Over the past decade, the importance of the extracellular matrix (ECM) composition surrounding the endothelial cells (ECs) of the blood-brain barrier (BBB) has been increasingly recognized not only in barrier development and maintenance, but also in dysfunction. The tight junction protein claudin-5 (CLDN5) is critical for sealing paracellular pores between ECs to prevent the passage of fluids, solutes, and cells, thereby forming the de facto BBB. Major gaps in knowledge include what ECM alterations occur during inflammatory injuries and how they contribute to barrier disruption, plus how ECM-EC interactions regulate CLDN5. Our long-term goal is to elucidate the endothelial-specific signaling pathways responsible for BBB dysfunction during inflammation. The overall objective of this proposal is to define the role of ECM-mediated dysregulation of CLDN5-dependent BBB function during inflammation. Emphasis is placed on a novel role for the isoform-specific function of AKT2 in maintaining maximal CLDN5 expression during homeostasis, plus a proinflammatory role of two small leucine- rich proteoglycans (SLRPs), decorin and biglycan, in CLDN5 downregulation during neuroinflammation. The central hypothesis is that inflammation triggers a release of endothelial-derived SLRPs which act in an autocrine fashion to interfere with constitutive ECM-dependent regulation of CLDN5, contributing to BBB dysfunction. This hypothesis was derived from preliminary findings generated in the applicant?s laboratory. The rationale for the proposed research is that a better understanding of ECM pathobiology will translate into increased insights of the pathogenic role of BBB dysfunction in a multitude of inflammation-associated diseases in the central nervous system (CNS) which collectively account for the suffering of approximately 9 million people in the United States alone and bear a cost burden of 300 billion dollars annually. Guided by robust preliminary data, this hypothesis will be tested by two specific aims: 1) Determine the role of endothelial-derived SLRPs in BBB dysfunction during inflammation; and 2) Define the role of impaired ?1-ILK-AKT2 signaling in ECM-dependent BBB dysfunction. The approach will be multifaceted, combining in vivo physiological analyses in a relevant animal model with comparable ex vivo and in vitro experiments using primary ECs isolated from CNS tissue. Innovative experimental models include transgenic mice with inducible BBB-specific SLRP deficiency or AKT2 overexpression; new molecular tools such as Tet-On constructs for gene transfer of decorin, biglycan, ILK, and AKT2; targeted screening of pharmacologic agents; and state-of-the-art histopathology techniques to detect inflammatory-mediated ECM alterations likely to only be present surrounding a small percentage of CNS microvessels (perivenular inflammatory lesions). The proposed research is significant, as data derived from these studies will not only establish novel concepts in ECM-dependent regulation of the endothelium but will also provide new mechanistic insights into the pathophysiology of BBB dysfunction with the potential to provide a basis for the development of new therapeutictargets.
Dysfunction of the inner lining of blood vessels that form the blood-brain barrier (BBB) is implicated in progression and perpetuation of inflammation-associated diseases in the central nervous system, including multiple sclerosis, stroke, traumatic brain injury, dementia, encephalopathies, and brain metastases, that impact the lives of 9 million individuals in the United States and carry an annual cost burden of 300 billion dollars. This study will investigate how changes in the composition of the perivascular extracellular matrix, a dense meshwork of proteins that immediately surrounds the blood vessels, contribute to BBB dysfunction and will focus primarily on the two small leucine-rich proteoglycans (SLRP) decorin and biglycan, which have been observed at abnormally high levels in the perivascular matrix of patients with multiple sclerosis. Understanding the roles of these SLRPs in BBB dysfunction as well as the mechanisms by which they act may allow for the development of novel therapeutic targets for use in the treatment of a wide variety of CNS diseases.
