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From the Department of Bioengineering,*
Clemson
University, Clemson, South Carolina; and the Division of Pediatric
Cardiology Research,
Children’s Hospital of
Philadelphia and the Department of Pediatrics, University of
Pennsylvania School of Medicine, Philadelphia, Pennsylvania
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Although the elastic fibers can be considered physiologically inert during adult life, a wide range of insults to elastic tissue can result in either chronic loss or excess accumulation.6 Matrix metalloproteinases (MMPs) are involved in elastolysis. In particular, both MMP-2 and MMP-9 are known to bind to insoluble elastin,7 and each has been shown to be actively involved in elastin degradation.8,9 Exuberant production of MMPs is a hallmark of many destructive diseases, such as arthritis, chronic ulceration, and tumor formation.10-12 With respect to calcification, MMPs have also been detected in association with calcification of bioprostheses.13,14 For example, subdermally implanted glutaraldehyde-treated bovine parietal pericardium contains an array of extracellular matrix protein-degrading proteinases including serine proteinases and MMPs.13,14 High concentrations of MMPs are also present in atherosclerotic plaques15 and in restenotic lesions.16
Tenascin-C (TN-C) is an extracellular matrix glycoprotein with a highly
restricted pattern of gene expression, but it is prominently expressed
in embryonic and adult tissues that are actively
remodeling.17
A number of studies indicate that MMPs
regulate TN-C expression.18,19
For example, after arterial
injury, TN-C and MMPs20
are up-regulated during the
development of occlusive neointimal lesions, whereas inhibition of MMP
activity attenuates this process.21
In addition, both
MMP-222
and TN-C23,24
are able to bind the
same cell surface receptor, the 
vß3 integrin, further indicating
that their regulation and functions may be interdependent. In fact, we
have recently shown that extracellular matrix protein proteolysis by
MMPs activates TN-C transcription via an ERK1/2 MAPK-dependent
signaling pathway.18,25
With respect to calcification, a number of studies indicate that there is a strong relationship between TN-C expression and calcification in normal and dystrophic mineralization. For example, TN-C is expressed in developing bone26 and co-localizes with the calcium-binding protein S-100ß in the cranium.27 During tooth development, TN-C is expressed by the peridontoblast at the inner enamel mineralization front.28 In addition, tissue culture studies demonstrate that osteoblast adhesion to TN-C up-regulates alkaline phosphatase, a well-established marker of bone differentiation.29 Other studies suggest that TN-C may act as a mediator of TGF-ß-dependent bone formation,30 as well as pericyte differentiation/mineralization during neovascularization.31 Moreover, physical loading and the resulting increased strain imposed on rat ulnae leads to early increases in osteoblast TN-C expression, indicating that this protein may act as a mediator of osteoregulatory responses to altered biomechanics.32 Despite these studies, the mechanistic and functional links that may exist between MMP and TN-C expression during elastin-oriented calcification in vivo have not been examined.
In the present study, we investigated the production and activity of MMPs and TN-C during early (3 and 7days) elastin implant calcification in rats by immunohistochemistry and gelatin substrate zymography. Furthermore, using a hydroxamate-based MMP inhibitor (BB-1101), we tested the hypothesis that systemic or site-specific inhibition of MMP activity would attenuate elastin calcification. Inhibition of MMP activity not only resulted in a reduction of elastin calcification, but also reduced TN-C production within elastin implants. Moreover, site-specific delivery of BB-1101 was more effective in reducing both TN-C and calcification. These studies indicate that MMPs are important mediators of both TN-C production and elastin-oriented calcification.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-smooth muscle actin (Sigma) were used in
immunohistochemistry studies. An alkaline phosphatase staining kit was
obtained from Boehringer-Mannheim (Mannheim, Germany). Ketamine
HCl (Ketaset; Fort Dodge Lab, Fort Dodge, IA) and xylazine (Rompun;
Miles Inc., Shawnee Mission, KS) were used for rat anesthesia. Rat Subdermal Implantation
Male Sprague-Dawley rats (21 to 24 days old, 50 to 65 g; Charles River Laboratories, Burlington, MA) were anesthetized by intramuscular injections of ketamine HCl and xylazine (4:3), 0.001 ml/g of body weight. Using a sterile technique, a small incision was made on the back of the rats and two subdermal pouches (one in front and one in back) were created. Two samples of elastin fibers were then implanted subdermally in the pouches. The rats (five per group) were sacrificed by CO2 asphyxiation at 3 days and 7 days and samples were retrieved. The samples (10 per group) were used for quantitative calcium and phosphorus analyses, and three samples per group were snap-frozen over dry-ice in frozen OCT (tissue embedding medium) and three samples per group were fixed in phosphate-buffered formalin fixative for morphological and immunohistochemical studies. All immunohistochemical-staining studies were performed with at least three different samples in each group.
MMP Inhibitor Studies
BB-1101 (30 mg) was suspended in 0.6 ml of ethanol and then 11.4 ml of phosphate-buffered saline (PBS) was added drop-wise with sonication to obtain a stable suspension. The control solution was made without BB-1101. This suspension was then administered preimplantation to ensure adequate drug levels in rats by subdermal injections (10-mg/kg body weight/day), either at the implant site (the back of the rats) or on the abdominal side (systemic). One day after initial administration of BB-1101, elastin samples were implanted (five rats per group, two samples in each rat) as described above. The drug therapy was continued once per day for 7 days. The drug suspension was sonicated each day before administration. The doses were chosen according to the manufacturer’s suggestion. Plasma concentrations of the drug were not determined. Rats were sacrificed by CO2 asphyxiation at 3 days and 7 days, and elastin samples along with the tissue capsule were retrieved and used for quantitative calcium and phosphorus determination and immunohistochemistry and zymography studies.
Immunostaining for MMPs and TN-C
Explanted elastin samples were fixed overnight in neutral-buffered formalin and embedded in paraffin. Sections (6 µm) were taken for histology, deparaffinized using xylene, and sequentially rehydrated in graded ethanol. Vectastain ABC kit (Vector Laboratories, Burlington, Ontario, Canada) was used for immunoperoxidase staining according to the manufacturer’s instructions. Mouse anti-MMP-2 monoclonal antibody and human TN-C monoclonal antibody diluted 1:100 in wash buffer were used for 2 hours at 37°C. Immune complexes were then stained by incubation in a solution of 3, 3'-diaminobenzidine tetrahydrochloride dihydrate, (diaminobenzidine substrate kit, Vector Laboratories), and hydrogen peroxide. Sections were then counterstained with hematoxylin and eosin. For all immunohistochemistry experiments, negative controls included omission of the primary antibody and substitution of nonspecific mouse antisera.
Assessment of Cellular Invasion
Similar immunohistochemical protocols as described above were
performed to characterize T cell, fibroblast, pericyte, and macrophage
infiltration within the elastin implant using monoclonal antibodies
against rat CD3 cells (mouse anti-rat CD3), vimentin (cell marker for
fibroblasts), -smooth muscle actin (for activated fibroblasts,
smooth muscle cells and pericytes), and rat macrophages (mouse anti-rat
macrophages). Anti-rat CD3 recognizes the rat T cell homologue of human
CD3.
Zymography
Explanted elastin samples were homogenized in extraction buffer containing 50 mmol/L Tris, 0.2% Triton X-100, 10 mmol/L CaCl2, and 2 mol/L guanidine.HCl, pH 7.5. The extracts were centrifuged at 10,000 x g for 10 minutes and the supernatants were dialyzed overnight against 0.2% Triton X-100, 50 mmol/L Tris, pH 7.5. The protein content of the extract was determined using the biocinchoninic acid method according to the manufacturer’s procedure (Pierce Inc., Rockford, IL). Samples containing equal amounts of protein (5 to 20 µg per lane) were electrophoresed on a 10% sodium dodecyl sulfate-polyacrylamide gel with 0.1% (wt/vol) gelatin (Bio-Rad, Richmond, CA) under nonreducing conditions. After electrophoresis, the gels were washed in 2.5% Triton X-100 for 30 minutes to remove sodium dodecyl sulfate and then incubated in the substrate buffer at 37°C (50 mmol/L Tris buffer, pH 7.8, containing 10 mmol/L CaCl2) for 70 hours. The gels were then stained with 0.02% Coomassie brilliant blue (Sigma Chemical Co.). MMPs appear as areas of lysis on a dark blue Coomassie-stained gelatin background. For selected gels, 100 µl BB-1101 (100 µg/ml) was added to the substrate buffer to study the MMP inhibitory effects of BB-1101.
Alkaline Phosphatase Staining
The alkaline-phosphate staining solution was prepared by adding 50 µl 4-nitro blue tetrazolium solution and 37.5 µl X-phosphate solution (50 mg/ml in dimethyl formamide) to 10 ml 0.1 mol/L of Tris buffer (pH 9.5, 0.05 mol/L MgCl2, 0.1 mol/L NaCl). The frozen sections were rinsed with the same buffer used for the staining solution. The sections were then incubated in the staining solution in the dark for 1 hour at room temperature. After development, the sections were washed in water, and then were fixed in 4% paraformaldehyde solution for 5 minutes at room temperature. Sections were washed with PBS and mounted with a mounting medium.
Calcification Assessment
Quantitative calcium and phosphorus analyses were performed on the explanted samples as per established procedures.33,34 Briefly, explanted elastin samples were lyophilized, weighed, and then digested in 6 N HCl at 95°C for 24 hours in a closed container. The solution was then completely evaporated at 70°C with a continuous flow of nitrogen in the tube. The residue left in the test tube was then dissolved in 1 ml of 0.01 N HCl. The calcium content of each sample was based on the aliquot concentration of each hydrolysate using atomic absorption spectroscopy according to the established procedures.33 The phosphorus levels were determined by molybdate complexation assay.34 Representative frozen sections of explants were stained with Alizarin Red staining for localization of calcium phosphate deposits.
Data Analyses
Quantitative results were expressed as the means ± SE. Differences between control means and treated groups were assessed using the unpaired Student’s t-test. Data were termed significant when P < 0.05.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Elastin fibers, when implanted subdermally in rats, underwent
progressive calcification as determined by quantitative calcium and
phosphorus analysis (Figure 1)
. At 3
days, calcification was evident at the peripheral elastin fibers and at
7 days, calcification was evident throughout the elastin implant as
shown by Alizarin Red staining (Figure 2F)
. Calcium and phosphorus levels within
the elastin implants were increased at 7 days. The molar ratio of
calcium to phosphorus, 
1.8, suggested deposition of poorly
crystalline hydroxyapatite.
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At 3 days after implantation, host cells began to invade the
surface of the elastin implant. This was associated with moderate
staining for MMP-2 and low immunoreactivity for TN-C (Figure 2, A and B)
. At 7 days, extensive cellular infiltration had occurred and this
corresponded with intense staining for MMP-2 and TN-C throughout the
implant (Figure 2, D and E)
. IgG-stained controls were negative for
MMP-2 and TN-C (Figure 2C)
. Moreover, the intense MMP-2 and TN-C
staining was apparent near the calcific deposits on elastin as seen by
Alizarin Red staining at 7 days (Figure 2F)
.
Characterization of Cellular Infiltrates
At 7 days, elastin implants were encapsulated by host tissue, and
were extensively invaded by host cells (Figure 3A)
. Immunostaining indicated that
numerous CD3-positive T cells were present in the outer capsule and
within the implant (Figure 3B)
. Alpha smooth muscle cell actin antibody
recognizes pericytes and activated fibroblasts. This staining showed
the presence of pericytes in small blood vessels forming within the
capsule and an occasional staining for activated fibroblasts close to
elastin fibers (Figure 3C)
. Vimentin staining for fibroblasts showed
heterogeneous staining throughout the capsule with strong staining in
close proximity of elastin fibers (Figure 3D)
. Only occasional cells
were stained, if any, with the macrophage antibody suggesting low
levels of macrophages surrounding the implant (Figure 3E)
.
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To determine whether MMPs regulate TN-C and elastin calcification,
rats were treated daily with an MMP inhibitor, BB-1101, either locally
or at the distal site (systemically) for 7 days. Gelatin substrate
zymography was used to assess the type and activity of MMPs in elastin
implants. In the control vehicle group, gelatinolytic bands were seen
at approximate molecular weights 90, 68, and 57 kd (Figure 4)
. The band at 90 kd most likely
represents MMP-9. Under the denaturing and nonreducing conditions used
for zymography, the pro-form of MMP-2 has an apparent molecular weight
of 68 kd, whereas the active form of MMP-2 appears at 57 kd. Thus,
our data indicates that both MMP-2 and MMP-9 are present within elastin
implants. When BB-1101, an MMP inhibitor was added to the substrate
buffer while performing zymography, no lytic bands were seen confirming
that the zymography bands seen were because of MMPs, and BB-1101
nonspecifically inhibits all MMP activity (Figure 4)
. When rats were
administered BB-1101, either at the implant site or systemically, the
MMP activity within the implant was not decreased as shown by
immunohistochemistry (data not shown) and zymography (Figure 4)
, which
is expected, as this drug does not act at the expression level. As the
BB-1101 chelation is reversible, it is possible that BB-1101 would be
removed from elastin explants during extraction and washing procedures
before gelatin zymography.
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. However, TN-C presence within the
elastin implants was almost completely suppressed by BB-1101 in the
site-specific delivery group, and was significantly reduced in the
systemic delivery group (Figure 5)
, strongly indicating that TN-C
production is dependent on MMP activity. Both control and BB-1101
groups showed comparable alkaline phosphatase activity within the
implant (Figure 6)
, however, at this time
we do not know which cell types show alkaline phosphatase activity.
Nevertheless, alkaline phosphatase activity within the elastin implant
was not altered by BB-1101 administration indicating that MMPs and TN-C
may regulate calcification via an alternative pathway.
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Site-specific delivery of BB-1101 significantly inhibited
calcification of elastin implants at 7 days (Table 1)
with a fourfold decrease in the
elastin calcium content in the BB-1101 group explants as compared to
control injections (5.43 ± 1.03 µg/mg Ca for BB-1101 group
versus 21.71 ± 1.19 for control group,
P < 0.001). Alizarin Red staining clearly showed that
the elastin fibers were heavily calcified in the control group, whereas
in BB-1101 local therapy group the calcification was scarce, with few
fibers showing initial calcification deposits (Figure 7)
. The systemic administration of
BB-1101 also significantly reduced elastin calcification (28.07 ±
5.81 control versus 16.92 ± 2.56 BB-1101 group,
P < 0.05), although less than the site-specific
administration (Table 1)
, with obvious calcification per Alizarin Red
staining (data not shown). No adverse side effects of this drug therapy
were seen on the development of rats as assessed by body weights at the
time of explantation (Table 1)
. Furthermore, serum calcium levels of
the rats were not altered by BB-1101 therapy (Table 1)
.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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MMPs are a family of zinc-dependent enzymes involved in tissue morphogenesis and remodeling of connective tissue. In the present study, intense MMP activity within the elastin implant was seen at 7 days. This high MMP activity could lead to the degradation of elastin, thereby generating elastin peptides. Elastin peptides have been shown chemotactic for fibroblasts, smooth muscle cells, and monocytes.42,43 Elastin peptides contain no RGD sequences and elastin does not interact with integrins. A 67-kd elastin-binding protein, known as elastin-laminin receptor, recognizes a hydrophobic hexapeptide, VGVAPG, which repeats in the elastin molecule.44 This 67-kd elastin-binding receptor has been shown to be present on a variety of differentiated cells including chondrocytes, endothelial cells, vascular smooth muscle cells, fibroblasts, monocytes, and macrophages.45-49 Activation of this receptor (because of elastin binding) triggers several cellular reactions such as modulation of the biosynthesis of connective tissue macromolecules, increase synthesis and release of protease including MMPs, modification of ion fluxes, and cell proliferation and apoptosis.48-51 Thus, in our elastin implants, it is possible that initial release of elastin peptides because of MMPs can trigger activation of this receptor on the fibroblasts and monocytes and cause several down-stream events including induction of MMPs and TN-C expression, as well as an increase in calcium ion fluxes within the cells. It has already been shown that rabbits, after injected with elastin peptides, develop aortic calcification.52
When the elastin fibers were implanted subdermally, we observed a
low-level inflammatory response by 7 days as assessed by scant
macrophage staining. The surrounding tissue capsule and elastin implant
was infiltrated with microvessels and with numerous monocytes and
fibroblast-like cells (Figure 3)
. This represents a typical
wound-healing response at this early stage of implantation. In our
previous study, when the implant time was extended for 21 days, only
occasional monocytes were observed within the implant.5
In
the present study, MMP and TN-C activity observed throughout the
implant at 7 days might be because of the expression of these proteins
by fibroblasts or monocytes. The cell-specific expressions of MMP and
TN-C are currently under investigation. However, we have shown
previously that porcine aortic valve tissue when implanted subdermally
calcify to the same extent in congenitally athymic, T-cell deficient
(nude) mice as implants in immunologically competent
hosts.53
Thus, inflammatory response may play a limited
role in the calcification process.
In this study, we also determined whether early inhibition of MMP activity would affect elastin calcification. Thus, we decided to block the activity of MMPs and study the down-stream events including TN-C production, alkaline phosphatase activity, and calcification. Accordingly we used a nonspecific MMP inhibitor, BB-1101. Such hydroxamate-based MMP inhibitors have been successfully used to prevent MMP activity in animal models as well as in humans.54 Although BB-1101 inhibits activity of almost all MMPs, this was not of concern in our elastin implant studies as we see higher activities of only MMP-2 and MMP-9 in our implants as per immunohistochemistry and zymography. As BB-1101 modulates MMP activity, rather than expression level, we observed an intense activity of MMP-2 (both latent and active form) and MMP-9 in control group (vehicle treated) as well as in the BB-1101-treated group within elastin implants at 7 days as per immunohistochemistry and zymography. The experiments where BB-1101 was added to the substrate buffer while performing zymography clearly indicated that this drug blocks overall MMP activity.
Next, we decided to study TN-C within the elastin implants as TN-C up-regulation by MMPs has been demonstrated.18,19,25 Blocking MMP activity resulted in significant down-regulation of TN-C production within elastin implants. This is in agreement with a previous cell culture study, where a specific MMP inhibitor, GM6001 also suppressed TN-C expression.18 Although TN-C expression is associated with physiological mineralization, the exact role of TN-C in pathological calcification is unknown. Our elastin implant studies show that TN-C production is dependent on MMP activation. Higher TN-C production was seen in highly calcified samples, while inhibiting MMPs activation via administration of BB-1101 led to diminished TN-C production and significant reduction in calcification. Future studies will directly evaluate the role of TN-C on elastin calcification. This study was performed with purified elastin samples and most of the purification methods reported for elastin cause partial degradation of elastin.
In conclusion, we have shown that MMPs, TN-C, and alkaline phosphatase are associated with the initial calcific deposits within elastin implants. Furthermore, we have shown that blocking of MMP activity by administration of BB-1101, significantly reduces elastin-oriented calcification. We believe that this is the first study where MMP inhibition suppressed pathological calcification.
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Footnotes |
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Supported by a Scientist Development Grant from the National American Heart Association (to N. R. V.), by an endowment from the Children’s Hospital of Philadelphia, and a National Heart, Lung, and Blood Institute Grant 38118 (to R. J. L.). We thank St. Jude Medical, Inc., MN for financial assistance.
N. R. V. and P. L. J. contributed equally to this work.
Accepted for publication May 31, 2000.
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