contents

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chapter05

endothelial progenitor cells give rise to

smooth muscle progeny:

implications for a common progenitor status

Jan-Renier AJ Moonen*, Guido Krenning*, Marja GL BrinkerMarja JA van Luyn and Martin C Harmsen

Stem Cell & Tissue Engineering Research Group, Dept. Pathology & Medical Biology, University Medical Center Groningen, University of Groningen, The Netherlands

* Authors contributed equally

Manuscript Submitted

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abstract

Endothelial cells and smooth muscle cells have long been thought to have dinstinct progenitors. However, embryonic vascular progenitor cells with endothelial and smooth muscle progeny have been described and transdifferentiation between both cell types has been shown. Given the plasticity between both lineages we hypothesized that endothelial progenitor cells, known for their angiogenic properties, could transdifferentiate to smooth muscle cells and thereby gain a common vascular progenitor status. Indeed, umbilical cord blood-derived endothelial outgrowth cells (EOC) can give rise to smooth muscle cells through a process termed endothelial-to-mesenchymal transdifferentiation (EnMT). This EnMT was induced by TGF-β1 and completely inhibited by ALK5 kinase inhibition. After EnMT, expression of endothelial markers was lost, as was endothelial anti-thrombogenic function. Transdifferentiated EOC gained mesenchymal marker expression and acquired a contractile phenotype. Furthermore, transdifferentiated EOC showed bFGF and angiopoietin-1-mediated pro-angiogenic paracrine effects on untransdifferentiated EOC. Our study demonstrates that EOC are capable of TGF-β1-mediated EnMT, and thereby give rise to smooth muscle progeny. This implies a common vascular progenitor status for EOC, which clearly has implications for their therapeutical use.

introduction

During embryogenesis, endothelial cells arise from hemangioblasts [156]. Smooth muscle cells arise from local mesenchyme and the neural crest [157;158]. As both vascular cell types originate from different sources, it has long been thought that these cells have dinstinct progenitors. However, in 2000, Yamashita and collegues described embryonic vascular progenitor cells, that can differentiate into endothelial and smooth muscle cells [159]. These results were later confirmed by Ferreira and co-workers, who showed functional integration of vascular progenitor cell-derived endothelial cells and smooth muscle cells in preexisting vasculature [160].

In addition to endothelial- and smooth muscle maturation of embryonic vascular progenitor cells, transdifferentiation between endothelial and smooth muscle cells has also been described. During embryogenesis, endothelial-to-mesenchymal transdifferentiation (EnMT), takes place during the formation of the heart valves [143;161]. In vitro, EnMT of embryonic endothelial cells to smooth muscle actin expressing mesenchymal cells was also shown [151]. Furthermore EnMT is described for mature endothelial cells [144;155;162-165], and recent evidence supports a role for this process in cardiovascular fibrosis [166]. Although less well documented, mesenchymal-to-endothelial transdifferentiation has also been described [167;168]. Furthermore, endothelial cells and vascular smooth muscle cells have certain markers in common such as VEGFR-2 and the type 3 TGF-β receptor endoglin (CD105). These data indicate a potential plasticity of endothelial and smooth muscle cell differentiation.

Numerous studies have shown that endothelial progenitor cells (EPC) contribute to neovascularization, either by differentiation into cells of the endothelial lineage and local engraftment into to vasculature [61;63] or by secretion of pro-angiogenic factors [102;113;169]. In vitro EPC, termed endothelial outgrowth cells (EOC), can be derived from mononuclear cells from umbilical cord and adult blood [170]. These cells, with phenotypical and functional characteristics of endothelial cells, show stem cell-like properties, such as the ability of clonal expansion and telomerase activity [47].

Given the plasticity between endothelial- and mesenchymal lineages, common marker expression, and the stem-cell like properties of EOC, we hypothesized that EOC can (trans)differentiate into smooth muscle cells. This would imply a common vascular progenitor cell status for the archetype in vitro EPC.

materials & methods.

isolation and culture of umbilical cord blood-derived endothelial outgrowth cells (uc-eoc)

Umbilical cord blood was collected at the department of Gynecology, Medical Center Leeuwarden, the Netherlands. Cord blood was isolated directly after normal term delivery, with informed consent from the parents and according to institutional guidelines and the declaration of Helsinki. Cord blood mononuclear cells were separated by density gradient centrifugation on Lymphoprep (Nycomed Pharma, Norway) and cultured on fibronectin-coated (2 μg/cm2; Harbor Bio-Products, MA) culture flasks at a

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from Cell Signaling, MA), phosphorylated SMAD2 (pSMAD2, 2 µg/mL; Millipore, MA) and SMAD1/5/8 (4 µg/mL, Santa Cruz Biotechnology, CA) overnight and incubated the next day using fluorescein-conjugated donkey antibody fragments to rabbit IgG. Labeled samples were visualized using a Leica LMRXA fluorescent microscope and Leica software.

functional characterizations

To assess the uptake of acetylated low density lipoprotein (acLDL), 10 μg/mL DiI-labeled acLDL was added to the culture medium and incubated overnight. Subsequently, cells were fixed using 2% paraformaldehyde (Polysciences, PA), rehydrated and incubated with 3 μM DAPI for 10 minutes.

Binding of lectins from Ulex europaeus (UEA-1) was assessed by incubating 2% paraformaldehyde-fixed cells with fluorescein-conjugated UEA-1 lectins (5 μg/mL) dissolved in 3 μM DAPI for 1 hour. Subsequently, samples were analyzed on a Leica DMRXA fluorescent microscope at 40x lens magnification.

Capillary sprout formation by UC-EOC was assessed on MatriGel™ (BD Biosciences, CA). In short, 10 μL of MatriGel™ was solidified in µ-Slide Angiogenesis plates (Ibidi GmbH, Germany). Next, 15 000 UC-EOC were placed on the MatriGel™ and cultured in endothelial outgrowth medium overnight. Sprout formation was analyzed by microscopic analysis.

Thrombin generation was assessed using a Thrombin Generation Assay (HeamoScan, The Netherlands) as described previously [113;165]. In short, cells were incubated with fibrinogen-depleted plasma under normal culture conditions. A mixture of 30mM CaCl2 and phospholipids was added to induce the formation of thrombin. At regular intervals, samples were drawn, diluted in ice-cold TrisHCL and incubated with 3mM chromophore-releasing Thrombin substrate S2238. Change in color was assessed in a microtiter plate reader (BioRad, VA) and plotted against a calibration curve of known thrombin concentrations. Human umbilical cord endothelial cells and tissue culture-treated plastic served as negative and positive controls, respectively.

Gel contraction experiments were performed as described previously [165]. Briefly, a solution of collagen type I (8 mg/mL; BD Biosciences, MA) was supplemented with 10 mM NaOH, 2.5 mM HEPES (GIBCO/Invitrogen, CA) and transdifferentiated cells (1•106/mL). Aliquots of 50 μL (containing 50 000 cells and 0.2 mg collagen type I) were plated on plastic culture dishes and allowed to solidity for 30 minutes. Thereafter 1.5 mL culture medium was added, gels were released with a spatula, and imaged using a common flatbed-scanner (ScanJet 5370C; HP, CA). Thereafter, gels were allowed to contract for 48 hours. After 20 hours of spontaneous contraction, TGF-β1 was added to the culture medium.

immunoblot analysis

Whole cell lysates were prepared in RIPA buffer supplemented with 1% proteinase inhibitor cocktail and 1% phosphatase inhibitor cocktail (both Sigma-Aldrich, MA). Cell lysates (20 µg/lane) were separated by gel electrophoresis on a 10% non-denaturing polyacrylamide gel and subsequently blotted onto nitrocellulose according to

density of 1-2•106 cells/cm2 in endothelial outgrowth medium containing Medium-199 (Cambrex BioScience, ME), 20% fetal bovine serum (FBS; Invitrogen/GIBCO, CA), 1% Penicillin/Streptomycin, 2 mM L-glutamine (both Sigma Aldrich, MA), 5 U/mL heparin (Leo Pharma, Denmark), 10 ng/mL bFGF, 20 ng/mL HGF, 10 ng/mL IGF-1 and 10 ng/mL VEGFa (all Prepotech, NJ). Cord blood mononuclear cells were cultured for 14-21 days before passaging and culture medium was refreshed every third day.

For endothelial-to-mesenchymal transdifferentiation (EnMT), UC-EOC cultured up to passage 4 were used. EnMT was initiated by replacing the endothelial outgrowth medium to mesenchymal differentiation medium consisting of RPMI 1640 supplemented with 20% FBS, 1% Penicillin/Streptomycin, 2 mM L-glutamine, 5 U/mL heparin, with addition of 5 ng/mL TGF-β1 and 15 ng/mL PDGF-BB (both from Peprotech, NJ). Transdifferentiated cells (UC-EnMT) were subsequently cultured in basal medium consisting of RPMI 1640 supplemented with 20% FBS, 1% Penicillin/Streptomycin, 2 mM L-glutamine and 5 U/mL heparin, for up to 7 additional passages. When applicable, ALK5-kinase activity was inhibited by addition of 20µM SB431542 (Sigma, MA) to the culture media.

phenotypic characterization

Cells were detached using accutase (PAA Laboratories, Austria) and subsequently stained using fluorophore-conjugated antibodies to human CD31 (5 µg/mL; IQ Products, The Netherlands), CD34 (5 µg/mL; BD Biosciences, CA), CD105 (5 μg/mL), CD144 (2.5 μg/mL), VEGF-R2 (5 μg/mL), Tie-2 (2.5 μg/mL), αSMA (2.5 μg/mL), TGFβ-R2 (5 μg/mL), and PDGF-Rβ (5 μg/mL; all R&D Systems, MN), or unconjugated antibodies to human eNOS (2.5 μg/mL; BD Biosciences, CA), von Willebrand Factor (3 μg/mL; DakoCytomation, Denmark), thrombomodulin (10 μg/mL), SM22α (5 μg/mL), SM-MHC2 (5 μg/mL), Calponin (5μg/mL; all Abcam, UK), ALK1 (5 µg/mL; R&D Systems, MN) and ALK5 (2 μg/mL; Santa Cruz Biotechnology, CA). Unlabeled primary antibodies were subsequently stained using either fluorescein-conjugated donkey antibody fragments to rabbit IgG (5 μg/mL; Jackson ImmunoResearch, PA) or fluoroscein-conjugated rabbit antibodies to mouse IgG (5 μg/mL; DakoCytomation, Denmark).

For detection of intracellular proteins, cells were fixed using 2% paraformaldehyde and permeabilized using 0.1% Saponin (Sigma-Aldrich, MA) prior to staining procedures. Flow cytometric analysis was performed on a FACSCalibur™ (BD Biosciences, CA). Fluorophore-conjugated, isotype-matched nonsense antibodies, as well as fluorescein-conjugated donkey antibody fragments to rabbit IgG and fluorescein-conjugated rabbit antibodies to mouse IgG served as controls.

For visualization of cytoskeletal organizations, cells were fixed in ice-cold methanol:acetone (1:1), rehydrated and incubated with 0.1 μM fluorescein-conjugated phallotoxins (Molecular Probes/Invitrogen, OR) in phosphate buffered saline (PBS) containing 3 μM 4’,6-diamidino-2-phenylindole (DAPI; Sigma-Aldrich, MA) for 30 minutes.

For detection of (phosphorylated) SMAD proteins, cells were fixed using 2% paraformaldehyde and permeabilized using 0.1% Saponin (Sigma-Aldrich, MA) prior to staining procedures. Samples were subsequently incubated with antibodies against SMAD2/3 (2 µg/mL) and phosphorylated SMAD1/5/8 (pSMAD1/5/8, 2 µg/mL; both

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Activity of telomerase was determined using the TeloTAGGG Assay (Roche Diagnostics, the Netherlands) following manufacturers protocol. In short, whole cell extracts of 2 000 cells were incubated with reaction mixture containing telomerase substrate at 25°C for 60 minutes. After telomerase-mediated substrate elongation, PCR amplification was performed at 30 cycles at 94°C for 30s, 50°C for 30s and 72°C for 90s. Hybridization and enzyme-linked immunosorbent assay (ELISA) of amplicons were performed according to manufacturer’s instructions.

Amplicons of telomeric repeats were visualized using gel electrophoresis on a 12% non-denaturing acrylamide gel following standard protocols. Subsequently, amplicons were transferred to a positively charged nylon membrane (Hybond-N+, Amersham, UK) by standard blotting techniques. Blotting membranes were blocked in 5% BSA for 30 minutes and incubated with alkaline phosphatase-conjugated streptavidins (0.1 µg/mL; Southern Biotech, AL) for 1 hour. NBT/BCIP (Bio-Rad, VA) was used as substrate for the detection.

paracrine effects of uc-enmt

UC-EnMT at passage 5 were cultured in basal medium for 72h. The conditioned medium (CM) was filtered using 0.2 µm filters to remove cell debris. Capillary sprout formation was performed as described above. CM was used with- or without 10 µg/mL bFGF neutralizing antibodies or 10 µg/mL of recombinant human Tie-2/Fc (both from R&D systems, UK). Unconditioned basal medium or basal medium supplemented with 200 ng/mL bFGF and 20 ng/mL angiopoietin-1 (both from Peprotech, NJ), were used as controls. The number of branching points were manually scored and the cumulative length of sprouts was analyzed using ImageJ version 1.41.

statistical analysis

All data are expressed as mean ± standard error of mean (SEM). For multiple comparisons testing, one-way ANOVA followed by Tukey post hoc analyses were performed. Probabilities of P < 0.05 were considered to be statistically significant.

results

characterization of endothelial outgrowth cells (uc-eoc)

The first UC-EOC colonies were observed 9-21 days after plating the initial MNC fraction (Figure 1A) and the culture plates reached confluence within the next 5-8 days (Figure 1B). Cells were cultured up to an additional three passages after which EC characteristics were determined by binding of fluorescein-conjugated lectin from Ulex europaeus and uptake of DiI-acetylated low density lipoprotein (DiI-acLDL) (Figures 1C and D, respectively). The ability of UC-EOC to form capillary-like sprouts on Matrigel was also confirmed (Figure 1E). An important functional parameter of EC is their ability to prevent thrombin formation. Indeed, UC-EOC showed anti-thrombogenic function

standard protocols. Blots were blocked for one hour in Tris-buffered saline (TBS) containing 5% bovine serum albumin (BSA) and incubated at room temperature in TBS-Tween with antibodies to human pSMAD2 (0.3 µg/mL; Millipore, MA), SMAD2/3 (0.3 µg/mL), pSMAD1/5/8 (0.2 µg/mL; both Cell Signaling, MA), SMAD1/5/8 (1 µg/mL), Id2 (1 µg/mL), Id3 (1 µg/mL; all Santa Cruz Biotechnology, CA), or GAPDH (1 µg/mL; AbD Serotec, UK) overnight. Subsequently, blots were washed and incubated with alkaline phosphatase-conjugated antibodies to mouse or rabbit IgG (0.1 µg/mL). NBT/BCIP (Bio-Rad, VA) was used as substrate for detection. Densitometric analysis was performed using ImageJ version 1.41 (Research Services Branch, National Institute of Mental Health, Bethesda, MD).

gene transcript analysis

RNA isolation was performed using the RNeasy Mini Kit (Qiagen Inc., CA) according to manufacturer’s protocol. Subsequently, 1 μg of total RNA was reverse transcribed using the FirstStrand cDNA synthesis kit (Fermentas UAB, Lithuania) according to manufacturer’s instructions. The cDNA-equivalent of 5 ng RNA was used for amplification in 384-well microtiter plates in a TaqMan ABI7900HT cycler (Applied Biosystems, CA) in a final reaction volume of 10 μL containing 5 μL TaqMan universal PCR Master Mix (Applied Biosystems, CA) and 0.5 μL primer/probe mix. Applied Biosystems ‘assay on demand’ primer/probe sets were used to detect amplimers of β-2-Microglobulin (β2M; Hs99999907_m1), SM22α (Hs00162558_m1), Calponin (Hs00154543_m1), ALK1 (Hs00163543_m1), ALK5 (Hs00610319_m1), Collagen type I (Hs00164004_m1), Collagen type 3 (Hs00164103_m1), angiopoietin-1 (Hs00181613_m1), CCL2 (Hs00234140_m1), EGF (Hs00153181_m1), BFGF (Hs00266645_m1), HGF (Hs00300159_m1), IGF-1 (Hs00153126_m1) and VEGFa (Hs00173626_m1). Cycle threshold (CT) values for individual reactions were determined using ABI Prism SDS 2.2 data processing software (Applied Biosystems, CA). To determine differences in expression, CT-values were normalized against β2M-expression using the ΔCT-method (ΔCT(gene) = CT(gene) – CT(β2M)). To correct for interassay variance, ΔCT values were normalized against expression levels of an external calibrator (ΔΔCT(gene) = ΔCT(gene) – ΔCT(calibrator)). Relative expression levels were calculated as 2-(ΔΔCT). All cDNA samples were amplified in triplicate. Differences of a two-fold higher or greater were considered to be biologically relevant.

telomerase expression & activity

Gene expression of Telomerase Reverse Transcriptase (hTERT) was analyzed by RT-PCR. Total RNA was isolated and reverse transcribed as described above. RT-PCR for hTERT (sense 5’-TGGATGATTTCTTGTTGGTG-3’; antisense 5’-TCTTCCAAACT TGCTGATGA-3’) and GAPDH (sense 5’-CTGCCGTCTAGAAAAACCTG-3’; antisense 5’-GTCC AGGGGTCTTACTCCTT-3’) were performed in a final reaction volume of 25 μL under the following reaction conditions of 0.25 mM dNTPs, 1.5 mM MgCl2, 1μM primer-mix, and 1U Taq DNA Polymerase. Amplification was performed on a MyCycler (Bio-Rad, VA) in 96-well plates for 30 cycles. Amplimers were separated by gel electrophoresis in a 2% agarose gel.

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comparable to human umbilical vein endothelial cells (HUVEC) in an in vitro Thrombin Generation Assay (Figure 1F).

UC-EOC phenotype was further characterized by flow cytometric analysis. Marker proteins of the endothelial cell lineage, namely CD31 (99.68 ± 0.25%), CD144 (94.84 ± 1.31%), VEGFR-2 (97.26 ± 2.06%), eNOS (99.08 ± 0.28%), vWF (99.21 ± 0.75%), thrombomodulin (83.73 ± 9.50%) and Tie-2 (90.77 ± 0.85%) were abundantly expressed by UC-EOC. In contrast, marker proteins of the mesenchymal cell lineage, namely αSMA (0,82 ± 0,70%), SM22α (2.78 ± 2.00%), SM-MHC2 (3.36 ± 2.13%) and calponin (0.05 ± 0.03%), were virtually not expressed by UC-EOC. UC-EOC were further analyzed for the expression CD34 (18.84 ± 1.63%) and for the expression of TGFβ-related growth factor receptors. Abundant expression of ALK1 (99.77 ± 0.06%), CD105 (99.90 ± 0.05%) and TGFβ-R2 (78.12 ± 1.42%) was observed on UC-EOC. In contrast, expression levels of ALK5 (2.50 ± 0.05%) and PDGF-Rb (4.86 ± 1.92%) were virtually absent (Figure 1).

transdifferentiation of uc-eoc to smooth muscle cells

UC-EOC were cultured up to passage 4 in endothelial medium, after which they were cultured in mesenchymal differentiation medium containing TGF-β1 and PDGF-BB, for an additional 21 days. Transdifferentiated cells (UC-EnMT) had strong proliferative capacity and classical hill and valley morphology, combined with typical cytoskeletal organization, as shown by staining with phallotoxin (Figures 2A & B). EnMT also resulted in changes in functional properties, i.e. gain of contractile phenotype and loss of anti-thrombogenicity (Figure 1F). Cells were embedded in collagen type I gels and cultured for 24 hours after which a spontaneous contraction of 30,1 ± 5,9% could be observed by the collagen-embedded cells. In contrast, cell-free controls and the gels loaded with UC-EOC did not show contraction. Addition of TGF-β1 (5 ng/ml) after 24 hours showed additional contraction of the transdifferentiatied cell-embedded collagen gels to a total of 72.0 ± 2.3% of control, likely due to TGF-β1 induced Rho activation [171], whereas this had no effect on control gels (Figure 2C).

The expression of endothelial lineage markers CD144 (4.06 ± 1.83%), eNOS (0.10 ± 0.10%), vWF (0.50 ± 0.45%), thrombomodulin (4.79 ± 0.35%) and Tie-2 (3.54 ± 1.96%) had diminished after transdifferentiation (Figure 2C). About 32% of transdifferentiated cells (UC-EnMT) still showed CD31 expression, although the molecular density had

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97.26 ± 2.06%VEGFR-2

99.08 ± 0.28%eNOS

99.21 ± 0.75%von Willebrand Factor

83.73 ± 9.50%Thrombomodulin

90.77 ± 0.85%Tie-2

99.90 ± 0.05%CD105

99.77 ± 0.06%ALK1

2.50 ± 0.05%ALK5

78.12 ± 1.42%TGFß-R2

4.86 ± 1.92%PDGF-Rb

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Figure 1. Characterization of umbilical cord blood-derived endothelial outgrowth cells (UC-EOC). A, Cord blood mononuclear cells were cultured in endothelial outgrowth medium. After 9-21 days the first outgrowth colonies could be observed. B, UC-EOC cultures reached confluency within the next 5-8 days. C, Binding of fluorescein-conjugated Ulex europaeus lectin by UC-EOC was confirmed. D, UC-EOC took up DiI-acetylated low density lipoprotein. E, UC-EOC formed capillary-like sprouts on Matrigel. F, Anti-thrombogenic function of UC-EOC was assessed by measuring their inhibition of thrombin generation. Anti-thrombogenicity of UC-EOC was comparable to that of human umbilical vein endothelial cell (HUVEC) controls. Transdifferentiated cells showed loss of anti-thrombogenic properties. G, Flow cytometric analysis of UC-EOC phenotype. Endothelial markers (CD31 = platelet endothelial cell adhesion molecule-1, CD144 = vascular endothelial cadherin, VEGFR-2 = vascular endothelial growth factor receptor-2, eNOS = endothelial nitric oxide synthase, von Willebrand Factor, Thrombomodulin and TEK tyrosine kinase Tie-2) were abundantly expressed. Mesenchymal markers (αSMA = alpha smooth muscle actin, SM22α = smooth muscle protein 22 alpha, SM-MHC2 = smooth muscle myosin heavy chain 2 and calponin) were virtually absent. High expression levels were found for transforming growth factor beta (TGFβ) related growth factor receptors, ALK1 = activin like kinase 1, CD105 = endoglin and TGFβ-R2 = TGFβ receptor type 2. In contrast to expression of ALK5 = activin like kinase 5 and PDGF-Rb = platelet derived growth factor receptor b. TCP = Tissue culture plate, EnMT = Transdifferentiated UC-EOC, * P < 0,05.

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decreased by 50-fold. The expression of mesenchymal lineage markers αSMA (96.77 ± 1.43%), SM22α (98.78 ± 1.01%), SM-MHC2 (96.30 ± 2.18%) and calponin (90.27 ± 7.09%) was strongly induced during EnMT (Figure 2C). After 21 days of EnMT, TGF-β1 and PDGF-BB, were omitted from the culture media. UC-EnMT were cultured up to an additional 7 passages and maintained their mesenchymal phenotype and proliferative behavior. The expression of endothelial lineage marker CD31 was fully lost after this additional culture period (data not shown). The expression of ALK1 (87.63 ± 1.20%) was reduced after EnMT, while the expression of ALK5 (89.08 ± 6.08%) had increased, resulting in an shift in ALK1/ALK5 balance (Figure 2C). Furthermore, EnMT resulted in increased expression of PDGF-Rb (38,59 ± 6,42%).

transdifferentiation of uc-eoc is mediated by tgf-β1.

EnMT was induced by stimulating UC-EOC with TGF-β1 for 96h. SMAD signaling through type 1 TGF-β receptors, ALK1 and ALK5, was analyzed by immunoblotting of the receptor regulated SMADs, SMAD1/5/8 and SMAD2/3. Relative gene expression levels of ALK1 and ALK5, and of SMAD-target genes were determined.

UC-EnMT had lower gene transcript levels of ALK1, compared to uninduced UC-EOC, after 96h of TGF-β1 stimulation (Figure 3A). ALK1 is involved in activation of the SMAD1/5/8 route through phosphorylation of SMAD1/5/8. In contrast to signaling via ALK5, another type 1 TGF-β-receptor, which leads activation of the SMAD2/3 route [172]. ALK5 expression increased after TGF-β1 stimulation and completely diminished with addition of its kinase activity inhibitor, SB431542, indicative for a positive feedback system (Figure 3A).

UC-EOC cultured in MDM for 96 hours showed reduced levels of SMAD2/3 and SMAD1/5/8 protein compared to unstimulated controls. The addition of the ALK5 kinase inhibitor SB431542 had no effect on basal levels of SMAD2/3 and SMAD1/5/8, but resulted in lower levels of pSMAD2 (Figures 3B & C). Expression levels of pSMAD1/5/8 decreased after TGF-β1 stimulation and expression levels were comparable with UC-EnMT. Supplementing MDM with SB431542, had no effect on pSMAD1/5/8 levels, which implies an ALK5-independent effect. (Figures 3B & C).

Inhibitor-of-DNA-binding-proteins (Id proteins) are dominant negative regulators of basic helix-loop-helix transcriptional regulators which play a role in lineage commitment, cell cycle control and cell differentiation [173]. Expression of Id genes is mediated via phosphorylation of SMAD1/5/8 which bind to Smad responsive elements (SREs) within the Id promotors, thereby activating transcription. TGF-β1, via SMAD2/3, is known to inhibit expression of Id genes via by activation of transcriptional repressor ATF3, which binds to ATF/CREB site on the Id promotors and represses transcription [174]. Id2 and Id3 are known to antagonize SMAD2/3 signaling by repressing pSMAD2 mediated gene transcription [175]. Ectopic expression of Id2 and Id3 has been shown to inhibit transdifferentiation of epithelial cells [176].

Analysis of Id2 and Id3 protein expression showed stable levels of Id2, in contrast to Id3, which showed strongly reduced levels after stimulation with TGF-β1. This effect was not inhibited by addition of SB431542 (Figures 3B & C).

Gene transcript levels of SM22α and calponin increased after 96h of culture in MDM and reached levels similar to UC-EnMT (Figure 3C). SM22α and calponin both are target

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SM22 SM-MHC2 CalponinSMA96.77 ± 1.44% 98.78 ± 1.01% 96.30 ± 2.18% 90.27 ± 7.09%

CD31 CD34 CD144 VEGFR-231.65 ± 5.57% 5.25 ± 0.39% 4.06 ± 1.83% 77.47 ± 2.86%

eNOS von Willebrand Factor Thrombomodulin Tie-20.10 ± 0.05% 0.50 ± 0.45% 4.79 ± 0.35% 3.54 ± 1.96%

CD105ALK1 ALK5 TGFß-R2 PDGF-Rb97.31 ± 0.53%87.62 ± 1.20% 89.08 ± 6.08% 79.46 ± 5.29% 38.60 ± 6.42%

Figure 2. Characterization of transdifferentiated cells (UC-EnMT). UC-EOC were cultured in mesenchymal differentiation medium (MDM) containing TGF-β1 and PDGF-BB, for 21 days after which TGF-β1 and PDGF-BB were ommitted from the culture media and cells were cultured up to an additional 7 passages. A, UC-EnMT showed classical hill and valley morphology, characteristical of vascular smooth muscle cells. B, Staining with fluorescein-conjugated phallotoxin, showed typical cytoskeletal organization. C&D, UC-EnMT were embedded in collagen type 1 gels and cultured for 24h after which spontaneous contraction (30.1 ± 5.9%) of the UC-EnMT embedded gels could be seen. In contrast to the UC-EOC embedded gels or the cell-free controls which did not show contraction. After 24h, TGF-β1 (5 ng/ml) was added to the cultures. This induced additional contraction of the UC-EnMT-embedded collagen gels (to a total of 72.0 ± 2.3%). E, Flow cytometric analysis of UC-EnMT after 21 days of culture in MDM. Expression of endothelial markers (upper row) had diminished after transdifferentiation. Mesenchymal markers (αSMA, SM22α, SM-MHC2 and calponin) were strongly induced after endothelial-to-mesenchymal transdifferentiation. Expression of ALK1 was reduced after EnMT, while the expression levels of ALK5 had increased compared to UC-EOC controls (Figure 1G). Endoglin and TGFβ-R2 expression levels were virtually unchanged after transdifferentiation, whereas expression of PDGF-Rb had increased.

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genes of pSMAD2. However their increased expression is more likely due to reduced levels of Id3, which, as stated above, acts as a repressor of pSMAD2 mediated gene transcription. Inhibition of ALK5 kinase activity with SB431542 decreased pSMAD2 expression and completely abolished SM22α and calponin gene transcript expression (Figure 3C). Gene expression levels of collagen type I were low in cells stimulated with TGF-β1 for 96 hours compared to expression levels in UC-EnMT. Contradictory, gene transcription of collagen type III increased after 96h of culture with TGF-β1. Transcripts of collagen type I and III genes were low compared to UC-EnMT, and diminished by the addition of SB431542.

uc-enmt show increased responsiveness to tgf-β1

UC-EOC and UC-EnMT were stimulated with 50 ng/ml of TGF-β1 for 1 hour with or without addition of SB431542. Immunoblotting and immunofluorescent imaging was used to study the expression of inactive, and phosphorylated SMAD2/3 and SMAD1/5/8. Stimulation of UC-EOC and UC-EnMT with TGF-β1 had no effect on basal SMAD2/3 and SMAD1/5/8 levels (figures 4A & B), while pSMAD1/5/8 levels were clearly reduced in UC-EnMT compared to UC-EOC. Expression of pSMAD2 increased in TGF-β1-stimulated UC-EnMT, and completely diminished with addition of SB431542. UC-EOC did not show increased pSMAD2 levels after stimulation with TGF-β1 (Figures 4A & B). Differential response to TGF-β1 by UC-EOC and UC-EnMT is likely due to increased expression of ALK5 in UC-EnMT which leads to increased ALK5 kinase activity and thereby increased phosphorylation of SMAD2. Another possible explanation may be found in the observed high molecular weight SMAD2/3 complexes present in transdifferentiated cells (Figure 4A). Pretreatment of the protein extracts with 2M NaCl or 8M urea failed to dissociate these complexes (data not shown).

loss of telomerase activity of uc-eoc with increasing population doublings and after trans-differentiation.

Gene expression of Telomerase Reverse Transcriptase (hTERT) was analyzed by RT-PCR. UC-EOC at low population doublings showed a readily detectable gene expression of hTERT, indicating that these cells are capable of self-renewal (Figure 5A). Subsequently, activity of telomerase was determined in UC-EOC and UC-EnMT. Telomerase activity of UC-EOC at low population doubling numbers was similar to that of hTERT-transfected HUVEC controls (Figures 5B & C). Telomerase activity reduced with increased population doublings, which indicates that UC-EOC give rise to terminally differentiated progeny. Also after EnMT, telomerase activity was lost (data not shown).

pro-angiogenic effects of uc-enmt by paracrine signaling.

To study potential paracrine effects of UC-EnMT on UC-EOC, we analyzed gene transcript levels of several pro-angiogenic factors. Expression levels of HGF, IGF-1 and EGF were low in both UC-EOC and UC-EnMT. Expression levels of VEGFa were similar in

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Figure 3. Transdifferentiation of UC-ECFC is mediated by TGF-β1. Endothelial-to-mesenchymal transdifferentiation was induced by stimulating UC-ECFC with TGF-β1 for 96h. Activin like kinase 5 (ALK5) kinase inhibitor SB431542 was added to block ALK5 mediated TGFβ signaling. A, Real-time RT-PCR showed increased gene transcript expression levels of ALK5 after stimulation with TGF-β1. Addition of SB431542 completely inhibited this effect. B, Representative images of immunoblotting of TGFβ signaling components SMAD1/5/8, SMAD2/3, their phosphorylated forms, pSMAD1/5/8 and pSMAD2/3 and pSMAD1/5/8 regulated Id2 and Id3. C, Densitometric analysis of SMAD immunoblotting. Basal levels of SMAD1/5/8 were reduced after stimulation with TGF-β1 to levels comparable to those of transdifferentiated cells (EnMT). Phosphorylation of SMAD1/5/8 also decreased after TGF-β1 stimulation. SB431542 did not inhibit these effects. Id2 and Id3 are known to antagonize SMAD2/3 signaling by repressing pSMAD2 mediated gene transcription. Id2 levels were stable, in contrast to Id3, which showed strongly reduced levels after stimulation with TGF-β1. D, Quantitative RT-PCR showed upregulation of SM22α, calponin and collagen type III. This was completely blocked by SB431542. Expression of collagen type I remained low after 96h of TGF-β1 stimulation compared to levels of transdifferentiated cells, ***P<0.001, *P<0.05.

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5UC-EOC and UC-EnMT. Interestingly, gene transcript levels of bFGF and angiopoietin-1 were strongly increased in UC-EnMT compared to UC-EOC. In contrast to angiopoietin-1 expression, the increase of bFGF expression was induced within 96 hours of TGF-β1 stimulation (Figure 6A).

SDF-1 and MCP-1, are angiogenic factors which play an important role in recruitment and differentiation of EPC [102;177-179]. UC-EnMT expressed high levels of SDF-1, which were virtually absent in UC-EOC, despite TGF-β1 stimulation for 96 hours. MCP-1 expression, however, was induced by short term TGF-β1 stimulation and almost absent in (quiescent) UC-EnMT.

UC-EnMT conditioned medium (CM) was used to study the paracrine effects of UC-EnMT on capillary sprouting of UC-EOC. Given the increased gene transcript levels of bFGF and angiopoietin-1 found in UC-EnMT, neutralizing antibodies to bFGF and soluble Tie-2 (sTie-2) were added to CM. The total number of branching points was comparable between basal medium (BM), BM with bFGF and angiopoietin-1, and CM (46 ± 8, 52 ± 7 and 57 ± 3 respectively). Neutralization of bFGF or addition of sTie-2 to the culture media, resulted in strongly reduced numbers of branching points (Figures 6B & C). The cumulative sprout length was also determined. CM showed increased cumulative length (45.63 ± 1.18%) compared to unconditioned basal medium. BM supplemented with bFGF and angiopoietin-1 showed a similar increase in cumulative sprout length (42.66 ± 4.04%). Addition of bFGF neutralizing antibodies or sTie-2 completely diminished the observed increase, indicating a bFGF and angiopoietin-1 mediated effect (Figures 6B & C).

discussion

In the present study we show the intrinsic capacity of umbilical cord blood-derived endothelial outgrowth cells (UC-EOC) to transdifferentiate into smooth muscle cells; (1) Endothelial-to-mesenchymal transdifferentiation (EnMT) of EOC resulted in waning

pSMAD1/5/8

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Figure 4. Transdifferentiated cells (UC-EnMT) show increased responsiveness to TGF-β1. UC-EOC and UC-EnMT were stimulated with a pulse of 50 ng/ml TGF-β1 for 1h. ALK5 kinase inhibitor SB431542 was added to discriminate ALK5 mediated effects of TGF-β1. Immunoblotting was used to study the expression of basal and phosphorylated SMAD1/5/8 and SMAD2/3. A, Representative images of immunoblotting of TGF-β-signaling components SMAD1/5/8, SMAD2/3 and their phosphorylated forms, pSMAD1/5/8 and pSMAD2/3. Note the formation of high molecular weight (110 kDa) SMAD2/3 complexes, specifically in the transdifferentiated cells. B, Densitometric analysis of SMAD immunoblotting. Stimulation of UC-EOC and UC-EnMT with TGF-β1 had no effect on basal SMAD2/3 and SMAD1/5/8 expression. Levels of pSMAD1/5/8 were reduced in UC-EnMT compared to UC-EOC, irrespective of TGF-β1 stimulation or ALK5 kinase inhibition. TGF-β1 stimulation induced increased expression of pSMAD2 in transdifferentiated cells, which was completely blocked by SB431542. UC-EOC did not show increased pSMAD2 levels after stimulation with TGF-β1. C, Immunofluorescent images showing typical cytoskeletal organization in UC-EnMT by staining with Fluorescein-conjugated phallotoxins (green). Unphosphorylated SMAD2/3 and SMAD1/5/8 show cytoplasmic staining patterns (red) while their phosphorylated forms (red) show typical nuclear (blue) localization. , *** p < 0.001.

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Figure 5. Loss of telomerase activity of UC-ECFC with increasing population doublings. A, Representative image of Telomerase Reverse Transcriptase (hTERT) gene transcript analysis. UC-ECFC at low populations doublings show hTERT gene expression, in contrast to UC-ECFC at higher population doublings. B, Telomerase activity analysis clearly shows high telomerase activity in UC-ECFC at low population doublings, comparable to activity levels of hTERT-transfected human umbilical vein endothelial cell (HUVEC) controls (CMV-hTERT). Telomerase activity decreased with increasing population doublings.

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5

of endothelial markers and loss of endothelial functionality. (2) Mesenchymal marker expression and contractile function were gained. (3) EnMT is mediated by TGF-β1 and PDGF-BB and inhibition of ALK5 kinase activity completely abolished EnMT by the inhibition of SMAD2 phosphorylation. (4) Transdifferentiated cells (UC-EnMT) were phenotypically stable, self-sustaining and showed pro-angiogenic paracrine properties. To our knowledge, this study is the first to describe the plasticity of UC-EOC towards smooth muscle cell differentiation.

Since their first description [46], endothelial progenitor cells (EPC) have been extensively studied and their availablity and angiogenic properties have advocated them an interesting and promising cell source for therapeutic neovascularization [180]. Despite this large research effort, plasticity of EPC has received little attention.

Embryonic vascular progenitor cells have been described to differentiate to endothelial and smooth muscle cells [151]. Besides, EnMT plays a physiological role during development in formation of the heart valves [143;161], and has been described postnatally, both in vivo and in vitro [144;155;162-165]. Furthermore, mesenchymal-to-endothelial transdifferentiation has been reported [167;168]. This plasticity between endothelial and mesenchymal lineages is suggestive for a common vascular progenitor.

In vivo, bone marrow-derived smooth muscle progenitor cells (SMPC) have been shown to play a role in cardiovascular pathology [141;181]. SMPC have been cultured from umbilical cord and adult blood, although their origin remains largely unclear [139;182]. Our results indicate that EOC, the archetype in vitro EPC [170], can give rise to smooth muscle progeny.

Since our data concern in vitro EPC, extrapolation to the in vivo situation is somewhat speculative. One could however hypothesize that EPC fulfill a role in replenishing pools of progenitor cells present in the vascular wall as described by Zengin et al.[183]. These cells would require the capacity to give rise to endothelial and smooth muscle progeny. The fact that bone marrow-derived EPC have been described to be recruited to the vessel wall, rather than to the endothelium itself [184] adds to the hypothesis. Also, Purhonen and co-workers recently confirmed these findings by describing that EPC did not contribute to the vascular endothelium, but were found perivascularly [185].

The capacity of EOC to transdifferentiate to smooth muscle cells certainly has implications for their therapeutic use in cardiovascular disease. The majority of patients eligible for cardiovascular cell therapy commonly share an inflammatory vascular profile [186]. TGF-β and it’s downstream effector pSMAD2 have been shown to be highly expressed at atherosclerotic lesion sites [187]. As our data show, increased TGFβ signaling results in mesenchymal differentiation of EPC. This potentially has beneficial effects by limiting plaque formation and increasing plaque stability [188], but it could also contribute to atherogenesis [181]. Co-administration of smooth muscle progenitor cells and EPC has recently been shown to have synergetic angiogenic effects by increased paracrine signaling, mainly through the angiopoietin/Tie2 signaling pathway [189]. Our results also show angiopoietin 1 mediated pro-angiogenic effects of transdifferentiated EOC, in addition to bFGF mediated effects. EOC EnMT thereby provide a novel therapeutic strategy for treating ischemic disease. Ex vivo priming of EOC has recently been described to enhance their angiogenic potential [179]. Our

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Figure 6. Pro-angiogenic effects of UC-EnMT by paracrine signaling. A, Quantitative RT-PCR analysis of gene transcript levels of pro-angiogenic factors normalized to β2M expression. Gene transcript levels of basic fibroblast growth factor (bFGF) and angiopoietin-1 were strongly increased in UC-EnMT compared to UC-EOC. The increase of bFGF expression was induced within 96 hours of TGF-β1 stimulation. Capillary sprouting capacity of UC-EOC was studied using UC-EnMT conditioned medium (CM) with or without bFGF neutralizing antibodies (bFGF NAbs) or recombinant human Tie-2/Fc (sTie2). Unconditioned basal medium (BM) or BM supplemented with 200 ng/mL bFGF and 20ng/mL angiopoietin-1 (BM++ )were used as controls. The total numbers of branching points were determined and the cumulative lengths of spouts were analyzed. B, Representative images of capillary sprout formation on Matrigel. Note the reduced capillary formation in sTie2 and bFGF NAbs treated cultures. C, The total number of branching points was similar between BM, BM++, and CM. Numbers were reduced with addition of bFGF NAbs or sTie2 to CM. The cumulative sprout length was increased with CM compared to unconditioned BM, similar to that observed with BM++. Addition of bFGF NAbs or sTie2 completely diminished these effects.

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results illustrate how ex vivo stimulation can indeed direct EOC differentiation, thereby changing phenotypical and functional properties of the cells. This phenomenon can be used in tuning EOC, creating tailor-made therapy for use in a specific patient with specific underlying pathology and co-morbidity.

conclusion

In conclusion, our study demonstrates that EOC are capable of TGF-β1-mediated EnMT, thereby giving rise to smooth muscle progeny. This plasticity of EOC implies a common vascular progenitor status for EOC and clearly has implications for therapeutical use.

appendices

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references

207 App

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