Angiogenesis is a cellular process of capillary sprouting and configuring of neovasculatures from the existing blood vessels. It is in contrast to another process of blood vessel formation, called vasculogenesis, whereby the blood vessels are de novo formed by the coalescence of endothelial progenitor cells. During primate development, such as embryogenesis and tissue/organ morphogenesis, blood vessels are produced by both vasculogenesis and angiogenesis. However, new vessels are formed only through angiogenesis in the adult. Except during the female reproductive cycles (ovulation, menstruation, implantation and pregnancy), the endothelial cells are quiescent and thus long-lived in the normal adult mammals. Yet, they undergo an activation process, upon appropriate stimuli, to grow into new capillaries during episodic growth and remodeling of the blood vessel network.
Angiogenesis is of crucial importance in a variety of physiological and pathological conditions and diseases, including ischemia and hypoxia, atherosclerosis, leukocyte trafficking and recruitment, hemostasis, wound healing, vascular leaky syndrome, diabetic retinopathy, macular degeneration, neovascular glaucoma, psoriasis, rheumatoid arthritis, hemangioma, and cancer growth and metastasis (Hanahan, D. and Folkman, J. Cell 86:353-364 (1996); Carmeliet, P. and Jain, R. K. Nature 407:249-257 (2000)). The importance of angiogenesis for the growth of a variety of cancers is now well recognized. For instance, the growth of solid tumors requires concomitant expanding of the vascular networks for their blood supplies; an insufficient supply of blood (more than 100 to 200 μm away from blood vessels) is known to lead to the necrosis of cancer tissues. Although vascular endothelial cell growth factor (VEGF), fibroblast growth factor (FGF), angiopoitins and other molecules are indispensable for vessel formations (Hanahan, D. Science 277:48-50 (1997); Yancopoulos, G. D. et al. Nature 407:242-248 (2000)), the molecular and cellular mechanisms governing tumor angiogenesis are still poorly understood.
In vertebrates, the vascular system is mainly for material transportation while the nervous system is mainly for information communication; notably, both of them have the circuit properties anatomically and physiologically (Shima, D. T. and Mailhos, C. Curr. Opin. Genet. Dev. 10:536-542 (2000)). For example, (1) capillary vessels are formed by the endothelial cells ensheathed with pericytes while nerves are composed by neurons surrounded with glia; (2) both blood vessels and nerves ramify throughout almost all the parts of the body; and (3) the circulatory system is divided by arteries (sending the blood out of the heart) and veins (send the blood back to the heart) while the nervous system has both motor nerves (sending the impulse out of the brain or the spinal cord) and sensory nerves (send the impulse back to the brain or the spinal cord). In addition to sharing of these morphological and functional features, blood vessels and nerves have an intimately physical relationship, such as the autonomic nerves that regulates the vascular tones.
It is currently known that the pathfinding of the nervous networks requires several families of neurological migratory cues, such as semaphorin, ephrin, netrin, Slit and several others. Prominent among these molecules that simultaneously promote angiogenesis is neuropilin-1, a membrane receptor of the semaphorin family expressed on both developing neurons and endothelial cells. It binds to VEGF165, a splicing isoform of the VEGF gene (Soker, S. et al. Cell 92:735-45 (1998)), and functionally, its mutant mouse embryos manifest severe defects of vascular formations (Kawasaki, T. et al. Development 126:4895-902 (1999)). Tumor cells can also express neuropilin-1, resulting in substantially enhanced tumor angiogenesis and enlarged tumors. In analog, the cell-bound ephrin ligands and their cognate Eph receptor tyrosine kinases play essential roles in vascular development. Among them, Ephrin-B2, a transmembrane ligand specifically expressed on arterial endothelial cells and surrounding cells, interacts with multiple EphB class receptors. Conversely, EphB4, a specific receptor for ephrin-B2, is expressed on venous endothelial cells. The bidirectional signals between EphB4 and ephrin-B2 are thought to be specific for the development of the arteries and veins (Wang, H. U. et al. Cell 93:741-53 (1998)). Further, the EphA2 receptor is up-regulated in transformed cells and tumor vasculatures where they likely contribute to cancer pathogenesis (Brantley, D. M. et al. Oncogene 21:7011-26 (2002)). Likewise, the rat netrin1 receptor Unc5h2 mRNA is observed during the early blood vessel formation, implicating the potential involvement of netrin and its receptors in vasculogenesis (Engelkamp, D. Mech. Dev. 118:191-197 (2002)).
Slit2, a member of another family of “neurological” migratory cues, is expressed by midline cells and endothelial cells. It reacts with a cell surface transmembrane protein, Roundabout1 (Robo1), and functions as a repellent in axon guidance (Kidd, T. et al. Cell 92:201-215 (1998); Brose, K. et al. Cell 96:795-806 (1999); Li, H. S. et al. Cell 96:807-818 (1999)) and branching (Wang, K.-H. et al. Cell 96:771-784 (1999); Whitford, K. L. et al. Neuron 33:47-61 (2002)), neuronal migration (Wu, W. et al. Nature 400:331-336 (1999)), and as an endogenous inhibitor for leukocyte chemotaxis (Wu, J. Y. et al. Nature 410:948-952 (2001)). Currently, there are three slit genes, slit1, 2 and 3 and four robo genes, robo1, robo2, rig-1 and robo4, known in the mammals. Their expressions outside the nervous system have been found in the rodents (Holmes, G. P. et al. Mech. Dev. 79:57-72 (1998); Piper, M. et al. Mech. Dev. 94: 213-217 (2000)). For example, mRNAs for Slit2 and Slit3, but not for Slit1, are found in rat endothelial cells and Robo1 RNA is found in mouse leukocytes (Wu, J. Y. et al. Nature 410:948-952 (2001)). Further, human endothelial cells express Robo4 (Huminiecki, L. et al. Genomics. 79:547-552 (2002)). However, it is not determined whether human cancer cells can express these genes, especially at the protein levels.