Time Course and Quantification of Extracellular Matrix Maturation in the Chick Chorioallantoic Membrane and in Cultured Endothelial Cells

Neovascularization, i.e. the formation of new blood vessels either from pre-existing ones or from mesenchymal cells, is associated with the deposition of a subendothelial basement membrane. The sequential expression of extracellular matrix (ECM) proteins, such as fibronectin, laminin, collagen-IV and collagen-I, was quantified both in vivo, in the chick chorioallantoic membrane (CAM) using Western blotting techniques, and in vitro, in cultured rat adrenal medullary endothelial (RAME) cells, using indirect immunofluorescence and enzyme-linked immunoadsorption techniques; the combination of these techniques allowed discrimination between extracellularly and intracellularly located proteins. In the CAM, fibronectin expression increased transiently with the peak at day 7 of development, after which it decreased gradually. By contrast, deposition of laminin and collagen-I increased steadily during development. Quantification at later stages of CAM development showed the predominance of collagen-I, whereas laminin comprised only a minor component of the ECM proteins. A similar temporal sequence of ECM protein expression was observed with RAME cells in vitro. Fibronectin was the first protein to appeal extracellularly and its expression was also transient. The levels of laminin and collagen-IV increased steadily during culture, to reach a plateau after confluency. Collagen-IV was the most abundant ECM protein of those studied. Both laminin and collagen-IV were synthesized from the onset of culture, but they were deposited into the ECM only after cell-cell contacts were established. Ascorbate, a known promoter of in vitro angiogenesis, was shown to have a differential effect on ECM protein expression.