Entre Burgos y Granada La torre de Santa María del Campo (1527-1529)

2.3.2.1 Umbilical vein endothelial cells

Human umbilical vein endothelial cells were cultured from fresh umbilical cords using a modification of the method of Jaffe et al (1973). Briefly, fresh human umbilical cords were trimmed to remove excess fat and the umbilical vein was identified and cannulated. The vein was washed using warmed PBS (37°C) and then 10ml/20cm of cord of pre-warmed collagenase solution was added. The cord was incubated for 30 minutes at 37°C. It was then massaged gently and the resultant endothelial cell suspension was aspirated. This suspension was centrifuged (150g) and resuspended in serum supplemented (20%) M l99. Primary isolates were seeded into 25cm^ gelatin-coated tissue culture flasks. Medium was changed at 24h and cells trypsinised and plated into a 75cm^ flask at confluence. Cells were cultured in standard culture medium (M l99/20% FCS supplemented with 7mg/ml heparin ECGS [Sigma, St Louis, MO] and 0.02mg/ml ECGS [Sigma, St Louis, MO]) and each cord generally yielded 2 confluent 75cm^ culture flasks at the 2nd passage. Cells were subcultured in a ratio of 1:2, and used for experimental studies at the 2nd to 4th passage. The typical appearance of confluent umbilical vein endothelial cells is illustrated in Figure 2.2.

2.3.2.2 Endothelial cell lines

The extended lifespan SV40 transfected human umbilical vein (HUVEC) derived endothelial cell line lE-7 was used throughout. Cells were used between the 14th and 17th passage, when they have been shown to retain their differentiated endothelial properties (Tickling et a l

1992). Cultures were maintained in standard endothelial cell growth medium (M199/ECGS/heparin/20%FCS), although they grow well in a range of media including DMEM/10% FCS. The appearance of confluent lE-7 cells is shown in Figure 2.2, and was indistinguishable from that of native HUVEC. For some experiments the virally transformed human dermal microvascular cell line HMEC-1 was employed. This line was generated by Ades et al (1992) and has been shown to maintain at least some of the phenotypic characteristics of native dermal microvascular endothelial cell cultures (Xu et a l 1994).

2.3.2.S Confirmation of endothelial cell phenotype.

Both native human umbilical vein endothelial cells and the HUVEC derived cell line lE-7 assume a typical cobblestone morphology in monolayer culture. The endothelial cell phenotype was confirmed by the expression of a range o f endothelial cell related antigens including vWF and by the uptake of acetylated LDL. A series of experiments were undertaken to confirm that the properties o f the cell line lE-7 were similar to those of non­ transformed endothelial cells. Apart from the typical morphology, which was indistinguishable from native HUVEC, lE-7 also demonstrated uptake of acetylated LDL. Their transformed nature was confirmed by expression of the SV40 large T-antigen, which was readily demonstrated by indirect immunofiuorecence (Figure 2.3). HUVEC express a number of cell surface adhesion molecules including ICAM-1, VCAM-1 and E-selectin, which are important in mediating adherence of leucocytes, particularly after stimulation with pro- inflammatory cytokines or bacterial lipopolysaccheride. The time-course and dose-response characteristics for expression of these adhesins by umbilical vein endothelial cells are well known (Haraldsen et a l 1996). Studies o f their expression by lE-7 cells suggested higher constitutive expression of ICAM-1 but otherwise similar patterns of expression to those reported for HUVEC. Representative dose-response and time course data are shown in

Figures 2.4 and 2.5. These results provided important additional evidence that the cell line lE-7 was likely to represent a good model for native endothelial cells in my experiments. However, wherever possible control studies were performed using HUVEC to validate the results obtained with lE-7.

The cobblestone morphology of endothelial cells at confluence was in marked contrast to the appearance of fibroblast cultures, and was also distinct from the appearance of two epithelial

o (/) n 1.5 E-selectin (6h) VCAM-1 (lOh) ICAM-1 (24h) 1.3 1.1 0.9 0.7 0.5 0.3 0.1 -0.1 0.1 1 10 100 1000 TNF (U/ml)

Figure 2.4 Dose-response curve for TNFa-induced expression of ICAM-1, VCAM-1 and E-selectin by lE-7

Dose-response curve for adhesion molecule expression by the human endothelial cell line lE-7. Data are for TNFa but equivalent results were obtained for IL -la. Time points at which expression was measured are indicated. Expression of adhesion molecules was determined in a cell bound ELISA by absorbance at 450nm and each data point represents mean ±sd for 3-6 replicate wells. See section 2.7 for assay details.

0.8 0.7 _E-selectin 0.6

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Figure 2.5 Time course for cytokine induced expression of EC adhesion molecules by lE-7

Pro-inflammatory cytokines induced upregulation of the EC adhesion molecules ICAM- 1, VCAM-1 and E-selectin. Representative data (mean ±sd for triplicate wells) for 0 (cytokine added and immediately washed off) 4 and 24h time points incubation of TNFa at 100 U/ml are shown. Expression wasquantified in a cell bound ELISA by absorbance at 450nm in triplicate wells. Control wells had no cytokine added and reflect constitutive expression of the adhesion molecules. As with native HUVEC ICAM-1 is significantly expressed even without pro-inflammatory cytokines. Similar data were obtained using EL-la (lOOU/ml)

cell lines A549 or A431 arising from lung or epidermoid carcinoma respectively which were employed in control experiments.