Combination Therapy With VELCADE and Tissue Plasminogen Activator Is Neuroprotective in Aged Rats After Stroke and Targets MicroRNA-146a and the Toll-Like Receptor Signaling Pathway

Objective-Activation of the toll-like receptor (TLR) signaling pathway exacerbates ischemic brain damage. The present study tested the hypothesis that combination treatment with VELCADE and tissue plasminogen activator (tPA) modulates the TLR signaling pathway on cerebral vasculature, which leads to neuroprotection in aged rats after stroke. Methods and Results-Focal cerebral ischemia acutely increased TLR2, TLR4, and interleukin-1 receptor-activated kinases 1 immunoreactivity on fibrin/fibrinogen-positive vessels in aged rats. Monotherapy of tPA further amplified these signals. However, VELCADE in combination with tPA-blocked stroke- and tPA-potentiated vascular TLR signals, leading to robust reduction of infarct volume compared with respective monotherapies. Quantitative reverse transcription polymerase chain reaction analysis of cerebral endothelial cells isolated by laser capture microdissection revealed that the combination treatment increased miR-146a levels, which was inversely associated with the reduction of vascular interleukin-1 receptor-activated kinases 1 immunoreactivity. In vitro, fibrin upregulated interleukin-1 receptor-activated kinases 1 and TLR4 expression and downregulated miR-146a on primary human cerebral endothelial cells. VELCADE elevated miR-146 levels and abolished fibrin-increased interleukin-1 receptor activated kinases 1 proteins. Conclusion-Stroke acutely activates the TLR signaling pathway on cerebral vasculature. Upregulation of miR-146a and inactivation of ischemia and tPA-potentiated TLR signaling pathway by VELCADE may play an important role in the neuroprotective effect of the combination therapy of VELCADE and tPA for acute stroke. (Arterioscler Thromb Vasc Biol. 2012;32:1856-1864.)