The Novel SPARC Family Member SMOC-2 Potentiates Angiogenic Growth Factor Activity

SMOC-2 is a novel member of the SPARC family of matricellular proteins. The purpose of this study was to determine whether SMOC-2 can modulate angiogenic growth factor activity and angiogenesis. SMOC-2 was localized in the extracellular periphery of cultured human umbilical vein endothelial cells (HUVECs). Ectopically expressed SMOC-2 was also secreted into the tissue culture medium. In microarray profiling experiments, a recombinant SMOC-2 adenovirus induced the expression of transcripts required for cell cycle progression in HUVECs. Consistent with a growth-stimulatory role for SMOC-2, its overexpression stimulated DNA synthesis in a dose-dependent manner. Overexpressed SMOC-2 also synergized with vascular endothelial growth factor or with basic fibroblast growth factor to stimulate DNA synthesis. Ectopically expressed SMOC-2 stimulated formation of network-like structures as determined by in vitro matrigel angiogenesis assays. Fetal calf serum enhanced the stimulatory effect of overexpressed SMOC-2 in this assay. Conversely, small interference RNA directed toward SMOC-2 inhibited network formation and proliferation. The angiogenic activity of SMOC-2 was also examined in experimental mice by subdermal implantation of Matrigel® plugs containing SMOC-2 adenovirus. SMOC-2 adenovirus induced a 3-fold increase in the number of cells invading Matrigel® plugs when compared with a control adenoviral vector. Basic fibroblast growth factor and SMOC-2 elicited a synergistic effect on cell invasion. Taken together, our results demonstrate that SMOC-2 is a novel angiogenic factor that potentiates angiogenic effects of growth factors. SMOC-2 is a novel member of the SPARC family of matricellular proteins. The purpose of this study was to determine whether SMOC-2 can modulate angiogenic growth factor activity and angiogenesis. SMOC-2 was localized in the extracellular periphery of cultured human umbilical vein endothelial cells (HUVECs). Ectopically expressed SMOC-2 was also secreted into the tissue culture medium. In microarray profiling experiments, a recombinant SMOC-2 adenovirus induced the expression of transcripts required for cell cycle progression in HUVECs. Consistent with a growth-stimulatory role for SMOC-2, its overexpression stimulated DNA synthesis in a dose-dependent manner. Overexpressed SMOC-2 also synergized with vascular endothelial growth factor or with basic fibroblast growth factor to stimulate DNA synthesis. Ectopically expressed SMOC-2 stimulated formation of network-like structures as determined by in vitro matrigel angiogenesis assays. Fetal calf serum enhanced the stimulatory effect of overexpressed SMOC-2 in this assay. Conversely, small interference RNA directed toward SMOC-2 inhibited network formation and proliferation. The angiogenic activity of SMOC-2 was also examined in experimental mice by subdermal implantation of Matrigel® plugs containing SMOC-2 adenovirus. SMOC-2 adenovirus induced a 3-fold increase in the number of cells invading Matrigel® plugs when compared with a control adenoviral vector. Basic fibroblast growth factor and SMOC-2 elicited a synergistic effect on cell invasion. Taken together, our results demonstrate that SMOC-2 is a novel angiogenic factor that potentiates angiogenic effects of growth factors. Angiogenesis, the formation of new capillaries from existing vasculature, is a vital process in development, tumor formation, and in the restoration of the blood supply to ischemic tissues. Numerous factors are involved in the regulation of this process such as growth factors, oxygen levels, proteases, and extracellular matrix components. Over the past decade, matricellular proteins have gained more attention in their role in regulating cellular functions and angiogenesis. Matricellular proteins are extracellular proteins that do not contribute structurally to the extracellular milieu but regulate interactions between cells and the extracellular matrix (1Bornstein P. Sage E.H. Curr. Opin. Cell Biol. 2002; 14: 608-616Crossref PubMed Scopus (755) Google Scholar). Proteins that have been grouped as matricellular proteins include the thrombospondins, tenascins, osteopontin, and the SPARC 3The abbreviations used are: SPARC, secreted protein acidic and rich in cysteine/osteonectin/BM-40; PDGF, platelet-derived growth factor; PDGFβR, PDGF β receptor; VEGF, vascular endothelial growth factor; bFGF, basic fibroblast growth factor; SMOC-2, secreted modular calcium-binding protein-2; Ad-SMOC-2, SMOC-2 adenovirus; Ad-Myc-SMOC-2, Myc-tagged SMOC-2; siCTR, non-targeting control siRNA; siSMOC-2-1 and siSMOC-2-2, two siRNA molecules targeted to different regions of human SMOC-2; GFP, green fluorescent protein; EGM-2, endothelial cell growth medium-2; HUVEC, human umbilical vein endothelial cell; EBM-2, endothelial basal medium-2; FBS, fetal bovine serum; siRNA, small interference RNA; CMV, cytomegalovirus; DAPI, 4′,6-diamidino-2-phenylindole; BrdUrd, bromodeoxyuridine.3The abbreviations used are: SPARC, secreted protein acidic and rich in cysteine/osteonectin/BM-40; PDGF, platelet-derived growth factor; PDGFβR, PDGF β receptor; VEGF, vascular endothelial growth factor; bFGF, basic fibroblast growth factor; SMOC-2, secreted modular calcium-binding protein-2; Ad-SMOC-2, SMOC-2 adenovirus; Ad-Myc-SMOC-2, Myc-tagged SMOC-2; siCTR, non-targeting control siRNA; siSMOC-2-1 and siSMOC-2-2, two siRNA molecules targeted to different regions of human SMOC-2; GFP, green fluorescent protein; EGM-2, endothelial cell growth medium-2; HUVEC, human umbilical vein endothelial cell; EBM-2, endothelial basal medium-2; FBS, fetal bovine serum; siRNA, small interference RNA; CMV, cytomegalovirus; DAPI, 4′,6-diamidino-2-phenylindole; BrdUrd, bromodeoxyuridine. (secreted protein acidic and rich in cysteine/osteonectin/BM-40) family of proteins. These proteins are expressed in many cell types and are highly expressed during embryogenesis, wound healing, and other instances where there is extensive tissue remodeling.SPARC is highly expressed during embryogenesis, and its expression becomes more restricted in adult tissues (2Porter P.L. Sage E.H. Lane T.F. Funk S.E. Gown A.M. J Histochem. Cytochem. 1995; 43: 791-800Crossref PubMed Scopus (194) Google Scholar). It is highly expressed in adult bone tissues and during processes involving tissue remodeling such as tumorigenesis and wound repair. Mice that are homozygous-null for SPARC are able to develop a relatively normal phenotype but soon develop cataracts (3Gilmour D.T. Lyon G.J. Carlton M.B. Sanes J.R. Cunningham J.M. Anderson J.R. Hogan B.L. Evans M.J. Colledge W.H. EMBO J. 1998; 17: 1860-1870Crossref PubMed Scopus (209) Google Scholar), have impaired bone formation (4Delany A.M. Amling M. 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Gohring W. Mann K. Maurer P. Hohenester E. Knauper V. Murphy G. Timpl R. J. Biol. Chem. 1997; 272: 9237-9243Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar). SPARC has been shown to bind several growth factors and alter their activity. These include platelet-derived growth factor (PDGF) (10Raines E.W. Lane T.F. Iruela-Arispe M.L. Ross R. Sage E.H. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 1281-1285Crossref PubMed Scopus (326) Google Scholar) and vascular endothelial growth factor (VEGF) (11Kupprion C. Motamed K. Sage E.H. J. Biol. Chem. 1998; 273: 29635-29640Abstract Full Text Full Text PDF PubMed Scopus (245) Google Scholar). SPARC indirectly influences the effects of basic fibroblast growth factor (bFGF) (12Hasselaar P. Sage E.H. J. Cell. Biochem. 1992; 49: 272-283Crossref PubMed Scopus (135) Google Scholar), and transforming growth factor β (13Francki A. Bradshaw A.D. Bassuk J.A. Howe C.C. Couser W.G. Sage E.H. J. Biol. 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Biochem. 1993; 214: 347-350Crossref PubMed Scopus (107) Google Scholar), tsc36/FRP (25Shibanuma M. Mashimo J. Mita A. Kuroki T. Nose K. Eur. J. Biochem. 1993; 217: 13-19Crossref PubMed Scopus (224) Google Scholar), and the recently-described SMOC-2 (secreted modular calcium-binding protein)/SMAP2 (26Nishimoto S. Hamajima Y. Toda Y. Toyoda H. Kitamura K. Komurasaki T. Biochim. Biophys. Acta. 2002; 1576: 225-230Crossref PubMed Scopus (20) Google Scholar). SMOC-2 is expressed in nearly all tissues, with the highest expression found in the heart, muscle tissue, spleen, and ovary (27Vannahme C. Gosling S. Paulsson M. Maurer P. Hartmann U. Biochem. J. 2003; 373: 805-814Crossref PubMed Scopus (89) Google Scholar), but no function has been assigned to this protein. To date, the only clinically relevant role described for SMOC-2 is in the rat carotid artery of accelerated atherosclerotic lesion formation, which demonstrated an up-regulation of SMOC-2 mRNA in response to injury (26Nishimoto S. Hamajima Y. Toda Y. Toyoda H. Kitamura K. Komurasaki T. Biochim. Biophys. Acta. 2002; 1576: 225-230Crossref PubMed Scopus (20) Google Scholar).In a microarray screen for transcripts that are differentially regulated by the environmental carcinogen benzo[a]pyrene, we identified SMOC-2 as a cell cycle-regulated and benzo-[a]pyrene-suppressed transcript. Previous studies from other laboratories have suggested a role for the SMOC-2-related protein SPARC in regulation of angiogenesis (14Bradshaw A.D. Reed M.J. Carbon J.G. Pinney E. Brekken R.A. Sage E.H. Wound Repair Regen. 2001; 9: 522-530Crossref PubMed Scopus (55) Google Scholar, 15Lane T.F. Iruela-Arispe M.L. Johnson R.S. Sage E.H. J. Cell Biol. 1994; 125: 929-943Crossref PubMed Scopus (220) Google Scholar, 16Sage E.H. Reed M. Funk S.E. Truong T. Steadele M. Puolakkainen P. Maurice D.H. Bassuk J.A. J. Biol. Chem. 2003; 278: 37849-37857Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar). Therefore, in studies presented here we have examined the effect of SMOC-2 on growth factor-induced mitogenesis and angiogenesis of endothelial cells in vitro and in vivo.EXPERIMENTAL PROCEDURESMaterials—Myc tag antibody was purchased from Cell Signaling. CD31 antibody was purchased from BD Pharmingen. α-Tubulin antibody was purchased from Oncogene (Cambridge, MA). Recombinant human VEGF-165 and bFGF were purchased from R&D Systems. Matrigel® was purchased from Cambrex. Small inhibitor RNAs (siRNAs) were purchased from Dharmacon Inc. for a control non-targeting sequence (siCTR, cat. # D-001210-01), and two SMOC-2 targeting sequences, siSMOC-2-1 (sense: 5′-GAGAGUGGAUCAAGAUAAAdTdT-3′; antisense: 5′-UUUAUCUUGAUCCACUCUCdTdT-3′) and siSMOC-2-2 (sense: 5′-CAAAUCCAUCUCCGUACAAdTdT-3′; antisense: 5′-UUGUACGGAGAUGGAUUUGdTdT-3′) and used according to the manufacturer's specifications.Adenovirus Construction and Cell Culture—Replication-deficient adenovirus vectors were produced using a method originally established by Becker et al. (28Becker T.C. Noel R.J. Coats W.S. Gomez-Foix A.M. Alam T. Gerard R.D. Newgard C.B. Methods Cell Biol. 1994; 43: 161-189Crossref PubMed Scopus (561) Google Scholar). Briefly, SMOC-2 cDNA was cloned into the multicloning site of pACCMVpLpA plasmid. pACCMVpLpA-SMOC-2 was co-transfected with pJM17 plasmid into 293 cells to allow for homologous recombination to produce the SMOC-2 adenovirus (Ad-SMOC-2). The Myc-tagged SMOC-2 (Ad-Myc-SMOC-2) was constructed by the same protocol but using a vector that contained a Myc tag upstream from the SMOC-2 insert. Control adenoviruses were used that expressed either green fluorescent protein (Ad-GFP) or β-galactosidase (Ad-βgal) under the control of a CMV promoter. Human umbilical vein endothelial cell (HUVEC) cultures infected for 24 h with Ad-GFP at a multiplicity of infection of 10 achieved 90% transduction efficiency (data not shown).Pooled HUVECs were purchased from BD Bioscience Inc. and maintained according to the manufacturer's instructions in endothelial cell growth medium-2 (EGM-2). Cells were used in the fourth to fifth cell passages.Western Blot Analysis—HUVECs grown to 70% confluency on 100-mm dishes were infected with Ad-GFP or Ad-Myc-SMOC-2 and incubated for 24 h. Medium was removed and replaced with 3 ml of fresh EGM-2, and cultures were harvested 24 h later. Proteins were separated by SDS-PAGE as described previously (29Rocnik E.F. van der Veer E. Cao H. Hegele R.A. Pickering J.G. J. Biol. Chem. 2002; 277: 38571-38578Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). Membranes were incubated with the designated primary antibody (1:1000 for all antibodies) overnight at 4 °C. Bound primary antibody was reacted with anti-rabbit peroxidase-conjugated IgG or anti-mouse peroxidase-conjugated Fab fragments for 1 h and detected by chemiluminescence, according to the manufacturer's recommendations (Roche Applied Science). Densitometric analyses were made by using the Scion Image computer program (Scion Corp., Frederick, MD).SMOC-2 Antibody Production and Immunofluorescence— Genemed Synthesis, Inc. (San Francisco, CA) was contacted to produce a SMOC-2 antibody. A synthetic peptide sequence (C-YPTLWTEQVKSRQNK) corresponding to the SMOC-2-specific domain (27Vannahme C. Gosling S. Paulsson M. Maurer P. Hartmann U. Biochem. J. 2003; 373: 805-814Crossref PubMed Scopus (89) Google Scholar) was conjugated to KLH: keyhole limpet hemocyanin and used for immunizations.Immunofluorescence was performed by culturing HUVECs in 4-well chamber slides to confluence. Cells were transduced with adenovirus made with an empty vector (Ad-Con) or Ad-Myc-SMOC-2 for 48 h. Cells were fixed with 4% paraformaldehyde for 10 min and then permeabilized with 0.2% Triton X-100 for 5 min. Following incubation with anti-SMOC-2 antibody, bound primary antibody was detected with fluorescein isothiocyanate-conjugated anti-rabbit secondary antibody. After washing, the slides were DAPI-stained and mounted with Vectashield solution (Vector Laboratories). Slides were imaged and analyzed using a Delta Vision Image Restoration Microscopy System (dv1301421, Applied Precision).Microarray Analysis—HUVECs were transduced with Ad-SMOC-2 or Ad-GFP for 48 h. RNA was purified from the HUVECs using a TRIzol kit (Invitrogen). RNA samples were submitted to the microarray core facility at The Department of Genetics and Genomics, Boston University School of Medicine for labeling, hybridization to Affymetrix chips, and data analysis.Cell Cycle Analysis—Cell cycle distribution of DNA content was determined by flow cytometry as described previously (30Vaziri C. Saxena S. Jeon Y. Lee C. Murata K. Machida Y. Wagle N. Hwang D.S. Dutta A. Mol. Cell. 2003; 11: 997-1008Abstract Full Text Full Text PDF PubMed Scopus (332) Google Scholar). Serum-starved cells pulsed with 10 mm BrdUrd (Roche Applied Science) for 1 h were harvested with trypsin-EDTA, fixed in 65% Dulbecco's modified Eagle's medium/35% ethanol for 1 h at 4 °C, and resuspended in 1 ml of 2 m HCl for 20 min at room temperature. Cells were labeled with fluorescein isothiocyanate-conjugated anti-BrdUrd antibody (BD Pharmingen #33284X) using standard laboratory procedures. The labeled cells were analyzed for propidium iodide and BrdUrd staining on a BD Biosciences flow cytometer using CellQuest software.DNA Synthesis Assays—HUVECs were grown in 6-well plates until cells reached 70-80% confluency. Controls and experimental conditions were done in triplicate. The medium was changed to endothelial basal medium-2 (EBM-2), and the cells were infected with adenovirus using multiplicity of infection (m.o.i.) values of 10 and 50. 24 h later, the medium was changed to fresh EBM-2 and either VEGF or bFGF was added at the indicated doses. After 20 h, the medium was changed to 750 μl of EBM-2 containing 4 μCi/ml [3H]thymidine (PerkinElmer Life Sciences). The HUVECs were labeled for 4 h at 37 °C. Following washing and precipitation with ice-cold 10% trichloroacetic acid, the cells were dissolved in 500 μl of 0.1 m NaOH overnight at 4 °C. The amount of radioactivity in each sample was counted in duplicate using 100 μl of the dissolved cell sample and Ecolite scintillation fluid (ICN). Data were presented relative to Ad-GFP-transduced cells at an m.o.i. of 10 with no growth factors.Cell Proliferation Studies—HUVECs were plated at 10,000 cells/cm2 on 24-well plates. Culture medium was changed every day during the time course. Cells were allowed to adhere; the medium was changed and then transduced with adenoviruses for 24 h. For experiments involving siRNAs, subconfluent HUVECs were transfected with siRNAs according to the manufacturer's instructions. After 24 h, cultures were trypsinized and plated at 10,000 cells/cm2 on 24-well plates. This was designated as day 0. A hemocytometer was utilized to count the number of cells per well after adding 100 μl of trypsin-EDTA per well, and cell viability was determined by trypan blue exclusion.Migration Assays—Cell migration was assayed using a modified Boyden chamber (ChemoTx® plate, Neuroprobe, Inc., Gaithersburg, MD) according to the manufacturer's instructions. HUVECs were infected with adenoviral vectors overnight in EGM-2 and then labeled with Dil-labeled acetylated low density lipoprotein (10 μg/ml) for 4 h in EBM-2. Cells were then trypsinized and resuspended in phenol red-free EBM at 10,000 cells/25 μl. VEGF or bFGF in phenol red-free EBM was added into the lower chamber. Each experiment was performed in triplicate, and three separate experiments were performed in each experimental group.Cellular migration was also measured using a wounding assay. A grid patter was drawn on the underside of 6-well plates before HUVECs were plated on them to serve as landmarks for the start of the migration period. HUVECs were grown to confluency and allowed to remain so for a further 24 h. Cultures were then infected for 24 h with adenovirus or were transfected with siRNAs according to the manufacturer's instructions for 24 h. A cell scraper was used to wipe away the cell monolayer on one side of the start line that had been drawn on the bottom of the plate. Images were captured with a video graphic system (DEI-750 CE Digital Output Camera, Optronics, Goleta, CA) at 4× magnification, and the same areas were photographed at 24 and 48 h with the assistance of the landmarks drawn on the undersurface of the plate. Several fields of view were captured per well, and experiments were repeated three times. Migration was quantified by measuring the area of the cell migration front as they migrated into the scraped area at each time point. Area was quantified using Scion Image analysis software.In Vitro Cell-network Formation Assay—The formation of network-like structures by HUVECs on Matrigel® (BD Biosciences) was performed as previously described (31Leopold J.A. Walker J. Scribner A.W. Voetsch B. Zhang Y.Y. Loscalzo A.J. Stanton R.C. Loscalzo J. J. Biol. Chem. 2003; 278: 32100-32106Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar). The 12-well culture plates were coated with Matrigel® according to the manufacturer's instructions. The adenovirus-transduced or siRNA-transfected HUVECs were seeded on coated plates at 3 × 104 cells/cm2 in EGM containing 0%, 0.5%, or 5% fetal bovine serum and incubated at 37 °C for overnight. Cells were observed using an inverted phase-contrast microscope (Nikon). Images were captured with a video graphic system (DEI-750 CE Digital Output Camera, Optronics, Goleta, CA). The degree of network formation was quantified by capturing five high power field images, and the area of the cells was quantified using Scion Corp. (National Institutes of Health Image) area analysis software.Mouse Angiogenesis Assay—The formation of new vessels in vivo was evaluated by the Matrigel® plug assay (BD Biosciences) employing a modification of the procedures described previously (32Skurk C. Maatz H. Rocnik E. Bialik A. Force T. Walsh K. Circ. Res. 2005; 96: 308-318Crossref PubMed Scopus (136) Google Scholar). Prior to injection, 0.5 ml of Matrigel® was mixed with heparin (10 units/ml) in chilled tubes. bFGF (50 ng/ml, R&D Systems) was added to the indicated tubes. Adenoviral vectors encoding GFP or SMOC-2 were added to a final concentration of 2 × 108 plaque forming units of virus. Tissues were sectioned, and immunohistochemistry was performed with an anti-CD31 antibody (platelet endothelial cell adhesion molecule-1, 1:100 dilution, BD Pharmingen). Bound primary was detected with biotinylated rabbit anti-rat IgG antibody (1:200 dilution, Vector) and the ABC Elite kit from Vector Laboratories using DAB. The sections were counterstained with Harris' hematoxylin. Images were captured using an Olympus BX41 microscope with color digital video camera, and an angiogenic response was quantified by cell counts of CD31-positive cells from 10 high power fields per section.Statistical Analysis—All data were compared by Student's t test or analysis of variance with the Scheffé post-hoc test using StatView 4.5 (Abacus Software, Burlington, MA). Data are expressed as mean ± S.E. for the number of independent experiments indicated. A p value of <0.05 was considered to be significant.RESULTSCellular Localization of Overexpressed SMOC-2 in HUVECs— To analyze the effects of SMOC-2 growth regulation of endothelial cells we constructed adenoviral vectors for expressing SMOC-2 (Ad-SMOC-2) and an Myc-tagged SMOC-2 (Ad-Myc-SMOC-2). HUVECs were transduced with the Ad-Myc-SMOC-2 virus or a control adenoviral vector expressing green fluorescent protein (Ad-GFP). Lysates from AdGFP and Ad-Myc-SMOC-2-transduced HUVECs were subjected to Western blot analysis using anti-c-Myc antibody (Fig. 1A). Cell extracts from the Ad-Myc-SMOC-2-transduced cells showed an intense anti-c-Myc-reactive band of 56 kDa, which corresponded to the predicted size of SMOC-2 fusion protein (Fig. 1A, lane 2) (27Vannahme C. Gosling S. Paulsson M. Maurer P. Hartmann U. Biochem. J. 2003; 373: 805-814Crossref PubMed Scopus (89) Google Scholar). SMOC-2 fusion protein was also detected in the culture medium, which was expected, because SMOC-2 does contain a signal peptide for secretion (27Vannahme C. Gosling S. Paulsson M. Maurer P. Hartmann U. Biochem. J. 2003; 373: 805-814Crossref PubMed Scopus (89) Google Scholar) (Fig. 1A, lane 4). Endogenous levels of SMOC-2 transcripts were measured by real-time PCR in serum-deprived HUVECs and HUVECs in complete growth medium. As shown in Fig. 1B, serum deprivation led to an 18-fold increase in SMOC-2 mRNA levels.We also performed immunofluorescence microscopy to determine the subcellular distribution of SMOC-2. HUVECs were infected with a control "empty" vector adenovirus (Ad-Con) or Ad-Myc-SMOC-2. The resulting cells were fixed and stained with a c-Myc monoclonal antibody or with rabbit anti-SMOC-2 antisera. The specificity of the anti-SMOC-2 antibody was tested in Rat1 cells, which express low endogenous SMOC-2. Antibody binding to the adenovirus-encoded protein could be inhibited by addition of excess immunizing peptide (data not shown). As shown in Fig. 2A, the SMOC-2 antisera detected a weak signal in the cellular periphery of Ad-Con-infected HUVECs. In contrast, in Ad-Myc-SMOC-2-infected cells we detected very strong staining in the cell periphery and some diffuse staining within the cells. We also performed costaining experiments in which Ad-Myc-SMOC-2-infected cells were stained with anti-SMOC-2 and anti-c-Myc antisera. As shown in Fig. 2B, the merged images showed good overlap between Myc (red) and SMOC-2 (green) signals indicating that the staining pattern was specific for SMOC-2. Taken together, our immunoblotting and immunofluorescence experiments demonstrate that SMOC-2 is predominantly localized to the cellular periphery and that a fraction is secreted from cells. This expression pattern is similar to that reported by other workers for the SMOC-2-related factor SPARC (33Sage H. Vernon R.B. Funk S.E. Everitt E.A. Angello J. J. Cell Biol. 1989; 109: 341-356Crossref PubMed Scopus (314) Google Scholar) in that SMOC-2 appears to associate with edges of the cell membrane. This would be consistent with a putative role for SMOC-2 as a regulator of extracellular matrix interactions and/or growth factor receptor signaling.FIGURE 2A, confluent HUVECs transduced with either control Ad-Con or Ad-Myc-SMOC-2 were fixed and immunostained with anti-SMOC-2 antibody. Bound primary was detected with fluorescein isothiocyanate-labeled anti-rabbit secondary antibody. Nuclei were visualized with DAPI. Positive staining was predominantly localized to the extracellular periphery. B, SMOC-2 staining was confirmed by double immunolabeling for SMOC-2 and c-Myc tag. Bound anti-c-Myc antibody was detected with a Texas Red-labeled anti-mouse secondary antibody. Nuclei were visualized with DAPI. Positive staining was again predominantly localized to the extracellular periphery, and co-localization of SMOC-2 and the c-Myc tag is indicated by yellow in the merged image.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Regulation of Cell Cycle-related Transcript by SMOC-2—Because SMOC-2 has similarity to the angiogenic regulator and growth factor-binding protein SPARC, we performed a microarray screen to identify mRNAs that are differentially expressed in HUVECs transduced with Ad-SMOC-2 and Ad-GFP control virus. HUVECs were serum-starved for 24 h in EBM-2 with 0.5% FBS and then transduced with adenovirus for 24 h. RNA samples from transduced cells were subjected to labeling and hybridization to Affymetrix chips containing arrayed oligonucleotides corresponding to the human "transcriptome." A partial list of the mRNAs that were differentially expressed in Ad-GFP and Ad-SMOC-2-infected cells is presented in Table 1. From the full list it was apparent that numerous cell cycle-related mRNAs were up-regulated in SMOC-2-expressing cells. These include transcripts encoding MCM4 and MCM10 (DNA replication factors required for "licensing" and "initiation" steps of DNA synthesis, respectively), Aurora B kinase (required for G2/M progression), the centromeric protein CENP-F (required for