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

Sammanfattning: The improvement of cardiovascular prosthetic materials, to reducethrombus formation and inflammatory/immunological reactions, is a clinicalchallenge. Endothelial cell (EC) seeding using autologous cells to line the luminalsurface of prosthetic materials has been suggested.The emphasis of this thesis lies on how to culture autologous cells and achievesufficient amounts for endothelialization. It also investigates if the seeded ECsretain some of the functional properties that characterize the endothelium invivo.It was shown that 3-5 cm of the great saphenous vein taken from patientsundergoing coronary bypass surgery, was sufficient to initiate and culture largeamounts of ECs. The use of human serum together with cAMP elevatingcompounds (cholera toxin and IBMX) was proven to be useful and superior tofetal calf serum for mass culture of umbilical and great saphenous vein ECs. Theaddition of bFGF and heparin further improved EC proliferation, and whencombined with cAMP elevating compounds, the stimulatory effect onproliferation of adult ECs was additive. The saphenous vein ECs were shown toproduce prostacyclin (PGI2) spontaneously and in response to thrombin, and tocontain von Willebrand factor antigen during culture. Methods to rapidlyvisualise the confluency of viable ECs on various surfaces, e.g. ePTFE and porcineaorta were developed: dyes that become fluorescent after cellular metabolismwere used for this purpose. A modification of hematoxylin-eosin staining wasalso performed. The ECs secretion of PGI2, tissue type plasminogen activator (t-PA) and its inhibitor (PAI-1) when seeded on seven different matrices wasinvestigated. It was shown that the secretion varied depending on the underlyingmatrix. Seeded on ePTFE, ECs secreted the same amount of PGI2 as did ECs seededon fibrin glue and de-endothelialized porcine aorta. However, lower amounts ofboth t-PA and PAI-1 were noted. It was shown that ECs seeded on ePTFE couldinactivate thrombin coagulant activity, and that thrombin bound tothrombomodulin on the EC surface, could activate protein C. In addition,monocyte adhesion to native tissue and to endothelialized biological tissue 1, 3and 7 days after EC seeding was investigated. It was shown that the monocyteadhesion was reduced on the endothelialized tissue in a time-dependent andadhesion molecule-dependent manner. MHC class II expression by ECs wasshown to be lower 3 and 7 days after seeding than one day after. The ECsresponded with adequate increases in expression of E-selectin, ICAM-1, VCAM-1and MHC class II after stimulation with IL-1 b and IFN-y. In conclusion, the results from these studies in vitro indicate that ECs fromadult donors can 1) be cultured efficiently 2) be easily visualised pre-operativelyon synthetic and biological tissues 3) secrete compounds active in the regulationof coagulation and fibrinolysis and 4) be active in regulating theinflammatory/immunogenic reaction towards a biologic tissue. These findingssuggest the clinical applicability of the cultured human endothelial cell.Doctoral Thesis© 1995 Caroline Gillis-HaegerstrandISBN 91-628-1745-0