Tissue-type plasminogen activator (t-PA) is approved for treatment of ischemic stroke patients, but it increases the risk of intracranial bleeding (ICB). Previously, we have shown in a mouse stroke model that stromelysin-1 (matrix metalloproteinase-3 [MMP-3]) induced in endothelial cells was critical for ICB induced by t-PA. In the present study, using bEnd.3 cells, a mouse brain–derived endothelial cell line, we showed that MMP-3 was induced by both ischemic stress and t-PA treatment. This induction by t-PA was prevented by inhibition either of low-density lipoprotein receptor–related protein (LRP) or of nuclear factor-κB activation. LRP was up-regulated by ischemic stress, both in bEnd.3 cells in vitro and in endothelial cells at the ischemic damage area in the mouse stroke model. Furthermore, inhibition of LRP suppressed both MMP-3 induction in endothelial cells and the increase in ICB by t-PA treatment after stroke. These findings indicate that t-PA deteriorates ICB via MMP-3 induction in endothelial cells, which is regulated through the LRP/nuclear factor-κB pathway.

Tissue-type plasminogen activator (t-PA) is a thrombolytic agent that degrades fibrin clots through activation of plasminogen to plasmin.1  Although t-PA given within 3 hours from onset of ischemic stroke improves the clinical outcome in patients, it induces a 10-fold increase of symptomatic intracranial bleeding (ICB).2  Furthermore, delayed t-PA treatment beyond 3 hours is associated with an increased risk of hemorrhagic transformation and with enhanced brain injury.3  The increase of ICB by delayed treatment with t-PA was also observed in a mouse stroke model.4 

Matrix metalloproteinases (MMPs), a family of zinc endopeptidases, contribute to tissue remodeling through degradation of extracellular matrix proteins. For ICB associated with ischemic stroke, MMPs have a key role in the degradation of the barrier of blood vessels.5,6  Previously, we have shown that the increase in ICB caused by t-PA treatment was impaired in mice with gene deficiency of MMP-3 (stromelysin-1) and that a broad-spectrum MMP inhibitor suppressed ICB in wild-type but not in MMP-3–deficient mice.7  MMP-3 can be activated by plasmin8  and has a broad-spectrum substrate specificity.9  Furthermore, t-PA treatment induced MMP-3 selectively in endothelial cells at the ischemic damaged area in a mouse stroke model,7  suggesting that MMP-3 may be involved in degradation of the barrier of blood vessels and contribute to ICB. However, the mechanism underlying MMP-3 induction by t-PA remained unknown and is the subject of the present study.

Low-density lipoprotein receptor–related protein (LRP), a member of the lipoprotein receptor family, is a scavenger receptor that binds a variety of biologic ligands and is thought to be primarily involved in lipoprotein metabolism10  and in clearance of protease-inhibitor complexes in the adult brain.11  Recent reports have shown that t-PA induces MMP-9 in brain endothelial cells12  and increases the blood-brain barrier permeability via LPR activation,13  suggesting a role for LRP as a t-PA receptor. It has also been reported that LRP activation by t-PA stimulates the nuclear factor kappa-B (NF-κB) pathway.14 

In this study, we have evaluated whether the LRP/NF-κB pathway plays a role in MMP-3 induction by t-PA treatment. Therefore, we used bEnd.3 cells, a transformed endothelial cell line derived from mouse brain, as well as a mouse stroke model.

Reagents

Recombinant human t-PA (Alteplase; Activase) was purchased from Genentech Inc. It was inactivated by incubation with D-Phe-Pro-Arg-chloromethylketone (Calbiochem); inactivation was confirmed using S-2258 (Chromogenix). Receptor-associated protein (RAP), which antagonizes binding of ligands to members of the low-density lipoprotein receptor family, was purchased from Chemicon. The following antibodies were purchased: murine MMP-3 (R&D Systems), NF-κB (Calbiochem), histone deacetylase 1 (HDAC1; Sigma-Aldrich), LRP (Progen and Santa Cruz Biotechnology), and β-actin (Cell Signaling Technology). MG-132 (carbobenzoxy-L-leucyl-L-leucyl-L-norvalinal, a proteome inhibitor) was purchased from Chemicon. The probes for real-time polymerase chain reaction (PCR) of LRP mRNA (Lrp1; 5′-TCGGCAGACCATCATCCAAG-3′, forward primer: MA072132-F and 5′-ATTGTCCGAGTTGGTGGCGTA-3′, reverse primer: MA072132-R) were purchased from Takara bio Inc. The primers for mouse β-actin (Takara bio Inc) were 5′-CATCCGTAAAGACCTCTATGCCAAC-3′ (forward primer) and 5′-ATGGAGCCACCGATCCACA-3′ (reverse primer).

Cell culture

bEnd.3 cells (a transformed mouse brain endothelial cell line) were used for in vitro studies. After culturing in Dulbecco modified Eagle medium (DMEM) containing fetal calf serum to confluence, oxygen-glucose deprivation (OGD), which mimics ischemic stress, was applied using a combination of the Areopack KENKI system (Mitsubishi Gas) and cultured in DMEM without glucose (Invitrogen). After 6 hours of OGD, cells were further cultured in DMEM with 25 mM glucose under normoxia over 18 hours. Control cells were cultured in DMEM with 25 mM glucose for 24 hours under normoxia. t-PA (5-30 μg/mL), inactivated t-PA (20 μg/mL), or solvent was added at the end of 6 hours of OGD or of normoxia. To study the role of LRP in the induction of MMP-3, cells were treated with 200 nM of RAP or 2 μg/mL of anti-LRP antibody, 10 minutes before t-PA administration. To study the involvement of NF-κB, cells were treated with 10 μM MG-132 at the initiation of OGD.

Assays

MMP-3 levels were monitored by Western blotting. After the incubations, bathing media and cell extract were prepared15  and frozen at −20°C until used. MMP-3 was immunoprecipitated using polyclonal rabbit anti–mouse MMP-3 antiserum and eluted with sodium dodecyl sulfate (SDS) sample buffer.16  After SDS-polyacrylamide gel electrophoresis (PAGE) and transfer to nitrocellulose paper, it was treated with a rat anti–mouse MMP-3 antibody followed by peroxidase conjugated rabbit anti–rat IgG. Signals were visualized using the enhanced chemiluminescence detection system (GE Healthcare) and quantified by densitometry (LAS-3000 mini and Multi Gauge Version 3.0; Fuji film).

Translocation of NF-κB to the nucleus was measured by a combination of cell fractionation and Western blotting. First, the cytosolic fraction was removed by treatment with hypotonic buffer (10 mM N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid-KOH, pH 7.8, containing 10 mM KCl, 0.1 mM ethylenediaminetetraacetic acid, and 0.1% NP-40) containing proteinase inhibitors (1 mM dithiothreitol, 2 μg/mL aprotinin, 0.5 mM phenylmethanesulfonyl fluoride). Then the nuclear fraction was isolated by treatment with another hypertonic buffer (50 mM N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid-KOH, pH 7.8, containing 420 mM KCl, 5 mM MgCl2, and 2% glycerol) containing proteinase inhibitors. This fraction was subjected to SDS-PAGE, and after transfer to a nitrocellulose membrane treated with anti–NF-κB antibody followed by goat anti–rabbit IgG conjugated with peroxidase. Signals were quantified using the enhanced chemiluminescence system. To confirm the application of equivalent amount of sample in the experiments, subsequent Western blotting with anti-HDAC1, an internal control of nuclear protein, was performed with the nitrocellulose membrane.

LRP protein or mRNA was measured by Western blotting or real-time PCR, respectively. For Western blotting, immunoprecipitated cell extracts were subjected to SDS-PAGE,15  and after transfer to nitrocellulose paper treated with anti-LRP antibody, followed by peroxidase conjugated goat anti–rabbit IgG. For the in vivo study, subsequent Western blotting with anti–β-actin, an internal control, was performed with the nitrocellulose membrane. For real-time PCR, 75 ng of total RNA prepared from cells using the TRIzol reagent (Invitrogen) was subjected to reverse transcription (SuperScript II; Invitrogen) to generate cDNA. Real-time PCR was preformed with a Thermal Cycler Dice Real time System single instrument (Takara bio Inc) using SYBR Premix Ex Taq II reagent (Takara bio Inc). The thermal cycle conditions were as follows: hold for 30 seconds at 95°C, followed by 2-step polymerase chain reaction for 40 cycles of 95°C for 5 seconds, followed by 60°C for 30 seconds. β-Actin was used to normalize gene expression in all samples. Fold induction values were calculated by subtracting the mean difference of gene and β-actin cycle threshold Ct numbers for each treatment group from the mean difference of gene and β-actin Ct numbers for the control group and raising the difference to the power of 2 (2−ΔΔCt). The specific amplification of PCR was proven by the presence of only one peak in the dissociation curve and of a single band on 3% agarose gel electrophoresis.

To assess cell viability, bath media and cell lysate (using 2% Triton X-100) were collected and lactate dehydrogenase activity was measured (Roche Diagnostic). Viability was expressed as the percentage of activity in cell lysate versus total activity in bath media and cell lysate.

Thrombotic ischemic stroke and ICB

To evaluate ICB, we used a model of ischemic stroke induced photochemically by middle cerebral artery (MCA) occlusion, as described elsewhere.4,7,17  Male C57Bl/6 mice weighing 20 to 30 g were used. At 4 hours after MCA occlusion, 10 mg/kg of recombinant t-PA or the same volume of solvent was administered as a bolus in the tail or penile vein. To study the role of LRP in ICB, 1 or 2 mg/kg of RAP was administered as an intravenous bolus 5 minutes before t-PA administration. Animals were killed 20 hours after t-PA administration by overdose of Nembutal (Abbott) and perfused with saline. Brains were removed and placed in a matrix for sectioning into 1-mm-thick sections. The extent of ICB was determined as area (square millimeters) of hemorrhage in the caudal side of all brain sections, using planimetry.7  A good correlation between ICB determined with a biochemical method and by planimetry was shown previously.18 

All animal experiments were approved by the ethical committee of the Hamamatsu University School of Medicine and the University of Leuven, and performed in accordance with the National Institutes of Health.19 

Histologic analysis

At the end of the experiments, mice were perfused with 2% paraformaldehyde containing 75 mM lysine-HCl and 100 mM sodium meta-peroxide, and 7- to 10-μm cryosections of the brain were prepared. For analysis of MMP-3 or LRP expression, sections were stained with rat anti–mouse MMP-3 or anti–human LRP antibody in combination with goat anti–CD31 (Santa Cruz Biotechnology) as an endothelial marker, followed byfluorescein-conjugated anti–rat IgG or rodamine-conjugated anti–mouse or anti–goat IgG. Sections were mounted using VECTASHELD (Vector Laboratories) and analyzed by immunofluorescence microscopy (Olympus DP-70 and DP manager; Olympus). Photographs taken with a 20× objective lens at room temperature were merged by WinROOF 5.0 (Mitani Corporation).

Statistical analysis

Statistical analysis was performed by analysis of variance followed by Fisher Protected Least Significant Difference test. P values less than .05 were considered significant.

Effect of OGD and t-PA on MMP-3 synthesis in bEnd.3 cells

The (Pro)MMP-3 level in bathing media was significantly enhanced after 6 hours of OGD insult followed by 18 hours of normoxia (1.7 ± 0.14-fold increase), compared with 24 hours of normoxia (1.0 ± 0.20-fold). After 3 hours of OGD followed by 21 hours of normoxia, no effect was observed (0.89 ± 0.22-fold). Active MMP-3 was not detected in either of these experiments. Treatment with t-PA caused a significant dose-dependent increase in pro–MMP-3 levels, both after 24 hours of normoxia and after OGD and normoxia, with the maximum effect at a dose between 10 and 20 μg/mL (Figure 1A-B). This effect was not observed by treatment with inactivated t-PA (20 μg/mL; P = .07 under normoxia and P = .99 under OGD vs absence of t-PA; Figure 1B). Pretreatment with RAP (200 nM) prevented the increase in pro-MMP-3 secretion by active t-PA (10 μg/mL) under both normoxia and OGD conditions; in the absence of t-PA, however, RAP had no effect (Figure 1C-D). Treatment with an anti-LRP antibody (2 μg/mL) also prevented the increase in MMP-3 by active t-PA (Figure 1E-F). Both pro- and active MMP-3 in cell extracts were below detection in all experiments (data not shown).

Figure 1

Effect of OGD and of t-PA treatment on MMP-3 secretion by bEnd.3 cells. MMP-3 secretion from bEnd.3 cells after 6 hours of OGD followed by 18 hours of normoxia (O) in the absence or the presence of different concentrations of t-PA is monitored by Western blotting. Data are compared with bEnd.3 cells kept on normoxia (N) for 24 hours. Representative immunoblots (A,C,E) and quantitative data are as fold increase (B,D,F) of the effects of different concentrations of t-PA (A-B), active site-blocked t-PA (B), pretreatment with RAP (C-D), or an anti-LRP antibody (E-F) are also shown. Data represent mean ± SD of 4 to 9 experiments. *P < .05; **P < .01.

Figure 1

Effect of OGD and of t-PA treatment on MMP-3 secretion by bEnd.3 cells. MMP-3 secretion from bEnd.3 cells after 6 hours of OGD followed by 18 hours of normoxia (O) in the absence or the presence of different concentrations of t-PA is monitored by Western blotting. Data are compared with bEnd.3 cells kept on normoxia (N) for 24 hours. Representative immunoblots (A,C,E) and quantitative data are as fold increase (B,D,F) of the effects of different concentrations of t-PA (A-B), active site-blocked t-PA (B), pretreatment with RAP (C-D), or an anti-LRP antibody (E-F) are also shown. Data represent mean ± SD of 4 to 9 experiments. *P < .05; **P < .01.

Close modal

NF-κB activation

To evaluate the involvement of NF-κB, we studied the effect of MG-132 (a proteome inhibitor that inhibits degradation of I-κB and suppresses NF-κB translocation to the nucleus) and anti-LRP antibody on the MMP-3 induction. In bEnd.3 cells, OGD enhanced nuclear NF-κB levels (4.1- ± 1.7-fold) compared with only normoxia (1.0- ± 0.17-fold, Figure 2A-B). Addition of t-PA resulted in a further increase in nuclear NF-κB levels (to 8.1- ± 3.4-fold), which was reversed to the level with OGD alone by pretreatment with MG-132 (5.1- ± 2.8-fold). Similar reduction of NF-κB translocation was observed by treatment of anti-LRP antibody (1.0- ± 0.0-fold in normoxia, 3.7 ± 0.3 in OGD alone, 5.5 ± 0.9 in OGD/t-PA, 3.1 ± 1.0 in OGD/anti-LRP/t-PA, Figure 2C-D). The level of HDAC1 was comparable in all samples (Figure 2A,C). MMP-3 secretion paralleled NF-κB translocation (1.0- ± 0.20-fold in normoxia, 1.7- ± 0.57-fold with OGD alone, 2.3- ± 1.2-fold with OGD plus t-PA and, 1.7- ± 0.52-fold with the combination OGD/MG-132/t-PA; Figure 2E-F). These findings indicate that induction of MMP-3 by t-PA occurs through LRP and subsequent NF-κB activation.

Figure 2

Effects of MG-132 on NF-κB activation and MMP-3 secretion in bEnd.3 cells. NF-κB activation (A-D) or MMP-3 secretion (E-F) is shown on 6 hours of OGD followed by 18 hours of normoxia and t-PA treatment without or with addition of MG-132 (A,B,E,F) or anti-LRP antibody (C-D). Representative immunoblots against NF-κB and HDAC1 as an internal control (A,C) or pro–MMP-3 (E) are also shown. Quantitative data of NF-κB (B,D) and MMP-3 (F) are shown as fold increase and represent mean ± SD of 3 to 15 experiments. *P < .05; **P < .01; ***P < .001.

Figure 2

Effects of MG-132 on NF-κB activation and MMP-3 secretion in bEnd.3 cells. NF-κB activation (A-D) or MMP-3 secretion (E-F) is shown on 6 hours of OGD followed by 18 hours of normoxia and t-PA treatment without or with addition of MG-132 (A,B,E,F) or anti-LRP antibody (C-D). Representative immunoblots against NF-κB and HDAC1 as an internal control (A,C) or pro–MMP-3 (E) are also shown. Quantitative data of NF-κB (B,D) and MMP-3 (F) are shown as fold increase and represent mean ± SD of 3 to 15 experiments. *P < .05; **P < .01; ***P < .001.

Close modal

Effect of OGD on LRP expression in bEnd.3 cells

In bEnd.3 cells, 6 hours of OGD enhanced LRP mRNA expression (2.1- ± 0.28-fold vs 1.0- ± 0.05-fold; Figure 3A), the average Δ-Ct for β-actin and LRP was 19 and 28 cycles, respectively. The increase of LRP expression after 6 hours of OGD was confirmed at the protein level (Figure 3B-C). This increase could not be explained by an increase in cell count, because OGD for 6 hours reduced the bEnd.3 cell survival (to 75% ± 3.7% of normoxia).

Figure 3

Effect of OGD on LRP expression in bEnd.3 cells. Expression LRP mRNA (A) and protein (B-C) on 6 hours of OGD of bEnd.3 cells. Data are shown as fold increase and represent mean ± SD of 5 (A) or 6 (B-C) experiments. *P < .05; **P < .01.

Figure 3

Effect of OGD on LRP expression in bEnd.3 cells. Expression LRP mRNA (A) and protein (B-C) on 6 hours of OGD of bEnd.3 cells. Data are shown as fold increase and represent mean ± SD of 5 (A) or 6 (B-C) experiments. *P < .05; **P < .01.

Close modal

Role of LRP in MMP-3 induction and in enhanced ICB caused by t-PA

LRP expression in the ipsilateral hemisphere was significantly up-regulated by MCA occlusion at both 6 and 24 hours in the mouse stroke model, whereas LRP expression in the contralateral hemisphere was comparable with the naive condition (Figure 4A-B). Immunofluorescence microscopy revealed that LRP was mainly localized at globular CD31-positive endothelial cells (Figure 4F-H). In contrast, it was localized predominantly to neuron-like cells and not to endothelial cells in a normal brain area (Figure 4C-E).

Figure 4

Expression and localization of LRP in the damaged cortex of the mouse brain after MCA occlusion. (A) Representative immunoblots against LRP and β-actin as an internal control are shown. The expression of LRP is up-regulated in the ipsilateral hemisphere at 6 and 24 hours after MCA occlusion, whereas it is not changed in the contralateral hemisphere. (B) Quantitative data are shown as fold increase. Data are mean ± SD of 6 experiments. *P < .05; **P < .01. ipsi indicates ipsilateral hemisphere; contra, contralateral hemisphere. The time after MCA-O is indicated in hours. Localization of LRP in the normal area (C-E) or the damaged border area (F-H) at 24 hours after MCA-O is shown by immunofluorescence microscopy. In the normal area, LRP immunoreactivity (red: rodamine) localized at neuron-like cells (arrowheads in panel C) but not at CD31 (green: fluorescein) positive endothelial cells (arrows in panels D-E). At the damaged border area, it localized at both globular cells (arrowhead in panel F) and CD31-positive endothelial cells (arrow in panels G-H). The scale bar represents 50 μm.

Figure 4

Expression and localization of LRP in the damaged cortex of the mouse brain after MCA occlusion. (A) Representative immunoblots against LRP and β-actin as an internal control are shown. The expression of LRP is up-regulated in the ipsilateral hemisphere at 6 and 24 hours after MCA occlusion, whereas it is not changed in the contralateral hemisphere. (B) Quantitative data are shown as fold increase. Data are mean ± SD of 6 experiments. *P < .05; **P < .01. ipsi indicates ipsilateral hemisphere; contra, contralateral hemisphere. The time after MCA-O is indicated in hours. Localization of LRP in the normal area (C-E) or the damaged border area (F-H) at 24 hours after MCA-O is shown by immunofluorescence microscopy. In the normal area, LRP immunoreactivity (red: rodamine) localized at neuron-like cells (arrowheads in panel C) but not at CD31 (green: fluorescein) positive endothelial cells (arrows in panels D-E). At the damaged border area, it localized at both globular cells (arrowhead in panel F) and CD31-positive endothelial cells (arrow in panels G-H). The scale bar represents 50 μm.

Close modal

To explore a functional role of LRP in the induction of MMP-3 at endothelial cells in the damaged brain and in the increase in ICB induced by t-PA treatment, the effect of RAP was studied in the mouse stroke model. As shown in a previous report,7  MMP-3 immunoreactivity colocalized with the endothelial cell marker CD31 at the border of ischemic damage area in mice treated with t-PA (Figure 5A-C). The immunoreactivity was suppressed by pretreatment with 2 mg/kg of RAP (Figure 5D-F), which is in agreement with our in vitro data. Expression of MMP-3 was also observed in neurons in the normal area but was not affected by treatment with RAP (data not shown). Furthermore, pretreatment with 1 or 2 mg/kg of RAP suppressed the enhancement of ICB induced by t-PA (Figure 5G-K), indicating an association between increased ICB and MMP-3 induction by t-PA in endothelial cells, mediated via LRP.

Figure 5

Effect of RAP on up-regulation of MMP-3 expression and increased ICB induced by t-PA treatment after MCA occlusion in mice. Immunohistochemical analysis of MMP-3 and CD31 expression (A-F). MMP-3 immunoreactivity (green: fluorescein) colocalizing with CD31 immunoreactivity (red: rodamine) at the border zone of the damaged brain after t-PA treatment without (A-C) or with (D-F) RAP pretreatment. The arrows represent endothelial cells, and the scale bar represents 50 μm. Representative pictures are shown of a damaged brain section after MCA-O without t-PA treatment (G), with t-PA treatment (H), with t-PA and 1 mg/kg RAP (I), or with t-PA and 2 mg/kg RAP (J). (K) Quantitative data are also shown. Data represent mean ± SD of 8 experiments. *P < .05; **P < .01.

Figure 5

Effect of RAP on up-regulation of MMP-3 expression and increased ICB induced by t-PA treatment after MCA occlusion in mice. Immunohistochemical analysis of MMP-3 and CD31 expression (A-F). MMP-3 immunoreactivity (green: fluorescein) colocalizing with CD31 immunoreactivity (red: rodamine) at the border zone of the damaged brain after t-PA treatment without (A-C) or with (D-F) RAP pretreatment. The arrows represent endothelial cells, and the scale bar represents 50 μm. Representative pictures are shown of a damaged brain section after MCA-O without t-PA treatment (G), with t-PA treatment (H), with t-PA and 1 mg/kg RAP (I), or with t-PA and 2 mg/kg RAP (J). (K) Quantitative data are also shown. Data represent mean ± SD of 8 experiments. *P < .05; **P < .01.

Close modal

MMP-3 secretion by bEnd.3 cells, an endothelial cell line derived from mouse brain, is induced by both OGD and t-PA treatment. The induction by t-PA is suppressed either by inhibition of LRP with RAP or with an anti-LRP antibody, or by inhibition of the NF-κB pathway with MG-132. MMP-3 was not induced by inactivated t-PA, confirming the importance of the proteolytic activity. We also observed up-regulation of LRP expression under ischemic conditions in endothelial cells, both in vitro and in vivo; this apparently resulted in the selective induction by t-PA of MMP-3 in endothelial cell at ischemic damaged area in vivo. Furthermore, both MMP-3 expression in endothelial cells and increased ICB induced by t-PA in vivo were suppressed by pretreatment with RAP, supporting a critical role of the t-PA/LRP/NF-κB/MMP-3 pathway in ICB.

t-PA induces MMP-3 through the LRP/NF-κB pathway

t-PA is a serine proteinase that induces thrombolysis through plasmin generation from plasminogen. However, recent studies have indicated that t-PA also acts through plasmin(ogen)-independent mechanisms, including LRP activation under various physiologic and pathophysiologic conditions.20-22  LRP is known to participate not only in endocytosis of extracellular proteins, but also in intracellular signal transduction.23  Furthermore, coupling of the LRP and NF-κB pathways has been observed in macrophages and endothelial cells.24,25  Thus, the involvement of the LRP/NF-κB pathway was reported in the induction of MMP-9 by t-PA in an endothelial cell line.12  In the present study, we showed that the induction of NF-κB and MMP-3 by t-PA under ischemic stress was suppressed by MG-132 and that NF-κB induction was suppressed by anti-LRP antibody, demonstrating that induction of MMP-3 by t-PA also occurs via the LRP/NF-κB pathway. It cannot be excluded that activation of this pathway also induces other genes. The increase in MMP-3 by t-PA seen under ischemic stress was relatively modest in bEnd.3 cells (1.5- to 2.5-fold over normoxic control), which may be the result of a relatively high level of MMP-3 secretion under normoxic conditions in cell culture; in contrast, MMP-3 expression is very low in endothelial cells of the naive brain and is immunohistochemically undetectable.7 

Role of MMP-3 in endothelial cells

MMP-3 is induced by t-PA not only in bEnd.3 cells in vitro under ischemic stress but also in endothelial cells at the ischemic border zone in vivo. It is conceivable that the increase in ICB by t-PA treatment occurs via MMP-3 induction. Because MMP-3 has a broad substrate specificity for extracellular proteins,9  it plays a key role in tissue remodeling. Furthermore, previous reports showed that MMP-3 was induced in endothelial cells by stimulation of cytokines26  or by overexpression of EST-1,27  a transcriptional factor associated with endothelial cell proliferation, suggesting an involvement of MMP-3 in vessel remodeling associated with endothelial cell proliferation. Therefore, t-PA may contribute to the vascular remodeling process, as supported by the observation that it increases blood-brain barrier permeability,13  which could be associated with angiogenesis.28 

In addition, MMP-3 induction associated with NF-κB activation has been widely observed in pathologic processes, such as glioma,29  atherosclerosis,30  and rheumatoid arthritis,31  although no canonical NF-κB sites were identified in the promoter sequence of MMP-3.32  Because these pathologic processes are associated with tissue remodeling, it is possible that MMP-3 induction by t-PA results in the acceleration of cellular response on remodeling.

LRP up-regulation in endothelial cells under ischemic conditions

As shown in this study both in vitro and in vivo, LRP expression in endothelial cells is up-regulated by ischemic stress. Because LRP functions as a receptor for t-PA, the sensitivity of endothelial cells to t-PA will be increased under ischemic conditions. This may explain the observation that increased ICB7,12  or blood-brain barrier permeability13  is induced selectively at ischemic damage sites, even if t-PA is administered systemically. Furthermore, we also observed that LRP was increased only after 6 hours of OGD in bEnd.3 cells, suggesting that its induction was relatively delayed after exposure of endothelial cells to ischemic stress. This is consistent with the clinical or experimental findings that ICB after stroke is not increased by early but only by delayed treatment with t-PA.2,4  Up-regulation of LRP was also observed in neurons under ischemic conditions.14  Therefore, the increase in LRP at the ischemic hemisphere may result in increased LPR expression not only by endothelial cells but also by neurons surrounding the damage area.

LRP also participates in the scavenging of t-PA, the complex of t-PA with plasminogen activator inhibitor-1 and of MMP-9.33  Thus, the up-regulation of LRP may also affect matrix remodeling through regulation of the balance of these molecules in the intercellular space.

Proteolytic activity of t-PA and MMP-3 induction

Both the increase in ICB and the induction of MMP-3 via LRP by t-PA required its proteolytic activity, suggesting that receptor binding is not sufficient to trigger these effects. This in consistent with our previous report that plasminogen is essential for t-PA–mediated ICB increase, indicating that plasmin generation is required. However, it has been shown that plasminogen is not necessary for LRP activation,13  suggesting a direct proteolytic effect of t-PA on LRP activation. The functional link with MMP-3 induction remains, however, enigmatic. Possibly, protease-activated receptors (PARs), which are activated by proteolytic cleavage of their extracellular amino-terminal domain, are involved.34  Direct activation of PARs by t-PA has, however, not been shown. Because LRP and PARs are colocalized at lipid rafts,35,36  concentrations of t-PA by LRP at rafts may lead to activation of PARs and in turn to NF-κB activation.37  Thus, PARs represent intriguing potential targets for t-PA, although other substrates cannot be excluded at present.

Therapeutic perspectives

The increase in both ICB and MMP-3 induction by t-PA was suppressed by treatment with RAP, suggesting that ICB caused by t-PA could be suppressed by LRP inhibition. MMP-9 in human endothelial cells is also induced by t-PA through LRP.12  However, our previous studies indicated that MMP-9 may be involved in ICB induced by the ischemic stroke itself, rather than in the increased ICB caused by t-PA treatment. This is suggested by previous findings that MMP-9 expression was already increased at endothelial cells in the ischemic brain after stroke,7,12  whereas t-PA treatment did not alter either the amount or the distribution of MMP-9 in the brain.6,7  Clinically, suppression of both ICB induced by t-PA and that associated with ischemic stress itself would be beneficial. Interestingly, BB-94, a broad-spectrum MMP inhibitor, suppressed the enhancement of ICB induced by t-PA treatment of rats with embolic focal ischemia.6  It remains to be shown whether LRP antagonists, through impairment of both MMP-3 and MMP-9 induction, may have the potential to suppress ICB in patients with stroke.

An Inside Blood analysis of this article appears at the front of this issue.

The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked “advertisement” in accordance with 18 USC section 1734.

The authors thank B. Van Hoef (KU Leuven) for excellent technical assistance.

This study was supported by the Mitsubishi Pharma Research Foundation and the Ministry of Education Culture of Japan (grant 20790204 to Y.S.) and by Excellentie Financiering KU Leuven (EF/05/013 to N.N., H.R.L.).

Contribution: Y.S. and N.N. designed and performed research; K.Y. and J.K. performed LRP studies; H.R.L. supervised and performed MMP-3 studies; and K.U. supervised the study.

Conflict-of-interest disclosure: The authors declare no competing financial interests.

Correspondence: Nobuo Nagai, Department of Physiology, Kinki University School of Medicine, Ohnohigashi 377-2, Osakasayama, Osaka 589-8511, Japan; e-mail: [email protected].

1
Lijnen
 
HR
Collen
 
D
Tissue-type plasminogen activator.
Ann Biol Clin (Paris)
1987
, vol. 
45
 (pg. 
198
-
201
)
2
National Institute of Neurological Disorders and Stroke rt-PA Stroke Study Group
Tissue plasminogen activator for acute ischemic stroke.
N Engl J Med
1995
, vol. 
333
 (pg. 
1581
-
1587
)
3
Clark
 
WM
Wissman
 
S
Albers
 
GW
Jhamandas
 
JH
Madden
 
KP
Hamilton
 
S
Recombinant tissue-type plasminogen activator (Alteplase) for ischemic stroke 3 to 5 hours after symptom onset. The ATLANTIS Study: a randomized controlled trial. Alteplase Thrombolysis for Acute Noninterventional Therapy in Ischemic Stroke.
JAMA
1999
, vol. 
282
 (pg. 
2019
-
2026
)
4
Suzuki
 
Y
Nagai
 
N
Collen
 
D
Comparative effects of microplasmin and tissue-type plasminogen activator (tPA) on cerebral hemorrhage in a middle cerebral artery occlusion model in mice.
J Thromb Haemost
2004
, vol. 
2
 (pg. 
1617
-
1621
)
5
Lapchak
 
PA
Chapman
 
DF
Zivin
 
JA
Metalloproteinase inhibition reduces thrombolytic (tissue plasminogen activator)-induced hemorrhage after thromboembolic stroke.
Stroke
2000
, vol. 
31
 (pg. 
3034
-
3040
)
6
Sumii
 
T
Lo
 
EH
Involvement of matrix metalloproteinase in thrombolysis-associated hemorrhagic transformation after embolic focal ischemia in rats.
Stroke
2002
, vol. 
33
 (pg. 
831
-
836
)
7
Suzuki
 
Y
Nagai
 
N
Umemura
 
K
Collen
 
D
Lijnen
 
HR
Stromelysin-1 (MMP-3) is critical for intracranial bleeding after t-PA treatment of stroke in mice.
J Thromb Haemost
2007
, vol. 
5
 (pg. 
1732
-
1739
)
8
Lijnen
 
HR
Plasmin and matrix metalloproteinases in vascular remodeling.
Thromb Haemost
2001
, vol. 
86
 (pg. 
324
-
333
)
9
Nagase
 
H
Woessner
 
JF
Matrix metalloproteinases.
J Biol Chem
1999
, vol. 
274
 (pg. 
21491
-
21494
)
10
Herz
 
J
Hamann
 
U
Rogne
 
S
Myklebost
 
O
Gausepohl
 
H
Stanley
 
KK
Surface location and high affinity for calcium of a 500-kd liver membrane protein closely related to the LDL-receptor suggest a physiological role as lipoprotein receptor.
EMBO J
1988
, vol. 
7
 (pg. 
4119
-
4127
)
11
Bu
 
G
Williams
 
S
Strickland
 
DK
Schwartz
 
AL
Low density lipoprotein receptor-related protein/alpha 2-macroglobulin receptor is an hepatic receptor for tissue-type plasminogen activator.
Proc Natl Acad Sci U S A
1992
, vol. 
89
 (pg. 
7427
-
7431
)
12
Wang
 
X
Lee
 
SR
Arai
 
K
et al. 
Lipoprotein receptor-mediated induction of matrix metalloproteinase by tissue plasminogen activator.
Nat Med
2003
, vol. 
9
 (pg. 
1313
-
1317
)
13
Yepes
 
M
Sandkvist
 
M
Moore
 
EG
Bugge
 
TH
Strickland
 
DK
Lawrence
 
DA
Tissue-type plasminogen activator induces opening of the blood-brain barrier via the LDL receptor-related protein.
J Clin Invest
2003
, vol. 
112
 (pg. 
1533
-
1540
)
14
Zhang
 
X
Polavarapu
 
R
She
 
H
Mao
 
Z
Yepes
 
M
Tissue-type plasminogen activator and the low-density lipoprotein receptor-related protein mediate cerebral ischemia-induced nuclear factor-kappaB pathway activation.
Am J Pathol
2007
, vol. 
171
 (pg. 
1281
-
1290
)
15
Gu
 
Z
Kaul
 
M
Yan
 
B
et al. 
S-nitrosylation of matrix metalloproteinases: signaling pathway to neuronal cell death.
Science
2002
, vol. 
297
 (pg. 
1186
-
1190
)
16
Nagai
 
N
Suzuki
 
Y
Van Hoef
 
B
Lijnen
 
HR
Collen
 
D
Effects of plasminogen activator inhibitor-1 on ischemic brain injury in permanent and thrombotic middle cerebral artery occlusion models in mice.
J Thromb Haemost
2005
, vol. 
3
 (pg. 
1379
-
1384
)
17
Zhao
 
BQ
Ikeda
 
Y
Ihara
 
H
et al. 
Essential role of endogenous tissue plasminogen activator through matrix metalloproteinase 9 induction and expression on heparin-produced cerebral hemorrhage after cerebral ischemia in mice.
Blood
2004
, vol. 
103
 (pg. 
2610
-
2616
)
18
Choudhri
 
TF
Hoh
 
BL
Solomon
 
RA
Connolly
 
ES
Pinsky
 
DJ
Use of a spectrophotometric hemoglobin assay to objectively quantify intracerebral hemorrhage in mice.
Stroke
1997
, vol. 
28
 (pg. 
2296
-
2302
)
19
Institution of Laboratory Animal Research Comission of Life Science, National Research Council
Guide for the Care and Use of Laboratory Animals
1996
Washington, DC
National Academy Press
20
Hu
 
K
Wu
 
C
Mars
 
WM
Liu
 
Y
Tissue-type plasminogen activator promotes murine myofibroblast activation through LDL receptor-related protein 1-mediated integrin signaling.
J Clin Invest
2007
, vol. 
117
 (pg. 
3821
-
3832
)
21
Polavarapu
 
R
Gongora
 
MC
Yi
 
H
et al. 
Tissue-type plasminogen activator-mediated shedding of astrocytic low-density lipoprotein receptor-related protein increases the permeability of the neurovascular unit.
Blood
2007
, vol. 
109
 (pg. 
3270
-
3278
)
22
Zhuo
 
M
Holtzman
 
DM
Li
 
Y
et al. 
Role of tissue plasminogen activator receptor LRP in hippocampal long-term potentiation.
J Neurosci
2000
, vol. 
20
 (pg. 
542
-
549
)
23
Lillis
 
AP
Van Duyn
 
LB
Murphy-Ullrich
 
JE
Strickland
 
DK
LDL receptor-related protein 1: unique tissue-specific functions revealed by selective gene knockout studies.
Physiol Rev
2008
, vol. 
88
 (pg. 
887
-
918
)
24
Gaultier
 
A
Arandjelovic
 
S
Niessen
 
S
et al. 
Regulation of tumor necrosis factor receptor-1 and the IKK-NF-kappaB pathway by LDL receptor-related protein explains the antiinflammatory activity of this receptor.
Blood
2008
, vol. 
111
 (pg. 
5316
-
5325
)
25
Yu
 
G
Rux
 
AH
Ma
 
P
Bdeir
 
K
Sachais
 
BS
Endothelial expression of E-selectin is induced by the platelet-specific chemokine platelet factor 4 through LRP in an NF-kappaB-dependent manner.
Blood
2005
, vol. 
105
 (pg. 
3545
-
3551
)
26
Hanemaaijer
 
R
Koolwijk
 
P
le Clercq
 
L
de Vree
 
WJ
van Hinsbergh
 
VW
Regulation of matrix metalloproteinase expression in human vein and microvascular endothelial cells: effects of tumour necrosis factor alpha, interleukin 1 and phorbol ester.
Biochem J
1993
, vol. 
296
 (pg. 
803
-
809
)
27
Oda
 
N
Abe
 
M
Sato
 
Y
ETS-1 converts endothelial cells to the angiogenic phenotype by inducing the expression of matrix metalloproteinases and integrin beta3.
J Cell Physiol
1999
, vol. 
178
 (pg. 
121
-
132
)
28
Rigau
 
V
Morin
 
M
Rousset
 
MC
et al. 
Angiogenesis is associated with blood-brain barrier permeability in temporal lobe epilepsy.
Brain
2007
, vol. 
130
 (pg. 
1942
-
1956
)
29
Cheng
 
SM
Xing
 
B
Li
 
JC
Cheung
 
BK
Lau
 
AS
Interferon-gamma regulation of TNFalpha-induced matrix metalloproteinase 3 expression and migration of human glioma T98G cells.
Int J Cancer
2007
, vol. 
121
 (pg. 
1190
-
1196
)
30
Chase
 
AJ
Bond
 
M
Crook
 
MF
Newby
 
AC
Role of nuclear factor-kappa B activation in metalloproteinase-1, -3, and -9 secretion by human macrophages in vitro and rabbit foam cells produced in vivo.
Arterioscler Thromb Vasc Biol
2002
, vol. 
22
 (pg. 
765
-
771
)
31
Bondeson
 
J
Foxwell
 
B
Brennan
 
F
Feldmann
 
M
Defining therapeutic targets by using adenovirus: blocking NF-kappaB inhibits both inflammatory and destructive mechanisms in rheumatoid synovium but spares anti-inflammatory mediators.
Proc Natl Acad Sci U S A
1999
, vol. 
96
 (pg. 
5668
-
5673
)
32
Westermarck
 
J
Kahari
 
VM
Regulation of matrix metalloproteinase expression in tumor invasion.
FASEB J
1999
, vol. 
13
 (pg. 
781
-
792
)
33
Strickland
 
DK
Ranganathan
 
S
Diverse role of LDL receptor-related protein in the clearance of proteases and in signaling.
J Thromb Haemost
2003
, vol. 
1
 (pg. 
1663
-
1670
)
34
Ramachandran
 
R
Hollenberg
 
MD
Proteinases and signalling: pathophysiological and therapeutic implications via PARs and more.
Br J Pharmacol
2008
, vol. 
153
 
suppl 1
(pg. 
S263
-
S282
)
35
Bae
 
JS
Yang
 
L
Rezaie
 
AR
Lipid raft localization regulates the cleavage specificity of protease activated receptor 1 in endothelial cells.
J Thromb Haemost
2008
, vol. 
6
 (pg. 
954
-
961
)
36
Wu
 
L
Gonias
 
SL
The low-density lipoprotein receptor-related protein-1 associates transiently with lipid rafts.
J Cell Biochem
2005
, vol. 
96
 (pg. 
1021
-
1033
)
37
Rahman
 
A
True
 
AL
Anwar
 
KN
Ye
 
RD
Voyno-Yasenetskaya
 
TA
Malik
 
AB
Galpha(q) and Gbetagamma regulate PAR-1 signaling of thrombin-induced NF-kappaB activation and ICAM-1 transcription in endothelial cells.
Circ Res
2002
, vol. 
91
 (pg. 
398
-
405
)
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