Material and Methods

Material and methods Cell Culture ADSC were isolated from adipose. Inguinal adipose tissue was excised from C57Bl/6 mice and digested at 37°C in Hanks buffer containing 2% BSA and 2 mg/ml collagenase (Worthington, Lakewood, NJ) for 60 min. After elimination of undigested fragments by filtration through a 40-m cell strainer (BD Falcon, Bedford, MA), mature adipocytes were separated by centrifugation (280g, 10 minutes) and incubated in hemolysis buffer (160 mmol/L NH4Cl, 14 mmol/L NaHCO3, 0.1 mmol/L EDTA, pH 7.3) for 5 minutes. After centrifugation (280g, 10 minutes), ADSC were plated in DMEM medium containing 10% fetal bovine serum (FBS). We also obtained human subcutaneous adipose tissue and cultured ADSC as described above. Mouse bone marrow cells were obtained by flushing the tibias and femurs. Low-density mononuclear cells were isolated by density gradient centrifugation with Ficoll, and injected into the ischemic limb of mice as previously described. 14 This protocol was approved by the Osaka University Graduate School of Medicine Standing Committee on Ethics. For differentiation ADSC into EC, ADSC were plated on culture dishes with or without a collagen type IV coating (Becton Dickinson Biocoat Cell Culture Dishes) and maintained in EBM-2 (EC basal medium-2, Clonetics Corp. San Diego, CA) medium or modified EGM2-MV, consisting of EBM-2 supplemented with 5% FBS and 10 ng/ml human VEGF-A, 10 ng/ml human fibroblast growth factor-2, and 10 ng/ml human epidermal growth factor.

Cell viability and migration

ADSC (2 x 104 cells) and HAEC (5 x 104 cells) were co-cultured in 24 well culture dished using a culture insert (1.0 M pore size; BD Biosciences, Franklin Lakes,

NJ) in EBM-2 or EGM2-MV medium supplemented with 0.5% FCS. After 48 hours of co-culture, cell viability of HAEC was measured by MTS [3-(4,5-dimethylthiazol-2-yl)-

5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium] assay (CellTiter 96, Promega, Madison, WI) as previously described 18. CellTiter 96 One Solution Reagent was added to each well for 3 hours, and light absorbance at 490 nm was detected by a plate reader.

Migration of HAEC was estimated in a modified Boyden chamber as previously described 19. In brief, polyninylpyrolidone (PPV)-free polycarbonate filters with 8-m pores were coated with 0.1% gelatin overnight and washed with phosphate- buffered saline to remove the excess coating. After 27 L conditioned EGM2-MV or

EBM-2 medium containing 0.5% FBS from HAEC or ADSC was placed in the lower chamber, the filter was positioned above the wells of the lower chamber, and then 106 cells/ml suspended in 50 L medium containing 0.5% FBS were added to the upper chamber. These HAEC were pretreated with a co-culture system that is used to maintain cell viability as described above for 16 hours. The apparatus was incubated at 37°C for

4 hours. After incubation, the filter was removed and the upper surface of the filter was scraped off. The filter was fixed with methanol and stained with Diff-Quik (Sysmex

Corporation, Kobe). The number of cells was counted in 4 randomly chosen fields and experiments were performed in triplicate.

Tube Formation Assay An angiogenesis kit (Kurabo, Tokyo, Japan) was used for tube formation assay as described by the manufacturer. Briefly, human endothelial cells and fibroblasts were cultured in 24-well plates and treated daily with conditioned medium from 1) EC in

EGM2-MV medium (0.5% FCS), 2) ADSC in EBM-2 (0.5% FCS), or 3) ADSC in

EGM2-MV medium (0.5% FCS). After treatment for 4 days, the cells were stained with anti-human CD31 (PECAM) monoclonal antibody as described above. Tube-like structures consisting of stained cells were analyzed with respect to four parameters: area, length, joint and path, using an Angiogenesis Image Analyzer (Kurabo, Tokyo).

Reverse Transcription-Polymerase Chain Reaction (RT-PCR) Analysis

ADSC were cultured in collagen type IV coated dish (Corning) with EGM2-

MV or EBM-2 medium containing 5% FBS, and differentiated ADSC into EC were collected with anti-mice PECAM antibody-attached Dynabeads (Dynal Biotech, Oslo,

Norway). Total RNA from ADSC was isolated using Isogen (Wako, Osaka). For the

PCR reaction, first-strand cDNA (the equivalent of 50 ng reverse-transcribed RNA) was amplified in a final volume of 20 μl with 0.5 U LA-Taq (Takara, Otsu) and 20 pmol of each nucleotide primer. The conditions of the PCR reaction and design of oligonucleotide primers followed a previous report 20 with minor modification, as shown in table1

For quantitative real time PCR analysis, mRNA of human ADSC and aortic vascular smooth muscle cells was extracted using the RNeasy Mini kit (QIAGEN,

Valencia, CA). Complementary DNA was synthesized using the Thermo Script RT-PCR

System (Invitrogen, Carlsbad, CA). Relative gene copy numbers of hepatocyte growth factor (HGF: Hs00300159), vascular endothelial growth factor (VEGF: Hs00173626), fibroblast growth factor-2 (FGF-2 : Hs00266645), angiopoietin-1 (Ang-1:

Hs00375822), angiopoietin-2 (ANGPT-2: Hs00169867), placental growth factor (PGF:

Hs00182176), transforming growth factor- (TGF-: Hs00171257), and GAPDH

(Hs99999905) were quantified by real-time qRT-PCR using TaqMan Gene Expression

Assays (Applied Biosystems, Foster City, CA). The absolute number of gene copies was standardized by a sample standard curve. Results are expressed as fold-increase relative to the GAPDH for copy numbers of each mRNA.

Measurement of VEGF and HGF To document the secretion of VEGF or HGF by human ADSC and HAEC, we used an enzyme-linked immunoassay (VEGF: R&D System, Minneapolis, MN or HGF:

Institute of Immunology Co., Ltd., Tokyo) exactly as described by the manufacturer.

HAEC or ADSC were plated on culture dishes with or without a collagen type I or IV coating (Corning, ) and maintained in EBM-2 (EC basal medium-2, Clonetics Corp. San

Diego, CA) medium or modified EGM2-MV, consisting of EBM-2 supplemented with

5% FBS and 10 ng/ml human VEGF-A (omitted from culture medium for quantification of VEGF by EIA assay), 10 ng/ml human fibroblast growth factor-2, and 10 ng/ml human epidermal growth factor. The results were compared with a standard curve constructed with recombinant VEGF or HGF, and absorbance was measured at 450 nm or 490 nm by a plate reader, respectively.

Inhibition of VEGF and HGF

In vitro culture experiments, inhibition of VEGF or HGF activity in human

ADSC and EC was examined by means of VEGF neutralizing rabbit polyclonal IgG which cross-reacts with human and murine VEGF (Neomarkers Co., Fremont, CA) and

HGF neutralizing goat polyclonal IgG which only cross-reacts with human HGF (R&D

Systems, Minneapolis, MN)20 For the antibody, the IgG fraction at a concentration of 10

g/ml was able to neutralize a biological activity of 100 ng/ml HGF or VEGF. Normal goat or rabbit serum IgG fraction (10 g/ml) was employed as a control. These neutralizing antibodies or control were added to EGM2-MV medium, and MTS assay or migration assay was performed as described in method section.

Mouse hindlimb ischemic model and evaluation of angiogenesis

Wild-type C57Bl/6J mice (8 weeks old, male) were anesthetized with ketamine chloride (80 mg/kg) and xyladine sulfate (8 mg/kg) subcutaneously, and unilateral hindlimb ischemia was induced as described previously 22. Briefly, the entire left saphenous artery and external iliac artery with deep demoral and circumflex arteries were ligated, cut, and excised to set up a murine model of severe hind limb ischemia. At

10 days after surgery (day 10), all mice were evaluated by Laser Doppler Image (LDI:

Moor Instruments, Devon, United Kingdom), selected them in the range from 0.3 to 0.6 which are perfusion ratio of ischemic hindlimb to untreated opposite limbs, and separated into three groups, and ADSC (1 x 106 cells per body) or saline was carefully injected into the mouse ischemic limb with a 26-gauge needle. Three separate injections of cells locally (intramuscularly into the ischemic limb, near both the proximal and distal arterial stumps) were performed. We also prepared human HGF plasmid, cDNA

(2.2 kb) was inserted into a simple eukaryotic expression plasmid that uses the cytomegalovirus (CMV) promoter/enhancer. "Naked" HGF vector (200 µg/100ul per animal) was injected directly into the ischemic limb of mice as previous described. 22 All experimental protocols were approved by the Osaka University Graduate School of

Medicine Standing Committee on Animals.

Blood flow by laser Doppler imaging (LDI: Moor Instruments, Devon, United

Kingdom) was performed as described previously 23 before and on post-treatment days

14 and 28, because laser Doppler flow velocity correlates well with capillary density.

Consecutive measurements were obtained over the same regions of interest (leg and foot). Low or no perfusion was displayed as dark blue, whereas the highest perfusion was displayed as white. To avoid data variations due to ambient light and temperature changes, hindlimb blood flow was expressed as the ratio of left (ischemic) to right (non- ischemic) laser Doppler blood flow. In all experiments, investigators performing the follow-up examinations were blinded to the identity of the treatment administered.

Capillary density within the ischemic thigh adductor skeletal muscles was analyzed to obtain specific evidence of vascularity at the level of the microcirculation.

After fixation in cold acetone (-20°C for 15 minutes), capillary endothelial cells (EC) were identified by immunohistochemical staining with anti-mouse PECAM mAb

(Pharmingen, San Diego, CA). These frozen sections were also examined by fluoromicroscopy to detect GFP cells, or stained with hematoxylin and eosin (HE) for overall morphological observation. These samples are also immunostained with anti- mice PECAM (1:100 dilution), -smooth muscle actin (1:400 dilution, SIGMA, Saint

Louis, MO), von Willebrand factor (1:200 dilution, DAKO) or GFP antibody (1:200 dilution, Molecular Probes, Inc., Eugene, OR) after fixation with aceton.

Figure legends: Figure I. Murine ADSC differentiated toward EC in growth factor-rich EGM2- MV medium. Upper panels are representative phase contrast microscopic views (a: x40, b&c: x100 magnification) showing spindle-shaped ADSC. Lower panels show immunostaining with PECAM (a&b) or VE-cadherin (c).

Figure II. Secretion of VEGF and HGF from human ADSC. VEGF and HGF concentrations in conditioned medium from HAEC or ADSC are shown. N = 4 per group calculated from 4 independent experiments. “EBM (ADSC)” indicates conditioned medium from ADSC in EBM-2 medium (without growth factor), “EGM (ADSC)” indicates conditioned medium from ADSC in EGM2-MV medium (with growth factor), “EGM (ADSC) Col 1 dish” indicates conditioned medium from ADSC in EGM2-MV medium in collagen type 1-coated dish, “EGM (ADSC) Col 4 dish” indicates conditioned medium from ADSC in EGM2-MV medium in collagen-type 4 coated dish, “EGM (EC)” indicates conditioned medium from HAEC in EGM2-MV medium in collagen type 1-coated dish, *P<0.01 vs. EGM (EC), †P<0.01 vs. DMEM (ADSC).

Figure III. A) Capillary density in cross sections of ischemic tissue immunostained with anti-CD31 (PECAM) antibody (brown). A representative picture and quantitative analysis are shown. “Control” indicates PBS injection, “ADSC (EBM)” indicates ADSC maintained in EBM-2 medium (without growth factor), “ADSC (EGM)” indicates ADSC maintained in EGM2-MV medium (with growth factor) Bar = 100 m. B) Quantitative analysis of peripheral blood flow analyzed by LDI at 4 weeks after injection that is expressed as perfusion ratio of ischemic hindlimb to untreated opposite limb. Table . PCR reaction conditions and design of oligonucleotide primers. Primer Size Ann Cycles Ref. Tie-2 F: 5'-ATGTGGAAGTCGAGAGGCGAT-3' 277 55℃ 35 21 R:5'-CGAATAGCCATCCACTATTGTCC-3' bp 1 cycles min Flk-1 F : 5'-TCTGTGGTTCTGCGTGGAGA-3' 269 53℃ 35 21 R : 5'-GTATCATTTCCAACCACCCT-3' bp 1 cycles min PECAM F : 5'-GTCATGGCCATGGTCGAGTA-3' 260 57℃ 30 21 R : 5'-CTCCTCGGCGATCTTGCTGAA-3' bp 1 cycles min G3PDH F : 5'-ACCACAGTCCATGCCATCAC-3' 145 55℃ 30 21 R : 5'-TCCACCACCCTGTTGCTGTA-3' bp 1 cycles min

F: forward primer, R: reverse primer, Ann: annealing temperature