Cell culture methods

2.2.3.1 Isolation of HUVECs

HUVECs were isolated directly from umbilical cord veins using collagenase treatment. Briefly, the umbilical cord was freshly cut at both ends to obtain clean vein ends. The umbilical cord contains 2 main arteries and a single vein, larger in diameter than the arteries. The vein was washed with warm RPMI containing 10% FCS, streptomycin and ampicillin, and then filled with 0.1% collagenase in RPMI. The cord was left in the incubator at 37°C for no longer than 20 minutes. The HUVECs were collected and then the vein was washed again in RPMI plus 10% FCS to collect any more HUVECs. The HUVECs were then centrifuged for 7 minutes at 1200 rpm . The supernatant was poured away and the HUVECs were then resupended in growth medium and placed in 25 cm^ tissue culture flasks. Very often the HUVEC preparation was contaminated with blood cells and therefore the HUVECs were washed 4 hours after plating.

2.2.3.2 HUVEC culture and seeding

The HUVECs isolated directly from umbilical cords were cultured in TC Nunclon flasks in medium 199 modified Earle’s salt solution containing 1.25 g/litre NaHCOa and Glutamax and supplemented with 20% FCS, 100 /tg/ml endothelial cell growth supplement, 1% Nutridoma NS, 100 /ig/ml heparin, penicillin (10,000 lU/ml) and streptomycin (10,000 pg/ml). Cells were cultured at 37°C in humidified air containing 5% CO2. For experiments cells were used between 1 and 3 passages. Cells were generally

passaged once every week. Old medium was removed by aspiration and the cells washed briefly in PBS (without Ca^"^ and Mg^"^). An appropriate amount of trypsin/EDTA was then added to the cells (ie. 3 mis for 75 cm^ flask). After 3-5 minutes, the cells were

easily detached from the flask by gentle agitation. Trypsin/EDTA was inactivated by fresh growth medium. Alternatively, pooled HUVECs were purchased from Clonetics and grown in EGM-2 supplemented with 2% FCS.

For immunofluorescence experiments cells were grown on glass coverslips coated with 10 /xg/ml human fibronectin until confluent. For biochemical experiments cells were grown on 6 and 10 cm dishes coated with human fibronectin until confluent. To obtain quiescent starved cells, the culture medium was replaced by medium containing 10% FCS but no heparin or other growth factors. Cells were incubated in this medium for no longer than 24 hours. Cells grown in EGM-2 were starved in 1% FCS. No differences between HUVEC responses were observed between cells grown in different media.

2.2.3.3 Other cell culture

HeLa and Swiss 3T3 cells were cultured in tissue flasks and grown at 37°C in a humidified atmosphere containing 5% CO2. Cells were generally passaged every 3-5 days

and split before reaching confluence by dilution at a ratio of 1:10 in fresh growth medium (DMEM, 10% FCS, penicillin and streptomycin). For biochemistry experiments cells were seeded directly onto tissue culture dishes and grown until confluent. For immunofluorescence experiments cells were grown on glass coverslips until confluent. Human monocytes were purified by allutriation and used in EGM-2.

2.2.3.4 HUVEC stimulation, receptor clustering and immunofluorescence

HUVECs were stimulated with TN F-a (10 ng/ml) by placing the TN F-a directly into the starvation medium in which the cells were maintained. ICAM-1, lCAM-2, NCAM- ICAM-1 and VCAM-1 cross-linking was performed using antibodies. To induce receptor clustering, either mouse monoclonal anti-ICAM-2/NCAM antibodies was added to starved cells at a final concentration of 10 /xg/ml for 60 mins, or mouse monoclonal anti- ICAM-1/ ICAM-2 antibodies were added to cells that had been stimulated for 24 hours with 10 ng/ml TNF-a. After incubation with primary antibodies, TNF-a and the primary antibodies were removed from the cell medium and 10 /xg/ml of Alexa 488-labelled goat

anti-mouse (GAM) antibody was added to the cells for between 15 and 60 minutes. Cells were then washed three times in TBS (10 mM Tris pH7.5, 150 mM Na Cl) containing 0.25% BSA, fixed in 4% paraformaldehyde for 20 mins at room temperature, permeabilized for 6 mins with 0.2% Triton X-100, and then incubated with 1 /xg/ml TRITC-phalloidin for 45 minutes to stain actin filaments, or for 45 mins with rabbit polyclonal anti-moesin antibody diluted 1:200, or rabbit polyclonal anti-myosin II antibody diluted 1:25, followed by the appropriate TRITC-labeled secondary antibody. The specimens were mounted in moviol. To observe responses in cells without receptor clustering, cells were incubated with primary antibodies as before for 60 mins. These cells were then washed, fixed, and stained with the secondary antibody for 45 mins. Staining for intracellular epitopes was then carried out as described above. All incubations were carried out in TBS containing 0.25% BSA.

In order to use the phosphospecific anti-CPERM antibody a different fixation procedure was used as previously described (Hayashi et a/., 1999). In brief, after receptor clustering cells were fixed in 10% trichloroacetic acid (TCA) in distilled water at 4 “C for 20 mins. All further staining was carried out in TBS containing 0.25% BSA. Cells were incubated with rat monoclonal anti-CPERM antibody (297S) for 45 mins, followed by Cy5- conjugated goat anti-rat antibody for 45 mins.

In order to use the anti-MVP (rabbit polyclonal) antibody a different fixation protocol was used as previously described. In brief, cells were fixed in methanol for 7 minutes at - 20°C. No permeabilization was necessary and subsequent staining was all carried out in TBS containing 0.25% BSA. However, fixation with 4% paraformaldehyde was also used where indicated in figure legends.

Confocal laser scanning microscopy was carried out with an LSM 510 (Zeiss, Welwyn Garden City, UK) mounted over an affinity corrected Axioplan microscope (Zeiss) fitted with a X 10 eyepiece, using either a x 40 NA 1.3 or a x 63 NA 1.4 oil immersion objective. Image files were collected as a matrix of 1024 x 1024 pixels describing the average of 8 frames scanned at 0.062 Hz where FITC, TRITC, and Cy5 were excited at 488 nm, 543 nm, and 633 nm and visualized with a 540 +/- 25, 608+/- 32, and 690+/- 30nm bandpass filters, respectively, where the levels of interchannel crosstalk were insignificant.

2.2.3.5 Transfection of cells

HUVEC transfections were performed when cells were between 50 and 70% confluent. HUVECs were transfected using a mixture of P6 integrin peptide and Lipofectin (Gibco) as previously described (Hart et a l, 1998). Briefly, P6 integrin-targeting peptide, Lipofectin, and plasmid DNA were allowed to form a complex, which was incubated with sub-confluent cells in Optimem for 4 hours. Cells were then washed in Optimem and grown to confluence in their normal growth medium (medium 199 with supplements). Also, for NCAM ICAM-1 chimera studies, HUVECS were transfected using Effectene according to the manufacturers protocol (QIAGEN). Transfection complexes containing enhancer plus a DNA/effectene ratio of 1:25 were placed into fresh growth medium on HUVECs for 3 hours. 0.2 pg of DNA was used for each coverslip. Transfection complexes were removed and the cells were washed to removed any remaining lipids/DNA. Cells were allowed to express NCAM-ICAM-1 chimaeras for 48 hours

HeLa cells were transfected using Eugene 6 transfection reagent (Boehringer Mannheim). Transfection complexes were made using serum-free media and DNA/Fugene6 at a ratio of 1:3. 0.5 pg of DNA was used for each coverslip. Complexes were then placed onto cells in fresh growth medium drop by drop with swirling. Cells were left to express for 48 hours.

2.2.3.Ô Metabolic labelling of cells

HUVECs were labelled overnight, during TN F-a stimulation, with 20 pCi/ml [^^S]- methionine/cysteine labelling mix. The cells were incubated in DMEM with serum that had been dialysed for 3 hours at 4°C to remove any free amino acids. FCS was dialysed in PBS (100 X volume of FCS) using Spectr/Por dialysis tubing with a Mw cut-off of 8 kDa.

2.2.3.7 Time-Lapse Microscopy

Time-lapse experiments were performed using an LSM 510 confocal microscope as described above. TNF-a-stimulated HUVECs were incubated with anti-ICAM-1 or anti- ICAM-2 antibody for 60 mins as described above. TNF-a and primary antibody were removed from the medium and cells were incubated with a TRITC-conjugated secondary antibody for two minutes in normal starvation medium. This was removed and replaced with a HEPES/Hanks’ balanced salts based medium to allow air buffering during the time series. This medium contained Ix Hanks’ balanced salt solution (Gibco), 10% FCS, and 20 mM HEPES pH 7.4. Cells were visualized with a x 40 water immersion objective and a frame was grabbed every 62 seconds for 50 frames. A heated stage was utilized to stabilize the temperature of the cells between 30“C and 37°C. The image series was converted from a series of TIFF files to an AVI file using Video Manager, and from an AVI file to an MPEG file using an AVI to MPEG conversion program. Control cells were loaded with 5-carboxyfluorescein diacetate, acetoxymethyl ester (5-CFDA, AM; Molecular Probes) to show that monolayer integrity was unaffected by the experimental conditions.

2.2.3.8 Flow assavs and migration of monocvtes

HUVECs were grown on fibronectin-coated slides until confluent and then placed in a flow chamber and exposed to post-capillary physiological shear stress of 1.5 dynes/cm^ for 24 hours. Circulating medium was then supplemented with TN F-a at 20 ng/ml for 4 hours to induce expression of ICAM-I. Flow was then stopped and cells, still positioned in the flow chamber, were transferred to a time-lapse microscope stage supplemented with CO2 and kept at 37°C. Medium was removed and replaced with new starvation

medium containing IxIO^ primary human monocytes. Migration of monocytes over the endothelium was then filmed for a period of one hour, one frame taken every 60 seconds. HUVEC alignment and monocyte migration were tracked using Kinetic Imaging Tempus Video Software. For inclusion in statistical analysis, monocytes were required to migrate at least 75% of the length of an elongated endothelial cell. The angles of the cell tracks

were normalized against the angle of endothelial cell orientation. This gave angles of monocyte migration relative to an imaginary axis of endothelial cell alignment.