bmLEC isolation and culture
Methods for isolation and culture of bovine endothelial cells were adapted from previously established methods [13, 14]. Sections of the gut mesentery were taken from freshly slaughtered cattle at a local abattoir (Better Beef, Guelph, Ontario). The mesenteric sections were brought back to the laboratory in warm PBS. Connective and fatty tissues, which were superficially rinsed with 75% ethanol, were removed to expose lymph nodes and vessels. Sterile 0.1% Evan's Blue dye was injected into the lymph nodes to high light the lymphatic vessels. Dye was flushed from excised vessels with warm PBS supplemented with penicillin (100 Units/mL, Life Technologies), streptomycin (100 μg/mL, Life Technologies), and fungizone (2.5 μg/mL Gibco, Invitrogen). Distal ends of excised vessels were occluded with surgical suture in order to infuse a solution of dispase I (2.6 U/mL, Roche) and collagenase A (1 mg/mL, Roche). Vessels occluded at both ends to trap the enzyme solution were incubated in PBS at 37°C for 10 minutes. Cells dislodged by the treatment were grown on plates coated with 1% bovine gelatin (Sigma-Aldrich), in DMEM (Dulbecco's modified eagle's medium, Sigma-Aldrich), and standard conditions. Cells isolated from four animals and several vessels from each animal were used in these studies.
bmVEC and bmAEC isolation and culture
Isolation of cells from venous and arterial vessels from bovine mesentery followed a similar strategy as described for bmLECs. However, the use of dye was unnecessary. Cells isolated from four animals and several vessels from each animal were used in these studies.
Eliminating contaminating cells
In order to establish high-purity cultures of bovine mesenteric endothelial cells (bmECs), a combination of strategies were used. Firstly, cells that were uninhibited by contact and grew atop the monolayer of endothelial cells were removed by several washes with trypsin such that the monolayer underneath was not disturbed. Secondly, visible regions of the monolayer that did not appear cobblestoned at confluency were removed by direct aspiration. Finally, limited dilution into 96-well plates was used to isolate groups of homogenous endothelial cells. Groups of cells comprising of more than 90% endothelial cells were used in experiments. 90% estimates of purity were based on monolayer cobblestone appearance.
Detection of transcripts by RT-PCR
Total RNA was prepared from cells isolated from bovine gut mesentery using Tri-Reagent (Sigma-Aldrich) according to manufacturer's instructions. cDNA was synthesized from 1 μg of total RNA with Thermoscript reverse transcriptase (Invitrogen) according to manufacturer's instructions. PCR was performed with Taq polymerase (Qiagen) with primer sequences: Tie-1 (forward primer 5'-TGA CTT TGC GGG AGA ACT GG-3', reverse primer 5'-CTC CGA CCA GCA CGT TTC GG-3'), Tie-2 (forward primer 5'-GAT TTT GGA TTG TCC CGA GGT CAA G-3', reverse primer 5'-CAC CAA TAT CTG GGC AAA TGA TGG-3'), Neuropilin-1 (forward primer 5'-CAG AAC GCT GCC CAC TGC AT-3', reverse primer 5'-CTT TCT GGG TCC TTT TTA TC3'), VEGFR3 (forward primer 5'-CGG TGC CCA GTG CGT GGG ACG-3', reverse primer 5'-TTG ACT AGC CAT CGT AGG ACA-3'), Prox-1 (forward primer 5'-TTG TCA CCC AAT CAC TTG AAA-3', reverse primer 5'-CTT CCA GGA AGG ATC AAC ATC-3') and GAPDH (forward 5'-ACC ACA GTC CAT GCC ATC AC-3', reverse primer 5'-TCC ACC ACC CTG TTG CTG TA-3')
bmECs were grown to confluency on gelatin-coated glass coverslips, fixed with 4% paraformaldehyde in PBS for 10 minutes at room temperature, and permeabilized with 0.1% TritonX-100 (Sigma-Aldrich) for 1 minute at room temperature. Fixed and permeabilized cells were stained with haematoxylin and eosin (H&E) or with immunofluorescence using antibodies: Prox-1(RDI), LYVE-1 (RDI), and podoplanin. The following protocol was used for Rhodamine-Phalloidin staining: Fixed and permeabilized cells were incubated in Rhodamine-phalloidin (Molecular Probes, 1:40 dilution) in PBS for 15 minutes, and washed. Cells were mounted on microscope slides with aqua-polymount (Polysciences Inc.) or DAPI-containing Vectashield mounting medium (Vector Laboratories) where appropriate. Bright-field microscope images were produced with the Zeiss Axioplan 2 light microscope (Carl Zeiss). Confocal images were produced using Carl Zeiss LSM 510 laser scanning confocal microscope. Fluorescent images were produced using the Zeiss Axiovert 200 M.
Stimulation of bovine endothelial cells
BmECs grown on gelatinized plates were moved to bare plates 24 hours prior to stimulation. Cells were stimulated for 15 minutes with human recombinant Ang1 or Ang2 (800 ng/mL, R&D Systems) in 10% FBS. For stimulations with recombinant Ang1, which contains a polyhistidine tag, anti-polyhistidine monoclonal antibody (anti-His, R&D Systems) was used to cluster the ligand according to manufacturer's instructions (ligand to antibody ratio 1:20). Where indicated, mock treatment refers to cells incubated with anti-His antibody alone. Three independent experiments were used to compile the quantitative results of figure 3.
Cell Lysis and Tie-2 Immunoprecipitation
Bovine ECs were washed twice with ice-cold PBS supplemented with 2 mM activated sodium orthovanadate (Sigma-Aldrich, see  for activation protocol). Cells were lysed on ice for 30 minutes with RIPA lysis buffer supplemented with 2 mM sodium orthovanadate, and complete protease inhibitors (Roche). Tie-2 was immunoprecipitated from equal protein amounts of cleared whole cell lysates with 2 μg of anti-Tie-2 antibody C-20 (Santa Cruz Biotechnology) pre-coupled to 25 μl protein A-sepharose (Amersham Biosciences) for 1 hour at 4°C.
Proteins were resolved on 10% PAGE gels and transferred to PVDF (Perkin Elmer) membranes. Antibodies used in immunoblots were: anti- phosphotyrosine 4G10 antibody (1 μg/ml, Upstate Biotechnologies), phosphoTie-2-specific anti-pTyr992 antibody (1:1000, Cell Signalling), anti-Tie-2 antibody 33.1 (0.5 μg/ml, BD Biosciences Pharmingen), and phospho and pan Erk1/2 (44/42 MAPK, 1:1000, Cell Signalling).
3H-Thymidine uptake assay
Bovine ECs were seeded in 96-well plates at a density of 4500 cells/well in 100 μL 10% FBS DMEM. After 24 hours, 10% FBS DMEM was replaced with 1% FBS DMEM + anti-His antibody (16 ug/mL, "Mock"), with 1% FBS DMEM + Ang1 (800 ng/mL) clustered with anti-His antibody (1:20 ligand to antibody ratio), or with 1% FBS DMEM + Ang2 (800 ng/mL). Cells treated thus were pulsed for 6 hours with 2 μCi of 3H-thymidine (Amersham). After 6 hours, the 96-well plates were placed in -80°C for cell lysis before 3H-thymidine incorporation was measured with the TopCount NXT Microplate Scintillation and Luminescence Counter (Packard). 3H-thymidine incorporation by bmLECs stimulated by various growth factors: bFGF (1 ng/mL, Sigma-Alrich), EGF (5 μg/mL, Invitrogen), VEGF-A165 (10 ng/mL, R&D Systems), and VEGF-CCys156Ser (1 μg/mL, R&D Systems) were determined using the same procedure. Results from two independent experiments, each with four replicates were compiled for figure 4A.
Cell count assay
40 000 cells were plated in each well of 6-well plates at the start of the assay in 10%FBS media or in mock media supplemented with 800 ng/mL supper-clustered Ang1 or 800 ng/mL Ang2 and cell numbers were monitored every 24 hours for 96 hours via cell counting with the haemocytometer. Mock media of bmLECs consisted of 5% FBS, of bmVECs consisted of 2.5% FBS, and of bmAECs consisted of 1% FBS. These percentages of FBS were required to maintain the respective cells over 96 hours while maintaining a detectable level of cellular proliferation and minimizing cell death. All mock media contained 16 ug/mL clustering anti-His antibody. Results of figure 4B–D were compiled from one representative experiment of two independent experiments, each done in tripplicate. Both experiments showed the same results (data not shown).
Modified Boyden Chamber Migration Assay
Bovine ECs were grown for 24 hours before they were serum starved for 2 hours in 0.1% FBS DMEM. Serum starved bovine ECs were then trypsinized and seeded in 500 μL of 0.1% FBS DMEM at a density of 84 000 cells/well in the upper chamber of 6 well plates containing 8 μm pore-size inserts (Falcon). 1.5 mL of the following were placed in the bottom wells: 10% FBS DMEM (positive control); 0.1% FBS DMEM + anti-His antibody (16 μg/mL, "Mock"); 0.1% FBS DMEM + Ang1 (800 ng/mL) clustered with anti-His antibody (1:20 ligand to antibody ratio); 0.1% FBS DMEM + Ang2 (800 ng/mL,). BmECs migration occurred over a 4-hour time period. Membrane of inserts were fixed and stained with filtered Harris' haematoxilin prior to mounting on microscope slides with aqua-polymount (Polysciences Inc.). Results from two independent experiments were compiled for figure 5, ten random fields were counted for each of three replicates of each experiment.
Cell death ELISA
Bovine ECs were seeded in 6-well plates at a density of 100 000 cells/plate and grown for 24 hours in standard tissue culture conditions and DMEM supplemented with 10% FBS before the start of the assay. To examine cell death due to serum starvation, 10% FBS DMEM was replaced with 0.1% FBS DMEM + anti-His (16 μg/mL), with 0.1% FBS DMEM + Ang1 (800 ng/mL, clustered with anti-His antibody), or with 0.1% FBS DMEM + Ang2 (800 ng/mL). In order to achieve detectable levels of cell death, bmLECs were harvested after 24 hour serum starvation, bmVECs after 48 hours, and bmAECs after 72 hours. Cell death was determined by quantifying cytoplasmic histone-associated-DNA-fragments (mono- and oligonuclosomes) via the ELISA (enzyme-linked immunosorbent assay, Cell Death ELISAplus, Roche) according to manufacturer's specifications. Results of three independent experiments each done in tripplicate were compiled for figure 6.
Paired, one-tailed t-tests were performed for the x-ray film densitometry results of the three independent experiments compiled for figures 3B and 3C. These parameters were appropriate since compared samples were not randomly selected as in proliferation, survival and migration assays. Compared samples belonged to the same x-ray film with the same film exposure time and linearity, which were different in each of the three experiments. Furthermore, trends of Ang1 being more effective in activating Tie-2 in bmVECs and bmAECs, and in activating ERK1/2 in the three cells remained constant in all three experiments (data not shown). Unpaired, two-tailed t-tests were performed on 95% confidence intervals for the survival assay presented in figure 6. ANOVA was performed when ttest analysis of more than two samples was required as in the proliferation and migration assays presented in figures 4 and 5.