Tissue-type plasminogen activator and the low-density lipoprotein receptor-related protein mediate cerebral ischemia-induced nuclear factor-kappaB 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)-kappaB pathway. The present study investigated the role that the interaction between tPA and LRP plays on middle cerebral artery occlusion (MCAO)-induced NF-kappaB-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-kappaB 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-kappaB 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-kappaB 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.

Figure 1

Definition of the AOI. Animals were subjected to MCAO followed by continuous MRI monitoring of CBF and apparent diffusion coefficient (ADC) of water. A and B: Changes in CBF (A) and ADC of water (B) 180 minutes after MCAO. Arrows in A depict the area of the brain with a >80% decrease in CBF (dark zone). Arrows in B denote the area of irreversibly injured brain (dark area). C: T₂-weighted image of the same brain of A and B showing the area of the ischemic brain with >80% decrease in CBF (pseudocolored in yellow) and the zone of the ischemic tissue irreversibly affected (pseudocolored in red). The black squares represent the areas of the brain where the observations for the immunohistochemical studies were performed.

Figure 2

Effect of genetic deficiency of tPA on cerebral ischemia-induced NF-κB activation. A and B: Representative pictures of immunohistochemical analysis of p65 (A) and p65 phosphorylated at serine 536 (B) in the area of ischemic penumbra in WT (a–c) and tPA^−/− mice (d–f) 6 hours after MCAO. a and d: Combined immunostaining for glial fibrillary acidic protein and DAPI; b and e: immunostaining for either p65 (A) or phosphorylated p65 (B); c and f: merged images. Arrows in b depict cells with either nuclear translocation of p65 (A) or phosphorylated p65 (B) indicative of NF-κB activation. Arrows in e (A) point to cells where p65 is primarily located in the cytoplasm indicative of inactive NF-κB pathway. Green is glial fibrillary acidic protein, red is p65 (A) or phosphorylated p65 (B), and blue is DAPI. Each observation was repeated four times. C: Western blot analysis of phospho-p65 and total p65 in brain extracts of WT and tPA^−/− mice 6 hours after MCAO. Actin expression levels were assayed as a control for protein loading. Each experiment was repeated four times. D: Representative gel of an EMSA in brain nuclear extracts from WT sham (lane 1), WT sham and an excess of unlabeled probe (lane 2), WT 6 hours after stroke (lane 3), tPA^−/− mice 6 hours after MCAO (lane 4), and WT sham treated with an anti-NF-κB antibody (lane 5). Black arrow points toward NF-κB DNA-binding activity. Each observation was repeated four times. Original magnifications, ×100.

Figure 3

Effect of genetic deficiency of plasminogen on cerebral ischemia-induced NF-κB activation. A: Western blot analysis of phospho-p65 and total p65 in brain extracts of WT, tPA^−/−, and Plg^−/− mice 6 hours after MCAO. Actin expression levels were assayed as a control for protein loading. Each experiment was repeated four times. B: Representative gel of an EMSA in brain nuclear extracts from WT sham (lane 1), WT sham and an excess of cold probe (lane 2), WT 6 hours after stroke (lane 3), Plg^−/− mice 6 hours after MCAO (lane 4), and WT sham treated with an anti-NF-κB antibody (lane 5). Black arrow points toward NF-κB DNA-binding activity. Each observation was repeated three times.

Figure 4

tPA mediates cerebral ischemia-induced LRP expression in the ischemic brain. A: Quantitative RT-PCR analysis of LRP expression in brain extracts from WT (black bars) and tPA^−/− mice (white bars) 0.1 to 72 hours after MCAO. Error bars described SEM. n = 7 for each observation. *P < 0.05 when compared with WT animals sacrificed 0.1 hour after MCAO. ^P < 0.05 when compared with tPA^−/− mice at each time point. ^†P < 0.05 when compared with tPA^−/− mice sacrificed 0.1 to 48 hours after MCAO. B: RT-PCR analysis of LRP expression in primary murine astrocytic and neuronal cultures exposed to OGD conditions for 4 hours and either PBS (black bars) or PBS and neuroserpin (NS, white bars). n = 3 for each observation. *P < 0.05 when compared with NS-treated cells.

Figure 5

Effect of LRP inhibition on the volume of the ischemic lesion after MCAO. Quantitative analysis of infarct volume 48 hours after MCAO in WT mice treated with PBS (black bar), RAP (white bar), or anti-LRP antibodies (gray bar). n = 6. Lines denote SEM. *P < 0.05 compared with PBS-treated animals.

Figure 6

Effect of LRP inhibition on cerebral ischemia-induced NF-κB activation. A and B: Representative gel of a Western blot analysis of phospho-p65 and total p65 in brain extracts of WT (A) and tPA-deficient (tPA^−/−) mice 6 hours after MCAO and the intracortical injection of the RAP or anti-LRP IgG in WT mice (B), and recombinant tPA or a combination of recombinant tPA and RAP in tPA^−/− animals. Actin expression levels were assayed as a control for protein loading. Each experiment was repeated four times.

Figure 7

Role of tPA and LRP on cerebral ischemia-induced nitrotyrosine production. A: Representative pictures of immunohistochemical analysis of nitrotyrosine formation in the area of ischemic penumbra (AOI-2) in WT (a) and tPA^−/− (b) mice 48 hours after MCAO. Arrows point to examples of nitrotyrosine-immunoreactive cells. Blue is DAPI; red is nitrotyrosine. Each observation was repeated four times. B: Western blot analysis of nitrotyrosine formation 48 hours after MCAO in WT and tPA^−/− mice. A subgroup of WT mice was treated with either anti-LRP IgG (α-LRP) or the RAP. Each observation was repeated three times. Original magnifications, ×40.

Figure 8

Role of tPA and LRP on cerebral ischemia-induced iNOS expression. A: Representative picture of immunohistochemical analysis of iNOS expression in the area of ischemic penumbra (AOI-2) in WT (a and b) and tPA^−/− (c and d) mice 24 hours after MCAO. Green is iNOS, and blue is DAPI. NC, necrotic core. Each observation was repeated four times. B: Quantitative real-time PCR (RT-PCR) analysis of iNOS expression in brain extracts from WT (black bars), tPA^−/− (white bars), and WT mice treated with the RAP (gray bars) 0.1 to 72 hours after MCAO. Error bars described SEM. n = 7 for each observation. *P < 0.05 when compared with treated and untreated WT animals sacrificed at each time point after MCAO. ^†P < 0.05 when compared with untreated WT mice at each time point. C: RT-PCR analysis of iNOS expression in primary murine astrocytic cultures exposed to OGD conditions for 4 hours and incubation with either PBS (black bars) or PBS and neuroserpin (NS, white bars). n = 4 for each observation. *P < 0.05 when compared with NS-treated cells. Original magnifications, ×40.