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Blood vessel formation is a well orchestrated process where multiple components including different cells types, growth factors as well as extracellular matrix proteins act in synergistic and highly regulated manner to support the growth of new blood vessels. During embryonic development this process is marked as vasculogenesis and entails the differentiation of mesodermal cells into angioblasts and their subsequent fusion into a primitive vascular plexus. Angiogenesis, in contrast, describes the formation of new vessels from the pre-existing vasculature and it occurs in the embryo during remodeling of the primitive plexus into a mature vascular network. Furthermore, in the adult, angiogenic processes play a role in various physiological and pathological conditions. Angiogenesis is governed by a set of factors and molecular mechanisms whose identification has been a major focus of cardiovascular research for the past several decades. Most recently, Epidermal growth factor-like domain 7 (EGFL7) has been described as a novel molecular player in this context. This secreted protein is produced by endothelial cells and has been implicated in vessel development. Studies performed in zebrafish revealed an important role for EGFL7 in lumen formation during vasculogenesis although the underlying molecular mechanism has not been elucidated yet. In contrast, the investigation of EGFL7’s functions during angiogenic sprouting has faced several challenges and the role of EGFL7 in angiogenesis remained elusive. The purpose of this thesis was to identify the functions of EGFL7 during angiogenic mode of vessel formation in a systematic fashion using numerous in vitro as well as in vivo approaches.
Previously it has been suggested that EGFL7 might associate with the extracellular matrix from where it could exert its effects. Indeed, we could show that EGFL7 accumulates on the outer surface of endothelial cells in vivo by demonstrating its co-localization with collagen IV, a major constituent of the basal lamina. Furthermore, after its secretion to the extracellular matrix (ECM), EGFL7 seemed to interact with some components of the extracellular matrix including fibronectin and vitronectin, but not collagens and laminin.
A major group of receptors that mediate the interaction between the cells and the ECM are integrin receptors. Our co-immunoprecipitation studies revealed that EGFL7 associated with integrin αvβ3 which is highly expressed in endothelial cells and known to be important for vessel growth. Importantly, this EGFL7-αvβ3 integrin interaction was dependent on Arg-Gly-Asp (RGD) motif present within the second EGF-like domain of EGFL7 protein. Adhesion assays performed with human umbilical vein endothelial cells (HUVEC) revealed that EGFL7 promoted endothelial cell adhesion compared to BSA used as a negative control, however, adhesion seemed to be less efficient as compared to bona fide ECM proteins such as fibronectin and vitronectin. In addition, cultivation of endothelial cells on EGFL7 was characterized by the absence of mature focal adhesions and stress fibers, but was paralleled by increased phosphorylation of kinases typical for integrin activation signaling cascade such as FAK, Src and Akt. This led us to the hypothesis that EGFL7 creates an environment that supports a motile phenotype of endothelial cells by serving as a modulator of existing interactions between the cells and the surrounding matrix. Indeed, EGFL7 increased random migration of HUVEC on fibronectin in an αvβ3 integrin dependent manner as shown using a live cell imaging platform. Most importantly, this was paralleled by a decrease in endothelial cell adhesion to fibronectin which is consistent with previous reports on secreted proteins that support a medium strength of adhesion and such promote cellular migration. To assess the overall effect of EGFL7 on the process of blood formation several in vitro and in vivo approaches were employed. First, the addition of EGFL7 to Matrigel injected subcutaneously into mice significantly increased the invasion of endothelial cells into the plugs. Second, a spheroid-based sprouting assay in three-dimensional collagen matrix clearly demonstrated the ability of EGFL7 to support angiogenic sprouting in an integrin dependent manner. This is consistent with the observed effects of EGFL7 on endothelial cell migration. Third, using in vivo assays such as the chick chorioallantoic membrane (CAM) assay as well as a zebrafish model system we were able to validate the importance of the EGFL7-integrin interaction for the process of angiogenesis in vivo. Taken together, I identified some of the major cellular functions EGFL7 modulates during angiogenesis. In addition, with integrin αvβ3 I unraveled a novel interaction partner of EGFL7 that delivers a mechanistical explanation for EGFL7’s effects on blood vessel formation. Most importantly, data presented in this PhD thesis contribute substantially to the existing literature on EGFL7 unambiguously assigning a role for this protein in the process of angiogenesis.
Blood vessels form de novo through the tightly regulated programs of vasculogenesis and angiogenesis. Both processes are distinct but one of the steps they share is the formation of a central lumen, when groups of cells organized as vascular cords undergo complex changes to achieve a tube-like morphology. Recently, a protein termed epidermal growth factor-like domain 7 (EGFL7) was described as a novel endothelial cell-derived factor involved in the regulation of the spatial arrangement of cells during vascular tube assembly. With its impact on tubulogenesis and vessel shape EGFL7 joined the large family of molecules governing blood vessel formation. Only recently, the molecular mechanisms underlying EGFL7's effects have been started to be elucidated and shaping of the extracellular matrix (ECM) as well as Notch signaling might very well play a role in mediating its biological effects. Further, findings in knock-out animal models suggest miR-126, a miRNA located within the egfl7 gene, has a major role in vessel development by promoting VEGF signaling, angiogenesis and vascular integrity. This review summarizes our current knowledge on EGFL7 and miR-126 and we will discuss the implications of both bioactive molecules for the formation of blood vessels.