Bone morphogenetic proteins (BMPs), originally identified as osteoinductive components in extracts derived from bone, are now known to play important roles in a wide array of processes during formation and maintenance of various organs including bone, cartilage, muscle, kidney, and blood vessels. BMPs and the related “growth and differentiation factors” (GDFs) are members of the transforming growth factor β (TGF-β) family, and transduce their signals through type I and type II serine–threonine kinase receptors and their intracellular downstream effectors, including Smad proteins. Furthermore, BMP signals are finely tuned by various agonists and antagonists. Because deregulation of the BMP activity at multiple steps in signal transduction is linked to a wide variety of human diseases, therapeutic use of activators and inhibitors of BMP signaling will provide potential avenues for the treatment of the human disorders that are caused by hypo- and hyperactivation of BMP signals, respectively.

Members of the bone morphogenetic protein (BMP) family have been implicated in the development and maintenance of vascular systems. Whereas members of the BMP-2/4 and osteogenic protein-1 groups signal via activin receptor-like kinase (ALK)-2, ALK-3 and ALK-6, BMP-9 and BMP-10 have been reported to bind to ALK-1 in endothelial cells. However, the roles of BMP-9–ALK-1 signaling in the regulation of endothelial cells have not yet been fully elucidated. Here, using various systems, we examined the effects of BMP-9 on the proliferation of endothelial cells. Vascular-tube formation from ex vivo allantoic explants of mouse embryos was promoted by BMP-9. BMP-9, as well as BMP-4 and BMP-6, also induced the proliferation of in-vitro-cultured mouse embryonic-stem-cell-derived endothelial cells (MESECs) by inducing the expression of vascular endothelial growth factor receptor 2 and Tie2, a receptor for angiopoietin-1. A decrease in ALK-1 expression or expression of constitutively active ALK-1 in MESECs abrogated and mimicked the effects of BMP-9 on the proliferation of MESECs, respectively, suggesting that BMP-9 promotes the proliferation of these cells via ALK-1. Furthermore, in vivo angiogenesis was promoted by BMP-9 in a Matrigel plug assay and in a BxPC3 xenograft model of human pancreatic cancer. Consistent with these in vivo findings, BMP-9 enhanced the proliferation of in-vitro-cultured normal endothelial cells from dermal tissues of adult mice and of tumor-associated endothelial cells isolated from tumor xenografts in host mice. These findings suggest that BMP-9 signaling activates the endothelium tested in the present study via ALK-1.

The tumor microenvironment (TME) consists of various components including cancer cells, tumor vessels, cancer‐associated fibroblasts (CAFs), and inflammatory cells. These components interact with each other via various cytokines, which often induce tumor progression. Thus, a greater understanding of TME networks is crucial for the development of novel cancer therapies. Many cancer types express high levels of TGF‐β, which induces endothelial‐to‐mesenchymal transition (EndMT), leading to formation of CAFs. Although we previously reported that CAFs derived from EndMT promoted tumor formation, the molecular mechanisms underlying these interactions remain to be elucidated. Furthermore, tumor‐infiltrating inflammatory cells secrete various cytokines, including TNF‐α. However, the role of TNF‐α in TGF‐β‐induced EndMT has not been fully elucidated. Therefore, this study examined the effect of TNF‐α on TGF‐β‐induced EndMT in human endothelial cells (ECs). Various types of human ECs underwent EndMT in response to TGF‐β and TNF‐α, which was accompanied by increased and decreased expression of mesenchymal cell and EC markers, respectively. In addition, treatment of ECs with TGF‐β and TNF‐α exhibited sustained activation of Smad2/3 signals, which was presumably induced by elevated expression of TGF‐β type I receptor, TGF‐β2, activin A, and integrin αv, suggesting that TNF‐α enhanced TGF‐β‐induced EndMT by augmenting TGF‐β family signals. Furthermore, oral squamous cell carcinoma‐derived cells underwent epithelial‐to‐mesenchymal transition (EMT) in response to humoral factors produced by TGF‐β and TNF‐α‐cultured ECs. This EndMT‐driven EMT was blocked by inhibiting the action of TGF‐βs. Collectively, our findings suggest that TNF‐α enhances TGF‐β‐dependent EndMT, which contributes to tumor progression.

Endothelial–mesenchymal transition (EndMT) plays important roles in various physiological and pathological processes. While signals mediated by transforming growth factor (TGF)-β have been implicated in EndMT, the molecular mechanisms underlying it remain to be fully elucidated. Here, we examined the effects of TGF-β signals on the EndMT of mouse pancreatic microvascular endothelial cells (MS-1). By addition of TGF-β2, MS-1 cells underwent mesenchymal transition characterized by re-organization of actin stress fibre and increased expression of various mesenchymal markers such as α-smooth muscle actin (α-SMA) through activation of Rho signals. Whereas activation of Rho signals via TGF-β-induced non-Smad signals has been implicated in epithelial–mesenchymal transition (EMT), we found that Arhgef5, a guanine nucleotide exchange factor, is induced by Smad signals and contributes to the TGF-β2-induced α-SMA expression in MS-1 cells. We also found that TGF-β2 induces the expression of myocardin-related transcription factor-A (MRTF-A) in a Smad-dependent fashion and its nuclear accumulation in MS-1 cells and that MRTF-A is required and sufficient for TGF-β2-induced α-SMA expression. These results indicate that activation of Smad signals by TGF-β2 have dual effects on the activation of Rho signals and MRTF-A leading to the mesenchymal transition of MS-1 endothelial cells.

Most cardiac diseases caused by inflammation are associated with fibrosis in the heart. Fibrosis is characterized by the accumulation of fibroblasts and excess deposition of extracellular matrix (ECM), which results in the distorted organ architecture and function. Recent studies revealed that cardiac fibroblasts are heterogeneous with multiple origins. Endothelial-mesenchymal transition (EndMT) plays important roles in the formation of cardiac fibroblasts during pathological settings. EndMT is regulated by signaling pathways mediated by cytokines including transforming growth factor (TGF)-β. Better understanding of the mechanisms of the formation of cardiac fibroblasts via EndMT may provide an opportunity to develop therapeutic strategies to cure heart diseases.