Transforming growth factor beta1 modulates extracellular matrix organization and cell‐cell junctional complex formation during in vitro angiogenesis

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Abstract
Transforming growth factor‐beta1 (TGF‐β1) is angiogenic in vivo. In two‐dimensional (2‐D) culture systems microvascular endothelial cell proliferation is inhibited up to 80% by TGF‐β1 however, in three‐dimensional (3‐D) collagen gels TGF‐β1 is found to have no effect on proliferation while eliciting the formation of calcium and magnesium dependent tube‐like structures mimicking angiogenesis. DNA analyses performed on 3‐D cell cultures reveal no significant difference in the amount of DNA or cell number in control versus TGF‐β1 treated cultures. In 2‐D cultures TGF‐β1 is known to increase cellular fibronectin accumulation; however, in 3‐D cultures no difference is seen between control and TGF‐β1 treated cells as established by ELISA testing for type IV collagen, fibronectin, and laminin. In 3‐D cultures there is increased synthesis and secretion of type V collagen in both control and TGF‐β1 treated cultures over 2‐D cultures. Even though an equal amount of type V collagen is seen in both 3‐D conditions, there is a reorganization of the protein with concentration along an organizing basal lamina in TGF‐β1 treated cultures. EM morphological analyses on 3‐D cultures illustrate quiescent, control cells lacking cell contacts. In contrast, TGF‐β1 treated cells show increased pseudopod formation, cell‐cell contact, and organized basal lamina‐like material closely apposed to the “abluminal” plasma membranes. TGF‐β1 treated cells also appear to form junctional complexes between adjoining cells. Immunofluorescence using specific antibodies to the tight junction protein ZO‐1 results in staining at apparent cell‐cell junctions in the 3‐D cultures. Northern blots of freshly isolated microvascular endothelium, 2‐D and 3‐D cultures, using cDNA and cRNA probes specific for the ZO‐1 tight junction protein, reveal the presence of the 7.8 kb mRNA. Western blots of rat epididymal fat pad endo‐thelial cells (RFC) monolayer lysates probed with anti‐ZO‐1 label a 220 kd band which co‐migrates with the bonafide ZO‐1 protein. These data confirm and support the hypothesis that TGF‐β1 is angiogenic in vitro, eliciting microvascular endothelial cells to form tube‐like structures with apparent tight junctions and abluminal basal lamina deposition in three‐dimensional cultures.