PROJECT SUMMARY
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 β1-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.