By
From the * Vascular Medicine and Atherosclerosis Unit, Cardiovascular Division, Department of
Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts 02115; Ares Serono, 1228 Geneva, Switzerland; and the § Institut de Génétique et de Biologie Moléculaire
et Cellulaire, Centre National de la Recherche Scientifique/Institut National de la Santé et de la
Recherche Médicale/Université Louis Pasteur, BP163, 67404 Illkirch cedex, France
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Abstract |
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Stromelysin-3 is an unusual matrix metalloproteinase, being released in the active rather than
zymogen form and having a distinct substrate specificity, targeting serine proteinase inhibitors (serpins), which regulate cellular functions involved in atherosclerosis. We report here that human atherosclerotic plaques (n = 7) express stromelysin-3 in situ, whereas fatty streaks (n = 5)
and normal arterial specimens (n = 5) contain little or no stromelysin-3. Stromelysin-3 mRNA
and protein colocalized with endothelial cells, smooth muscle cells, and macrophages within
the lesion. In vitro, usual inducers of matrix metalloproteinases such as interleukin-1, interferon-, or tumor necrosis factor
did not augment stromelysin-3 in vascular wall cells. However, T cell-derived as well as recombinant CD40 ligand (CD40L, CD154), an inflammatory
mediator recently localized in atheroma, induced de novo synthesis of stromelysin-3. In addition, stromelysin-3 mRNA and protein colocalized with CD40L and CD40 within atheroma.
In accordance with the in situ and in vitro data obtained with human material, interruption of
the CD40-CD40L signaling pathway in low density lipoprotein receptor-deficient hyperlipidemic mice substantially decreased expression of the enzyme within atherosclerotic plaques.
These observations establish the expression of the unusual matrix metalloproteinase stromelysin-3 in human atherosclerotic lesions and implicate CD40-CD40L signaling in its regulation, thus
providing a possible new pathway that triggers complications within atherosclerotic lesions.
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Introduction |
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The proteinase stromelysin-3 (MMP-11) belongs to the matrix metalloproteinase (MMP)1 family, enzymes which participate in tissue remodeling in a variety of diseases, including tumor metastasis and invasion, arthritis, and atherogenesis (1). In contrast to "classical" MMPs, secreted as inactive zymogens, stromelysin-3 is released as an active enzyme (9, 10). The zymogen contains a unique 10- amino acid insert between the pro and catalytic domain that includes a recognition motif for furin, the Golgi-associated pro protein convertase, which processes stromelysin-3 to its enzymatically active form during traversal through the constitutive secretory pathway.
Stromelysin-3 was originally identified by differential screening of cDNA libraries derived from an invasive breast cancer surgical specimen and from benign breast fibroadenoma (11). Stromelysin-3 mRNA and protein expression correlates with the invasiveness of human carcinomas and also occurs in some sarcomas or other nonepithelial tumors (3, 12). These, as well as other studies (16), indicated a crucial role for the proteinase in processes characterized by extensive extracellular matrix turnover. Stromelysin-3, however, has a restricted substrate specificity distinct from all other matrix metalloproteinases. The enzyme only weakly, if at all, degrades extracellular matrix constituents such as laminin, fibronectin, and collagen (21, 22). Instead, substrates of stromelysin-3 include serine proteinase inhibitors, termed serpins (23, 24).
The serpin superfamily includes more than 60 proteins,
which can function as inhibitory mediators by acting as suicide substrates for the respective enzyme, with the vast majority targeted towards the serine proteinases known to be
involved in extracellular matrix remodeling, regulation of
blood pressure, modulation of inflammatory responses, cell
migration and differentiation, fibrinolysis, or blood coagulation (24). This group of serpins consists of single chain
proteins, such as 1-proteinase inhibitor (
1-PI),
1-antitrypsin (
1-AT),
2-antiplasmin (
2-AP),
2-macroglobulin
(
2-M), plasminogen activator inhibitor, antithrombin III
(AT-III), C1 inhibitor, angiotensinogen, etc. Most of these
serpins have known functions:
1-PI acts on elastolytic
proteases, such as leukocyte elastase, and cathepsin G (25,
26);
1-AT and
2-M are involved in lipoprotein catabolism (27);
1-AT also inhibits renin and thus the renin- angiotensinogen interaction (30);
2-AP and plasminogen
activator inhibitor are involved in the regulation of fibrinolysis; C1 inhibitor is essential for the regulation of activation of the complement and kinin generating system; and
AT-III as well as heparin cofactor II play a central role in
the regulation of blood coagulation (31). Interestingly, serpins also inhibit matrix degrading enzymes, such as cathepsins (32). The regulation of matrix degradation may thus
not only be promoted by tissue inhibitors of matrix metalloproteinases/MMP imbalance but also accelerated by decreased
levels of certain serpins such as
2-AP or
1-PI (33), inhibitors of matrix degrading activity. In this manner, stromelysin-3, despite possessing weak direct matrix degrading capabilities, might yet play a crucial role in matrix turnover.
Serpins play critical roles in maintaining homeostasis.
Therefore, any mechanism that reduces the functional level
of members of this superfamily, including inactivation by
proteinases, may result in substantial pathological problems
(23, 24). Several of the functions listed above relate to atherosclerosis, and atherosclerotic patients exhibit changes in
the levels of several of these serpins, including 1-AT,
2-M
(34, 35), and AT-III (36). In addition, animal studies indicate a potential role of serpins in the inhibition of arterial
intimal thickening (37) and atherosclerotic plaque development post injury (38) in vivo. For these reasons, the present
study analyzed the expression of stromelysin-3 within human atherosclerotic lesions.
Although the expression of stromelysin-3 at sites of pathologic processes such as Alzheimer's disease or cancer has prompted great interest in its regulation, little is known about the mechanisms involved (11, 39, 40). The stromelysin-3 gene promoter differs markedly from previously described matrix metalloproteinase promoters as it lacks a consensus activator protein 1 binding site and has a functional retinoic acid responsive element. We recently demonstrated the presence of the immune mediators CD40 ligand (CD40L) and its receptor CD40 on endothelial cells (EC), smooth muscle cells (SMC), and macrophages (MØ) within human atherosclerotic lesions (41) and showed that ligation of CD40 induces de novo synthesis of the classical MMP interstitial collagenase (MMP-1) and the gelatinases A and B (MMP-2 and -9, respectively) (42, 43) in these cells. Therefore, this study also tested the hypothesis that CD40 signaling regulates the expression of the constitutively active matrix metalloproteinase stromelysin-3 in human atheroma-associated cells and in mouse atheroma in vivo.
We report here that (a) stromelysin-3 (MMP-11) is present in human atherosclerotic lesions in situ, (b) cultured human vascular EC, SMC, and MØ synthesize stromelysin-3 de novo upon stimulation with CD40L, and (c) interruption of CD40-CD40L signaling in hyperlipidemic mice diminishes stromelysin-3 expression in atherosclerotic lesions in vivo.
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Materials and Methods |
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Materials.
Human recombinant IL-1Cell Isolation and Culture.
Human vascular EC were isolated from saphenous veins by collagenase treatment (1 mg/ml; Worthington Biochemicals) and cultured in dishes coated with fibronectin (1.5 µg/cm2; New York Blood Center Reagents). Cells were maintained in medium 199 (M199; BioWhittaker) supplemented with 1% penicillin/streptomycin (BioWhittaker), 5% fetal bovine serum (FBS) (Atlanta Biologicals), 50 µg/ml heparin (Sigma Chemical Co.), and endothelial cell growth factor (Pel-Freez Biological). SMC were isolated from human saphenous veins by explant outgrowth (47) and cultured in DMEM (BioWhittaker) supplemented with 1% L-glutamine (BioWhittaker), 1% penicillin/streptomycin, and 10% FBS. Both cell types were subcultured following trypsinization (0.5% trypsin [Worthington Biochemicals]/0.2% EDTA [EM Science]) in 75 cm2 culture flasks (Becton Dickinson) and used throughout passages two to four. Culture media and FBS contained <40 pg endotoxin/ml as determined by chromogenic Limulus amoebocyte assay analysis (QLC-1000; BioWhittaker). EC and SMC were characterized by immunostaining with anti von Willebrand factor and anti SMCImmunohistochemistry.
Surgical specimens of human carotid atheroma and aorta were obtained by protocols approved by the Human Investigation Review Committee at the Brigham and Women's Hospital. Serial cryostat sections (5 µm) were cut, air dried onto microscope slides (Fisher Scientific), and fixed in acetone atBiochemical Analysis of Human Atherosclerotic Lesions.
Frozen tissue from five nonatherosclerotic arteries and seven atheromatous carotid plaques were homogenized (IKA-Labortechnik, Ultra-turrax T 25) and lysed (0.3 mg tissue/ml lysis buffer) as described previously (5). The lysates were clarified (16,000 g, 15 min) and the protein concentration for each tissue extract as well as for the cell culture samples was determined using a bicinchoninic acid protein assay according to the instructions of the manufacturer (Pierce). 50 µg total protein were applied to Western blot analysis.In Situ Hybridization.
In situ hybridization was performed according to the instructions of the manufacturer (Hyb-ProbeTM; Shandon/Lipshaw). Frozen tissue sections, obtained as described above, were fixed in cold acetone, air-dried, and incubated with a mixture of FITC labeled stromelysin-3-specific (5'-GGTACCGTCAACCAGGTCCTCGTCCACG-3'; 5'-CTCAGAGTCGGGTCTACTGACCGTCC-3'; 5'-CCTACTGGTCCCGTGTCTGGACGACGTCCA-3'; 5'-ACGGTCCGGTGCTTATAGTCC-GATCTCCTGG-3'), CD40L-specific (5'-TTATGGGTGTCAA-GGCGGTTTGGAACGCCCGTT-3'; 5'-TGAAAAACGACA-CATAGAAGTATCTTCCAA-3'; 5'-TACTAGCTTTGTATGTTGGTTTGAAGAGGGGCT-3'; 5'-TGCAGGAAACCGAATGAGTTTGAGACTTGT-3'), or random oligomers in hybridization buffer (30% formamide, 0.6 M NaCl2, 10% dextran sulfate, 50 mM Tris [pH 7.5], 0.1% sodiumpyrophosphate, 0.2% Ficoll, 5 mM EDTA) for 10 min at 65°C and subsequently for 2 h at 37°C in a moist chamber. Thereafter, slides were immersed in TBS/ Triton (50 mM Tris, 150 mM NaCl, pH 7.6/0.1% Triton X-100) to allow coverslips to float off and were washed (TBS/ Triton) three times for 3 min at 37°C. The slides were forwarded to the immunological reaction by incubation with blocking solution (10 min, rt) and subsequent addition of the alkaline phosphatase-conjugated rabbit Fab' anti FITC (30 min, rt, moist chamber) as the primary detection reagent. Finally, the slides were washed twice in TBS (3 min, rt), covered with alkaline phosphatase substrate buffer (5 min, rt), and developed using the NBT/BCIP (nitro-blue-tetrazolium/5-bromo-4-chloro-3-indolyl phosphate) chromogen solution (1-2 h, rt, moist chamber).Western Blotting and Radioimmunoprecipitation.
Cell extracts (25 µg total protein/lane) and culture supernatants were separated by standard SDS-PAGE under reducing conditions and blotted to polyvinylidene difluoride membranes (Bio-Rad) using a semidry blotting apparatus (0.8 mA/cm2, 30 min; Bio-Rad). Blots were blocked and first and second monoclonal antibodies were diluted in 5% defatted dry milk/PBS/0.1% Tween 20. After 1 h of incubation with the respective primary antibody, blots were washed three times (PBS/0.1% Tween 20) and the secondary, peroxidase-conjugated, goat anti-mouse antibody (Jackson ImmunoResearch) was added for another hour. Finally, the blots were washed (20 min, PBS/0.1% Tween 20) and immunoreactive proteins were visualized using the Western blot chemiluminescence system (NENTM). For radioimmunoprecipitation experiments, cells were washed and further incubated with unlabeled medium lacking methionine/cysteine. Subsequently, medium containing 10% FBS and 50 mCi/ml [35S]methionine/cysteine (NENTM) was added to the cells for 24 h. Supernatants were harvested and concentrated (10×) using Centricon 3 devices (Amicon). Immunoprecipitation buffer (50 mM Tris-HCl, 0.1% SDS, 0.1% sodium deoxycholate, 1% NP-40, 150 mM NaCl, 5 mM EDTA, 20 µg/ml soybean trypsin inhibitor, 0.1% mM PMSF, 0.2 U/ml aprotinin, 0.025% sodium azide) was added to the cultures and cells were harvested by scraping. Subsequently, non immune mouse serum (Vector) was added (24 h, 4°C) to preclear the samples. Antigens in supernatants and cell extracts were immunoprecipitated with the specific anti stromelysin-3 antibody 5ST-4A9 (2 h, 4°C) and pelleted by subsequent addition of rabbit anti-mouse IgG (18 h, 4°C) as well as protein A-Sepharose beads (2 h, 4°C). The beads were washed four times in 50 mM Tris-HCl and finally 50 µl SDS-PAGE loading buffer (200 mmol/liter Tris, 5% glycerol, 0.1% SDS, 3%Isolation of RNA and PCR.
Total RNA from unstimulated or stimulated (24 h) EC, SMC, or MØ was isolated by a one-step preparation according to the method of Chomczynski and Sacchi (50). The cDNA was prepared by reverse transcription (RT) of total RNA (1 µg) with oligo(dT) using superscript reverse transcriptase (GIBCO BRL). The RT products were diluted in 480 µl twice distilled water and 10 µl of these cDNA preparations were mixed on ice with 10 µl primers (20 µM), 80 µl reaction mix (including 10 ml of PCR buffer, 2.5 mM MgCl), 4 µl dNTPs (200 µM; final concentration), and 0.5 µl Taq-polymerase (2.5 U; all GIBCO BRL). PCR was performed for 35 cycles at 95°C (120 s), 62°C (120 s), and 72°C (180 s, 2 s prolongation per cycle) after hot start. The sequences of primers for human stromelysin-3 were 5'-CTGCAGTCATCTGGGCTGAGACTC-3' and 5'-CCATGGCAGTTGGTGCAGGAGCAG-3'. The primers were obtained from Integrated DNA Technologies. Aliquots of the PCR products were run on 1.3% agarose gels and visualized by UV transillumination.In Vivo Analysis of CD40L-mediated Stromelysin-3 Expression.
LDLR ![]() |
Results |
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Immunohistochemical analysis of the expression of stromelysin-3 in normal aortic specimens (n = 5; Fig. 1 A, left) and human atherosclerotic fatty streaks (n = 5; data not shown) revealed little or no expression of the enzyme. In contrast, well-developed human carotid atherosclerotic lesions (n = 7) consistently showed strong stromelysin-3 immunoreactivity, most prominently at the luminal border and in the shoulder region of the plaque (Fig. 1 A, right). Western Blot analysis, performed on protein extracts of the surgical specimens and using the identical antibody used for the immunohistochemistry studies, revealed barely detectable immunoreactive stromelysin-3 in control specimens but markedly increased levels of the proteinase in atherosclerotic tissue (Fig. 1 B). The immunoreactive bands detected had molecular masses of ~64, 48, 35, and 28 kD, corresponding to the zymogen, intermediate, and active forms of stromelysin-3 (9, 10, 22, 23, 52, and 53). Higher magnifications of the immunohistochemical analysis, as well as immunofluorescent double staining with respective cell-selective antibodies, localized stromelysin-3 within EC, SMC, and MØ of the plaque (Fig. 2). Tissues showed no staining with the respective control IgG1 antibody (data not shown). Because we recently localized CD40 and CD40L in human atherosclerotic plaques and have shown that CD40 ligation induces interstitial collagenases and gelatinases in atheroma-associated cells (41), we investigated the possible colocalization of stromelysin-3 with CD40. Indeed, cells expressing stromelysin-3 also bear CD40 (Fig. 3). Furthermore, we analyzed the cellular localization of stromelysin-3 transcripts by in situ hybridization (Fig. 4). Human atheroma (Fig. 4, C-E), but not normal arteries (Fig. 4, A and B), contained stromelysin-3 mRNA. Within the atherosclerotic lesion, stromelysin-3 transcripts localized most prominently at the luminal border and the shoulder region of the plaques, areas described above as positive for the immunoreactive protein. The staining for the transcripts colocalized with smooth muscle cell- and macrophage-like cells (Fig. 4, D and E) as well as the endothelium (Fig. 4 E). Furthermore, transcripts for the immune mediator CD40L showed a similar distribution on adjacent sections (Fig. 4, G and H). In situ hybridization with negative control probes did not yield any signal (Fig. 4 F).
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To determine the mechanisms involved in stromelysin-3
expression in atheroma-associated cells, we analyzed the
expression of the enzyme in human vascular EC and SMC
as well as in human mononuclear phagocytes in vitro. Human vascular EC, SMC, and MØ released immunoreactive
stromelysin-3 constitutively in moderate amounts. Stimulation of the cells with the classical mediators of MMP regulation, IL-1 (1 and 10 ng/ml), TNF- (5 and 50 ng/ml), or
IFN
(100 and 1,000 U/ml), did not affect the expression
of stromelysin-3, as illustrated here for IL-1 and SMC (Fig.
5). However, the immunohistochemical studies presented
above indicated possible involvement of the CD40-
CD40L signaling pathway. Stimulation with both T cell-
derived as well as recombinant human CD40L induced stromelysin-3 expression in all three cell types (Figs. 5 and 6). Besides an increased intensity of the higher (~64 kD)
molecular mass band, which corresponds to the molecular
mass of the stromelysin-3 zymogen, CD40 ligation induced
the expression of immunoreactive proteins of lower molecular mass, corresponding to the molecular mass of the active cleavage products of the zymogen (Fig. 5). In control
experiments, addition of blocking anti-CD40L antibody during stimulation of the cells with native or recombinant
ligand inhibited induction of stromelysin-3 expression.
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Among the cell types analyzed, the immunoreactive forms of stromelysin-3 detected after CD40 ligation showed slight differences in the intensity of the respective bands but were identical in molecular mass. In vascular SMC, CD40 ligation concentration and time dependently elevated expression of the 64-kD protein, as well as induced immunoreactive proteins with molecular masses of ~48 and ~28 kD. The induction of the lower molecular mass forms of stromelysin-3 in cultures of SMC required stimulation with 3-10 µg/ml rCD40L (Fig. 6 A). An increase in the 64-kD protein, as well as in the ~48-kD immunoreactive protein, occurred after 1 h of stimulation, whereas detection of the 28-kD band required at least 6-12 h of exposure to CD40L, as determined by Western blot analysis (data not shown) and radioimmunoprecipitation experiments (Fig. 7). The patterns of immunoreactive proteins detected in cultures of vascular smooth muscle cells resembled those found in fibroblasts, an established source of stromelysin-3 (data not shown). The pattern of immunoreactive bands observed with supernatants of CD40L-stimulated EC (Fig. 6 B) resembled that obtained with cultures of SMC, except that the 64-kD form was much less abundant in EC.
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Macrophages, derived from monocytes cultured for 9 d,
showed an additional immunoreactive protein at ~35 kD.
Stimulation with 0.3 µg/ml rCD40L increased levels of
immunoreactive proteins with apparent molecular masses
of 64, 48, 35, and 28 kD (Fig. 6 C). Induction of these immunoreactive bands required at least 6 h of stimulation
(data not shown). Specificity of the antibody used was confirmed by Western blot analysis performed with the antistromelysin-3 antibody preincubated with recombinant
stromelysin-3 (Fig. 6, blocking). In contrast to monocyte-derived MØ cultured for 9 d, freshly isolated peripheral
blood monocytes expressed stromelysin-3 neither constitutively nor when stimulated with rCD40L (Fig. 6 D). Responsiveness of monocyte-derived cells to CD40 ligation
required a minimum of three days of in vitro culture (data
not shown).
The stromelysin-3 expression induced by CD40 ligation in human vascular EC, SMC, and MØ resulted from de novo synthesis of the protein. Metabolic labeling and immunoprecipitation experiments yielded autoradiographic bands resembling the patterns of immunoreactive proteins observed by Western blot analysis as shown here for vascular SMC (Fig. 7). In accordance with our protein analysis, RT-PCR experiments showed increased product corresponding to stromelysin-3 transcript after CD40 ligation in EC and SMC as well as MØ (data not shown).
Interruption of CD40-CD40L Signaling Diminished Stromelysin-3 Expression in Mouse Atherosclerotic Lesions.The in
situ observations of stromelysin-3 expression in human
atherosclerotic plaques, in combination with the in vitro
findings that CD40 ligation selectively mediates the expression of stromelysin-3, suggested an in vivo evaluation of
the importance of CD40-CD40L signaling for the expression of this enzyme within atherosclerotic plaques. For this
purpose, an established animal model of arteriosclerosis was
used: LDLR/
mice were fed a high cholesterol (1.25%)
diet to develop atherosclerotic lesions (51). Immunohistochemical analysis of lesions within the aortic arch as well
as the thoracic portion of the aorta revealed a stromelysin-3
expression pattern similar to that found in human atherosclerotic plaques. All three vascular cell types
EC, SMC and MØ
within the lesions stained for the proteinase.
Treatment of the mice with rat IgG (n = 8) did not affect
the stromelysin-3 expression (Fig. 8 A) compared to controls. In contrast, mice treated with the anti-mouse CD40L
antibody (n = 8) showed substantially reduced immunoreactivity for stromelysin-3 within the atherosclerotic lesions
(Fig. 8 B). Since the anti-CD40L antibody treatment also resulted in a decrease of total plaque number and area (51), lesions of similar sizes within the treatment groups were
compared. No immunoreactivity was observed in tissues
stained with the control IgG1 antibody (data not shown).
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Discussion |
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This study establishes the expression of stromelysin-3 in
advanced human atheroma and furthermore provides evidence for (a) colocalization of this matrix metalloproteinase
with CD40 on lesional EC, SMC, and MØ in situ and (b)
regulation of de novo expression of stromelysin-3 by
CD40 ligation, rather than by the classical soluble mediators of MMPs such as IL-1, TNF-, or IFN-
. We also demonstrated in vivo that the interruption of CD40-
CD40L interaction markedly reduced the expression of
stromelysin-3 in mouse atheroma. The finding that EC,
SMC, and MØ express stromelysin-3 establishes atheroma-associated cells as novel sources of stromelysin-3. Thus, this
report provides evidence that stromelysin-3, whose expression correlates with the invasiveness of malignancies (3, 11, 12, 16, 17, 54), might also participate in the pathogenesis of
another common human disease, atherosclerosis. In view of
its unusual substrate specificity, stromelysin-3 might participate in several pathways. The pattern of stromelysin-3 expression within tissues that undergo extensive remodeling
in pathological (carcinomas; 11, 12, 16, 17, 54) as well as
physiological (placenta and uterus; 11, 18) events, indicates
a role for stromelysin-3 in these processes. Although related
by sequence to the MMP family, stromelysin-3 does not
hydrolyze many of the extracellular matrix components that are substrates for other MMPs such as fibronectin,
laminin, elastin, or collagen type I and type IV (23). From
this perspective, the enzyme has been considered a nonmatrix-degrading metalloproteinase. Instead, stromelysin-3
appears to act as a predominant regulator of serpin function. Interestingly, a recent report postulated that extracellular matrix degradation might depend not only on an
imbalance between matrix metalloproteinases and their
inhibitors, but might accelerate in face of decreased levels
of certain serpins such as
2-M,
2-AP, or
1-PI (33),
preferred substrates of stromelysin-3 (23). Pathways by
which this enzyme might regulate matrix degradation include (a) inactivation of matrix-degrading, enzyme-inhibiting serpins as described for cathepsins (32) or for elastolytic activity by
1-PI, shown to simultaneously augment the proliferative and invasive activity of cells (23); (b)
activation of other members of the MMP family (17) copiously expressed in atheroma, such as interstitial collagenase,
gelatinases A and B, and stromelysin-1 (5) (at sites of
chronic inflammation, such as atherosclerosis, constitutively
active stromelysin-3 [9] might act proximally to promote
conversion of the zymogen forms of these classical MMPs
to their active forms); and (c) degradation of matrix molecules not cleaved by the classical MMPs such as extracellular proteins containing amino acids with unusual long side
chains, including those generated in vivo by certain posttranslational modifications (55). Thus, matrix degradation,
a critical step in the progression from the stable atheroma
to one prone to rupture and capable of causing thrombotic
complications, might indeed involve the action of stromelysin-3. The serpin-degrading function of stromelysin-3
may thus have significance beyond tumor invasion and metastasis.
Furthermore, the action of stromelysin-3 on serpins also
might affect atherosclerosis by pathways other than extracellular matrix remodeling. Serpins regulate multiple functions associated with the disease, including (a) blood pressure (e.g., the serpin 1-antitrypsin inhibits renin and thus
renin-angiotensinogen interaction, as well as angiotensinogen itself [30]), (b) fibrinolysis (e.g., the serpin
1-antiplasmin targets plasmin, a key effector of fibrinolysis [23]), (c)
blood coagulation (e.g., the serpins anti-thrombin III and
heparin cofactor II rapidly interact with thrombin in the
presence of heparin [31]), or (d) lipoprotein uptake (cleavage of the serpins
1-antitrypsin and
2-macroglobulin is
associated with increased low density lipoprotein uptake
into cells, indicating that those cleaved serpins disturb the
intracellular cholesterol homeostasis [27-29]). Aside from
these effects, stromelysin-3 might further affect atherosclerosis via the insulin-like growth factor-insulin-like growth
factor binding protein (IGF-IGFBP) system. Human atheroma contain IGF-1 and IGFBPs (56). These mediators are
associated with cardiovascular pathophysiology particularly via their critical role in vascular growth (57). IGF-1 directly accelerates arteriosclerosis in rat aorta allografts via myointimal proliferation and intimal thickening (58). The level of
free, biologically active IGF-1 depends on the degree of
complex formation with the respective binding proteins. A
recent study identified IGFBP-1 as a potential physiological
substrate for human stromelysin-3 (59). Finally, IGF also acts
as a survival factor for human vascular SMC derived from
normal vessels as well as coronary atherosclerotic plaques
(60), suggesting stromelysin-3 may prevent apoptosis of vascular SMC by augmenting IGF-1 levels.
The potential relevance of serpin degradation for the
pathogenesis of atherosclerosis in humans is supported by
reports showing that levels of several serpins, including
AT-III (36), 2-M, and
2-AP (35), decrease in this prevalent disease. In addition, recent in vivo studies demonstrated that serpins inhibit coronary restenosis in atherosclerotic swine (37) and atherosclerotic plaque development in
rabbits post injury (38). Our findings that (a) differentiated
atherosclerotic lesions but not their precursor, the fatty
streak, bear stromelysin-3; (b) the immunoreactive bands
detected within the atheromatous plaques resembled
mostly the pattern obtained with MØ in vitro; and (c) only
differentiated MØ, but not peripheral blood monocytes,
exhibit stromelysin-3 induction; support a role of this enzyme in the late rather than the early states of atherosclerosis. Interestingly, the Rotterdam study showed increased
AT-III levels in moderate peripheral arterial atherosclerosis
and decreased levels in more severe atherosclerosis (61).
Future studies will have to establish whether these various functions apply to human atherosclerosis and whether stromelysin-3 is the crucial mediator in reduction of all or only certain atheroma-associated serpin activities. However, based on the recently burgeoning background information on stromelysin-3, our findings suggest functions of this proteinase in regulation of plaque progression and (in)stability by mechanisms distinct from those ascribed to members of the MMP family, including augmented extracellular matrix degradation, promotion of blood coagulation, decreased fibrinolytic activity, and dysregulation of blood pressure as well as lipoprotein catabolism. The colocalization of stromelysin-3 with CD40 and CD40L within the human atherosclerotic lesion, as well as the demonstration that CD40L, rather than the classical mediators of MMPs, induce stromelysin-3 expression in atheroma-associated cells demonstrate CD40-CD40L signaling as a new, and, as indicated by the in vivo studies, probably crucial pathway of stromelysin-3 induction.
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Footnotes |
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Address correspondence to Peter Libby, Vascular Medicine and Atherosclerosis Unit, Cardiovascular Division, Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, 221 Longwood Ave., LMRC 307, Boston, MA 02115. Phone: 617-732-6628; Fax: 617-732-6961; E-mail: plibby{at}rics.bwh.harvard.edu
Received for publication 22 October 1998 and in revised form 21 December 1998.
1 Abbreviations used in this paper:We thank Maria Muszynski, Eugenia Shvartz, and Elissa Simon-Morrissey (Brigham and Women's Hospital) for their skillful technical assistance.
This work was supported in part by grants from the National Heart, Lung, and Blood Institute to Dr. Peter Libby (HL-43364) and the Swiss National Research Fund to Dr. François Mach and was performed during the tenure of the Paul Dudley White fellowship of the American Heart Association by Dr. Uwe Schönbeck.
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