Bavisotto L M, Schwartz S M, Heimark R L
Department of Medicine, University of Washington, Seattle 98195.
J Cell Physiol. 1990 Apr;143(1):39-51. doi: 10.1002/jcp.1041430106.
Cultured endothelial cells have been shown to possess two mechanisms of intercellular adhesion: Ca2(+)-dependent and Ca2(+)-independent. We report here that growth of bovine aortic endothelial cells (BAEC) in complete medium containing purified basic fibroblast growth factor (bFGF, 6 ng/ml) results in loss of Ca2(+)-dependent intercellular adhesion. In the presence of heparin (90 micrograms/ml), this effect is reproduced upon treatment with acidic fibroblast growth factor (aFGF, 6 ng/ml) or endothelial cell growth supplement (ECGS, 100 micrograms/ml), in both human umbilical vein endothelial cells (HUVEC) and BAEC. Treatment at these doses with aFGF in the absence of heparin or with heparin alone is without significant effect. Loss of Ca2(+)-dependent adhesion following treatment of cells with heparin-binding growth factors (HBGFs) is prevented by pre-treatment of cell layers with cycloheximide. The Ca2(+)-independent adhesion mechanism is unaffected by HBGF treatment. Exposure of endothelial cells to HBGFs, moreover, prevents the eventual establishment of quiescence in growing cultures and restimulates replication in confluent cultures that have reached a final density-inhibited state. Addition of bFGF alone or aFGF + heparin at these doses results in a 4-fold increase in DNA synthesis over untreated control cultures at saturation density as reflected by thymidine index. A single addition of bFGF (6 ng/ml) to untreated quiescent confluent BAEC monolayers results in an increase in 3H-TdR incorporation reaching a peak at 22 hours with a parallel loss of Ca2(+)-dependent adhesiveness. Fluorescent staining with rhodamine-phalloidin demonstrates an altered distribution of polymerized F-actin in the bFGF-treated monolayers, marked by disruption of the dense peripheral microfilament bands retained by untreated confluent monolayers. Together, these results indicate that the mitogenic effect of HBGFs in cultured endothelial cells is associated with a "morphogenic" set of responses, perhaps dependent on breakdown of calcium-dependent cell-cell contacts.
钙离子依赖型和非钙离子依赖型。我们在此报告，在含有纯化碱性成纤维细胞生长因子（bFGF，6 ng/ml）的完全培养基中培养牛主动脉内皮细胞（BAEC）会导致钙离子依赖型细胞间黏附丧失。在肝素（90 μg/ml）存在的情况下，用酸性成纤维细胞生长因子（aFGF，6 ng/ml）或内皮细胞生长补充剂（ECGS，100 μg/ml）处理人脐静脉内皮细胞（HUVEC）和BAEC时，都会出现这种效应。在无肝素的情况下以这些剂量用aFGF处理或单独用肝素处理均无显著影响。用环己酰亚胺预处理细胞层可防止在用肝素结合生长因子（HBGFs）处理细胞后钙离子依赖型黏附丧失。非钙离子依赖型黏附机制不受HBGF处理的影响。此外，内皮细胞暴露于HBGFs可防止生长中的培养物最终进入静止状态，并在已达到最终密度抑制状态的汇合培养物中重新刺激复制。以这些剂量单独添加bFGF或aFGF + 肝素，与未处理的对照培养物相比，在饱和密度下DNA合成增加了4倍，这由胸苷指数反映出来。向未处理的静止汇合BAEC单层细胞中单次添加bFGF（6 ng/ml）会导致3H-TdR掺入增加，在22小时达到峰值，同时钙离子依赖型黏附性平行丧失。用罗丹明 - 鬼笔环肽进行荧光染色显示，在bFGF处理的单层细胞中，聚合的F - 肌动蛋白分布发生改变，其特征是未处理的汇合单层细胞保留的致密周边微丝带受到破坏。总之，这些结果表明，HBGFs在培养的内皮细胞中的促有丝分裂作用与一组“形态发生”反应相关，这可能依赖于钙依赖型细胞 - 细胞接触的破坏。
