Human Endothelial Colony Forming Cells From Adult Peripheral Blood Have Enhanced Sprouting Angiogenic Potential Through Up-regulating VEGFR2 Signaling Hyung Joon Joo, Sukhyun Song, Ha-Rim Seo, Jennifer H. Shin, SeungCheol Choi, Jae Hyoung Park, Cheol Woong Yu, Soon Jun Hong, Do-Sun Lim PII: DOI: Reference:

S0167-5273(15)01314-5 doi: 10.1016/j.ijcard.2015.06.013 IJCA 20669

To appear in:

International Journal of Cardiology

Received date: Revised date: Accepted date:

2 January 2015 3 May 2015 12 June 2015

Please cite this article as: Joo Hyung Joon, Song Sukhyun, Seo Ha-Rim, Shin Jennifer H., Choi Seung-Cheol, Park Jae Hyoung, Yu Cheol Woong, Hong Soon Jun, Lim Do-Sun, Human Endothelial Colony Forming Cells From Adult Peripheral Blood Have Enhanced Sprouting Angiogenic Potential Through Up-regulating VEGFR2 Signaling, International Journal of Cardiology (2015), doi: 10.1016/j.ijcard.2015.06.013

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ACCEPTED MANUSCRIPT ECFC as a sprouting angiogenic endothelial cell

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Human Endothelial Colony Forming Cells From Adult Peripheral Blood Have Enhanced

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Sprouting Angiogenic Potential Through Up-regulating VEGFR2 Signaling

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Hyung Joon Joo1, Sukhyun Song2, Ha-Rim Seo1, Jennifer H. Shin2, Seung-Cheol Choi1, Jae Hyoung

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Park1, Cheol Woong Yu1, Soon Jun Hong1, Do-Sun Lim1 1

Department of Cardiology, Cardiovascular Center, Korea University Anam Hospital, Seoul, Korea

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Department of Mechanical Engineering, KAIST, Daejeon, Korea

Running Title: ECFC as a sprouting angiogenic endothelial cell

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Corresponding author’s contact information: Dr. Do-Sun Lim

Department of Cardiology, Cardiovascular Center, Korea University Anam Hospital, 126-1, 5ka,

Republic of Korea

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Anam-dong, Sungbuk-ku, Seoul, 136-705

Phone: +82-2-920-5445; Fax: +82-2-927-1478

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E-mail:[email protected]

ACKNOWLEDGEMENTS

This research was supported by the Basic Science Research Program through the National Research

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Foundation of Korea (NRF) and funded by the Ministry of Education (2013R1A1A2005655). The authors thank Ji-Hyun Choi and Tae Yeon Kim for their technical assistance.

CONFLICTS OF INTEREST The authors declare that they have no conflict of interest.

Keywords: Endothelial progenitor cell; Angiogenesis; Vascular endothelial growth factor receptor; Microfluidic system

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ABSTRACT Background: Endothelial colony forming cells (ECFCs), a subtype of endothelial progenitor cells,

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have been studied as a promising cellular source for therapeutic angiogenesis. Although ECFCs are

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very similar to mature endothelial cells, details regarding the role of ECFCs during angiogenesis are

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not known. We compared the cellular and angiogenic properties of ECFCs and mature endothelial cells (HUVECs).

Methods: HUVECs was used as control. Quatitative RT-PCR, western blotting, immunofluorescence

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staining, flow cytometric analyses and angiogenic cytokine array were performed. 3D-microfluidic

was assessed by Matrigel plug assay.

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angiogenesis assay system was adopted for in vitro angiogenic potential. in vivo angiogenic potential

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Results: ECFCs had higher expression of activated endothelial tip cell markers (Dll4, CXCR4, CD34,

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and VCAM1) and arterial genes (DLL4 and CX40), but lower expression of venous and lymphatic genes (COUP-TFII and PROX1). In 3D-microfluidic angiogenesis assay system, ECFCs induced

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robust sprouting vascular structures. Co-cultivation of both ECFCs and HUVECs gave rise to lumenformed hybrid vascular structures, with the resulting ECFCs predominantly localized to the tip portion. This finding suggests that the ECFC has a role as a sprouting endothelial tip cell. Interestingly, VEGF-

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A phosphorylated VEGFR2 and its downstream signaling molecules more strongly in ECFCs than in HUVECs. ECFC induced sprouting angiogenesis even with small amounts of VEGF-A stimulation. Finally, co-administration of ECFCs and human dermal fibroblasts successfully induced lumenformed maturated neovessels in vivo. Conclusion: ECFCs derived from adult peripheral blood had enhanced sprouting angiogenic potential in vitro and in vivo through up-regulation of the VEGFR2 signaling pathway.

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INTRODUCTION

Cell therapy using various kinds of stem cells and/or progenitor cells is a novel, but

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challenging, strategy to achieve therapeutic neovascularization for many ischemic diseases, such as

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ischemic heart disease, peripheral occlusive disease, and diabetic ulcers[1]. Of the various cell types, endothelial progenitor cells (EPCs) have been extensively studied for vascular regeneration during the

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last few decades. Translational and clinical research has revealed that EPCs and EPC-enriched fractions directly and indirectly contribute to neovascularization and improve clinical outcomes in

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ischemic diseases[2, 3].

Recently, endothelial colony-forming cells (ECFCs), a subpopulation of circulating EPCs, have shown robust clonogenic proliferative potential and have displayed immunophenotypical

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characteristics similar to those of mature endothelial cells[4]. Although ECFCs form vessel-like structures and become incorporated into the pre-existing circulating system when they are implanted in vivo[5, 6], the detailed mechanisms of how ECFCs participate in angiogenesis and the cellular

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differences between ECFCs and mature endothelial cells are still largely unknown. Before ECFCs can

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be applied clinically for therapeutic purposes, it is essential to investigate their unique cellular

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properties related to angiogenesis and neovasculogenesis.

Moreover, there are several limitations to the therapeutic application of ECFCs. First, circulating ECFCs are extremely rare in peripheral blood and the ability to induce ECFCs from adult peripheral blood is very limited (less than one ECFC colony per 1 million peripheral blood

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mononuclear cells). Second, fetal bovine serum in culture medium should be replaced with serumalternatives of human origin for therapeutic ECFC transplantation. Recently, G-CSF and AMD3100 were reported to increase the mobilization of ECFCs into the blood stream and improve the ECFC induction rate[7]. A humanized protocol using pooled human platelet lysate for induction and expansion of ECFCs has been introduced[8].

Here, we developed an animal serum-free culture protocol for ECFC induction from adult peripheral blood using autologous serum. We attempted to address the phenotypic cellular characteristics of ECFCs as endothelial cells. The unique angiogenic properties of ECFCs were precisely analyzed using a novel 3D-microfluidic system. Lastly, we also characterized ECFCs derived in vivo formed neovessels. 3

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MATERIALS AND METHODS Ethical approval The study protocol was approved by the Institutional Review Board at Korea University

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Anam Hospital (IRB NO: ED12196). All procedures were performed in accordance with the Helsinki

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declaration and its later amendments. Informed consent was obtained from all individual participants included in the study. And, all procedures involving animals were also performed in accordance with

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the ethical standards of the institution after protocol approval.

Blood sampling

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Adult peripheral blood samples were obtained from 18 patients who underwent coronary angiography (13 men, 5 women; ages 35-77 years old). Blood sampling (23 ml) was performed before coronary angiography. Among those 18 patients, 9 patients had obvious coronary artery disease. Four

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patients had history of previous percutaneous coronary intervention and 5 patients had angiographically significant de novo coronary lesions of more than 50% diameter stenosis in major

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coronary arteries.

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Cell culture

ECFC induction was performed as described previously[9]. Briefly, adult peripheral blood

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was separated into an upper serum layer, a middle mononuclear cell (MNC) layer, and a bottom red blood cell layer after Ficoll-Paque (GE-Healthcare) density gradient centrifugation (Figure 1A, left). We carefully collected the serum and MNCs. After washing the collected MNCs twice, 8.0 x 10 6 MNCs were plated on the collagen (Stem Cell Technologies)-coated 12-well plates and cultured in

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EGM-2 (Lonza) supplemented with autologous serum instead of fetal bovine serum (FBS). The medium was changed daily for 7 days and then every other day. After ECFC induction, the medium was changed into EGM-2 containing 5% fetal bovine serum (FBS) for further experiments. HUVECs were also cultured in EGM-2 supplemented containing 5% FBS. All cells were used prior to passage 8.

Angiogenesis assay using 3D-microfluidic system To examine the angiogenic properties of ECFCs, we applied a microfluidic device that has previously been described[10]. Briefly, a polydimenthylsiloxane(PDMS)-based microfluidic platform containing three channels was fabricated with polydimethylsiloxane using a soft lithography process. Two empty spaces between three parallel channels were filled with a mixture of type I collagen (3 mg/ml, rat tail, BD Bioscience) and fibronectin (50 μg/ml, Invitrogen), which together acted as an ECM scaffold (colored in pink). The center channel was coated with fibronectin (10 μg/ml), and was 4

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seeded with 40-50 μl of cell suspension (1 x 107 cells/ml). The device was tilted vertically for 1 hr to allow the cells to attach to the sidewall of the scaffold by gravity. The cells were then seeded onto the other sidewall by flipping the device upside down. The EGM-2 medium containing the indicated

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concentrations of VEGF-A165 in the side channels was exchanged with fresh medium every 12 hr. Quantitative RT-PCR

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Total RNA was extracted from the cells using a RNeasy® Plus Mini Kit (Qiagen) according to the manufacturer’s instructions. 1 g of the RNA was reverse transcribed into cDNA using M-MLV reverse transcriptase (Invitrogen). Synthesized cDNA was applied for quantitative real-time PCR

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using iQTM SYBR® Green Supermix (Bio-Rad) and a MyiQTM2 real-time PCR detection system (BioRad) with the indicated primers (Supplemental table I). GAPDH was used as the reference gene and

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the results were presented as a relative expression to control using the ddCt method.

Immunofluorescence staining

Cells were fixed with 2% paraformaldehyde and blocked with 5% goat serum in PBST (0.1%

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TritonX-100 in PBS) for 20 minutes. The cells were incubated at 4C with the following primary

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antibodies: anti-human CD31 (clone WM59, BD Biosciences Pharmingen), anti-human CD144 (clone 55-7H1, BD Biosciences Pharmingen), anti-human von Willebrand Factor (vWF, Sigma), and anti-

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human CD34 (clone 563, BD Biosciences Pharmingen). After washing in PBST 3 times, the cells were incubated for 1 hr at room temperature with the following secondary antibodies: FITCconjugated anti-rabbit IgG (Biosciences), Alexa Fluor® 594-conjugated anti-mouse IgG (H+L) (Invitrogen), and Alexa Fluor® 488-conjugated anti-mouse IgG (H+L) (Invitrogen). Nuclei were

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stained with 4’,6-diamidino-2-phenylindole (DAPI, Invitrogen). The cells were then mounted in fluorescent mounting medium (DAKO Corporation). Immunofluorescent images were acquired using a TE-FM Epi-Fluorescence system attached to an Olympus Inverted Microscope (Olympus, Tokyo, Japan) or Zeiss LSM510 confocal fluorescence microscope (Carl Zeiss).

Western blotting Cell lysates were performed as previously described using the following antibodies: antiVEGFR2 antibody (#2479, Cell Signaling), and anti-pVEGFR2 antibody (#2478, Cell Signaling).

Flow cytometric analyses Cells were harvested using dissociation buffer (Invitrogen) and resuspended in HBSS/2% FBS at 1 x 106 cells per 100 l. The cells were incubated for 20 min with the following antibodies: 5

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phycoerythrin (PE)-conjugated anti-human CD31 (clone WM59, BD Biosciences Pharmingen), PEconjugated anti-human CD34 (clone 581, Biolegend), allophycocyanin (APC)-conjugated anti-human CD144 (clone BV9, Biolegend), PE-conjugated anti-human Tie2 (Clone 33.1 (Ab33), Biolegend), PE-

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conjugated anti-human Endoglin (clone 43A3, Biolegend), PE-conjugated anti-human Delta-like

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protein 4 (Dll4, clone MHD4-46, Biolegend), APC-conjugated anti-human Notch4 (clone MHN4-2, Biolegend), Alexa Fluor® 647-conjugated anti-human CD309 (VEGFR2, KDR, clone HKDR-1,

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Biolegend), APC-conjugated anti-human CXCR4 (clone 12G5, Biolegend), APC-conjugated antihuman CD106 (clone STA, Biolegend), PE-conjugated anti-human CD117 (clone YB5.B8, BD Biosciences Pharmingen), PE-conjugated anti-human CD133 (clone AC133, Miltenyi Biotech), Alexa

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Fluor® 647-conjugated anti-human STRO-1(clone STRO-1, Biolegend), anti-human CD90 (clone 5E10, BD Biosciences Pharmingen), biotin-conjugated anti-human CD140a (clone16A1, Biolegend), PE-conjugated anti-human CD140b (clone 18A2, Biolegend), FITC-conjugated anti-human CD45

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(clone 2D1, BD Biosciences Pharmingen), and FITC-conjugated anti-human Lineage Cocktail (CD3, CD14, CD16, CD19, CD20, CD56, clone UCHT1, HCD14, 3G8, HIB19, 2H7, HCD56, Biolegend). After washing in HBSS/2% FBS twice, the cells were incubated with the following secondary

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antibodies: FITC-conjugated streptavidin (Biolegend), PE-conjugated streptavidin (Biolegend), APC-

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conjugated streptavidin (Biolegend) and Alexa Fluor® 488-conjugated anti-mouse IgG (H+L) (Invitrogen). Analyses were performed using a FACS Caliber flow cytometer (Becton Dickinson).

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Dead cells were excluded by 7-aminoactinomycin D (7-AAD, Invitrogen). Data were analyzed using FlowJo software (Tree Star Inc.).

Matrigel tube forming assay

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Cells were plated on Matrigel-filled plates at a density of 2-4 x 104 cells/cm2, and were incubated in EGM-2 medium supplemented with or without VEGF-A165 (100ng/ml). After 12 hrs, the tube structures were observed using an Olympus Inverted Microscope (Olympus, Tokyo, Japan) attached to the TE-FM Epi-Fluorescence system. Phase-contrast images were taken using a C-5060 Olympus digital camera.

In vivo Matrigel plug assay Total one million cells (HUVEC + human dermal fibroblast [hDF, Lonza Inc., Rockland ME, USA][1:1 ratio], ECFC + hDF [1:1 ratio]) mixed with 100 l Matrigel supplemented with VEGF-A (500 ng/ml) were implanted subcutaneously into the dorsal side of eight-week-old nude-SCID mice (n = 3). After two weeks, the mice were sacrificed after injection of an anesthetic mixture (80 mg/kg 6

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ketamine and 12 mg/kg xylazine), and the implanted Matrigel was fixed by systemic vascular

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perfusion with 1% PFA, harvested, and whole-mounted for histologic analyses.

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Angiogenic cytokine array

The experiments were performed using the RayBio Human Angiogenesis Cytokine Antibody

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Array C Series 1000 kit (AAH-ANG-1000, RayBiotech Inc.). Cell lysates were prepared and processed according to the manufacturer’s instructions. HUVEC and ECFC cell lysates were put on the Array A and B membranes for 2 hours. After washing, array antibody and HRP-conjugated streptavidin were added to the membrane for 2 hours. Membranes were detected by 1X detection

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buffer C and D and the array was exposed to X-ray film. Densitometric quantification of blotting spots was performed using Quantity One software (Bio-Rad). Different membranes were normalized

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using positive controls.

Statistics

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Values presented are means  standard deviation (SD). Significant differences between the

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means were determined by analysis of variance followed by the Student-Newman-Keuls test.

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Significance was set at p < 0.05.

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RESULTS

ECFCs induced from adult human PBMC using autologous serum have typical endothelial cell-

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like characteristics

ECFCs can be induced from adult peripheral blood and umbilical cord blood[4, 9]. Although

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a faster and more productive induction of ECFC colonies has been demonstrated by using umbilical cord blood, adult peripheral blood is more feasible and acceptable for clinical use. Based on previous ECFC induction protocols using adult peripheral blood, we established a new ECFC induction

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protocol using autologous human serum (Figure 1A, right) and compared the ECFC induction rates of 2%, 5%, 10%, 20%, and 50% from autologous serum with that from 10% fetal bovine serum (Figure 1B). Five percent autologous serum in culture medium yielded a comparable ECFC induction rate to

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the 10% fetal bovine serum. The higher concentrations of autologous serum failed to increase the ECFC induction rate and allowed more monocyte/macrophage attachment to the culture plates (data not shown). Therefore, all ECFCs used in subsequent experiments were induced with 5% autologous

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serum. The ECFCs induced with autologous serum were first detected after 13.6  3.7 days (the

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earliest appearance was 9 days) (Supplemental table II).

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ECFCs were induced as cell colonies with cobblestone-like endothelial cell morphology (Figure 1C). Typical endothelial cell-specific markers were expressed by the ECFCs (Figure 1D); endothelial cell-specific adhesion molecules such as CD144 and CD31 were localized to the cell-cell junction. vWF was expressed as a granular pattern in the cytoplasm. CD34 was expressed in the

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cytoplasm of some ECFCs. ECFCs also successfully induced network formation as early as 12 hours on the Matrigel and were able to uptake acetylated LDL (Figure 1E). Network-formed tubular structures expressed CD144 in the cell-to-cell junction and vWF in the perinuclear region (Figure 1F). These data suggest that ECFCs induced by autologous serum also have very similar characteristics of typical endothelial cells and have the potential to form primary vascular plexus-like network in vitro.

Immunophenotypic and transcriptional comparison between ECFCs and HUVECs

To investigate the cellular characteristics of ECFCs, we performed phenotypic analyses of various markers and gene expression and compared these analyses with those of HUVECs, the most widely accepted mature endothelial cells. Flow cytometric analysis revealed that ECFCs expressed endothelial cell-specific markers (CD31, CD144, CD105, CD54, VEGFR2, Tie2, c-Met, Notch1, 8

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Notch 4) (Figure 2A, 2B, and Supplemental figure I). ECFCs rarely expressed hematopoietic markers (Lineage, CD45, CD117 or CD133), mural cell markers (CD140a or CD140b), or other mesenchymal cell markers (CD90 or STRO1). ECFCs showed much higher positivity for VECAM1 (14.3-fold),

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CXCR4 (2.2-fold), CD34 (4.6-fold), and Dll4 (20.1-fold) compared to HUVEC. Recently, Dll4,

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CXCR4, and CD34 have been revealed to be sprouting angiogenic tip cell markers[11-13]. VCAM1 has been known to be expressed in the TNF-a-induced activated endothelial cell and associated with

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its migration and angiogenesis[14]. Collectively, these data suggest that ECFCs could have similar cellular properties to the activated and sprouting endothelial tip cells.

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Next, we compared endothelial cell-specific gene expression between ECFCs and HUVECs (Figure 2C). ECFCs expressed more DLL4 (9.9-fold), HEY2 (23.1-fold), VEGFR3 (1.9-fold), CX40 (6.1-fold), EFNB2 (3.8-fold), and EPHB4 (8.3-fold), whereas they showed lower expression of HEY1

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(0.7-fold), HES1 (0.6-fold), TIE2 (0.6-fold), NRP1 (0.6-fold), NRP2 (0.4-fold), Prox1 (0.2-fold), LYVE1 (0.5-fold), and COUP-TFII (0.3-fold). HEY2 expression showed the most drastic differences compared to other DLL4 downstream molecules (HEY1 and HES1), suggesting that HEY2 could be

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the transcription factor responsible for higher expression of DLL4 in ECFCs. Interestingly, ECFCs

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showed higher expression of arterial endothelial cell genes (DLL4 and CX40) and lower expression of venous and lymphatic endothelial genes (venous; COUP-TF II, lymphatic; PROX1 and LYVE1).

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However, expression of other arterial and venous specific genes (NRP1, NRP2, EFNB2, EPHB4, and VEGFR3) was inconsistent and insufficient to support the identity of ECFCs as arterial endothelial cells. In fact, those genes were associated with other endothelial functions as well as having arterialvenous-lymphatic specifications. Considering that Notch-Dll, COUP-TFII, and Prox1 are the most

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powerful master regulators for endothelial cell fate specification[15, 16], our interpretation is that ECFCs are an unique endothelial cell type that is more similar to arterial endothelial cells than to venous or lymphatic endothelial cells.

ECFCs are induced as cell colonies from blood and their clonogenic character gives rise to questions about the cellular differences between each of the colonies. Fortunately, we successfully induced 9 separate ECFC colonies from a single peripheral blood sample and compared the DLL4, COUP-TFII, VWF, and PROX1 expression of each (Supplemental figure II). Each ECFC colony showed higher DLL4 and VWF expression and lower COUP-TFII and PROX1 expression compared to the HUVEC colonies. These data again suggest that ECFCs have similar characteristics to arterial endothelial cells. 9

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ECFC has stronger angiogenic potential in vitro compared to HUVEC

To explore the angiogenic potential of ECFCs, we adopted a novel 3D-microfluidic system

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(Supplemental figure III A). Briefly, ECFCs were plated onto the sidewall of an extracelluar matrix

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(ECM) scaffold in the center channel. Different VEGF-A concentrations in the culture medium of each channel provided a VEGF-A gradient in the ECM scaffold. ECFCs successfully induced

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sprouting vessel-like structures under a VEGF-A gradient and invaded into the ECM scaffold toward the higher concentration of VEGF-A (Supplemental figure III B). Immunofluorescence staining clearly showed CD144+ lumen-formed sprouting vascular structures (Figure 3A). We compared the

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angiogenic properties of ECFCs and HUVECs. Vascular densities were 1.7-fold higher in the ECFC group compared to the HUVEC group (Figure 3B and 3C). Sprouting filopodia in tip cells were also 1.4-fold more abundant in the ECFC group compared to the HUVEC group (Figure 3D and 3E). In

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addition, Z-stack images also clearly showed lumen-formed vascular structures induced from ECFCs (Figure 3F). When ECFCs and HUVECs were co-cultured after labeling, the induced vascular structures were almost hybrids consisting of both ECFCs and HUVECs; there were no clear areas or

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vascular structures consisting of a single cell type (Figure 3G). This suggests that angiogenesis could

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come from overall cell expansion of the stimulated endothelial cells rather than from clonal cell expansion. To investigate the localization of ECFCs during angiogenesis, ECFCs were labeled and co-

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cultured with unlabeled HUVECs. Interestingly, ECFCs were predominantly distributed in the tip region (Figure 3H and 3I), suggesting that ECFCs might prefer to be endothelial tip cells rather than stalk cells.

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ECFCs are more sensitive to VEGF-A stimulation than HUVECs

Sprouting angiogenesis is induced by VEGF-A stimulation. Therefore, we assumed that the response to VEGF-A stimulation could be different between ECFCs and HUVECs. In the 3Dmicrofluidic system, 20 ng/ml of VEGF-A successfully induced sprouting angiogenesis from ECFCs, whereas it failed to induce vascular sprouting from HUVECs (Figure 4A and 4B). This suggests that ECFCs could be more sensitive to VEGF-A stimulation.

There are 3 possible mechanisms for the higher VEGF-A sensitivity of ECFCs: (1) higher VEGFR2 expression in ECFCs, (2) higher endogenous VEGF-A expression in ECFCs, and (3) stronger phosphorylation of VEGFR2 in ECFCs. 10

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Quantitative RT-PCR and FACS analysis showed similar VEGFR2 expression between ECFCs and HUVECs (Figure 2A, 2C, 4C and Supplemental figure IV). Furthermore, quantitative RT-PCR failed to detect endogenous VEGF-A expression in both ECFCs and HUVECs (data not shown). Angiogenic

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cytokine array also showed no VEGF-A expression in both ECFCs and HUVECs (Figure 4D and 4E,

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Supplemental figure V). Interestingly, cell lysates revealed higher expressions of GRO, bFGF, IL-8, Ang2, and μPAR in ECFCs compared to HUVECs. Conditioned medium of ECFCs also contained

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higher levels of GRO, IL-8, Leptin, IFN-γ and µPAR compared to that of HUVECs. These data suggested that different expressions of angiogenic cytokines in ECFCs might contribute to their higher angiogenic potential. Moreover, we compared the phosphorylation of VEGFR2 between

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ECFCs and HUVECs in both a dose-dependent and a time-dependent manner (Figure 4F-4I). Western blotting demonstrated that a smaller dose of VEGF-A induced much stronger phosphorylation of VEGFR2 in ECFCs compared to HUVECs and its effects were maximized at 10 to 30 ng/ml of

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VEGF-A. However, the duration of VEGFR2 phosphorylation state was maintained similarly between ECFCs and HUVECs. These data suggested that the augmented phosphorylation of VEGFR2 might

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have roles in higher VEGF-A responsiveness of ECFCs.

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ECFCs successfully form the functional vessels in vivo

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To explore the in vivo neovasculogenic potential of ECFCs, Matrigel plugs with ECFCs or HUVECs were implanted into nude-SCID mice. However, both cell types failed to induce neovessels in vivo (data not shown). It appears that endothelial cells themselves might be insufficient to induce

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neovessels spontaneously in vivo. Previous studies have also demonstrated that cell-to-cell interactions, such as cell spheroids and mixing with other surrounding cells, might play important roles in in vivo neovascularization[17, 18].

We mixed ECFCs and HUVECs with human dermal fibroblasts (hDFs) in Matrigel and transplanted the Matrigel plug into nude-SCID mice (Figure 5A). The implanted Matrigel plug was harvested 2 weeks later. Interestingly, ECFCs with hDFs in the implanted Matrigel induced neovessels containing red blood cells, whereas, HUVECs with hDFs failed to induce neovessels (Figure 5B and 5C). Immunohistochemical and immunofluorescent staining clearly showed the implanted ECFCderived hCD31+ vessels in the Matrigel plug (Figure 5D (cross-section) and 5E (whole mount)). Noticeably, abundant sprouting filopodia were noted in the hCD31+ neovessels (Figure 5F) and some of the hCD31+ neovessels were connected to the recipient-derived mCD31+ blood vessels (Figure 11

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5G). In addition, most of the hCD31+ neovessels were covered with ɑSMA+ mural cells. In addition, some maturated neovessels clearly showed a vascular lumen-containing DAPI+ cell, suggesting a recipient hematopoietic cell (Figure 5H). Based on these observations, we concluded that ECFCs have

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strong neovasculogenic potential in vivo as well as in vitro.

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DISCUSSION

This study demonstrated that ECFCs are an endothelial cell-like cell population that

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originates from the blood. ECFCs express endothelial cell markers (CD34+/CD146+/CD31+/CD105+)

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and do not express hematopoietic (stem) cell markers (CD45-/CD133-)[4, 19]. ECFCs in this study did not express Lineage (including CD45) or CD133, but expressed CD31, CD144, and CD105.

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Previous studies have shown variable CD34 expression in both ECFCs and HUVECs[20, 21]. The CD34 positivity in ECFCs was 14.4 ± 11.35 % in this study. Another recent paper demonstrated variable CD34 expression in ECFCs derived from patients with acute coronary syndrome and the

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induction rate in this case was only 13%[22]. Interestingly, the ECFC induction rate correlates with aging and the severity of cardiovascular disease[23, 24]. Among 18 blood donors in this present study, 9 patients had angiographically significant coronary artery disease (CAD) (Supplemental table II).

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The average ages were 50.6 ± 9.23 year-old in non-CAD group and 56.4 ± 9.75 year-old in CAD group. There were no significant differences in ECFC colony forming rate, ECFC colony appearance day, and further expansion rate between CAD group and non-CAD group. Considering that feasible

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ECFC induction rates from healthy donors are approximately 80%[9], the ECFC induction protocol

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using autologous serum in this study would be quite reasonable.

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Previous studies, which compared immunophenotypic characteristics between ECFCs and HUVECs, produced conflicting results. Some studies demonstrated up-regulation of VEGFR2 expression between ECFCs and HUVECs[25, 26], whereas other studies showed no difference[20, 21]. The present study demonstrated no significant difference in VEGFR2 expression by FACS and

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quantitative RT-PCR. The expression of VECAM1, CXCR4, CD34, and Dll4 in ECFCs was upregulated compared to the expression in HUVECs. VECAM1 and CXCR4 expression in ECFCs has been reported to be upregulated compared to HUVECs[20, 22, 27]. Interestingly, those markers have been identified as arterial markers recently.

When we characterized the arterial-venous-lymphatic specifications of ECFC, qRT-PCR showed that higher expression of arterial markers (Dll4, Cx40) and lower expression of venous (COUP-TFII) and lymphatic (Prox1) markers were shown in ECFCs compared to HUVECs, suggesting that ECFCs may be more similar to arterial endothelial cells rather than to venous and lymphatic endothelial cells. However, expression of other markers such as NRPs and ephrins failed to show ECFC as arterial endothelial cell type specification. NRP1 (arterial marker) expression of ECFC was lower than that of HUVEC and EPHB4 (venous marker) expression of ECFC was higher than 13

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that of HUVEC. Recently, Boyer-Di Ponio J et al. also demonstrated that some venous marker gene expressions (COUP-TFII and SELP) of ECFC were similar to those of HAEC and other venous maker gene expressions (NRP2 and EPHB4) of ECFC were similar to those of HUVEC[28]. Similarly, some

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arterial marker gene expressions (PLGF, HES2, CD34, and CXCR4) of ECFC were similar to those of

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HUAEC and other arterial marker gene expressions (ANG2, EFNB2, JAG1, LMOD1, and HEY2) of ECFC were similar to those of HUVEC. In addition, high concentration of VEGF-A stimulation

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increased the arterial marker gene expression in ECFC. Therefore, ECFC might have a unique hierarchy or plasticity of endothelial cell.

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A major strength of this study was the detailed comparison of the angiogenic and neovasculogenic characteristics of ECFCs and HUVECs. Previously, Finkenzeller et al. demonstrated that ECFCs have similar angiogenic potential to HUVECs in 2D-matrigel assays and 3D-spheroid

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sprouting assays[29]. In contrast, Sieminski et al. and Smadja et al. demonstrated an enhanced microvascular network formation of ECFCs compared to HUVECs[26, 30]. The authors’ in vitro methods for angiogenesis only led to primitive sprouting and potential network formations, which

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were insufficient to confirm vascular angiogenesis or neovasculogenesis. Recently, a 3D-microfluidic

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system has been widely adopted for in vitro angiogenesis[10, 31, 32]. We adopted the 3D-microfluidic system to investigate the angiogenic potential of ECFCs compared to HUVECs. To the best of our

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knowledge, this study is the first to demonstrate in vitro ECFC-derived lumen-forming vascular structures, as well as hybrid vascular structures derived from ECFCs and mature endothelial cells in vitro. Moreover, we propose that ECFCs are unique, forming sprouting endothelial tip cells with

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abundant sprouting filopodia and a predominant localization within the sprouting tip region.

We propose that the VEGF-A hypersensitivity of ECFCs is the mechanism behind its enhanced angiogenic potential. We clearly demonstrate in this study that low dose VEGF-A stimulation significantly induced sprouting angiogenesis. As mentioned above, there has been some controversy surrounding VEGFR2 expression. Although there was no significant difference of VEGFR2 expression between ECFCs and HUVECs and no endogenous VEGF-A was detected in both the ECFCs and HUVECs in this study, a small dose of VEGF-A significantly phosphorylated VEGFR2 in ECFCs compared to HUVECs. Interestingly, western blotting showed that Notch signaling inhibition by DAPT (γ-secretase inhibitor) significantly inhibited the VEGFR2 phosphorylation in ECFCs compared to HUVECs, suggesting that Dll4-Notch signaling pathway might have roles in VEGF-A responsiveness of ECFCs (Supplemental figure VI). Previously, the cross-talk between Notch signaling and BMP signaling has been reported to contribute to the 14

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angiogenic sprouting process of endothelial cells[33]. Moreover, Smadja DM et al. demonstrated that ECFC expressed higher levels of BMP2 and BMP4 compared to other cell types (‘early endothelial progenitor cell (CFU-Hill)’ and embryonic stem cell), and that BMP4 rather than BMP2 enhanced

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ECFC angiogenesis in vitro and in vivo[34]. When we checked DLL4, BMP2 and BMP4 expressions

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in both ECFC and HUVEC, ECFC showed higher DLL4 (4.1-fold) and BMP4 (2.0-fold) expressions, but lower BMP2 (0.6-fold) expression compared to HUVEC (Supplemental figure VII). It might

contribute to the VEGF-A responsiveness of ECFCs.

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implicate that expression of BMP4 in ECFC could interplay with Dll4-Notch signaling pathway and

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On the other hand, the maintenance of VEGFR2 phosphorylation was similar between ECFCs and HUVECs. In the 3D-microfluidic system, there was no significant difference between ECFCs and HUVECs on vascular regression induced by VEGF-A depletion (Supplemental figure

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VIII).

The present study showed different angiogenic growth factor and cytokine expressions

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between ECFC and HUVEC. For the cell lysates, GRO, bFGF, IL-6, Ang2, and uPAR expressions

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were significantly higher in ECFC compared to HUVEC. For the conditioned media, levels of GRO, IL-8, Leptin, IFN-γ and µPAR in ECFC conditioned medium were higher compared to those in

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HUVEC conditioned medium. Previously, Liu Y et al. also reported that high concentration of angiogenin, angiopoietin-2, GRO, IL6, IL8, MCP1, MIP-3a, PDGF-BB, TIMP2 in ECFC conditioned medium[35]. Therefore, different angiogenic growth factor and cytokine expressions between ECFC and HUVEC could affect their angiogenic sprouting potential through paracrine or autocrine manner.

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Moreover, considering that the angiogenic sprouting responses of both ECFC and HUVEC were totally dependent to VEGF-A stimulation (angiogenic sprouting was not induced without VEGF-A stimulation), the angiogenic growth factor and cytokine expression in both ECFC and HUVEC could be modulated by VEGF-VEGFR2 signaling pathway, and contribute to their angiogenic potentials. For example, Marghri F et al. reported that VEGF-dependent angiogenesis was associated with the increased uPAR accumulation in ECFC caveolae[36].

In the present study, we demonstrated that only ECFCs, and not HUVECs induced in vivo neovessels when mixed with human dermal fibroblasts. The similar results were observed when mixed with other mesenchymal supporting cells including 10T1/2 cells (Supplemental figure IX). Our experiments clearly resulted in smooth muscle cell-covered, maturated, lumen-formed vascular structures that were derived from the implanted ECFCs. Previous studies have also demonstrated that 15

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ECFCs can induce neovessels in vivo when co-implanted with stromal cells (in this case, vascular smooth muscle cells)[18, 37]. ECFCs have been found to have comparable and perhaps even superior in vivo neovasculogenic potential compared to human microvascular endothelial cells and HUVECs.

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On the other hand, Finkenzeller et al. reported that ECFCs demonstrated inferior in vivo

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neovasculogenic potential compared to HUVECs[38]. In this study, the authors implanted the ECFC and HUVEC spheroids without other stromal cells into immunodeficient mice and demonstrated a

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decreased number of in vivo neovessels in ECFC spheroids compared to HUVEC spheroids. These conflicting results between various studies could have originated from cellular differences in the neovasculogenic mechanisms between ECFCs and mature endothelial cells. While a detailed study of

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the in vivo differences between ECFCs and other mature endothelial cells must be further investigated, we postulated that the VEGF-A hypersensitivity of ECFCs could contribute to its neovasculogenic

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and angiogenic potential in vivo.

In summary, ECFCs demonstrated distinct endothelial cell phenotypes. DLL4, VCAM1, CXCR4, and CD34 expression were higher in ECFCs compared to HUVECs. In 3D-microfluidic

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system, ECFCs dominated the tip region during angiogenic sprouting process when co-cultured with

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HUVECs, and induced more robust angiogenic sprouts with abundant filopodia compared to HUVECs. VEGFR2 phosphorylation and angiogenic sprouts induction on low-dose VEGF-A

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stimulation in ECFCs (VEGF-A hypersensitivity) might contribute to their stronger angiogenic potential. Interestingly, attenuation of VEGFR2 phosphorylation by DAPT in ECFCs suggested the involvement of Dll4-Notch signaling pathway on ECFC angiogenic potential. Finally, the coadministration of ECFCs and dermal fibroblasts successfully induced profound in vivo

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neovascularization.

STUDY LIMITATION In the present study, ECFCs were induced and expanded successfully in 11 donors from total 18 donors. Each donor had different cardiovascular risk factors. Cellular differences of ECFCs derived from the donors with different medical conditions should be further explored for their clinical applications in the future.

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FIGURE LEGENDS Figure 1. ECFCs were successfully induced from adult peripheral blood using autologous serum

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and revealed similar cellular characteristics to mature endothelial cells. (A) ECFC induction

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scheme from adult peripheral blood using autologous serum. (B) Dose dependency of autologous serum on the number of the induced ECFC colonies at day 14. n = 3 (C) Phase contrast images of an

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ECFC colony induced from adult peripheral blood using autologous serum at day 13. Bars = 100 μm. (D) Immunofluorescence images showing CD31+, CD144+, vWF+, CD34+ ECFCs. Bars = 50 μm. Asterisks indicate CD34+ cell. (E) Network formation of ECFCs with Alexa594-conjugated acLDL uptake after 24 hrs of culture in Matrigel. Bars = 500 μm. (F) Cord-like sprouting structure of

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CD144+/vWF+ ECFCs in Matrigel. Bars = 100 μm.

Figure 2. Phenotypic comparison of surface markers and gene expression between ECFCs and

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HUVECs. (A) Percent positivity of each cell surface marker from ECFCs and HUVECs. n = 7. *p < 0.05 vs. HUVEC. (B) Representative FACS analyses of Dll4, VECAM1, CD34, CXCR4, Notch1, and Notch4 in ECFCs and HUVECs. (C) Relative changes of endothelial cell-related gene expression in

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ECFCs compared to HUVECs. n = 7. *p < 0.05 Figure 3. Characterization of sprouting angiogenic potential between ECFCs and HUVECs. To evaluate the sprouting angiogenic properties of ECFCs and HUVECs, a 3D-microfluidic angiogenesis

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assay system was applied. Analyses were performed at day 6. Bar = 100 μm. (A) Phase contrast and immunofluorescence images showing the angiogenic sprouting of ECFCs into the ECM scaffold. The ECM scaffold provided a gradient of VEGF-A (20 to 50 μM). CD144 (green) and F-actin (red) were

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immunostained. (B, D, and F) Immunofluorescence images of F-actin-stained (red) sprouting vascular structures induced from ECFCs and HUVECs. Arrowheads indicate sprouting filopodia in the tip cell. Asterisks indicate vascular lumens formed in the ECM scaffold. (C) Vascular density (% area) of sprouting vascular structures. n = 4. *p < 0.05 (E) Number of sprouting filopodia in tip cells. n = 4. *p < 0.05 (G) Phase contrast and immunofluorescence images of sprouting vascular structure after coculture of Alexa488 (green) labeled ECFCs and Alexa594 (red) labeled HUVECs. Arrowheads indicate lumen-forming ECFCs in hybrid vascular structures. (H) Immunofluorescence images of Alexa594-labeled (red) ECFCs in F-actin-stained (green) sprouting vascular structures after co-culture of ECFCs and HUVECs. (I) Distribution of ECFC in the sprouting vascular structures induced from co-culture of ECFCs and HUVECs. n = 4. *p < 0.05 vs. Stalk. Figure 4. ECFCs induced stronger VEGFR2 phosphorylation and sprouting angiogenesis under a small dose of VEGF-A stimulation than HUVECs. (A) Immunofluorescence images of sprouting 21

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vascular structures induced from ECFCs and HUVECs at day 6 under different VEGF-A concentration gradients. F-actin (red) was immunostained. Bars = 100 μm. (B) Vascular density (% area) of sprouting vascular structures under different VEGF-A concentration gradients. n = 4. *p