Studies on the cultured human endothelial cell with special regard to clinical applicability

Abstract

The improvement of cardiovascular prosthetic materials, to reduce thrombus formation and inflammatory / immunological reactions, is a clinical challenge. Endothelial cell (EC) seeding using autologous cells to line the luminal surface of prosthetic materials has been suggested. The emphasis of this thesis lies on how to culture autologous cells and achieve sufficient amounts for endothelialization. It also investigates if the seeded ECs retain some of the functional properties that characterize the endothelium in vivo. It was shown that 3-5 cm of the great saphenous vein taken from patients undergoing coronary bypass surgery, was sufficient to initiate and culture large amounts of ECs. The use of human serum together with cAMP elevating compounds (cholera toxin and IBMX) was proven to be useful and superior to fetal calf serum for mass culture of umbilical and great saphenous vein ECs. The addition of bFGF and heparin further improved EC proliferation, and when combined with cAMP elevating compounds, the stimulatory effect on proliferation of adult ECs was additive. The saphenous vein ECs were shown to produce prostacyclin (PGI2) spontaneously and in response to thrombin, and to contain von Willebrand factor antigen during culture. Methods to rapidly visualise the confluency of viable ECs on various surfaces, e.g. ePTFE and porcine aorta were developed: dyes that become fluorescent after cellular metabolism were used for this purpose.

A modification of hematoxylin-eosin staining was also performed. The ECs secretion of PGI2, tissue type plasminogen activator (t-PA) and its inhibitor (PAI-1) when seeded on seven different matrices was investigated. It was shown that the secretion varied depending on the underlying matrix. Seeded on ePTFE, ECs secreted the same amount of PGI2 as did ECs seeded on fibrin glue and de-endothelialized porcine aorta. However, lower amounts of both t-PA and PAI-1 were noted. It was shown that ECs seeded on ePTFE could inactivate thrombin coagulant activity, and that thrombin bound to thrombomodulin on the EC surface, could activate protein C. In addition, monocyte adhesion to native tissue and to endothelialized biological tissue 1, 3 and 7 days after EC seeding was investigated. It was shown that the monocyte adhesion was reduced on the endothelialized tissue in a time-dependent and adhesion molecule-dependent manner. MHC class II expression by ECs was shown to be lower 3 and 7 days after seeding than one day after. The ECs responded with adequate increases in expression of E-selectin, ICAM-1, VCAM-1 and MHC class II after stimulation with IL-1 b and IFN-y.

In conclusion, the results from these studies in vitro indicate that ECs from adult donors can 1) be cultured efficiently 2) be easily visualised pre-operatively on synthetic and biological tissues 3) secrete compounds active in the regulation of coagulation and fibrinolysis and 4) be active in regulating the inflammatory / immunogenic reaction towards a biologic tissue. These findings suggest the clinical applicability of the cultured human endothelial cell.