CHANGES IN 3-DIMENSIONAL ARCHITECTURE OF MICROFILAMENTS IN CULTURED VASCULAR SMOOTH-MUSCLE CELLS DURING PHENOTYPIC MODULATION

To investigate changes in the three-dimensional microfilament architec ture of vascular smooth muscle cells (SMC) during the process of pheno typic modulation, rabbit aortic SMCs cultured under different conditio ns and at different time points were either labelled with fluorescein- conjugated probes to cytoskeletal and contractile proteins for observa tion by confocal laser scanning microscopy, or extracted with Triton X -100 for scanning electron microscopy. Densely seeded SMCs in primary culture, which maintain a contractile phenotype, display prominent lin ear myofilament bundles (stress fibres) that are present throughout th e cytoplasm with alpha-actin filaments predominant in the central part and beta-actin filaments in the periphery of the cell. Intermediate f ilaments form a meshed network interconnecting the stress fibres and l inking directly to the nucleus. Moderately and sparsely seeded SMCs, w hich modulate toward the synthetic phenotype during the first 5 days o f culture, undergo a gradual redistribution of intermediate filaments from the perinuclear region toward the peripheral cytoplasm and a part ial disassembly of stress fibres in the central part of the upper cort ex of the cytoplasm, with an obvious decrease in alpha-actin and myosi n staining. These changes are reversed in moderately seeded SMCs by da y 8 of culture when they have reached confluence. The results reveal t wo changes in microfilament architecture in SMCs as they undergo a cha nge in phenotype: the redistribution of intermediate filaments probabl y due to an increase in synthetic organelles in the perinuclear area, and the partial disassembly of stress fibres which may reflect a degra dation of contractile components.