Bone Morphogenetic Protein 9 Regulates Early Lymphatic-Specified Endothelial Cell Expansion during Mouse Embryonic Stem Cell Differentiation.

Exogenous cues involved in the regulation of the initial steps of lymphatic endothelial development remain largely unknown. We have used an in vitro model based on the co-culture of vascular precursors derived from mouse embryonic stem cell (ESC) differentiation and OP9 stromal cells to examine the first steps of lymphatic specification and expansion. We found that bone morphogenetic protein 9 (BMP9) induced a dose-dependent biphasic effect on ESC-derived vascular precursors. At low concentrations, below 1 ng/mL, BMP9 expands the LYVE-1-positive lymphatic progeny and activates the calcineurin phosphatase/NFATc1 signaling pathway. In contrast, higher BMP9 concentrations preferentially enhance the formation of LYVE-1-negative endothelial cells. This effect results from an OP9 stromal cell-mediated VEGF-A secretion. RNA-silencing experiments indicate specific involvement of ALK1 and ALK2 receptors in these different BMP9 responses. BMP9 at low concentrations may be a useful tool to generate lymphatic endothelial cells from stem cells for cell-replacement strategies.

Bone morphogenetic protein 9 (BMP9) controls lymphatic vessel maturation and valve formation.

Lymphatic vessels are critical for the maintenance of tissue fluid homeostasis and their dysfunction contributes to several human diseases. The activin receptor-like kinase 1 (ALK1) is a transforming growth factor-ß family type 1 receptor that is expressed on both blood and lymphatic endothelial cells (LECs). Its high-affinity ligand, bone morphogenetic protein 9 (BMP9), has been shown to be critical for retinal angiogenesis. The aim of this work was to investigate whether BMP9 could play a role in lymphatic development. We found that Bmp9 deficiency in mice causes abnormal lymphatic development. Bmp9-knockout (KO) pups presented hyperplastic mesenteric collecting vessels that maintained LYVE-1 expression. In accordance with this result, we found that BMP9 inhibited LYVE-1 expression in LECs in an ALK1-dependent manner. Bmp9-KO pups also presented a significant reduction in the number and in the maturation of mesenteric lymphatic valves at embryonic day 18.5 and at postnatal days 0 and 4. Interestingly, the expression of several genes known to be involved in valve formation (Foxc2, Connexin37, EphrinB2, and Neuropilin1) was upregulated by BMP9 in LECS. Finally, we demonstrated that Bmp9-KO neonates and adult mice had decreased lymphatic draining efficiency. These data identify BMP9 as an important extracellular regulator in the maturation of the lymphatic vascular network affecting valve development and lymphatic vessel function.

A new function of the splicing factor SRSF2 in the control of E2F1-mediated cell cycle progression in neuroendocrine lung tumors.

The transcription factor E2F1 belongs to the E2F family and plays a crucial role during cell cycle progression and apoptosis. Ser/Arg-Rich (SR) proteins are a family of RNA-binding phosphoproteins that control both constitutive and alternative pre-mRNA splicing events. We previously identified the SR protein SRSF2 as a new transcriptional target of E2F1 and demonstrated that both proteins cooperate to induce apoptosis in non-small cell lung carcinoma. In this study, we postulated that SRSF2 is also involved in the proliferative functions of E2F1. Using IHC, we first demonstrate that SRSF2 and its phosphorylated form (P-SRSF2) are overexpressed in neuroendocrine lung tumors that are highly proliferative tumors expressing high levels of E2F1. Importantly, we show a direct correlation between cyclin E, an E2F1-target gene controlling S phase, and P-SRSF2 proteins levels (p = 0.0083), suggesting a role of SRSF2 in E2F1-mediated cellular proliferation. Accordingly, using neuroendocrine lung carcinoma cell lines, we demonstrate that SRSF2 is a cell cycle-regulated protein involved in entry and progression into S phase. We also provide evidence that SRSF2 interacts with E2F1 and stimulates its transcriptional control of cell cycle target genes such as cyclin E. Finally, we show that inhibition of AKT signaling pathway prevents SRSF2 phosphorylation and activity toward E2F1 transcriptional function. Taken together, these results identify a new role of SRSF2 in the control of cell cycle progression and reinforce the functional link between SRSF2 and E2F1 proteins.

TGFß1 inhibits lymphatic endothelial cell differentiation from mouse embryonic stem cells.

The lymphatic vasculature is essential for the maintenance of tissue fluid, immune surveillance, and dissemination of metastasis. Recently, several models for lymphatic vascular research and markers specific for lymphatic endothelium have been characterized. Despite these significant achievements, our understanding of the early lymphatic development is still rather limited. The purpose of the study was to further define early lymphatic differentiation regulatory pathways. In the present study, we have developed conditions leading to lymphatic endothelial cell differentiation under both serum-rich and serum-free conditions, using the coculture system of Flk-1-positive vascular precursors derived from murine embryonic stem (ES) cells grown on an OP9 stromal cell layer. In this work, we also identified Transforming Growth Factor-ß1 (TGFß1) as a negative regulator of lymphvasculogenesis from ES-derived vascular progenitors. Finally, we could show that TGFß1 addition decreases COUP-TFII and Sox18 mRNA levels, which are two transcription factors known to be involved in early lymphatic endothelial differentiation. Taken together these findings support the concept that manipulating the TGFß signaling pathway may represent an interesting target to favor lymphatic endothelial cell expansion for cell replacement strategies.