Cognitive impairment is a well-known complication of diabetes mellitus (DM). Microvascular compromise was described one DM complication. Recently we showed blood brain barrier (BBB) permeability and memory loss are associated with diminution of tight junctions (TJ) in brain endothelium and pericyte coverage and inflammation in cerebral microvessels and brain tissue paralleling hyperglycemia in mice of both DM types. The current study demonstrates that exposure of brain microvessels to hyperglycemic conditions or advanced glycation end products (AGEs) ex vivo resulted in significant abnormalities in membranous distribution of TJ proteins. We found significant increase in the amount of extracellular vesicles (EVs) isolated from DM mice and enhanced presence of TJ proteins, occludin and claudin-5, on EVs. Exposure of BMVECs to high glucose and AGEs led to significant augmentation of ICAM and VCAM expression, elevated leukocyte adhesion to and migration across BMVEC monolayers, and increased BBB permeability in vitro. Pericytes exposed to hyperglycemia and AGEs displayed diminished expression of integrin α1, PDGF-R1β and connexin-43. Our findings indicate BBB compromise in DM ex vivo, in vitro and in vivo models in association with BMVEC/pericyte dysfunction and inflammation. Prevention of BBB injury may be a new therapeutic approach to avert cognitive demise in DM.

Neuroinflammatory conditions often involve dysfunction of the Blood-Brain Barrier (BBB). Therefore, identifying molecular targets that can maintain barrier fidelity is of clinical importance. We have previously reported on the anti-inflammatory effects that glycogen synthase kinase 3β (GSK3β) inhibition has on primary human brain endothelial cells. Here we show that GSK3β inhibitors also promote barrier tightness by affecting tight junction (TJ) protein stability. Transendothelial electrical resistance (TEER) was used to evaluate barrier integrity with both pharmacological inhibitors and mutants of GSK3β. Inhibition of GSK3β produced a gradual and sustained increase in TEER (as much as 22% over baseline). Analysis of subcellular membrane fractions revealed an increase in the amount of essential tight junction proteins, occludin and claudin-5, but not claudin-3. This phenomenon was attributed to a decrease in TJ protein turnover and not transcriptional regulation. Using a novel cell-based assay, inactivation of GSK3β significantly increased the half-life of occludin and claudin-5 by 32% and 43%, respectively. A correlation was also established between the enhanced association of β-catenin with ZO-1 as a function of GSK3β inhibition. Collectively, our findings suggest the possibility of using GSK3β inhibitors as a means to extend the half-life of key tight junction proteins to promote re-sealing of the BBB during neuroinflammation.

MicroRNA‐mediated regulation of gene expression appears to be involved in a variety of cellular processes, including development, differentiation, proliferation, and apoptosis. Mir‐146a is thought to be involved in the regulation of the innate immune response, and its expression is increased in tissues associated with chronic inflammation. Among the predicted gene targets for mir‐146a, the chemokine CCL8/ MCP‐2 is a ligand for the CCR5 chemokine receptor and a potent inhibitor of CD4/CCR5‐mediated HIV‐1 entry and replication. In the present study, we have analyzed changes in the expression of mir‐146a in primary human fetal microglial cells upon infection with HIV‐1 and found increased expression of mir‐146a. We further show that CCL8/MCP‐2 is a target for mir‐146a in HIV‐1 infected microglia, as overexpression of mir‐146a prevented HIV‐induced secretion of MCP‐2 chemokine. The clinical relevance of our findings was evaluated in HIV‐encephalitis (HIVE) brain samples in which decreased levels of MCP‐2 and increased levels of mir‐146a were observed, suggesting a role for mir‐146a in the maintenance of HIV‐mediated chronic inflammation of the brain.—Rom, S., Rom, I., Passiatore, G., Pacifici, M., Radhakrishnan, S., Del Valle, L., Piña‐Oviedo, S., Khalili, K., Eletto, D., Peruzzi, F. CCL8/MCP‐2 is a target for mir‐146a in HIV‐1 infected human microglial cells. FASEB J. 24, 2292–2300 (2010). www.fasebj.org