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From the Department of Bioengineering, Clemson University, Clemson, South Carolina
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Abstract |
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-1, an osteoblast-specific transcription factor, increased in parallel with elastin calcification and attained 
9.5-fold higher expression at 21 days compared to 3 days after implantation. Similarly, mRNA levels of the bone markers osteopontinand alkaline phosphatasealso increased progressively, but osteocalcinlevels remained unchanged. We conclude that degenerative and osteogenic processes may be involved in elastin calcification.
Elastin is a major component of the extracellular matrix in cardiovascular connective tissues. Matrix metalloproteinases (MMPs) are expressed in normal physiological processes such as wound healing and angiogenesis, but increased levels of MMPs have also been identified in many cardiovascular pathologies.6 MMP-9 and MMP-2 bind and degrade insoluble elastin to generate soluble peptides.7 These elastin peptides can interact with a 67-kd transmembrane protein, the elastin laminin receptor (ELR),8 which is present on the surface of most cells. Activation of ELR by elastin peptides triggers diverse biological activities in various cell types including synthesis and release of elastase, liberation of free radicals, increased Ca2+ influx in endothelial cells, NO-dependent vasorelaxation, proliferation of arterial smooth muscle cells, chemotaxis of monocytes and fibroblasts, and apoptosis.9-13 Taken together, these data suggest a possible correlation between elastin degradation, activation of ELR, and elastin calcification, but the mechanisms that link these processes are as yet unknown. We have reported that MMP-mediated elastin degradation is the initial step in elastin calcification in the rat subdermal implantation model and that inhibition of MMPs leads to significant reduction in calcification.14,15 Moreover, we have recently shown in an abdominal aorta injury model in rats and MMP-knockout mice that MMP-mediated elastin degradation precedes elastin calcification and that the presence of active MMP-2 and MMP-9 are required for elastin calcification.16
We hypothesized that pathological elastin calcification is governed by two processes: enzyme-mediated elastin degeneration and cell-facilitated ectopic osteogenesis. As a result of elastin degradation, soluble peptides are released locally, where they activate quiescent cells, resulting in ELR and MMP up-regulation and enhanced matrix degradation. The degraded elastin-rich extracellular matrix becomes a calcification-prone substrate. We further hypothesized that elastin degradation also induces differentiation of nonbone cells into osteoblast-like cells, which secrete bone proteins that promote elastin calcification.17
To evaluate this hypothesis, we examined the expression of genes implicated in elastin degeneration such as MMP-2, MMP-9, ELR, and transforming growth factor(TGF-ß1) as well as osteogenesis-associated genes such as core binding factor--1(CBFA-1), osteocalcin(OCN), alkaline phosphatase(ALP), and osteopontin(OPN) using real-time reverse transcription-polymerase chain reaction (RT-PCR) in a time-course study in a rat subdermal elastin calcification model. We also evaluated soluble elastin peptide production and MMP activity, as markers of elastin degradation, as well as ALP enzyme activity and OCN secretion, as markers of osteoblastic differentiation. In the present study, we report that elastin remodeling and osteogenesis may be involved in elastin calcification.
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Materials and Methods |
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Porcine hearts were obtained from a local slaughterhouse and transported to the laboratory on ice. A supravalvular aortic segment (3 to 4 cm) was dissected, cleaned to remove fat and adherent tissues, and rinsed in cold saline. Aortic elastin was purified by autoclaving, as previously described by Partridge and Keeley18 Briefly, each aorta was cut into 2-mm strips, rinsed with distilled water, and shredded using a blender. The shredded aorta was washed with cold saline to remove soluble proteins until no protein was detected by BCA assay (Pierce, Rockford, IL). The washed aorta was autoclaved four times in distilled water (1 hour per cycle) and washed with distilled water until no protein was detected in the solution by BCA assay. This was then followed by defatting the aorta with ethanol and diethyl ether and lyophilization. This procedure extracts all cellular materials, collagenous and noncollagenous components, including elastin-associated glycoproteins, leaving pure elastin intact.
Subdermal Implantation of Elastin
Juvenile male Sprague-Dawley rats (21 days old, 35 to 40 g; Harlan, Indianapolis, IN) were anesthetized with acepromazine (0.5 mg/kg; Ayerst Laboratories, Inc., Rouse Point, NJ) and maintained on isoflurane gas (2 to 2.5%) throughout surgery. A small incision was made on the back of the rats, and three subdermal pouches were formed by blunt dissection. Each rat received three 30- to 40-mg elastin implants (one per pouch), which were rehydrated in sterile saline 1 to 2 hours before implantation. Four rats were sacrificed by CO2 asphyxiation at each time point (3, 7, 14, and 21 days after implantation), and the implants were retrieved along with the surrounding fibrous capsule. One explant from each rat was frozen in OCT (Sakura Finetek, Zoeterwoude, The Netherlands) on dry ice and stored at –80°C for immunohistochemistry; another explant was frozen on dry ice for protein analysis. The third explant from each rat was stored in RNAlater (Ambion, Austin, TX) for quantitative real-time RT-PCR. Small segments from each explant were frozen on dry ice for calcium and phosphorous analysis. As a control, uninjured subdermal tissue samples were collected from four rats and divided into two segments. One from each rat was stored in RNAlater for quantitative real-time RT-PCR and the other was frozen on dry ice for zymography. As a control medical grade polyester fabric (Dacron) samples (30 to 40 mg each, 4 x 4 mm pieces) were implanted subdermally in juvenile rats (n = 4) and explanted at 21 days as described above. Animal experiments were conducted according to National Institutes of Health guidelines for the care and use of laboratory animal (NIH publication no. 86-23, revised 1996). The animal protocol was approved by the Animal Research Committee at Clemson University.
Calcium and Phosphorous Determination
Analysis of calcium and phosphorous content in rat subdermal explants was performed by previously described procedures.19 Briefly, lyophilized elastin explants (16 to 23 mg) were placed in 1 ml of 6 N HCl and hydrolyzed in a boiling water bath for 8 hours. Samples were evaporated under a continuous stream of nitrogen gas and residual material dissolved in 1 ml of 0.01 N HCl. Calcium content in explants was determined (n = 8 per time point) with an atomic absorption spectrophotometer (model 3030; Perkin-Elmer, Norwalk, CT). Phosphorous content (n = 8 per time point) was measured on the same acid hydrolysates using the molybdate complexation assay.20
Immunohistochemical (IHC) Staining for Characterization of Cell Infiltrates
Explants embedded in OCT were cryosectioned (6 µm) and mounted onto poly-L-lysine-coated glass slides. Sections were fixed in cold acetone for 5 minutes and blocked with 0.3% hydrogen peroxide in 0.3% normal sera in Tris-buffered saline for 5 minutes to neutralize endogenous peroxidase activity. Sections were incubated with mouse-derived primary antisera directed against rat monocytes/macrophages (1:200 dilution, MAB1435; Chemicon, Temecula, CA), vimentin (1:5000 dilution; Sigma, St. Louis, MO) diluted in TNB blocking buffer (Perkin Elmer Life Sciences, Boston, MA), and -smooth muscle actin (Sigma) for 1 hour at room temperature. TNB buffer alone was used as negative control. Staining was visualized using rat-adsorbed biotinylated anti-mouse IgG secondary antibody (5 µg/ml; Vector Laboratories, Burlingame, CA), avidin-biotin-horseradish peroxidase complex (Vectastain Elite kit, Vector Laboratories), and diaminobenzidine substrate (Vector Laboratories). Sections were counterstained with hematoxylin and mounted. IHC for macrophages, fibroblasts, and pericytes was also performed on paraffin-embedded sections of uninjured rat skin as a control.
Alizarin Red Staining for Mineralization
Mineralization in elastin implants and Dacron implants was examined by Alizarin red staining.14 Frozen sections were warmed to room temperature, fixed in 95% ethanol for 30 seconds, stained for 3 minutes at room temperature with 1% Alizarin red solution, and rinsed with distilled water. Sections were counterstained with 1% light green solution for 8 to 10 seconds and rinsed with distilled water and mounted.
Gelatin Zymography for MMP Detection
Proteins from elastin explants with associated capsules and control rat subdermal tissue were extracted in a guanidine buffer, dialyzed, and analyzed in triplicates by gelatin zymography using 10 µg of protein per lane (as per BCA assay), as described before.21 Intensity of MMP bands (white on dark background) were evaluated by densitometry using LabImage software (Labsoft Diagnostics AG, Halle, Germany) and expressed as relative density units (RDUs) normalized to protein content.
Enzyme-Linked Immunosorbent Assay (ELISA) for Elastin Peptides
Quantitation of soluble elastin peptides was performed in the same extracts that were used for zymography by a competitive ELISA as described by Wei and colleagues22 with some modifications. Wells of microtiter plates were coated with 2 µg/ml of elastin peptides (CB573; Elastin Products Company Inc., Owensville, MO). Simultaneously, in a separate plate (precoated with 0.5% bovine serum albumin), samples were mixed 1:1 (v/v) with a 1:500 dilution of rabbit anti-bovine neck elastin antibody (PR403, Elastin Products Company Inc.). This primary antibody is specific for bovine and porcine elastin but does not cross-react with rat elastin.
After overnight incubation of both plates at 4°C, contents of each sample-antibody mixture well were transferred to corresponding wells in the elastin peptide-coated plate and allowed to react for 30 minutes at 37°C, followed by secondary antibody (anti-rabbit IgG peroxidase conjugate, diluted 1:2000). After 60 minutes at 37°C, peroxidase activity was detected using o-phenylene diamine hydrochloride as a substrate at 490 nm. All washes between steps and dilutions of samples and antibodies were performed in phosphate-buffered saline containing 0.05% Tween 20 and 0.5% bovine serum albumin. Extracts obtained from unimplanted elastin served as controls. All assays were done in triplicates and concentrations of soluble elastin peptides were calculated from a standard curve obtained with 0 to 2 µg/ml of competing elastin peptides and expressed as µg per mg protein.
Assays for ALP, OCN, and CBFA Protein Expression
Elastin explants with associated capsules were homogenized in RIPA extraction buffer (50 mmol/L Tris, 150 mmol/L NaCl, 1 mmol/L ethylenediaminetetraacetic acid, 1% Triton X-100, 1% sodium deoxycholate, 0.1% sodium dodecyl sulfate, pH 7.4) including protease inhibitor cocktail (Sigma). Homogenized explants were centrifuged at 13,000 rpm for 15 minutes, supernatants were collected, and protein concentrations were determined using a BCA assay kit (Pierce). ALP activities were examined with p-nitrophenyl phosphate as a substrate (procedure no. 104; Sigma Diagnostic Inc., St. Louis, MO). Enzyme activity was calculated using a p-nitrophenol standard curve and expressed as Sigma U per mg protein. The OCN ELISA was performed using a rat OCN EIA kit (Biomedical Technologies Inc., Stoughton, MA) and following the manufacturer’s instructions. Rat OCN antibody-precoated well strips were incubated with samples at 4°C overnight and reacted with OCN antiserum at 37°C for 1 hour. Donkey anti-goat IgG peroxidase conjugate was used as secondary antibody and 3,3',5,5'-tetra-methyl benzidine was used as the peroxidase substrate. Levels of OCN were calculated using a rat OCN standard curve and normalized to total protein concentration. CBFA-1 was assayed in elastin explants using the TransAM AML-3/Runx2 transcription factor assay kit (Active Motif, Carlsbad, CA) according to the manufacturer’s instructions. Levels of CBFA-1 were calculated using an osteoblast nuclear extract standard curve and normalized to total protein.
RNA Isolation and Characterization
Total RNA was isolated at predetermined time points from elastin-explanted and uninjured rat subdermal tissue using RNeasy fibrous tissue kit (Qiagen, Valencia, CA). RNA quality and quantity were evaluated for each sample using the RNA 6000 Nano Assay, Agilent Bioanalyzer 2100 (Agilent Technologies, Inc., Germantown, MD). The purity of RNA was determined by RIN (RNA integrity number) software algorithm (Agilent Technologies, Inc.), which allows for the classification of eukaryotic total RNA based on a numbering system from 1 to 10, with 1 being the most degraded RNA and 10 being the most intact RNA. The RINs of all RNAs extracted were greater than 9 (data not shown).
Real-Time RT-PCR Analysis
Reverse transcription reactions were performed with 1 µg of total RNA for each 20-µl RT reaction using Moloney murine leukemia virus reverse transcriptase with oligo (dT) primers (RetroScript Kit; Ambion). Target-specific PCR primers were designed using Primer3 software and synthesized by Integrated DNA Technologies Inc. (Coralville, IA). Primer sets, gene accession numbers, and PCR product sizes are listed in Table 1
. Real-time PCR amplifications were performed using SYBR Green PCR kit (Qiagen) in a Rotorgene 3000 thermal cycler (Corbett Research, Mortlake, NSW, Australia), and the cycle number at which the amplification plot crosses the threshold was calculated (CT). The 2–
CT method was used to analyze the relative changes in gene expression from real-time quantitative PCR using ß2-microglobulin (ß2-MG) as a housekeeping gene. Minus RT reactions performed on a representative subset of samples demonstrated that genomic DNA contamination was insignificant (data not shown). Reaction specificities were routinely verified by product purification, agarose gel electrophoresis, as well as routine melting curve analysis. The identities of PCR products were determined by automated sequencing at Arizona State University DNA Analysis Facility. The levels of gene expression were presented as the change in expression of a given time point (X) relative to 3 days as time 0 in a time-course study using the 2–CT method23
as follows: CT = (CT target gene – CT ß2-MG)Time X – (CT target gene – CT ß2-MG)Time 0.
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Data are reported as mean ± SEM. Statistical significance of value at a given time point as compared to the 3-day time point was determined by Student’s t-test with statistical significance defined as P < 0.05.
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Results |
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Calcium content in elastin explants increased progressively with time (P < 0.05 for all time points) and reached 133.65 ± 5.82 µg Ca/mg dry explant at 21 days. Phosphorous content also increased in parallel with calcium content (Figure 1)
. The molar ratios of Ca/P were 2.5, 1.6, 1.5, and 1.4 at 3, 7, 14, and 21 days, respectively, suggesting the presence of poorly crystalline hydroxyapatite deposition. We have previously analyzed the mineral associated with elastin in this animal model by X-ray diffraction and have shown that it resembles hydroxyapatite.24
Control Dacron implants did not show any calcification in this model (0.8 ± 0.09 µg Ca/mg dry explants at 21 days).
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To evaluate the incidence of infiltrating cells in the implants, IHC was performed to identify macrophages, fibroblasts, and pericytes/activated myofibroblasts. Vimentin-positive fibroblasts were observed throughout the capsule and within the elastin implants and they were apparently the dominant cells alongside with infiltrating macrophages (Figure 2, A and B)
. These cells appeared to make intimate contact with the elastin fibers. -Smooth muscle cell actin staining showed the presence of pericytes in small blood vessels forming within the capsule (Figure 2C)
. Normal skin samples of rats showed strong staining for fibroblasts throughout and for pericytes around the vasculature but weak staining for few scattered macrophages (Figure 2, D–F)
. Alizarin red staining showed that elastin fibers started to calcify at the edge of elastin implants as early as 3 days after implantation (data not shown), and significant calcium deposition was noted throughout the entire elastin implant at 21 days after implantation (Figure 2G)
whereas no calcification was observed in Dacron implant at 21 days after implantation (Figure 2H)
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Relative gene expression levels for MMP-2, MMP-9, and TGF-ß1were up-regulated in elastin subdermal implants with increasing elastin calcification (Figure 3)
. mRNA levels of TGF-ß1and MMP-9significantly increased at 7 days and remained at high levels at 2 and 3 weeks, respectively (P < 0.05), in parallel with increasing elastin calcification. In the case of MMP-2and ELR, mRNA levels were significantly increased (P < 0.05) at 7 and 14 days as compared to 3 days after implantation and then decreased at 21 days; overall the levels were lower than control rat subdermal tissue. To evaluate MMP activities, gelatin zymography was performed on protein extracts obtained at various time points from elastin explants and control rat subdermal tissue (Figure 4A)
. At 3 days, MMP-9 activity was the strongest and then decreased slightly with time. MMP-2 activity was also increased at earlier time points and then decreased with time. Overall MMP levels were significantly higher in elastin implants as compared to control subdermal tissue. The content of soluble elastin peptides as a consequence of elastin degradation in elastin explants was determined by ELISA assay and showed the highest levels at 7 days, in parallel with the MMP activities in elastin explants (Figure 4B)
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Real-time RT-PCR was performed on the isolated RNA from elastin explants at different time points to evaluate osteogenic gene expression in cells associated with elastin implants. Relative gene expression of CBFA-1, OPN, and ALPincreased in elastin subdermal implants with increasing elastin calcification, but OCNlevels, a late marker of calcification in osteogenesis, remained unchanged (Figure 5)
. In particular, mRNA levels of CBFA-1, an osteoblast-specific transcription factor, increased significantly at all time points compared to 3 days and attained 9.5-fold higher expression at 21 days than that at 3 days (P < 0.05). ELISA quantitation detected CBFA-1 protein levels in elastin explants at 14 and 21 days after implantation (12.62 ± 0.66 and 6.88 ± 3.27 µg nuclear extract equivalent of osteoblasts/mg protein, respectively).
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1.32-, 5.44-, and 4.07-fold at 7, 14, and 21 days, respectively, compared to 3 days (Figure 6A)
. OCN levels were, however, not significantly changed (P > 0.05) in parallel with mineralization (Figure 6B)
. Overall, protein secretion of ALP and OCN matched with mRNA levels at various time points.
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Discussion |
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MMPs have been identified in pathological conditions in many cardiovascular diseases.6
Longo and colleagues28
reported that macrophage-derived MMP-9 and MMP-2 derived from mesenchymal cells (smooth muscle cells and fibroblasts) work in concert to produce abdominal aortic aneurysms, which involves elastic lamina degeneration. MMP-9 and MMP-2 degrade insoluble elastin to soluble peptides. Elastin peptides have been shown to activate the ELR.8
The ELR was identified on lymphocytes, macrophages, granulocytes, smooth muscle cells, and endothelial cells.29,30
Activation of this receptor by binding of elastin peptides triggers diverse biological activities. These peptides stimulate fibroblast adhesion to elastin fibers,31
regulate cellular proliferation,32
and are chemotactic for several cell types, such as monocytes33
and fibroblasts.9
Substantial infiltration of elastin implants by macrophages/monocytes and fibroblasts, observed by IHC staining (Figure 2, A and B)
, resulted in higher cell densities than those observed in normal rat skin (Figure 2, D and E)
. Increased mRNA expression of MMP-9and MMP-2was observed at all time points compared to 3 days as an earliest time point of calcification (Figure 3)
. Both MMP-9 and MMP-2 showed high enzyme activities at early time points in calcification (Figure 4A)
, and the level of degraded elastin peptides in elastin explants was also increased in parallel with MMP expression (Figure 4B)
. The mRNA level of ELRwas also increased significantly at 7 and 14 days compared to 3 days although the expression levels in implants were lower than those found in normal skin. Thus, it is possible that MMP-mediated degradation of elastin after implantation leads to binding of soluble elastin peptides to ELR on both fibroblasts and macrophages in our implants. The mRNA level of TGF-ß1was also increased in parallel with MMP-2and MMP-9gene expression. Several studies have demonstrated multiple interactions between MMPs and TGF-ß1 that suggest simultaneous expression of these molecules may promote elastin calcification through a positive-feedback mechanism. MMP-2 and MMP-9 can activate TGF-ß1 by cleaving its latent form,34
and TGF-ß1 has been shown to induce an elevated level of MMPgene expression.35,36
TGF-ß1 promotes osteoprogenitor cell proliferation and osteogenesis. TGF-ß1 also stimulates ALP activity in cells.37
Thus, increased expression of TGF-ß1 in our implants may promote osteogenesis.
The occurrence of ectopic osteogenesis process was confirmed by the up-regulated expression of bone-specific genes such as CBFA-1, OCN, OPN, and ALPin elastin implants. Of particular interest, progressive and sustained increases in the expression of the osteoblast-specific transcription factor CBFA-1were observed, attaining 9.5-fold higher expression at 21 days compared to 3 days. Moreover, CBFA-1gene expression was observed 2.3-, 5.7-, 8.7-, and 20.2-fold higher expression at 3, 7, 14, and 21 days, respectively, than that of rat subdermal tissue. Detectable levels of CBFA-1 protein were found at 14 days and 21 days. CBFA-1is the earliest and most specific marker of developmental osteogenesis. CBFA-1acts as an activator of transcription and can induce osteoblast-specific gene expression in fibroblasts and myoblasts.38,39
Significantly increased OPNgene expression was also observed at 7 days and 21 days compared to 3 days and control rat subdermal tissue. OPN is a phosphorylated protein of wide tissue distribution that is found in calcified vascular tissues.40
Giachelli and colleagues41
reported that OPNexpression is increased under injury and disease in many tissues and it is closely related to calcified deposits found in numerous pathologies. We also demonstrated that production of bone marker proteins such as ALP was increased in parallel with calcification on days 14 and 21. OCN content did not significantly change when normalized to total protein.
In the present studies, IHC analysis showed that infiltration by pericytes/activated myofibroblasts was increased in a time-dependent manner along with fibroblast and macrophage invasion. Moreover, RT-PCR and protein analysis clearly indicated the presence of osteoblast-like cells in the vicinity of calcifying elastin. Although the origin of osteoblast-like cells in calcified elastin is not clearly defined in our study, we speculate that activated fibroblasts may undergo cell differentiation and express an osteoblast-like phenotype. In support of this speculation, we have recently observed increased gene and protein expression of CBFA-1 in primary rat skin fibroblast cell cultures exposed to elastin peptides and TGF-ß1 for several days (data not shown). However, more research is needed to identify what specific types of cells are involved in osteogenesis in the rat subdermal implantation model.
The present work involved study of elastin calcification in the rat subdermal implantation model, which may provide some insights for elastin-specific medial calcification seen in vasculature. Elastin in native arteries is surrounded by acidic glycoproteins, including fibrillin, fibulin, and latent TGF-ß1-binding protein among others.42 These glycoproteins may protect elastin from calcification. For example, fibrillin mutant mice develop severe elastin-specific vascular calcification.43 Glycoprotein levels in the arteries decrease with age, in parallel with the propensity for vascular calcification.44 Physical (plaque formation) or biochemical (inflammatory reactions) injury may expose elastin to cells. Cells may then produce MMPs and other serine elastases to degrade elastin. Elastin peptides would then activate ELR on a variety of cells, which in turn up-regulates MMP expression by positive feedback mechanisms. MMPs are also known to degrade glycoproteins surrounding elastin, including fibrillin,45 thus exposing more elastin for degradation. Several stimuli induce up-regulation of MMPgene expression and increase the degradation of matrix. The elastin degradation may release matrix-bound cytokines such as TGF-ß1, which are chemotactic for several cells and induce inflammatory reactions.46,47 These released cytokines may also induce the differentiation of smooth muscle cells or fibroblasts into osteoblast-like cells, which would produce bone-specific proteins and aid the calcification process.35 Vascular smooth muscle cells and pericytes, considered resting mesenchymal stem cells, can assume osteoblastic/chondrocytic phenotypes,3,48,49 implying a phenotypic modulation of resident vascular cells in a permissive matrix environment. TGF-ß1 has also been demonstrated to have osteogenic activity50,51 and to promote the calcification of aortic smooth muscle cells in culture.52
There is also a possibility of cross-talk between the ELR and TGF-ß/BMP-Smad intracellular pathways. The signal transduction pathway of ELR involves activation of phospholipase C by a pertussis toxin-sensitive G-protein. Phospholipase C induces the production of inositol triphosphate (IP3) leading to the increase in the intracellular free calcium.53 Free calcium would then bind to calmodulin. Calmodulin has been shown to bind Smads 1 to 4 in a calcium-dependent manner.54 The Smads were shown to play an important role in transducing specific TGF-ß1 signaling pathways. Recently, Smads were also shown to have links to CBFA-1.55 Thus, interrelations between ELR activation and osteogenic differentiation may exist. However, more research is needed to confirm these interactions.
In conclusion, initiation and progression of elastin calcification in the rat subdermal model involves MMP-mediated elastin degeneration and localized ectopic osteogenesis. The present study may shed some light on the mechanisms of elastin calcification in medial pathological vascular calcification. Ongoing gene silencing studies will extend our investigations of these mechanisms by using delivery of MMP siRNA and CBFA-1 siRNA to block elastin degradation and osteogenesis processes in the rat subdermal model and circulatory aortic injury models.
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Acknowledgements |
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Footnotes |
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Supported by the National Institutes of Health (grant HL-61652).
Accepted for publication October 6, 2005.
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