Lecture 18, Angiogenesis and axon guidance, Slit-Robo March 30, 2011.doc

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University of Toronto St. George
Cell and Systems Biology
Maurice Ringuette

Lecture 18 Notes March 30, 2011 Text taken verbatim from cited articles with minor modifications Angiogenesis and endothelial cell migration Eiken and Adams (2010) Dynamics of endothelial cell behavior in sprouting angiogenesis Current Opinion in Cell Biology 22:617-625 Introduction Endothelial cells (ECs) form two extensive tubular networks, the blood vasculature and the lymphatic vasculature, that transport a large variety of molecular and cellular cargo. The formation and function of these vessels supports the growth of organs during development and early postnatal life, but also helps to defend diseases, stabilize the body temperature and maintain homeostatic balance in the adult. Blood vessels form a closed tubular system for blood circulation, which consists of hierarchically organized arteries, capillaries and veins. In contrast, lymphatic vessels are blind-ending tubules that transport protein-rich fluid (lymph) and immune cells unidirectionally from distal tissues into the venous branch of the circulation. All vessels consist of interconnected, tube-forming ECs. However, ECs are highly heterogeneous in terms of functional properties and gene expression profiles, which depend on their differentiation and proliferation status, identity as blood vascular or lymphatic endothelium, location within a specific part of the vasculature, and organ-specific specialization. In addition, mural cells (smooth muscle cells and pericytes) are attached to the abluminal (basal) surface of certain vessels and help, for example, to stabilize the vessel wall and regulate blood pressure. Whereas vasculogenesis, the de novo formation of vessels from mesoderm-derived endothelial precursor cells (angioblasts), is responsible for the formation of the first, primitive blood vessels in the embryo, physiological and pathological blood vessel growth in later life is predominantly, Most of our knowledge about the morphological processes and molecular regulation of angiogenesis results from studies in developing zebrafish embryos or the vascularization of the retina in the postnatal mouse. Its great experimental accessibility and the possibility of life imaging are main advantages of the zebrafish system. Key benefits of the mouse retina model are the progressive expansion of the growing retinal blood vessels from the center to the periphery, their initial organization as a flat two-dimensional network, the spatial separation of growing vs. maturing areas, and the relatively easy identification of sprouts and tip cells (Figure 1). Growth of the vascular network is highly dynamic and requires a multitude of individual processes such as the proliferation of ECs, their directional migration, the establishment of appropriate connections, tube formation (tubulogenesis), remodeling and pruning, specification into arteries, veins and capillaries, and the recruitment of mural cells. Sprouting and the emergence of tip cells are early steps in this complex program. Tip cells possess many long and motile filopodia that extend towards the source of pro-angiogenic growth factors and respond to other positive or negative guidance cues to enable directional and prevent unorganized and random vessel growth (Figures 1 and 2). VEGF family growth factors master regulators of angiogenesis A key molecule for the initiation and direction of sprouting is vascular endothelial growth factor A (VEGF-A). In the postnatal mouse retina, VEGF-A is secreted by astrocytes in the avascular region, which leads to the formation of a VEGF gradient. This role of VEGF-A as a directional, chemoattractive cue for angiogenic sprouting has been also observed in the embryonic spinal cord of the mouse or during the vessel growth in the developing zebrafish. A second VEGF family member, the growth factor VEGF-C, is 1 also strong promoter of angiogenic growth in the retina, in the zebrafish embryo and in tumors. While VEGF-A binds to and signals though the receptor tyrosine kinase (RTK) VEGFR2/KDR/Flk1, the effects of VEGF-C are mainly mediated by the receptor VEGFR3/Flt4. Both VEGFR2 and VEGFR3 are strongly expressed in the angiogenic endothelium. High levels of VEGFR3 in lymphatic ECs are the basis for the important role of VEGF-C as a promoter of lymphangiogenesis. Another related RTK, VEGFR1/Flt1, which has high affinity for VEGF-A but weak tyrosine kinase activity and also exists in a secreted, catalytically inactive form, acts as a negative regulator of angiogenesis. Soluble VEGFR1 expression in the vicinity of emerging sprouts, presumably in stalk cells, appears to enhance the steepness of the VEGF- A signaling gradient and has been found to control the guidance of tip cells and filopodia. Tip and stalk cells angiogenic ECs are not all alike When vessels are exposed to pro-angiogenic signals such as VEGF, only a fraction of the ECs acquire the tip cell phenotype, whereas others form the stalk of the vascular sprout or stay behind to maintain the integrity and perfusion of the growing vascular bed. This decision is strongly controlled by the Notch pathway, a key regulator of cell fate, differentiation and patterning processes in a large variety of tissues and organisms. Tip cells contain relatively high levels of Delta-like 4 (Dll4), a transmembrane protein and Notch ligand, whereas stalk cells preferentially express a different ligand, namely Jagged-1. Activation of Notch signaling by Dll4 is thought to occur predominantly in stalk ECs and leads to the downregulation of VEGFR2 and VEGFR3 expression in these cells. Accordingly, the blockade of Notch signaling or insufficient expression of the Notch ligand Delta-like 4 (DLL4) increases the number of tip-like cells and EC proliferation in the postnatal mouse retina and in sprouting intersegmental vessels in zebrafish. Likewise, disruption of Dll4Notch interactions in experimental tumors causes extensive endothelial sprouting. On the basis of these results, it is currently believed that cells at the angiogenic front compete with each other through Notch signaling. In response to VEGF, all cells upregulate Dll4 and signal to their neighbors. Factors such as high exposure to Dll4-expressing cells in their vicinity or limited access to VEGF might favor stalk cell behavior. Conversely, cells with higher levels of Dll4, low Notch activity and stronger VEGF receptor transcription are thought to convert into tip cells (Figure 2). Mediated by lateral inhibition effects, Notch signaling presumably becomes increasingly directional (between signal- sending tip and signal-receiving stalk cells) so that differences in EC behavior and gene expression get enhanced further (Figure 2). The ligand Jagged1 is a pro- angiogenic regulator that antagonizes Dll4Notch signaling and thereby positively controls the number of sprouts and tips. Postranslational modification of Notch receptors by Fringe family glycosyltransferases, which enhances Delta-mediated Notch activation but strongly weakens the signaling capability of Jagged1, are critical for the opposing roles of the two ligands. Jagged1 and Dll4 not only maintain differential Notch signaling among tip and stalk cells, they also control the levels of VEGF receptor expression and thereby EC growth and proliferation throughout the angiogenic vasculature. The phenotypic specialization of ECs as tip or stalk cells is presumably very transient. Tips form connections with other sprouts or nearby capillaries and thereby get incorporated into the endothelial lining of new vessels. Conversely, local changes in VEGF and Notch signaling can trigger the conversion of stalk cells into tips and induce a new round of sprouting (Figure 4). The Notch pathway can act in a very transient, oscillating modus, which might well promote the dynamic interconversion of tip and stalk cells. Thus, tip and stalk cell determination is probably transient, reversible and depends on the balance between pro-angiogenic factors (like VEGF and Jagged1) and signals suppressing EC sprouting and proliferation (e.g., Dll4Notch activity). 2Meet the leaders similarities between tip cells and axonal growth cones The filopodia-extending and motile endothelial tip cells share many structural and functional features with growth cones found at the distal end of extending axons in the nervous system. Both are responsible for the formation of new connections and respond to similar cues that they encounter in their vicinity. While vascular sprouts or axonal growth are attracted by guidance cues, they are blocked by repulsive signals leading to either growth inhibition or changes in the direction of growth. Eph/ephrin molecules, which provide mostly repulsive signals for growing nerve cells, are one such example. The receptor EphB4 and its ligand ephrin-B2 are critical regulators of vascular morphogenesis in the early mouse embryo. EphB4/ephrin-B2 signaling also controls vein and artery segregation in the developing zebrafish, where highly migratory EphB4-positive cells leave a common precursor vessel to form the cardinal vein, whereas ephrin-B2-expressing cells form the dorsal aorta. Recent work has linked ephrin-B2 to motile and invasive tip cell behavior as well as to VEGF receptor signaling. ECs lacking or expressing a mutant form ephrin- B2 fail to internalize VEGF receptors in response to growth factor stimulation, which also compromises the activation of downstream signaling pathways. In addition, ephrin-B2 also regulates the recruitment of mural cells to endothelial tubes. Matrix reloaded reorganizing the subendothelial basement membrane Endothelial sprouting is just an early step in the formation of new vessels.
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