TGF-β₁, -β₂ and -β₃ cooperate to facilitate tubulogenesis in the explanted quail heart

Background: Transforming growth factor-β (TGF-β) isoforms have been implicated as both pro- and anti-angiogenic modulators. In this study we addressed the roles of TGF-β isoforms on coronary tubulogenesis. Methods: Embryonic (E6) quail ventricular specimens were explanted onto collagen gels allowing endothelial cells to migrate and form vascular tubes. Growth factors and/or neutralizing growth factor antibodies were added to the cultures. Endothelial cells were identified using a quail endothelial cell marker, QH1. Image analysis was used to quantify aggregate tube length. Results: Addition of any isoform (TGF-β₁, TGF-β₂ or TGF-β₃) virtually prevented tubulogenesis (>95% inhibition), while stimulation of tubulogenesis occurred by adding neutralizing antibodies to TGF-β₃, but not to TGF-β₁ or -β₂. When all three isoforms were added, tubulogenesis was enhanced, indicating the key role of TGF-β₃. Documentation of the inhibitory effect of TGF-β isoforms on tubulogenesis is further supported by our experiments in which the marked enhancement of tube formation by bFGF and VEGF was negated when exogenous TGF-β₁, -β₂, or -β₃ were added to the cultures. Conclusions: (1) TGF-β₁, -β₂ and -β₃ each inhibits angiogenesis; (2) cooperation between the three TGF-β isoforms and other angiogenic factors is essential for the regulation of normal tubulogenesis and (3) the stimulatory effect of VEGF or bFGF on tubulogenesis is negated by exogenous TGB-βs.