L28	TGF-beta in endothelial cell function and vascular development.
1P.ten Dijke, 2R.Carvalho, 3M-J.Goumans, 2F.Lebrin, 4H.Arthur, 2Chr.Mummery
1Leiden University Medical Center, Leiden, NL; 2Institute for Developmental Biology, Utrecht, NL; 3Experimental Cardiology, Utrecht University, Utrecht, NL; 4Institute for Human Genetics, University of Newcastle upon Tyne, Newcastle upon Tyne, GB.

Transforming growth factor b1 (TGF-b1) is a prototype of a large family of structurally related pleiotropic cytokines with pivotal roles in development and tissue homeostasis. TGF-b initiates its diverse cellular responses by signaling via specific type I and type II serine/threonine kinase receptors and downstream Smad effector proteins. Genetic studies in mice and humans have revealed an important role for TGF-b in vascular development and maintenance. Mice deficient for various TGF-b signaling components develop an embryonic lethality due to vascular defects. In patients, mutations in the TGF-b type I receptor ALK1 or in the accessory TGF-b receptor endoglin are linked to an autosomal dominant disorder of vascular dysplasia termed Hereditary Haemorrhagic Telangiectasia (HHT). The highly context dependent properties and multifunctional actions of TGFb and its actions on many cell types distinct from vascular cells, e.g. epithelial and immune cells, have made the roles of TGFb in vascular biology difficult to interpret. TGF-b has potent direct effects on endothelial cells and smooth muscle cells. TGF-b, produced by endothelial cells, can be activated from its latent form upon contact between pericytes or smooth muscle cells with endothelial cells. TGF-b is critical for differentiation of mural precurors into pericytes and smooth muscle cells. TGFb can induce the expression of various smooth muscle-specific markers and extracellular matrix genes in mesenchymal cells. Recently, we have proposed a model in which TGF-b regulates the activation state of the endothelium via two opposing type I receptor/Smad pathways: ALK1 induces Smad1/5 phosphorylation, leading to an increase in endothelial cell proliferation, migration and invasion, while ALK5 promotes Smad2/3 activation and inhibits these processes. Interestingly, we found that ALK5 is important for TGF-b/ALK1 signaling; endothelial cells lacking ALK5 are deficient in TGF-b/ALK1-induced responses. Interestingly, ALK1 not only induces a biological response opposite to that of ALK5, it potently inhibited ALK5-induced transcriptional responses. The requirement for ALK5 in ALK1 signaling and counteractive interplay between ALK5 and ALK1 provides the endothelial cell with a TGF-b controlled switch. Endoglin is predominantly expressed on proliferating endothelial cells in culture and on angiogenic blood vessels in vivo. Consistent with the notion that mutations in endoglin and ALK1 lead to HHT, we observed that endoglin is required for efficient TGF-b/ALK1 signaling and indirectly inhibits ALK5 signaling. To provide more insights into the underlying mechanism of multisystemic vascular dyplasia and recurrent haemorrhage phenotype of HHT caused by mutations in endoglin (or ALK1), we have analyzed TGF-b signaling in yolk sacs from endoglin knockout mice and from mice with endothelial-specific deletion of the TGF-b type II receptor (TbRII) or ALK5. We found that TGF-b/ALK5 signaling from endothelial cells to adjacent mesothelial cells is defective in these mice, as evidenced by reduced Smad2 phosphorylation. This results in the failure of vascular smooth muscle cells to differentiate and associate with endothelial cells so that blood vessels remain fragile and become dilated. Smad2 phosphorylation and differentiation of smooth muscle can be rescued by culture of the yolk sac with exogenous TGF-b1. These results may provide a possible explanation for weak vessel walls associated with HHT.

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