Senescence of aortic endothelial cells in vitro: influence of culture conditions and preliminary characterization of the senescent phenotype.

The replicative lifespan of a cloned strain of adult bovine aortic endothelial cells has been examined by serial passage in culture using conditions where the cells were either dependent on fibroblast growth factor (FGF) for rapid growth or relatively independent on FGF for growth. In FGF-dependent cultures a replicative lifespan of 130 generations was attained with a phase III period which spanned approximately 20 generations. Withdrawal of FGF at generation 55 and repeated passage of such cultures in the absence of the growth factor resulted in the loss of proliferative potential within 15 generations. The morphological changes occurring upon FGF withdrawal were different than those that occurred when cultures senesced in the presence of FGF, FGF withdrawn cells showed a homogeneous increase in cell size and after several passages overgrew one another at confluency. Endothelial cells which senesced in the presence of FGF showed a very heterogeneous distribution of enlarged cells, many of which were binucleated but continued to form a confluent monolayer at high cell densities. Under FGF-independent conditions (begun at generation 48) a replicative lifespan of 105 generations was attained in the presence of the growth factor. FGF withdrawal under these conditions only decreased the replicative lifespan to 95 generations. Under these conditions the morphological changes occurring during phase III were identical in the presence and absence of FGF. Examination of the sensitivity of endothelial cells to FGF as they entered phase III showed that their dose-response characteristics were not qualitatively altered after the onset of phase III, although the number of cells responding to FGF progressively dropped. Comparison of the patterns of proteins synthesized in phase II and phase III cultures showed that phase III cultures even when plated at sparse densities continued to synthesize proteins which were normally observed in phase II confluent cultures. The results indicate that onset of the phase III period in aortic endothelial cells cloned and maintained in the presence of fibroblast growth factor can be delayed to a greated extent if the cells are maintained under conditions where they are made dependent on FGF for rapid growth in culture.