CULTURED MICROVASCULAR ENDOTHELIAL-CELLS (MVEC) DIFFER IN CYTOSKELETON, EXPRESSION OF CADHERINS AND FIBRONECTIN MATRIX - A STUDY UNDER THE INFLUENCE OF INTERFERON-GAMMA

Endothelial cells are known to undergo transitions in cell shape durin g long-term culture. Thus, the assumption that the separate phenotypes of microvascular endothelial cells (MVEC) recently isolated from bovi ne corpus luteum represent constitutively different cell strains canno t automatically be made. For this reason, particular morphological qua lities from four of five reported MVEC types were studied. Confluent c ultures of MVEC types 1, 3, 4 and 5 were either left untreated or expo sed to recombinant bovine interferon-gamma (IFN-gamma 200 units/0.5 ml culture medium) for 3 days. Paraformaldehyde-fixed monolayers were pe rmeabilized with Triton(R) X-100 prior to the detection of filamentous actin, using phalloidin-FITC. Vimentin filaments, cytokeratin filamen ts, microtubules, E- and N-cadherins as molecules of cell adhesion pla ques, and fibronectin filaments were localized by the application of s pecific antibodies in combination with epifluorescence microscopy. Cel ls from untreated single cultures uniformly and reproducibly showed an actin cytoskeleton that distinguished the particular MVEC type. MVEC type 1 presented a circular band of fine actin filaments. MVEC type 3 preferentially had developed a starburst-like actin pattern. MVEC type 4 mainly exhibited a polygonal network. MVEC type 5 showed a prominen t circular band of thick microfilament bundles from which short filame nts radiated. Cytokeratin filaments were noted in MVEC type 1 only. Vi mentin filaments occurred as a dense network constricted to the centra l area in MVEC type 1, while they were spread out in MVEC types 3 and 4. A wavy path comparable to the course of microtubules was apparent i n MVEC type 5. Fibronectin assembled into two differently shaped layer s at the basal cell side of each MVEC type. Under IFN-gamma treatment, cytoskeletal diversities were maintained between the MVEC types, yet each MVEC type showed specific modulations to its cytoskeleton and to its fibronectin matrix. Upregulation of anti-E-cadherin labelling was detected in MVEC type 1, showing a fluorescent cell border of linear c ontour. The upregulation of E-cadherin by IFN-gamma treatment could al so be demonstrated by western blotting, which revealed a 135 kDa full- sized molecule and a 95 kDa tryptic fragment characteristic of cadheri ns. Anti-N-cadherin labelling was evident for MVEC type 5, giving rise to a fluorescent punctate cell margin. Our investigations support the existence of truly separate MVEC types.