SEARCH

SEARCH BY CITATION

Keywords:

  • Alzheimer's disease;
  • amyloid β protein;
  • β-amyloid precursor protein;
  • gelatinase;
  • human cerebrovascular smooth muscle cells;
  • matrix metalloproteinase

Abstract

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. HCSM cell culture
  5. MMP inhibitors
  6. mRNA isolation and RNase protection assay
  7. Quantitative immunoblotting
  8. Gelatin substrate zymography
  9. Cell viability assay
  10. Results
  11. MMP-2 mRNA expression is increased in HCSM cells treated with Aβ40D
  12. MMP-2 protein levels and activity are increased in HCSM cells treated with Aβ40D
  13. MT1-MMP mRNA is increased in HCSM cells treated with Aβ40D
  14. Certain MMP inhibitors decrease MMP-2 activation and increase HCSM cell viability upon treatment with Aβ40D
  15. Discussion
  16. Acknowledgement
  17. References

Cerebral amyloid angiopathy (CAA) is a major pathological feature of Alzheimer's disease and related disorders. Human cerebrovascular smooth muscle (HCSM) cells, which are intimately associated with CAA, have been used as an in vitro model system to investigate pathologic interactions with amyloid β protein (Aβ). Previously we have shown that pathogenic forms of Aβ induce several pathologic responses in HCSM cells including fibril assembly at the cell surface, increase in the levels of Aβ precursor, and apoptotic cell death. Here we show that pathogenic Aβ stimulates the expression and activation of matrix metalloproteinase-2 (MMP-2). Furthermore, we demonstrate that the increase in MMP-2 activation is largely caused by increased expression of membrane type-1 (MT1)-MMP expression, the primary MMP-2 activator. Finally, treatment with MMP-2 inhibitors resulted in increased HCSM cell viability in the presence of pathogenic Aβ. Our findings suggest that increased expression and activation of MMP-2 may contribute to HCSM cell death in response to pathogenic Aβ. In addition, these activities may also contribute to loss of vessel wall integrity in CAA resulting in hemorrhagic stroke. Therefore, further understanding into the role of MMPs in HCSM cell degeneration may facilitate designing therapeutic strategies to treat CAA found in AD and related disorders.

Abbreviations used

amyloid β protein

Aβ40D

Dutch variant Aβ40

AβPP

amyloid β precursor protein

AD

Alzheimer's disease

BBB

blood–brain barrier

BSA

bovine serum albumin

CAA

cerebral amyloid angiopathy

DMEM

Dulbecco's modified Eagle's medium

HCSM

human cerebrovascular smooth muscle

MMP

matrix metalloproteinase

MMP-2

matrix metalloproteinase-2

MT1-MMP

membrane type-1 matrix metalloproteinase

MT3-MMP

membrane type-3 matrix metalloproteinase

PVDF

polyvinylidene difluoride

SDS

sodium dodecyl sulfate

TIMP

tissue inhibitor of matrix metalloproteinase

Cerebral amyloid angiopathy (CAA) is a key pathological feature of Alzheimer's disease (AD) and related disorders such as hereditary cerebral hemorrhage with amyloidosis-Dutch type (Vinters 1987; Wattendorf et al. 1995). In this condition deposition of amyloid β protein (Aβ) results in the degeneration of cerebrovascular smooth muscle cells (Kawai et al. 1993) and in severe cases leads to loss of vessel wall integrity and hemorrhagic stroke. We have used human cerebrovascular smooth muscle (HCSM) cells as an in vitro model to investigate the pathogenic mechanisms of the cerebrovascular pathology of CAA. Previous reports from us and others have demonstrated that pathogenic forms of Aβ can induce several key pathologic responses in HCSM cells including fibril assembly at the cell surface and robust increases in the levels of Aβ precursor which precede an apoptotic death (Davis-Salinas et al. 1995; Van Nostrand et al. 1998; Melchor and Van Nostrand 2000). The downstream alterations in proteolytic mechanisms that are induced by Αβ deposition in HCSM cells, particularly the role of matrix metalloproteinases (MMPs), have not been defined.

MMPs are a large family (> 25 members) of neutral, zinc-dependent proteinases that degrade a wide array of extracellular matrix components responsible for maintaining homeostasis of the extracellular environment (for reviews see Birkedal-Hansen et al. 1993; Stetler-Stevenson et al. 1993; Nagase and Woessner 1999). Members of the MMP family are distinguished by the substrates they degrade and can be categorized into various subgroups: collagenases, gelatinases, stromelysins, and membrane-type MMPs (MT-MMPs). The activities of MMPs are tightly controlled by three mechanisms that include gene expression, zymogen activation and endogenous tissue inhibitors of metalloproteinases (TIMPs). MMPs are expressed as zymogens (latent pro-form) that are activated through limited proteolysis by serine proteinases, growth factors, cell–cell or cell–matrix interactions and some activated MMPs within the amino-terminal propeptide domain, with the exception of MMP-2 which is primarily activated at the cell surface by MT-MMPs (Sato et al. 1994; Lewalle et al. 1995; Strongin et al. 1995; Takino et al. 1995; Yu et al. 1995). This results in a conformational change in the MMP molecule disrupting an unpaired Cys–Zn2+ interaction known as the ‘cysteine switch’ (Springman et al. 1990; Van Wart and Birkedal-Hansen 1990). The liberated Zn2+ then participates in cleavage of the propeptide in an autolytic manner producing a lower molecular mass active MMP (Nagase 1997).

MMPs play a role in tissue remodeling under various physiologic and pathologic conditions. Physiologically, MMPs are involved in embryonic development, morphogenesis, angiogenesis, tissue repair, and the immune response. Two gelatinolytic MMPs, MMP-2 and MMP-9, are known to be expressed in smooth muscle cells and endothelial cells in blood vessels and, through their turnover of basement membrane extracellular matrix (ECM) proteins such as collagen type IV, collagen type I, laminin, and fibronectin, are believed to play important physiological roles in angiogenesis and blood vessel integrity. However, when MMP regulation goes awry, they can participate in pathological conditions such as arthritis, cancer, neurological disease, breakdown of the blood–brain barrier (BBB), and cerebral hemorrhage (for reviews see Stetler-Stevenson et al. 1993; Yong et al. 1998, 2001). In this regard, MMP-2 and MMP-9 have been implicated in the deterioration of BBB integrity under certain pathological conditions. For example, evidence from both animal models and human studies support a role for MMP-9 during stroke and neuroinflammatory conditions such as multiple sclerosis and AD as well as cerebrovascular diseases (Backstrom et al. 1992; Gijbels et al. 1992; Lim et al. 1996; Anthony et al. 1997; Chandler et al. 1997; Lim et al. 1997; Rosenberg and Navratil 1997; Mun-Bryce and Rosenberg 1998; Yong et al. 1998, 2001). Direct intracerebral injection of MMP-2 has been shown to cause opening of the BBB and cause intracerebral hemorrhage by disrupting the ECM (Rosenberg et al. 1990, 1993, 1995). These deleterious effects of MMP-2 were reduced by administering its natural inhibitor TIMP-2 (Rosenberg et al. 1992). In addition to a direct loss of vessel wall integrity MMP-mediated degradation of ECM components may lead to loss of specific ECM–integrin interactions resulting in apoptotic vascular cell death (Frisch and Ruoslahti 1997). Despite the strong association between cerebrovascular Αβ deposition and cerebrovascular cell death, loss of vessel wall integrity and hemorrhage, virtually nothing is known about the role of MMPs in these pathological processes in CAA. In this report, we examined the downstream alterations in proteolytic mechanisms that are induced by Αβ deposition in the HCSM cell model. In particular, we investigated the role of matrix metalloproteinases (MMPs) in the degeneration of HCSM cells induced by pathogenic Αβ and the effect of MMP inhibitors on HCSM cell viability.

HCSM cell culture

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. HCSM cell culture
  5. MMP inhibitors
  6. mRNA isolation and RNase protection assay
  7. Quantitative immunoblotting
  8. Gelatin substrate zymography
  9. Cell viability assay
  10. Results
  11. MMP-2 mRNA expression is increased in HCSM cells treated with Aβ40D
  12. MMP-2 protein levels and activity are increased in HCSM cells treated with Aβ40D
  13. MT1-MMP mRNA is increased in HCSM cells treated with Aβ40D
  14. Certain MMP inhibitors decrease MMP-2 activation and increase HCSM cell viability upon treatment with Aβ40D
  15. Discussion
  16. Acknowledgement
  17. References

Primary cultures of HCSM cells were established and characterized as previously described (Van Nostrand et al. 1994). The HCSM cells used in the present studies were derived from leptomeningeal vessels of control adult subjects and showed strong expression of the specific markers vascular smooth muscle cell α-actin, vascular smooth muscle cell myosin, and SM22α. HCSM cell cultures were > 95% free of contaminating cell types. HCSM cells were cultured in 24-well plates in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (Gemini Bio-Products, Woodland, CA, USA), 1 mm nonessential amino acids, 2 mm glutamine, 1000 U/mL penicillin, 1000 µg/mL streptomycin (Life Technologies, Rockville, MD, USA). For experiments, cells were grown to near-confluence (90%) and replaced with serum-free DMEM containing 0.1% bovine serum albumin (BSA) for 24 h prior to treatment. Cells were treated, in triplicate, with freshly solubilized monomeric Dutch variant β-amyloid (Αβ40D, 25 µm, American Peptide Co., Sunnyvale, CA, USA) for 6 days in the presence or absence of MMP inhibitors. Cells were examined routinely using an inverted system Olympus IX70 phase contrast microscope (Olympus America Inc., Lake Success, NY, USA).

MMP inhibitors

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. HCSM cell culture
  5. MMP inhibitors
  6. mRNA isolation and RNase protection assay
  7. Quantitative immunoblotting
  8. Gelatin substrate zymography
  9. Cell viability assay
  10. Results
  11. MMP-2 mRNA expression is increased in HCSM cells treated with Aβ40D
  12. MMP-2 protein levels and activity are increased in HCSM cells treated with Aβ40D
  13. MT1-MMP mRNA is increased in HCSM cells treated with Aβ40D
  14. Certain MMP inhibitors decrease MMP-2 activation and increase HCSM cell viability upon treatment with Aβ40D
  15. Discussion
  16. Acknowledgement
  17. References

The purified MMP inhibitors TIMP-1, TIMP-2 and CT-1847 were the kind gifts of Dr Stanley Zucker (Department Medicine, Stony Brook University and Veteran Affairs Memorial Center, Northport, NY, USA). The purified endogenous inhibitors TIMP-1 and TIMP-2 were used at 20 nm. The synthetic hydroxamic acid inhibitor CT-1847 (C15H29N3O4S, MW = 347) was found to be effective at 500 nm in our cell culture model (non-toxic to cells). Lower concentrations were found to be ineffective in inhibiting MMP-2 activity as assayed by gelatin zymography.

mRNA isolation and RNase protection assay

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. HCSM cell culture
  5. MMP inhibitors
  6. mRNA isolation and RNase protection assay
  7. Quantitative immunoblotting
  8. Gelatin substrate zymography
  9. Cell viability assay
  10. Results
  11. MMP-2 mRNA expression is increased in HCSM cells treated with Aβ40D
  12. MMP-2 protein levels and activity are increased in HCSM cells treated with Aβ40D
  13. MT1-MMP mRNA is increased in HCSM cells treated with Aβ40D
  14. Certain MMP inhibitors decrease MMP-2 activation and increase HCSM cell viability upon treatment with Aβ40D
  15. Discussion
  16. Acknowledgement
  17. References

Primary cultures of HCSM cells were grown to ∼90% confluency in T225 cm2 flasks. The cells were then incubated under serum-free conditions either in the absence or presence of 25 µmΑβ40D for 6 days. Messenger RNA was isolated using the mRNA Isolation Kit according to the manufacturer's instructions (Roche Molecular Biochemicals, Indianapolis, IN, USA) and stored at − 70°C. Complementary DNA templates for riboprobes were generated using the following primers: MMP-2 forward: 5′-GAGGAGGCTGTGTTCTTTGCAG-3′, MMP-2 reverse: 5′-CACGCTCTTCAGACTTTGGTTC-3′ (GenBank accession number NM004530), MT1-MMP forward: 5′-CTGATGCAGACACCATGAAGGC-3′, MT1-MMP reverse: 5′-GATCATGATGTCGGCCTGCTTC-3′ (GenBank accession number NM004995), MT3-MMP forward: 5′-CATATCATGCCGCCGCAGTCCATGAACTGGGACATG-3′, MT3-MMP reverse: 5′-CATATGATGGATCCTGTCATTTTTCCTTGGGTCAGC-3′ (GenBank accession number D85511) and AMV reverse transcriptase (Roche). PCR products of 208, 297, and 262 bp for MMP-2, MT1-MMP, and MT3-MMP, respectively, were made using the High Pure PCR Product Purification Kit (Roche), digested with NotI and BamHI and purified by electrophoretic separation and subsequent extraction from a 0.8% agarose gel using the GeneClean II kit (BIO101, Vista, CA, USA). The fragment was ligated into pBluescript (KS +) (Stratagene, La Jolla, CA, USA) at the BamHI-NotI site in an antisense direction using a rapid DNA Ligation Kit (Roche). The integrity of all constructs was verified by DNA sequencing analysis. The MT1-MMP/Bluescript or MMP-2/Bluescript construct was digested with PvuII and SacI. A 511, 580, and 506 bp fragment for MMP-2, MT1-MMP, and MT3-MMP, respectively, was purified by electrophoretic separation and subsequent extraction from a 0.8% agarose gel using the GeneClean II Kit (BIO101).

For RNase protection assay, an antisense radiolabeled riboprobe was generated by using InVitro Transcription Kit (Promega, Madison, WI, USA) with approximately 50 ng of MT1-MMP or MMP-2 fragment, T3 RNA polymerase (Roche), and 100 µCi of [α-32P]UTP (NEN Life Science Products, Boston, MA, USA). As a control, the GAPDH housekeeping gene was made using 50 ng GAPDH template (PharMingen, San Diego, CA, USA) with T7 RNA polymerase (PharMingen). The radiolabeled antisense riboprobes were ethanol precipitated and resuspended in 50 µL of hybridization buffer. Using an RNase Protection Assay Kit (RiboQuant, PharMingen), 300–500 ng of HCSM mRNA or 4 µg negative control tRNA was hybridized at 56°C overnight with 100 000 cpm/µL of either MMP-2, MT1-MMP, MT3-MMP, or GAPDH radiolabeled antisense riboprobe. Free probe and single-stranded mRNA was digested with RNaseA and T1 for 45 min at 30°C. Subsequent digestion was done with proteinase K at 37°C for 15 min. The hybridized samples were purified by phenol/chloroform extraction and electrophoresed on a 6% Tris-borate-EDTA (TBE)-urea gel (Invitrogen) at 180 V for 1 h. The resulting gel was fixed in 30% methanol/5% glycerol for 30 min followed by dehydration under vacuum at 80°C for 2 h, and analyzed by autoradiography. Bands were quantitated from films by densitometric scanning using the NIH Image software.

Quantitative immunoblotting

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. HCSM cell culture
  5. MMP inhibitors
  6. mRNA isolation and RNase protection assay
  7. Quantitative immunoblotting
  8. Gelatin substrate zymography
  9. Cell viability assay
  10. Results
  11. MMP-2 mRNA expression is increased in HCSM cells treated with Aβ40D
  12. MMP-2 protein levels and activity are increased in HCSM cells treated with Aβ40D
  13. MT1-MMP mRNA is increased in HCSM cells treated with Aβ40D
  14. Certain MMP inhibitors decrease MMP-2 activation and increase HCSM cell viability upon treatment with Aβ40D
  15. Discussion
  16. Acknowledgement
  17. References

Αβ40D-treated cells were lysed in 1% sodium dodecyl sulfate (SDS) lysis buffer containing a cocktail of protease inhibitors (Roche, Indianapolis, IN, USA). Equal amounts of protein were loaded onto a 10–20% Tris-Tricine mini-gel (Invitrogen, Carlsbad, CA, USA), electrophoresed and transferred to a polyvinylidene difluoride (PVDF) membrane (Millipore, Bedford, MA, USA). Membranes were blocked in 5% BSA/PBS-T overnight at RT. Primary antibody was added (Anti-MMP-2, Oncogene Research Products, Boston, MA, USA; 0.4 µg/mL) for 1 h at 22°C, washed 3 × 15 min with PBS-T. Horseradish peroxidase-conjugated sheep anti-mouse IgG (SAM-HRP, 1 : 5000 Amersham-Pharmacia, Piscataway, NJ, USA) was used as the secondary antibody for 1 h at room temperature, and washed 3 × 15 min with PBS-T. Bands were visualized using the ECL detection method (Amersham-Pharmacia) and Kodak X-Omat blue film (Kodak, Rochester, NY, USA). Densitometric analysis was performed using a Bio-Rad Fluor-S MultiImager and the manufacturer's Quantity One software (Hercules, CA, USA).

Gelatin substrate zymography

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. HCSM cell culture
  5. MMP inhibitors
  6. mRNA isolation and RNase protection assay
  7. Quantitative immunoblotting
  8. Gelatin substrate zymography
  9. Cell viability assay
  10. Results
  11. MMP-2 mRNA expression is increased in HCSM cells treated with Aβ40D
  12. MMP-2 protein levels and activity are increased in HCSM cells treated with Aβ40D
  13. MT1-MMP mRNA is increased in HCSM cells treated with Aβ40D
  14. Certain MMP inhibitors decrease MMP-2 activation and increase HCSM cell viability upon treatment with Aβ40D
  15. Discussion
  16. Acknowledgement
  17. References

Conditioned media samples from HCSM cells treated with 25 µmΑβ40D in the absence and presence of MMP inhibitors were electrophoresed on 8% SDS–polyacrylamide gels containing 0.1% gelatin at 100 V for 2 h at 22°C. The gels were removed and incubated in rinse buffer (50 mm Tris, pH 7.5, 200 mm NaCl, 5 mm CaCl2, 2.5% Triton X-100) for 3 h with several changes, washed 3 × 10 min with ddH2O, then incubated in assay buffer (50 mm Tris, pH 7.5, 200 mm NaCl, 5 mm CaCl2) overnight at 37°C, washed 3 × 10 min with ddH20, stained with 0.25% Coomassie Brilliant Blue R-250 and then destained. Gelatinolytic MMP activity was observed as clear zones of lysis.

Cell viability assay

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. HCSM cell culture
  5. MMP inhibitors
  6. mRNA isolation and RNase protection assay
  7. Quantitative immunoblotting
  8. Gelatin substrate zymography
  9. Cell viability assay
  10. Results
  11. MMP-2 mRNA expression is increased in HCSM cells treated with Aβ40D
  12. MMP-2 protein levels and activity are increased in HCSM cells treated with Aβ40D
  13. MT1-MMP mRNA is increased in HCSM cells treated with Aβ40D
  14. Certain MMP inhibitors decrease MMP-2 activation and increase HCSM cell viability upon treatment with Aβ40D
  15. Discussion
  16. Acknowledgement
  17. References

Cell viability was assessed using a fluorescent live/dead cell assay following the manufacturer's protocol (Molecular Probes, Eugene, OR, USA). Briefly, after treatment with Αβ40D in the presence or absence of MMP inhibitor, cells were exposed to both calcein AM and ethidium homodimer-1 and viewed using an inverted system Olympus IX70 fluorescence microscope. Intracellular esterases in living cells convert the substrate calcein AM into a brightly green fluorescent product calcein. Dead cells are observed when the DNA chelator ethidium homodimer-1 enters cells with damaged cell membranes resulting in a bright red fluorescence. Live and dead cells were counted from several fields (n = 4–6) from at least three separate wells for each experiment.

MMP-2 mRNA expression is increased in HCSM cells treated with Aβ40D

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. HCSM cell culture
  5. MMP inhibitors
  6. mRNA isolation and RNase protection assay
  7. Quantitative immunoblotting
  8. Gelatin substrate zymography
  9. Cell viability assay
  10. Results
  11. MMP-2 mRNA expression is increased in HCSM cells treated with Aβ40D
  12. MMP-2 protein levels and activity are increased in HCSM cells treated with Aβ40D
  13. MT1-MMP mRNA is increased in HCSM cells treated with Aβ40D
  14. Certain MMP inhibitors decrease MMP-2 activation and increase HCSM cell viability upon treatment with Aβ40D
  15. Discussion
  16. Acknowledgement
  17. References

We examined the effects of pathogenic Αβ treatment on MMP expression and activity in primary HCSM cells. In our experiments we used the Dutch mutant Αβ40 (Aβ40D) since our earlier studies clearly showed that this form of peptide consistently induces more robust pathologic responses in cultured HCSM cells compared to wild-type Aβ peptides (Davis and Van Nostrand 1996). Preliminary cDNA microarray analysis suggested that MMP-2 gene expression was upregulated in Αβ40D-treated HCSM cells (data not shown). To explore this further HCSM cells were treated in the absence or presence of Αβ40D. Messenger RNA was isolated from the cells and analyzed for MMP-2 expression using the RNase protection assay. This technique revealed that in HCSM cells, MMP-2 mRNA levels are elevated ∼ four-fold in response to treatment with Αβ40D (Figs 1a and b).

image

Figure 1. Increased expression of MMP-2 mRNA in HCSM cells treated with pathogenic Aβ. HCSM cells were treated in the absence or presence of 25 µm Aβ40D for 6 days, mRNA was isolated and then subjected to RNase protection analysis using a 208-bp fragment corresponding to nucleotides 1879–2087 of the MMP-2 cDNA. (a) A representative RNase protection experiment. Lane 1, MMP-2 probe; lane 2, hGAPDH probe; lane 3, untreated cells; and lane 4, Aβ40D-treated cells. (b) Quantitation of MMP-2 (lane 1) and hGAPDH (lane 2) expression in HCSM cells. Data represent the mean ± SEM of four separate experiments and are expressed as fold increase over untreated HCSM cells.

Download figure to PowerPoint

MMP-2 protein levels and activity are increased in HCSM cells treated with Aβ40D

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. HCSM cell culture
  5. MMP inhibitors
  6. mRNA isolation and RNase protection assay
  7. Quantitative immunoblotting
  8. Gelatin substrate zymography
  9. Cell viability assay
  10. Results
  11. MMP-2 mRNA expression is increased in HCSM cells treated with Aβ40D
  12. MMP-2 protein levels and activity are increased in HCSM cells treated with Aβ40D
  13. MT1-MMP mRNA is increased in HCSM cells treated with Aβ40D
  14. Certain MMP inhibitors decrease MMP-2 activation and increase HCSM cell viability upon treatment with Aβ40D
  15. Discussion
  16. Acknowledgement
  17. References

We next determined if MMP-2 protein levels are similarly elevated in HCSM cells treated with pathogenic Αβ by performing quantitative immunoblotting analysis of culture media samples. We found that MMP-2 protein was increased in Αβ40D-treated HCSM cells approximately eight-fold compared to untreated HCSM cells (Figs 2a and b). Gelatin zymography was subsequently performed to identify inactive precursor and active forms of MMP-2 in the media samples after exposure to increasing concentrations of Aβ40D or increasing lengths of time. Both latent (inactive, pro-MMP-2) and active forms of MMP-2 were observed in the experiments. In untreated HCSM cells, only the presence of pro-MMP-2 was observed at 72 kDa (Fig. 3a, lane 1). Upon treatment with increasing concentrations of Αβ40D, both pro-MMP-2 at 72 kDa and active MMP-2 at 66 kDa were observed (Fig. 3a, lanes 2–5). Increased expression and activation of MMP-2 were first observed at approximately 5 µm Aβ40D and increased through 25 µm. Furthermore, when HCSM cells treated with 25 µm Aβ40D over 6 days increased expression and activation of MMP-2 in HCSM cells was first observed at about 3 days and increased over the 6-day treatment period. These results are consistent with our previous studies identifying the concentrations of Aβ40D and length of exposure to peptide that induce degenerative effects on HCSM cells (Davis and Van Nostrand 1996; Melchor and Van Nostrand 2000). It is noteworthy that the zymography experiments revealed a large increase in total MMP-2 in the Αβ40D-treated HCSM cells consistent with the above RNase protection and immunoblotting studies (Figs 1 and 2).

image

Figure 2. Increased expression of MMP-2 protein in HCSM cells treated with pathogenic Aβ. HCSM cells were treated in the absence (lanes 1) or presence (lanes 2) of 25 µm Aβ40D for 6 days. Conditioned culture media samples were collected and subjected to quantitative immunoblotting for MMP-2 as described in Materials and methods. (a) Representative immunoblot experiment. (b) Quantitation of MMP-2 protein levels secreted by HCSM cells. Data represents the mean ± SEM of three separate experiments.

Download figure to PowerPoint

image

Figure 3. Increased activation of MMP-2 in HCSM cells treated with pathogenic Aβ. HCSM cells were treated in the absence or presence of increasing concentrations of Aβ40D for 6 days (a) or treated with 25 µm Aβ40D for 1–6 days (b). Conditioned media were collected and aliquots were subjected to gelatin zymography as described in Materials and methods. Gelatinolytic activity is observed as clear zones of lysis. Pro-MMP-2 and active MMP-2 migrate at 72 kDa and 66 kDa, respectively.

Download figure to PowerPoint

MT1-MMP mRNA is increased in HCSM cells treated with Aβ40D

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. HCSM cell culture
  5. MMP inhibitors
  6. mRNA isolation and RNase protection assay
  7. Quantitative immunoblotting
  8. Gelatin substrate zymography
  9. Cell viability assay
  10. Results
  11. MMP-2 mRNA expression is increased in HCSM cells treated with Aβ40D
  12. MMP-2 protein levels and activity are increased in HCSM cells treated with Aβ40D
  13. MT1-MMP mRNA is increased in HCSM cells treated with Aβ40D
  14. Certain MMP inhibitors decrease MMP-2 activation and increase HCSM cell viability upon treatment with Aβ40D
  15. Discussion
  16. Acknowledgement
  17. References

The activation of MMP-2 occurs primarily via the MT-MMPs including MT1- and MT3-MMP. RNase protection assay showed that MT1-MMP mRNA was increased approximately five-fold in HCSM cells treated with pathogenic Αβ(Fig. 4, lane 5). In contrast, MT3-MMP mRNA was barely detected in the cultured HCSM cells and its expression was not influenced by treatment with Αβ40D (Fig. 4, lanes 6 and 7).

image

Figure 4. Increased expression of MT1-MMP mRNA in HCSM cells treated with pathogenic Aβ. HCSM cells were treated in the absence or presence of 25 µmΑβ40D for 6 days, mRNA was isolated and then subjected to RNase protection analysis using a 297-bp fragment corresponding to nucleotides 471–768 of the MT1-MMP cDNA or a 236-bp fragment corresponding to nucleotides 765–1001 of the MT3-MMP cDNA. (a) A representative RNase protection experiment. Lane 1, MT1-MMP probe; lane 2, MT3-MMP probe; lane 3, hGAPDH probe; lanes 4 and 6, untreated cells; and lanes 5 and 7, Aβ40D-treated cells. (b) Quantitation of MT1-MMP (lane 1) and hGAPDH (lane 2) expression in HCSM cells. Data represent the mean ± SEM of five separate experiments and are expressed as fold increase over untreated HCSM cells.

Download figure to PowerPoint

Certain MMP inhibitors decrease MMP-2 activation and increase HCSM cell viability upon treatment with Aβ40D

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. HCSM cell culture
  5. MMP inhibitors
  6. mRNA isolation and RNase protection assay
  7. Quantitative immunoblotting
  8. Gelatin substrate zymography
  9. Cell viability assay
  10. Results
  11. MMP-2 mRNA expression is increased in HCSM cells treated with Aβ40D
  12. MMP-2 protein levels and activity are increased in HCSM cells treated with Aβ40D
  13. MT1-MMP mRNA is increased in HCSM cells treated with Aβ40D
  14. Certain MMP inhibitors decrease MMP-2 activation and increase HCSM cell viability upon treatment with Aβ40D
  15. Discussion
  16. Acknowledgement
  17. References

We showed above that MMP-2 activity was greatly increased in HCSM cells treated with pathogenic Αβ. MMP-2 activity results in increased degradation of extracellular matrix proteins. As this activity may impact on the viability of HCSM cells, we examined whether inhibition of MMP-2 could diminish the loss of cell viability in response to treatment with pathogenic Αβ. We used the endogenous MMP inhibitors TIMP-1 (20 nm), TIMP-2 (20 nm) or the synthetic hydroxamate inhibitor CT-1847 (500 nm) and examined MMP-2 activation in Αβ40D-treated HCSM cells using gelatin zymography. TIMP-1, which does not inhibit MT-MMP activation of pro-MMP-2, had little effect in inhibiting MMP-2 activation (Fig. 5a, lane 3). On the other hand, TIMP-2 and CT-1847, which inhibit MT-MMP activation of pro-MMP-2, resulted in nearly complete inhibition of MMP-2 activation (Fig. 5a, lanes 4 and 8, respectively). The inhibitors alone did not affect the normal expression levels of MMP-2 (data not shown). Similarly, TIMP-2 and CT-1847 were effective in significantly lowering the extent of HCSM cell death upon treatment with Αβ40D whereas TIMP-1 was ineffective (Fig. 5b, lanes 4 and 5, respectively). CT-1847 was the more effective inhibitor resulting in > 85% cell viability. The inhibitors alone did not have any effect on HCSM cell viability (data not shown). These findings suggest that increased activation of MMP-2 may, in part, play a role in pathogenic Αβ induced loss of HCSM cell viability.

image

Figure 5. MT1-MMP inhibitors reduce MMP-2 activation and decrease cell death in HCSM cells treated with pathogenic Aβ. HCSM cells were treated with Aβ40D for 6 days in the absence or presence of various MMP inhibitors. (a) Conditioned media samples were collected from the cells and subjected to gelatin zymography. Lanes 1 and 5, untreated cells; lanes 2 and 7, cells treated with 25 µm Aβ40D alone; lane 3, cells treated with 25 µm Aβ40D + 20 nm purified TIMP-1; lane 4, cells treated with 25 µm Aβ40D + 20 nm purified TIMP-2; and lane 6, cells treated with 500 nm CT-1847 alone; lane 8, cells treated with 25 µm Aβ40D + 500 nm CT-1847. (b) A fluorescence-based cell viability assay was performed at the end of 6 days. Lane 1, untreated cells; lane 2, cells treated with 25 µm Aβ40D alone; lane 3, cells treated with 25 µm Aβ40D + 20 nm purified TIMP-1; lane 4, cells treated with 25 µm Aβ40D + 20 nm purified TIMP-2; and lane 5, cells treated with 25 µm Aβ40D + 500 nm CT-1847. Data represent the mean ± SEM of four separate experiments. Tukey–Kramer multiple comparison post-tests showed that TIMP-2 treatment (*p < 0.001) and CT-1847 treatment (**p < 0.001) significantly lowered HCSM cell death.

Download figure to PowerPoint

Discussion

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. HCSM cell culture
  5. MMP inhibitors
  6. mRNA isolation and RNase protection assay
  7. Quantitative immunoblotting
  8. Gelatin substrate zymography
  9. Cell viability assay
  10. Results
  11. MMP-2 mRNA expression is increased in HCSM cells treated with Aβ40D
  12. MMP-2 protein levels and activity are increased in HCSM cells treated with Aβ40D
  13. MT1-MMP mRNA is increased in HCSM cells treated with Aβ40D
  14. Certain MMP inhibitors decrease MMP-2 activation and increase HCSM cell viability upon treatment with Aβ40D
  15. Discussion
  16. Acknowledgement
  17. References

MMPs are best known for their pathological role in cancer metastasis (Stetler-Stevenson et al. 1993), but also play a significant role in ischemic stroke and neurodegenerative diseases (Rosenberg 1995; Yong et al. 1998, 2001). MMPs have been implicated in stroke due to the findings that MMP-2 and MMP-9 are rapidly upregulated after focal cerebral ischemia in rats (Rosenberg et al. 1996; Romanic et al. 1998; Planas et al. 2001). MMP-9 has been described to contribute to neuroinflammatory diseases such as multiple sclerosis (Gijbels et al. 1992), amyotrophic lateral sclerosis (Lim et al. 1996) and AD (Backstrom et al. 1992, 1996; Lim et al. 1997). Furthermore, when mixed glial and neuronal hippocampal cultures were exposed to Αβ, MMP-2 and MMP-9 were found to be greatly increased (Deb and Gottschall 1996).

In the present study, we examined the role of MMPs in primary HCSM cells treated with pathogenic Αβ, an in vitro cell culture model for CAA. We found that HCSM cells demonstrated an increase in expression and activation of a single gelatinase, MMP-2, in response to Αβ40D treatment. Increased MMP-2 mRNA levels were demonstrated by an RNase protection assay (Fig. 1). MMP-2 is unique from other metalloproteinases in that it is constitutively expressed – it does not contain an AP-1 site or TATA box in the promoter region of the gene (Uhm et al. 1997). Therefore, induction of MMP-2 overexpression is generally not controlled by an up-regulation of gene transcription. In fact, it has been described that MMP-2 mRNA levels do not fluctuate, but rather the stability of its mRNA increases (Overall et al. 1991). In light of these findings, the increased levels of MMP-2 mRNA observed in HCSM cells likely reflects enhanced stability.

Some reports have shown that cytokines can induce the overexpression of MMP-2 in certain cell types (Janowska-Wieczorek et al. 1999; Li et al. 1999; Kouwenhoven et al. 2001). It is possible that Aβ induces HCSM cells to produce cytokines, which may be responsible for the up-regulation of MMP-2 expression. Since our cultures are > 95% smooth muscle cells, it is unlikely that any contaminating cells (such as microglia) would contribute any cytokine production in response to Aβ40D and cause an increase in MMP-2 in this fashion.

Quantitative immunoblotting and gelatin zymography confirmed increased MMP-2 protein levels and activation in the conditioned media of Αβ-treated HCSM cells. MMP-2 activation is also unique from the other MMPs in that it is primarily activated by MT-MMPs (Sato et al. 1994; Lewalle et al. 1995; Strongin et al. 1995; Takino et al. 1995; Yu et al. 1995) and not by serine proteinases, growth factors, phorbol esters, cell–cell or cell–matrix interactions which is the case for other MMPs (Nagase 1997). In rat vascular smooth muscle cells MT1- and MT3-MMP have been mostly implicated in the activation of MMP-2 (Shofuda et al. 1997, 1998). Using RNase protection assay to quantitatively examine mRNA levels, we found that MT1-MMP mRNA is expressed in untreated HCSM cells and markedly increased in response to treatment with pathogenic Αβ. In contrast, MT3-MMP was barely detected in the HCSM cells. This suggests that the major activator of MMP-2 in HCSM cells is MT1-MMP. Further support for this finding came from the fact that the natural inhibitor of MT1-MMP, TIMP-2, was highly effective in inhibiting MMP-2 activation. TIMPs bind non-covalently, but irreversibly, to the active site of MMPs. TIMP-1 demonstrated a slight effect in inhibition of MMP-2 activation; however, not nearly as dramatic as that of TIMP-2. This is in agreement with previous findings that have found that TIMP-2 is at least 10-fold more effective than TIMP-1 in inhibiting activation of MMP-2 (Howard et al. 1991). The synthetic hydroxamate inhibitor, CT-1847, which chelates the Zn2+ ion from the active site of MMPs, was also found to be effective in inhibiting MMP-2 activation. Hydroxamate inhibitors have been shown to bind to the same site as that of TIMP-2, i.e. the catalytic domain of MT1-MMP (Zucker et al. 1998).

We have previously shown that the first signs of cell death in HCSM cells occur after 3 days of Αβ40D exposure (Melchor and Van Nostrand 2000). Activation of MMP-2 was first observed after 3 days of exposure to pathogenic Αβ and continued to increase to through 6 days (Fig. 3b). In parallel to the above findings, we observed that by inhibiting the activation of MMP-2 in Αβ40D-treated HCSM cells this resulted in a notable increase in cell viability (> 85%). This result suggests that activation of MMP-2 in the HCSM cells may contribute to the loss of viability that is promoted by pathogenic Αβ. The mechanism by which MMP-2 activity could lead to loss in cell viability remains unclear. The degradation of specific matrix components could be involved. However, this idea needs further investigation.

Clearly, the role of MMPs in vivo is much more complicated than in vitro. For example, there is a growing body of evidence linking the activities of the plasminogen activator/plasmin system and MMP functions in vascular alterations. Although the plasminogen activator/plasmin system can degrade some extracellular matrix components directly it is likely more effective in activating pro-MMPs. For instance, it was reported that urokinase-type plasminogen activator can directly activate pro-MMP-2 (Keski-Oja et al. 1992). Plasmin can directly activate pro-MMP-9 and an intermediate form of MMP-2 (Okada et al. 1992). In addition, increased expression of plasminogen activators and MMPs has been found in atherosclerotic plaques and associated with smooth muscle cell destruction and vessel wall rupture (Sakalihasan et al. 1996; Andreasen et al. 2000). Recent data in our laboratory has demonstrated that pathogenic Αβ causes a robust increase in the expression of urokinase-type plasminogen activator in cultured HCSM cells (Van Nostrand et al. 2003). These results suggest that enhanced plasminogen activator and MMP expression in cerebrovascular cells in response to pathogenic Αβ deposition may directly or indirectly contribute to loss of vessel wall integrity and hemorrhaging in CAA.

In summary, using primary HCSM cells as an in vitro model for CAA, we found increased levels and activation of MMP-2 and its activator MT1-MMP in response to pathogenic Αβ treatment. MMP-2 may be, in part, responsible for Αβ-mediated cell death in HCSM cells. These findings implicate the expression and activation of MMP-2 in the pathology associated with CAA. Since MMP inhibitors are effective in diminishing MMP-2 activation and rescuing HCSM cell viability this suggests that MMP inhibitors may have potential therapeutic value in the treatment of CAA, AD and other neurological conditions.

References

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. HCSM cell culture
  5. MMP inhibitors
  6. mRNA isolation and RNase protection assay
  7. Quantitative immunoblotting
  8. Gelatin substrate zymography
  9. Cell viability assay
  10. Results
  11. MMP-2 mRNA expression is increased in HCSM cells treated with Aβ40D
  12. MMP-2 protein levels and activity are increased in HCSM cells treated with Aβ40D
  13. MT1-MMP mRNA is increased in HCSM cells treated with Aβ40D
  14. Certain MMP inhibitors decrease MMP-2 activation and increase HCSM cell viability upon treatment with Aβ40D
  15. Discussion
  16. Acknowledgement
  17. References
  • Andreasen P. A., Egelund R. and Petersen H. H. (2000) The plasminogen activation system in tumor growth, invasion, and metastasis. Cell Mol. Life Sci. 57, 2540.
  • Anthony D. C., Ferguson B., Matyzak M. K., Miller K. M., Esiri M. M. and Perry V. H. (1997) Differential matrix metalloproteinase expression in cases of multiple sclerosis and stroke. Neuropathol. Appl. Neurobiol. 23, 406415.
  • Backstrom J. R., Miller C. A. and Tokes Z. A. (1992) Characterization of neutral proteinases from Alzheimer-affected and control brain specimens: identification of calcium-dependent metalloproteinases from the hippocampus. J. Neurochem. 58, 983992.
  • Backstrom J. R., Lim G. P., Cullen M. J. and Tokes Z. A. (1996) Matrix metalloproteinase-9 (MMP-9) is synthesized in neurons of the human hippocampus and is capable of degrading the amyloid-beta peptide (1–40). J. Neurosci. 16, 79107919.
  • Birkedal-Hansen H., Moore W. G., Bodden M. K., Windsor L. J., Birkedal-Hansen B., De-Carlo A. and Engler J. A. (1993) Matrix metalloproteinases: a review. Crit. Rev. Oral Biol. Med. 4, 197250.
  • Chandler S., Miller K. M., Clements J. M., Lury J., Corkill D., Anthony D. C., Adams S. E. and Gearing A. J. (1997) Matrix metalloproteinases, tumor necrosis factor and multiple sclerosis: an overview. J. Neuroimmunol. 72, 155161.
  • Davis J. and Van Nostrand W. E. (1996) Enhanced pathologic properties of Dutch-type mutant amyloid β-protein. Proc. Natl Acad. Sci. USA 93, 29963000.
  • Davis J., Wagner M. R., Zhang W., Xu F. and Van Nostrand W. E. (2003) Amyloid β-protein stimulates the expression of urokinase-type plasminogen activator (uPA) and its receptor (uPAR) in human cerebrovascular smooth muscle cells. J. Biol. Chem. in press.
  • Davis-Salinas J., Saporito-Irwin S. M., Cotman C. W. and Van Nostrand W. E. (1995) Amyloid beta-protein induces its own production in cultured degenerating cerebrovascular smooth muscle cells. J. Neurochem. 65, 931934.
  • Deb S. and Gottschall P. E. (1996) Increased production of matrix metalloproteinases in enriched astrocyte and mixed hippocampal cultures treated with beta-amyloid peptides. J. Neurochem. 66, 16411647.
  • Frisch S. M. and Ruoslahti E. (1997) Integrins and anoikis. Curr. Opin. Cell Biol. 9, 701706.
  • Gijbels K., Masure S., Carton H. and Opdenakker G. (1992) Gelatinase in the cerebrospinal fluid of patients with multiple sclerosis and other inflammatory neurological disorders. J. Neuroimmunol. 41, 2934.
  • Howard E. W., Bullen E. C. and Banda M. J. (1991) Preferential inhibition of 72- and 92-kDa gelatinases by tissue inhibitor of metalloproteinases-2. J. Biol. Chem. 266, 1307013075.
  • Janowska-Wieczorek A., Marquez L. A., Nabholtz J. M., Cabuhat M. L., Montano J., Chang H., Rozmus J., Russell J. A., Edwards D. R. and Turner A. R. (1999) Growth factors and cytokines upregulate gelatinase expression in bone marrow CD34(+) cells and their transmigration through reconstituted basement membrane. Blood 93, 33793390.
  • Kawai M., Kalaria R. N., Cras P., Siedlak S. L., Velasco M. E., Shelton E. R., Chan H. W., Greenberg B. D. and Perry G. (1993) Degeneration of vascular muscle cells in cerebral amyloid angiopathy of Alzheimer disease. Brain Res. 623, 142146.
  • Keski-Oja J., Lohi J., Tuuttila A., Tryggvason K. and Vartio T. (1992) Proteolytic processing of the 72,000 Da type IV collagenase by urokinase plasminogen activation. Exp. Cell Res. 202, 471476.
  • Kouwenhoven M., Carlstrom C., Ozenci V. and Link H. (2001) Matrix metalloproteinase and cytokine profiles in monocytes over the course of stroke. J. Clin. Immunol. 21, 365375.
  • Lewalle J. M., Munaut C., Pichot B., Cataldo D., Baramova E. and Foidart J. M. (1995) Plasma membrane-dependent activation of gelatinase A in human vascular endothelial cells. J. Cell Physiol. 165, 475483.
  • Li Z., Froehlich J., Galis Z. S. and Lakatta E. G. (1999) Increased expression of matrix metalloproteinase-2 in the thickened intima of aged rats. Hypertension 33, 116123.
  • Lim G. P., Backstrom J. R., Cullen M. J., Miller C. A., Atkinson R. D. and Tokes Z. A. (1996) Matrix metalloproteinases in the neocortex and spinal cord of amyotrophic lateral sclerosis patients. J. Neurochem. 67, 251259.
  • Lim G. P., Russell M. J., Cullen M. J. and Tokes Z. A. (1997) Matrix metalloproteinases in dog brains exhibiting Alzheimer-like characteristics. J. Neurochem. 68, 16061611.
  • Melchor J. P. and Van Nostrand W. E. (2000) Fibrillar amyloid beta-protein mediates the pathologic accumulation of its secreted precursor in human cerebrovascular smooth muscle cells. J. Biol. Chem. 275, 97829791.
  • Mun-Bryce S. and Rosenberg G. A. (1998) Matrix metalloproteinases in cerebrovascular disease. J. Cerebr. Blood Flow Metabol. 18, 11631172.
  • Nagase H. (1997) Activation mechanisms of matrix metalloproteinases. Biol. Chem. 378, 151160.
  • Nagase H. and Woessner J. F. Jr (1999) Matrix metalloproteinases. J. Biol. Chem. 274, 2149121494.
  • Okada Y., Gonoji Y., Naka K., Tomita K., Nakanishi I., Iwata K., Yamashita K. and Hayakawa T. (1992) Matrix metalloproteinase 9 (92 kDa gelatinase/type IV collagenase) from HT1080 human fibrosarcoma cells. Purification and activation of the precursor and enzymatic properties. J. Biol. Chem. 267, 2171221719.
  • Overall C. M., Wrana J. L. and Sodek J. (1991) Transcriptional and post-transcriptional regulation of 72-kDa gelatinase/type IV collagenase by transforming growth factor-beta 1 in human fibroblasts. Comparisons with collagenase and tissue inhibitor of matrix metalloproteinase gene expression. J. Biol. Chem. 266, 1406414071.
  • Planas A. M., Sole S. and Justicia C. (2001) Expression and activation of matrix metalloproteinase-2 and -9 in rat brain after transient focal cerebral ischemia. Neurobiol. Dis 8, 834846.
  • Romanic A. M., White R. F., Arleth A. J., Ohlstein E. H. and Barone F. C. (1998) Matrix metalloproteinase expression increases after cerebral focal ischemia in rats: inhibition of matrix metalloproteinase-9 reduces infarct size. Stroke 29, 10201030.
  • Rosenberg G. A. (1995) Matrix metalloproteinases in brain injury. J. Neurotrauma 12, 833842.
  • Rosenberg G. A. and Navratil M. (1997) Metalloproteinase inhibition blocks edema in intracerebral hemorrhage in the rat. Neurology 48, 921926.
  • Rosenberg G. A., Mun-Bryce S., Wesley M. and Kornfeld M. (1990) Collagenase-induced intracerebral hemorrhage in rats. Stroke 21, 801807.
  • Rosenberg G. A., Kornfeld M., Estrada E., Kelley R. O., Liotta L. A. and Stetler-Stevenson W. G. (1992) TIMP-2 reduces proteolytic opening of blood–brain barrier by type IV collagenase. Brain Res. 576, 203207.
  • Rosenberg G. A., Estrada E., Kelley R. O. and Kornfeld M. (1993) Bacterial collagenase disrupts extracellular matrix and opens blood–brain barrier in rat. Neurosci. Lett. 160, 117119.
  • Rosenberg G. A., Estrada E. Y., Dencoff J. E. and Stetler-Stevenson W. G. (1995) Tumor necrosis factor-alpha-induced gelatinase B causes delayed opening of the blood–brain barrier: an expanded therapeutic window. Brain Res. 703, 151155.
  • Rosenberg G. A., Navratil M., Barone F. and Feuerstein G. (1996) Proteolytic cascade enzymes increase in focal cerebral ischemia in rat. J. Cerebr. Blood Flow Metabol. 16, 360366.
  • Sakalihasan N., Delvenne P., Nusgens B. V., Limet R. and Lapiere C. M. (1996) Activated forms of MMP2 and MMP9 in abdominal aortic aneurysms. J. Vasc. Surg. 24, 127133.
  • Sato H., Takino T., Okada Y., Cao J., Shinagawa A., Yamamoto E. and Seiki M. (1994) A matrix metalloproteinase expressed on the surface of invasive tumour cells. Nature 370, 6165.
  • Shofuda K., Yasumitsu H., Nishihashi A., Miki K. and Miyazaki K. (1997) Expression of three membrane-type matrix metalloproteinases (MT-MMPs) in rat vascular smooth muscle cells and characterization of MT3-MMPs with and without transmembrane domain. J. Biol. Chem. 272, 97499754.
  • Shofuda K., Nagashima Y., Kawahara K., Yasumitsu H., Miki K. and Miyazaki K. (1998) Elevated expression of membrane-type 1 and 3 matrix metalloproteinases in rat vascular smooth muscle cells activated by arterial injury. Lab. Invest. 78, 915923.
  • Springman E. B., Angleton E. L., Birkedal-Hansen H. and Van Wart H. E. (1990) Multiple modes of activation of latent human fibroblast collagenase: evidence for the role of a Cys73 active-site zinc complex in latency and a ‘cysteine switch’ mechanism for activation. Proc. Natl Acad. Sci. USA 87, 364368.
  • Stetler-Stevenson W. G., Aznavoorian S. and Liotta L. A. (1993) Tumor cell interactions with the extracellular matrix during invasion and metastasis. Annu. Rev. Cell Biol. 9, 541573.
  • Strongin A. Y., Collier I., Bannikov G., Marmer B. L., Grant G. A. and Goldberg G. I. (1995) Mechanism of cell surface activation of 72-kDa type IV collagenase. Isolation of the activated form of the membrane metalloprotease. J. Biol. Chem. 270, 53315338.
  • Takino T., Sato H., Shinagawa A. and Seiki M. (1995) Identification of the second membrane-type matrix metalloproteinase (MT-MMP-2) gene from a human placenta cDNA library. MT-MMPs form a unique membrane-type subclass in the MMP family. J. Biol. Chem. 270, 2301323020.
  • Uhm J. H., Dooley N. P., Villemure J. G. and Yong V. W. (1997) Mechanisms of glioma invasion: role of matrix-metalloproteinases. Can. J. Neurol. Sci. 24, 315.
  • Van Nostrand W. E., Rozemuller A. J., Chung R., Cotman C. W. and Saporito-Irwin S. M. (1994) Amyloid beta-protein precursor in cultured leptomeningeal smooth muscle cells. Amyloid 1, 17.
  • Van Nostrand W. E., Melchor J. P. and Ruffini L. (1998) Pathologic amyloid beta-protein cell surface fibril assembly on cultured human cerebrovascular smooth muscle cells. J. Neurochem. 70, 216223.
  • Van Wart H. E. and Birkedal-Hansen H. (1990) The cysteine switch: a principle of regulation of metalloproteinase activity with potential applicability to the entire matrix metalloproteinase gene family. Proc. Natl Acad. Sci. USA 87, 55785582.
  • Vinters H. V. (1987) Cerebral amyloid angiopathy. A critical review. Stroke 18, 311324.
  • Wattendorff A. R., Frangione B., Luyendijk W. and Bots G. T. A. M. (1995) Hereditary cerebral haemorrhage with amyloidosis, Dutch type (HCHWA-D): clinicopathological studies. J. Neurol. Neurosurg. Psychiat. 59, 699705.
  • Yong V. W., Krekoski C. A., Forsyth P. A., Bell R. and Edwards D. R. (1998) Matrix metalloproteinases and diseases of the CNS. Trends Neurosci. 21, 7580.
  • Yong V. W., Power C., Forsyth P. and Edwards D. R. (2001) Metalloproteinases in biology and pathology of the nervous system. Nat. Rev. Neurosci. 2, 502511.
  • Yu M., Sato H., Seiki M. and Thompson E. W. (1995) Complex regulation of membrane-type matrix metalloproteinase expression and matrix metalloproteinase-2 activation by concanavalin A in MDA-MB-231 human breast cancer cells. Cancer Res. 55, 32723277.
  • Zucker S., Drews M., Conner C., Foda H. D., DeClerck Y. A., Langley K. E., Bahou W. F., Docherty A. J. and Cao J. (1998) Tissue inhibitor of metalloproteinase-2 (TIMP-2) binds to the catalytic domain of the cell surface receptor, membrane type 1-matrix metalloproteinase 1 (MT1-MMP). J. Biol. Chem. 273, 12161222.