Progressive modulation of endothelial phenotype during in vitro angiogenesis

Cultures of endothelial cells expressing the sprouting phenotype were used as an in vitro model system to study the progressive morphologic and biosynthetic changes associated with the formation of tubular, capillary-like structures. In vitro, sprouting endothelial cells formed spontaneously from cultures of cloned endothelium exhibiting a confluent polygonal phenotype without the addition of any exogenous factors. These phenotypically variant endothelial cells progressed to form reticular networks which gradually reorganized into tubular structures. Concomitant with these morphologic alterations, the biosynthesis of extracellular matrix proteins was modulated, as determined by Northern blot analysis, metabolic labeling and immunocytochemistry. The initial sprouting phase was characterized by the induction of type I collagen synthesis and the appearance of a fibronectin isoform containing the ED-A domain. Analysis of RNAs from sprouting versus polygonal cultures revealed the preferential synthesis of the ED-A and ED-B fibronectin isoforms by sprouting endothelial cells. Sprouting cells also expressed the $\alpha$1 (I) polypeptide chain of type I collagen which is not synthesized by nonsprouting cells. Translation of type I collagen and the ED-A containing fibronectin isoform RNAs were confirmed at the protein level by gel electrophoresis of newly synthesized proteins and/or immunohistochemistry. The organizational stage where sprouting cells organized into tubular structures was additionally characterized by the expression of type IV collagen and the ED-A containing isoform of fibronectin. Additionally, the formation of tubular structures by endothelial cells could be blocked by incubation of the cells with antibodies either to type I collagen or the ED-A domain of fibronectin. Heparin specifically inhibited the synthesis of type III collagen in endothelial cell cultures, regardless of their morphologic phenotype and additionally appeared to stabilize the normal polygonal morphology. However, in the presence of type I collagen fibers, heparin enhanced the formation of tubular structures. These studies demonstrate that the progression from polygonal to sprouting cultures and subsequent tubular organization involves major alterations in extracellular matrix protein expression, emphasizing the importance of these macromolecules in the angiogenic process