The Extracellular Matrix Produced during Angiogenesis in Culture
Rings of rat aorta embedded in thick plasma clot and cultured either in Eagle’s minimum essential medium or in Waymouth’s MB 752/1, each supplemented with 20% fetal bovine serum, 50 μg/ml ascorbic acid, and antibiotics, gave rise to cellular outgrowths composed of branching microvessels and spindly mesenchymal cells. Affinity-purified rabbit antibodies specific for fibronectin, laminin, collagen types I, III, IV, and V were used to study the composition and distribution of the extracellular matrix produced in these cultures. Frozen sections obtained from 1-to 4-week-old cultures were mounted on albumin-coated histology slides and stained using indirect immunofluoresence. immunoperoxidase and peroxidase-antiperoxidase techniques. Fibronectin, laminin, and collagen types IV and V were demonstrated both in immature endothelial sprouts and in mature endothelial channels. The staining of fibronectin was particularly intense in 7-day-old cultures, suggesting an increased production of fibronectin during the early stages of angiogenesis. The staining of laminin and collagen type IV was more intense in older cultures, indicating the formation of a continuous basement membrane around the mature endothelial channels. Immunoperoxidase stain demonstrated fibrils of collagen type I and III around the endothelial outgrowths and in the surrounding fibrin. The amount of collagen type I and III increased as the cultures aged, and it was greatest in 28-day-old cultures. In summary, this study demonstrates that the formation of new endothelial channels in plasma clot cultures of rat aorta is associated with the production of an abundant and heterogeneous extracellular matrix that strongly resembles the extracellular matrix of microvessels in vivo. Future studies with this model may provide new insights into the role played by extracellular matrix proteins in angiogenesis and may help us elucidate the effect of these proteins on the perivascular spread of carcinoma cells.
KeywordsCollagen Type Capillary Endothelial Cell Plasma Clot Type Versus Collagen Vascular Sprout
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