Abstract 2966: Role of the Cerebral Vascular Toll-Like Receptor Signaling Pathway in Neuroprotection Induced by Combination Therapy of VELCADE and TPA

Abstract

Background and Purpose: Toll-like receptors (TLRs) and their family members including IL-1 receptor-associated kinase-1 (IRAK1) mediate ischemic cell damage. MicroRNAs (miRNAs) including miR-146a regulate TLRs. The present study investigated whether treatment of stroke in older animals with VELCADE in combination with tPA affects expression of TLRs and miR-146a in cerebral endothelial cells. Methods: Wistar rats at the age of 16-18 months were subjected to right embolic middle cerebral artery occlusion (MCAo). VELCADE (0.1mg/kg) and tPA (5mg/kg) were intravenously administered 2h after MCAo (n=16). Ischemic rats treated with tPA alone (n=14) or saline (n=15) were used as control groups. Levels of TLRs and miR-146a in cerebral endothelial cells were measured. Cultured primary cerebral endothelial cells (PCECs) were used for investigating the direct effect of VELCADE on expression of miR-146a. Results: Quantitative RT-PCR analysis revealed that VELCADE in combination with tPA significantly increased miR-l46a levels (11±3 fold) in the cerebral endothelial cells isolated by laser capture microdissection compared with that in ischemic rats treated with tPA monotherapy (2±1). Concurrently, immunostaining showed that the combination therapy suppressed tPA monotherapy-upregulated TLR2 (44±7 vs 93±8/mm 2 in tPA), TLR4 (39±6vs 91±8), IRAK1 (16±3 vs 46±5), fibrin (17±6 vs 40±3), and NF-kB (9±2 vs 22±3) immunoreactive vessels, leading to reduction of infarct volume (14±2 vs 28±3% in tPA, and 30±3% in saline, p<0.05). These in vivo data indicate that combination therapy-upregulated miR-146a is inversely related with TLRs levels and ischemic cell damage. In vitro, Western blots showed that incubation of PCECs with fibrin at 1.5 µg/ml increased levels of IRAK1 (1.4 fold) and myeloid differentiation factor 88 (MyD88, 1.4 fold). RT-PCR analysis of PCECs showed that fibrin reduced miR-146a levels by 30 %. However, PCECs treated with VELCADE (10 ng/ml) in the presence of fibrin elevated miR-146 levels by 80% and abolished fibrin-elevated IRAK1 and MyD88 proteins. Moreover, overexpression of miR146a in PCECs downregulated IRAK1 protein, a miR-146a target, by 40%. Conclusion: Our data demonstrate that fibrin activates endothelial TLRs and that VELCADE-upregulated miR-146a abolishes tPA-induced TLRs, suggesting that miRNAs and the TLR signaling pathway in cerebral endothelial cells play an important role in the neuroprotective effect of the combination therapy of VELCADE and tPA for acute stroke.