UBC Theses and Dissertations
SPARC expression during development and inflammation at the blood-brain barrier Alkabie, Samir
SPARC (secreted protein acidic and rich in cysteine) is a cell-matrix modulating protein involved in angiogenesis and endothelial barrier function, yet a potential role in cerebrovascular repair and inflammatory responses in the central nervous system (CNS) has not previously been characterized. The inflammatory demyelinating disease, multiple sclerosis (MS) is characterized pathologically by inflammatory infiltrates, demyelination and axonal damage/loss and aberrant alterations in blood-brain barrier (BBB) integrity. We hypothesize that SPARC expression may be influenced by inflammatory or repair processes during MS, and that SPARC itself may influence BBB integrity. This study examined SPARC expression in cultured human cerebral microvascular endothelial cell (hCMEC/D3), an in vitro model of the BBB, under steady state conditions or those modeling an inflammatory milieu by immunoblotting and immunocytochemistry. hCMEC/D3s constitutively express SPARC during proliferative growth and downregulate SPARC as cells establish a BBB phenotype. SPARC expression in cerebral endothelia directly correlated with the cell proliferation marker Ki-67, consistent with a role for SPARC in CNS angiogenesis. Proinflammatory cytokines associated with inflammation and immune activation differentially regulate SPARC expression in cerebral endothelia. Tumor necrosis factor alpha (TNF-α) cytokine or lipopolysaccharide (LPS) endotoxin treatment significantly increased SPARC protein levels. TNF-α and interferon gamma (IFN-γ) cotreatment abrogated SPARC induction compared to TNF-α alone, suggesting divergent roles for each cytokine in regulating SPARC expression in cerebral endothelia. Compared to cultures replenished with media lacking exogenously supplied SPARC, addition of a physiological SPARC concentration observed in healthy individuals (0.1μg/ml) increased tight junction protein expression of zonula occludens 1 (ZO-1) and occludin by approximately thirty percent, suggesting a role in BBB maintenance. Paradoxically, functional assays show recombinant human SPARC applied exogenously increased the transendothelial permeability of hCMEC/D3 monolayers. In agreement, barrier hCMEC/D3s exposed to increased SPARC concentrations (1-10 μg/ml) associated with pathological conditions in vivo, reduced ZO-1 and occludin by one-third. Together, these data support a role for SPARC in BBB maintenance under normal physiological conditions and BBB alterations during inflammatory conditions. In this regard, SPARC levels may play a key role in regulating BBB integrity and serve to alter processes of CNS inflammation and repair.
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