Yasuhiro Yoshimatsu

COUP-TFII regulates the functions of Prox1 in lymphatic endothelial cells through direct interaction

During embryonic lymphatic development, Prox1 homeobox transcription factor is expressed in a subset of venous blood vascular endothelial cells (BECs) in which COUP‐TFII orphan nuclear receptor is highly expressed. Prox1 induces differentiation of BECs into lymphatic endothelial cells (LECs) by inducing the expression of various LEC markers including vascular endothelial growth factor receptor 3 (VEGFR3). However, the molecular mechanisms of how transcriptional activities of Prox1 are regulated are largely unknown. In the present study, we show that COUP‐TFII plays important roles in the regulation of the function of Prox1. In BECs and LECs, Prox1 promotes the proliferation and migration toward VEGF‐C by inducing the expression of cyclin E1 and VEGFR3, respectively. Gain‐of‐function studies showed that COUP‐TFII negatively regulates the effects of Prox1 in BECs and LECs whereas loss‐of‐function studies showed that COUP‐TFII negatively and positively regulates Prox1 in BECs and LECs, respectively. We also show that endogenous Prox1 and COUP‐TFII physically interact in LECs and that both Prox1 and COUP‐TFII bind to the endogenous cyclin E1 promoter. These results suggest that COUP‐TFII physically and functionally interact during differentiation and maintenance of lymphatic vessels.

TGF-β-induced mesenchymal transition of MS-1 endothelial cells requires Smad-dependent cooperative activation of Rho signals and MRTF-A

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.

Roles of TGF-β Signals in Endothelial-Mesenchymal Transition during Cardiac Fibrosis

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.

Bone morphogenetic protein-9 inhibits lymphatic vessel formation via activin receptor-like kinase 1 during development and cancer progression

Significance Because lymphatic vessels (LVs) play critical roles not only in physiological processes such as maintenance of fluid homeostasis but also in pathological conditions including cancer metastasis, identification of factors that control LV formation is crucial. Signals mediated by activin receptor-like kinase 1 (ALK-1), a receptor for bone morphogenetic protein 9 (BMP-9), have been implicated in the formation of blood vessels because of its linkage to a human vascular disease. However, their roles in LV formation largely remain to be elucidated. Here, we show that BMP-9/ALK-1 signals inhibit LV formation in physiological and pathological conditions. Furthermore, we elucidated its molecular mechanisms in detail, using both in vivo and in vitro systems. These findings will help develop therapeutic strategies for LV-related diseases such as lymphedema and cancer. Lymphatic vessels (LVs) play critical roles in the maintenance of fluid homeostasis and in pathological conditions, including cancer metastasis. Although mutations in ALK1, a member of the transforming growth factor (TGF)-β/bone morphogenetic protein (BMP) receptor family, have been linked to hereditary hemorrhagic telangiectasia, a human vascular disease, the roles of activin receptor-like kinase 1 (ALK-1) signals in LV formation largely remain to be elucidated. We show that ALK-1 signals inhibit LV formation, and LVs were enlarged in multiple organs in Alk1-depleted mice. These inhibitory effects of ALK-1 signaling were mediated by BMP-9, which decreased the number of cultured lymphatic endothelial cells. Bmp9-deficient mouse embryos consistently exhibited enlarged dermal LVs. BMP-9 also inhibited LV formation during inflammation and tumorigenesis. BMP-9 downregulated the expression of the transcription factor prospero-related homeobox 1, which is necessary to maintain lymphatic endothelial cell identity. Furthermore, silencing prospero-related homeobox 1 expression inhibited lymphatic endothelial cell proliferation. Our findings reveal a unique molecular basis for the physiological and pathological roles of BMP-9/ALK-1 signals in LV formation.

Ets family members induce lymphangiogenesis through physical and functional interaction with Prox1

Prox1 plays pivotal roles during embryonic lymphatic development and maintenance of adult lymphatic systems by modulating the expression of various lymphatic endothelial cell (LEC) markers, such as vascular endothelial growth factor receptor 3 (VEGFR3). However, the molecular mechanisms by which Prox1 transactivates its target genes remain largely unknown. Here, we identified Ets-2 as a candidate molecule that regulates the functions of Prox1. Whereas Ets-2 has been implicated in angiogenesis, its roles during lymphangiogenesis have not yet been elucidated. We found that endogenous Ets-2 interacts with Prox1 in LECs. Using an in vivo model of chronic aseptic peritonitis, we found that Ets-2 enhanced inflammatory lymphangiogenesis, whereas a dominant-negative mutant of Ets-1 suppressed it. Ets-2 also enhanced endothelial migration towards VEGF-C through induction of expression of VEGFR3 in collaboration with Prox1. Furthermore, we found that both Prox1 and Ets-2 bind to the VEGFR3 promoter in intact chromatin. These findings suggest that Ets family members function as transcriptional cofactors that enhance Prox1-induced lymphangiogenesis.

Identification of targets of Prox1 during in vitro vascular differentiation from embryonic stem cells: Functional roles of HoxD8 in lymphangiogenesis

During lymphatic development, Prox1 plays central roles in the differentiation of blood vascular endothelial cells (BECs) into lymphatic endothelial cells (LECs), and subsequently in the maturation and maintenance of lymphatic vessels. However, the molecular mechanisms by which Prox1 elicits these functions remain to be elucidated. Here, we identified FoxC2 and angiopoietin-2 (Ang2), which play important roles in the maturation of lymphatic vessels, as novel targets of Prox1 in mouse embryonic-stem-cell-derived endothelial cells (MESECs). Furthermore, we found that expression of HoxD8 was significantly induced by Prox1 in MESECs, a finding confirmed in human umbilical vein endothelial cells (HUVECs) and human dermal LECs (HDLECs). In mouse embryos, HoxD8 expression was significantly higher in LECs than in BECs. In a model of inflammatory lymphangiogenesis, diameters of lymphatic vessels of the diaphragm were increased by adenovirally transduced HoxD8. We also found that HoxD8 induces Ang2 expression in HDLECs and HUVECs. Moreover, we found that HoxD8 induces Prox1 expression in HUVECs and that knockdown of HoxD8 reduces this expression in HDLECs, suggesting that Prox1 expression in LECs is maintained by HoxD8. These findings indicate that transcriptional networks of Prox1 and HoxD8 play important roles in the maturation and maintenance of lymphatic vessels.

Expression of platelet‐derived growth factor receptor β is maintained by Prox1 in lymphatic endothelial cells and is required for tumor lymphangiogenesis

The lymphatic system plays important roles not only in the physiological processes, such as maintenance of tissue fluid homeostasis, but also in pathological processes including the lymph node metastasis of tumor cells. Therefore, understanding of the molecular mechanisms underlying lymphatic vessel formation is crucial. Previous studies have shown that proliferation and migration of lymphatic endothelial cells (LECs) are activated by multiple types of signals mediated by tyrosine kinase receptors such as vascular endothelial growth factor receptor (VEGFR) 3. Although signals mediated by platelet‐derived growth factor receptor β (PDGFRβ) have been implicated in lymphangiogenesis, the mechanisms explaining how PDGFRβ expression is maintained in LECs remain to be fully elucidated. In the present study, we show that PDGFRβ expression in LECs is maintained by Prox1 transcription factor. Knockdown of Prox1 expression in human dermal LECs decreased the expression of PDGFRβ, leading to the lowered migration of human dermal LECs towards PDGF‐BB. Furthermore, we found that PDGF signals play important roles in inflammatory lymphangiogenesis in a chronic aseptic peritonitis model. Intraperitoneal administration of imatinib, a potent inhibitor of PDGFRβ, and transduction of PDGFRβ/Fc chimeric protein by an adenoviral system both reduced inflammatory lymphangiogenesis induced by thioglycollate in mice. We also found that the expression of PDGFRβ/Fc reduced tumor lymphangiogenesis in a BxPC3 human pancreatic cancer xenograft model. These findings suggest that PDGFRβ is one of the key mediators of lymphatic vessel formation acting downstream of Prox1.

Vasohibin-2 is required for epithelial-mesenchymal transition of ovarian cancer cells by modulating TGF-β signaling.

Vasohibin‐2 (VASH2) is a homolog of VASH1, an endothelium‐derived angiogenesis inhibitor. Vasohibin‐2 is mainly expressed in cancer cells, and has been implicated in the progression of cancer by inducing angiogenesis and tumor growth. Although VASH2 has been recently reported to be involved in epithelial–mesenchymal transition (EMT), its precise roles are obscure. The aim of the present study was to clarify the role of VASH2 in the EMT of cancer cells in relation to transforming growth factor‐β (TGF‐β) signaling, which is a major stimulator of EMT. Decreased expression of VASH2 in ovarian cancer cells significantly repressed the expression of TGF‐β type I receptor, namely activin receptor‐like kinase 5. Transforming growth factor‐β1‐induced phosphorylation of Smad2 and Smad3 was markedly decreased in VASH2 knockdown cells while the expression of Smad2 and Smad3 was unchanged. Accordingly, the responses to TGF‐β1 shown by promoter assay and plasminogen activator inhibitor type 1 expression were significantly attenuated in VASH2 knockdown cells. Furthermore, knockdown of VASH2 in cancer cells abrogated the TGF‐β1‐induced reduced expression of epithelial markers including E‐cadherin, and the elevated expression of mesenchymal markers including fibronectin, ZEB2, and Snail2, suggesting that endogenous VASH2 is required for TGF‐β1‐induced EMT. In accordance with these results, the effects of TGF‐β1 on cell morphology, migration, invasion, and MMP2 expression were also abrogated when VASH2 was knocked down. These results indicate that VASH2 played a significant role in the EMT by modulating the TGF‐β signaling. We propose that VASH2 would be a novel molecular target for the prevention of EMT in cancers.

Dual targeting of vascular endothelial growth factor and bone morphogenetic protein-9/10 impairs tumor growth through inhibition of angiogenesis

Clinical development of anti‐angiogenic agents has been a major landmark in cancer therapy for several types of cancers. Signals mediated by both vascular endothelial growth factor (VEGF) and bone morphogenetic protein (BMP)‐9 and 10 have been implicated in tumor angiogenesis. However, previous studies have shown that targeting the individual signals was not sufficiently effective in retarding tumor growth in certain preclinical and clinical conditions. In the present study, we developed a novel decoy chimeric receptor that traps both VEGF and BMP‐9/10. Single targeting of either VEGF or BMP‐9/10 signals significantly reduced the formation of tumor vessels in a mouse xenograft model of human pancreatic cancer; however, it did not show significant therapeutic effects on tumor growth. In contrast, dual targeting of the angiogenic signals resulted in more significant inhibition of tumor angiogenesis, leading to delay of tumor growth. Our findings suggest that simultaneous blockade of VEGF and BMP‐9/10 signals is a promising therapeutic strategy for the cancers that are resistant to anti‐VEGF and BMP‐9/10 therapies.

Abstract 5276: Roles of BMP-9 signals during the formation of tumor vessels

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Angiogenesis plays critical roles during development and progression of cancer. Members of the bone morphogenetic protein (BMP) family are involved in TGF-beta superfamily, and have been implicated in the development and maintenance of vascular systems. While BMP-9 and 10 have been recently reported to bind to ALK-1 in endothelial cells, the roles of BMP-9/ALK-1 signaling in the regulation of endothelial cells have not yet been fully elucidated. Here, we examined the effects of BMP-9 on the proliferation of endothelial cells using various systems. Vascular tube formation from ex vivo allantoic explants of mouse embryos was promoted by BMP-9. BMP-9 also induced the proliferation of in vitro-cultured mouse embryonic stem cell-derived endothelial cells (MESEC) by inducing the expression of vascular endothelial growth factor receptor 2 and Tie2, a receptor for Angiopoietin-1. Decrease in ALK-1 expression and expression of constitutively active ALK-1 in MESEC abrogated and mimicked the effects of BMP-9 on the proliferation of MESEC, respectively, suggesting that BMP-9 promotes their proliferation via ALK-1. Furthermore, in vivo angiogenesis was promoted by BMP-9 in a Matrigel plug assay and a BxPC3 human pancreatic cancer xenograft model. In 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 tumor-associated endothelial cells isolated from tumor xenografts in host mice. These findings suggest that BMP-9 signaling activates the endothelium via ALK-1. In this presentation, the roles of BMP-9 signals in the formation of lymphatic vessels will also be discussed. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5276. doi:1538-7445.AM2012-5276