Targeting the cerebrovasculature in sepsis: A focus on the brain microvascular endothelium

The blood-brain barrier (BBB) is a critical interface between the systemic circulation and the brain. It is a specialized multicellular unit composed of brain microvascular endothelial cells (BMECs), pericytes, a basement membrane, and astrocytic end foot processes. BMECs are a principal component of the BBB that provide the structural framework needed for the stringent transport of molecules into the brain. BMEC dysfunction permits the trafficking of neurotoxins from systemic circulation into the brain, which ultimately exacerbates BBB dysfunction and neuroinflammation. Studies have shown that BBB dysfunction is a key determinant of cognitive decline in sepsis. However, there are critical knowledge gaps that exist in understanding the molecular and cellular mechanisms underpinning BMEC physiology and function as it relates to the maintenance of BBB integrity. A strategy for bridging this critical knowledge gap requires an examination of proteins and enzymes localized to BMECs. Tissue-nonspecific alkaline phosphatase (TNAP) has been historically used as a brain endothelial histological marker due to its abundant expression on BMECs. Yet, the role of TNAP in BMECs remains unclear. The objective of this project was to understand the function of BMEC TNAP at the BBB in normal physiology and sepsis. Our central hypothesis is that BMEC TNAP is critical for maintenance of BBB integrity in normal physiology and in sepsis through molecular mechanisms that preserve BMEC structure and function. The studies presented in this dissertation demonstrated a novel anti-inflammatory role for TNAP in cerebral microvessels. We also demonstrated that BMEC TNAP plays an important role in maintaining paracellular barrier integrity during systemic inflammation via cytoskeletal reorganization. Importantly, our data elucidated a molecular target (Rho-associated protein kinase) through which loss of TNAP activity in cerebral microvessel in sepsis could be mitigated. Finally, we utilized the cre-lox system to generate a genetic mouse model with an endothelial VE-cadherin conditional knockout of the Alpl gene (VE-cKO). We showed that VE-cKO mice exhibited whole brain size selectively increased BBB permeability that is worsened in sepsis and stroke. Collectively, this body of work demonstrated that TNAP activity in cerebral microvessel is important for maintaining BBB integrity. These findings lay the groundwork needed to stimulate the discovery of a therapeutic target whose activity can be manipulated to mitigate long-term neurological dysfunction in sepsis. Moreover, our results will impact not only sepsis but will improve the quality of life and medical outcomes in other neurodegenerative and inflammatory diseases such as stroke and Alzheimer’s disease (AD)