216 Loss of Endothelial Tissue-Nonspecific Alkaline Phosphatase Exacerbates Microhemorrhages and Neuroinflammation in a Mouse Model of Ischemic Stroke

Abstract

INTRODUCTION: Despite significant progress in treatment strategies, stroke remains a leading cause of mortality and morbidity. Blood-brain barrier dysfunction is coupled to neuroinflammation and poor outcomes post-stroke. Tissue-nonspecific alkaline phosphatase (TNAP) is a glycophosphatidylinositol-linked protein localized to brain microvascular endothelial cells (BMECs). However, TNAP’s role in BMECs remains unclear. Recent in vitro data from our laboratory demonstrates that conditional loss of BMEC TNAP significantly worsened barrier integrity. Importantly, we have shown that TNAP activity is significantly reduced on cerebral microvessels in the penumbra of wild-type stroke mice. METHODS: To test this, we generated mice with a conditional deletion of TNAP in endothelial cells (VE-cKO) and compared the results to controls (Alplfl/fl). Six-month-old mice underwent photothrombotic ischemic stroke (PTS) or sham surgery. RESULTS: Laser speckle flowmetry confirmed that PTS mice exhibited a sustained loss of cerebral blood flow compared to sham mice (p < 0.0001). No significant differences in cerebral blood flow and infarct size were noted between VE-cKO PTS and Alplfl/fl PTS mice. Seven days post-PTS, mice were euthanized, and brains were evaluated for BBB dysfunction and neuroinflammation. Preliminary evidence demonstrates a significant increase (p < 0.0001) in GFAP-positive astrogliosis in the cortical penumbra of VE-cKO PTS compared to Alplfl/fl PTS mice. Astrogliosis was also coupled to a significant increase in the number of microhemorrhages (p < 0.0001) in VE-cKO PTS compared to Alplfl/fl PTS mice. Interestingly, large molecule BBB permeability evaluation with immunoglobulin G (150 kDa) showed no significant differences between VE-cKO PTS and Alplfl/fl PTS mice. CONCLUSIONS: Ongoing studies will quantitatively assess the extent of BBB permeability utilizing fluorescent tracers of varying sizes. Collectively, these results support a novel mechanism through which loss of TNAP activity is linked to brain microvascular dysfunction post-stroke.