubes, washed with HBSS, and cultured in a 24-well plate with Bfs medium: DMEM containing 5% fetal bovine serum, 5% Nu-Serum (BD Biosciences, Bedford, MA), 0.5 µg/ml testosterone, 5 µg/ml insulin, 100 units/ml penicillin, and 100 µg/ml streptomycin (Sigma). The explants were cultured at 37°C in 5% CO2, and culture medium was changed every 48 h. Stromal cells migrated out of the tissue, attached to the culture dish, and were grown to confluence. The remaining tissue was removed and the cells passaged by routine procedures. Standard immunocytochemistry procedures were used to characterize the HPS-19B cells as cytokeratin negative (1–124-161; Boehringer Mannheim, Indianapolis, IN), and vimentin positive (sc-7557; Santa Cruz Biotechnology, Santa Cruz, CA). Approximately 60% of the cells were sm 
-actin positive, similar to our stromal cell lines reported previously (2)
. Cultures between passage 10 and 15 were used for experiments.
Our PS-1 stromal cell line was derived from an adult rat (Harlan Sprague Dawley) prostate organ explant as described previously (17) and maintained in Bfs medium.
Preparation of Conditioned Medium.
GeneSwitch-CHO-ps20pGENE/V5-His or GeneSwitch-CHO-pGENE/V5-His control cells were plated and grown to confluence. Cells were pulsed with 100 pM mifepristone for 24 h, then incubated in Ham’s F-12 medium supplemented with 1% fetal bovine serum and 2 mM L-glutamine for 48 h. Conditioned medium was collected and cleared by low-speed centrifugation. Approximate concentration of ps20-V5-His in conditioned medium was determined by Western analysis using an anti-V5 antibody comparing conditioned medium with serial dilutions of purified insect-expressed ps20-V5-His. Conditioned medium contained 
5 µg/ml ps20-V5-His protein.
Migration Assays.
BAE cell migration assays were carried out in transwell chambers (Costar-Corning Inc., Corning, NY). Polycarbonate filter inserts for 24-well plates with 8-µm pores were coated with 17 µg/cm2 rat tail collagen I (BD Biosciences). Confluent BAE cells were incubated in DMEM-Low Glucose with 0.1% BSA (Sigma) for 2 h, then 8.25 x 104 cells in 150 µl medium were plated onto each insert. To each insert, 50 µl ps20 conditioned medium (final concentration of 50 nM ps20-V5-His protein) or control conditioned medium were added, whereas each lower chamber received 600 µl DMEM-Low Glucose with 0.1% BSA. Assays were carried out at 37°C in 5% CO2 for 16 h. Cells that had not migrated through the filter were removed, and those that had were stained and fixed with crystal violet for 10 min [0.5% (w/v) crystal violet, 3.2% formaldehyde (Sigma), 0.17% NaCl (Fisher Scientific, Pittsburgh, PA), and 22% ethanol (AAPER Alcohol and Chemical, Co., Shelbyville, KY)], washed three times with water, and allowed to dry. From each filter, four random fields at x200 were photographed, and an observer blinded to experimental conditions counted and averaged migrated cells. Assays were performed at least three times in triplicate.
COS-1 cell migration assays were carried out using standard wounding assays. The parent cell line COS-1, control line COS-1 pBKCMV-
lac-4, and experimental lines COS-1 pBKCMV-lac-rps20–3/-4 were plated on glass coverslips at 3.58 x 104 cells/cm2 and allowed to attach overnight. Triplicate scrapes were made in the cell monolayers, and coverslips were harvested at multiple time points from 0 to 96 h. Coverslips were stained and fixed with crystal violet for 10 min, washed three times with water, and allowed to dry. Images were analyzed using the public domain NIH Image program (developed at the U.S. NIH and available on the Internet)6
with the density of wound area filled measured in arbitrary units. Experiments were repeated in triplicate three times.
Proliferation Assay.
BAE cells or HUVECs were plated at 3000 cells/cm2 in 24-well plates and allowed to attach overnight. Cells were incubated in serum-free medium for 24 h. Serum-free medium was removed and replaced with 250 µl of DMEM-Low Glucose supplemented with 2% calf serum (BAE cells) or endothelial cell growth medium-2 (HUVECs) and 250 µl of either ps20 (final concentration of 100 nM ps20-V5-His protein) or control conditioned medium. BAE cells were incubated for 48, 72, or 96 h, harvested, and counted using a hemacytometer. HUVECs were counted at the 48-h time point only. Each experiment was carried out three independent times in triplicate.
Animals.
Athymic NCr-nu/nu male homozygous nude mice, 6–8 weeks of age, were purchased from Charles River Laboratories (Wilmington, MA). Experiments were conducted in accordance with the NIH Guide for the Care and Use of Laboratory Animals.
Preparation of Xenograft Tumors.
DRS model xenograft tumors were generated under three-way conditions (LNCaP + stromal cells + Matrigel) as described previously (2)
with some modifications. Briefly, frozen aliquots of cells (32 x 106 LNCaP and 8 x 106 HPS-19B cells) were thawed, mixed, and washed with 10-ml culture medium. Cells were pelleted at 400 x g for 2 min, then resuspended in 12 ml culture medium and divided into two 6-ml aliquots. Cells were pelleted and resuspended in 300 µl of either ps20 (final concentration of 80 nM ps20-V5-His protein) or control conditioned medium. Matrigel ECM (Becton Dickinson, Bedford, MA) was stored in 500-µl aliquots at -20°C and thawed on ice at 4°C for 3 h before use. Cells were incubated on ice for 1.5 min, then mixed with Matrigel and drawn into a 1-ml syringe with a 20-gauge needle. Using a 25-gauge needle, 100 µl of cell-Matrigel suspension (2 x 106 LNCaP and 0.5 x 106 HPS-19B cells; 4:1 ratio) were injected s.c. in each lateral flank. Six sites (three animals) were injected per treatment group for each experiment, and tumors were harvested on day 10. Experiments were carried out four times.
GeneSwitch-3T3-ps20pGENE/V5-His cell tumors were generated in a similar manner. Frozen cells (4 x 106) were thawed and washed twice with 10 ml DMEM supplemented with 10% fetal bovine serum. Cells were pelleted and resuspended in a total of 300-µl wash medium. Matrigel preparation and cell injections were carried out as described above, resulting in injections of 0.5 x 106 stromal cells per site. To induce expression of ps20-V5-His, animals received mifepristone (RU486; Sigma) at 0.5 mg/kg administered as 100 µl i.p. injections at the time of tumor injection and repeated every 48 h until tumors were harvested on day 11. Animal dosage of mifepristone was based on protocols shown previously to induce gene expression in vivo and not affect tumor weight or volume (2 , 18 , 19) . Control mice were treated with vehicle alone. A duplicate experiment with similar results was carried out with tumors harvested on day 15.
All of the tumors were weighed and measured in three dimensions using calipers. Tumor volume was calculated with the formula: V = 0.52 x length x width x height (20) . Tissues were fixed in 4% paraformaldehyde overnight at 4°C and then washed three times with PBS. Tumors were paraffin embedded, and 5-µm sections were cut and mounted onto ProbeOn Plus slides (Fisher Scientific). All of the sections were stained with H&E for histological analysis.
Immunohistochemistry.
Immunostaining for ps20 and sm -actin was performed using the EnVision + System for rabbit primary antibodies or mouse primary antibodies (Dako, Carpinteria, CA) following the manufacturer’s recommendations. For ps20 staining, our previously characterized affinity purified rabbit polyclonal ps20ab-1 was used (4)
. Tissue sections were incubated with ps20ab-1 diluted 1:150 for 1 h at 37°C, and with labeled polymer for 45 min at room temperature. For sm -actin staining, we used the mouse monoclonal 1A4 antibody (Sigma). Tissue sections were incubated with primary antibody diluted 1:5000 for 30 min and labeled polymer for 30 min, both at room temperature.
Immunostaining for CD31 and V5 was performed with the MicroProbe Staining System (Fisher Biotech) following our protocol published previously (2) . Reagents used with this capillary gap system were purchased from Research Genetics (Huntsville, AL). Antimouse CD31 (platelet endothelial cell adhesion molecule-1) antibody (MEC13.3, rat monoclonal), and biotin-conjugated antirat IgG secondary antibody were purchased from BD PharMingen (San Diego, CA). Anti-V5 antibody was purchased from Invitrogen (46–0705, mouse monoclonal). Antigen retrieval was necessary for both antibodies; sections were incubated in 0.1% trypsin (Zymed, South San Francisco, CA) for 10 min at 37°C (CD31) or subjected to high temperature steamer treatment in 10 mM sodium citrate buffer (pH 6.0) for 20 min (V5). For V5 staining, sections were incubated in 20 µg/ml goat-antimouse Fab fragment (Jackson ImmunoResearch, West Grove, PA) for 30 min at 37°C to block host immunoglobulins. Sections were incubated with primary antibody overnight at 4°C (CD31, 1:50; V5, 1:200) and then with secondary antibody (antirat, 1:100; Research Genetics Universal Secondary) for 45 min at 37°C (CD31) or 4 min at 50°C (V5).
Microvessel Density Analysis.
Tumor sections were stained for CD31, and four random high-power fields (x400) were photographed for each tumor. Vessels were counted by a blinded observer using standard criteria (21)
. For DRS-LNCaP experiments, 12 tumors (48 fields) from duplicate experiments were analyzed for each treatment group. For 3T3 cell tumors, 4 tumors (16 fields) were analyzed for each treatment group at each time point.
Northern Analysis.
ps20 mRNA expression was analyzed by Northern blotting as described previously (4)
. Briefly, PS-1 cells were incubated in DMEM with 5 µg/ml insulin, 5 µg/ml transferrin, and 5 ng/ml selenium (ITS; Sigma), supplemented with either vehicle control, 25 pM, or 50 pM TGF-ß1 (porcine; R&D Systems, Minneapolis, MN) for 24 h. Total RNA was prepared using RNA Stat-60 total RNA isolation reagent (Tel-Test, Inc., Friendswood, TX), and electrophoresed through 1% agarose gels containing 1x 4-morpholinepropanesulfonic acid and 5.1% of a 37% formaldehyde solution and transferred to neutral charged nylon membranes (Schleicher & Schuell, Keene, NH). Inserts of ps20 cDNA segments from T340pCRII and 42T7pCRII were excised, gel-purified, labeled by random priming with [-32P]dCTP, purified, and denatured. Blots were UV cross-linked and exposed to probe at 1–5 x 106 counts/ml overnight at 42°C, and washed at 60°C in 0.1% SDS containing 2–0.1x SSC. Blots were exposed to film for 24 h to 2 weeks, as necessary. RNA transferred to the blot was stained with methylene blue as a loading control. Similar results were obtained in duplicate experiments. NIH Image was used to quantitate data, and ps20 mRNA expression was normalized to 18S rRNA.
Statistical Analysis.
Two-way ANOVA, followed by Bonferroni post tests, was used to compare treatment groups over time in both the COS-1 migration and the cell proliferation studies. BAE cell migration, tumor weights and volumes, and tumor microvessel density were each evaluated with an unpaired t test. Statistical analysis was performed with Prism version 3 (GraphPad Software, San Diego, CA). Error bars represent SE, and P < 0.05 was considered statistically significant.
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that consists predominately of smooth muscle cells (1)
, as shown by sm -actin staining (Fig. 1B)
. Intense ps20 staining was also detected in the smooth muscle cell layer of vessel walls (Fig. 1A
, arrowheads). To date, our staining of human prostate cancer samples has revealed variable degrees of ps20 expression. However, strong ps20 expression was consistently observed in the vascular smooth muscle cells in Gleason 3 cancer foci evaluated in this study (Fig. 1C
, arrowhead), whereas the reactive stroma compartment had little to no ps20 immunoreactivity. These data suggested that ps20 may function in vessel wall biology. Accordingly, to additionally characterize ps20 function, we examined the effects of ps20 on endothelial cell biology in vitro and angiogenesis in vivo in a prostate cancer xenograft model.
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; P < 0.05, unpaired t test), representing a 104% increase.
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lac-rps20-3 and 4, exhibited increased cell migration and wound closure over time when compared with either the COS-1 parent cell line or control COS-1 cells stably transfected with control vector (Fig. 2B
; P < 0.05, two-way ANOVA with Bonferroni post-test). At 48 h, the average migration of ps20 expressing cell lines showed a 75% increase compared with the average migration of the control cell lines. Our previous reports describe ps20 as a growth inhibitor of the prostate epithelial carcinoma cell line PC-3 (4 , 5) . Factors involved in angiogenesis often exhibit endothelial cell growth regulating properties. For this reason, we examined the effect of ps20 on BAE cell and HUVEC proliferation in vitro. BAE cells and HUVECs exposed to ps20 showed no statistically significant differences in proliferation when compared with control cells (data not shown). This finding indicates that the ps20-induced increase in the number of migrated cells does not reflect a combined migration and growth stimulation effect.
ps20 Stimulates Angiogenesis and Tumor Growth in Vivo.
We have shown previously that stromal cells promote tumorigenesis and early angiogenesis in our DRS xenograft tumors composed of Matrigel, LNCaP human prostate carcinoma cells, and various human prostate stromal cell lines (2
, 3)
. To determine the effect of ps20 on DRS tumor growth and angiogenesis, we mixed either ps20 or control conditioned medium with Matrigel used for injections of LNCaP and HPS-19B human prostate stroma cells. Our previous studies have shown that day 10 of tumor growth was an optimal time point to assess early angiogenesis in DRS tumors (2)
. Accordingly, cells were injected and tumors were harvested on day 10. To evaluate tumor vasculature, sections were stained with an anti-CD31 (PECAM-1) antibody (Fig. 3A
, control, and B, ps20), a marker for endothelial cells, and microvessel density was determined following procedures we have reported previously (2
, 3)
. Tumors containing ps20 exhibited a microvessel density of 20 ± 2 compared with 12 ± 2 vessels/high power field in control tumors (Fig. 3C
; P < 0.001, unpaired t test), representing a 67% increase in vessel density. In addition, tumors containing ps20 exhibited a 29% increase in growth as determined by wet weights, 22 ± 0.76 mg for ps20 tumors compared with 17 ± 0.99 mg for control tumors, as well as a 58% elevation in tumor volumes, 19 ± 1 mm3 for ps20 tumors compared with 12 ± 1 mm3 for control tumors (Fig. 3, D and E
; P < 0.0001, unpaired t tests).
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control, and B, mifepristone). In addition, we noted that ps20-V5-His expressing cells in the tumors from the mifepristone-treated mice were often localized immediately adjacent to newly formed vessels in a pericyte position (Fig. 4B
, inset, arrows). This suggests a possible direct role in new vessel formation or stabilization. Tumors expressing ps20-V5-His (mifepristone-treated) exhibited a 129% elevation in microvessel density relative to control tumors, 48 ± 5 and 21 ± 3, respectively (Fig. 4, C–E
; P < 0.0001, unpaired t test).
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. The ps20 mRNA expression was elevated 4.5- and 11.3-fold above basal levels with 25 and 50 pM TGF-ß1, respectively. Similar results with reverse transcription-PCR analysis of ps20 mRNA expression after TGF-ß1 induction were noted (data not shown).
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DISCUSSION |
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As a secreted protein, ps20 may affect several cell types including endothelial cells. Stimulation of endothelial cell migration in vitro often correlates to elevated angiogenesis in vivo (24) . We determined that ps20 significantly increased BAE cell motility compared with control cells. In addition, we report that ps20 expression in stably transfected COS-1 cells promotes migration when compared with either the parent COS-1 cell line or to control cells stably transfected with empty vector. Whereas a specific mechanism of action remains to be determined, we hypothesize that ps20 functions as a serine protease inhibitor to stabilize the ECM and alter cell-matrix interactions with a resulting increase in migration. Whether this specific effect accounts for the increase in microvessel density observed here is not known.
Many factors that alter angiogenesis in vivo affect endothelial cell proliferation in vitro. The original description of ps20 characterized it as a growth inhibitor of prostate epithelial cells, as well as stably transfected COS-1 cells (4 , 5) . In the current study, ps20 did not affect the growth of BAE cells or HUVECs in culture. The WAP family member elafin is capable of inhibiting cell proliferation by preventing elastase-dependent release of the growth factor basic fibroblast growth factor from the ECM (25) . The growth inhibition activity of ps20 may rely on a similar indirect mechanism and be significant only in specific cell types under certain physiological conditions.
A number of human epithelial cancers, including prostate cancer, are known to involve a stromal reaction where increases are observed in stromal myofibroblast type cells, ECM deposition, and angiogenesis (26 , 27) . The current study examined the effect of ps20 on DRS tumors generated with LNCaP human prostate carcinoma cells and HPS-19B human prostate stromal cells. We determined that ps20 increased tumor wet-weight, volume, and microvessel density in day 10 tumors. We also assessed the effect of ps20 on angiogenesis in 3T3 cell tumors to determine whether the ps20-induced increase of angiogenesis in the DRS model is specific to prostate cancer xenograft tumors or is a more generalized effect. 3T3 cell tumors with induced expression of ps20 were found to have an increased microvessel density, similar to the DRS tumors. Together, these results suggest that ps20-increased angiogenesis is because of a generalized effect. Because ps20 is a serine protease inhibitor family member, its observed properties may reflect inhibition of protease activity, and resultant regulation of ECM remodeling and cell-matrix interactions culminating in enhanced endothelial cell migration into developing tumors or stabilization of newly formed vessels.
Whereas a suspected protease inhibitor may initially be predicted to inhibit angiogenesis, as many proteases are proangiogenic, there is emerging evidence that some protease inhibitors may stimulate angiogenesis. Plasminogen activator inhibitor 1, a urokinase plasminogen activator serine protease inhibitor, promotes angiogenesis by its protease inhibition properties, and high levels of plasminogen activator inhibitor 1 are predictive of poor survival rates in cancer patients (28, 29, 30, 31) . Another protease-dependent cascade that affects angiogenesis is the release of antiangiogenic protein fragments from the basement membrane. The antiangiogenic factor, endostatin, is generated from collagen XVIII in a cleavage reaction involving the serine protease elastase (32) . Because of the shared WAP-type serine protease inhibitor domain, ps20 is related to the potent elastase inhibitor elafin and, thus, may inhibit the production of endostatin-like antiangiogenic peptides. Accordingly, protease inhibitors may balance or control excessive ECM degradation that would otherwise hinder new vessel formation.
In addition to possibly affecting the endothelial cell sprouting and migration phase of angiogenesis, ps20 may function to stabilize newly formed vascular structures. Our localization of ps20 secreting stromal cells to tumor neovasculature indicates that these stromal cells are potentially recruited as pericyte-type cells. Pericyte coverage is known to stabilize vessel structures and protect against regression (33 , 34) . Our observation that Wfdc1 (ps20) is a TGF-ß1 inducible gene relates directly to the potential vessel stabilizing effect of ps20, as the direct interaction of endothelial cells with mural cells results in the activation of latent TGF-ß1 (35) . Whereas a role for protease inhibition in pericyte-induced vessel stabilization and maturation has yet to be defined, the balance between protease activity and inhibition is known to be central in the pathophysiology of large vessel destabilization, as seen in abdominal aortic aneurysms (36 , 37) .
Whereas ps20 is a small secreted protein of the WAP-type four-disulfide core domain family, a target protease of ps20 inhibition has not yet been identified. Although the WAP family member SLPI inhibits a broad range of target proteases, elafin is a specific inhibitor of elastase and proteinase 3 (11) . For these reasons, we suspect that ps20 inhibits a single protease or a narrow range of serine proteases. Our laboratory is currently screening for a potential ps20 target protease.
Although additional studies are necessary to determine the exact mechanisms of action for ps20, this report indicates that ps20 is a smooth muscle cell secreted protein capable of increasing endothelial cell migration, and promoting tumor growth and angiogenesis. Moreover, the ps20 effects described in this report make the functional inhibition of ps20 activity a possible target for antivascular tumor therapy.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported by NIH Grants RO1-CA58093, RO1-DK45909, Specialized Programs of Research Excellence CA58204, and UO1-CA84296. S. J. M. is a Hudson Scholar of the Baylor College of Medicine Medical Scientist Training Program. 
2 Present address: Craniofacial Developmental Biology and Regeneration Branch, National Institute of Dental and Craniofacial Research, NIH, 30 Convent Drive, MSC 4370, Bethesda, MD 20892-4370.
3 Present address: Wyle Laboratories, Life Sciences Systems and Services, 1290 Hercules Drive, Houston, TX 77058.
4 To whom requests for reprints should be addressed, at Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, TX 77030. Phone: (713) 798-6220; Fax: (713) 790-1275; E-mail: drowley{at}bcm.tmc.edu
5 The abbreviations used are: ECM, extracellular matrix; DRS, differential reactive stroma; BAE, bovine aortic endothelial; HUVEC, human umbilical vein endothelial cell; ps20, prostate stromal 20 kDa; SLPI, secretory leukocyte protease inhibitor; sm -actin, smooth muscle -actin; TGF, transforming growth factor; WFDC1, whey acidic protein-type four-disulfide core-1; WAP, whey acidic protein; CHO, Chinese hamster ovary.
6 Internet address: http://rsb.info.nih.gov/nih-image/.
Received 4/24/03. Revised 6/25/03. Accepted 6/30/03.
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and COS-1 pBKCMV-
) or empty vector (COS-1 pBKCMV-
), and nontransfected COS-1 cells (
) evaluated in an in vitro wound closure assay to assess cell migration (n = 3 for each cell line at each time point; experiment was repeated three times). *, statistically significant increase in migration of COS-1 cells stably expressing ps20 when compared with either control cell line (P < 0.05).
