SPARC expression by cerebral microvascular endothelial cells in vitro and its influence on blood-brain barrier properties

Background: SPARC (secreted protein acidic and rich in cysteine) is a nonstructural, cell-matrix modulating protein involved in angiogenesis and endothelial barrier function, yet its potential role in cerebrovascular development, inflammation, and repair in the central nervous system (CNS) remains undetermined. Methods: This study examines SPARC expression in cultured human cerebral microvascular endothelial cells (hCMEC/D3)-an in vitro model of the blood-brain barrier (BBB)-as they transition between proliferative and barrier phenotypes and encounter pro-inflammatory stimuli. SPARC protein levels were quantified by Western blotting and immunocytochemistry and messenger RNA (mRNA) by RT-PCR. Results: Constitutive SPARC expression by proliferating hCMEC/D3s is reduced as cells mature and establish a confluent monolayer. SPARC expression positively correlated with the proliferation marker Ki-67 suggesting a role for SPARC in cerebrovascular development. The pro-inflammatory molecules tumor necrosis factor-alpha (TNF-alpha) and endotoxin lipopolysaccharide (LPS) increased SPARC expression in cerebral endothelia. Interferon gamma (IFN-gamma) abrogated SPARC induction observed with TNF-alpha alone. Barrier function assays show recombinant human (rh)-SPARC increased paracellular permeability and decreased transendothelial electrical resistance (TEER). This was paralleled by reduced zonula occludens-1 (ZO-1) and occludin expression in hCMEC/D3s exposed to rh-SPARC (1-10 mu g/ml) compared with cells in media containing a physiological dose of SPARC. Conclusions: Together, these findings define a role for SPARC in influencing cerebral microvascular properties and function during development and inflammation at the BBB such that it may mediate processes of CNS inflammation and repair.