Modeling perfusable human brain microvasculature: Multimodal investigation of cerebrovasculature in dementia, trauma and development.

Abstract

The brain vasculature is a dense, hierarchical network responsible for ensuring the transport of oxygen and metabolic precursors essential for normal neurological function. Unlike blood vessels in other organ systems, the brain vasculature is uniquely characterized by the neurovascular unit (NVU), a specialized vascular subunit composed of dynamic cellular interactions between endothelial cells and perivascular cells, including mural cells, astrocytes, and glial cells. These components work together to tightly regulate the blood-brain barrier (BBB), a semi-permeable barrier critical for maintaining brain homeostasis. The integrity of the BBB is maintained through the formation of tight junctions, a continuous basement membrane, and the expression of transmembrane proteins that facilitate the selective transport of molecules and metabolites. This tightly regulated barrier effectively sequesters neurotoxic substances, preventing them from entering the brain parenchyma. Dysfunction of the NVU plays a key role in the pathogenesis of numerous cerebrovascular diseases. Therefore, understanding the mechanisms regulating the BBB under normal conditions, as well as its pathophysiological alterations in disease states, is essential for advancing our knowledge of cerebrovascular diseases and developing effective therapeutic interventions. However, most studies to date have relied on animal models, which fail to fully capture the complexities of human cerebrovascular biology and often exhibit intrinsic molecular differences in cerebrovascular development and disease progression. Recent advances in engineered brain vasculature models and human stem cell technologies provide promising opportunities to overcome species-specific differences and enhance our ability to study human brain microvasculature with greater accuracy. This body of work presents the development of a 3D perfusable brain microvascular model that enables spatiotemporal investigation of vascular changes in response to perfused stimuli. Each chapter explores increasing complexity of the 3D perfusable brain microvessel model, employing the model to study cerebrovascular dysfunctions in neurodegenerative diseases and culminating in the creation of a novel brain vascular model that mimics the hierarchical vascular bed found in the brain. The first section focuses on the use of stem-cell-derived cortical neurons and the perfusion of conditioned media from differentiated cortical neurons to study endothelial dysfunctions in Alzheimer’s disease (AD). This work highlights the potential of perfusing disease-mimicking stimuli, such as amyloid beta (Aβ) peptides, to examine how brain endothelial dysfunction manifests in brain microvessels. Through this study, we demonstrated that a familial AD genotype-dependent increase in Aβ levels can trigger pro-inflammatory and pro-thrombotic responses within cerebral microvessels, which may exacerbate AD pathology even before clinical symptoms appear. The second section challenges the functionality of 3D perfusable vessels as an endothelial barrier model, specifically for investigating BBB integrity and its breakdown in traumatic brain injury (TBI). By perfusing fluorescent particles through these microvessels after inducing TBI-mimicking BBB disruption, the model serves as a screening platform to identify potential therapeutic targets for TBI-induced endothelial barrier dysfunction. Specifically, this study explored the role of kinase inhibitors—known to be heavily implicated in endothelial barrier modulation—as therapeutic agents. Furthermore, by incorporating a machine-learning-based kinase regression model, a novel kinase target for barrier restoration was identified. This work highlights the feasibility of leveraging functional parameters derived from 3D microvascular models within a systems biology framework for therapeutic discovery and screening. Finally, recognizing the need for more complex and physiologically relevant models of brain vasculature, the last section presents the development of a novel brain vascular model using human induced pluripotent stem cell (hiPSC)-derived vascular organoids. These organoids integrate astrocytes, neurons, and hemodynamic stimuli to create hierarchical vascular networks that replicate the molecular signatures of the blood-brain barrier (BBB), advancing the field of vascular engineering. By mimicking both the cellular diversity and geometric organization of the brain vasculature, this sophisticated model offers a more comprehensive platform for investigating neurovascular interactions and cerebrovascular disease mechanisms. In summary, this work leverages advanced brain vascular engineering techniques to generate physiologically relevant models of brain vasculature and conduct multifaceted investigations into endothelial dysfunction in Alzheimer’s disease and traumatic brain injury (TBI). Additionally, it presents an innovative model of the hierarchical brain vasculature, incorporating relevant neurovascular cell types and replicating the complex hierarchical vascular bed found in the brain. This model serves as a novel platform for investigating the mechanisms underlying cerebrovascular diseases.