Tissue-Type Plasminogen Activator and the Low-Density Lipoprotein Receptor-Related Protein Mediate Cerebral Ischemia-Induced Nuclear Factor-κB Pathway Activation

Abstract

Tissue-type plasminogen activator (tPA) is a serine proteinase found in the intravascular space and the central nervous system. The low-density lipoprotein receptor-related protein (LRP) is a member of the low-density lipoprotein receptor gene family found in neurons and astrocytes. Cerebral ischemia induces activation of the nuclear factor (NF)-B pathway. The present study investigated the role that the interaction between tPA and LRP plays on middle cerebral artery occlusion (MCAO)-induced NF-B-mediated inflammatory response. We found that MCAO increased LRP expression primarily in astrocytes and that this effect was significantly decreased in the absence of tPA. The onset of the ischemic insult induced activation of the NF-B pathway in wild-type and plasminogen (Plg ؊/؊ )-deficient mice, and this effect was attenuated after inhibition of LRP or genetic deficiency of tPA. Moreover, administration of tPA to tPA ؊/؊ mice resulted in activation of the NF-B pathway comparable with that observed in wild-type and Plg ؊/؊ mice. We also report that inhibition of either tPA activity or LRP or genetic deficiency of tPA resulted in a significant decrease in MCAO-induced nitric oxide production and inducible nitric-oxide synthase expression. In conclusion, our results demonstrate that after MCAO the interaction between tPA and LRP results in NF-B activation in astrocytes and induction of inducible nitric-oxide synthase expression in the ischemic tissue , suggesting a cytokine-like plasminogen-independent role for tPA during cerebral ischemia.

The Low-Density Lipoprotein Receptor-Related Protein 1 Mediates Tissue-Type Plasminogen Activator-Induced Microglial Activation in the Ischemic Brain

“…27 Thus, we decided to investigate whether the observed effect of tPA on microglial activation was mediated by LRP1. We performed immunohistochemical analysis for F4/80 and ␤-isolectin and DAPI in wild-type and macLRP Ϫ mice 24 hours after MCAO ( Figure 3A, a-f).…”

“…Indeed, our earlier work indicates that after MCAO there is accumulation of nitrotyrosine in the ischemic tissue of wild-type mice, and that this effect is significantly attenuated in both tPA Ϫ/Ϫ mice and in wild-type animals treated with the receptor-associated protein. 27 Thus, to investigate the role of microglial LRP1 on peroxynitrate (ONOO Ϫ ) production and nitrotyrosine accumulation during cerebral ischemia we performed Western blot analysis and immunohistochemical staining for nitrotyrosine in both wild-type and macLRP Ϫ mice 24 hours after MCAO. A subgroup of macLRP Ϫ mice was treated with tPA immediately after the onset of the ischemic insult.…”

“…19,27 In brief, each coronal section of the brain was divided into 16 square areas (150 mm 2 each one) that involved the necrotic core and the area of ischemic penumbra, and comparable areas in the nonischemic hemisphere.…”

“…22,23 In the CNS, LRP1 is found in perivascular astrocytes, neurons, and microglia 19,24 where it has been implicated in cellular signal transduction pathways. 25 After middle cerebral artery occlusion (MCAO) there is an increase in endogenous tPA activity within the ischemic tissue, 17,18,20 and the association of this tPA with LRP1 has an effect on cerebrovascular tone, 26 NF-B activation, 27 Akt phosphorylation, 28 and regulation of the permeability of the neurovascular unit. 19,20 To gain insight into mechanisms leading to microglial activation during cerebral ischemia in vivo, we have generated a mouse model in which LRP1 has selectively been deleted in macrophages and microglia.…”

Neuroserpin Protects Neurons from Ischemia-Induced Plasmin-Mediated Cell Death Independently of Tissue-Type Plasminogen Activator Inhibition

“…17 Briefly, either the cortex or the hippocampal tissue was dissected, transferred into Hanks' balanced salt solution containing 100 units/ml penicillin, 100 g/ml streptomycin, and 10 mm HEPES, and incubated in trypsin containing 0.02% DNase at 37°C for 15 minutes. Tissue was then triturated, and the supernatant was resuspended in B27-supplemented neurobasal medium containing 2 mmol/L l-glutamine and plated onto 0.1 mg/ml polyl-lysine-coated wells.…”