Induction of Caspase-Mediated Cell Death by Matrix Metalloproteinases in Cerebral Endothelial Cells after Hypoxia

Abstract

Matrix metalloproteinases (MMPs) may contribute to the pathophysiology of cerebral ischemia by degrading matrix components in the neurovascular unit. In this study, the authors document a pathway by which MMPs interfere with cell—matrix interactions and trigger caspase-mediated cytotoxicity in brain endothelial cells. Hypoxia—reoxygenation induced endothelial cytotoxicity. Cytoprotection with zDEVD-fmk confirmed that cell death was partly caspase mediated. The temporal profile of caspase-3 activation was matched by elevations in MMP-2 and MMP-9. MMP inhibitors significantly decreased caspase-3 activation and reduced endothelial cell death. Degradation of matrix fibronectin confirmed the presence of extracellular proteolysis. Increasing integrin-linked kinase signaling with the β1 integrin-activating antibody (8A2) ameliorated endothelial cytotoxicity. The results suggest that MMP-9 and MMP-2 contribute to caspase-mediated brain endothelial cell death after hypoxia—reoxygenation by disrupting cell—matrix interactions and homeostatic integrin signaling.

Keywords

Matrix metalloproteinases Caspase Cerebral ischemia Blood—brain barrier

Matrix metalloproteinases (MMPs) constitute a family of extracellular proteases that are involved in the remodeling of extracellular matrix (Hartung and Kieseier, 2000; Yong et al., 1998). Although normal MMP activity is required in development and wound healing, a pathologic role for MMPs has also been suggested in acute and chronic neurodegenerative disorders such as stroke, spinal cord injury, Alzheimer's disease, and multiple sclerosis (Campbell and Pagenstecher, 1999; Lukes et al., 1999; Rosenberg 2002; Yong et al., 2001). Dysregulated MMP activity may damage brain by degrading matrix substrates and interrupting cell—cell or cell—matrix homeostatic interactions necessary for cell survival (Chen and Strickland, 1997; Mun-Bryce and Rosenberg, 1998; Tsirka et al., 1997). In particular, two members of the MMP family, MMP-2 and MMP-9, can degrade critical neurovascular substrates such as collagen IV, laminin, and fibronectin, all of which are major components of vascular basal lamina. Neurovascular matrix degradation then leads to blood—brain barrier perturbations, edema, inflammatory infiltration and parenchymal injury (Asahi et al., 2001a; Mun-Bryce and Rosenberg, 1998; Rosenberg 2002; Yong et al., 2001). More recently, it has been shown that MMP-9 may also directly trigger anoikislike neuronal cell death by interrupting cell—matrix survival signaling (Gu et al., 2002).

In a nonhuman primate model of focal cerebral ischemia, regional neuronal injury was statistically correlated with local loss of microvascular integrity (Tagaya et al., 2001), suggesting an important link between vascular and parenchymal compartments in tissue homeostasis. Presumably, stroke disrupts these homeostatic interactions thus triggering cascades of cell death in brain (del Zoppo 2003; Lo et al., 2002, 2003). Although neuronal and glial cell death ultimately mediates brain tissue injury, proximal interactions with endothelial dysfunction play a critical role. However, the molecular mechanisms of cerebral endothelial cell injury after stroke remain to be fully elucidated. Here, we examined the role of MMPs, hypothesizing that upregulation of MMP-2 and MMP-9 after hypoxia—reoxygenation triggers caspase-3–mediated cytotoxicity by interrupting cell—matrix interactions and perturbing homeostatic integrin signaling in human cerebral endothelial cells.

MATERIALS AND METHODS

Cell culture

Human brain microvascular endothelial cells (Cell Systems Corporation, Kirkland, WA, U.S.A.) were grown and exposed to hypoxia—reoxygenation as previously described (Lee and Lo, 2003). For matrix experiments, plates or dishes were treated with 10 μg/mL human plasma fibronectin (Sigma, St. Louis, MO, U.S.A.) and incubated for 2 hours at 37°C before seeding. For inhibitor experiments, BB-94 (British Biotech, London, United Kingdom), GM6001 (Chemicon, Temecula, CA, U.S.A.), human TIMP-1 (Chemicon), and zDEVD-fmk (Enzyme Systems, Aurora, OH, U.S.A.) were added 30 minutes before hypoxia. 8A2 antibody for β1 integrin activation and mouse IgG (Sigma) for control were used.

Gelatin zymography

Culture medium was centrifuged at 10,000g for 5 minutes at 4°C to remove cells and debris. The cleared medium was concentrated using Microcon (Millipore, Bedford, MA, U.S.A.) with a 10-kd pore diameter cutoff, then each sample was mixed with equal amounts of sodium dodecyl sulfate (SDS) sample buffer (Novex, Carlsbad, CA, U.S.A.) and electrophoresed on 10% SDS-polyacrylamide gels (Novex) containing 1 mg/mL gelatin as the protease substrate. Following electrophoresis, gels were placed in 2.7% TritonX-100 for 1 hour to remove SDS, and then incubated for 20 hours at 37°C in developing buffer (50 mmol/L Tris base, 40 mmol/L HCl, 200 mmol/L NaCl, 5 mmol/L CaCl₂, and 0.2% Briji 35; Novex). After incubation, gels were stained in 30% methanol, 10% acetic acid, and 0.5% w/v Coomassie Brilliant Blue for 1 hour followed by destaining. To confirm pro and processed active forms of MMP-2 or MMP-9, conditioned medium were treated for 2 hours at 37°C with p-aminophenylmercuric acetate (APMA), which converts zymogen to active enzyme (data not shown). Mixed human MMP-2 and MMP-9 standards (Chemicon) were used as positive controls. Relative gelatinolytic activity (MMP-2 and MMP-9) was quantified and expressed as a ratio of the loaded positive controls via measurement of optical density using the NIH image analysis software.

Western blot analysis

Cultures were rinsed twice with ice-cold phosphate-buffered saline (PBS) and the cells were collected into cell lysis buffer (20 mmol/L Tris, pH 7.5; 150 mmol/L NaCl, 1 mmol/L EDTA, 1 mmol/L EGTA, 1% Triton X-100, 2.5 mmol/L sodium pyrophosphate, 1 mmol/L β-glycerophosphate, 1 mmol/L Na₃VO₄, 1 μg/mL leupeptin, and 1 mmol/L PMSF). Cell lysates were then homogenized and centrifuged at 10,000g for 15 minutes at 4°C and protein concentration was determined with the Bradford assay (Bio-Rad, Hercules, CA, U.S.A.). Samples were boiled in SDS sample buffer (Novex) with 10 mmol/L DTT and proteins (30 μg/lane) were separated on 4% to 20% SDS-polyacrylamide gels. The separated proteins were transferred to polyvinylidene difluoride membranes (Novex), treated with blocking buffer (Tris-buffered saline containing 0.2% I-block [Tropix, Bedford, MA, U.S.A.] and 0.1% Tween 20). Membranes were probed overnight at 4°C with anticleaved caspase-3 (Cell Signaling Technology, Beverly, MA, U.S.A.) and antifibronectin (Sigma) antibody. Anti—rabbit immunoglobulin G conjugated with horseradish peroxidase was used as the secondary antibody and immune complexes were visualized by enhanced chemiluminescence (Amersham, Piscataway, NJ, U.S.A.).

Lactate dehydrogenase release assay

Hypoxia—reoxygenation induced cytotoxicity was quantified by a standard measurement of lactate dehydrogenase (LDH) release using the LDH assay kit (Roche, Indianapolis, IN, U.S.A.).

DNA fragmentation assay

Genomic DNA isolation was performed using the apoptotic DNA ladder detection kit (Chemicon). Lysed cells with TE buffer were incubated with solution containing RNase A at 37°C for 10 minutes and Proteinase K at 55°C for 30 minutes. DNA was precipitated at -20°C for 2 hours with 3 mol/L ammonium acetate and the DNA ladder was visualized under ultraviolet light with ethidium bromide staining.

Fibronectin fragment analysis

Fibronectin (1 μg) from human plasma (Sigma) was incubated with purified human MMP-2 or MMP-9 (Chemicon) in 50 mmol/L Tris-HCl (pH 7.5), 0.1 mol/L NaCl, and 10 mmol/L CaCl₂ buffer at 37°C for 6 hours. For enzyme activation, 10 μmol/L APMA was added in the buffer before reaction. The MMP inhibitor BB-94 was prepared at 1 mmol/L in dimethyl-sulfoxide (DMSO) and used at 1 μmol/L. The reactions were stopped with SDS sample buffer (Novex) with 10 mmol/L DTT and SDS polyacrylamide gel electrophoresis was performed using precast 4% to 20% gels followed by Western blotting.

Integrin-linked kinase (ILK) assay

Equivalent amounts of protein (200 μg) from cell lysates were incubated for 4 hours at 4°C in the presence of anti-ILK antibody (2 μg/mL; Upstate, Lake Placid, NY, U.S.A.). The protein/antibody complex was then precipitated with protein A and centrifuged. The complex was then washed three times with lysis buffer, and two times with kinase buffer containing 50 mmol/L HEPES (pH 7.0), 10 mmol/L MnCl₂, 10 mmol/L MgCl₂, 2 mmol/L sodium fluoride, and 1 mmol/L Na₃VO₄. Myelin basic protein (2 μg; Upstate) and ATP were added in kinase buffer, incubated for 20 minutes at 30°C. The reaction was stopped by adding sample buffer and proteins were separated on a 16% SDS polyacrylamide gels followed by Western blot analysis with anti—phospho-MBP antibody (Upstate).

Statistical analysis

Quantitative data were analyzed using analysis of variance followed by Tukey honestly significant difference tests between individual groups. Data were expressed as mean ± SD. A value of P < 0.05 was considered significant.

RESULTS

Hypoxia—reoxygenation induces caspase-3-mediated cytotoxicity in human cerebral endothelial cells

Cell death was quantified by measurement of LDH, a stable cytoplasmic enzyme released into the cell culture medium. Although this assay does not distinguish between necrotic or apoptotic cell death, it is a generally accepted cumulative index of cytotoxicity in cell culture systems where eventually cell lysis occurs independent of the precise death pathways involved. Cell death increased upon reoxygenation for periods of more than 12 hours after 4 hours hypoxia (Fig. 1A). The presence of DNA laddering suggested the role of a caspase-mediated apoptoticlike mechanism (Fig. 1B). Concomitantly, caspase-3 cleavage and activation occurred over the reoxygenation period (Fig. 1C). To confirm the involvement of caspase-3 in this model of hypoxia—reoxygenation-induced endothelial cell death, we tested the effects of caspase-3 inhibition. Pretreatment with zDEVD-fmk 30 minutes before the start of hypoxia significantly ameliorated cytotoxicity in a dose-dependent manner (Fig. 1D).

FIG. 1.

Involvement of caspase-3 in brain endothelial cell death after hypoxia—reoxygenation. (A) Increasing lactate dehydrogenase (LDH) release indicates cytotoxicity after 4 hours hypoxia (H4) alone (R0) or hypoxia followed by various periods of reoxygenation; R6 for 6 hours, R12 for 12 hours, and R24 for 24 hours (mean ± SD of four independent experiments). *P < 0.05 versus normal cells. (B) DNA electrophoresis showing DNA laddering after 4 hours hypoxia and 24 hours reoxygenation. (C) Western blots showing elevated levels of cleaved/active caspase-3 (17 and 19 kd). (D) Pretreatment with the caspase-3 inhibitor zDEVD-fmk (20–50 μmol/L) reduces endothelial cell death (mean ± SD of three independent experiments). *P < 0.05 versus H4/R24 cells.

Endothelial cell death is mediated by MMPs after hypoxia—reoxygenation

Gelatin zymography showed that baseline levels of MMP-2 but not MMP-9 were detectable in conditioned media under normal conditions (Fig. 2A, lane 2). Hypoxia—reoxygenation increased both MMP-2 and MMP-9 production. Total MMP-2 levels including both pro and processed active forms continuously increased during reoxygenation for up to 24 hours (Fig. 2A and Fig. 2B). In contrast, only processed active forms of MMP-9 were elevated, reaching a level of twofold over baseline levels after 24 hours (Fig. 2A and Fig. 2C).

Since the promoter regions of many MMP genes are known to contain nuclear factor kB (NF-kB) transcription factor sites, we tested the effects of the relatively selective NF-kB inhibitor SN50 in our system. Gelatin zymography showed that pretreatment with SN50 significantly reduced MMP-9 in a dose-dependent manner (Fig. 2D). No changes were observed for MMP-2 (data not shown).

FIG. 2.

Upregulation of endothelial MMP-2 and MMP-9 after hypoxia—reoxygenation. (A) Gelatin zymography showing that MMP-2 and MMP-9 progressively increases after 4 hours hypoxia followed by reoxygenation. PC indicates positive controls loaded with MMP-2 and MMP-9 standards. (B) Densitometry quantitation of pro and active MMP-2 in conditioned media (fold increase over baseline, mean ± SD). (C) Densitometry quantitation of active MMP-9 in conditioned media (fold increase over baseline, mean ± SD). *P < 0.05 versus normal cells. (D) The nuclear factor kB inhibitor SN50 reduces MMP-9 levels in a dose-dependent manner (mean ± SD of three independent experiments). *P < 0.05 versus H4/R24 cells.

To show that MMPs were involved in brain endothelial cytotoxicity, we tested two broad-spectrum metalloproteinase inhibitors, BB-94 (1 μmol/L) and GM6001 (5 μmol/L), as well as the endogenous tissue inhibitor of metalloproteinase recombinant TIMP-1 (2 μg/mL). All inhibitors significantly reduced caspase-3 activation (Fig. 3A) and cell death (Fig. 3B) after hypoxia—reoxygenation.

FIG. 3.

Effects of MMP inhibitors on caspase-3–mediated endothelial cell death. Cells were pretreated with 1 μmol/L BB-94 (BB), 5 μmol/L GM6001 (GM), and 2 μg/mL TIMP1 (T1) for 30 minutes before hypoxia. All MMP inhibitors (A) reduce caspase-3 cleavage/activation on Western blots and (B) significantly protect against cell death (mean ± SD of four independent experiments). Actin serves as a protein loading control. *P < 0.05 versus H4/R24 cells. H4, 4 hours hypoxia; R24, 24 hours reoxygenation.

MMPs degrade extracellular matrix and disrupt integrin signaling

Endothelial cells are dependent on adhesion to extracellular matrix for their continued survival. Thus, disruption of appropriate cell—matrix interactions can disrupt homeostasis and initiate cell death. In a cell-free system, exogenously added recombinant MMP-2 (Fig. 4A, left lanes) and MMP-9 (Fig. 4A, right lanes) degraded the matrix protein fibronectin. The metalloproteinase inhibitor BB-94 reduced fibronectin degradation caused by exogenous MMPs, thus confirming the specificity of the response. Next, we tested whether similar events occurred in our brain endothelial cell cultures grown on fibronectin-coated plates. After cells were damaged by 4 hours hypoxia and 24 hours reoxygenation, fibronectin degradation was detected (Fig. 4B). The metalloproteinase inhibitor BB-94 ameliorated fibronectin degradation (Fig. 4B).

FIG. 4.

MMP-mediated degradation of matrix fibronectin. (A) Western blots show that in a cell-free system, addition of recombinant MMP-2 (left lanes) and MMP-9 (right lanes) degrade fibronectin. Fibronectin (1 μg) incubated with buffer alone (lanes 1 and 4), with MMP enzyme (lanes 2 and 5), or with MMP plus the metalloproteinase inhibitor BB-94 (lanes 3 and 6). (B) In the brain endothelial cell culture model, conditioned media after 4 hours hypoxia (H4) and 24 hours reoxygenation (R24) show that matrix fibronectin is degraded by MMPs, and pretreatment with BB-94 reduces fibronectin degradation. Lower blot demonstrates cleaved fibronectin bands that show up more clearly after longer exposure times. Arrows indicate fibronectin fragments.

Integrin-mediated signaling is dependent on homeostatic interactions with extracellular matrix components. Hence, matrix-degrading enzymes such as MMPs may induce anoikislike cell death in our model by disrupting cell—matrix interactions. A major pathway involves matrix fibronectin-mediated integrin signaling. In our brain endothelial model, the β1 integrin-activating antibody 8A2 upregulated integrin-linked kinase (Fig. 5A). Pretreatment with 8A2 (10 μg/mL) at 30 minutes before hypoxia reduced caspase-3 cleavage and activation after hypoxia—reoxygenation (Fig. 5B). Concomitantly, rescue of integrin signaling with 8A2 also significantly ameliorated endothelial cytotoxicity (Fig. 5C).

FIG. 5.

Protective effects of β1 integrin activation. (A) Integrin linked kinase activity, as measured by phosphorylation of the substrate myelin basic protein (MBP), is upregulated in endothelial cells exposed to 10 micrograms/mL beta-1 integrin-activating antibody 8A2, confirming the involvment of integrin signaling pathways in our model. (B) Western blots show that pretreatment with 8A2 decreases cleavage/activation of caspase-3 after 4 hours hypoxia (H4) and 24 hours reoxygenation (R24). (C) Pretreatment with 8A2 significantly ameliorates brain endothelial cytotoxicity after hypoxia—reoxygenation (mean ± SD of three independent experiments). *P < 0.05 versus H4/R24 cells.

DISCUSSION

A singular focus on neuron death pathways alone has not yielded successful stroke therapies (Fisher and Ratan, 2003; Gladstone et al., 2002). An emerging concept in stroke research now proposes that all cells of the neurovascular unit (typically comprising endothelium, astrocytes, and neurons) provide the functional substrate that mediates an integrative tissue response to ischemia and infarction (Lo et al., 2003). This modular concept emphasizes cell—cell and cell—matrix interactions, and further suggests that microvascular reactions to ischemia form a proximal trigger for parenchymal injury (del Zoppo and Mabuchi, 2003; Lo et al., 2003). Therefore, investigations into cerebral endothelial responses in stroke are urgently needed. Here, we used a human brain endothelial cell model to demonstrate that extracellular proteolysis due to dysregulated MMPs degrade cell—matrix interactions, and disruption of homeostatic integrin signaling then promotes caspase-mediated cell death (Fig. 6).

FIG. 6.

Schematic diagram shows extracellular proteolysis due to dysregulated MMPs that degrade cell—matrix interactions. Subsequent disruption in integrin signaling promotes caspase-mediated cell death in our model.

MMPs, especially the gelatinases MMP-2 and MMP-9, are upregulated in animal models of cerebral ischemia and hemorrhage (Lo et al., 2002; Rogenberg, 2002; Yong et al., 2001), and MMP-9 gene knockout protects against blood—brain barrier leakage (Asahi et al., 2001a; Wang et al., 2000). These findings are consistent with the idea that dysregulated MMPs degrade basal lamina substrates (e.g., collagen, laminin, fibronectin), impair microvascular function, and lead to parenchymal edema and injury. Although vascular matrix degradation and edema are usually considered to be the major pathways for MMP-mediated pathology, direct actions on cells may also occur. For example, MMP inhibition and MMP-9 gene knockout reduced hippocampal neuronal injury after transient global cerebral ischemia, even though blood—brain barrier leakage does not occur in this model (Lee et al., 2004). Extracellular proteolysis promotes anoikislike death in epithelial cells (Frisch and Francis, 1994). It is increasingly recognized that similar anoikislike pathways may operate in neurons as well (Lesay et al., 2001). Loss of neuron—matrix interactions promotes neurotoxicity by downregulating cell-survival integrin-signaling pathways (Gary et al., 2003; Gary and Mattson, 2001). In mouse hippocampus, kainate excitotoxicity is induced by tPA-mediated degradation of neuron—laminin interactions (Indyk et al., 2003; Tsirka et al., 1997). In addition to the plasminogen system, MMPs comprise the other major extracellular protease system in mammalian brain (Lo et al., 2002). MMP-9–mediated disruption of cell—matrix interactions promotes neuronal death (Gu et al., 2002). Here, we showed that similar mechanisms may be involved, insofar as endogenous apoptotic machinery has been previously demonstrated to be present in endothelial cells (Aoudjit and Vuori, 2001; Lin and Maiese, 2001; Meguro et al., 2001; Xu et al., 2001). After hypoxia—reoxygenation, MMP-2 and MMP-9 were upregulated in human brain endothelial cells, and subsequent degradation of extracellular matrix fibronectin appeared to trigger a form of caspase-mediated cytotoxicity. In this study, both cell-free and cell culture systems showed that MMP-2 and MMP-9 can degrade matrix fibronectin.

Fibronectin signaling is required to stave off apoptosis in many cell types (Di Matola et al., 2000; Jeong et al., 2001). A recent study showed that fibronectin knockout mice suffered increased neuronal apoptosis and infarction after cerebral ischemia (Sakai et al., 2001). Although the precise survival signals conveyed by matrix fibronectin remain to be fully dissected, interactions with various integrins are involved. Loss of constitutive integrin signaling recruits caspase-8 and triggers apoptosis (Stupack et al., 2001). The intermediary pathway may involve ILK, which activates the prosurvival signal Akt (Gary et al., 2003). Indeed, ILK protects a venous endothelial cell line against oxidized low-density-lipoprotein—induced cytotoxicity (Zhang et al., 2001). Here, we showed that directly activating β1 integrin with 8A2 antibody (Seki et al., 1996) increased ILK signaling, decreased caspase-3 levels, and ameliorated cell death.

The present finding linking MMPs to endothelial cytotoxicity is important because clinical data suggest that patients with elevated MMP-9 levels are prone to cerebral hemorrhage and injury after thrombolytic therapy (Horstmann et al., 2003; Montaner et al., 2003). Nevertheless, there may be a few caveats worth considering. First, although gel zymograms showed elevations in active MMP-2 and MMP-9 protein, we did not measure mRNA and so cannot define the mechanisms of MMP upregulation. However, hypoxia—reoxygenation induces transcriptional responses in gelatinases (Ben-Yosef et al., 2002; Kolev et al., 2003). NF-kB mediates MMP-9 regulation (Horstmann et al., 2003; Wang et al., 2003), and we showed that the NF-kB inhibitor SN50 downregulated MMP-9 in our model. Secondly, we cannot distinguish between MMP-2 and MMP-9 effects. Active forms of both gelatinases are increased in our model, and the MMP inhibitors used here (BB-94, GM6001 and recombinant TIMP1) can inhibit both proteases. Future studies using specific RNAi strategies may be required to clarify this point. However, it should be noted that MMP-9–knockout mice are protected against focal ischemia (Asahi et al., 2000, 2001a), whereas MMP-2 knockouts are not (Asahi et al., 2001b), suggesting a dominance for MMP-9 in this system. A third question relates to quantitation. Exactly how much of brain endothelial cell death seen here is accounted for by MMPs and caspase? MMP inhibitors reduced caspase activation in our model, but neither MMP inhibitors nor caspase inhibitors completely prevented cytotoxicity. Only about 30% to 40% of cell death was ameliorated. These data suggest that although MMP-mediated phenomena comprise an important portion of the lethal events after brain endothelial injury, other parallel pathways operate as well, further underscoring the need for combination therapies in stroke. For example, cross-talk between calpain and caspase proteolytic systems have been implicated in neuronal cell death (Chen et al., 2002; Rami, 2003; Neumar et al., 2003; Wang, 2000), and the use of calpain inhibitors may have significant cytoprotective properties here. In addition to the outside-in integrin signals delineated here, activation of extrinsic death pathways via Fas and tumor necrosis factor-α receptors may also amplify intrinsic mitochondrial death mechanisms (Eldadah and Faden, 2000; Ferrer and Planas, 2003; Plesnila et al., 2001; Wallach et al., 1999). Once death cascades are initiated, other caspase-independent signals may contribute, including the recently defined apoptosis-inducing factor (AIF) and poly-ADP-ribose polymerase (PARP) pathways (Ferrer and Planas, 2003; Susin et al., 1999; Yu et al., 2002, 2003). A fourth potential limitation concerns our cell culture model. Although the human brain endothelial system has been used before, the focus here on cell—matrix interactions may be hampered by the two-dimensional nature of the model. It is impossible to recapitulate all aspects of three-dimensional extracellular matrix and brain cell structure in cell culture. In vivo, more complex and redundant pathways may operate.

Finally, although our simplified schema in Fig. 6 suggests a linear pathway for brain endothelial injury, we aware that multiple mechanisms become activated after stroke. Endothelial cells undergoing apoptosis become more prone to interact with platelets and leukocytes (Bombeli et al., 1999; Schwartz et al., 1999), which further perturbs cerebral perfusion. MMPs may also cleave and liberate proinflammatory signals such as tumor necrosis factor-α and ICAM-1 (Rosenberg, 2002; Yong et al., 2001), hence promoting secondary tissue injury. Here, we showed that MMP inhibition decreased levels of caspase-3, but caspases can in turn cleave and activate MMPs (Levkau et al., 2002), so that positive feedback loops may occur. Furthermore, kinase signaling pathways are notorious for having complex cross-talk so that much more than ILK will be involved, including prosurvival or prodeath MAP kinase cascades (Griendling et al., 2000). Ultimately, however, these data show a central role for MMP-mediated caspase cell death in brain endothelium, which may comprise an important upstream trigger in stroke.

In summary, we document a role for MMPs in caspase-mediated brain endothelial cell death after hypoxia—reoxygenation. Extracellular proteolysis degrades cell—matrix interactions and disrupts integrin signaling. Further investigations into how MMP's disrupt cell—cell and cell—matrix homeostasis within all cells of the neurovascular unit are warranted. A deeper understanding of how endothelial injury and death in brain affect neurovascular function may reveal new therapeutic opportunities not only for stroke, but perhaps also for other neurodegenerative conditions where vascular dysfunction might be involved.

Footnotes

Acknowledgments

The authors thank Ken Arai and Xiaoying Wang for many helpful discussions, and are extremely grateful to John Harlan for kindly providing the 8A2 integrin-activating antibody.

References

Aoudjit

Vuori

(2001) Matrix attachment regulates Fas-induced apoptosis in endothelial cells: a role for c-flip and implications for anoikis. J Cell Biol 152:633–643

Asahi

Jung

del Zoppo

Fini

(2000) Role for matrix metalloproteinase 9 after focal cerebral ischemia: effects of gene knockout and enzyme inhibition with BB-94. J Cereb Blood Flow Metab 20:1681–1689

Asahi

Sumii

Fini

Itohara

(2001a) Matrix metalloproteinase 2 gene knockout has no effect on acute brain injury after focal ischemia. Neuroreport 12:3003–3007

Asahi

Wang

Mori

Sumii

Jung

Moskowitz

Fini

(2001b) Effects of matrix metalloproteinase-9 gene knock out on the proteolysis of blood-brain barrier and white matter components after cerebral ischemia. J Neurosci 21:7724–7732

Ben-Yosef

Lahat

Shapiro

Bitterman

Miller

(2002) Regulation of endothelial matrix metalloproteinase-2 by hypoxia/reoxygenation. Circ Res 90:784–791

Bombeli

Schwartz

Harlan

(1999) Endothelial cells undergoing apoptosis become proadhesive for nonactivated platelets. Blood 93:3831–3838

Campbell

Pagenstecher

(1999) Matrix metalloproteinases and their inhibitors in the nervous system: the good, the bad and the enigmatic. Trends Neurosci 22:285–287

Chen

Won

Krajewski Gottlieb

(2002) Calpain and mitochondria in ischemia/reperfusion injury. J Biol Chem 277:29181–29186

Chen

Strickland

(1997) Neuronal death in the hippocampus is promoted by plasmin-catalyzed degradation of laminin. Cell 91:917–925

10.

del Zoppo

Mabuchi

(2003) Cerebral microvessel responses to focal ischemia. J Cereb Blood Flow Metab 23:879–894

11.

Di Matola

Mueller

Fenzi

Rossi

Bifulco

Marzano

Vitale

(2000) Serum withdrawal-induced apoptosis in thyroid cells is caused by loss of fibronectin-integrin interaction. J Clin Endocrinol Metab 85:1188–1193

12.

Eldadah

Faden

(2000) Caspase pathways, neuronal apoptosis, and CNS injury. J Neurotrauma 17:811–829

13.

Fisher

Ratan

(2003) New perspectives on developing acute stroke therapy. Ann Neurol 53:10–20

14.

Ferrer

Planas

(2003) Signaling of cell death and cell survival following focal cerebral ischemia: life and death struggle in the penumbra. J Neuropathol Exp Neurol 62:329–339

15.

Frisch

Francis

(1994) Disruption of epithelial cell-matrix interactions induces apoptosis. J Cell Biol 124:619–626

16.

Gary

Mattson

(2001) Integrin signaling via the PI3-kinase-Akt pathway increases neuronal resistance to glutamate-induced apoptosis. J Neurochem 76:1485–1496

17.

Gary

Milhavet

Camandola

Mattson

(2003) Essential role for integrin linked kinase in Akt-mediated integrin survival signaling in hippocampal neurons. J Neurochem 84:878–890

18.

Gladstone

Black

Hakim

(2002) Toward wisdom from failure: lessons from neuroprotective stroke trials and new therapeutic directions. Stroke 33:2123–2136

19.

Griendling

Sorescu

Lassegue

Ushio-Fukai

(2000) Modulation of protein kinase activity and gene expression by reactive oxygen species and their role in vascular physiology and pathophysiology. Arterioscler Thromb Vasc Biol 20:2175–2183

20.

Kaul

Yan

Kridel

Cui

Strongin

Smith

Liddington

Lipton

(2002) S-nitrosylation of matrix metalloproteinases: signaling pathway to neuronal cell death. Science 297:1186–1190

21.

Hartung

Kieseier

(2000) The role of matrix metalloproteases in autoimmune damage to the central and peripheral nervous system. J Neuroimmunol 107:140–147

22.

Horstmann

Kalb

Koziol

Gardner

Wagner

(2003) Profiles of matrix metalloproteinases, their inhibitors, and laminin in stroke patients: influence of different therapies. Stroke 34:2165–2170

23.

Indyk

Chen

Tsirka

Strickland

(2003) Laminin chain expression suggests that laminin-10 is a major isoform in the mouse hippocampus and is degraded by the tissue plasminogen activator/plasmin protease cascade during excitotoxic injury. Neuroscience 116:359–371

24.

Jeong

Han

Lim

Kim

Park

Woods

Couchman

(2001) Rat embryo fibroblasts require both the cell-binding and the heparin-binding domains of fibronectin for survival. Biochem J 356:531–537

25.

Kolev

Skopal

Simon

Csonka

Machovich

Nagy

(2003) Matrix metalloproteinase-9 expression in post-hypoxic human brain capillary endothelial cells: H2O2 as a trigger and NF-kappaB as a signal transducer. Thromb Haemost 90:528–537

26.

Lee

(2003) Interactions between p38 mitogen-activated protein kinase and caspase-3 in cerebral endothelial cell death after hypoxia-reoxygenation. Stroke 34:2704–2709

27.

Lee

Tsuji

Lee

(2004) Role of matrix metalloproteinases in delayed neuronal damage after transient global cerebral ischemia. J Neurosci 13:671–678

28.

Lesay

Hickman

Gibson

(2001) Disruption of focal adhesions mediates detachment during neuronal apoptosis. Neuroreport 12:2111–2115

29.

Levkau

Kenagy

Karsan

Weitkamp

Clowes

Ross

Raines

(2002) Activation of metalloproteinases and their association with integrins: an auxiliary apoptotic pathway in human endothelial cells. Cell death and differentiation 9:1360–1367

30.

Lin

Maiese

(2001) The metabotropic glutamate receptor system protects against ischemic free radical programmed cell death in rat brain endothelial cells. J Cereb Blood Flow Metab 21:262–275

31.

Dalkara

Moskowitz

(2003) Mechanisms, challenges and opportunities in stroke. Nat Rev Neurosci 4:399–415

32.

Wang

Cuzner

(2002) Extracellular proteolysis in brain injury and inflammation: role for plasminogen activators and matrix metalloproteinases. J Neurosci Res 69:1–9

33.

Lukes

Mun-Bryce

Lukes

Rosenberg

(1999) Extracellular matrix degradation by metalloproteinases and central nervous system diseases. Mol Neurobiol 19:267–284

34.

Meguro

Chen

Lancon

Zhang

(2001) Oxyhemoglobin induces caspase-mediated cell death in cerebral endothelial cells. J Neurochem 77:1128–1135

35.

Montaner

Molina

Monasterio

Abilleira

Arenillas

Ribo

Quintana

Alvarez-Sabin

(2003) Matrix metalloproteinase-9 pretreatment level predicts intracranial hemorrhagic complications after thrombolysis in human stroke. Circulation 107:598–603

36.

Mun-Bryce

Rogenberg

(1998) Matrix metalloproteinases in cerebrovascular disease. J Cereb Blood Flow Metab 18:1163–1172

37.

Neumar

Gada

Guttmann

Siman

(2003) Cross-talk between calpain and caspase proteolytic systems during neuronal apoptosis. J Biol Chem 278:14162–14167

38.

Plesnila

Zinkel

Amin-Hanjani

Qiu

Chiarugi

Thomas

Kohane

Korsmeyer

Moskowitz

(2001) BID mediates neuronal cell death after oxygen/ glucose deprivation and focal cerebral ischemia. Proc Natl Acad Sci U S A 98:15318–15323

39.

Rami

(2003) Ischemic neuronal death in the rat hippocampus: the calpain-calpastatin-caspase hypothesis. Neurobiol Dis 13:75–88

40.

Rosenberg

(2002) Matrix metalloproteinases in neuroinflammation. Glia 39:279–291

41.

Sakai

Johnson

Murozono

Sakai

Magnuson

Wieloch

Cronberg

Isshiki

Erickson

Fassler

(2001) Plasma fibronectin supports neuronal survival and reduces brain injury following transient focal cerebral ischemia but is not essential for skin-wound healing and hemostasis. Nat Med 7:324–330

42.

Schwartz

Karsan

Bombeli

Harlan

(1999) A novel beta 1 integrin-dependent mechanism of leukocyte adherence to apoptotic cells. J Immunol 162:4842–4848

43.

Seki

Koyama

Kovach

Yednock

Clowes

Harlan

(1996) Regulation of beta1-integrin function in cultured human vascular smooth muscle cells. Circ Res 78:596–605

44.

Stupack

Puente

Boutsaboualoy

Storgard

Cheresh

(2001) Apoptosis of adherent cells by recruitment of caspase-8 to unligated integrins. J Cell Biol 155:459–470

45.

Susin

Lorenzo

Zamzami

Marzo

Snow

Brothers

Mangion

Jacotot

Costantini

Loeffler

Larochette

Goodlett

Aebersold

Siderovski

Penninger

Kroemer

(1999) Molecular characterization of mitochondrial apoptosis-inducing factor. Nature 397:441–446

46.

Tagaya

Haring

Stuiver

Wagner

Abumiya

Lucero

Lee

Copeland

Seiffert

del Zoppo

(2001) Rapid loss of microvascular integrin expression during focal brain ischemia reflects neuron injury. J Cereb Blood Flow Metab 21:835–846

47.

Tsirka

Rogove

Bugge

Degen

Strickland

(1997) An extracellular proteolytic cascade promotes neuronal degeneration in the mouse hippocampus. J Neurosci 17:543–552

48.

Wallach

Varfolomeev

Malinin

Goltsev

Kovalenko

Boldin

(1999) Tumor necrosis factor receptor and Fas signaling mechanisms. Annu Rev Immunol 17:331–367

49.

Wang

(2000) Calpain and caspase: can you tell the difference? Trends Neurosci 23:20–26

50.

Wang

Jung

Asahi

Chwang

Russo

Moskowitz

Dixon

Fini

(2000) Effects of matrix metalloproteinase-9 gene knock-out on morphological and motor outcomes after traumatic brain injury. J Neurosci 20:7037–7042

51.

Wang

Lee

Arai

Lee

Tsuji

Rebeck

(2003) Lipoprotein receptor-mediated induction of matrix metalloproteinase by tissue plasminogen activator. Nat Med 9:1313–1317

52.

Chen

Ahmed

Chen

Hsu

(2001) Amyloid beta peptide-induced cerebral endothelial cell death involves mitochondrial dysfunction and caspase activation. J Cereb Blood Flow Metab 21:702–710

53.

Yong

Krekoski

Forsyth

Bell

Edwards

(1998) Matrix metalloproteinases and disease of the CNS. Trends Neurosci 21:75–80

54.

Yong

Power

Forsyth

Edwards

(2001) Metalloproteinases in biology and pathology of the nervous system. Nat Rev Neurosci 2:502–511

55.

Wang

Dawson

(2003) Poly(ADP-ribose) polymerase-1 and apoptosis inducing factor in neurotoxicity. Neurobiol Dis 14:303–317

56.

Wang

Poitras

Coombs

Bowers

Federoff

Poirier

Dawson

(2002) Mediation of poly-(ADP-ribose) polymerase-1-dependent cell death by apoptosis-inducing factor. Science 297:259–263

57.

Zhang

(2001) Protection against oxidized low-density lipoprotein-induced vascular endothelial cell death by integrin-linked kinase. Circulation 104:2762–2766

Induction of Caspase-Mediated Cell Death by Matrix Metalloproteinases in Cerebral Endothelial Cells after Hypoxia—Reoxygenation

Abstract

Keywords

MATERIALS AND METHODS

Cell culture

Gelatin zymography

Western blot analysis

Lactate dehydrogenase release assay

DNA fragmentation assay

Fibronectin fragment analysis

Integrin-linked kinase (ILK) assay

Statistical analysis

RESULTS

Hypoxia—reoxygenation induces caspase-3-mediated cytotoxicity in human cerebral endothelial cells

Endothelial cell death is mediated by MMPs after hypoxia—reoxygenation

MMPs degrade extracellular matrix and disrupt integrin signaling

DISCUSSION

Footnotes

Acknowledgments

References