From the Department of Medicine, Health Sciences Center, Stony Brook University, Stony Brook, New York 11794-8153
Received for publication, February 10, 2003
, and in revised form, March 21, 2003.
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ABSTRACT |
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INTRODUCTION |
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Cerebrovascular A deposition, known as cerebral amyloid angiopathy (CAA), is accompanied by smooth muscle cell degeneration suggesting a toxic effect of A
to these cells in vivo (16, 17, 18). The degenerating smooth muscle cells have been implicated in the overproduction of A
PP and A
in the cerebral vessel wall further suggesting the active involvement of these cells in the progression of this cerebrovascular pathology (17, 18, 19). Similar to these in vivo observations, we have reported that A
42, the more pathogenic form of the wild-type peptide, causes cellular degeneration accompanied by a marked increase in the level of cell-associated A
PP in cultured human cerebrovascular smooth muscle (HCSM) cells, an in vitro model of CAA (20, 21, 22). In more recent studies we demonstrated that mutations within A
that are associated with familial forms of CAA (E22Q Dutch, E22K Italian, and D23N Iowa) markedly enhance both the fibrillogenic and cerebrovascular pathogenic properties of A
toward cultured HCSM cells (23, 24, 25, 26). These investigations showed that CAA mutant forms of A
assemble into an extensive network of fibril-like structures on the surfaces of HCSM cells. Furthermore, fibril assembly of pathogenic A
on the cell surface is required for inducing downstream pathologic responses in HCSM cells, including cell surface accumulation of sA
PP
, degradation of vascular smooth muscle cell
actin, and ultimately an apoptotic cell death (24, 25, 26, 27).
Urokinase-type plasminogen activator (uPA) and tissue-type plasminogen activator (tPA) are serine proteinases that mediate the conversion of the proenzyme plasminogen into the active enzyme plasmin. Both tPA and uPA function locally as plasminogen activators because of their interactions with specific proteins that restrict their activities. For example, tPA is normally localized to fibrin deposits because of a direct tight binding interaction, where it mediates plasminogen activation for dissolution of the fibrin clot. It is noteworthy that fibrillar A can substitute for fibrin in binding and stimulating tPA-mediated plasminogen activation (28, 29, 30). On the other hand, the activity of uPA appears to be localized to cell surfaces via its binding to the uPA receptor (uPAR), a glycosylphosphatidylinositol-anchored protein (31). The binding of uPA to uPAR also enhances its activity generating high local levels of plasmin at cell surfaces. This has implied an important role for uPA in the induction of proteolytic events at the cell surface such as extracellular matrix protein degradation and activation of latent proteinases such as matrix metalloproteinases (MMPs). Components of the uPA/plasmin system are expressed in brain-derived neurons, astrocytes, and microglia in vitro (30, 32, 33, 34, 35). Similarly, expression of uPA, its receptor uPAR, and plasminogen have been reported in these cell types in brain, as well as in the cerebral vessel wall in vivo (30, 33, 34).
The uPA/plasmin system has been implicated in several pathophysiological processes including thrombosis, atherosclerosis, and vessel rupture (36, 37). There is a growing body of evidence linking the activities of the uPA/plasmin system and MMP functions in vascular alterations. Although the uPA/plasmin system can degrade some extracellular matrix components directly it is likely more effective in activating pro-MMPs. For example, it was reported that uPA can directly activate pro-MMP-2 (38). Plasmin can directly activate pro-MMP-9 and an intermediate form of MMP-2 (39). In addition, there are several lines of in vivo evidence to further support the interaction between uPA/plasmin and MMPs in vascular pathological conditions. For instance, increased expression of uPA and MMPs has been found in atherosclerotic plaques and associated smooth muscle cells (40, 41). Smooth muscle cell destruction and vessel wall rupture were reduced in a mouse atherosclerosis model developed on a uPA knockout background suggesting that uPA may contribute to these pathologic events (42). Similarly, uPA-deficient mice were protected from cardiac rupture in a mouse model of acute myocardial infarction (43). Despite the number of studies investigating the uPA/plasmin system in vascular alterations and vessel rupture in the periphery there have been no investigation into its potential role in loss of vessel wall integrity and cerebral hemorrhage in CAA.
Here we show that pathogenic A deposition causes a robust increase in the expression and cell surface localization of uPA in cultured HCSM cells. The present results suggest that enhanced uPA expression and subsequent plasminogen activation may initially be a protective response by HCSM cells to promote localized degradation of A
, the pathogenic stimulus, and inhibit the progression of CAA. However, prolonged activation of plasminogen may have its own deleterious effects and contribute to loss of vessel wall integrity and hemorrhage in CAA.
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EXPERIMENTAL PROCEDURES |
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HCSM Cell CulturePrimary cultures of HCSM cells were established as described previously (45) and shown to robustly express vascular smooth muscle cell actin, vascular smooth muscle myosin, and SM22
. Two lines of HCSM cells were used in these studies. One was derived from a 70-year-old male Alzheimer's disease patient, and the other was derived from a 37-year-old female control. All HCSM cells were used between passages 4 and 7 and maintained in 24-well tissue culture dishes with Dulbecco's minimum essential medium containing 10% fetal bovine serum (Gemini Bio-Products, Calabasas, CA), non-essential amino acids, and antibiotics (Invitrogen). For experiments, near confluent cultures of HCSM cells were placed in serum-free medium containing 0.1% bovine serum albumin, non-essential amino acids, and antibiotics overnight prior to treatment. Freshly solubilized monomeric wild-type or Dutch mutant A
40, at a final concentration of 25 µM, was added to the cultures in serum-free medium and incubated at 37 °C for up to 6 days. Cells were viewed and photographed using an Olympus IX70 phase-contrast microscope. Cell viability was quantified using a fluorescent live/dead cell assay following the manufacturer's protocol (Molecular Probes, Eugene, OR). The number of live and dead cells were counted from several fields (n = 4) from at least three separate wells for each experiment. To determine the percent of adherent HCSM cells calcein probe was added to the cells, and the amount of fluorescence was measured at excitation wavelength 485 nm and emission wavelength 535 nm using a Cytofluor II fluorescence plate reader (PerkinElmer Life Sciences). Each measurement was performed in duplicate, and five fields were scanned for each well from three separate experiments.
Cell Culture Plasminogen Activation AssayHCSM cells were grown in 48-well tissue culture plates and incubated for 2 to 6 days in phenol red-free, serum-free culture medium in absence or presence of A peptides. The conditioned medium samples were collected, and the cells were rinsed two times with 500 µl of fresh phenol-red free and serum-free culture medium and then replaced with 150 µl of fresh phenol red-free, serum-free medium. Purified Glu-plasminogen was added at a final concentration of 0.5 µM to the collected conditioned medium samples and the fresh phenol red-free, serum-free culture medium on the HCSM cells. The medium samples and cells were incubated for 30 min at 37 °C, and the generated plasmin activity in the samples was measured using the chromogenic substrate tosyl-Gly-Pro-Lys-p-nitroanilide in a Vmax microtiter plate reader as described previously (29).
HCSM Cell Plasminogen Activator ZymographyNear confluent cultures of HCSM cells were incubated in the absence or presence of 25 µM wild-type or Dutch mutant A40 for 2 to 6 days. The culture medium was removed, and the cells were rinsed with PBS three times and then solubilized in a lysis buffer containing 50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% Triton X-100, and 5 mM EDTA. Aliquots of culture medium and cell lysates and known quantities of purified tPA and uPA were subjected to SDS-PAGE under non-reducing conditions. The completed gels were soaked in 2.5% Triton X-100 for 20 min at room temperature to remove the SDS. The gels were then soaked in water two times for 30 min at room temperature to remove the Triton X-100. The polyacrylamide gels were then overlaid on plasminogen/substrate gels composed of 1% agarose, 2.5% non-fat dry milk, 100 mM Tris-HCl, pH 7.5, and 2 µg/ml Glu-plasminogen. The stacked gels were placed in a sealed, humidified container and incubated for 1216 h at 37 °C. Plasminogen activator activity was visualized as clearing zones in the underlying plasminogen/substrate gel and documented using a Fluor-S Multi-Imager (Bio-Rad) with the manufacturer's Quantity One software.
mRNA Isolation and RNase Protection AssayHCSM cells were grown to 90% confluency in T-225 cm2 flasks. The cells were incubated under serum-free conditions either in the absence or presence of 25 µM Dutch mutant A
40 for 6 days. Messenger RNA was isolated using the mRNA isolation kit according to the manufacturer's instructions (Roche Molecular Biochemicals) and stored at 70 °C. Using avian myeloblastosis virus reverse transcriptase (Roche Molecular Biochemicals), cDNA was made using
500 ng of HCSM cell mRNA, 60 pg/µl oligo(dT), 500 nM dNTP, incubated for 1 h at 41 °C. cDNA templates for riboprobes were generated using the following primers for uPA (GenBankTM accession number X02419
[GenBank]
; nucleotides 33504459): forward, 5'-GCTCTGTCACCTACGTGTGTG-3'; and reverse, 5'-GGAACCTTGCCTAGAAGT-3'; or for uPAR (GenBankTM accession number XM009232; nucleotides 6601014): forward, 5'-CTATCGGACTGGCTTGAAGATCAC-3'; and reverse, 5'-GGTGGTGTTGCAGCATTTCAGGAAG-3'.
PCR products of 244 and 354 bp for uPA and uPAR, respectively, were made and purified using a High Pure PCR product purification kit (Roche Molecular Biochemicals) and digested with NotI and BamHI. The digested PCR products were purified by electrophoretic separation and subsequent extraction from a 0.8% agarose gel using the Gene-Clean II kit (BIO101, Vista, CA). The fragment was ligated into pBluescript (KS+) (Stratagene) at the BamHI-NotI site in an antisense direction using a rapid DNA ligation kit (Roche Molecular Biochemicals). DNA sequencing analysis verified the integrity of all the constructs. The uPA/Bluescript or uPAR/Bluescript construct was digested with PvuII and SacI. A 464and 540-bp fragment for uPA and uPAR, respectively, was purified by electrophoretic separation and subsequent extraction from a 0.8% agarose gel using the GeneClean II kit.
For RNase protection assay, an antisense radiolabeled riboprobe was generated by using an in vitro transcription kit (Promega, Madison, WI) with 50 ng of uPA or uPAR fragment, T3 RNA polymerase (Roche Molecular Biochemicals), and 100 µCi of [
-32P]UTP (PerkinElmer Life Sciences). As a control, the GAPDH housekeeping gene was made using 50 ng of GAPDH template (PharMingen) 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), 300500 ng of HCSM cell mRNA or 4 µg of negative control tRNA was hybridized at 56 °C overnight with 100,000 cpm/µl of uPA, uPAR, 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 with phenol/chloroform extraction and electrophoresed on a 6% TBE-urea gel (Invitrogen) under 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.
Quantitative ImmunoblottingNear confluent cultures of HCSM cells were incubated in the absence or presence of 25 µM Dutch mutant A40 in serum-free medium for 6 days. In some experiments increasing concentrations of Glu-plasminogen were included. After incubation, the medium was collected, and the cells were rinsed with PBS three times and then solubilized in a lysis buffer containing 50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% Triton X-100, 5 mM EDTA, and proteinase inhibitor mixture (Roche Molecular Biochemicals). Aliquots of the medium or cell lysate samples were electrophoresed in either SDS 10% polyacrylamide gels or Tris-Tricine 1020% polyacrylamide gels, and the proteins were transferred onto Hybond nitrocellulose membranes (Amersham Biosciences). Unoccupied sites on the membranes were blocked overnight with 5% non-fat milk in PBS with 0.05% Tween 20. The membranes were probed with a monoclonal antibody to uPAR (American Diagnostica, Greenwich, CT) or monoclonal antibody 6E10 to A
(Signet Laboratories, Dedham, MA) and then incubated with a secondary peroxidase-coupled sheep anti-mouse IgG antibody at a dilution of 1:1000. The peroxidase activity on the membranes was detected using a Supersignal Dura West (Pierce), and bands were measured using a Fluor-S Multi-Imager (Bio-Rad) with the manufacturer's Quantity One software.
Solid-phase Binding AssayDutch mutant A40 was assembled into fibrils as described above, and 2 µg/well in 100 µl of PBS was dried down in triplicate wells of a 96-well microtiter plate (Corning Glass) overnight at 37 °C. The wells were rinsed with PBS three times and blocked with 100 µl/well PBS containing 1 mg/ml BSA for 1 h at room temperature. After rinsing three times with PBS, purified tPA (2 nM)or uPA (2 nM) in PBS containing 0.1 mg/ml BSA were incubated in triplicate (100 µl/well) for 1 h at room temperature. After rinsing the wells with PBS three times, 100 µl of rabbit anti-tPA or rabbit anti-uPA (1:1000) in PBS containing 0.1 mg/ml BSA was added for 1 h at room temperature. After rinsing the wells with PBS three times, 100 µl of HRP-conjugated donkey anti-rabbit IgG (1:1000) in PBS containing 0.1 mg/ml BSA was added for 1 h at room temperature. The binding of secondary antibody was detected using the TMB Microwell Peroxidase substrate (Kirkegaard & Perry Laboratories, Gaithersburg, MD) as described by the manufacturer. The conversion of the colorimetric substrate was measured at a wavelength of 450 nm using a Molecular Dynamics Vmax kinetic plate reader (Sunnyvale, CA).
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RESULTS |
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Fibrillar A Does Not Bind uPA or Stimulate Its Activity Previous studies (28, 29) showed that fibrillar A
binds tPA and stimulates its activation of plasminogen. Soluble pathogenic Dutch mutant A
40 rapidly assembles into fibrils on the surfaces of HCSM cells (24, 25, 26). Because the levels of HCSM cell surface-associated uPA dramatically increase in response to treatment with Dutch mutant A
40 (see Figs. 1 and 3), we determined whether A
fibrils could be responsible for this localized increase in HCSM cell surface uPA. Solid-phase binding assays showed that tPA exhibited robust binding to immobilized A
fibrils whereas uPA showed little binding (Fig. 4A). Similarly, fibrillar A
potently stimulated the activity of tPA whereas it had no effect on the activity of uPA (Fig. 4B). These findings suggest that HCSM cell surface fibrillar A
is not responsible for directly facilitating the localization of cell surface uPA.
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Pathogenic A Stimulates the Expression of uPAR in HCSM CellsuPA is normally localized to the surfaces of cells through binding to its receptor uPAR (31). Therefore, we determined whether pathogenic A
influences the expression of uPAR in HCSM cells. RNase protection assays showed that Dutch mutant A
40 increased the expression of uPAR mRNA severalfold in HCSM cells (Fig. 5, A and B). More striking, the levels of uPAR protein were increased nearly 8-fold in HCSM cells treated with pathogenic A
(Fig. 5, C and D). This is in close agreement with the nearly 8-fold increase in HCSM cell surface uPA activity observed in Fig. 1. These findings indicate that pathogenic A
stimulates the expression of both uPA and uPAR in HCSM cells and that the increase in uPAR leads to the localized increase in uPA activity on the HCSM cell surface.
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Increased Plasminogen Activation Leads to Robust Degradation of A and Decreased Toxicity in HCSM CellsAlthough plasminogen activators do not directly cleave A
plasmin can effectively degrade this pathogenic peptide in vitro (29, 30, 32, 46). Therefore, we determined the consequences of administering plasminogen to HCSM cells in the presence of pathogenic A
. In the presence of increasing amounts of plasminogen there was a concomitant increased degradation of monomeric soluble A
in the culture medium and deposited A
on the HCSM cell surface (Fig. 6, A and B, respectively). It is noteworthy that activated plasminogen was also effective in degrading the larger oligomeric forms of A
associated with the HCSM cells. Because the fibril-like oligomeric forms of A
on the HCSM cell surface are responsible for mediating subsequent cellular degeneration and loss in cell viability we measured A
-induced HCSM cell death in the presence of increasing concentrations of plasminogen. As shown in Fig. 7, the addition of 0.1 µM plasminogen increased HCSM cell viability from
20% to nearly 50% in the presence of Dutch mutant A
40. Higher concentrations of plasminogen, which led to robust degradation of A
(Fig. 6), completely restored HCSM cell viability. Plasminogen alone at any of the concentrations did not affect HCSM cell viability in this 6-day experimental paradigm. These findings suggest that uPA-mediated activation of plasminogen can lead to effective degradation of pathogenic A
and protection from its cytotoxic properties in HCSM cells.
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Prolonged Activation of Plasminogen Leads to Loss of HCSM Cell AdhesionThe experiments described above and shown in Figs. 6 and 7 showed that activation of plasminogen may initially be protective via its degradation of pathogenic A. However, when HCSM cells were incubated for 12 days in the presence of plasminogen, conditions that would lead to prolonged activation of plasminogen, there was a profound effect on cell attachment. As shown in Fig. 8 in the presence of increasing amounts of plasminogen there was obvious retraction of cell bodies and loss of cell adhesion. At 1 µM plasminogen many detached HCSM cells were observed. Continuous exposure to 0.1 µM and 1 µM plasminogen resulted in a 34 and 64% loss in cell adhesion, respectively, compared with HCSM cells incubated in the absence of plasminogen (Fig. 8D). These findings suggest that chronic activation of plasminogen can undermine HCSM cell attachment by degradation of cell adhesion components.
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DISCUSSION |
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Our initial findings demonstrated the levels of secreted and, particularly, cell surface plasminogen activator activity were indeed increased in HCSM cells in response to treatment with pathogenic Dutch mutant A (Fig. 1). However, it was unclear whether this increase in plasminogen activator activity was attributable to tPA, uPA, or both. RNase protection assays (Fig. 2) and plasminogen zymography (Fig. 3) unequivocally demonstrated that the plasminogen activator activity produced in HCSM cells was uPA. Wild-type A
42 and Dutch mutant A
40, both of which are pathogenic for cultured HCSM cells (20, 21, 22, 23), caused increased levels of secreted and cell-associated uPA in these cells. However, wild-type A
40, which is devoid of pathogenic properties to HCSM cells (20, 21, 23), had no effect. Previously, it was shown that pathogenic A
stimulates apoptotic cell death in HCSM cells (27, 54). We performed parallel experiments using the protein phosphatase inhibitor okadaic acid, which also induces apoptotic cell death (55, 56), to examine the specificity of the effect on uPA expression by pathogenic A
in HCSM cells. Although okadaic acid induced HCSM cell death it had no appreciable effect on uPA expression (Fig. 3C). This result demonstrates that the observed increase in HCSM cell uPA expression in response to pathogenic forms of A
is not a general response to cytotoxic insult.
The more robust accumulation of uPA on the HCSM cell surface suggested its binding to some component at this location. Our earlier studies demonstrated that pathogenic forms of A assemble into an extensive network of fibrillar structures on the HCSM cell surface and that this assembly is required for subsequent pathologic responses in these cells (24, 25, 26). Furthermore, it was shown previously (28, 29) that fibrillar A
binds and stimulates the activity of tPA in vitro. This finding stemmed from earlier observations that anti-A
antibodies cross-react with conformational epitopes on human fibrinogen and that anti-fibrinogen antibodies cross-react with A
suggesting structural similarities (57). The fibrin-mimicry of A
may help explain why patients with CAA that are administered therapeutic tPA for acute myocardial infarction suffer from a high incidence of intracerebral hemorrhage (51, 52). The subsequent activation of plasminogen could result in excessive proteolysis leading to loss of vessel integrity and rupture. In fact, this consequence of increased cerebral hemorrhage by systemic tPA administration has been confirmed recently (53) in a transgenic mouse model that develops cerebrovascular fibrillar A
deposits. These previous results raised the possibility that HCSM cell surface A
fibrils similarly mediated the binding and perhaps stimulation of uPA. However, solid-phase binding assays failed to show appreciable binding of uPA to fibrillar pathogenic A
(Fig. 4A). As well, unlike tPA the plasminogen activator activity of uPA was not stimulated by fibrillar A
(Fig. 4B). This suggested that another surface component must be responsible for the accumulation of uPA in pathogenic A
-treated HCSM cells. In fact, further investigation revealed that the expression of the cell surface uPA receptor, uPAR, was markedly increased when HCSM cells were treated with pathogenic A
(Fig. 5).
The parallel increase in expression of uPAR along with uPA appears to serve to localize much of the increased uPA and its plasminogen activator activity to the HCSM cell surface. Initially, this may be highly beneficial to the HCSM cell that is encountering pathogenic A. As mentioned earlier, pathogenic A
fibril assembly on the HCSM cell surface is a required primary event for the subsequent cellular degeneration. By simultaneously increasing expression of uPA and uPAR this would result in increased plasminogen activation at the HCSM cell surface, the site where the pathogenic A
fibrils have assembled. Previously it was shown that plasmin can effectively degrade both unassembled and assembled fibrillar A
in vitro (29, 30, 32, 46). Similarly, we show here that the addition of plasminogen and its subsequent conversion to plasmin by uPA results in effective degradation of soluble A
in the culture medium and, more importantly, oligomeric A
associated with the HCSM cell surface (Fig. 6). Moreover, the effective degradation of HCSM cell surface oligomeric A
by the generated plasmin provided striking protection from the cytotoxic effects of pathogenic A
in these cells (Fig. 7). Together, these findings suggest that uPA/uPAR expression and subsequent plasminogen activation may initially be increased as a mechanism to protect the vulnerable HCSM cells from the downstream pathologic consequences resulting from pathogenic A
deposition. Even though uPA was the only plasminogen activator detected in cultured HCSM cells tPA is likely present in cerebral vessels as well through production by the endothelium (58, 59). The present results are very similar to the recent findings of Tucker et al. (30, 32, 60) which showed that A
stimulated the expression of tPA and uPA in cultured primary neurons, and in the presence of plasminogen, led to degradation of A
and protection from its neurotoxic effects. This implies that pathologic deposition of A
in senile plaques and in the cerebral vessel wall may promote similar localized protective responses from neurons and cerebrovascular smooth muscle cells, respectively, involving plasminogen activator/plasmin systems to degrade A
.
Although the initial activation of plasminogen may be a protective response by HCSM cells to degrade and clear pathogenic A chronic activation of plasminogen may have its own pathologic consequences. Plasmin can directly degrade certain extracellular matrix components and may influence cell-cell contacts and cell adhesion. In fact we found that prolonged plasminogen activation led to a marked loss of HCSM cell morphology and adhesion (Fig. 8) suggesting that plasmin may have a direct influence the integrity of these cells in the cerebral vessel wall. Furthermore, activation of the uPA/plasmin system may further promote cerebral vessel wall instability in CAA by activation of certain MMPs. It is noteworthy that we found recently (61) that pathogenic A
stimulates the expression and activation of MMPs in HCSM cells. Several reports have implicated interactions between the uPA/plasmin and MMP systems in loss of vascular integrity and vessel wall rupture in the periphery (36, 37, 40, 41, 42, 43). The present study suggests that a parallel situation may occur in the cerebral vessel wall in CAA where the chronic activation of plasminogen over time may directly, and/or indirectly through interactions with MMPs, undermine the integrity of the cerebral vessel wall contributing to spontaneous hemorrhage in this condition.
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FOOTNOTES |
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Both authors contributed equally to this work.
To whom correspondence should be addressed: Dept. of Medicine, HSC T-15/081, Stony Brook University, Stony Brook, NY 11794-8153. Tel.: 631-444-1661; Fax: 631-444-7518; E-mail: William.VanNostrand{at}stonybrook.edu.
1 The abbreviations used are: A, amyloid
-protein; A
PP, amyloid
-protein precursor; uPA, urokinase-type plasminogen activator; tPA, tissue-type plasminogen activator; uPAR, uPA receptor; MMP, matrix metalloproteinase; HCSM, human cerebrovascular smooth muscle; CAA, cerebral amyloid angiopathy; PBS, phosphate-buffered saline; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; BSA, bovine serum albumin; hGAPDH, human GAPDH.
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REFERENCES |
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