DERMAL FIBROBLAST-CULTURE AS A MODEL SYSTEM FOR STUDIES OF FIBRILLIN ASSEMBLY AND PATHOGENETIC MECHANISMS - DEFECTS IN DISTINCT GROUPS OF INDIVIDUALS WITH MARFANS-SYNDROME

Most patients with Marfan's syndrome (>95%) and 75% of patients with u ncertain diagnosis can be classified into four groups (Aoyama et al, 1 994, 1995) based on abnormal patterns of synthesis, intracellular tran sport, and/or matrix deposition of fibrillin-1 in fibroblast cultures. Herein we report a systematic study of fibrillin assembly in normal a nd Marfan's syndrome fibroblasts and correlations between pulse-chase, immunofluorescence, and immunoelectron microscopic data. Normal contr ol fibroblasts were grown at confluent conditions from 2 to 10 days be fore passage and then maintained at hyperconfluent cell densities for an additional period of 1 to 6 days before assaying. Maximum depositio n in the extracellular matrix of pulse-labeled fibrillin required at l east 6 days of confluent and 4 to 5 days of hyperconfluent culture. Th is result is explained by immunofluorescence studies with fibrillin-1- specific antibodies, because 1 day after seeding cells at hyperconflue ncy, patches of irregular immunostained structures were already presen t. Within these patches, fluorescence intensity and fibrillar material increased over 3 to 4 days, and after only 5 days, fibrillar networks extended throughout the culture. We propose that fibrillin-containing microfibrillar material is passaged together with the cells, newly sy nthesized fibrillin molecules are deposited onto preexisting microfibr illar assemblies, and several additional days of culture at high cell density are necessary for the cells to construct a sufficient microfib rillar network for binding and detection of pulse-labeled fibrillin mo lecules in insoluble form during a 20-hour chase period. This fraction is decreased to a varying extent in fibroblast cultures of four biosy nthetically distinct groups of Marfan's syndrome patients, but only Gr oups II and IV clearly showed reduction in immunostainable microfibril s. In long-term cultures, immunoelectron microscopy of the extracellul ar matrix with fibrillin antibodies also detected differences among th ese groups and in comparison to normal controls with respect to the ar rangement of fibrillin-containing microfibrils, thickness of microfibr illar bundles, and the presence of amorphous material. The data suppor t the idea of different pathogenetic mechanisms for each biosynthetica lly defined group of Marfan's syndrome, which depends on the nature of fibrillin-1 mutations.