Doctor of Philosophy (PhD), Ohio University, 2022, Biological Sciences (Arts and Sciences)

The mammalian vasculature caters to tissue specific gaseous exchange and metabolic needs. The brain accounts for the largest consumption of oxygen and glucose, for which a structurally organized and functional cerebrovasculature is essential. Brain arteriovenous malformations (BAVM) are characterized by abnormally enlarged blood vessels, which direct blood through arteriovenous (AV) shunts, bypassing the normal artery-capillary-vein network. High-pressure, low-resistance AV shunts disrupt healthy blood flow and can result in cerebrovascular hemorrhage. BAVM is the leading cause of intracerebral hemorrhage in children, and accounts for 50% of stroke incidences in children and young adults. Clinically, BAVM treatments are invasive and not applicable to all cases; thus, there is critical need to understand BAVM mechanisms and develop targeted therapeutics. Using a mouse model of BAVM, that is deficient in Notch effector Rbpj from endothelial cells, from birth – we show that isolated Rbpj-deficient (RbpjiΔEC) brain endothelial cells (BECs) elicit altered whole-genome transcriptomic profile, early at Postanal day (P)7 when expansion of AV diameters – the most prominent BAVM phenotype is not observed, suggesting contribution of Rbpj regulated effector molecules in triggering onset of BAVM pathogenesis. Cellular studies over the course of characterized developmental time-periods at P7, P10, and P14 revealed that AV expansion in RbpjiΔEC mice do not originate from hyperplastic or hypertrophic mechanisms; but RbpjiΔEC BECs acquired atypical morphology and increased BEC density along AV shunts, as compared to controls, in postnatal mice. RbpjiΔEC mice also showed reduced regression of BECs over AV connections when studied through empty basement membrane collagen sleeve (EBMS) dynamics, suggesting lack of remodeling and accumulation of BECs over capillary like vessels in vivo. Using isolated postnatal mouse BECs, we found altered small GTPase activity in (open full item for complete abstract)

Committee: Corinne Nielsen (Advisor); Mark Berryman (Committee Member); Fabian Benencia (Committee Chair); Monica Burdick (Committee Co-Chair); Soichi Tanda (Committee Member) Subjects: Biochemistry; Cellular Biology; Genetics; Molecular Biology

Bachelor of Science (BS), Ohio University, 2022, Biological Sciences

The intestinal tract is highly vascularized, as nutrients from digested food are absorbed into vascular and lymphatic capillary beds of intestinal villi for distribution in the body. Therefore, it is important that blood vessels are properly formed and the intestine receives adequate blood flow for optimal function. Compromised vasculature in the intestine can lead to numerous disease states, including arteriovenous malformation, ischemia, ulceration, perforation, and hemorrhage. Little is known about the pathways that drive vascular development or vascular disorders in the intestine. Notch signaling is an evolutionarily conserved pathway that has been uncovered as a key regulator of vascular morphogenesis and vessel stability. Rbpj, a transcriptional mediator of Notch signaling, is genetically deleted from endothelial cells in our mouse model, effectively knocking down the Notch signaling pathway. In neonatal mice, just two weeks post-Rbpj deletion, vascular density was increased in intestinal villus tips in all three anatomical/functional regions of the small intestine following the endothelial deletion of Rbpj. Cell proliferation assays revealed that endothelial cell proliferation cannot explain the change in vascular density. Further, we determined through LYVE1 and Cdh5 immunostaining that the Rbpj deletion was isolated to vascular endothelium when using Cdh5-CreERT2 as the driver line. In conclusion, this thesis identifies a novel role for Notch/Rbpj signaling in the proper vascular development of intestinal villi in neonatal mice.

Committee: Soichi Tanda (Advisor); Corinne Nielsen (Advisor) Subjects: Biology; Developmental Biology

Bachelor of Science (BS), Ohio University, 2019, Neuroscience

Brain arteriovenous malformation (AVM) is a disease characterized by multiple vascular abnormalities, including arteriovenous (AV) shunting – formation of direct connections between arteries and veins at the expense of capillary networks. Blood flows rapidly through these abnormal connections; thus, these vessels are prone to rupture, which can lead to devastating outcomes. Treatment methods for brain AVM are limited; therefore, it is important to understand the mechanisms of disease pathogenesis in order to develop novel therapies. Using genetically engineered mice, we have previously shown that deletion of Rbpj from endothelial cells – cells that form the inner lining of blood vessels – during the developmental period just after birth, leads to characteristic AVM features. Using our AVM model, I tested the hypothesis that, following deletion of endothelial Rbpj, pericyte coverage of endothelium is affected. Pericytes are specialized vascular cells that enwrap capillaries and are important for the development and maintenance of the vascular system. By analyzing pericyte area, endothelial area, and percent endothelium covered by pericytes in control and Rbpj-mutant mice, I found increased areas of pericytes and endothelium in select regions of the brain and at select timepoints. I also determined that the percent endothelium covered by pericytes was not significantly different between mutants and controls. These results suggest that endothelial Rbpj regulates total pericyte and endothelial area in the brain, and in Rbpj-mediated brain AVM, pericytes keep pace with the pathological increase of endothelium. The increased pericyte area may result from a variety of changes, including increased pericyte cell number and/or alterations in pericyte morphology. Results showed, in mutant vs. control brains, that pericyte number may be increased but that pericyte proliferation was not increased, suggesting another mechanism of pericyte recruitment. Additionally, altered pericy (open full item for complete abstract)

Committee: Corinne Nielsen (Advisor) Subjects: Biology; Neurosciences

Bachelor of Science (BS), Ohio University, 2018, Biological Sciences

Brain Arteriovenous Malformation (AVM) is a human disease with clinically defined vascular abnormalities, such as arteriovenous shunting. This shunting allows arterial blood to flow directly to a vein; bypassing capillaries and affecting gas exchange in the affected tissue. We use a genetic mouse model of brain AVM to study the disease pathogenesis, which proceeds during the early postnatal period, just after birth. This time window coincides with postnatal cerebellum development and includes events such as cerebellar lobulation, lamination, and circuitry formation. Interestingly, 100% of our brain AVM mice have similar cerebellar abnormalities. While the cerebellar defects may be a secondary consequence of the vascular abnormalities (e.g. impaired oxygen delivery to brain tissue), they may also be a primary consequence of impaired Notch/Rbpj signaling between endothelial cells (ECs) and other cell types in the brain. We hypothesize that endothelial Rbpj is required for postnatal cerebellar morphogenesis and laminar organization. To study this, we deleted Rbpj selectively in ECs and examined cerebellar defects. We found significantly decreased cerebellar area, decreased molecular layer thickness, along the sides and tips of select cerebellar lobules, as well as decreased fissure length between lobules, suggesting impaired outward growth of postnatal cerebellum. We also found reduced number of Purkinje neurons in the Purkinje cell layer of the cerebellum, suggesting that abnormal cerebellar lamination. Our data suggest that Rbpj is required regionally in postnatal ECs for proper cerebellar morphogenesis and laminar organization.

Committee: Corinne Nielsen Ph.D. (Advisor) Subjects: Biology; Cellular Biology; Molecular Biology