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Outgrowing endothelial progenitor-derived cells display high sensitivity to angiogenesis modulators and delayed senescence
FEBS Letters 581 (2007) 2663–2669
Outgrowing endothelial progenitor-derived cells display high sensitivity to angiogenesis modulators and delayed senescence Jung-Min Haa, Myoung-Rae Kima, Ho-Kyun Oha, Byung Hun Leea, Hyun-Young Ahnb, Jong-Chul Shinb, Sang Hong Baekc, Young Ae Joea,* a
Cancer Research Institute and Department of Biomedical Science, College of Medicine, The Catholic University of Korea, Banpo-dong 505, Seocho-ku, Seoul 137-701, Republic of Korea b Department of Obstetrics and Gynecology, Division of Maternal-Fetal Medicine, College of Medicine, The Catholic University of Korea, Seoul 137-701, Republic of Korea c Department of Internal Medicine, Division of Cardiovascular Medicine, College of Medicine, The Catholic University of Korea, Seoul 137-701, Republic of Korea Received 12 March 2007; revised 25 April 2007; accepted 3 May 2007 Available online 15 May 2007 Edited by Francesc Posas
Abstract Outgrowing endothelial progenitor-derived cells (EPDCs) originate from a novel hierarchy of endothelial progenitor cells. In this study, EPDCs isolated from human cord blood were examined for phenotype and functional features upon aging. Young or aged EPDCs were similar to human umbilical vein endothelial cells (HUVECs), in exhibiting typical endothelial phenotypes. However, EPDCs were more sensitive to angiogenesis inducers or inhibitors in proliferation and migration. In addition, EPDCs underwent senescence markedly slowly with sustained endothelial NO synthase expression and activation, and their ability to undergo capillary morphogenesis was retained throughout longterm culture. Thus, these results suggest that a homogenous population of EPDCs derived from clonogenic expansion may provide an effective vasculogenesis tool. 2007 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. Keywords: Vasculogenesis; Endothelial progenitor cell; Senescence; Angiogenesis; Inhibitor
1. Introduction Endothelial progenitor cells (EPCs) can contribute to postnatal vasculogenesis in tissue ischemia, wound healing, and tumor growth [1–3]. Early EPCs with the limited proliferating potential (for less than 3 weeks) were initially characterized as a cell type with postnatal vasculogenic capacity by Asahara et al. [1,2]. These observation led to clinical trials of therapeutic application of EPCs in cardiovascular disease [4,5]. Later, several groups reported a different cell type with high expansion capacity that appears at a late stage [6–9]. The cells were named endothelial progenitor-derived cells (EPDCs) or late EPCs. In in vitro culture, early EPCs form adherent spindleshaped cells at day 7–14, and die after 3 weeks. In the case of EPDCs, they have been reported to differentiate from a subset of EPCs after 2 weeks, show exponential growth at 4–8 weeks and live up to 12 weeks. In particular, cord * Corresponding author. Fax: +82 2 532 0575. E-mail address: [email protected] (Y.A. Joe).
blood-derived EPDCs have been shown to have a greater proliferative rate and expandability than those of adult peripheral blood-derived EPDCs [6]. For in vitro angiogenesis assays, human umbilical vein endothelial cells (HUVECs) have been used frequently and commonly. However, HUVECs, mature endothelial cells, that are primarily cultured from human cord vein vessel, are changed in phenotypes and signal pathways during long term culture [10,11]. The growth of HUVECs slows down after about 19–22 population doublings and the cells are used between passages 2 and 8 (about 4 weeks). In addition, the culture of these cells is sometimes complicated by contamination with fibroblasts. In the case of EPDCs, they constitute a homogenous population by clonogenic expansion. Moreover, EPDCs have been shown to have tissue repair capability through neovasculogenesis in a limb ischemia model of mouse, suggesting a potential for cell therapy of vascular grafts [8]. However, the shape of cultured EPDCs is very similar to HUVECs and the isolation of EPDCs is not always successful, resulting in a frequency of about 30% of total blood samples under our experimental conditions. So, considering that HUVECs and EPDCs are derived from cord vessel wall and cord blood, respectively, the possibility cannot be excluded that EPDCs might appear from an in vitro artifact, like immortalization or in vitro transformation of endothelial cells that emerged from vessel walls. Therefore, we addressed the question of how EPDCs are different from HUVECs in phenotypes and functional features during long-term culture. In these experiments, we show that EPDCs, in contrast to HUVECs, retain endothelial characteristics in phenotype and function during a long-term culture period, and only slowly undergo senescence, not being immortalized, thereby suggesting that EPDCs may be an effective vasculogenesis tool for in vitro assays and cell therapy.
2. Materials and methods 2.1. Cell culture The Institutional Review Board at the Catholic University of Korea College of Medicine approved all protocols. Cord blood and cords were obtained from donors with informed consent. Mononuclear cells (MNCs) were isolated from human cord blood using Ficoll-Hypaque
0014-5793/$32.00 2007 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.febslet.2007.05.010
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density gradient centrifugation as described previously [12]. Isolated MNCs were plated into a dish coated with 10 lg/ml human fibronectin (Sigma, St. Louis, MO) and incubated in Medium 199 (M199, JBI, Daegu, Korea) supplemented with 20% fetal bovine serum (FBS, Gibco BRL, Grand Island, NY), 30 lg/ml endothelial cell growth supplement (ECGS, Sigma), 90 lg/ml heparin (Sigma) and 1% penicillin– streptomycin (Gibco BRL). After changing to fresh media every 3–4 days for 15–21 days, an emerging EPDC clone was trypsinized, and transferred to 1% gelatin-coated dishes for subculture. Human umbilical vein endothelial cells (HUVECs) were cultured as described previously [12]. 2.2. Cell proliferation potential At subconfluence, cells were detached and counted. The number of divisions was estimated using the following equation: (Ln (number of cells counted/number of cells at the beginning of the assay)/Ln 2) [7]. 2.3. Reverse transcriptase polymerase chain reaction (RT-PCR) RNA of EPDCs or HUVECs was extracted by use of the Trizol reagent (Life Technologies, San Diego, CA). First-strand cDNA was synthesized using the reverse transcription system (Promega, Madison, WI), and was used for the amplification of each cDNA by Super Taq PLUS version (Super Bio Co. LTD, Seoul, Korea). The PCR primer sequences used for RT-PCR are listed in Table 1. PCR conditions were 95 C for 5 min, followed by 30 cycles (25 cycles for GAPDH) of 95 C for 1 min, 55–63 C for 1 min, and 72 C for 1 min, and ending with 72 C for 10 min. 2.4. FACS analysis FACS analysis was performed on EPDCs and HUVECs collected after trypsinization as described previously, except vWF and VE-cadherin [12]. For detection of VE-cadherin, cells were detached by treatment of 4 mM EDTA for less than 5 min, washed twice, and blocked with 0.5% BSA and 1 lg human immunoglobulin (IgG) in PBS. vWF was detected as reported by Lin et al. [13]. Binding of anti-vWF (mouse monoclonal, Dako, Glostrup, Denmark) antibody was visualized with Cy3-conjugated goat anti-mouse IgG (Chemicon, Temecula, CA). Anti-CD31, anti-CD34 (mouse monoclonal, Becton Dickinson, San Jose, CA), anti-CD105 (mouse monoclonal, Serotec, UK), and anti-VE-cadherin (mouse monoclonal, R&D, Minneapolis, MN) antibodies were used directly as the PE- or FITC-conjugated form.
Table 1 List of primer sequence Gene
Nucleotide sequence
PK5
5 0 -CAGTATCGATCACTCCTTCCGAAGAAGAC 3 0 -CTGATCTAGATCAAAATGAAGGGGCCGC 5 0 -CAGTATCGATACATACTCATCAGGACTTTC 3 0 -CTGATCTAGACTATTTGGAGAAAGAGGTC F-GTGATTGCCATGTTCTTCTG R-GAGGATCTTGAGTTCAGACA F-GGTCTTTGCCTGAAATGGTGAG R-GACTGTTGCTTCACAGGTCAGA F-ATGCGTCAACAAGCTTCCTTCC R-CACCTAAGCTTACAATCTGGCC F-ACGCTCCTTCTCGATTATTGGG R-CAGAGGAGATGTTGCATGAGC F-CTCAGTCATCTCTGAGAC R-AATCCAGAGGCTTCATGG F-CGTCTTCCCGGATGTTTCTTCT R-CTGCTCTTGGAACTCCTGAAAC F-TACCAGTCCTTGCAGTGGTTCG R-CCACTGAATTCACCAACAGCCA F-CAGGAAGGAATGCCTACTTCTG R-CCCATCTCCAGCAAAGTGAAAC F-CCTGAAGGAGAATCTGCTGAAG R-GATGCATCATTCCTCACGCCAA F-AAAGTGGAGTTGTCAGTCTGGG R-ACAGGAGAAGTTGTCGCACTCA F-ATGTTCGTCATGGGTGTGAACC R-GGCAGGTTTTTCTAGACGGCAG
Endostatin KDR Flt-1 Tie-2 vWF VE-cadherin Integrin av Integrin a5 Integrin b1 Integrin b3 Integrin b5 GAPDH
2.5. Expression and purification of angiogenesis inhibitors The recombinant proteins of PK5 and endostatin were expressed in Pichia using EasySelectPichia Expression Kit (Invitrogen, Carlsbad, CA) as follows. cDNA fragments were obtained from Dr. Yeup Yoon (PK5, Mogam Biotechnology Research Institute, Korea) and Dr. SooIk Chang (mouse endostatin, Chungbuk National University, Korea). DNA fragments encoding the amino acids of human plasminogen kringle 5 (PK5, Thr456–Phe546 of plasminogen) and endostatin (His132–Lys315 of mouse a1 (XVIII)) were amplified by PCR with Pfu polymerase (Stratagene, La Jolla, CA) and PK5 primers or endostatin primers (Table 1), and subcloned into the Cla I–Xba I site of pPICZa-C. The insert DNA was cut with SacI and introduced into Pichia pastoris GS115 (pPICZa-C/PK5) or X-33 (pPICZa-C/endostatin) by electroporation. After the selection of PK5 or endostatinexpressing clones, expression in a large-scale culture and purification were performed by a similar method to that reported previously for the recombinant kringles 1–2 of tissue-type plasminogen activator (TK1–2) protein [14]. 2.6. Cell proliferation assay Cell proliferation assays were performed using a thymidine incorporation assay as described previously [15]. 2.7. Cell migration assay The migration of EPDCs and HUVECs was evaluated in a modified Boyden chamber assay as described previously [14]. 2.8. In vitro tube formation on Matri-gel Two hundred and fifty microliters of chilled Matri-gel (BD) was added in each well of a chilled 24 well plate. After 30 min of incubation at 37 C, 250 ll M199 medium containing 4 · 104 cells/well was added onto the solidified Matri-gel. After 18 h of incubation, the cells were stained with hematoxylin and five representative fields were photographed. Images were analyzed in Image J v.1.34s (http://rsb.info.nih.gov/ij/) for determining tube density. 2.9. Senescence associated (SA)-b-galactosidase assay The cells were washed, fixed, and incubated overnight at 37 C with X-gal chromogenic substrate at pH 6.0 following the conventional protocol for SA-b-gal staining [16]. After taking pictures of five randomly selected fields, the cells were washed with PBS and 500 ll DMSO was added to each well for dissolving the stain, and incubated at 37 C for 30 min, followed by measurement of absorbance at 595 nm. 2.10. Telomerase assay Telomerase activity was measured with 5 lg protein by the TeloTAGGG Telomerase PCR ELISAPLUSKit according to the manufacturer’s instructions (Roche Applied Science, Germany). 2.11. Western blot Cell lysate (50 lg protein) was resolved on SDS–PAGE gel and transferred to a nitrocellulose membrane. The membrane was incubated with an antibody against phospho-eNOS, eNOS, ERK 1/2 (Cell Signaling, Danvers, MA), b-actin, b-tubulin (Sigma) or GAPDH (R&D, Minneapolis, MN), followed by binding with the corresponding secondary antibody conjugated to horseradish peroxidase. The binding was detected by enhanced chemiluminescence (ECL plus, Amersham, Germany).
3. Results and discussion 3.1. Comparison of growth kinetics and phenotypes Under our experimental conditions, we were able to isolate EPDCs at a frequency of 3 donors out of a total of 9 donors from the culture of mononuclear cells (MNCs) isolated from the cord blood of several infants. EPDCs started to appear in culture dishes 17 days after seeding of MNCs and about ten individual colonies were obtained at once. As shown in Fig. 1A, the clone became larger over time and grew exponen-
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Fig. 1. The comparison of growth kinetics and phenotypes. (A) Population doubling of EPDCs isolated from different individual cord blood samples was compared with that of HUVECs (left). Ex vivo cultivation day 0 (D0) indicates the day when MNCs or HUVECs were isolated for initiating in vitro culture. Representative photomicrographs of an EPDC colony were captured (right, 100·). (B) The representative pictures of each cell type were captured upon aging (200·). RT-PCR (C) and FACS analyses (D) were performed on various endothelial cell markers and vascular integrins.
tially. When the growth kinetics of EPDCs and HUVECs were compared, the population doubling curves demonstrate that both EPDCs, derived from two donor bloods, have a higher proliferation potential than HUVECs (Fig. 1A). EPDCs reached growth arrest after about 40 population doublings, whereas HUVECs reached growth arrest faster, after 27 population doublings, in about 50 days. Subculture of EPDCs was maintained for more than 3 months without any apparent signs of replicative senescence. However, the growth rate of EPDCs decreased after 110 days, thereby indicating that EPDCs eventually stop growing, not being immortalized. Both young HUVECs (D10, 10 days after initiating ex vivo culture) and young EPDCs (D50) appeared as similar cobblestoneshapes. In accordance with several established studies showing that cells entering senescence become enlarged and flattened, aged HUVECs (D50) became larger and flatter than aged EPDCs (D100) (Fig. 1B).
When we examined the expression of typical endothelial cell markers, including KDR, Flt-1, Tie-2, vWF, VE-cadherin, CD34, CD31, and CD105, by RT-PCR and FACS analyses, we could not detect any marked difference between EPDCs and HUVECs (Fig. 1C and D). Thus, at present, the difference in phenotype between EPDCs and HUVECs is not clear. More precise analysis methods including microarray analysis, which allows analysis of many genes at once, will be desirable to differentiate between HUVEC and EPDC phenotypes. 3.2. EPDCs are sensitive to various angiogenesis modulators When we tested the functional properties of EPDCs, we found that basal levels of endothelial proliferation and migration were higher in young cells than in aged cells in both EPDCs and HUVECs (Fig. 2A and C). In particular, EPDCs, young or aged, showed high sensitivity to growth and movement stimuli, whereas aged HUVECs of the same ex vivo
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Fig. 2. Comparison of sensitivities to angiogenesis inducers or inhibitors in cell proliferation and migration. The [3H] thymidine-incorporation assay was performed on young or aged EPDCs and HUVECs, for evaluating cell growth stimulated by bFGF (A) and the anti-proliferative effect of PK5 (B). VEGF-stimulated cell migration (C), and anti-migrative effect of PK5, TK1–2 and endostatin (D) were assessed. The inhibitory effects were shown as a percentage of stimulated growth (cpm) (B) or migration (cell number) (D) in the absence of inhibitor. Values represent the mean of three independent experiments (means ± S.E.). *P < 0.05, **P < 0.01, compared with non-treated cells.
cultivation day as the young EPDCs, showed marginal induction of proliferation and marked induction of migration by bFGF or VEGF. In both cell types, young cells displayed more sensitivity in cell proliferation to the bFGF stimulus than did aged cells. Next, we tested if EPDCs are sensitive to endothelial-selective angiogenesis inhibitors including plasminogen kringle 5 (PK5), tissue-type plasminogen activator kringles 1–2 (TK1– 2), and endostatin. Interestingly, PK5 dose-dependently inhibited cell proliferation significantly only for young EPDCs (IC50 = 60 nM) and HUVECs (D10, IC50 = 250 nM) (Fig. 2B). Aged HUVECs (D50) did not show any significant inhibition of proliferation by PK5. In the migration assay, all the inhibitors tested showed inhibition in young EPDCs (PK5, IC50 = 100 nM; TK1–2 IC50 = 80 nM; endostatin, IC50 = 80 nM) (Fig. 2D). Aged HUVECs showed a little higher IC50’s than young EPDCs at the same age (D50) (PK5, IC50 > 500 nM; TK1–2 IC50 = 100 nM; endostatin, IC50 = 100 nM). Upon aging, the sensitivity of EPDCs to the inhibitors in cell migration decreased. Interestingly, inhibitors showed inhibitory activity in migration of aged cells of both cell types, unlike the difference in effects on proliferation. Overall, EPDCs at day 50, the same age as aged HUVECs, showed high sensitivity to angiogenesis inducers and inhibitors in both proliferation and migration, suggesting that EPDCs have more potentiality for vasculogenesis, and that they can be used more effectively in migration and proliferation assays.
3.3. Different capacity for capillary tube formation between aged EPDCs and HUVECs In the in vitro tube formation assay, young EPDCs formed capillary-like structures on Matri-gel (Fig. 3). When we compared the capacity for capillary tube formation between young and aged cells, aged HUVECs showed a low capacity for capillary tube formation, although they showed a high motility upon VEGF induction. On the other hand, aged EPDCs were able to form capillary tubes fully, suggesting that EPDCs retain the capability of capillary morphogenesis through long-term culture. In previous reports, senescence has been reported to progress more increased adhesion to the extracelluar matrix while losing cell–cell contacts [17,18]. Therefore, the progression of senescence in HUVECs seems to result in a decrease in their capacity to form tubes.
3.4. Markedly delayed senescence in EPDCs We investigated the senescence levels of EPDCs and HUVECs through SA-b-galactosidase staining, which is widely used as a biomarker of senescence. Aged EPDCs showed much fewer SA-b-gal positive cells than aged HUVECs, while their young cells showed few stained cells (Fig. 4A). When EPDCs were stained at the growth-arrest point (at day 130), EPDCs were also strongly stained (data not shown), suggesting that EPDCs also undergo senescence eventually, but do so much more slowly than HUVECs. Next, telomerase activity was
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Fig. 3. Formation of tube-like structure on Matri-gel. After young or aged EPDCs (D50, D100) and HUVECs (D10, D50) were incubated for 18 h on Matri-gel, the representative pictures were captured (100·), and tube density was analyzed.
determined since cellular senescence is critically influenced by telomerase. As shown in Fig. 4B, telomerase activity was reduced drastically in HUVECs upon aging, whereas aged EPDCs showed telomerase activity at a certain level, albeit at a reduced one, thereby corresponding well with the data obtained from SA-b-galactosidase staining. Thus, these results indicate that EPDCs are not derived from artifacts of in vitro cell culture, such as immortalization or transformation of vessel cells, and that they undergo senescence slowly. Next, we investigated the expression of eNOS and Akt and its phosphorylation status since aging of endothelial cells has been reported to be associated with reduced eNOS expression and decreased NO synthesis [19]. Indeed, eNOS protein expression and phosphorylation levels were drastically reduced in aged HUVECs (Fig. 5A and B). However, aged EPDCs, as well as young EPDCs, showed certain levels of expression and phosphorylation of eNOS, indicating no signs of senescence. Interestingly, the expression level of Akt and ERK 1/2 did not change upon aging, but the mean expression level of b-tubulin and b-actin decreased in HUVECs only, although there was some variation in the expression level of b-tubulin and b-actin among the donor types of HUVECs. This result corresponds well with a previous report that b-actin expression was down-regulated in rat gastric mucosa upon senescence [20]. At present, albeit with some questionable points, the results could suggest that the decreased expression of cytoskeleton proteins b-actin and b-tubulin, which are involved in cell movement and shape, might be related to the loss of tube for-
Fig. 4. Cellular senescence of EPDCs and HUVECs. (A) Young or aged cells were stained by the SA-b-galactosidase reaction. (B) The cells were assessed for relative telomerase activity (RTA).
mation ability and can be a characteristic of endothelial cells during senescence. Very recently, the inhibition of cellular senescence in HUVECs by nitric oxide has been reported [21]. Therefore, we tested whether inhibition of eNOS causes progression of senescence in EPDCs, as it does in HUVECs. Indeed, treatment with L -NAME (a NOS inhibitor) decreased the proliferation of EPDCs as they got older and showed more SA-b-gal stained cells, when compared with treatment with D -NAME (negative control) (Fig. 5C). Such an effect was also detected in two other EPDC clones, clones 2 and 3. Telomerase activity was also reduced upon L -NAME treatment compared to D -NAME treatment (Fig. 5D). Thus, all the results suggest that the delay of senescence in EPDCs may be due to a steady eNOS expression and activation (and possibly NO production). In summary, we found that EPDCs show high sensitivity to angiogenesis inducers and inhibitors and also undergo delayed senescence, even though both EPDCs and HUVECs are derived from infant cord blood and tissue. In particular, a large difference in senescence levels between EPDCs and HUVECs is manifested in in vitro tube formation capability, expression of eNOS and b-tubulin, and phosphorylation of eNOS. Moreover,
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Fig. 5. Differential expression of eNOS, b-tubulin and b-actin in aged EPDCs and HUVECs and effect of NO on EPDC senescence. (A) Phosphoryation of eNOS Ser1177 and expression of eNOS, Akt, ERK1/2, b-tubulin, b-actin and GAPDH were determined by Western blot analysis. GAPDH was used as a loading control. (B) The densitometric quantification of the Western blots was performed. The density of each protein band was normalized with that of GAPDH. Values represent the mean of three independent Western blot experiments (means ± S.E.). (*P < 0.01 compared with young cells). (C and D) EPDCs were cultivated in the presence of L -NAME or D -NAME (Sigma) at 100 lM from ex vivo cultivation day 50 to day 82, with replacement of new medium every 48 h. SA-b-galactosidase staining (C) and assessment of relative telomerase activity (D) were performed at day 82 after cultivation in the presence of L -NAME or D -NAME. Each point represents the mean ± S.E. of results from three experiments.
EPDCs constitute a homogenous population originating from clonogenic expansion of EPCs, not from transformed endothelial cells. In addition, cultured EPDCs can be stored more safely in a liquid nitrogen tank than can HUVECs, because thawing and freezing procedures do not influence their growth kinetics or cell adhesion significantly, in contrast to HUVECs (data not shown). Therefore, all the present findings suggest that cord blood-derived EPDCs may be used efficiently in studies of endothelial cell function and possibly in cell therapy. Acknowledgements: This work was supported by a grant of the Korea Science and Engineering Foundation (KOSEF) through the Vascular System Research Center at Kangwon National University (R112001-090-01004-0), by the KOSEF grant funded by the Ministry of Science & Technology (No. M10641000066-06N4100-06610), and by a grant of the High-Performance Cell Therapy R&D Project (0405DB01-0104-0006), Ministry of Health and Welfare, Republic of Korea. We also thank Dr. Edith C. Wolff (NIDCR, NIH, USA) and Eunju O. for helpful comments on the manuscript.
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Outgrowing endothelial progenitor-derived cells display high sensitivity to angiogenesis modulators and delayed senescence Jung-Min Haa, Myoung-Rae Kima, Ho-Kyun Oha, Byung Hun Leea, Hyun-Young Ahnb, Jong-Chul Shinb, Sang Hong Baekc, Young Ae Joea,* a
Cancer Research Institute and Department of Biomedical Science, College of Medicine, The Catholic University of Korea, Banpo-dong 505, Seocho-ku, Seoul 137-701, Republic of Korea b Department of Obstetrics and Gynecology, Division of Maternal-Fetal Medicine, College of Medicine, The Catholic University of Korea, Seoul 137-701, Republic of Korea c Department of Internal Medicine, Division of Cardiovascular Medicine, College of Medicine, The Catholic University of Korea, Seoul 137-701, Republic of Korea Received 12 March 2007; revised 25 April 2007; accepted 3 May 2007 Available online 15 May 2007 Edited by Francesc Posas
Abstract Outgrowing endothelial progenitor-derived cells (EPDCs) originate from a novel hierarchy of endothelial progenitor cells. In this study, EPDCs isolated from human cord blood were examined for phenotype and functional features upon aging. Young or aged EPDCs were similar to human umbilical vein endothelial cells (HUVECs), in exhibiting typical endothelial phenotypes. However, EPDCs were more sensitive to angiogenesis inducers or inhibitors in proliferation and migration. In addition, EPDCs underwent senescence markedly slowly with sustained endothelial NO synthase expression and activation, and their ability to undergo capillary morphogenesis was retained throughout longterm culture. Thus, these results suggest that a homogenous population of EPDCs derived from clonogenic expansion may provide an effective vasculogenesis tool. 2007 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. Keywords: Vasculogenesis; Endothelial progenitor cell; Senescence; Angiogenesis; Inhibitor
1. Introduction Endothelial progenitor cells (EPCs) can contribute to postnatal vasculogenesis in tissue ischemia, wound healing, and tumor growth [1–3]. Early EPCs with the limited proliferating potential (for less than 3 weeks) were initially characterized as a cell type with postnatal vasculogenic capacity by Asahara et al. [1,2]. These observation led to clinical trials of therapeutic application of EPCs in cardiovascular disease [4,5]. Later, several groups reported a different cell type with high expansion capacity that appears at a late stage [6–9]. The cells were named endothelial progenitor-derived cells (EPDCs) or late EPCs. In in vitro culture, early EPCs form adherent spindleshaped cells at day 7–14, and die after 3 weeks. In the case of EPDCs, they have been reported to differentiate from a subset of EPCs after 2 weeks, show exponential growth at 4–8 weeks and live up to 12 weeks. In particular, cord * Corresponding author. Fax: +82 2 532 0575. E-mail address: [email protected] (Y.A. Joe).
blood-derived EPDCs have been shown to have a greater proliferative rate and expandability than those of adult peripheral blood-derived EPDCs [6]. For in vitro angiogenesis assays, human umbilical vein endothelial cells (HUVECs) have been used frequently and commonly. However, HUVECs, mature endothelial cells, that are primarily cultured from human cord vein vessel, are changed in phenotypes and signal pathways during long term culture [10,11]. The growth of HUVECs slows down after about 19–22 population doublings and the cells are used between passages 2 and 8 (about 4 weeks). In addition, the culture of these cells is sometimes complicated by contamination with fibroblasts. In the case of EPDCs, they constitute a homogenous population by clonogenic expansion. Moreover, EPDCs have been shown to have tissue repair capability through neovasculogenesis in a limb ischemia model of mouse, suggesting a potential for cell therapy of vascular grafts [8]. However, the shape of cultured EPDCs is very similar to HUVECs and the isolation of EPDCs is not always successful, resulting in a frequency of about 30% of total blood samples under our experimental conditions. So, considering that HUVECs and EPDCs are derived from cord vessel wall and cord blood, respectively, the possibility cannot be excluded that EPDCs might appear from an in vitro artifact, like immortalization or in vitro transformation of endothelial cells that emerged from vessel walls. Therefore, we addressed the question of how EPDCs are different from HUVECs in phenotypes and functional features during long-term culture. In these experiments, we show that EPDCs, in contrast to HUVECs, retain endothelial characteristics in phenotype and function during a long-term culture period, and only slowly undergo senescence, not being immortalized, thereby suggesting that EPDCs may be an effective vasculogenesis tool for in vitro assays and cell therapy.
2. Materials and methods 2.1. Cell culture The Institutional Review Board at the Catholic University of Korea College of Medicine approved all protocols. Cord blood and cords were obtained from donors with informed consent. Mononuclear cells (MNCs) were isolated from human cord blood using Ficoll-Hypaque
0014-5793/$32.00 2007 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.febslet.2007.05.010
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density gradient centrifugation as described previously [12]. Isolated MNCs were plated into a dish coated with 10 lg/ml human fibronectin (Sigma, St. Louis, MO) and incubated in Medium 199 (M199, JBI, Daegu, Korea) supplemented with 20% fetal bovine serum (FBS, Gibco BRL, Grand Island, NY), 30 lg/ml endothelial cell growth supplement (ECGS, Sigma), 90 lg/ml heparin (Sigma) and 1% penicillin– streptomycin (Gibco BRL). After changing to fresh media every 3–4 days for 15–21 days, an emerging EPDC clone was trypsinized, and transferred to 1% gelatin-coated dishes for subculture. Human umbilical vein endothelial cells (HUVECs) were cultured as described previously [12]. 2.2. Cell proliferation potential At subconfluence, cells were detached and counted. The number of divisions was estimated using the following equation: (Ln (number of cells counted/number of cells at the beginning of the assay)/Ln 2) [7]. 2.3. Reverse transcriptase polymerase chain reaction (RT-PCR) RNA of EPDCs or HUVECs was extracted by use of the Trizol reagent (Life Technologies, San Diego, CA). First-strand cDNA was synthesized using the reverse transcription system (Promega, Madison, WI), and was used for the amplification of each cDNA by Super Taq PLUS version (Super Bio Co. LTD, Seoul, Korea). The PCR primer sequences used for RT-PCR are listed in Table 1. PCR conditions were 95 C for 5 min, followed by 30 cycles (25 cycles for GAPDH) of 95 C for 1 min, 55–63 C for 1 min, and 72 C for 1 min, and ending with 72 C for 10 min. 2.4. FACS analysis FACS analysis was performed on EPDCs and HUVECs collected after trypsinization as described previously, except vWF and VE-cadherin [12]. For detection of VE-cadherin, cells were detached by treatment of 4 mM EDTA for less than 5 min, washed twice, and blocked with 0.5% BSA and 1 lg human immunoglobulin (IgG) in PBS. vWF was detected as reported by Lin et al. [13]. Binding of anti-vWF (mouse monoclonal, Dako, Glostrup, Denmark) antibody was visualized with Cy3-conjugated goat anti-mouse IgG (Chemicon, Temecula, CA). Anti-CD31, anti-CD34 (mouse monoclonal, Becton Dickinson, San Jose, CA), anti-CD105 (mouse monoclonal, Serotec, UK), and anti-VE-cadherin (mouse monoclonal, R&D, Minneapolis, MN) antibodies were used directly as the PE- or FITC-conjugated form.
Table 1 List of primer sequence Gene
Nucleotide sequence
PK5
5 0 -CAGTATCGATCACTCCTTCCGAAGAAGAC 3 0 -CTGATCTAGATCAAAATGAAGGGGCCGC 5 0 -CAGTATCGATACATACTCATCAGGACTTTC 3 0 -CTGATCTAGACTATTTGGAGAAAGAGGTC F-GTGATTGCCATGTTCTTCTG R-GAGGATCTTGAGTTCAGACA F-GGTCTTTGCCTGAAATGGTGAG R-GACTGTTGCTTCACAGGTCAGA F-ATGCGTCAACAAGCTTCCTTCC R-CACCTAAGCTTACAATCTGGCC F-ACGCTCCTTCTCGATTATTGGG R-CAGAGGAGATGTTGCATGAGC F-CTCAGTCATCTCTGAGAC R-AATCCAGAGGCTTCATGG F-CGTCTTCCCGGATGTTTCTTCT R-CTGCTCTTGGAACTCCTGAAAC F-TACCAGTCCTTGCAGTGGTTCG R-CCACTGAATTCACCAACAGCCA F-CAGGAAGGAATGCCTACTTCTG R-CCCATCTCCAGCAAAGTGAAAC F-CCTGAAGGAGAATCTGCTGAAG R-GATGCATCATTCCTCACGCCAA F-AAAGTGGAGTTGTCAGTCTGGG R-ACAGGAGAAGTTGTCGCACTCA F-ATGTTCGTCATGGGTGTGAACC R-GGCAGGTTTTTCTAGACGGCAG
Endostatin KDR Flt-1 Tie-2 vWF VE-cadherin Integrin av Integrin a5 Integrin b1 Integrin b3 Integrin b5 GAPDH
2.5. Expression and purification of angiogenesis inhibitors The recombinant proteins of PK5 and endostatin were expressed in Pichia using EasySelectPichia Expression Kit (Invitrogen, Carlsbad, CA) as follows. cDNA fragments were obtained from Dr. Yeup Yoon (PK5, Mogam Biotechnology Research Institute, Korea) and Dr. SooIk Chang (mouse endostatin, Chungbuk National University, Korea). DNA fragments encoding the amino acids of human plasminogen kringle 5 (PK5, Thr456–Phe546 of plasminogen) and endostatin (His132–Lys315 of mouse a1 (XVIII)) were amplified by PCR with Pfu polymerase (Stratagene, La Jolla, CA) and PK5 primers or endostatin primers (Table 1), and subcloned into the Cla I–Xba I site of pPICZa-C. The insert DNA was cut with SacI and introduced into Pichia pastoris GS115 (pPICZa-C/PK5) or X-33 (pPICZa-C/endostatin) by electroporation. After the selection of PK5 or endostatinexpressing clones, expression in a large-scale culture and purification were performed by a similar method to that reported previously for the recombinant kringles 1–2 of tissue-type plasminogen activator (TK1–2) protein [14]. 2.6. Cell proliferation assay Cell proliferation assays were performed using a thymidine incorporation assay as described previously [15]. 2.7. Cell migration assay The migration of EPDCs and HUVECs was evaluated in a modified Boyden chamber assay as described previously [14]. 2.8. In vitro tube formation on Matri-gel Two hundred and fifty microliters of chilled Matri-gel (BD) was added in each well of a chilled 24 well plate. After 30 min of incubation at 37 C, 250 ll M199 medium containing 4 · 104 cells/well was added onto the solidified Matri-gel. After 18 h of incubation, the cells were stained with hematoxylin and five representative fields were photographed. Images were analyzed in Image J v.1.34s (http://rsb.info.nih.gov/ij/) for determining tube density. 2.9. Senescence associated (SA)-b-galactosidase assay The cells were washed, fixed, and incubated overnight at 37 C with X-gal chromogenic substrate at pH 6.0 following the conventional protocol for SA-b-gal staining [16]. After taking pictures of five randomly selected fields, the cells were washed with PBS and 500 ll DMSO was added to each well for dissolving the stain, and incubated at 37 C for 30 min, followed by measurement of absorbance at 595 nm. 2.10. Telomerase assay Telomerase activity was measured with 5 lg protein by the TeloTAGGG Telomerase PCR ELISAPLUSKit according to the manufacturer’s instructions (Roche Applied Science, Germany). 2.11. Western blot Cell lysate (50 lg protein) was resolved on SDS–PAGE gel and transferred to a nitrocellulose membrane. The membrane was incubated with an antibody against phospho-eNOS, eNOS, ERK 1/2 (Cell Signaling, Danvers, MA), b-actin, b-tubulin (Sigma) or GAPDH (R&D, Minneapolis, MN), followed by binding with the corresponding secondary antibody conjugated to horseradish peroxidase. The binding was detected by enhanced chemiluminescence (ECL plus, Amersham, Germany).
3. Results and discussion 3.1. Comparison of growth kinetics and phenotypes Under our experimental conditions, we were able to isolate EPDCs at a frequency of 3 donors out of a total of 9 donors from the culture of mononuclear cells (MNCs) isolated from the cord blood of several infants. EPDCs started to appear in culture dishes 17 days after seeding of MNCs and about ten individual colonies were obtained at once. As shown in Fig. 1A, the clone became larger over time and grew exponen-
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Fig. 1. The comparison of growth kinetics and phenotypes. (A) Population doubling of EPDCs isolated from different individual cord blood samples was compared with that of HUVECs (left). Ex vivo cultivation day 0 (D0) indicates the day when MNCs or HUVECs were isolated for initiating in vitro culture. Representative photomicrographs of an EPDC colony were captured (right, 100·). (B) The representative pictures of each cell type were captured upon aging (200·). RT-PCR (C) and FACS analyses (D) were performed on various endothelial cell markers and vascular integrins.
tially. When the growth kinetics of EPDCs and HUVECs were compared, the population doubling curves demonstrate that both EPDCs, derived from two donor bloods, have a higher proliferation potential than HUVECs (Fig. 1A). EPDCs reached growth arrest after about 40 population doublings, whereas HUVECs reached growth arrest faster, after 27 population doublings, in about 50 days. Subculture of EPDCs was maintained for more than 3 months without any apparent signs of replicative senescence. However, the growth rate of EPDCs decreased after 110 days, thereby indicating that EPDCs eventually stop growing, not being immortalized. Both young HUVECs (D10, 10 days after initiating ex vivo culture) and young EPDCs (D50) appeared as similar cobblestoneshapes. In accordance with several established studies showing that cells entering senescence become enlarged and flattened, aged HUVECs (D50) became larger and flatter than aged EPDCs (D100) (Fig. 1B).
When we examined the expression of typical endothelial cell markers, including KDR, Flt-1, Tie-2, vWF, VE-cadherin, CD34, CD31, and CD105, by RT-PCR and FACS analyses, we could not detect any marked difference between EPDCs and HUVECs (Fig. 1C and D). Thus, at present, the difference in phenotype between EPDCs and HUVECs is not clear. More precise analysis methods including microarray analysis, which allows analysis of many genes at once, will be desirable to differentiate between HUVEC and EPDC phenotypes. 3.2. EPDCs are sensitive to various angiogenesis modulators When we tested the functional properties of EPDCs, we found that basal levels of endothelial proliferation and migration were higher in young cells than in aged cells in both EPDCs and HUVECs (Fig. 2A and C). In particular, EPDCs, young or aged, showed high sensitivity to growth and movement stimuli, whereas aged HUVECs of the same ex vivo
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Fig. 2. Comparison of sensitivities to angiogenesis inducers or inhibitors in cell proliferation and migration. The [3H] thymidine-incorporation assay was performed on young or aged EPDCs and HUVECs, for evaluating cell growth stimulated by bFGF (A) and the anti-proliferative effect of PK5 (B). VEGF-stimulated cell migration (C), and anti-migrative effect of PK5, TK1–2 and endostatin (D) were assessed. The inhibitory effects were shown as a percentage of stimulated growth (cpm) (B) or migration (cell number) (D) in the absence of inhibitor. Values represent the mean of three independent experiments (means ± S.E.). *P < 0.05, **P < 0.01, compared with non-treated cells.
cultivation day as the young EPDCs, showed marginal induction of proliferation and marked induction of migration by bFGF or VEGF. In both cell types, young cells displayed more sensitivity in cell proliferation to the bFGF stimulus than did aged cells. Next, we tested if EPDCs are sensitive to endothelial-selective angiogenesis inhibitors including plasminogen kringle 5 (PK5), tissue-type plasminogen activator kringles 1–2 (TK1– 2), and endostatin. Interestingly, PK5 dose-dependently inhibited cell proliferation significantly only for young EPDCs (IC50 = 60 nM) and HUVECs (D10, IC50 = 250 nM) (Fig. 2B). Aged HUVECs (D50) did not show any significant inhibition of proliferation by PK5. In the migration assay, all the inhibitors tested showed inhibition in young EPDCs (PK5, IC50 = 100 nM; TK1–2 IC50 = 80 nM; endostatin, IC50 = 80 nM) (Fig. 2D). Aged HUVECs showed a little higher IC50’s than young EPDCs at the same age (D50) (PK5, IC50 > 500 nM; TK1–2 IC50 = 100 nM; endostatin, IC50 = 100 nM). Upon aging, the sensitivity of EPDCs to the inhibitors in cell migration decreased. Interestingly, inhibitors showed inhibitory activity in migration of aged cells of both cell types, unlike the difference in effects on proliferation. Overall, EPDCs at day 50, the same age as aged HUVECs, showed high sensitivity to angiogenesis inducers and inhibitors in both proliferation and migration, suggesting that EPDCs have more potentiality for vasculogenesis, and that they can be used more effectively in migration and proliferation assays.
3.3. Different capacity for capillary tube formation between aged EPDCs and HUVECs In the in vitro tube formation assay, young EPDCs formed capillary-like structures on Matri-gel (Fig. 3). When we compared the capacity for capillary tube formation between young and aged cells, aged HUVECs showed a low capacity for capillary tube formation, although they showed a high motility upon VEGF induction. On the other hand, aged EPDCs were able to form capillary tubes fully, suggesting that EPDCs retain the capability of capillary morphogenesis through long-term culture. In previous reports, senescence has been reported to progress more increased adhesion to the extracelluar matrix while losing cell–cell contacts [17,18]. Therefore, the progression of senescence in HUVECs seems to result in a decrease in their capacity to form tubes.
3.4. Markedly delayed senescence in EPDCs We investigated the senescence levels of EPDCs and HUVECs through SA-b-galactosidase staining, which is widely used as a biomarker of senescence. Aged EPDCs showed much fewer SA-b-gal positive cells than aged HUVECs, while their young cells showed few stained cells (Fig. 4A). When EPDCs were stained at the growth-arrest point (at day 130), EPDCs were also strongly stained (data not shown), suggesting that EPDCs also undergo senescence eventually, but do so much more slowly than HUVECs. Next, telomerase activity was
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Fig. 3. Formation of tube-like structure on Matri-gel. After young or aged EPDCs (D50, D100) and HUVECs (D10, D50) were incubated for 18 h on Matri-gel, the representative pictures were captured (100·), and tube density was analyzed.
determined since cellular senescence is critically influenced by telomerase. As shown in Fig. 4B, telomerase activity was reduced drastically in HUVECs upon aging, whereas aged EPDCs showed telomerase activity at a certain level, albeit at a reduced one, thereby corresponding well with the data obtained from SA-b-galactosidase staining. Thus, these results indicate that EPDCs are not derived from artifacts of in vitro cell culture, such as immortalization or transformation of vessel cells, and that they undergo senescence slowly. Next, we investigated the expression of eNOS and Akt and its phosphorylation status since aging of endothelial cells has been reported to be associated with reduced eNOS expression and decreased NO synthesis [19]. Indeed, eNOS protein expression and phosphorylation levels were drastically reduced in aged HUVECs (Fig. 5A and B). However, aged EPDCs, as well as young EPDCs, showed certain levels of expression and phosphorylation of eNOS, indicating no signs of senescence. Interestingly, the expression level of Akt and ERK 1/2 did not change upon aging, but the mean expression level of b-tubulin and b-actin decreased in HUVECs only, although there was some variation in the expression level of b-tubulin and b-actin among the donor types of HUVECs. This result corresponds well with a previous report that b-actin expression was down-regulated in rat gastric mucosa upon senescence [20]. At present, albeit with some questionable points, the results could suggest that the decreased expression of cytoskeleton proteins b-actin and b-tubulin, which are involved in cell movement and shape, might be related to the loss of tube for-
Fig. 4. Cellular senescence of EPDCs and HUVECs. (A) Young or aged cells were stained by the SA-b-galactosidase reaction. (B) The cells were assessed for relative telomerase activity (RTA).
mation ability and can be a characteristic of endothelial cells during senescence. Very recently, the inhibition of cellular senescence in HUVECs by nitric oxide has been reported [21]. Therefore, we tested whether inhibition of eNOS causes progression of senescence in EPDCs, as it does in HUVECs. Indeed, treatment with L -NAME (a NOS inhibitor) decreased the proliferation of EPDCs as they got older and showed more SA-b-gal stained cells, when compared with treatment with D -NAME (negative control) (Fig. 5C). Such an effect was also detected in two other EPDC clones, clones 2 and 3. Telomerase activity was also reduced upon L -NAME treatment compared to D -NAME treatment (Fig. 5D). Thus, all the results suggest that the delay of senescence in EPDCs may be due to a steady eNOS expression and activation (and possibly NO production). In summary, we found that EPDCs show high sensitivity to angiogenesis inducers and inhibitors and also undergo delayed senescence, even though both EPDCs and HUVECs are derived from infant cord blood and tissue. In particular, a large difference in senescence levels between EPDCs and HUVECs is manifested in in vitro tube formation capability, expression of eNOS and b-tubulin, and phosphorylation of eNOS. Moreover,
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Fig. 5. Differential expression of eNOS, b-tubulin and b-actin in aged EPDCs and HUVECs and effect of NO on EPDC senescence. (A) Phosphoryation of eNOS Ser1177 and expression of eNOS, Akt, ERK1/2, b-tubulin, b-actin and GAPDH were determined by Western blot analysis. GAPDH was used as a loading control. (B) The densitometric quantification of the Western blots was performed. The density of each protein band was normalized with that of GAPDH. Values represent the mean of three independent Western blot experiments (means ± S.E.). (*P < 0.01 compared with young cells). (C and D) EPDCs were cultivated in the presence of L -NAME or D -NAME (Sigma) at 100 lM from ex vivo cultivation day 50 to day 82, with replacement of new medium every 48 h. SA-b-galactosidase staining (C) and assessment of relative telomerase activity (D) were performed at day 82 after cultivation in the presence of L -NAME or D -NAME. Each point represents the mean ± S.E. of results from three experiments.
EPDCs constitute a homogenous population originating from clonogenic expansion of EPCs, not from transformed endothelial cells. In addition, cultured EPDCs can be stored more safely in a liquid nitrogen tank than can HUVECs, because thawing and freezing procedures do not influence their growth kinetics or cell adhesion significantly, in contrast to HUVECs (data not shown). Therefore, all the present findings suggest that cord blood-derived EPDCs may be used efficiently in studies of endothelial cell function and possibly in cell therapy. Acknowledgements: This work was supported by a grant of the Korea Science and Engineering Foundation (KOSEF) through the Vascular System Research Center at Kangwon National University (R112001-090-01004-0), by the KOSEF grant funded by the Ministry of Science & Technology (No. M10641000066-06N4100-06610), and by a grant of the High-Performance Cell Therapy R&D Project (0405DB01-0104-0006), Ministry of Health and Welfare, Republic of Korea. We also thank Dr. Edith C. Wolff (NIDCR, NIH, USA) and Eunju O. for helpful comments on the manuscript.
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