The latent transforming growth factor-beta-binding protein-1 promotes in vitro differentiation of embryonic stem cells into endothelium

The latent transforming growth factor-beta -binding protein-1 (LTBP-1) belo ngs to a family of extracellular glycoproteins that includes three addition al isoforms (LTBP-2 -3, and -4) and the matrix proteins fibrillin-1 and -2. Originally described as a TGF-P-masking protein, LTBP-1 is involved both i n the sequestration of latent TGF-P in the extracellular matrix and the reg ulation of its activation in the extracellular environment. Whereas the exp ression of LTBP-1 has been analyzed in normal and malignant cells and roden t and human tissues, little is known about LTBP-1 in embryonic development. To address this question, we used murine embryonic stem (ES) cells to anal yze the appearance and role of LTBP-1 during ES cell differentiation. In vi tro, ES cells aggregate to form embryoid bodies (EBs), which differentiate into multiple cell lineages. We analyzed LTBP-1 gene expression and LTBP-1 fiber appearance with respect to the emergence and distribution of cell typ es in differentiating EBs. LTBP-1 expression increased during the first 12 d in culture, appeared to remain constant between d 12 and 24, and declined thereafter. By immunostaining, fibrillar LTBP-1 was observed in those regi ons of the culture containing endothelial, smooth muscle, and epithelial ce lls. We found that inclusion of a polyclonal antibody to LTBP-1 during EB d ifferentiation suppressed the expression of the endothelial specific genes ICAM-2 and von Willebrand factor and delayed the organization of differenti ated endothelial cells into cord-like structures within the growing EBs. Th e same effect was observed when cultures were treated with either antibodie s to TGF-beta or the latency associated peptide, which neutralize TGF-beta. Conversely, the organization of endothelial cells was enhanced by incubati on with TGF-beta1. These results suggest that during differentiation of ES cells LTBP-1 facilitates endothelial cell organization via a TGF-beta -depe ndent mechanism.