Immortalized Endothelial Cells and Tumor Cells
The generation of endothelioma cell lines (polyoma middle T-immortalized mouse endothelial cells) from mouse embryos (Balb/c and C57Bl/6) has been described previously . As a model for mouse tumor cells we used pancreatic beta cell tumor (βTC) .
All studies involving the use of mice were approved by our local institutional animal care committee. The protocols were also approved by the regional council on animal care and correspond to the requirements of the American Physiology Society.
Isolation and culture of mouse lung microvascular endothelial cells
Mouse lung microvascular endothelial cells (MLMVECs) were isolated from mouse lungs using a magnetic cell separation method and cultured as previously described . Briefly, after euthanasia, three mice were subjected to thoracotomy and the lungs were rinsed with PBS via the right cardiac ventricle and excised. After removal of the larger blood vessels the lungs were minced under sterile conditions with scissors and placed in a 60-mm dish containing DMEM. Tissue was digested with collagenase A (Cat.no. 10103586001; Roche, Germany), rinsed with DMEM and the collected cells were strained through a 40 μm cell strainer (BD Bioscience, Germany). Cells were incubated with anti-CD31-coated Dynabeads (rat, clone MEC13.3 BD; Cat.no.110.035, Dynal, Norway) and separated in a magnetic field. After the washing away of the unbound cells, the beads were released by trypsin/EDTA treatment or cells were seeded with the microbeads. Cell-culture medium was replaced with DMEM enriched with 20% FBS (Cat. no. S1810; Biowest, France). After 8 -10 days, cells were separated again by FACS sorting (FACS Aria, BD Bioscience, Germany) using the same CD31 antibody as before. Immunophenotyping of CD31+ cells was carried out by flow cytometry after cell expansion (passage 4-6) using a previously described multipanel of cell surface and cytoslic markers . Additionally, cells were characterized for markers more specific for blood or lymph endothelial cells . Passages 6 -20 were used for consecutive experiments.
MLMVEC cells were seeded at 1 × 104 cells/cm2 in gelatinised separate six-well plates or later in small cell culture flasks (25 cm2). The medium contained DMEM and 15-20% FCS (MBE medium) and has been described previously . Cells were fed at least twice a week and split 1:4 up to 1:16 after confluence. Growth studies were performed in duplicate or triplicate and for studies with growth factors, endothelial cell growth supplement (ECGF, Cat. no. 300-090; Reliatech, Germany) was omitted from the media (ECGF-free cell growth protocol). Growth was determined by electronic cell counting from day 0, at 48-h intervals. Results show the average of duplicate/triplicate measurements repeated at least twice.
MLMVECs and endothelioma cells were seeded at 1.5 × 104/well in separate 48 well plates (1,1 cm2/well) in MBE medium. Following 72 h incubation at 37°C with 5% CO2, the medium was replaced and this time point was designated day 3. Proliferation studies were performed in duplicate. Cell growth was determined after day 3 at 24 h intervals, using an electronic cell counter (Casy Systems, Germany). Results denote the average of duplicate measurements repeated at least once. For the proliferation assay in the presence of growth factors, cells were seeded in minimal medium (in 5% FCS w/o ECGF) supplemented with the growth factors and measured over 4-6 days with one growth factor exchange after three days. For proliferation assays in the presence of VEGF-A (mouse VEGF164) we used 5 and 20 ng/ml growth factor.
Limiting dilution analysis
Limiting dilution analysis was performed as described earlier with some modifications . The single-cell clonogenic assay was performed with MLMVECs from one isolation at 80-100% confluence. Briefly, cells were trypsinized, and from the suspension the cell number was electronically determined (Casy 1 Cell Counter). We diluted our cells to one cell per 100 μl of standard culture medium and then dispended 100 μl/well into two and a half gelatine-coated 96-well plates. Cells were examined daily under the microscope to study their colony-forming ability. After one week, wells were supplemented with another 100 μl of standard culture medium. Cells were further cultured at 37°C with 5% CO2 and 21% O2 for another three weeks. After 4 weeks all wells, which contained at least one cell, were counted. Quantification of moderately and highly proliferating colonies was performed independently by two persons by visual inspection at x40 magnification. Results denoted the average from 240 wells. Highly proliferating, clonogenic MLMVECs were obtained after further expanding colonies from confluent wells.
Immunophenotyping by FACS Analysis
MLMVECs and other mouse endothelial cells were treated with accutase (Cat.no. L11-007, PAA, Austria) and suspended at 1 × 105 cells per tube in 1 ml microtubes containing 200 μl of cold PBS (calcium and magnesium free) supplemented with 2% FCS. For the immunostaining, cells were incubated for 15 min on ice. All cells were rinsed with cold PBS/2%FCS and incubated with the primary antibody or with fluorescein-conjugated lectin or isotype control antibody for 30 min on ice in the dark. Cells were rinsed in cold PBS/2% FCS and then incubated with the corresponding fluorescent dye-conjugated secondary antibody. At the end of antibody incubation, cells were transferred to flow cytometric tubes (Micronic, The Netherlands) and subjected to flow analysis using FACS Calibur (Becton Dickinson). We used the following primary antibodies: CD31 (rat clone MEC13.3; BD), CD34 (rat clone RAM34; BD), CD45-FITC (rat clone 104, BD Pharmingen), CD102 (rat clone 3C4(mlC2/4); BD), CD105 (rat #6J9; Reliatech), CD144 (rat clone 11D4.1; BD), VEGFR-2 (rat clone Avas12α1; BD), VEGFR-3 (rat Cat. no. 103-M36, Reliatech), Lyve1 (rabbit Cat. no. 103-PA50AG; Reliatech), podoplanin (hamster Cat. no. 103-M40; Reliatech), PV1/Meca32 (rat; U. Deutsch), LA102 (rat Cat.no. 103-M152; Reliatech) and LA5 (rat, Cat.no. 103-M154; Reliatech). The following primary antibodies for HSC were used: CD117-PE (rat 120-002-406; Miltenyi Biotech), CD133 (rat clone 13A4; eBioscience, U.S. and rat clone MB9-3Ga, Miltenyi Biotech, Germany) and CD41-PE (rat clone MWReg30; eBioscience, US). For lectin binding we used the FITC-conjugated Griffonia simpliciflolia (Cat. no. FL-1101; Biozol, Germany). For immunodetection of the von Willebrand factor, cells were incubated with the primary and secondary antibodies in the presence of 0.15% TritonX-100. We used the following conjugated secondary antibodies: goat anti rat-FITC (Jackson IR, U.S.), goat-anti rabbit-FITC (Jackson IR), goat-anti-hamster-PE (Jackson IR). As a control, cells were incubated with secondary antibodies exclusively (negative control). Confirmation of protein expression or lectin interaction in MLMVECs was the right-shift of the curve as compared with that of a corresponding control.
Measurement of NO production
The cell-permeable diacetate derivate, DAF-2 DA (4,5-Diaminofluorescein.diacetate) was used to measure NO production (Cat. no. ALX-620-056; Alexis, Switzerland). After loading and subsequent hydrolysis by cytosolic esterases, DAF-2 is released, which is relatively non-fluorescent at physiological pH. In the presence of NO and O2, DAF-2 is converted to the fluorescent triazole derivated, DAF-2T . Briefly, EPCs grown in 6-well plates overnight were than incubated with 5 μM fresh DAF-DA in 1 ml PBS for 30 min. After washing, cells were detached and transferred to FACS buffer for FCS analysis (see above). NIH/3T3 cells have been initially used as control cells and have been found to be negative for NO.
Matrigel in vitro outgrowth assay
To study sprouting angiogenesis in vitro, mouse endothelium spheroids of a defined cell number were generated as described previously . Briefly, ECs were suspended in MBE culture medium containing 0.25% (wt/vol) methyl cellulose and cultured overnight in hanging drops of 50 μl. The cells formed single spheroids with a defined cell number. Matrigel (BMM Matrigel; BD Bioscience, Germany) was diluted 1:3 with serum-free endothelial cell medium (Cat. no. U15-002, PAA, Austria), supplemented with various growth factors (HGF, ECGF, VEGF-A, bFGF, VEGF-C, TNF-α) and 150 μl was mixed with each spheroid and seeded in 48-well plates (as duplicates). Wells were analysed for sprout formation using phase-contrast microscopy at 1, 5 and 14 days after seeding. The average length of sprouts was measured with a digitalized Zeiss photomicroscope and compared to the negative controls. Results denote the average of multiple measurements of at least two separate experiments.
Green fluorescent protein and luciferase transfection
Lentiviral constructs encoding green fluorescent protein (GFP; pPREGFPSIN) and luciferase (LUC; pJSCMVLuc) without antibiotica resistance, were produced as previously described . Low passage MLMVECs and high passage MLMVECs (> 20) were seeded in a 12-well plate, supplemented with standard culture medium and incubated to about 60-70% confluence. The medium was removed and culture supernatant containing lentiviral particles constructs, was added to the cells in the presence of 8 μg/ml Polybrene for 8 h (Sigma). The infected cells were cultivated as a pool of infected cells. The infection efficiency varied between 40-60% depending on the expression cassette and the cell type. For maintenance the medium was changed twice a week until cells were confluent and used for liquid nitrogen freezing and for in vivo Matrigel injections.
Matrigel-Mediated de novo Vessel Formation in vivo
MLMVECs were trypsinized, counted and cultured overnight in hanging drops to allow predifferentiation of cells in spheroids (~ 10,000 cells/spheroid). Matrigel was supplemented with 500 ng/ml bFGF and VEGF-A and used with or without spheroids. For each injection, about 30-50 spheroids were mixed in 300-400 μl unpolymerized Matrigel at 4°C. The mixed solution was injected subcutaneously (left and right dorsal region, two plugs per animal) into mildly sedated (ketamine 100 mg/kg ip) Balb/c female mice using a 20-gauge needle. The injected Matrigel polymerizes at body temperature and becomes a plug. As controls, Matrigel was injected on the left side without cells but with growth factors. 10-12 days after injection, Matrigel plugs were excised and treated overnight with methanol/10% DMSO. Plugs were further immersion-fixed in 4% paraformaldehyde, treated with saccharose and embedded in frozen section medium (Neg50; Richard-Allan Scientific, U.S.).
Immunodetection of MLMCECs in vivo
Excised Matrigel plugs embedded in frozen section medium were used to prepare cryosections of 16-20 μm thickness. Non-specific binding of antibodies was blocked by incubation with 1% bovine serum albumin (BSA) for 1 h before incubation with primary antibodies. The following primary antibodies were used for single and double-staining: Anti-mouse CD31 (rat clone MEC13.3; BD), anti-mouse CD45 (rat clone 104; BD Pharmingen) anti-mouse podoplanin (hamster Cat. no. 103-M40; Reliatech), anti-mouse CD105 (rat #6J9; Reliatech), anti-GFP (rabbit Cat. no. ab6556-25, Abcam, U.K.) and anti-mouse LA102 (rat Cat. no 103-M152).
The sections were incubated with primary antibodies for 1 h. After rinsing, secondary antibodies were applied at 1:100 or 1:200 dilution for 1 h. We used goat anti-rat TRITC-conjugated, (Jackson IR, U.S.), donkey anti-rabbit Alexa488 (Pierce, Rockford, U.S.), goat anti-hamster Alexa594, goat anti-rabbit Alexa594 and goat anti-rat Alexa350-labeled (MoBiTec, Göttingen, Germany). DAPI reagent (Hoechst Dye) was added to stain nuclei.
After being rinsed, the sections were mounted under coverslips with Fluoromount-G (Southern Biotechnology, U.S.). They were studied with a DM500 B epifluorescent microscope and Axioplan 2 LSM 510 laser-scanning microscope (Zeiss, Germany).
Anchorage-independent growth on soft agar
MLMVECs and mouse tumour cells (βTC) as positive control were suspended in MBE medium and seeded at a density of ~1 × 103 cells/cm2 in 48 well plates coated with 0.8% soft agar in MBE medium. Cells were incubated at 37°C and 5% CO2. After 8-10 days, plates were studied with phase-contrast microscopy. Efficiency of anchorage-independent growth was determined by the capacity of cells to form colonies (> 100 cells) within the agar. Experiments were performed as duplicates and repeated at least once.