Primary antibodies used for immunoblot and immunofluorescence analysis were goat anti-human VEGFR1 (AF321), rabbit anti-human phospho-VEGFR1 (Y1213) (AF4170), and goat anti-human hairy enhancer of split-1 (HES-1) (AF3317), all from R&D Systems. Mouse anti-human von Willebrand factor (vWF) (3H3126) (sc-73267), mouse anti-human vascular cell adhesion molecule-1 (VCAM-1) (P3C4) (sc-20070), mouse anti-human phosphotyrosine (PY99) (sc-7020), rabbit anti-human smoothelin B (sc-28562), rabbit anti-human Notch 3 (m-134) (sc-5593), and goat anti-human Notch 4 (E-12) (sc-32613) were all from Santa-Cruz Biotechnology. Rabbit anti-human Notch 1 (C37C7), rabbit anti-human Notch 2 (8A1), mouse anti-human platelet/endothelial cell adhesion molecule-1 (PECAM-1) (89C2) (3528), rabbit anti-human vascular endothelial cadherin (VE-cadherin) (2158) and rabbit anti-human VEGFR2 (55B11) (2479) were all from Cell Signaling Technology. Mouse anti-human smooth muscle alpha actin (αSMA) (clone 1A4), mouse anti-human calponin (clone Calp), mouse anti-human smooth muscle myosin heavy chain 1 (SM-MHC-1) (clone SMMS-1), and negative control mouse IgG1 (X093101) were purchased from DAKO.
Growth factors and inhibitors
VEGF-A165 (298-VS) and VEGF neutralising monoclonal antibody (clone 26503) were obtained from R&D Systems. VEGFR tyrosine kinase inhibitor (KRN633) was purchased from Calbiochem. The gamma secretase inhibitor N-[N-(3,5-difluorophenacetyl-L-alanyl)]-S-phenylglycine t-butyl ester (DAPT) was obtained from Sigma-Aldrich.
Human bone marrow stromal cells were obtained from the bone marrow of a 28-year-old female and from a 21-year-old male (Lonza). The HBMSCs had a typical spindle-shaped morphology (Supplementary Fig. S1A), were positive for CD73, CD29, CD44, CD51, and CD105, but negative for the haematopoietic cell marker CD34 (Supplementary Fig. S1B), and had the potential to differentiate toward adipocyte, osteocyte, or chondrocyte lineages (Supplementary Fig. S1C). HBMSCs cultured on 0.1% gelatin (Sigma-Aldrich) were maintained in basal medium (Invitrogen) supplemented with 2% L-glutamine and 0.1% penicillin/streptomycin. HBMSCs were cultured at 37 °C in a humidified atmosphere of 20% O2, 5% CO2, and used at passage 4–5. Human umbilical vein endothelial cells from 43-year-old and 29-year-old females (Invitrogen) were maintained in endothelial cell basal medium (Lonza), containing singleQuot supplements, and used at passage 4. Human dermal fibroblasts (HDFs) from a 51-year-old male, obtained from the European Collection of Cell Cultures, were maintained in Dulbecco's minimal essential medium (DMEM) (Invitrogen) containing 10% fetal bovine serum, 1% L-glutamine, 0.2% penicillin/ streptomycin, and used at passage 8.
For standard culture conditions, cells were seeded at 70 000 cells in 9.6 cm2 at initial plating (corresponding to 70% confluence). For high cell density experiments, HBMSCs were seeded at 100 000 cells in 9.6 cm2 at initial plating. For low density experiments, HBMSCs were seeded at 10 000 cells in 9.6 cm2 at initial plating.
Semiquantitative and quantitative reverse-transcription polymerase chain reaction
Total RNA was isolated from cultured cells using Trizol reagent. Total RNA (1 μg) was reverse-transcribed and subjected to polymerase chain reaction (PCR). Quantitative PCR was performed using a SYBR green quantitative PCR core kit (Eurogentec), with each sample run in triplicate. Data were normalized to GAPDH, and relative expression was calculated according to the 2-[delta]CT formula. Oligonucleotide primers for PCR were designed using Primer3 software. Each primer pair was designed using the same parameters (70- to 100-bp product size and Tm of 60 °C). PCR were repeated using a second primer set (sequences not shown). Reaction products were resolved using 2.5% ultrapure agarose (Invitrogen) gel electrophoresis run at 100 V for 2 h in 1X TAE (Tris base, acetic acid, EDTA) buffer containing 2.5 μl Gel Red (Biotium), and visualised using a transilluminator.
Primer sequences were as follows: GAPDH (71 bp), forward (5′-AAGGGCATCCTGGGCTAC-3′) and reverse (5′-GTGGAGGAGTGGGTGTCG-3′); VEGFR1 (99 bp), forward (5′-GCGACGTGTGGTCTTACG-3′) and reverse (5′-GGCGACTGCAAAAGTCCT-3′); VEGFR-2 (81 bp), forward (5′-CATCCAGTGGGCTGATGA-3′) and reverse (5′-TGCCACTTCCAAAAGCAA–3′); VEGFR-3 (87 bp), forward (5′-GATGCGGGACCGTATCTG-3′) and reverse (5′-ATCCTCGGAGCCTTCCAC-3′); HES1 (91 bp), forward (5′-CCAAAGACAGCATCTGAGCA-3′) and reverse (5′-TCAGCTGGCTCAGACTTTCA-3′); VEGF-A (98 bp), forward (5′-CACCCATGGCAGAAGGAG-3′) and reverse (5′-CACCAGGGTCTCGATTGG-3′); VE-cadherin (74 bp), forward (5′-GGAGCCGAGCATGTGTCT-3′) and reverse (5′-TCTGCAAGGTGTGCCTGA-3′).
Cells were washed with phosphate-buffered saline (PBS), incubated with lysis buffer for 15 min on ice, and then isolated. Total protein lysates were quantitated using a bicinchoninic acid (BCA) assay (Pierce), electrophoresed under reducing conditions using NuPAGE 4–12% Bis-Tris gels at 200 V for 2 h, and then transferred to a nitrocellulose membrane using a NuPAGE Western transfer system (Invitrogen). Membranes were blocked in 4% milk solids and then incubated with the primary antibody overnight at 4 °C (1:500–1:1000 dilution). Membranes were incubated with horseradish peroxidise (HRP)-conjugated secondary antibodies (DAKO, UK) for 2 h and then developed with SuperSignal West Dura Extended Duration Chemiluminescence (Thermo Fisher Scientific). Proteins were visualised using KODAK X-AR film and density of bands was determined using Gene Tools software (Syngene), and then the corresponding loading control was normalised.
After culturing cells on gelatin-coated 1.7 cm2 coverslips, cells were washed with PBS and fixed in 4% paraformaldehyde for 20 min, quenched in 0.2 M glycine (Fisher Scientific) for 20 min, and then permeabilised in 0.5% Triton X-100 (Sigma-Aldrich) for 4 min. Nonspecific binding was blocked with 2% fish skin gelatin solution (Sigma-Aldrich) for 1 h, and then cells were incubated overnight at 4 °C with primary antibodies (1:50–1:100 dilution) in blocking solution. After washing with PBS, cells were incubated with Alexafluor secondary antibodies (Alexa-Fluor 488 conjugated or Alexa-Fluor 546/555 conjugated (Invitrogen) (1:200 dilution) for 2 h at room temperature. For F-actin staining, cells were also incubated with rhodamine-conjugated phalloidin (Invitrogen) (1:500 dilution) for 2 h at room temperature. Cells were then washed and coverslips mounted using Prolong Gold Antifade solution containing 4′,6-diamidino-2-phenylindole (DAPI) (Invitrogen). At least 20 representative images of each sample were captured for each analysis. All immunofluorescence experiments included no primary antibody, no secondary antibody, and isotype-specific IgG1 controls to confirm specificity (data not shown).
For fluorescence microscopy, images were collected on an Olympus Widefield BX51 upright microscope using a 20x/UPlanFLN objective and captured using a Coolsnap HQ camera (Photometrics) through MetaVue Software (Molecular Devices). Specific band pass filter sets for DAPI, FITC, and tetramethylrhodaminoisothiocyanate (TRITC) were used to prevent bleed-through from one channel to the next. For confocal microscopy, images were collected using a Nikon C1 confocal on an upright 90i microscope with a 60x/1.40 Plan Apo objective and 3x confocal zoom. Images for DAPI, FITC, and Texas red were excited with the 405, 488, and 543 nm laser lines, respectively. Images were then processed and analysed using Nikon EZ-C1 FreeViewer v3.3 software. For Delta Vision microscopy, a Delta Vision restoration microscope (Applied Precision) 40x/0.85 UPlan Apo objective and Coolsnap (Photometrics) camera was utilised. Raw images were deconvolved using the Softworx software; maximum intensity projections of these deconvolved images are shown.
Following trypsinisation, cells were left to recover in growth medium for 1 h, washed twice in wash buffer (0.5% bovine serum albumin in PBS) and 1 × 105 cells incubated with either phycoerythrin (PE) conjugated or unconjugated primary antibody for 1 h, and then washed three times with wash buffer. For an unconjugated antibody, cells were additionally incubated with a species-specific fluorescein isothiocyanate (FITC)-conjugated secondary antibody (1:200) (Dako) for 45 min and then washed three times with wash buffer. For each analysis, 100 000 cells were counted using a FACscan cytometer at a flow rate less than 200 events per second.
Enzyme-linked immunosorbent (ELISA) assays were performed using the Quantikine human VEGF-A immunoassay (R&D Systems), according to the manufacturer's protocol.
Notch was activated either by ethylenediaminetetraacetic acid (EDTA) (Rand et al., 2000
) or recombinant Jagged-1. For EDTA experiments, cells were stimulated for 15 min with 5 mM EDTA, washed twice with basal medium, and left to recover for 24 h, before isolation. For Notch activation using Jagged-1, recombinant human Jagged-1/Fc chimera (1277-JG) (R&D Systems) was immobilised on culture plates by incubating plates with a solution of Jagged-1 (5 μg/ml) in PBS for 2 h at 37 °C. Control plates did not contain recombinant human Jagged-1. For these experiments, HBMSCs were seeded onto plates at low or high density and cultured for 24 h.
HBMSC differentiation assay
HBMSCs were induced to differentiate toward adipocyte, osteocyte, or chondrocyte lineages using an MSC functional identification kit (R&D Systems), according to the manufacturer's protocol, and then cells were prepared for immunofluorescence analysis as previously described using the antibodies provided. In addition, lipid droplets were stained using boron-dipyrromethene (Bodipy) 493/503 (Invitrogen) and F-actin filaments stained using phalloidin.
siRNA knockdown transfections
HBMSCs (5 × 105 cells), together with 3 μg small interfering RNAs (siRNAs), were transfected by electroporation using a human Nucleofector kit (Lonza), and cultured overnight in growth medium. Validated siRNAs, which were functionally tested to provide ≥ 70% target gene knockdown, were used for VEGF-A knockdown (Qiagen). For Notch knockdowns, siRNAs for Notch1 (sc-36095), Notch2 (sc-40135), and Notch3 (sc-37135) (Santa Cruz, USA) were employed, with scrambled siRNA as a control (Qiagen).
Cell lysates were precleared using 10% (w/v) protein A-Sepharose (GE Healthcare), and then precleared lysates were incubated with primary antibody overnight at 4 °C. Immune complexes were isolated by incubation with 10% (w/v) protein A-Sepharose for 2 h at 4 °C. Protein A-Sepharose beads were washed three times using ice-cold lysis buffer, and then resuspended in NuPAGE gel loading buffer (Invitrogen).
Endothelial functional tests
LDL uptake assay
ECs readily take up 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine-labeled acetylated low density lipoprotein (Dil-Ac-LDL), where the lipoprotein is degraded by lysosomes and fluorescent Dil accumulates within intracellular membranes. Cells were incubated with 10 μg/ml Dil-Ac-LDL (BT-906) (Biomedical Technologies Ltd) for 3 h at 37 °C, washed with PBS, fixed in 4% paraformaldehyde for 20 min, and then quenched in 0.2 M glycine for 20 min. Uptake of DiI-ac-LDL was assessed by mounting coverslips using Prolong Gold Antifade solution containing DAPI (Invitrogen) and examination using an Olympus Widefield BX51 microscope.
Induction of vascular cell adhesion molecule-1 in response to TNFα
Recombinant human tumour necrosis factor α (TNFα) (10 ng/ml) (R&D Systems; 210-TA-010) was added to cells for 24 h, and then cells were prepared for immunofluorescence analysis as previously described.
Matrigel tubule formation assay
A thin layer of growth factor-reduced Matrigel (BD Biosciences) was used to coat round glass coverslips and allowed to set for 30 min at 37 °C. Cells (20 000) in 0.5% serum containing DMEM were seeded onto the surface of Matrigel and incubated at 37 °C for up to 48 h to allow tubule formation, and then prepared for immunofluorescence analysis as previously described.
Chorioallantoic membrane (CAM) angiogenesis assay
Fertilized white chicken eggs were incubated for 6 days at 37.5 °C and 43% humidity in a specialised egg incubator. To distinguish implanted cells from chick cells on the CAM, implanted cells were prelabeled with 10 μg/ml 1,1-dioctadecyl-3,3,3,3-tetramethylindocarbocyanine perchlorate DiI (Sigma-Aldrich) for 30 min at 37 °C, seeded onto Matrigel-coated coverslips, and then left to adhere for 1 h. Eggs were cleaned with 70% ethanol and a square window was cut into the shell with dissecting scissors to reveal the underlying embryo and CAM blood vessels. The membrane covering the CAM surface was carefully removed with forceps and coverslips were implanted cell face down onto the CAM. The window was sealed with transparent tape and the egg was returned to the incubator for 48 h at 37.5 °C and 65% humidity, after which coverslips were removed and prepared for immunofluorescence as previously described.
In all quantitation experiments, results were expressed as the mean ± standard deviation. Statistical differences between sets of data were determined using a paired t test on SigmaPlot 8.0 software, with P < 0.05 considered significant.