Coordinate Induction of the Actin Cytoskeletal Regulatory Proteins Gelsolin, Vasodilator-Stimulated Phosphoprotein, and Profilin during Capillary Morphogenesisin Vitro

Abstract

The formation of capillaries during development and tissue repair is likely to involve active reorganization of the actin cytoskeleton, although few studies have addressed this issue. Here, we have utilized anin vitromodel of capillary morphogenesis whereby human umbilical vein endothelial cells are suspended within three-dimensional type I collagen gels. The cells undergo dramatic morphogenic changes to develop capillary lumens, tubes, and networks over 72 h of culture. Western blots using cell extracts of these gels over this time frame were performed using antibodies directed to various proteins associated with the actin cytoskeleton. Three proteins showed altered expression during the time course, and they were gelsolin, which increased fivefold; vasodilator-stimulated phosphoprotein (VASP), which increased twofold; and profilin, which increased threefold in expression between the 24- and the 72-h time points. Reverse transcriptase-polymerase chain reaction and Northern blot analysis revealed a similar increase in mRNA expression of the three proteins. After the onset of network formation, the differentiated endothelial cells (dECs) undergoing capillary morphogenesis were removed from collagen gels at 48 h of culture to compare their properties with untreated endothelial cells (uECs). These dECs showed two- to threefold increased spontaneous migration in Boyden chamber assays compared to uECs. The dECs also displayed a prominent spindle-shaped morphology and the novel presence of intranuclear gelsolin compared to uECs when both cell types were replated on type I collagen-coated microwells and glass coverslips. These data suggest that increased gelsolin, VASP, and profilin expression may play an important role in the regulation of capillary tube and network formation in three-dimensional extracellular matrix.