Endothelial Rbpj Deficiency Induces Pathological Alterations to the Neurovascular Unit in a Mouse Model of Brain Arteriovenous Malformation

The neurovascular unit (NVU) is an anatomical and functional feature of the mammalian brain that encompasses the brain microvasculature and is pivotal for maintaining healthy nutrient, waste, and ion exchange between the blood supply and parenchymal tissue. Specializations of the NVU tightly regulate this exchange, thereby restricting the entry/exit of solutes to/from brain tissue and forming a functional blood-brain barrier (BBB). This complex network is composed of tightly interlinked cellular components – including vascular endothelial cells (ECs), pericytes, neurons, astrocytes, and microglia – making the brain vulnerable to pathologies affecting any single NVU element. Disruption in one part can lead to wider dysfunction, exacerbating neurological disease pathogenesis and symptoms. Cells of the NVU interact closely to maintain overall brain health. Pericytes are vital for vascular development and homeostasis, regulating cerebral blood flow and establishing the BBB. Astrocytes are also critical to the development and maintenance of the BBB and allow for vessel-parenchyma crosstalk through perivascular astrocytic end-feet. Microglia serve as regulators of immunogenic function and vascular development and are key players in maintenance of vessel integrity and diameter. Brain arteriovenous malformations (AVMs) present as high-flow vascular anomalies, distinguished by dilated vascular channels that forego a capillary bed, resulting in direct arterial-to-venous connections. This abnormality predisposes patients to spontaneous intracerebral hemorrhages. Currently, therapeutic options are restricted to invasive procedures, including stereotactic radiosurgery, endovascular embolization, and surgical excision. Such limitations underscore the urgent demand for the development of preventive strategies and less invasive treatment approaches. This dissertation reports on the molecular and cellular impacts to the NVU during pathogenesis of brain AVMs. Our lab uses a genetic mouse model of brain AVM in which the Rbpj gene, which encodes a Notch signaling effector and transcription factor, is deleted from ECs during the early postnatal period. Using this model, I tested the hypothesis that endothelial deletion of Rbpj perturbed other cellular and functional components of the NVU during brain AVM pathogenesis. This hypothesis was tested in three experimental aims focused on consequences to pericytes, microglia, and astrocytes. We observed that Rbpj deletion in ECs led to (1) the pathological expansion of pericytes to maintain coverage of abnormal vascular endothelium in the brain, (2) pericytes potentially adapting morphological features of smooth muscle cells, and (3) abnormal gene expression, with pericytes showing decreased expression of Pdgfrβ, N-cadherin, and CD146, factors involved in healthy EC-pericyte interactions. Microglia in Rbpj mutants displayed signs of pathological activation, including heightened Iba1 expression, proliferation, morphological changes, and gene expression changes consistent with inflammation- induced cells polarization and dysfunction. Astrocytes in Rbpj-mutants showed increased GFAP expression, hypertrophy, and proliferation, suggesting progression to a pathological reactive state. Glial scar formation, abnormal gene expressions, metabolic alterations and atypical perivascular endfeet morphology further demonstrated astrocyte reactivity and dysfunction in mutant brain tissue. This dissertation research identified cellular and molecular consequences to pericytes, microglia, and astrocytes of the NVU, following endothelial Rbpj deletion. By providing such mechanistic insight into how the NVU is affected during Rbpj-mutant brain AVM, this research will help identify potential therapeutic targets for treating Rbpj/Notch-related brain AVMs. Findings of this work can be extrapolated to other neurovascular conditions that share analogous pathological mechanisms, such as strokes (e.g. ischemic stroke) and different types of vascular malformations (e.g. cerebral cavernous malformations). The insights gained from studying brain AVMs may inform the development of treatment strategies for these related disorders, emphasizing the potential for cross-applicability of therapeutic approaches. Given the similar underpinnings of disrupted vascular integrity and abnormal blood flow, interventions successful in brain AVMs could pave the way for novel therapies in a spectrum of neurovascular diseases, thereby broadening the scope of neurovascular medicine and patient care.