ANIMAL CELLS AND SYSTEMS, 2016 VOL. 20, NO. 3, 118–129 http://dx.doi.org/10.1080/19768354.2016.1189961 Angiogenic activity of endothelial progenitor cells through angiopoietin-1 and angiopoietin-2 a a b c d Vahid Siavashi , Reyhaneh Sariri , Seyed Mahdi Nassiri , Masoumeh Esmaeilivand , Simin Asadian , b e f Hadi Cheraghi , Mazyar Barekati-Mowahed and Reza Rahbarghazi a b Department of Biology, Faculty of Sciences, University of Guilan, Rasht, Iran; Department of Clinical Pathology, Faculty of Veterinary Medicine, c d University of Tehran, Tehran, Iran; School of Nursing and Midwifery, Kermanshah University of Medical Sciences, Kermanshah, Iran; Imam Reza Hospital, Kermanshah University of Medical Sciences, Kermanshah, Iran; Department of Human Biology, University of Toronto, Toronto, ON, Canada; Stem Cell Research Center, Tabriz University of Medical Sciences, Tabriz, Iran ABSTRACT ARTICLE HISTORY Received 11 February 2016 Angiogenesis is a regulated process involving the proliferation, migration, and remodeling of Revised 29 April 2016 different cell types particularly mature endothelial cells and recently discovered progenitor cells, Accepted 6 May 2016 named as endothelial progenitor cells (EPCs). Up to now, many attempts have been made to understand the dynamic balance of pro- and anti-angiogenic factors on EPCs on different milieu. KEYWORDS It has been accepted that Ang-1, -2 and Tie-1, -2 signaling play a key role on angiogenesis Angiopoietin-1; angiopoietin- pathways in endothelial lineage cells. In the current experiment, the angiogenic/angio- 2; endothelial progenitor modulatory potency of Ang-1 and -2 was investigated on isolated EPCs. Freshly isolated EPCs cells; angiogenic activity were exposed to different concentrations of Ang-1 and -2 (25 and 50 ng/ml) over a course of 7 and 14 days. Corroborating to our results, a superior effect of Ang-1 on angiogenic properties, including an increased concentration of vascular endothelial growth factor, in vitro tubulogenesis, EPC migratory, Tie-2 expression and clonogenicity, was determined. A large amount of positive mature endothelium markers was achieved in EPCs being-exposed to Ang-1 peptide. Nonetheless, the number of CD133 positive cells increased in the presence of Ang-2. Collectively, we conclude that Ang-1 potentially induces functional and mature vascular-like behavior in EPCs more than Ang-2. Introduction ligands would determine the vascular assembly and Blood vessel development is a regulated process invol- endothelial quiescence status (Scharpfenecker et al. ving the proliferation, migration, and remodeling of 2005). In the literature it was demonstrated that sustained endothelial cells (ECs) from the existing vasculature increase of Ang-2, coupled with reciprocal decrease in (angiogenesis) or following differentiation of endothelial Ang-1 content contributed to proliferation and migration progenitor cells (EPCs) from mesodermal precursors (vas- of ECs in angiogenic milieu (Lobov et al. 2002). It has been culogenesis) (Jain 2003). Both vasculogenesis and angio- previously shown that phosphorylation of Tie-2 is genesis occur during embryonic development (Asahara induced by the stimulation of Ang-1, while Ang-2 acts & Kawamoto 2004; Kawamoto et al. 2006). EPCs are a in the opposite manner (Felcht et al. 2012). However, subtype of stem cells with high proliferative potential some authorities reported the context dependence of that are capable of differentiating into mature ECs and Ang-1/2 and Tie-2 receptor (either phosphorylated or contributing to neovascularization (Yoder 2012). non-phosphorylated forms) axis (Dumont et al. 2006). Recently, a novel endothelial-speciﬁc receptor tyrosine Follow-up studies demonstrated vascular endothelial kinase, Tie2 also termed Tek, was identiﬁed, which was growth factor receptor-2 (VEGFR-2, also known as demonstrated to play a role in vascular maintenance Kinase insert domain receptor), Tie-2 and CD34 as puta- and angiogenesis. Tie2 is a receptor tyrosine kinase tive biomarkers for early EPCs in the bone marrow (Frie- +/– cloned from ECs (Dumont et al. 2006) as well as from drich et al. 2006; Grigoras et al. 2014), and CD133 , bone marrow Hematopoietic stem cells (Iwama et al. CD34, VEGFR-2, CD146, CD144 and CD31 for circulating 1993). Angiopoietin-1 (Ang-1) and angiopoietin-2 (Ang- EPCs (Cheng et al. 2013). Recently, the Angs (Ang-1– 2) were identiﬁed as ligands of the Tie2 receptor, with Ang-4) have been shown to be important mediators of Ang-2 acting as a natural Ang-1/Tie2 inhibitor (Hawighorst angiogenesis by their regulation of EC survival in malig- et al. 2002). The dynamic balance of both Ang-1 and Ang-2 nant and nonmalignant tissues (Martin et al. 2008). Also, CONTACT Reyhaneh Sariri email@example.com; Reza Rahbarghazi Rezarahbarghazidvm@gmail.com © 2016 Korean Society for Integrative Biology DEVELOPMENTAL BIOLOGY ANIMAL CELLS AND SYSTEMS 119 in the literature, two different classic subpopulations of different concentrations of Ang-1 and Ang-2 (25 and EPCs, namely early EPCs and late EPCs were deﬁned 50 ng) were added to the culture medium. The cells (Hur et al. 2004). In addition, the critical role of both were cultured for 7 days. Ang-1 and Ang-2 has been shown in embryonic, neonatal and mature conditions in which targeted inactivation of EPC characterization the Ang-1 or Ang-2 genes renders inappropriate form of complex vascular network (Suri et al. 1996). For EPC characterization and distinguishing these cells In this study, we focused on the effects of the Tie-2 from hematopoietic cells, cells were analyzed by ﬂow ligands, Ang-1 and Ang-2 on the vasculogenic properties cytometry using a panel of antibodies, including of EPCs. Phycoerythrin (PE) conjugated anti-mouse CD45 (Cat No: 12-0451-81; eBioscience, Inc., USA), PE-conjugated anti-mouse CD14 (Cat No: 123309; Biolegend, Inc., USA), Materials and methods PE-conjugated anti-mouse CD11b (Cat No: 12-0112-81; eBioscience, Inc., USA) and PE-conjugated anti-mouse Animal CD105 antibodies (Cat No: 120407; Biolegend, Inc., Male non-transgenic and green ﬂuorescent protein (GFP) USA). An isotype control was used for each assay. To positive transgenic C57BL/6 mice were purchased from further distinguish the EPCs pre-expanded on ﬁbronectin Royan Institute Animal Facility. The mice were treated from hematopoietic progenitor cells, 4 × 10 EPCs were in accordance with the published guideline of The Care plated in the methylcellulose semi-solid medium con- and Use of Laboratory Animals (NIH Publication, 8th taining 100 ng/ml VEGF and 5 ng/ml Granulocyte-macro- edition, revised 2011). All procedures of this study were phage colony-stimulating factor (GM-CSF) for two also approved by the Animal Care Committee of the Uni- weeks, as described previously (Salter et al. 2009). The versity of Tehran. colonies were ﬁnally imaged and counted. Isolation, expansion and cell culture procedure Cell proliferation assay After cervical dislocation, the femurs and tibiae were 1×10 EPCs were seeded in 24-well culture plates in the aseptically opened and bone marrow was harvested EGM-2 MV medium supplemented with 25 and 50 ng/ml by repeated ﬂushing with sterile phosphate buffered Ang-1 (Cat No: 130-06; Peprotech) and Ang-2 (Cat No: saline (PBS, Cat No: 15140-122; Gibco), then ﬁltered 130-07; Peprotech). The effects of increasing concen- through sterile 200 µm mesh (Cat No: 0.075; Damavand, trations of Ang-1 and Ang-2 on the proliferative rate of Iran). Single-cell suspension was added to Ficoll (Sigma) cultured EPCs were assessed using a growth curve analy- at a ratio of 1:1. The mixture was then centrifuged at sis. Cells cultured in each well were harvested at days 7 2000 rpm for 20 min to separate the cells into three and 14, and then counted using a cell counter machine layers. The middle layer, which is white and cloudy (Model: MEK-6450k; Nihon Kohden). The numbers of and consists of the mononuclear cells, was gently cells were then plotted against culture time to generate removed and collected in 50 ml conical tubes, the the growth curve. cells were washed three times with PBS. Prior to the seeding, the total cell count was performed using a Functional analysis of EPCs under Ang-1 and Ang-2 cell counter machine (Model: MEK-6450k, Nihon Kohden). Before cell seeding, 24-well culture plates To analyze the functionality of cultured EPCs treated with were coated with a total volume of 300 µl ﬁbronectin Ang-1 and Ang-2, 7-day-cultured, EPCs recognized as (Cat No: C-43050; Promocell, Germany with concen- attaching spindle-shaped cells were assessed by staining tration of 1 µg/ml). The plates were then exposed to the cells with acetylated low-density lipoprotein (Ac-LDL) ultraviolet radiation under laminar ﬂow hood for 20 labeled with ﬂuorescent Dil dye (Dil-Ac-LDL, Cat No: L- min and incubated at 37°C for 10 min. Finally, bone 3485; Gibco). Brieﬂy, adherent cells were washed three marrow mononuclear cells were seeded and incubated times with PBS and incubated in the M199 medium con- at 37°C, with 5% CO , and 95% relative humidity in taining Dil-Ac-LDL at 10 µg/ml concentration for 4 h. M199 containing EGM-2 Supplement Pack (Cat No: C- Then, the medium was removed and the cells were 39211; Promocell, Germany) containing Fetal calf washed with PBS. Finally, the cells were observed under serum, VEGF, Insulin-like growth factor, Basic ﬁbroblast a ﬂuorescent microscope (Olympus, Tokyo, Japan) and growth factor, hydrocortisone, ascorbic acid, heparin also further subjected to the ﬂow cytometry analysis. To and Epidermal growth factor. For our experiments, analyze the phenotype of mature ECs, EPCs under Ang- 120 V. SIAVASHI ET AL. 1 and Ang-2 were incubated with anti-mouse CD31 (Cat assigned to each colony as follows: 0, aggregate colony No: Sc-80913; Santa Cruz Biotechnology, Inc.) followed with no sprouting; 1, colony sprouting without arboriza- with goat anti-mouse IgG-Texas red (Cat No: ab6787; tion; 2, with arborization; 3, with anastomosis; and 4, Abcam) and analyzed by BD FACSAria II (BD Biosciences). development of a complex tubular network. For each Data were analyzed using FlowJo v7.6.5 software. well, the total score was calculated by adding all the 25 scores to attain a maximum possible score of 100. EPC colony formation assay Migration assay Murine bone marrow mononuclear cells were seeded on ﬁbronectin (3 × 10 cells/well) in M199-EGM-2 with and For the migration assay, 24-well polycarbonate mem- without Ang-1 and Ang-2 for 7 days. Then, the total brane cell culture plate inserts with 8-μm-pore size number of colonies with more than 50 aggregated were obtained (Cat No. 35224; SPL Life Science). In cells was manually counted under a phase contrast brief, EPCs were serum-starved overnight. After trypsini- microscope. zation, 5 × 10 cells were suspended in 100 μl serum- free M199 and seeded on the top chamber of the trans- well inserts (triplicate per one sample). The bottom Flow cytometry chambers contained 750 μl serum-free M199 with Bone marrow mononuclear cells and EPCs cultured for 7 different concentrations of Ang-1 and Ang-2 (25 and days with different concentration of Ang-1 and Ang-2 (25 50 ng/ml). The serum-free M199 was used as control. and 50 ng/ml) were immunophenotyped with a panel of After incubation for 24 h in a CO incubator, the cell antibodies, including Allophycocyanin-conjugated rat were stained with 4 ,6-diamidino-2-phenylindole (1 µg/ anti-mouse CD133/AC133 (Cat No: 17-1331; eBioscience), ml, Cat No: D9542; Sigma–Aldrich) and counted. All PE-conjugated rat anti-mouse Tie2 (Cat No: 12-5987-81; experiments were performed in triplicate (Vasa et al. eBioscience), paciﬁc blue-conjugated rat anti-mouse 2001; Chen et al. 2004). VEGFR2 (Cat No: 121914; Biolegend, USA) and Alexa Fluor 488 anti-mouse CD45 antibodies (Cat No: Measurement of VEGF protein in the conditioned 103121; Biolegend, USA). For each antibody, a relevant medium isotype control was used. Brieﬂy, cells were blocked with 5% Bovine serum albumin (BSA) solution at 4°C for To analyze VEGF secreted into the EPC culture medium, 20 min and incubated with above-mentioned antibodies cells were ﬁrst cultured in EGM-2MV + M199 for 7 days at 4°C for 30 min. Finally, the cells were washed twice then exposed to serum-free M199 with different concen- with PBS and analyzed by BD FACSAria II (BD Biosciences). trations of Ang-1 and Ang-2 (25 and 50 ng/ml) for 48 h. Data were analyzed using FlowJo v7.6.5 software. Afterwards, conditioned media were collected and ana- lyzed for VEGF by enzyme-linked immunosorbent assay (Cat No: MMV00; R&D system) according to the manufac- Effects of Ang-1 and Ang-2 on matrigel turer’s protocol. tubulogenesis of cultured EPCs Matrigel tube formation assay was performed as pre- Immunocytochemistry viously described with some modiﬁcations (Mukai et al. 2008). In brief, growth factor-reduced matrigel (Cat No: GFP-positive EPCs were subjected to an immunocyto- 354230; BD) was thawed at 4°C overnight, then added chemistry assay as previously described (Takakura et al. to a 96-well plate (50 µl for each well), and allowed to 1997). In brief, mice were sacriﬁced under anesthesia solidify at 37°C for 30 min. EPCs under Ang-1 and Ang- by cervical dislocation; the femurs of mice were ﬂushed 2 (50 ng/ml) were ﬁrst trypsinized, then 1 × 10 EPCs with PBS and cultured on ﬁbronectin-coated 24-well were seeded in each well and incubated in the EGM-2 plates in EGM-2 Supplement Pack with Ang-1 and Ang- MV medium with 1% Fetal bovine serum at 37°C for 2 for 7 days. Cells were washed with PBS, blocked with 24 h. Afterwards, the generation of tube-like structures BSA and then incubated with a mouse monoclonal was inspected under an inverted light microscope anti-Tie2 antibody (Cat No: ab24859; Abcam) for 2 h at (100×) and analyzed by ImageJ Ver. 1.44p (NIH, USA) to 4°C. After washing with PBS, the cells were incubated quantify the tube length. Tubulogenesis was scored with goat anti-mouse IgG-Texas red (1:1000, Cat No: using a colony scoring system according to our previous ab6787; Abcam) for 1 h at room temperature, and then publications (Mohammadi et al. 2015). Brieﬂy, 25 colo- washed with PBS. Finally, the cells were investigated nies were examined in each well and a 0–4 score was under an inverted ﬂuorescence microscope (Olympus). ANIMAL CELLS AND SYSTEMS 121 Figure 1. Effect of Ang1 and Ang2 on cell proliferation of EPCs. Representative bright-ﬁeld images of EPCs expanded on the ﬁbronectin surface 7 days post-seeding (a). Ang1 signiﬁcantly increase the proliferation of EPCs, whereas Ang2 suppressed EPCs proliferation 7 and 14 days after culture (b). Data are presented as mean ± SEM. Three independent experiments were performed in triplicate. *P < .05, **P < .01, ***P < .001 (one-way ANOVA with Tukey post hoc test). Western blot analysis EPCs with or without Ang-1 and Ang-2 (25 and 50 ng/ ml) treatment were lysed in a tissue lysis buffer (50 mM Tris-HCl, 1% Triton X-100, 150 mM NaCl, 1 mM Ethylene glycol tetraacetic acid, 0.25% sodium deoxycholate, and 1 mM NaF) containing 1 mM Na VO , 1 mM phenyl- 3 4 methane sulfonyl ﬂuoride or phenylmethylsulfonyl ﬂu- oride, and protease inhibitor pellets. Quantitation of proteins was measured using the Bradford assay. Lysates (20 µg) were then loaded on sodium dodecyl sulfate polyacrylamide gel electrophoresis gels (5% stacking and 10% separating gels) after a 5 min boiling and subsequently transferred to 0.2 μm immune-Blot™ polyvinylidene diﬂuoride membranes (Cat No: 162-017777; Bio-Rad Laboratories, CA, USA). The membranes were blocked with 3% non-fat dry milk (Cat No: 1.15363.0500; Merck KGaA, Darmstadt, Germany) or 5% BSA (Cat No: A-7888; Sigma–Aldrich, MO, USA) in Tris-buffered saline containing 0.1% Tween 20 (TBST) for 1 h. They were thereafter incu- bated with individual primary antibodies for 1 h at room temperature, washed thrice (3× 10 min) with TBST, and incubated with HRP-conjugated secondary Figure 2. Immunophenotypic negativity of EPCs for CD11b, CD14, CD45 and CD105 (a). Outgrowth of CFU-EC after seeding antibodies for 1 h at room temperature, followed by of EPCs on VEGF and GM-CSF containing methylcellulose incubation in a solution containing 0.006% 3,3 -diami- medium (b). Number of CFU-EC developed by EPCs on the nobenzidine (Cat No: D5637; Sigma–Aldrich, MO, USA) methylcellulose medium (c). EPCs seeding on the methylcellulose to visualize immunoreactive bands. The Western blot medium did not give rise to CFU-GM. analysis was performed with a panel of antibodies, including mouse anti-Tie2 (1 μg/ml; Cat No: ab24859; mouse IgG-HRP (1:4000; Cat No: ab6728; Abcam) and Abcam), rabbit antiphospho-Tie2 (1:1000, Cat No: sheep anti-rabbit IgG-HRP (1:5000, Cat No: ab6795; ab78142; Abcam), mouse anti-beta actin-loading Abcam). The membranes were scanned using an HP control (1 μg/ml, Cat No: ab8224; Abcam), rabbit anti- Scanjet G3110 apparatus (Hewlett-Packard Company, 122 V. SIAVASHI ET AL. Figure 3. Effect of Ang1 and Ang2 on colony formation of EPCs. Colony formation of GFP-positive murine EPCs on ﬁbronectin (a). Ang1 signiﬁcantly augmented the colonogenic potential of EPCs in a dose-dependent manner (b). Three independent experiments were per- formed in triplicate. *P < .05, **P < .01, ***P < .001 (one-way ANOVA with Tukey post hoc test). Figure 4. Effect of Ang-1 and Ang-2 on maturation and Dil-ac-LDL uptake by EPCs derived from murine bone marrow mononuclear cells. Immunoﬂuorescence analysis of EPCs labeled with Dil-Ac-LDL (a). The level of Dil-Ac-LDL uptake was greater in EPCs treated with Ang1 than in cells with Ang2 or in non-treated EPCs (b). CD31-positive mature ECs increased through Ang1 induction of EPCs (c). Three independent experiments were performed. ANIMAL CELLS AND SYSTEMS 123 Figure 5. Flow cytometry analysis of EPCs for CD133, VEGFR-2 and Tie-2 under Ang1 and Ang2 induction. Flow cytometry dot plots for CD133, VEGFR-2 and Tie-2 for freshly harvested bone marrow mononuclear cells and for EPCs cultured on ﬁbronectin-coated plates with Ang1 and Ang2 (a). Ang-2 induction of EPCs led to the increased percentage of CD133+ cells, whereas Ang-1 induction led to the enrichment of Tie2+ and VEGFR-2 positive cells in cultured EPCs (b). Data are expressed as mean ± SD (three independent experiments were performed in triplicate). *P < .05, **P < .01, ***P < .001 (one-way ANOVA with Tukey post hoc test). CA, USA). Protein expression was normalized to β-actin. groups and one-way Analysis of variance (ANOVA) was Densitometry of proteins was performed using the used in experiments with more than two groups. The ImageJ Version 1.44p software (NIH, USA). The percen- Tukey test was used as post hoc. The data analysis was tage area under the curve of each band was divided performed with GraphPad InStat software version 2.02. by the corresponding percentage area under the Values of P ≤ .05 were considered statistically signiﬁcant. curve of the actin band, and the calculated values Statistical analyses were carried out using y signiﬁcant. In were compared statistically between the C and T histograms, statistical difference between groups is groups (Mohammadi et al. 2015). shown by brackets with *P < .05, **P < .01 and ***P < .001. Statistical analysis Results Data are expressed as mean ± SD. After analyzing the Morphological characteristics of EPCs normal distribution of data and homogeneity of var- iances, Student’s t-test was used to assess the signiﬁ- Mononuclear cells isolated from murine bone marrow cance of differences in experiments with only two were cultured in M199 containing EGM-2 Supplement 124 V. SIAVASHI ET AL. GM) (Figure 2(b) and 2(c)). Overall, these results showed that the cells expanded on all extracellular matrix (ECM) were not hematopoietic after 7 days. EPCs characterization and the expression level of CD markers used for characterization in this study were consistent with previous reports of murine EPC features (Salter et al. 2009; Siavashi et al. in press). Mononuclear cells were seeded on ﬁbronectin- coated wells in the EGM-2 MV medium with different concentrations of Ang-1 and Ang-2. Clonogenic potency of the seeded cells was monitored every day. We found that numerous round cell clusters appeared by spindle-shaped cells within 3–7 days post-plating. Induction of EPCs by Ang-1 led to earlier appearance of EPC colonies and signiﬁcantly greater number of EPC colonies (Figure 3(a) and 3(b)). Induction of mature endothelial phenotype after treatment of EPCs with Ang-1 After 7 days of culture, EPCs were functionally character- ized using labeling with (Ac-LDL. The level of Dil-Ac-LDL uptake was greater in EPCs treated with Ang-1 than in cells with Ang-2 or in non-treated EPCs (Figure 4(a) and 4(b)). Also the ﬂow cytometry analysis of CD31 expression by EPCs showed that the percentage of Figure 6. Matrigel tube formation of EPCs induced with Ang-1 CD31 mature ECs were augmented after induction of and Ang-2. Representative images of matrigel tubulogenesis EPCs with Ang-1 (Figure 4(c)). by EPCs cultured with Ang-1 and Ang-2 (a). Tube length (b) and colony tubulogenesis score (c) was augmented after induc- tion of EPCs with Ang-1. Data are expressed as mean ± SD Flow cytometry analysis of stemness phenotype (three independent experiments were performed in triplicate). of EPCs under induction by Ang-1 and Ang-2 *P < .05, **P < .01, ***P < .001 (one-way ANOVA with Tukey post hoc test). We detected low levels of CD133 (2.55 ± 0.006%), FLK-1 (0.601 ± 0.007%) and Tie2 (0.202 ± 0.010%) on freshly har- Pack with different concentrations of Ang-1 and Ang-2 vested bone marrow MNCs, but by day 7 of culture on ﬁbro- (25 and 50 ng/ml). The cells immediately aggregated 1 nectin, the expression levels of these markers were day post-seeding (data not shown), then they exhibited signiﬁcantly enriched (Figure 5(a)). Intriguingly, the cultivation a spindle-like morphology at day 7 (early EPC) (Figure 1 of EPCs under Ang-1 induction resulted in increased percen- (a)). There was no discernible difference between the tage of Tie2+ cells compared with Ang-2 and non-treated morphology of EPCs with Ang-1 and Ang-2 after 7 EPCs (p < .001). On Ang-1 versus Ang-2, fresh MNCs or non-treated EPCs days. Meanwhile, the EPCs number cultured with Ang- the other hand, a signiﬁcantly increased number of CD133 1 was markedly higher than with Ang-2 and non- cells were obtained after induction of EPCs with Ang-2 treated EPCs 7 and 14 days after seeding (p (p < .001) (Figure 5(b)). Ang-1 versus Ang-2 versus Ang-1, fresh MNCs or non-treated EPCs < .001) and this was independent Ang-2 and non-treated EPC of concentration (Figure 1(b)). EPCs expanded on ﬁbro- EPCs under Ang-1 and Ang-2 demonstrate nectin were found to be negative for CD11b, CD14, angiogenic potential by in vitro tubule formation CD45 and CD105 after 7 days (Figure 2(a)). Moreover, seeding the cells pre-expanded on ﬁbronectin sub- To determine the effects of Ang-1 and Ang-2 on tubulo- strates onto the VEGF/GM-CSF containing the methyl- genic potential of EPCs, the cells were expanded for 21 cellulose semi-solid medium consistently resulted in days under Ang-1 or Ang-2 induction, and then sub- the outgrowth of colony-forming units of endothelial jected to the matrigel tubulogenesis assay for 24 h. We cells (CFU-EC), whereas none of the substrates yielded found that EPCs pre-cultured with Ang-1 exhibited colony forming unit - granulocyte/macrophage (CFU- superior tube formation properties as was evidenced ANIMAL CELLS AND SYSTEMS 125 Figure 7. Effect of Ang-1 and Ang-2 on the migratory activity of EPCs. Ang-1 and Ang-2 were used in 25 and 50 ng/ml concentrations (a). Superior migratory activity of EPCs by Ang-1 induction (b). by signiﬁcantly increased tube length and tubulogenesis scoring of EPCs with Ang-1 induction compared with Ang-2 or non-treated cells (Figure 6(a)–(c)). Ang-1 induced EPCs migration EPC migratory activity under Ang-1 and Ang-2 treatment was analyzed in the modiﬁed Boyden chamber assay. We found that Ang-1 with 50 ng/ml concentration remarkably prompted the migration of EPCs (P < .001) while Ang-2 had an inferior effect on EPC migration (Figure 7(a) and 7(b)). Moreover, our results showed increased concen- tration of VEGF in the EPC conditioned medium after treatment with Ang-1 (50 ng/ml) (Figure 8). Ang-1 treatment promotes Tie-2 and Tie2-pho receptor in EPCs Immunocytochemistry was performed to investigate the distinct effects of Ang-1 versus Ang-2 on Tie-2 receptor in EPCs. The results showed that the addition of Ang-1 to the culture medium dramatically activated the Tie-2 receptor content more than that of Ang-2 or non- Figure 8. VEGF concentration in the conditioned medium of treated controls (Figure 9). Ang-1 presumably enhanced EPCs with or without Ang-1 and Ang-2 induction. 126 V. SIAVASHI ET AL. Figure 9. Representative immunoﬂuorescence images of EPCs stained for Tie-2 following induction with Ang-1 and Ang-2. Enrichment of Tie-2 positive cells was detected after treatment of EPCs with Ang-1. Representative images from three independent experiments are shown. the adhesion of Tie2-expressing to EPCs than of Ang-2 excluding the possibility of hematopoietic mixture. and non-treated cells (Figures 9 and 5(b)). Moreover, EPCs expanded on all ECMs yielded only The expression of Tie-2 and Tie2-pho on EPCs after CFU-EC after seeding on the methylcellulose medium exposure to Ang-1 and Ang-2 (25 and 50 ng/ml) or supplemented with VEGF and GM-CSF, further substan- non-treated EPC was measured by Western blotting. tiating that these cells were not from an hematopoietic EPCs under Ang-1 (25 and 50 ng/ml) treatment increased origin (Salter et al. 2009). Tie-2 and tie2-pho expression level compared to the EPCs, known as an alternative cell source for partici- non-treated EPC and EPC with Ang-2, which was statisti- pating in angiogenesis process, play a key role in both cally signiﬁcant (p < .001) pathological and physiological procedures (Rae et al. Ang-1 versus Ang-2 and non-treated EPCs (Figure 10(a)–(c)). 2011). These cells have also putative potency to mobilize, circulate and recruit to sites of neovascularization in response to chemotactic factors (Urbich & Dimmeler Discussion 2004). Many authorities enumerated EPCs based on expression of different cell surface markers, including The cells expanded under the culture conditions used in CD133, CD34 and VEGFR-2, Tie-2, etc. (Povsic et al. this study were negative for hematopoietic markers, ANIMAL CELLS AND SYSTEMS 127 Figure 10. Western blot analysis (a), changes in Tie-2 (b), Tie2-pho (c) and Tie2-pho/Tie-2 ratio (d) on EPCs after exposure to Ang-1 and Ang-2 treatment or non-treated EPCs. Western blot analysis showed that Tie2-pho protein levels were markedly higher in the EPCs with Ang-1 treatment (n = 3) compared with EPCs with Ang-2 treatment and non-treated EPCs. Molecular weights of the immunoreactive bands are as follows: Tie-2 ≃ 124, and Tie2-pho ≃ 124. After band densitometry, the area under the curve of each band was divided by the area under the curve of the corresponding actin band, and the calculated values were compared between the groups. Data are expressed as mean ± SD. *P < .05, **P < .01 and ***P < .001. 2009). Two distinct EPC populations, including early and our results, Ang-1 initiated EPC to EC maturation by the late cells, have been identiﬁed based on identiﬁed differ- upregulation of CD31 and subsequent high capacity of ences on outgrowth capability and speciﬁc cell surface Ac-LDL uptake. It was deciphered that the overexpres- markers (Chen et al. 2007). Recent evidence also unveiled sion of Ang-1 increased signiﬁcantly the number of the critical role of Tie-2 receptor on functional and in the CD31+ and smooth muscle-like cells population and development of deﬁnitive angiogenesis process (Rein- VEGF expression in bone marrow (Zeng et al. 2012). ardy et al. 2015). In this experiment, we scrutinized the Tie-2 positive cells increased in our study after treat- role of the Tie-2 ligands, Ang-1 and Ang-2, on the vascu- ment by Ang-1 which possibly could be related to conco- logenic properties of EPCs. According to our result, no mitant Tie2 upregulation either in mRNA or protein levels evidence of Ang-1 or Ang-2 associate morphological (Yu et al. 2001). Binding of Ang-1 to the extracellular changes was found (Figure 1). On the other hand, the domain of Tie2 in ECs results in receptor auto-phos- total number of cells under EPC-speciﬁc medium mark- phorylation and the activation of several intracellular sig- edly increased in a dose-independent manner following naling pathways, leading to EC migration, tube a period of 14 days (Figure 1(b)). Ang-1 treatment formation, sprouting and survival (Dumont et al. 2006). increased the bioactivity of cultured EPCs to form great Additionally, the increase in the number of CD133 posi- number of colonies in an early stage of current exper- tive cells orchestrated by Ang-2 peptide may correlate iment (Figure 2). Lee et al. (2008) previously showed an with the activation of the JAK2/STAT3 pathway, as increase in the number of bone marrow cell colonies recently reported in cancer stem cell-like phenotype derived from irradiated mice under Ang-1 treatment. It induction (Abubaker et al. 2014). We found that Ang-1 was also notiﬁed that exogenous Ang-1 could preserve but not Ang-2 stimulated EPC migration, which is con- the mix feature colony-forming capacity of hematopoie- sistent with the results previously described for mature tic stem cells by the Ang-1/Tie-2 signaling pathway (Arai ECs (Witzenbichler et al. 1998; Smadja et al. 2006). It et al. 2004; Jones and Dumont, 1998). Corroborating to has also been demonstrated that the Ang-1-mediated 128 V. SIAVASHI ET AL. Arai F, Hirao A, Ohmura M, Sato H, Matsuoka S, Takubo K, Ito K, pathway is involved in the differentiation of ECs from Koh GY, Suda T. 2004. Tie2/angiopoietin-1 signaling regu- EPCs (Hildbrand et al. 2004). Overall, from the clinical per- lates hematopoietic stem cell quiescence in the bone spective, it seems that Ang-1 may augment the angio- marrow niche. Cell. 118:149–161. genic activity of EPCs after implantation by stimulating Asahara T, Kawamoto A. 2004. Endothelial progenitor cells for migration and differentiation of progenitor cells into postnatal vasculogenesis. Am J Physiol Cell Physiol. 287: the endothelial lineage (Kobayashi et al. 2006). C572–C579. Brindle NP, Saharinen P, Alitalo K. 2006. Signaling and functions Angiopoietin-1 has powerful vascular protective of angiopoietin-1 in vascular protection. Circ Res. 98:1014– effects. Ang1 binds to and activates Tie2, then Ang1– Tie2 regulates signals for angiogenesis. The diversity of Chen JZ, Zhu JH, Wang XX, Zhu JH, Xie XD, Sun J. 2004. Effects downstream signaling molecules activated by Tie2 of homocysteine on number and activity of endothelial pro- depends on the presence or absence of cell–cell con- genitor cells from peripheral blood. J Mol Cell Cardiol. 36:233–239. tacts. Ang2, on the other hand, has similar afﬁnity to Chen YH, Lin SJ, Lin FY, Wu TC, Tsao CR, Huang PH, Liu PL, Chen Tie2 to compete with the binding of Ang1 to Tie2, YL, Chen JW. 2007. High glucose impairs early and late endo- thereby, resulting in inhibition of Ang1-mediated vascu- thelial progenitor cells by modifying nitric oxide-related but lar protection (Brindle et al. 2006; Fukuhara et al. 2009). not oxidative stress-mediated mechanisms. Diabetes. Our results also showed a superior tube formation 56:1559–1568. activity of EPC through Ang-1 induction. It seems that Cheng CC, Chang SJ, Chueh YN, Huang TS, Huang PH, Cheng SM, Tsai TN, Chen JW, Wang HW. 2013. Distinct angiogenesis Ang-1 induced and stabilized tube-like structures via roles and surface markers of early and late endothelial pro- EPC intracellular Tie2-FAK-AKT complex and subsequent genitor cells revealed by functional group analyses. BMC changes in pp38, pSAPK/JNK, and phosphorylated extra- Genom. 14:182. cellular signal–regulated kinases mediated Mitogen-acti- Dumont DJ, Bogdanovic E, Nguyen VP. 2006. Activation of Tie2 vated protein kinases activation (Moon et al. 2015). by angiopoietin-1 and angiopoietin-2 results in their release and receptor internalization. J Cell Sci. 119:3551–3560. Felcht M, Luck R, Schering A, Seidel P, Srivastava K, Hu J, Bartol A, Kienast Y, Vettel C, Loos EK. 2012. Angiopoietin-2 differen- Conclusion tially regulates angiogenesis through TIE2 and integrin sig- Taken together, our data showed that Ang-1 has an naling. J Clin Invest. 122:1991–2005. Friedrich EB, Walenta K, Scharlau J, Nickenig G, Werner N. 2006. augmentary effect on the angiogenic properties of − + + CD34 /CD133 /VEGFR-2 endothelial progenitor cell sub- EPCs through induction of maturation, clonogenicity, population with potent vasoregenerative capacities. Circ migration and upregulation of Tie-2 receptor in these Res. 98:e20–e25. progenitors. Fukuhara S, Sako K, Noda K, Nagao K, Miura K, Mochizuki N. 2009. Tie2 is tied at the cell–cell contacts and to extracellular matrix by angiopoietin-1. Exp Mol Med. 41:133–139. Acknowledgements Grigoras D, Pirtea L, Ceausu RA. 2014. Endothelial progenitor cells contribute to the development of ovarian carcinoma We extend our appreciation to Dr S. Ebrahimi and A.R. Ghovati tumor blood vessels. Oncol Lett. 7:1511–1514. for technical assistance. Hawighorst T, Skobe M, Streit M, Hong Y-K, Velasco P, Brown LF, Riccardi L, Lange-Asschenfeldt B, Detmar M. 2002. 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Animal Cells and Systems
– Taylor & Francis
Published: May 3, 2016
Keywords: Angiopoietin-1; angiopoietin-2; endothelial progenitor cells; angiogenic activity