TISSUE-SPECIFIC STEM CELLS TGFBIp Regulates Differentiation of EPC (CD1331C-kit1Lin2 cells) to EC Through Activation of the Notch Signaling Pathway YONG-SUN MAENG,a YEON JEONG CHOI,a EUNG KWEON KIMa,b Key Words. EPC • TGFBIp • Differentiation • Notch

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Department of Ophthalmology, Corneal Dystrophy Research Institute and bBrain Korea 21 Plus Project for Medical Science, Institute of Vision Research, Severance Biomedical Science Institute, Yonsei University College of Medicine, Seoul, South Korea Correspondence: Eung Kweon Kim, M.D., Ph.D. Department of Ophthalmology, Yonsei University College of Medicine, 250 Seongsanno, Seodaemungu, Seoul, 120-752, Korea. Telephone: 82-2-2228-0824; Fax: 82-2-2227-8129; e-mail: [email protected] Received October 7, 2015; accepted for publication February 6, 2015; first published online in STEM CELLS EXPRESS March 18, 2015. C AlphaMed Press V

1066-5099/2015/$30.00/0 http://dx.doi.org/ 10.1002/stem.2003

ABSTRACT Endothelial progenitor cells (EPCs) in the circulatory system have been suggested to maintain vascular homeostasis and contribute to adult vascular regeneration and repair. These processes require that EPCs recognize the extracellular matrix (ECM), migrate, differentiate, and undergo tube morphogenesis. The ECM plays a critical role by providing biochemical and biophysical cues that regulate cellular behavior. Here, we tested the importance of transforming growth factor-beta-induced protein (TGFBIp) in regulation of the differentiation and angiogenic potential of human cord blood-derived EPCs (CD1331C-kit1Lin2 cells). EPCs displayed increased endothelial differentiation when plated on TGFBIp compared to fibronectin. EPCs also exhibited increased adhesion and migration upon TGFBIp stimulation. Moreover, TGFBIp induced phosphorylation of the intracellular signaling molecules SRC, FAK, AKT, JNK, and ERK in EPCs. Using integrin-neutralizing antibodies, we showed that the effects of TGFBIp on EPCs are mediated by integrins a4 and a5. Furthermore, TGFBIp increased the adhesion, migration, and tube formation of CD341 mouse bone marrow stem cells in vitro. Gene expression analysis of EPCs plated on TGFBIp revealed that EPCs stimulated by TGFBIp exhibit increased expression of Notch ligands, such as delta-like 1 (DLL1) and Jagged1 (JAG1), through nuclear factor-kappa B signaling activation. Collectively, our findings demonstrate, for the first time, that locally generated TGFBIp at either wounds or tumor sites may contribute to differentiation and angiogenic function of EPCs by augmenting the recruitment of EPCs and regulating the expression of endothelial genes DLL1 and JAG1. STEM CELLS 2015;33:2052–2062

INTRODUCTION Postnatal vasculogenesis has been implicated as an important mechanism for neovascularization via circulating endothelial progenitor cells (EPCs) derived from bone marrow (BM) [1, 2]. In addition, EPCs have been proposed as a potential therapeutic tool for treating vascular disease either via infusion to the site of vascularization [3, 4] or via ex vivo expansion for engineering of vascularized tissue constructs [5, 6]. Understanding the molecular mechanism that regulates neovascularization by EPCs will provide insight for such therapeutic vascularization. Differentiation, mobilization, and recruitment of EPCs and outgrowing endothelial cells (OECs) have been found to be regulated by vascular endothelial growth factor (VEGF), stromal cell-derived factor-1 (SDF-1), and insulinlike growth factor 2 (IGF2) [7–10]. Administration of these factors into the site of ischemia induces EC mobilization and blood flow restoration [9–12]. The extracellular matrix (ECM) provides critical support for ECs; their adhesion to the ECM is required for proliferation,

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migration, morphogenesis, and survival as well as for the stabilization of blood vessels [13] through both biochemical and mechanical functions [14–16]. Matrix elasticity has been reported to induce stem cell differentiation and morphological changes [17, 18]. Changes in physical interactions between cell surface integrins and the ECM, due to alterations in ECM elasticity, regulate cell shape, and cytoskeletal structure [19–21]. Mechanical forces exerted by ECs on the ECM stimulate capillary growth in vivo [22] and formation of capillarylike structures in vitro [23, 24]. Recently, matrix elasticity was found to modulate the adhesion and differentiation of EPCs [25], and biomechanical forces alone were sufficient to mediate vascular growth in vivo in a manner independent of endothelial sprouting [26]. The Notch pathway, which is evolutionarily conserved from invertebrates to mammals, was first recognized as a determinant of cell fate decisions between epithelial and neural lineages in Drosophila [27–29]. Notch signaling also plays an important role in the regulation of endothelial and hematopoietic cell fates in C AlphaMed Press 2015 V

Maeng, Choi, Kim both invertebrates and mice [30, 31]. Perturbation of Notch signaling has deleterious effects on embryonic hematopoiesis and vascular development [32–37]. In humans, a critical role for Notch signaling through the Notch ligand Jagged1 (JAG1) has been demonstrated for the proliferation and differentiation of normal primitive human hematopoietic progenitors [38–40]. Transforming growth factor-beta-induced protein (TGFBIp) has been identified as a major TGF-beta-responsive gene in the lung adenocarcinoma cell line A549 [41, 42] and is also upregulated by lysophosphatidic acid in mesenchymal stem cells and fibroblasts [43, 44]. TGFBIp, which is a secreted protein that has been detected in various human cell types [41, 45], is a 68-kDa protein that consists of four fasciclin-1 domains and a C-terminal arginyl-glycyl-aspartic acid sequence, both of which can bind integrins [46]. As one of the ECM components, TGFBIp interacts with other ECM molecules, including collagens, fibronectin, laminins, and glycosaminoglycans [42], and functions as a linker protein in the interaction between ECM and integrins [47]. Little is known, however, about the impact of TGFBIp on adhesion, migration, and differentiation of stem/progenitor cells during neovascularization. In this study, we defined the role of TGFBIp in adhesion, migration, and differentiation of human EPCs (CD1331 C-kit1Lin2 cells) and more specifically in regulation of Notch ligand expression. TGFBIp increased adhesion, migration, and differentiation of EPCs and CD341 mouse BM mononuclear cells (CD341 mBMMNC) through binding to integrins a4 and a5. Moreover, TGFBIp increased expression of Notch ligands, such as delta-like 1 (DLL1) and JAG1, through nuclear factorkappa B (NF-jB) signaling pathway activation and ultimately promoted differentiation of EPC to EC. Thus, we conclude that the regulation of TGFBIp expression in neovascular regions may control EPC differentiation and neovasculogenesis and serve as a useful target for the development of novel means to treat angiogenesis-dependent diseases.

MATERIALS

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METHODS

Study Population and Sample Collection Between June 2007 and March 2008, patients who had an uncomplicated pregnancy and were pending elective Cesarean section between 37–41 weeks of gestation were enrolled in the study. Umbilical cord blood sampling was performed immediately after infant delivery. Pregnancies associated with premature rupture of membranes, fetal malformation, chromosome anomaly, multiple pregnancies, preeclampsia, hypertension, connective tissue disease, and renal or endocrine diseases were excluded. This study was approved by the Institutional Review Board at Yonsei University (4-2005-0186), and informed consent to participate in the study was obtained from all patients.

Isolation and Cultivation of EPCs (CD1331C-kit1Lin2 Cells: CKL2 Cells) Preparation of EPCs was performed as described previously [10]. Briefly, EPCs were isolated from human umbilical cord blood samples (50 ml each) by density gradient centrifugation using Biocoll (Biochrom, Berlin, Germany) for 30 minutes at 4003g, and samples were then washed three times in

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phosphate-buffered saline (PBS; Biochrom). CKL2 cells were purified by positive and negative selection with anti-CD133/ C-kit/Lin2 microbeads (Miltenyi Biotec, Bergisch-Gladbach, Germany) using a magnetic cell sorter device (Miltenyi Biotec.). Purity, as assessed by fluorescence-activated cell sorting (FACS) analysis, was > 98%. CKL2 cells were seeded into 6well plates coated with human fibronectin (Sigma, St. Louis, MO) in endothelial basal medium-2 (EBM-2; Clonetics, Cell Systems, St. Katharinen, Germany). The medium was supplemented with endothelial growth medium-2 (EGM-2; Clonetics, Cell Systems) containing fetal bovine serum, human VEGF-A, human fibroblast growth factor-B, human epidermal growth factor, IGF1, and ascorbic acid in appropriate amounts. Cultures were maintained for 7 days, and phenotypic analyses of the cells were performed on days 3, 5, and 7. EPC identification and estimation of culture purity (90–95%) were determined by staining cells with fluorescein isothiocyanate (FITC)-labeled Ulex europaeus lectin I (Vector Laboratories, Burlingame, CA) and measuring the uptake of Dil-conjugated acetylated low-density lipoprotein (acLDL; Molecular Probes, Leiden, The Netherlands).

EPC Differentiation Assay EPCs were cultured on fibronectin (10 mg/ml; Sigma), TGFBIp (10 mg/ml; Sino Biological Inc., Beijing, China), or fibronectin and TGFBIp (each 10 mg/ml), which were coated on 6-well plates. Morphological assessment of EPC differentiation to OECs was then performed by light microscopy. Differentiation days and the number of colonies formed in each set were determined by light microscopy. At least three assays were performed for each sample.

Isolation of CD341 mBMMNC After administering an overdose of sodium pentobarbital, both tibial and femoral bones from C57/BL6 mice (Orient Company, Seoul, Korea) were removed. The ends of the bones were cut, and BM was removed by irrigation with PBS. Irrigates were fractionated with Histopaque-1083, and mononuclear layers were harvested and washed three times with PBS. The CD341 mBMMNCs were purified by positive selection with anti-CD34 microbeads (Miltenyi Biotec) using a magnetic cell sorter device (Miltenyi Biotec.). Purity, as assessed by FACS analysis, was >98%. CD341 mBMMNCs were cultured in EGM-2 BulletKit medium for 7 days to obtain spindle-shaped, attached cells. The identity of cultured cells as EPCs was confirmed by staining with Bandeiraea simplicifolia-1 (Sigma) and measuring the uptake of DiI-acLDL.

CD341 mBMMNC Tube Formation Assay Tube formation was assayed as previously described [48]. Briefly, 250 ml Matrigel (BD Biosciences, Bedford, MA) was added to a 16-mm diameter tissue culture dish and allowed to polymerize for 30 minutes at 37 C. After trypsinization, the harvested CD341 mBMMNCs were resuspended in EBM containing TGFBIp (5–10 mg/ml) and plated onto the layer of Matrigel (1.2 3 105 cells/well). Matrigel cultures were incubated at 37 C for 10 days and photographed at various time points (2003 magnification). The area covered by the tube network was determined using an optical imaging technique. R Pictures of the tubes were scanned into Adobe PhotoshopV and quantified using ImageJ software (National Institutes of Health, http://rsb.info.nih.gov/ij/). C AlphaMed Press 2015 V

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Cell Migration Assay Cell migration was assayed using the Transwell system (Corning Costar, Acton, MA) with 6.5-mm diameter polycarbonate filters (5-mm pore size). Briefly, the lower surface of the filter was coated with 10 mg/ml fibronectin (Sigma-Aldrich), 2.5–10 mg/ml recombinant human TGFBIp (rhTGFBIp; Sino Biological Inc.), or 3% (w/v) bovine serum albumin as a control for nonspecific binding. EPCs (105) were seeded onto chemotaxis filters in EBM plus 0.5% fetal bovine serum. After the 24-hours migration period, non-migrating cells were removed from the top surface of the membrane. Migrating cells adhering to the undersurface of the filters were examined by hematoxylin and eosin (H&E) staining and quantified using the Kodak 1D software (Eastman Kodak, Rochester, NY). Results are representative of four independent experiments.

Cell-Matrix Adhesion

Oligonucleotide Microarrays Total RNA (10 lg) from EPCs treated with TGFBIp (10 mg/ml) for 6 h and control EPCs were hybridized to the HG-U133A 2.0 microarray (54675 human genes; Affymetrix, Santa Clara, CA). The standard protocol for sample preparation and microarray processing from Affymetrix was used. Expression data were analyzed using Microarray Suite version 5.0 (Affymetrix) and GenPlex v2.4 software (ISTECH Inc., Seoul, Korea, http://istech21.tradekorea.com/).

Immunofluorescence Staining

Cell-matrix adhesion assays were performed as described previously [49]. The 96-well plates were coated overnight at 4 C with 2.5–10 mg/ml rhTGFBIp or 10 mg/ml human fibronectin. EPCs in adhesion buffer were seeded at 105 cells/well in a volume of 100 mL and then incubated for 30 minutes at 37 C. After the removal of nonadherent cells by two washes, adherent cells were measured by H&E staining and quantified in triplicate by counting adherent cells in five randomly selected fields per well (Axiovert 100; Carl Zeiss Micro-Imaging, Thornwood, NY). For blocking experiments, 1 mg/ml anti-integrin a4 antibody (Chemicon Inc., CA, USA), anti-integrin a5 antibody (Abcam, Cambridge, England), anti-integrin avb3 antibody (Abcam), anti-integrin b3 antibody (Chemicon Inc.), or a nonspecific IgG were added to the EPCs 30 minutes before starting the migration experiments. After the 30-minutes adhesion period, nonadhering cells were completely removed, and adherent cells were measured by H&E staining and quantified in triplicate by counting adherent cells in five randomly selected fields per well (Axiovert 100; Carl Zeiss Micro-Imaging). Results are representative of three different experiments performed in duplicate.

OEC Tube Formation Assay Tube formation was assayed as previously described [48]. In brief, 250 ml Matrigel (BD Biosciences) was added to a 16-mm diameter tissue culture well and allowed to polymerize for 30 minutes at 37 C. After trypsinization, the harvested OECs were resuspended in EBM and plated onto the layer of Matrigel (1.2 3 105 cells/well). Matrigel cultures were incubated at 37 C and photographed at various time points (2003 magnification). The area covered by the tube network was determined using an optical imaging technique. Pictures of the tubes were scanned into Adobe PhotoshopV and quantified using ImageJ software. R

Real-Time Quantitative RT-PCR (RT-qPCR) Total RNA was isolated from EPCs and OECs by extraction in TRIZOL reagent (Invitrogen, Carlsbad, CA). Using the Power SYBR Green RNA-to-CTTM 1-Step kit (Applied Biosystems, Foster City, CA) and StepOnePlusTM (Applied Biosystems), mRNA expression of human genes was measured according to the manufacturer’s instructions. The PCR conditions for all genes were as follows: 48 C for 30 minutes and then 95 C for 10 C AlphaMed Press 2015 V

minutes followed by 40 cycles of 95 C for 15 s and 60 C for 1 minute. Results are based on cycle threshold (Ct) values. We calculated differences between the Ct values for experimental and reference (GAPDH) genes and graphed the results as the ratio of each RNA to the calibrated sample. Primers used for gene amplification are shown in Supporting Information Table S1.

Cells were fixed in 3.7% formaldehyde for 20 minutes and permeabilized with 0.1% Triton X-100 in PBS. Cells were then pre-incubated with a blocking solution of PBS containing 5% normal donkey serum and 0.05% Tween-20. Cells were incubated with primary antibody (anti-mouse CD31 antibody [Angiobio Co, Del Mar, CA, USA], anti-mouse CD34 antibody [BD PharMingen, San Diego, CA], anti-human CD31 antibody [DAKO, Denmark], or anti-human NF-jB antibody [Cell Signaling, Beverly, MA]) for 2 hours at room temperature and then labeled with a fluorescein-conjugated secondary antibody (Molecular Probes, Eugene, OR). Nuclei were counterstained with 40 ,6diamidino-2-phenylindole (DAPI). Samples were observed with a fluorescence microscope (Olympus, Tokyo, Japan, www.olympusglobal.com/).

Transfections and Analysis of Luciferase Activity EPCs were transfected with 1 lg of the pEZX-DLL1 promoter luciferase plasmids (21.2 Kbp) (Genecopoeia, Rockville, MD) and 0.5 lg of the control pCMV-b-gal plasmid using electroporation (Amaxa Biosystems, Gaithersburg, MD). After 24 hours, Cells were stimulated with TGFBIp (10 mg/ ml) for 24 hours, and cell extracts were prepared. Luciferase assays were performed using the Luciferase Assay System (Promega, Madison, WI, USA). Luciferase activities were normalized with respect to parallel b-galactosidase activities to correct for differences in transfection efficiency, and the b-galactosidase assays were performed using the b-Galactosidase Enzyme Assay System (Promega). Each experiment was performed in at least quadruplicate.

EPC Differentiation Assay in Matrigel Seven-week-old male C57/BL6 mice (Orient Company, Seoul, Korea) were injected subcutaneously with 0.6 mL Matrigel containing 5 3 104 human EPCs and TGFBIp (10 mg/ml) or Fibronectin (10 mg/ml). After 5 days, the plugs were removed and washed with PBS. Hemoglobin was measured using the Drabkin method and the Drabkin reagent kit 525 (SigmaAldrich) for the quantification of blood vessel formation. The concentration of hemoglobin was calculated from a known amount of hemoglobin assayed in parallel. Differentiated EPCs and blood vessels in the Matrigel plugs were identified by staining with anti-human CD31 antibody [DAKO, Denmark] and viewed using fluorescence microscopy. STEM CELLS

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Figure 1. TGFBIp promotes differentiation of EPCs to ECs. (A): Morphological assessment of EPC differentiation to OECs upon culture with TGFBIp (10 mg/ml) or fibronectin (10 mg/ml). Arrowheads indicate differentiated OEC colonies. Images were viewed at 1003 magnification. (B): Quantitative analysis of differentiation days. In all figures, data are presented as mean 6 standard error (SE). **, p < .01 vs. control. (C): Morphological assessment of EPC differentiation to OECs upon culture with TGFBIp (10 mg/ml) or fibronectin (10 mg/ml). Dashed-line circles indicate differentiated OEC colonies. Images were viewed at 403 magnification. (D): Quantitative analysis of differentiated OEC colonies. In all figures, data are presented as mean 6 standard error. **, p < .01 vs. control. (E): Tube formation of OECs differentiated by TGFBIp stimulation. Immunofluorescence staining of tube-forming OECs with anti-CD31 antibody. Images were viewed at 403 magnification. Abbreviation: EPC, endothelial progenitor cell; OEC, outgrowing endothelial cell; TGFBIp, transforming growth factor-beta-induced protein.

Western Blot Analysis Cell lysates were fractionated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride (PVDF) membranes. The blocked membranes were incubated with the appropriate antibody, and the immunoreactive bands were visualized with a chemiluminescent reagent as recommended by Amersham Biosciences, Inc.

Statistical Analyses All experiments were repeated at least three times. Data are presented as the means 6 standard error, and statistical com-

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parisons between groups were performed by one-way analysis of variance (ANOVA) followed by Tukey’s test.

RESULTS TGFBIp Promotes Differentiation of EPCs to ECs To assess the effect of TGFBIp on differentiation of EPCs to OECs, EPCs were cultured on TGFBIp-coated plates, and then differentiation was analyzed. EPCs cultured on TGFBIp displayed accelerated differentiation to OECs (Fig. 1A, 1B) and significantly C AlphaMed Press 2015 V

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Figure 2. TGFBIp increases EPC differentiation and blood vessel formation in vivo. Seven-week-old male C57/BL6 mice (Orient Company, Seoul, Korea) were injected subcutaneously with 0.6 ml Matrigel containing 5 3 104 human EPCs and TGFBIp (10 mg/ml) or Fibronectin (10 mg/ml). After 5 days, the plugs were removed and washed with PBS. (A): Representative Matrigel plugs. (B): Hemoglobin levels in the Matrigel. (C): Differentiated EPCs and blood vessels in the Matrigel plugs were identified by staining with anti-CD31 antibody (red) and viewed using fluorescence microscopy. Scale bars 5 100 mm. **, p < .01 vs. control. Abbreviation: DAPI, 40 ,6-diamidino-2-phenylindole; EPCs, endothelial progenitor cells; PBS, phosphate-buffered saline; TGFBIp, transforming growth factor-beta-induced protein.

enhanced OEC colony formation (Fig. 1C, 1D). The magnitude of EPC differentiation induced by TGFBIp was higher than that induced by fibronectin, which is a known EPC differentiationinducing factor (Fig. 1A–1D). The cells that differentiated from EPCs in the presence of TGFBIp were characterized by an endothelial phenotype based on tube formation on Matrigel and expression of endothelial cell markers (Fig. 1E and Supporting Information Fig. S1, S2A). Also, expression of progenitor cell markers (CXCR4 and c-Kit) decreased during stimulation with TGFBIp at early time points, whereas expression of endothelial specific markers (vWF) increased (Supporting Information Fig. S2B). Next, we analyzed the effect of TGBFIp on EPC differentiation and blood vessel formation in vivo. Matrigel plugs containing EPCs with TGFBIp showed increased angiogenesis and hemoglobin content compared to control Matrigel plugs (Fig. 2A, 2B). In addition, the density of CD31-postive blood vessels and differentiated EPCs increased in matrigel plugs containing TGFBIp (Fig. 2C). In particular, the fibronectin-induced increases in hemoglobin content and angiogenesis in matrigel plugs were less than those induced by TGFBIp (Fig. 2A–2C). Taken together, these results suggest that TGFBIp plays an important role in promoting the differentiation of EPCs to OECs in vivo.

TGFBIp Increases Adhesion and Migration of EPCs Through Integrins a4 and a5 Next, we analyzed the effect of TGFBIp on EPC adhesion and migration. As shown in Figure 3A–3D, TGFBIp increased EPC C AlphaMed Press 2015 V

adhesion and migration in a dose-dependent manner. Several lines of evidence have identified integrins as the cellular receptors for TGFBIp [50, 51]; however, the integrin subtype responsible for mediating EPC binding to TGBFIp remains unknown. To identify integrins that interact with TGFBIp, we first analyzed the expression levels of integrin genes in EPCs and OECs by microarray analysis. As shown in Fig. 3E, expression of integrins a4, a5, a9, aM, and aX was higher in EPCs compared with those in OECs, and these findings were confirmed by RT-qPCR (Fig. 3F). Next, we investigated the integrin subtype that mediated the important interaction between EPCs and TGFBIp. We pretreated the cells with integrin a4, a5, avb3, or b3 blocking antibody and then performed an adhesion assay. As shown in Figure 3G, TGFBIp-induced EPC adhesion was inhibited by both a4 and a5 blocking antibodies, and a4 integrin blocking antibody exerted a greater inhibitory effect on cellular function compared to integrin a5 blocking antibody, suggesting an important role for a4 in facilitating TGFBIp-mediated EPC activation. These results suggest that TGFBIp promotes EPC migration and adhesion through specific binding of a4 and a5 integrins. Stimulation of EPCs by TGFBIp may activate intracellular signaling pathways, as has been reported for IGF2 [10]. We thus investigated the phosphorylation of intracellular angiogenic signaling molecules in response to TGFBIp. TGFBIp activated the phosphorylation of ERK, AKT, p38, SRC, and JNK in a time-dependent manner (Fig. 3H). These data demonstrate STEM CELLS

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Figure 3. TGFBIp increases adhesion and migration of EPCs via binding to integrins a4 and a5. (A, B): EPCs were treated with TGFBIp (2.5, 5, and 10 mg/ml) or fibronectin (10 mg/ml). After 24 hours, migration was quantified by counting the cells that migrated to the lower side of the filter using optical microscopy at 2003 magnification. (C, D): EPCs were treated with TGFBIp (2.5, 5, and 10 mg/ml) or fibronectin (10 mg/ml). Cell adhesion was quantified by counting the cells that attached to the TGFBIp- or fibronectin-coated matrix using optical microscopy at 2003 magnification. All data are presented as the mean 6 SE from three independent experiments performed in duplicate. **, p < .01 vs. control. (E): Total mRNA was isolated from EPCs and OECs, and gene expression profiles were assessed using an Affymetrix gene chip. Expression of integrin genes (integrin a4, a5, a9, aM, and aX) were presented with the appropriate expression value. (F): The mRNA levels of each gene in EPCs and OECs were determined by RT-qPCR. (G): EPCs were preincubated for 30 minutes with or without anti-integrin a4-, a5-, avb3-, or b3-neutralizing antibody (1 mg/ml) and then stimulated with TGFBIp (10 mg/ml). Adhesion was quantified. All data are presented as the mean 6 SE from three independent experiments performed in duplicate. **, p < .01 vs. TGFBIp alone. (H): After serum-starved EPCs were treated with TGFBIp (10 mg/ml) for the indicated periods of time (5, 10, 15, 30, or 60 minutes), cell lysates were subjected to Western blot analysis using IgGs against pERK, pAKT, p-p38, pSRC, and pJNK. The membranes were then stripped and reprobed with IgGs against ERK, AKT, p38, SRC, JNK, and SRC to estimate the total protein loaded. Abbreviation: EPCs, endothelial progenitor cells; OECs, outgrowing endothelial cells; RT-qPCR, reverse transcription quantitative polymerase chain reaction; TGFBIp, transforming growth factor-beta-induced protein.

that TGFBIp induces phosphorylation of intracellular signaling molecules in a pathway that may be necessary for TGFBIpmediated angiogenic activity of EPCs.

results suggest that TGFBIp may induce adhesion, migration, and differentiation in circulating EPCs.

TGFBIp Increases the Expression of Notch Ligands in EPCs TGFBIp Increases Adhesion, Migration, and Tube Formation of CD341 mBMMNCs Several lines of evidence have indicated that mononuclear cells derived from BM contribute to new vessel formation in ischemia and tumor models [10, 52]. To further investigate whether TGFBIp induces angiogenic activity of BM-derived EPCs, we isolated CD341 mBMMNCs from C57/BL6 mice and analyzed the angiogenic potential of these cells in response to TGFBIp. TGFBIp increased the adhesion and migration of CD341 mBMMNCs (Supporting Information Fig. S3A–S3C). In addition, CD341 mBMMNCs stimulated with TGFBIp exhibited a significantly enhanced ability to form capillary-like tubes compared with the ability of unstimulated cells (Supporting Information Fig. S3D). The capillary-like tube-forming cells were confirmed to be differentiated ECs by immunofluorescent staining with CD31 and CD34. Taken together, these

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According to our experiments, TGFBIp promotes differentiation of EPCs to ECs. Thus, we investigated whether EPCs stimulated with TGFBIp display different gene expression profiles compared with those of control cells using microarray analysis. As shown in Figure 4A–4C, many genes were differentially expressed (upregulated and downregulated) in EPCs treated with TGFBIp. Differentially expressed genes were clustered according to expression pattern (Fig. 4B) and classified by gene ontology (Fig. 4C). Interestingly, among the differentially expressed genes, Notch signaling molecules were highly expressed in TGFBIptreated EPCs as compared with those of control cells (Fig. 4D). Notch signaling plays an important role in the regulation of endothelial cell fates and in vasculogenesis during embryo development and in adults [30, 31]. To better elucidate the role of TGFBIp in the regulation of Notch signaling molecule expression, we analyzed the expression of the TGFBIp-induced Notch C AlphaMed Press 2015 V

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Figure 4. Gene expression microarray analysis of EPCs activated by TGFBIp. (A): MA plot and scatter plot analysis of microarray data. (B): Hierarchical cluster analysis of differentially expressed (twofold or fourfold) genes in EPCs following treatment with TGFPIp for 6 hours and control EPCs. (C): Gene ontology analysis of differentially expressed (fourfold) genes in EPCs treated with TGFBIp for 6 hours compared with control EPCs. (D): Cluster of Notch signaling related upregulated (fourfold) genes in EPCs treated with TGFBIp for 6 hours compared with control EPCs. Red indicates high expression. Abbreviations: EPCs, endothelial progenitor cells; TGFBIp, transforming growth factor-beta-induced protein.

signaling molecules in EPCs. As shown in Figure 5A, of the Notch signaling molecules examined, expression of Notch ligand DLL1 and JAG1 mRNA was significantly increased in EPCs by

TGFBIp. Western blot analysis confirmed increased expression of DLL1 and JAG1 protein following TGFBIp stimulation (Fig. 5B and Supporting Information Fig. S4). Increased expression of

Figure 5. TGFBIp increases the expression of Notch ligands in EPCs. (A, C): The mRNA levels of each gene in EPCs treated with TGFBIp for the indicated times were determined by RT-qPCR. All results were normalized to GAPDH. **P < 0.01 vs. control. (B): Serum-depleted EPCs were stimulated with TGFBIp (10 mg/ml) for the indicated times, and the protein levels of delta-like 1 and Jagged1 were analyzed by Western blot analysis. Abbreviations: EPCs, endothelial progenitor cells; RT-qPCR, Real-time quantitative RT-PCR; TGFBIp, transforming growth factor-beta-induced protein. C AlphaMed Press 2015 V

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Figure 6. TGFBIp increases the expression of Jag1 and DLL1 through the NF-jB signaling pathway in EPCs. (A): Serum-depleted EPCs were stimulated with TGFBIp (10 mg/ml) for the indicated times, and cell lysates were subjected to Western blot analysis using IgGs against pIjBa and pGSKa/b. The membranes were then stripped and reprobed with IgGs against IjBa and GSKa/b to estimate the total protein loaded. (B): Immunostaining analysis of NF-jB p65 localization. EPCs were preincubated for 1 hour in the presence or absence of 100 lM PDTC, then stimulated with 10 mg/ml TGFBIp for 15 minutes, and subjected to immunofluorescence staining. (C, D): Serum-depleted EPCs were preincubated for 1 hour in the presence or absence of 100 lM PDTC and then stimulated with 10 mg/ml TGFBIp for 6 or 24 hours. The mRNA and protein levels of DLL1 and Jagged1 were analyzed by RT-qPCR (C) and by Western blot analysis (D), respectively. All data are presented as the mean 6 SE from three independent experiments performed in duplicate. **, p < .01 vs. TGFBIp alone. Abbreviations: EPCs, endothelial progenitor cells; DLL1, delta-like 1; RT-qPCR, Real-time quantitative RT-PCR; TGFBIp, transforming growth factor-beta-induced protein.

DLL1 and JAG1 by TGFBIp suggests that the Notch signaling pathway can be activated and that expression of Notch target genes may ultimately be increased by TGFBIp. Therefore, we investigated the expression of Notch target genes in response to TGFBIp. RT-qPCR analysis revealed that TGFBIp increased the expression of the Notch targets MYC, HES1, 4, and 5, and HEY1 (Fig. 5C). These results suggest that TGFBIp may promote the differentiation of EPCs to ECs through activation of the Notch signaling pathway.

TGFBIp Increases the Expression of Jag1 and DLL1 in EPCs Through the NF-jB Signaling Pathway The Wnt/b-catenin and NF-jB signaling pathways are involved in modulating the expression of Notch ligands in lymphatic ECs and tumor cells [53, 54]. To determine whether these pathways are also involved in modulating DLL1 and JAG1 expression in the TGFBIp-stimulated EPCs, we first examined the phosphorylation of IjBa, an upstream signaling molecule of NF-jB. TGFBIp significantly increased the phosphorylation of IjBa in a time-dependent manner but did not induce GSK3a/b activation (Fig. 6A). The dissociation of NF-jB from IjBa results in translocation of NF-jB to the nucleus, where this factor binds to specific sequences in the promoter regions of target genes. We next determined the effect of TGFBIp on NF-jB nuclear translocation and DNA-binding activity. TGFBIp induced significant nuclear translocation of the NF-jB p65 subunit, and this translocation was significantly decreased by pretreatment of cells

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with the NF-jB inhibitor PDTC (Fig. 6B and Supporting Information Fig. S5). We further explored the role of the NF-jB pathway on TGFBIp-induced expression of DLL1 and JAG1 in EPCs. As expected, the mRNA and protein levels of DLL1 and JAG1 were increased by TGFBIp, and these increases were almost markedly inhibited by pretreatment of PDTC (Fig. 6C, 6D and Supporting Information Fig. S6). To investigate whether transcription factor NF-jB controls DLL1 expression in EPCs at the transcriptional level, a human DLL1 promoter assay was performed using EPCs transfected with a plasmid encoding a human DLL1 promoter-driven luciferase reporter gene. The DLL1 promoter region of this luciferase construct has previously been shown to contain a binding site for NF-jB [55]. Indeed, DLL1 promoter activity was substantially increased in EPCs treated with TGFBIp compared with control cells (Fig. 7A), and these responses were significantly abrogated by pretreatment of cells with PDTC before incubation with TGFBIp (Fig. 7B). Collectively, these data demonstrate that TGFBIp activates the NF-jB signaling pathway, and that this activation is essential for the TGFBIp-induced expression of DLL1 and JAG1 in EPCs.

DISCUSSION The ECM, which is a noncellular component present within all tissues and organs, not only provides an essential physical scaffold for the cellular constituents but also initiates crucial biochemical and biomechanical cues that determine cell differentiation, proliferation, survival, polarity, and migration. C AlphaMed Press 2015 V

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Figure 7. TGFBIp increases the expression of Jag1 and DLL1 through the NF-jB signaling pathway in EPCs. (A): EPCs were cotransfected with a b-galactosidase plasmid and the pEZX-DLL1 promoter luciferase plasmid as depicted. Twenty-four hours after transfection, cells were incubated with 10 mg/ml TGFBIp for 24 hours. Luciferase activity was normalized to b-galactosidase activity. (B): EPCs were cotransfected with a b-galactosidase plasmid and the pEZX-DLL1 promoter luciferase plasmid as depicted. Twenty-four hours after transfection, cells were preincubated for 1 hour in the presence or absence of 100 lM PDTC and then stimulated with 10 mg/ml TGFBIp for 24 hours. Luciferase activity was normalized to b-galactosidase activity. Data are presented as the mean 6 SE from three independent experiments performed in duplicate. **, p < .01 vs. control or TGFBIp alone. (C): Proposed model describes the differentiation of EPCs to ECs by TGFBIp. (1) EPCs incorporated into the neovascular region recognize TGFBIp secreted by environment cells, such as fibroblasts, via binding to integrins a4 and a5. (2) Binding of TGFBIp to integrins in EPCs activates the NF-jB signaling pathway that induces expression of DLL1 and JAG1. Interaction between JAG1 and DLL1 and NOTCH stimulates adjacent cells to become signal-receiving cells, in which Notch target genes are upregulated. (3) Finally, EPCs activated by TGFBIp through Notch signaling differentiate to ECs. Abbreviations: DLL1, delta-like 1; EPCs, endothelial progenitor cells; EC, endothelial cell; JAG1, Jagged1; OEC, outgrowing endothelial cell; NF-jB, nuclear factor-kappa B; TGFBIp, transforming growth factor-beta-induced protein.

Thus, the ECM plays an essential role in tissue morphogenesis, differentiation, and homeostasis [56]. The direct effect of ECM components on cell behavior is derived mainly from two ECM properties: the ability to bind directly to their cellular receptors, integrins, and discoidin domain tyrosine kinase receptors, which in turn are signal transduction receptors and the ability to bind and present growth factors in an organized, solid phase [56]. In this study, we provide evidence that the secreted ECM protein TGFBIp acts as an EPC differentiationinducing factor. First, we showed that TGFBIp promotes migration and adhesion of human EPCs (CD1331C-kit1Lin2 cells) in a dosedependent manner and that these effects are mediated by the binding of TGFBIp to integrins a4 and a5 specifically. This finding is consistent with an increase of migration, adhesion, and tube formation of CD341 mBMMNCs by TGFBIp. In addition, TGFBIp induces phosphorylation of ERK, AKT, p38, and JNK in a time-dependent manner. These results suggest that TGFBIp secreted by environment cells binds to integrins a4 and a5 and activates intracellular signaling pathways that may be essential for TGFBIp-mediated migration, adhesion, and tube formation of EPCs. C AlphaMed Press 2015 V

EPC adhesion to the ECM is an essential step during differentiation, allowing cells to attach to a substrate and acquire proliferative and survival signals from the underlying matrix. Moreover, direct interactions between integrins and ECM regulate EPC paracrine factor production. Interestingly, our data indicate that EPCs cultured on TGFBIp exhibit accelerated differentiation to OECs and result in significantly increased OEC colony formation. The magnitude of EPC differentiation induced by TGFBIp was greater than that induced by fibronectin. Interestingly, EPCs cultured on matrix combined with TGFBIp and fibronectin showed less OEC colony formation than that induced by TGFBIp or fibronectin alone. These effects suggest that the combination of TGFBIp and fibronectin may induce interinhibitory signaling pathways to properly activate EPCs in order to inhibit the over-differentiation of EPCs induced by TGFBIp or fibronectin alone. The Notch pathway is an evolutionarily conserved signaling mechanism that transduces signals between adjacent cells and plays established roles in cell fate determination during development, tissue homeostasis, and stem cell maintenance [27–31]. Distinct spatial expression of DLLs and JAGs in the normal developing vasculature suggests that ligand-specific STEM CELLS

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outcomes of Notch signaling are required for normal development [57, 58]. Here, we demonstrated that TGFBIp specifically increases the expression of the Notch ligands DLL1 and JAG1 in EPCs. The outcome of Notch signaling, including the signaling strength, can be influenced by the type of ligand expressed. Using gene expression profiling, we found that the TGFBIp-induced expression of DLL1 and JAG1 increased the expression of Notch target genes MYC, HESs, and HEY. HEY and HES are basic helix-loop-helix transcription factors that form heterodimers to enhance Notch signaling effects [59–61]. Furthermore, these studies revealed that TGFBIp significantly increased the phosphorylation of IjBa and nuclear translocation of the p65 subunit of NF-jB and that inhibition of NF-jB signaling pathway by pretreatment with PDTC markedly abrogated TGFBIp-mediated DLL1 and JAG1 expression and transcription activity. These findings provide a new example of the manipulation of the EPC signaling mechanism by TGFBIp. TGFBIp is an ECM protein, but here, we show that the induction of DLL1 and JAG1 by TGFBIp is specifically NFjB-dependent. Although the relationship between NF-jB signaling and Notch pathway activation in EPCs has previously been unclear, our data indicate a direct link between NF-jB and DLL1 and JAG1 expression in EPCs. Interestingly, our data also indicate that the induction of DLL1and JAG1 in EPCs is unlikely to occur as a result of VEGF stimulation. In conclusion, this report is the first to describe a functional association between TGFBIp and Notch signaling. In our study, we described the molecular mechanisms through which TGFBIp activates the Notch signaling pathway by directly increasing the expression of two Notch ligands (DLL1 and JAG1) through an NF-jB-dependent signaling pathway. We also suggest an effect of TGFBIp-stimulated DLL1 and JAG1 signaling on gene expression in adjacent cells and show that both ligands affect differentiation-associated genes and may co-operate to permit functional signaling in the context of EPC differentiation to ECs. In summary, our findings clearly show that TGFBIp is an EPC differentiation-inducing factor that functions based on three probable steps. First, EPCs incorporated into the neo-

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vascular region recognize the TGFBIp secreted by cells in the environment via binding to integrins a4 and a5. Second, binding of TGFBIp to integrins in EPCs induces phosphorylation of intracellular signaling molecules in a pathway necessary for TGFBIp-mediated angiogenic activity of EPCs. In addition, binding of TGFBIp to integrins activates the NF-jB signaling pathway that induces expression of DLL1 and JAG1 in EPCs. Interaction between JAG1 and DLL1 and NOTCH stimulates adjacent cells to become signal-receiving cells, in which Notch target genes are upregulated. Finally, EPCs activated by TGFBIp through Notch signaling differentiate to ECs (Fig. 7C). Our data suggest, for the first time, that locally generated TGFBIp at either wounds or tumor sites may contribute to differentiation and angiogenic function of EPCs by augmenting the recruitment of EPCs and regulating the expression of endothelial genes DLL1 and JAG1. Overall, our finding suggests that the regulation of TGFBIp expression in neovascular regions may control EPC differentiation and neovasculogenesis and serve as a useful target for the development of novel mechanisms to treat angiogenesis-dependent diseases.

ACKNOWLEDGMENTS This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MEST) (No. 2011-0028699).

AUTHOR CONTRIBUTIONS Y.-S. M.: Conception and design, collection and assembly of data, data analysis and interpretation, provision of study materials, manuscript writing. Y. J. C.: Conception and design, collection and assembly of data, data analysis and interpretation. E. K. K.: Provision of study materials, manuscript writing.

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The authors indicate no potential conflicts of interest.

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