Novel Regulators of Angiogenesis and Cerebrovascular Integrity

Judah Folkman first hypothesized in 1971 that tumor growth depends on angiogenesis, the growth of new blood vessels from pre-existing ones. Eighteen years later, in 1989, Harold Dvorak and Napoleana Ferrara independently isolated Vascular Endothelial Growth Factor (VEGF), which Dr. Ferrara went on to prove to be one of the most important endothelial cell mitogens ever discovered. In 2004, the FDA approved the use of Bevacizumab, a monoclonal antibody against VEGF, for the treatment of colon cancer after the drug showed superiority when combined with a standard chemotherapy regimen. While the use of VEGF inhibitors in the clinic has shown benefit in treating patients, there is room for clinical improvement. This thesis was initially established on the concept that the identification of other novel regulators of angiogenesis could provide additional targets for anti-angiogenic therapy to be used either alone or in combination with VEGF inhibition to achieve results superior to VEGF blockade alone. Against this background, mouse models of conditional Egfl7, Egfl8, miR-126, and GPR124 genetic deletion were designed and generated to determine the function of these novel molecules in the context of regulating angiogenesis. We discovered that the previously described Egfl7 mouse knockout phenotypes of 50% embryonic lethality with edema and delayed cranial blood vessel formation and post-natal vascularization of the retina were actually due to disruption of the microRNA miR-126 residing in the seventh intron of Egfl7. We further showed that miR-126 regulates angiogenesis through suppressing expression of p85[beta] and Spred1 which inhibit the downstream signaling cascade of VEGF signaling. Surprisingly, the Egfl7 mice were phenotypically normal even with simultaneous deletion of the paralog Egfl8. We noted that the previous study inaccurately described the miR-126 angiogenic phenotypes as those of Egfl7 after using insertional deletion ("knockin") strategies to develop mouse models of Egfl7 gene deletion which inadvertently disrupted expression of miR-126. We highlight that this concept complicates the design of mouse genetic deletion studies and recommend that strategies which provide minimal deletion of a region of the locus are superior to "knockin" strategies. GPR124 is a known requirement of developmental angiogenesis specifically within the Central Nervous System (CNS). As GPR124 is expressed by endothelial cells and pericytes in the CNS, it was unknown at the start of my graduate career if endothelial cell or pericyte expression was required for blood vessel migration into the CNS. Using an endothelial cell specific driver, we were able to delete GPR124 specifically within endothelial cells and prove that endothelial cell expression of GPR124 drove vascularization of the CNS during development, thus proving that GPR124 functions endothelial cell autonomously. Further, as embryos lacking GPR124 die with 100% penetrance before birth, I crossed my conditional allele with an inducible driver to delete GPR124 in adulthood in order to bypass the window of embryonic lethality. I found that GPR124 was not required for homeostasis of the quiescent cerebrovasculature. Using orthotopic glioma and transient middle cerebral artery occlusion studies, we found that mice lacking GPR124 were susceptible to intracranial hemorrhage after insult. These results showed that GPR124 plays an important role in maintaining cerebrovacular integrity in the context of disease within the CNS. Overall, this thesis has identified, clarified or further described the functional role of several novel regulators of angiogenesis. Future studies beyond the scope of this thesis will use the mouse models of conditional gene deletion built for this dissertation to further investigate the mechanisms through which these genes regulate angiogenesis. Hopefully, further understanding of how each of these novel genes regulates endothelial cell physiology will provide valuable contributions towards improving out ability to treat cancer through manipulation of its blood supply.