Actin-Tethered Junctional Complexes in Angiogenesis and Lymphangiogenesis in Association with Vascular Endothelial Growth Factor

Vasculature is present in all tissues and therefore is indispensable for development, biology, and pathology of multicellular organisms. Endothelial cells guarantee proper function of the vessels and are the original component in angiogenesis. Morphogenesis of the vascular system utilizes processes like cell adhesion, motility, proliferation, and survival that are closely related to the dynamics of actin filaments and actin-tethered adhesion complexes. Here we review involvement of actin cytoskeletonassociated junctional molecules of endothelial cells in angiogenesis and lymphangiogenesis. Particularly, we focus on F-actin binding protein afadin, an adaptor protein involved in broad range of signaling mechanisms. Afadin mediates the pathways of vascular endothelial growth factor-(VEGF-) and sphingosine 1-phosphate-triggered angiogenesis and is essential for embryonic development of lymph vessels in mice. We propose that targeting actin-tethered junctional molecules, including afadin, may present a new approach to angiogenic therapy that in combination with today used medications like VEGF inhibitors will benefit against development of pathological angiogenesis

Actin-Tethered Junctional Complexes in Angiogenesis and Lymphangiogenesis in Association with Vascular Endothelial Growth Factor

Vasculature is present in all tissues and therefore is indispensable for development, biology, and pathology of multicellular organisms. Endothelial cells guarantee proper function of the vessels and are the original component in angiogenesis. Morphogenesis of the vascular system utilizes processes like cell adhesion, motility, proliferation, and survival that are closely related to the dynamics of actin filaments and actin-tethered adhesion complexes. Here we review involvement of actin cytoskeleton-associated junctional molecules of endothelial cells in angiogenesis and lymphangiogenesis. Particularly, we focus on F-actin binding protein afadin, an adaptor protein involved in broad range of signaling mechanisms. Afadin mediates the pathways of vascular endothelial growth factor- (VEGF-) and sphingosine 1-phosphate-triggered angiogenesis and is essential for embryonic development of lymph vessels in mice. We propose that targeting actin-tethered junctional molecules, including afadin, may present a new approach to angiogenic therapy that in combination with today used medications like VEGF inhibitors will benefit against development of pathological angiogenesis

Vascular Endothelial Growth Factor-A Exerts Diverse Cellular Effects via Small G Proteins, Rho and Rap

Vascular endothelial growth factors (VEGFs) include five molecules (VEGF-A, -B, -C, -D, and placental growth factor), and have various roles that crucially regulate cellular functions in many kinds of cells and tissues. Intracellular signal transduction induced by VEGFs has been extensively studied and is usually initiated by their binding to two classes of transmembrane receptors: receptor tyrosine kinase VEGF receptors (VEGF receptor-1, -2 and -3) and neuropilins (NRP1 and NRP2). In addition to many established results reported by other research groups, we have previously identified small G proteins, especially Ras homologue gene (Rho) and Ras-related protein (Rap), as important mediators of VEGF-A-stimulated signaling in cancer cells as well as endothelial cells. This review article describes the VEGF-A-induced signaling pathways underlying diverse cellular functions, including cell proliferation, migration, and angiogenesis, and the involvement of Rho, Rap, and their related molecules in these pathways

An Adaptor Molecule Afadin Regulates Lymphangiogenesis by Modulating RhoA Activity in the Developing Mouse Embryo.

Afadin is an intracellular binding partner of nectins, cell-cell adhesion molecules, and plays important roles in the formation of cell-cell junctions. Afadin-knockout mice show early embryonic lethality, therefore little is known about the function of afadin during organ development. In this study, we generated mice lacking afadin expression in endothelial cells, and found that the majority of these mice were embryonically lethal as a result of severe subcutaneous edema. Defects in the lymphatic vessels of the skin were observed, although the morphology in the blood vessels was almost normal. Severe disruption of VE-cadherin-mediated cell-cell junctions occurred only in lymphatic endothelial cells, but not in blood endothelial cells. Knockout of afadin did not affect the differentiation and proliferation of lymphatic endothelial cells. Using in vitro assays with blood and lymphatic microvascular endothelial cells (BMVECs and LMVECs, respectively), knockdown of afadin caused elongated cell shapes and disruption of cell-cell junctions among LMVECs, but not BMVECs. In afadin-knockdown LMVECs, enhanced F-actin bundles at the cell periphery and reduced VE-cadherin immunostaining were found, and activation of RhoA was strongly increased compared with that in afadin-knockdown BMVECs. Conversely, inhibition of RhoA activation in afadin-knockdown LMVECs restored the cell morphology. These results indicate that afadin has different effects on blood and lymphatic endothelial cells by controlling the levels of RhoA activation, which may critically regulate the lymphangiogenesis of mouse embryos

Genotype and number of viable progeny from crosses between afadin^flox/+;Tie2-Cre and afadin^flox/flox mice.

<p>The percentage of each genotype relative to the total progeny at each embryonic and post natal day is shown in parentheses (). The number of mice with an abnormal phenotype is shown in brackets [ ].</p

Disruption of cell-cell junctions on lymphatic, but not blood, vessel walls in afadin cKO mice.

<p>Back skins from control and afadin cKO embryos at E14.5 were fixed and immunostained with the indicated combination of Abs. Magnified images of the dashed boxes in (<b>B</b>) and (<b>E</b>) are also depicted. Arrows in (<b>A</b>) and (<b>D</b>) indicate the afadin-negative and VE-cadherin-positive area with patchy staining of VE-cadherin (<b>A</b>) and the afadin-negative and podoplanin-positive area with abnormal staining of podoplanin (<b>D</b>). White and yellow arrowheads in (<b>C</b>) indicate the arterial and venous vasculatures, respectively. Arrowheads in (<b>E</b>) indicate numerous punctures on lymphatic vessel walls. Bar graphs in (<b>B</b>) and (<b>E</b>) show the blood vessel density determined by VE-cadherin-positive and podoplanin-negative staining in the randomly selected areas (<b>B, left</b>), the percentage of VE-cadherin-positive staining in the podoplanin-positive areas (<b>B, right</b>) and the number of punctures in the LYVE-1-positive areas (<b>E</b>). Error bars indicate the mean ±S.D. at least three different samples. *, p<0.01 vs. Control. N.S., not statistically significant. Scale bars in (<b>A</b>), (<b>B</b>), (<b>C</b>), (<b>D</b>) and (<b>E</b>) represent 100 µm, 200 µm, 20 µm, 20 µm and 200 µm, respectively.</p