Furin-Like Proprotein Convertases Are Central Regulators of the Membrane Type Matrix Metalloproteinase–Pro-Matrix Metalloproteinase-2 Proteo
http://www.100md.com
《循环学杂志》
the Department of Medicine/Cardiology, Deutsches Herzzentrum Berlin, Berlin, Germany (P.S., H.M., D.S., N.B.P.S., M.R., U.T., E.F., K.G.)
Diseases of Aging and Regional Protein Chemistry Centers, Ottawa Health Research Institute, University of Ottawa, Ottawa, Ontario, Canada (J.P.V., M.C.)
Laboratory of Biochemical Neuroendocrinology, Clinical Research Institute, Montreal, Quebec, Canada (N.G.S.).
Abstract
Background— Accumulation of macrophages and their in situ expression of matrix metalloproteinases (MMPs) are important determinants of plaque stability. Activation of membrane-bound MT1-MMP, the major activator of pro-MMP-2, requires intracellular endoproteolytic cleavage of its precursor protein. This type of activation typically requires suitable furin-like proprotein convertases (PCs), specifically furin and PC5. The present study was done to investigate the function of MT1-MMP as well as furin-like PCs in mononuclear inflammatory cells.
Methods and Results— Macrophage differentiation of human monocytic THP-1 cells was accompanied by increased expression of furin, PC5, and MT1-MMP. Some pro-MMP-2 activation was found in macrophages, but pro-MMP-2 level or activation was not enhanced after stimulation with the proinflammatory mediators tumor necrosis factor- or lipopolysaccharide. However, culturing of macrophages in conditioned medium from serum-starved vascular smooth muscle cells, which constitutively secrete pro-MMP-2, resulted in a strong pro-MMP-2 activation. Inhibition of furin-like PCs with the specific pharmacological inhibitor decanoyl-RVKR-chloromethylketone (dec-CMK) inhibited MT1-MMP activation in macrophages. Dec-CMK or furin-specific small interfering RNA significantly inhibited macrophage MT1-MMP–dependent activation of vascular smooth muscle cell–derived pro-MMP-2. Flow cytometry demonstrated that human circulating monocytes express furin and PC5, and MT1-MMP and immunohistochemistry revealed their colocalization in macrophages in advanced human atherosclerotic lesions.
Conclusions— Furin-like PCs (furin and PC5) play a central role in a MT-MMP–MMP-2 proteolytic cascade, involving provision of macrophage MT1-MMP for the activation of pro-MMP-2 synthesized by other cells. Furin and PC5 are expressed in human peripheral blood mononuclear cells and colocalize with MT1-MMP in macrophages in the atherosclerotic plaque, supporting the hypothesis that they are potential targets in atherosclerosis.
Key Words: metalloproteinases ; atherosclerosis ; inflammation ; proprotein convertases
Introduction
Expression of matrix metalloproteinases (MMPs) by monocytes/macrophages is a key mechanism for the initiation, progression, and complications of atherosclerosis.1,2 MMPs facilitate mononuclear cell entry of the vessel wall, which is important for the chronic inflammatory response in atherosclerosis,1 as well as vascular smooth muscle cell (VSMC) migration/invasion, which is required for neointima formation and restenosis.3 Rupture-prone atherosclerotic lesions are characterized by accumulation of macrophages and their in situ expression of MMP activity,4 likely contributing to weakening of the plaque cap through the breakdown of the extracellular matrix (ECM).5 In mononuclear cells, MMPs and their tissue inhibitor of metalloproteinases (TIMPs) are regulated by proinflammatory mediators, such as interleukin-1, tumor necrosis factor- (TNF-), and oxidized LDL (ox-LDL).6–8 Regulation of MMPs in mononuclear cells involves prostaglandin E2/cAMP-dependent signaling,9 and secretion of MMPs from macrophages/foam cells is inhibited by statins in vitro and in vivo.10
The family of mammalian MMPs now includes >20 members, with overlapping cleavage activity toward the pericellular matrix (eg, laminin, vitronectin) and ECM.11 According to their mode of activation, MMPs can be grouped into soluble MMPs and membrane-bound MT-MMPs. Monocytes/macrophages express soluble MMPs (eg, MMP-1, -3, -9)4,10,12–14 and MT-MMPs.6,7 Soluble MMPs (eg, MMP-2, MMP-9) are released from the cells as zymogens and require extracellular posttranslational cleavage to gain biological activity.11 In contrast, MT-MMPs are activated intracellularly and then tethered to the cell surface as active enzymes.11,15 Moreover, MT-MMPs amplify a proteolytic cascade in that they are the major pro-MMP-2 activators.11,15 In human atherosclerotic lesions, MT-MMPs are found in the media underlying fibrous and lipid regions, localizing to VSMCs and macrophages.6,7
Activation of the archetypical MT1-MMP has been demonstrated to require endoproteolytic cleavage by furin-like proprotein convertases (PCs).16 Furin is the prototypical member of the 7 thus far identified mammalian subtilisin/kexin-like serine proteinases (including PC1, PC2, PACE4, PC4, furin, PC5A and PC5B, and PC7).17,18 These enzymes activate precursor proteins via endoproteolytic cleavage at di-basic amino acid residues.17,18 Each of these PCs, alone or in combination with others, has its own set of substrates, and substrate specificity is further determined by differences in organ, cell, and subcellular distribution. Whereas PC4 is exclusively confined to germinal cells19 and PC1 and PC2 are mostly found in secretory granules of neuroendocrine cells, furin and PC5 display a more widespread cell and tissue distribution.17,18 Recently, we reported upregulation of PC5 in the neointima after balloon injury in rodents20 and demonstrated that integrin v endoproteolytic activation by PC5 is required for v-dependent outside-in signaling and subsequent activation of tyrosine kinases in VSMCs.21 However, we21 and others22,23 did not find inhibition of MT1-MMP–dependent pro-MMP-2 activation in VSMCs after inhibition of PCs. Indeed, a cell type–specific requirement of furin-like PCs for MT1-MMP activation has been suggested,24 and MT1-MMP may potentially function as a self-convertase.25 Thus, the present study was done to investigate the function and regulation of furin-like PCs for the MT1-MMP–MMP-2 proteolytic cascade in mononuclear inflammatory cells.
Methods
Materials
Cell culture medium and materials were from Invitrogen. The broad-spectrum hydroxamate class MMP-inhibitor GM6001 (Ilomastat) was purchased from Chemicon, and the furin-like PC inhibitor decanoyl-RVKR-chloromethylketone (dec-CMK) from Bachem. All other chemicals were from Sigma. TNF- was from TEBU, and bacterial lipopolysaccharides (LPS) (from Salmonella enteriditis) were from Sigma. Recombinant TIMP-2 was from Chemicon. The following primary antibodies were used: -smooth muscle actin (-SMA), actin, and TIMP-2 from Sigma. Antibodies to CD31 and CD68 were from Dako. The MT1-MMP antibody directed to the hinge region (AB815) was from Chemicon. Antibodies to furin and PC5 were from the Clinical Research Institute.
Cell Cultures
The human monocytic THP-1 cell line was obtained from Deutsche Sammlung von Mikroorganismen und Zellkulturen (Germany). Cells were routinely propagated in RPMI 1640, supplemented with 10% FCS, 2 mmol/L L-glutamine, 100 U/mL penicillin, and 100 μg/mL streptomycin at 95% relative humidity and 5% CO2 at 37°C. Differentiation of THP-1 monocytes to macrophages (THP/M) occurred in the presence of 100 nmol/L phorbol 12-myristate 13-acetate (PMA) for 48 hours.26 After differentiation to THP/M, cells were washed, PMA was removed, and further experiments were done in serum-free medium. Cells were cultured in VSMC-conditioned medium with or without dec-CMK (50 μmol/L), TIMP-2 (200 ng/mL), or GM6001 (50 μmol/L) for 24 hours. VSMCs were cultured as described previously,20 and conditioned medium was obtained after 48-hour culture in serum-free DMEM. Cell viability was monitored by propidium iodide analysis and trypan blue exclusion. Experiments were done at least in triplicate.
Small Interfering RNA
Small interfering RNA (siRNA) for furin and a nonsilencing control were purchased from Qiagen. The following furin sequence was used: sense 5' GGACUAAACGGGACGUGUA 3'; antisense 5' UACACGUCCCGUUUAGUCC 3'. After macrophage differentiation, transfections were performed with siRNA (100 nmol/L) with the use of OligofectAMINE (GIBCO), as recommended by the manufacturer. Twenty-four hours after transfection, cells were used in experiments.
Immunoblotting
Proteins were extracted in a 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 Na3VO4) containing freshly dissolved protease inhibitors (Complete EDTA-free, Boehringer). Up to 50 μg of proteins was then subjected to 10% reducing SDS-PAGE as described previously.20 Semiquantitative densitometry was done with the use of the National Institutes of Health program 1.62 and is expressed in arbitrary units.
Gelatin Zymography
Conditioned media were mixed 1:1 with 2x Novex buffer (Invitrogen) without reducing agents. Samples were electrophoresed in 10% SDS-PAGE containing 0.1% gelatin. After migration, gels were renatured by exchanging SDS with Triton X-100 (2.5%) and then incubated for 24 hours at 37° in activation buffer (50 mmol/L Tris, pH 7.6, 5 mmol/L CaCl2, 0.2 mol/L NaCl, and 0.02% Brij). Gels were subsequently stained with Coomassie staining solution (0.5% Coomassie R250, 30% MeOH, 10% acetic acid) for 2 hours, followed by destaining (50% MeOH and 10% acetic acid). Supernatants from the human fibrosarcoma cell line (HT1080) were used as standard.
Flow Cytometry Analysis
Cells (200 000 cells per sample) or 100 μL of freshly drawn heparinized blood (obtained from healthy volunteers) was washed once with PBS/0.2% NaN3/0.15% BSA/1 mg/mL hIgG (500g, 5 minutes, 5°C) with a final volume of 300 μL. After addition of 2.5 μg control rabbit IgG (Sigma, I-5006) or specific antibody, samples were incubated for 30 minutes at 5°C and washed again, as described above. Then 8.4 μg goat anti-rabbit IgG(H+L)-FITC (Vector; # FI-1000) was added to both samples, followed by incubation for 30 minutes at 5°C in the dark. In experiments with samples from human blood, fixation of peripheral blood mononuclear cells with simultaneous lysis of erythrocytes was achieved by 10-minute incubation at 5°C with FACS Lysing Solution (BD Biosciences). Finally, samples were washed once with PBS/0.2% NaN3/0.15% BSA/1 mg/mL hIgG (500g, 5 minutes, 5°C). The fluorescence of the cells was measured with the FACScan (BD Biosciences) in the fluorescence-1 channel (20 000 events per sample). Generation of the fluorescence histograms and calculation of the median and mean fluorescence values of the different populations were done with the use of WinMDI 2.8 software after gating in experiments with samples from human blood after gating for lymphocytes (4800 per sample), granulocytes (11 000/sample), and monocytes (900 per sample).
Immunohistochemistry and Immunofluorescence
Fifteen human atherosclerotic carotid artery plaques obtained by endarterectomy were investigated. Single- and double-label immunohistochemical stainings were performed as described recently.21 Specificity controls were done by omission of the primary antibody and/or incubation with nonimmune IgG. Immunofluorescence analysis of CD68 was done as described.20 Microscopy was performed with an Olympus BX61 with the use of analySIS Imagine software.
Statistical Analysis
Data are expressed as mean±SD. Comparison between groups was performed with the use of 1-way ANOVA followed by Bonferroni/Dunn multiple comparison test. Statistical significance was designated at a probability value of <0.05.
Results
Cell Differentiation of Human Monocytic THP-1 Cells to Macrophages Is Characterized by Increased Expression of Furin, PC5, and MT1-MMP
To investigate the regulation of furin-like PCs and MT1-MMP during monocyte to macrophage differentiation, we used THP-1–derived macrophages, which behave comparably to native human monocyte-derived macrophages.26,27 Macrophage maturation was achieved by phorbol ester (PMA) treatment (48 hours; 100 nmol/L)26; PMA was then removed, and cells were cultivated for an additional 24 hours. Macrophage phenotype was assessed by typical morphology of adherent cells and immunofluorescence analysis of CD68 expression (data not shown) as well as Western blot analysis of vimentin, a marker protein of macrophage differentiation28 (Figure 1A). Immunoblotting revealed that differentiation of monocytes to macrophages was accompanied by increased expression of furin and PC5 (Figure 1B). Likewise, MT1-MMP was upregulated after macrophage maturation. Whereas monocytes displayed mostly pro-MT1-MMP (68 kDa), increased levels of the mature (63-kDa) MT1-MMP were detected in macrophages (Figure 1C). This was accompanied by increased levels of the physiological MMP inhibitor TIMP-2 (21 kDa) (Figure 1C). Flow cytometry demonstrated the cell surface expression of furin and PC5 on macrophages (Figure 1D, 1E).
Furin-like Proprotein Convertases Are Required for MT1-MMP Activation in Human THP-1/Macrophages and Regulate Pro-MMP-2 Activation Derived From VSMCs
The specific pharmacological furin-like PC inhibitor dec-CMK29 (50 μmol/L, 24 hours) was used to investigate the function of furin/PC5 in THP/M. Whereas MT1-MMP was almost entirely present in its cleaved, mature 63-kDa form in THP/M, dec-CMK significantly inhibited MT1-MMP maturation, evident by the increases of the 68-kDa pro-MT1-MMP (Figure 2A; densitometry of the percentage of pro- (black) and active (white) MT1-MMP equilibrated to actin reblotting is depicted in Figure 2B). Whereas flow cytometry demonstrated increased cell surface expression of MT1-MMP on monocyte to macrophage differentiation (Figure 2C, 2D), dec-CMK did not affect MT1-MMP cell surface expression (Figure 2E). Furthermore, incubation of THP-1 monocytes with dec-CMK did not affect PMA-induced monocyte to macrophage maturation (Figure 2F, 2G, 2H).
Regulation of MMP-2 and MMP-9 activity was further investigated by gelatin zymography. All experiments were done in serum-free medium because FCS contains large amounts of pro-MMP-2 (data not shown). Whereas monocytes expressed little pro-MMP-2 and pro-MMP-9, pro-MMP-9 synthesis was greatly increased on macrophage maturation. MMP-2 was entirely present in its latent form and did not increase in monocytes, but some increase and activation of pro-MMP-2 (72 kDa) to its intermediate form (68 kDa) was evident in macrophages (Figure 3A). Activation of the 72-kDa pro-MMP-2 requires MT1-MMP, which activates it to a 68-kDa intermediate MMP-2, which then autocatalytically converts to its 62-kDa form.30,31 In human monocyte-derived macrophages, MT1-MMP has been reported to be increased after TNF- or ox-LDL stimulation.6 We investigated the effects of TNF- or LPS on pro-MMP-2 activation, the indicator of MT1-MMP activity. Neither TNF- (10 ng/mL; 24 hours) nor LPS (1 μg/mL; 24 hours) affected pro-MMP-2 activation in monocytes, but both increased pro-MMP-9 synthesis in these cells (Figure 3B). In macrophages, pro-MMP-2 level/activation (Figure 3B, bottom) or pro-MMP-9 synthesis did not increase further (data not shown). However, pro-MMP-2 is known to be constitutively secreted by VSMCs.32 Thus, we tested the hypothesis that activation of pro-MMP-2 from other cells is a function of macrophage MT1-MMP. Therefore, we cultivated (24-hour) monocytes or macrophages in conditioned medium from serum-starved VSMCs. No pro-MMP-2 activation was found in monocytes, but in macrophages strong activation of VSMC-derived pro-MMP-2 to its intermediate and fully active form was evident (Figure 3C, 3D).
To investigate the function of furin-like PCs in this proteolytic cascade, we inhibited furin/PC5 with dec-CMK or used a furin-specific siRNA. Macrophages were successfully transfected with siRNA (Figure 4A, 4B; transfection rate 60%), and immunoblotting demonstrated the reduction of furin protein levels with the furin-specific siRNA (Figure 4C, 4D; P<0.05 versus controls). We found that VSMC-derived pro-MMP-2 activation by macrophages MT1-MMP was virtually abolished by dec-CMK (50 μmol/L, 24 hours), comparable to the MMP inhibitor GM6001 (50 μmol/L, 24 hours) or the addition of an excess of recombinant TIMP-2 (200 ng/mL, 24 hours; all P<0.05 versus controls). Furthermore, silencing furin with a furin-sepcific siRNA (100 nmol/L) significantly inhibited pro-MMP-2 activation (P<0.05 versus controls), whereas the non-furin silencing control siRNA (100 nmol/L) had no effect (Figure 4E, 4F).
Furin, PC5, and MT1-MMP Are Expressed on Human Peripheral Blood Mononuclear Cells and Colocalize With Macrophages/Foam Cells in Carotid Atherosclerotic Plaques
Fifteen human carotid plaques obtained by endarterectomy were investigated. Figure 5A shows a typical atheromatous plaque with lipid-laden macrophages, mononuclear inflammatory cells, and adjacent fibrous tissue (hematoxylin-phloxine saffron staining). In the plaque shoulder, single-label immunohistochemistry demonstrates CD68-positive macrophages (inset; brown staining), and these cells express PC5 (Figure 5B; blue). Furthermore, PC5 (blue) colocalized with CD68-positive macrophages (brown; Figure 4C and inset) and was coexpressed with MT1-MMP (brown; PC5=blue) in macrophages in the plaque shoulder (Figure 5D). Furin (brown) was also found in CD68-positive macrophages (blue) and colocalized with MT1-MMP as well (data not shown). Negative control by omission of the primary antibody demonstrated no significant staining (Figure 5F).
To investigate the expression of furin, PC5, and their substrate MT1-MMP on human peripheral blood mononuclear cells, flow cytometry was done with blood obtained from healthy volunteers. Both furin and PC5 were significantly expressed on human monocytes and granulocytes, but only slight cell surface expression of furin and PC5 was detectable on lymphocytes. Comparable cell surface expression of their substrate MT1-MMP was found on human monocytes and granulocytes, with little expression on lymphocytes (Table).
Flow Cytometry Analysis of Furin, PC5, and MT1-MMP in Peripheral Blood Mononuclear Cells
Discussion
The present study demonstrates that activation of MT1-MMP, a membrane-bound MMP that activates pro-MMP-2, is regulated by furin-like PCs in macrophages. Our study shows that furin-like PCs control a proteolytic activation cascade, initiated by macrophage MT1-MMP activating pro-MMP-2 provided by other cell types.
Mononuclear cells are major contributors to the chronic inflammatory process in atherosclerosis.33 Macrophage maturation occurs concomitant to monocyte transmigration of the endothelial cell barrier and renders the cells ready for active participation in tissue inflammation.34 Using the human monocytic THP-1 cell line to investigate macrophage maturation,26,27 we demonstrate that monocyte to macrophage differentiation is characterized by increased levels of furin, PC5, and MT1-MMP.
Among the MMPs, MT-MMPs are unique in that they are activated intracellularly and then anchored to the cell membrane, which targets their activity to the pericellular space.11,15 MT1- and MT3-MMP are found in human atherosclerotic lesions, localizing predominantly to VSMCs in the media and macrophages within the plaque.6,7 MMP activity may not only contribute to weakening of the plaque cap via cleavage of the ECM but could also determine lesion stability through the cleavage of nonmatrix substrates, including growth factors, cytokines, and cell surface receptors.2,11 Increased macrophage density and MMP gelatinolytic activity have been reported at rupture-prone sites in atherosclerotic lesions,4 and levels of MMP-2 (gelatinase A) and MMP-9 (gelatinase B) are reported to be increased in patients with acute coronary syndromes.35 In contrast to MMP-9, MMP-2 may directly contribute to lesion instability via its ability to readily degrade collagen type I,36 a major constituent of the plaque.37
However, macrophages synthesize little MMP-2 but large amounts of pro-MMP-9,12,14,38 whereas VSMCs constitutively secrete latent MMP-2,32 potentially stored in the matrix.39 Pro-MMP-2 is not readily induced by cytokines that regulate other MMPs, and thus latent MMP-2 activation is an important step in controlling its activity.15 Whereas pro-MMP-9 is activated extracellularly by plasmin and other serine proteases,11 pro-MMP-2 activation depends on the formation of a MT1-MMP/pro-MMP-2/TIMP-2 trimer at the cell surface.30,31 Apart from MT4-MMP,40 all of the 6 identified MT-MMPs can activate pro-MMP-2; however, MT5-MMP is brain specific and MT6-MMP is primarily found in peripheral blood leukocytes and cancer cells.11
Comparable to human macrophages,12,14,38 monocytic cell line–derived macrophages express primarily pro-MMP-9.14,41 MT1-MMP, the major pro-MMP-2 activator, increases in human macrophages after TNF- or ox-LDL stimulation.6 We found that TNF- or LPS stimulated pro-MMP-9 synthesis in THP-1 monocytes and that macrophages abundantly expressed pro-MMP-9. MMP-2 was entirely present in its latent form in monocytes, but some increase/activation of pro-MMP-2 was evident in MT1-MMP–expressing macrophages. Still, levels of pro-MMP-2 or its activation could not be further increased by TNF- or LPS stimulation of macrophages. Thus, we tested the hypothesis that activation of pro-MMP-2, potentially provided by neighboring cells, such as VSMCs,32 is a major function of macrophage MT1-MMP. Culturing of THP-1 macrophages in VSMC-conditioned medium led to a strong pro-MMP-2 activation, whereas no pro-MMP-2 activation was found when monocytes were cultured in VSMC-conditioned medium. This demonstrates that pro-MMP-2 activation derived from VSMCs depends on MT1-MMP provided by macrophages.
Regulation of the prototype MT1-MMP activity involves trafficking, internalization, and shedding as well as inhibition by TIMPs, but zymogen activation remains a critical step.11,15 Reconstituting furin with MT1-MMP mutants in furin-deficient cell lines, Yana and Weiss16 demonstrated that activation of pro-MT1-MMP depends on endoproteolytic cleavage of its pro-peptide at a RRKR111 motif by furin-like convertases. Furin is the prototype convertase found within the secretory pathway, which cleaves precursor proteins with narrow specificity following di-basic residues.17,18 However, some MT1-MMP maturation occurs in furin-deficient cell lines,16 indicating that other PCs potentially substitute. We have recently demonstrated that activation of MT1-MMP occurs most likely in the trans-Golgi network,42 where furin, PC5, and PC7 are localized.17,18 Comparable to furin, PC5 can process RXKR-, RXXR-, and KXXR-containing sites,17,18 whereas PC7 requires R or K at P6 for optimal activity,43 which is not found in the MT1-MMP pro-peptide.16 Thus, furin and PC5, which cycle between the trans-Golgi network and the cell surface,17,18 are likely the major MT1-MMP convertases.
To investigate the role of these PCs for MT1-MMP activation in macrophages, we used the specific pharmacological furin-like PC inhibitor dec-CMK.29 Incubation of THP-1 macrophages with dec-CMK inhibited MT1-MMP maturation from its pro- to active form and virtually abolished VSMC-derived pro-MMP-2 activation, the indicator of MT1-MMP activity. This inhibition was comparable to the MMP inhibitor GM6001 or the use of recombinant TIMP-2, a natural MMP inhibitor.11 However, flow cytometry revealed that inhibition of MT1-MMP maturation did not affect its sorting/routing to the cell membrane. Still, this uncleaved but membrane-expressed MT1-MMP is ineffective in pro-MMP-2 activation, presumably because of impaired TIMP-2 binding,44 mandatory for pro-MMP-2 activation.45 Because dec-CMK inhibits furin and PC5, we transfected macrophages with a furin-specific siRNA. In contrast to the complete inhibition of MT1-MMP–dependent pro-MMP-2 activation by dec-CMK, silencing furin with siRNA resulted in a significant but incomplete inhibition of pro-MMP-2 activation. This may be attributable to an incomplete transfection rate but may also point out that other PC isozymes, most likely PC5, are required for MT1-MMP activation in macrophages. Our study also demonstrates that furin, PC5, and MT1-MMP are expressed on human peripheral blood mononuclear cells, mainly monocytes, and colocalize with MT1-MMP in CD68-positive macrophages/foam cells in human endarterectomy plaques, supporting their in vivo participation in MT1-MMP maturation.
Taken together, our study demonstrates that the furin-like PCs furin and PC5 are potential targets to modulate the initiation of a macrophage MT-MMP–driven proteolytic cascade of pro-MMP-2 activation derived from other cells, which may be important for the progression of atherosclerosis.
Acknowledgments
This work was supported by a grant from the Bundesministerium für Bildung und Forschung (CAN02/005) to P.S. and E.F. and an independent research grant from Philip Morris Inc, Virginia to P.S., E.F., and K.G. J.P.V. and M.C. were supported by the Heart and Stroke Foundation of Ontario (NA4337 and T4891) and the Canadian Stroke Network. N.G.S. was supported by the Canadian Institute of Health Research (MGP-44363). The authors thank W. Prichett-Pejic (Ottawa) for technical assistance with immunohistochemistry.
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Diseases of Aging and Regional Protein Chemistry Centers, Ottawa Health Research Institute, University of Ottawa, Ottawa, Ontario, Canada (J.P.V., M.C.)
Laboratory of Biochemical Neuroendocrinology, Clinical Research Institute, Montreal, Quebec, Canada (N.G.S.).
Abstract
Background— Accumulation of macrophages and their in situ expression of matrix metalloproteinases (MMPs) are important determinants of plaque stability. Activation of membrane-bound MT1-MMP, the major activator of pro-MMP-2, requires intracellular endoproteolytic cleavage of its precursor protein. This type of activation typically requires suitable furin-like proprotein convertases (PCs), specifically furin and PC5. The present study was done to investigate the function of MT1-MMP as well as furin-like PCs in mononuclear inflammatory cells.
Methods and Results— Macrophage differentiation of human monocytic THP-1 cells was accompanied by increased expression of furin, PC5, and MT1-MMP. Some pro-MMP-2 activation was found in macrophages, but pro-MMP-2 level or activation was not enhanced after stimulation with the proinflammatory mediators tumor necrosis factor- or lipopolysaccharide. However, culturing of macrophages in conditioned medium from serum-starved vascular smooth muscle cells, which constitutively secrete pro-MMP-2, resulted in a strong pro-MMP-2 activation. Inhibition of furin-like PCs with the specific pharmacological inhibitor decanoyl-RVKR-chloromethylketone (dec-CMK) inhibited MT1-MMP activation in macrophages. Dec-CMK or furin-specific small interfering RNA significantly inhibited macrophage MT1-MMP–dependent activation of vascular smooth muscle cell–derived pro-MMP-2. Flow cytometry demonstrated that human circulating monocytes express furin and PC5, and MT1-MMP and immunohistochemistry revealed their colocalization in macrophages in advanced human atherosclerotic lesions.
Conclusions— Furin-like PCs (furin and PC5) play a central role in a MT-MMP–MMP-2 proteolytic cascade, involving provision of macrophage MT1-MMP for the activation of pro-MMP-2 synthesized by other cells. Furin and PC5 are expressed in human peripheral blood mononuclear cells and colocalize with MT1-MMP in macrophages in the atherosclerotic plaque, supporting the hypothesis that they are potential targets in atherosclerosis.
Key Words: metalloproteinases ; atherosclerosis ; inflammation ; proprotein convertases
Introduction
Expression of matrix metalloproteinases (MMPs) by monocytes/macrophages is a key mechanism for the initiation, progression, and complications of atherosclerosis.1,2 MMPs facilitate mononuclear cell entry of the vessel wall, which is important for the chronic inflammatory response in atherosclerosis,1 as well as vascular smooth muscle cell (VSMC) migration/invasion, which is required for neointima formation and restenosis.3 Rupture-prone atherosclerotic lesions are characterized by accumulation of macrophages and their in situ expression of MMP activity,4 likely contributing to weakening of the plaque cap through the breakdown of the extracellular matrix (ECM).5 In mononuclear cells, MMPs and their tissue inhibitor of metalloproteinases (TIMPs) are regulated by proinflammatory mediators, such as interleukin-1, tumor necrosis factor- (TNF-), and oxidized LDL (ox-LDL).6–8 Regulation of MMPs in mononuclear cells involves prostaglandin E2/cAMP-dependent signaling,9 and secretion of MMPs from macrophages/foam cells is inhibited by statins in vitro and in vivo.10
The family of mammalian MMPs now includes >20 members, with overlapping cleavage activity toward the pericellular matrix (eg, laminin, vitronectin) and ECM.11 According to their mode of activation, MMPs can be grouped into soluble MMPs and membrane-bound MT-MMPs. Monocytes/macrophages express soluble MMPs (eg, MMP-1, -3, -9)4,10,12–14 and MT-MMPs.6,7 Soluble MMPs (eg, MMP-2, MMP-9) are released from the cells as zymogens and require extracellular posttranslational cleavage to gain biological activity.11 In contrast, MT-MMPs are activated intracellularly and then tethered to the cell surface as active enzymes.11,15 Moreover, MT-MMPs amplify a proteolytic cascade in that they are the major pro-MMP-2 activators.11,15 In human atherosclerotic lesions, MT-MMPs are found in the media underlying fibrous and lipid regions, localizing to VSMCs and macrophages.6,7
Activation of the archetypical MT1-MMP has been demonstrated to require endoproteolytic cleavage by furin-like proprotein convertases (PCs).16 Furin is the prototypical member of the 7 thus far identified mammalian subtilisin/kexin-like serine proteinases (including PC1, PC2, PACE4, PC4, furin, PC5A and PC5B, and PC7).17,18 These enzymes activate precursor proteins via endoproteolytic cleavage at di-basic amino acid residues.17,18 Each of these PCs, alone or in combination with others, has its own set of substrates, and substrate specificity is further determined by differences in organ, cell, and subcellular distribution. Whereas PC4 is exclusively confined to germinal cells19 and PC1 and PC2 are mostly found in secretory granules of neuroendocrine cells, furin and PC5 display a more widespread cell and tissue distribution.17,18 Recently, we reported upregulation of PC5 in the neointima after balloon injury in rodents20 and demonstrated that integrin v endoproteolytic activation by PC5 is required for v-dependent outside-in signaling and subsequent activation of tyrosine kinases in VSMCs.21 However, we21 and others22,23 did not find inhibition of MT1-MMP–dependent pro-MMP-2 activation in VSMCs after inhibition of PCs. Indeed, a cell type–specific requirement of furin-like PCs for MT1-MMP activation has been suggested,24 and MT1-MMP may potentially function as a self-convertase.25 Thus, the present study was done to investigate the function and regulation of furin-like PCs for the MT1-MMP–MMP-2 proteolytic cascade in mononuclear inflammatory cells.
Methods
Materials
Cell culture medium and materials were from Invitrogen. The broad-spectrum hydroxamate class MMP-inhibitor GM6001 (Ilomastat) was purchased from Chemicon, and the furin-like PC inhibitor decanoyl-RVKR-chloromethylketone (dec-CMK) from Bachem. All other chemicals were from Sigma. TNF- was from TEBU, and bacterial lipopolysaccharides (LPS) (from Salmonella enteriditis) were from Sigma. Recombinant TIMP-2 was from Chemicon. The following primary antibodies were used: -smooth muscle actin (-SMA), actin, and TIMP-2 from Sigma. Antibodies to CD31 and CD68 were from Dako. The MT1-MMP antibody directed to the hinge region (AB815) was from Chemicon. Antibodies to furin and PC5 were from the Clinical Research Institute.
Cell Cultures
The human monocytic THP-1 cell line was obtained from Deutsche Sammlung von Mikroorganismen und Zellkulturen (Germany). Cells were routinely propagated in RPMI 1640, supplemented with 10% FCS, 2 mmol/L L-glutamine, 100 U/mL penicillin, and 100 μg/mL streptomycin at 95% relative humidity and 5% CO2 at 37°C. Differentiation of THP-1 monocytes to macrophages (THP/M) occurred in the presence of 100 nmol/L phorbol 12-myristate 13-acetate (PMA) for 48 hours.26 After differentiation to THP/M, cells were washed, PMA was removed, and further experiments were done in serum-free medium. Cells were cultured in VSMC-conditioned medium with or without dec-CMK (50 μmol/L), TIMP-2 (200 ng/mL), or GM6001 (50 μmol/L) for 24 hours. VSMCs were cultured as described previously,20 and conditioned medium was obtained after 48-hour culture in serum-free DMEM. Cell viability was monitored by propidium iodide analysis and trypan blue exclusion. Experiments were done at least in triplicate.
Small Interfering RNA
Small interfering RNA (siRNA) for furin and a nonsilencing control were purchased from Qiagen. The following furin sequence was used: sense 5' GGACUAAACGGGACGUGUA 3'; antisense 5' UACACGUCCCGUUUAGUCC 3'. After macrophage differentiation, transfections were performed with siRNA (100 nmol/L) with the use of OligofectAMINE (GIBCO), as recommended by the manufacturer. Twenty-four hours after transfection, cells were used in experiments.
Immunoblotting
Proteins were extracted in a 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 Na3VO4) containing freshly dissolved protease inhibitors (Complete EDTA-free, Boehringer). Up to 50 μg of proteins was then subjected to 10% reducing SDS-PAGE as described previously.20 Semiquantitative densitometry was done with the use of the National Institutes of Health program 1.62 and is expressed in arbitrary units.
Gelatin Zymography
Conditioned media were mixed 1:1 with 2x Novex buffer (Invitrogen) without reducing agents. Samples were electrophoresed in 10% SDS-PAGE containing 0.1% gelatin. After migration, gels were renatured by exchanging SDS with Triton X-100 (2.5%) and then incubated for 24 hours at 37° in activation buffer (50 mmol/L Tris, pH 7.6, 5 mmol/L CaCl2, 0.2 mol/L NaCl, and 0.02% Brij). Gels were subsequently stained with Coomassie staining solution (0.5% Coomassie R250, 30% MeOH, 10% acetic acid) for 2 hours, followed by destaining (50% MeOH and 10% acetic acid). Supernatants from the human fibrosarcoma cell line (HT1080) were used as standard.
Flow Cytometry Analysis
Cells (200 000 cells per sample) or 100 μL of freshly drawn heparinized blood (obtained from healthy volunteers) was washed once with PBS/0.2% NaN3/0.15% BSA/1 mg/mL hIgG (500g, 5 minutes, 5°C) with a final volume of 300 μL. After addition of 2.5 μg control rabbit IgG (Sigma, I-5006) or specific antibody, samples were incubated for 30 minutes at 5°C and washed again, as described above. Then 8.4 μg goat anti-rabbit IgG(H+L)-FITC (Vector; # FI-1000) was added to both samples, followed by incubation for 30 minutes at 5°C in the dark. In experiments with samples from human blood, fixation of peripheral blood mononuclear cells with simultaneous lysis of erythrocytes was achieved by 10-minute incubation at 5°C with FACS Lysing Solution (BD Biosciences). Finally, samples were washed once with PBS/0.2% NaN3/0.15% BSA/1 mg/mL hIgG (500g, 5 minutes, 5°C). The fluorescence of the cells was measured with the FACScan (BD Biosciences) in the fluorescence-1 channel (20 000 events per sample). Generation of the fluorescence histograms and calculation of the median and mean fluorescence values of the different populations were done with the use of WinMDI 2.8 software after gating in experiments with samples from human blood after gating for lymphocytes (4800 per sample), granulocytes (11 000/sample), and monocytes (900 per sample).
Immunohistochemistry and Immunofluorescence
Fifteen human atherosclerotic carotid artery plaques obtained by endarterectomy were investigated. Single- and double-label immunohistochemical stainings were performed as described recently.21 Specificity controls were done by omission of the primary antibody and/or incubation with nonimmune IgG. Immunofluorescence analysis of CD68 was done as described.20 Microscopy was performed with an Olympus BX61 with the use of analySIS Imagine software.
Statistical Analysis
Data are expressed as mean±SD. Comparison between groups was performed with the use of 1-way ANOVA followed by Bonferroni/Dunn multiple comparison test. Statistical significance was designated at a probability value of <0.05.
Results
Cell Differentiation of Human Monocytic THP-1 Cells to Macrophages Is Characterized by Increased Expression of Furin, PC5, and MT1-MMP
To investigate the regulation of furin-like PCs and MT1-MMP during monocyte to macrophage differentiation, we used THP-1–derived macrophages, which behave comparably to native human monocyte-derived macrophages.26,27 Macrophage maturation was achieved by phorbol ester (PMA) treatment (48 hours; 100 nmol/L)26; PMA was then removed, and cells were cultivated for an additional 24 hours. Macrophage phenotype was assessed by typical morphology of adherent cells and immunofluorescence analysis of CD68 expression (data not shown) as well as Western blot analysis of vimentin, a marker protein of macrophage differentiation28 (Figure 1A). Immunoblotting revealed that differentiation of monocytes to macrophages was accompanied by increased expression of furin and PC5 (Figure 1B). Likewise, MT1-MMP was upregulated after macrophage maturation. Whereas monocytes displayed mostly pro-MT1-MMP (68 kDa), increased levels of the mature (63-kDa) MT1-MMP were detected in macrophages (Figure 1C). This was accompanied by increased levels of the physiological MMP inhibitor TIMP-2 (21 kDa) (Figure 1C). Flow cytometry demonstrated the cell surface expression of furin and PC5 on macrophages (Figure 1D, 1E).
Furin-like Proprotein Convertases Are Required for MT1-MMP Activation in Human THP-1/Macrophages and Regulate Pro-MMP-2 Activation Derived From VSMCs
The specific pharmacological furin-like PC inhibitor dec-CMK29 (50 μmol/L, 24 hours) was used to investigate the function of furin/PC5 in THP/M. Whereas MT1-MMP was almost entirely present in its cleaved, mature 63-kDa form in THP/M, dec-CMK significantly inhibited MT1-MMP maturation, evident by the increases of the 68-kDa pro-MT1-MMP (Figure 2A; densitometry of the percentage of pro- (black) and active (white) MT1-MMP equilibrated to actin reblotting is depicted in Figure 2B). Whereas flow cytometry demonstrated increased cell surface expression of MT1-MMP on monocyte to macrophage differentiation (Figure 2C, 2D), dec-CMK did not affect MT1-MMP cell surface expression (Figure 2E). Furthermore, incubation of THP-1 monocytes with dec-CMK did not affect PMA-induced monocyte to macrophage maturation (Figure 2F, 2G, 2H).
Regulation of MMP-2 and MMP-9 activity was further investigated by gelatin zymography. All experiments were done in serum-free medium because FCS contains large amounts of pro-MMP-2 (data not shown). Whereas monocytes expressed little pro-MMP-2 and pro-MMP-9, pro-MMP-9 synthesis was greatly increased on macrophage maturation. MMP-2 was entirely present in its latent form and did not increase in monocytes, but some increase and activation of pro-MMP-2 (72 kDa) to its intermediate form (68 kDa) was evident in macrophages (Figure 3A). Activation of the 72-kDa pro-MMP-2 requires MT1-MMP, which activates it to a 68-kDa intermediate MMP-2, which then autocatalytically converts to its 62-kDa form.30,31 In human monocyte-derived macrophages, MT1-MMP has been reported to be increased after TNF- or ox-LDL stimulation.6 We investigated the effects of TNF- or LPS on pro-MMP-2 activation, the indicator of MT1-MMP activity. Neither TNF- (10 ng/mL; 24 hours) nor LPS (1 μg/mL; 24 hours) affected pro-MMP-2 activation in monocytes, but both increased pro-MMP-9 synthesis in these cells (Figure 3B). In macrophages, pro-MMP-2 level/activation (Figure 3B, bottom) or pro-MMP-9 synthesis did not increase further (data not shown). However, pro-MMP-2 is known to be constitutively secreted by VSMCs.32 Thus, we tested the hypothesis that activation of pro-MMP-2 from other cells is a function of macrophage MT1-MMP. Therefore, we cultivated (24-hour) monocytes or macrophages in conditioned medium from serum-starved VSMCs. No pro-MMP-2 activation was found in monocytes, but in macrophages strong activation of VSMC-derived pro-MMP-2 to its intermediate and fully active form was evident (Figure 3C, 3D).
To investigate the function of furin-like PCs in this proteolytic cascade, we inhibited furin/PC5 with dec-CMK or used a furin-specific siRNA. Macrophages were successfully transfected with siRNA (Figure 4A, 4B; transfection rate 60%), and immunoblotting demonstrated the reduction of furin protein levels with the furin-specific siRNA (Figure 4C, 4D; P<0.05 versus controls). We found that VSMC-derived pro-MMP-2 activation by macrophages MT1-MMP was virtually abolished by dec-CMK (50 μmol/L, 24 hours), comparable to the MMP inhibitor GM6001 (50 μmol/L, 24 hours) or the addition of an excess of recombinant TIMP-2 (200 ng/mL, 24 hours; all P<0.05 versus controls). Furthermore, silencing furin with a furin-sepcific siRNA (100 nmol/L) significantly inhibited pro-MMP-2 activation (P<0.05 versus controls), whereas the non-furin silencing control siRNA (100 nmol/L) had no effect (Figure 4E, 4F).
Furin, PC5, and MT1-MMP Are Expressed on Human Peripheral Blood Mononuclear Cells and Colocalize With Macrophages/Foam Cells in Carotid Atherosclerotic Plaques
Fifteen human carotid plaques obtained by endarterectomy were investigated. Figure 5A shows a typical atheromatous plaque with lipid-laden macrophages, mononuclear inflammatory cells, and adjacent fibrous tissue (hematoxylin-phloxine saffron staining). In the plaque shoulder, single-label immunohistochemistry demonstrates CD68-positive macrophages (inset; brown staining), and these cells express PC5 (Figure 5B; blue). Furthermore, PC5 (blue) colocalized with CD68-positive macrophages (brown; Figure 4C and inset) and was coexpressed with MT1-MMP (brown; PC5=blue) in macrophages in the plaque shoulder (Figure 5D). Furin (brown) was also found in CD68-positive macrophages (blue) and colocalized with MT1-MMP as well (data not shown). Negative control by omission of the primary antibody demonstrated no significant staining (Figure 5F).
To investigate the expression of furin, PC5, and their substrate MT1-MMP on human peripheral blood mononuclear cells, flow cytometry was done with blood obtained from healthy volunteers. Both furin and PC5 were significantly expressed on human monocytes and granulocytes, but only slight cell surface expression of furin and PC5 was detectable on lymphocytes. Comparable cell surface expression of their substrate MT1-MMP was found on human monocytes and granulocytes, with little expression on lymphocytes (Table).
Flow Cytometry Analysis of Furin, PC5, and MT1-MMP in Peripheral Blood Mononuclear Cells
Discussion
The present study demonstrates that activation of MT1-MMP, a membrane-bound MMP that activates pro-MMP-2, is regulated by furin-like PCs in macrophages. Our study shows that furin-like PCs control a proteolytic activation cascade, initiated by macrophage MT1-MMP activating pro-MMP-2 provided by other cell types.
Mononuclear cells are major contributors to the chronic inflammatory process in atherosclerosis.33 Macrophage maturation occurs concomitant to monocyte transmigration of the endothelial cell barrier and renders the cells ready for active participation in tissue inflammation.34 Using the human monocytic THP-1 cell line to investigate macrophage maturation,26,27 we demonstrate that monocyte to macrophage differentiation is characterized by increased levels of furin, PC5, and MT1-MMP.
Among the MMPs, MT-MMPs are unique in that they are activated intracellularly and then anchored to the cell membrane, which targets their activity to the pericellular space.11,15 MT1- and MT3-MMP are found in human atherosclerotic lesions, localizing predominantly to VSMCs in the media and macrophages within the plaque.6,7 MMP activity may not only contribute to weakening of the plaque cap via cleavage of the ECM but could also determine lesion stability through the cleavage of nonmatrix substrates, including growth factors, cytokines, and cell surface receptors.2,11 Increased macrophage density and MMP gelatinolytic activity have been reported at rupture-prone sites in atherosclerotic lesions,4 and levels of MMP-2 (gelatinase A) and MMP-9 (gelatinase B) are reported to be increased in patients with acute coronary syndromes.35 In contrast to MMP-9, MMP-2 may directly contribute to lesion instability via its ability to readily degrade collagen type I,36 a major constituent of the plaque.37
However, macrophages synthesize little MMP-2 but large amounts of pro-MMP-9,12,14,38 whereas VSMCs constitutively secrete latent MMP-2,32 potentially stored in the matrix.39 Pro-MMP-2 is not readily induced by cytokines that regulate other MMPs, and thus latent MMP-2 activation is an important step in controlling its activity.15 Whereas pro-MMP-9 is activated extracellularly by plasmin and other serine proteases,11 pro-MMP-2 activation depends on the formation of a MT1-MMP/pro-MMP-2/TIMP-2 trimer at the cell surface.30,31 Apart from MT4-MMP,40 all of the 6 identified MT-MMPs can activate pro-MMP-2; however, MT5-MMP is brain specific and MT6-MMP is primarily found in peripheral blood leukocytes and cancer cells.11
Comparable to human macrophages,12,14,38 monocytic cell line–derived macrophages express primarily pro-MMP-9.14,41 MT1-MMP, the major pro-MMP-2 activator, increases in human macrophages after TNF- or ox-LDL stimulation.6 We found that TNF- or LPS stimulated pro-MMP-9 synthesis in THP-1 monocytes and that macrophages abundantly expressed pro-MMP-9. MMP-2 was entirely present in its latent form in monocytes, but some increase/activation of pro-MMP-2 was evident in MT1-MMP–expressing macrophages. Still, levels of pro-MMP-2 or its activation could not be further increased by TNF- or LPS stimulation of macrophages. Thus, we tested the hypothesis that activation of pro-MMP-2, potentially provided by neighboring cells, such as VSMCs,32 is a major function of macrophage MT1-MMP. Culturing of THP-1 macrophages in VSMC-conditioned medium led to a strong pro-MMP-2 activation, whereas no pro-MMP-2 activation was found when monocytes were cultured in VSMC-conditioned medium. This demonstrates that pro-MMP-2 activation derived from VSMCs depends on MT1-MMP provided by macrophages.
Regulation of the prototype MT1-MMP activity involves trafficking, internalization, and shedding as well as inhibition by TIMPs, but zymogen activation remains a critical step.11,15 Reconstituting furin with MT1-MMP mutants in furin-deficient cell lines, Yana and Weiss16 demonstrated that activation of pro-MT1-MMP depends on endoproteolytic cleavage of its pro-peptide at a RRKR111 motif by furin-like convertases. Furin is the prototype convertase found within the secretory pathway, which cleaves precursor proteins with narrow specificity following di-basic residues.17,18 However, some MT1-MMP maturation occurs in furin-deficient cell lines,16 indicating that other PCs potentially substitute. We have recently demonstrated that activation of MT1-MMP occurs most likely in the trans-Golgi network,42 where furin, PC5, and PC7 are localized.17,18 Comparable to furin, PC5 can process RXKR-, RXXR-, and KXXR-containing sites,17,18 whereas PC7 requires R or K at P6 for optimal activity,43 which is not found in the MT1-MMP pro-peptide.16 Thus, furin and PC5, which cycle between the trans-Golgi network and the cell surface,17,18 are likely the major MT1-MMP convertases.
To investigate the role of these PCs for MT1-MMP activation in macrophages, we used the specific pharmacological furin-like PC inhibitor dec-CMK.29 Incubation of THP-1 macrophages with dec-CMK inhibited MT1-MMP maturation from its pro- to active form and virtually abolished VSMC-derived pro-MMP-2 activation, the indicator of MT1-MMP activity. This inhibition was comparable to the MMP inhibitor GM6001 or the use of recombinant TIMP-2, a natural MMP inhibitor.11 However, flow cytometry revealed that inhibition of MT1-MMP maturation did not affect its sorting/routing to the cell membrane. Still, this uncleaved but membrane-expressed MT1-MMP is ineffective in pro-MMP-2 activation, presumably because of impaired TIMP-2 binding,44 mandatory for pro-MMP-2 activation.45 Because dec-CMK inhibits furin and PC5, we transfected macrophages with a furin-specific siRNA. In contrast to the complete inhibition of MT1-MMP–dependent pro-MMP-2 activation by dec-CMK, silencing furin with siRNA resulted in a significant but incomplete inhibition of pro-MMP-2 activation. This may be attributable to an incomplete transfection rate but may also point out that other PC isozymes, most likely PC5, are required for MT1-MMP activation in macrophages. Our study also demonstrates that furin, PC5, and MT1-MMP are expressed on human peripheral blood mononuclear cells, mainly monocytes, and colocalize with MT1-MMP in CD68-positive macrophages/foam cells in human endarterectomy plaques, supporting their in vivo participation in MT1-MMP maturation.
Taken together, our study demonstrates that the furin-like PCs furin and PC5 are potential targets to modulate the initiation of a macrophage MT-MMP–driven proteolytic cascade of pro-MMP-2 activation derived from other cells, which may be important for the progression of atherosclerosis.
Acknowledgments
This work was supported by a grant from the Bundesministerium für Bildung und Forschung (CAN02/005) to P.S. and E.F. and an independent research grant from Philip Morris Inc, Virginia to P.S., E.F., and K.G. J.P.V. and M.C. were supported by the Heart and Stroke Foundation of Ontario (NA4337 and T4891) and the Canadian Stroke Network. N.G.S. was supported by the Canadian Institute of Health Research (MGP-44363). The authors thank W. Prichett-Pejic (Ottawa) for technical assistance with immunohistochemistry.
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