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Axon Guidance Cues in Tumor and Developmental Angiogenesis
Hints for such targets have unexpectedly come from the developmental patterning mechanism for the nervous system. Increasing evidence is emerging that similar patterning mechanisms are used during the development of neural and vascular networks in vertebrates. Molecules that guide axons to their targets appear to have counterparts that guide the patterning of vessels. Four families of guidance molecules, namely netrins, semaphorins, ephrins, and slits, and their cognate receptors, mediate axon guidance. Each of these guidance families has recently been shown to contain at least one ligand-receptor pair that plays a functional role in patterning of the vasculature. Our study focuses on the slit-robo signaling system in vascular development and investigates one member of the Roundabout (Robo) family, robo4, in vessel guidance. Robos were originally found in Drosophila to mediate repulsive cues for slit ligands by preventing the re-crossing of axons once they have crossed the midline. Four members of the Robo family have been identified. Robo1, robo2, and robo3 are primarily expressed in the central nervous system (CNS) and help guide axons. Outside the nervous system, robos have been thought to play a role in leukocyte trafficking and kidney branching morphogenesis. However, until the discovery of the fourth member of this family, robo4, and its expression patterns in developing mouse vasculature, robos were unappreciated in vascular development. Besides developmental angiogenesis, robo4 is expressed in sites of active angiogenesis in tumor vessels. Because robo4 is both selective to tumor vasculature and is a cell-surface receptor, it is an attractive target for tumor endothelium. To investigate robo4 function, we studied its ortholog in zebrafish during embryonic development. We characterized robo4 gene expression and pursued functional studies by gene knockdown approaches using morpholinos (antisense oligonucleotides). Robo4’s in situ expression showed three interesting features. First, its expression in the embryonic zebrafish vasculature was transient and was seen in both angioblasts (Figure 1, parts A and B, white asterisk) and intersomitic vessels (ISVs) (Figure 1, parts C and D, black asterisks). Second, robo4 expression in notochord and ISVs of the trunk region overlapped, such that a concomitant decrease in robo4 expression in notochord in a rostral-to-caudal manner was immediately followed by expression in ISVs, suggesting a mechanistic role for robo4 in the ISV sprouting process (Figure 1, parts B and C). Third, robo4 expression in rostral sprouts ceased first, prior to its expression in caudal sprouts, suggesting a temporal regulation for the ISV sprouting process. To assess phenotype, we performed in situ studies with both endothelial (flk) and neuronal (acetylated tubulin) markers in robo4 knockdown embryos. Compared with wild-type embryos (Figure 1, part E), robo4 knockdown embryos displayed either a lack of ISV sprouts (Figure 1, part F) or linear ISV sprouts, albeit weak ones in the zebrafish trunk region. Figure 1. Robo4 expression patterns in embryonic zebrafish vascular development. Panels A through D show robo4 in situ (blue) across 20 (A) and 22 (D) somites in embryos. Panels B and C are high-power images of the trunk region of (A) and (D), respectively. (E) Wild-type embryo and (F) morpholino-injected embryo. Panels E and F depict trunk regions of 22-somite embryos double stained for flk RNA (blue, endothelial marker) and antibody to acetylated tubulin (brown, neuronal marker). N, notochord; ISV, intersomitic vessels. Black asterisks depict the location of ISVs, and the white asterisk depicts angioblasts. To visualize the ISV vessel growth in a live embryo, we pursued time-lapse imaging of the trunk vasculature in embryos injected with morpholinos. Using live time-lapse imaging in robo4 knockdown vascular-specific transgenic embryos, we demonstrated that when vessel sprouting occurs incorrectly, vessel formation is often aborted. Also, embryos often displayed a loss of temporal and spatial regulation of ISV sprouting from the dorsal aorta (DA). The surprising finding was that prior to our gene knockdown study, it was widely assumed that robos were primarily negative regulators of guidance; however, our study suggests that removal of a presumptive negative guidance cue resulted in the collapse of vessels as opposed to the expected increase in sprouts. This result suggests that in the absence of guidance molecules, vessels normally have a default mechanism that prevents them from growing into incorrect tissue sites. This helps clarify why robo4 would be upregulated in tumor vasculature since, from a tumor standpoint, a cancer cell would subvert or override normal developmental guidance checkpoints and utilize this mechanism to form a chaotic vascular network, which is seen routinely in tumor vasculature. Another interpretation of our knockdown result is that besides traditional repulsive cues, robos mediate attractive cues as well. In fact, preliminary unpublished evidence from our laboratory suggests that this cannot be excluded, and perhaps, tumors use axon guidance molecules, which are known to have bi-functional properties, to fulfill their growing needs.
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decades of angiogenesis research has culminated with the first U.S. Food and
Drug Administration (FDA)–approved anti-angiogenic drug, Avastin, which
is showing promise in the clinic. Avastin, a monoclonal antibody to vascular
endothelial growth factor (VEGF), sops up VEGF secreted by tumors and thus denies
them the ability to grow new blood vessels. However, VEGF has other physiological
functions, which are compromised in Avastin-treated patients. Because of this,
discovering novel targets of vascular endothelium has taken on a new sense of
urgency and prompted the search for the next generation of anti-angiogenic drug
targets that are tumor-vasculature specific. Cell-surface moieties are of particular
interest; historic evidence suggests that they might be suitable as targets.
