Fig. 7.
Expression of ANXA1 is selectively lost from the cerebral capillaries of patients with MS, but not those with other neurodegenerative conditions. (A) Confocal microscopic analysis of ANXA1 expression within the endothelium of a typical human cortical capillary, immunofluorescently labeled for CD31 (green) and ANXA1 (red). (Scale bar: 10 µm.) (B) Representative immunohistochemical analysis of ANXA1 expression within cortical capillaries of human male and female postmortem brains. Note in particular the strong expression of ANXA1 in circulating leukocytes of patients with MS, but the absence of endothelial ANXA1 immunostaining (typical images from cases described in Table S1; sections are 8 µm thick). (Scale bar: 10 µm.) (C) Immunohistochemical analysis of ANXA1 expression within the ependyma of the lateral ventricles, the choroid plexus, and the meningeal blood vessels of aged control subjects and patients with MS. Note in particular the expression of ANXA1 in meningeal vessel endothelial cells, but not parenchymal vessel endothelial cells (arrow) in patients with MS (sections are 8 µm thick). (Scale bar: 20 µm.) (D) Plasma ANXA1 content from patients with MS and healthy age- and sex-matched control subjects (lines indicate mean values; *P < 0.05, Student t test). (E) Paracellular permeability coefficient (expressed as percentage of the untreated control) of monolayers of human hCMEC/D3 cells following exposure for 24 h to medium containing 20% plasma from patients with MS, correlated with total levels of ANXA1 in the same hCMEC/D3 cells following exposure (linear regression line and 95% CIs are marked; coefficient of correlation is −0.797; P < 0.01). (F) Administration of exogenous hrANXA1 to plasma from patients with MS limits the increase in permeability caused upon application of the plasma to WT hCMEC/D3 cells (24 h exposure to 20% MS plasma in normal medium); data are representative of three independent experiments, using serum from at least 10 different patients with MS, performed in triplicate, and expressed as mean ± SEM (*P < 0.05, +P < 0.05 vs. control).