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Mucopolysaccharidosis I (MPS I), known as Hurler syndrome in the severe form, is a lysosomal storage disease due to α-l-iduronidase (IDUA) deficiency. It results in fragmentation of elastin fibers in the aorta and heart valves via mechanisms that are unclear, but may result from the accumulation of the glycosaminoglycans heparan and dermatan sulfate. Elastin fragmentation causes aortic dilatation and valvular insufficiency, which can result in cardiovascular disease. The pathophysiology of aortic disease was evaluated in MPS I mice. MPS I mice have normal elastic fiber structure and aortic compliance at early ages, which suggests that elastin assembly is normal. Elastin fragmentation and aortic dilatation are severe at 6 months, which is temporally associated with marked increases in mRNA and enzyme activity for two elastin-degrading proteins, matrix metalloproteinase-12 (MMP-12) and cathepsin S. Upregulation of these genes likely involves activation of STAT proteins, which may be induced by structural stress to smooth muscle cells from accumulation of glycosaminoglycans in lysosomes. Neonatal intravenous injection of a retroviral vector normalized MMP-12 and cathepsin S mRNA levels and prevented aortic disease. We conclude that aortic dilatation in MPS I mice is likely due to degradation of elastin by MMP-12 and/or cathepsin S. This aspect of disease might be ameliorated by inhibition of the signal transduction pathways that upregulate expression of elastase proteins, or by inhibition of elastase activity. This could result in a treatment for patients with MPS I, and might reduce aortic aneurism formation in other disorders.
Mucopolysaccharidosis I (MPS I) is an autosomal recessive lysosomal storage disease due to α-l-iduronidase (IDUA; EC 220.127.116.11) deficiency that results in the accumulation of the glycosaminoglycans (GAG) heparan and dermatan sulfate . The severe (Hurler syndrome), intermediate (Hurler–Scheie syndrome), and mild (Scheie syndrome) forms of MPS I can all result in fragmented elastin fibers in the ascending aorta and cardiac valves in humans [2,3], which can result in aortic insufficiency due to reduced structural integrity of the valve. Dog [4,5] and mouse [3,6,7] models of MPS I also have elastin fragmentation of the ascending aorta and heart valves, which can result in aortic dilatation in addition to aortic insufficiency.
Elastic fibers are complex structures that consist primarily of tropoelastin monomers organized into a highly crosslinked polymer in a process that involves elastin binding protein (EBP), components of extracellular matrix microfibrils, and crosslinking enzymes . Abnormalities in any of the molecules or assembly steps could result in the fragmented elastic fibers seen in MPS I tissues. Indeed, EBP levels were low and elastin biogenesis was reduced in fibroblasts from patients with MPS I, and it was proposed that defects in elastin assembly resulted in fragmented elastic fibers in the aorta .
An alternative explanation for elastin fragmentation is that MPS I results in activation of elastin-degrading proteins such as metalloproteinases (MMPs), cathepsins, or neutrophil elastase [10,11]. The metal-dependent MMP-2, 7, 8, 9, 10, 12, and 14 can degrade elastin and/or play a role in aortic aneurisms [12–15]. Furthermore, MMP-9 or MMP-12 deficiency reduced elastin fragmentation within atherosclerotic lesions in hypercholesterolemic mice  or reduced CaCl2-induced aneurism formation [12,16]. In addition, deficiency of MMP-9, but not MMP-12, reduced aneurism formation after elastase perfusion . MMPs are activated proteolytically by urokinase plasminogen activator (uPA), or without cleavage by SIBLINGs (small integrin-binding ligand N-linked glycoproteins) such as osteopontin . uPA deficiency reduced aneurism formation  while osteopontin is upregulated in aortic aneurisms . Tissue inhibitor of metalloproteinases (TIMP) 1 binds and inactivates MMPs, while TIMP-2 protects MMPs from inactivation by TIMP-1; TIMP-1 overexpression  and TIMP-2  deficiency reduced aneurism formation in mice.
Cathepsins are lysosomal cysteine proteases that can degrade elastin and are upregulated in aneurisms . Although most are rapidly inactivated at the neutral pH found in the extracellular space, cathepsin S maintains 64% of its peak activity at pH 7.5 . Cathepsin S RNA is increased in atherosclerotic lesions in mice  and its deficiency reduced elastin fiber fragmentation in hypercholesterolemic mice . Cystatin C is a cathepsin inhibitor, and its mRNA is reduced in human aortic aneurisms, and its deficiency increases elastin fragmentation in hypercholesterolemic mice .
The goal of this study was to determine the pathogenesis of elastic fiber fragmentation in the ascending aorta of MPS I mice. We demonstrate here that elastin structure and function are normal in young mice, suggesting that MPS I does not adversely affect elastin synthesis and assembly in vivo. However, increases in mRNA and enzyme activity for the elastolytic proteases MMP-12 and cathepsin S correlated with progressive elastin fragmentation and aortic dilatation in older mice, suggesting that the mechanism underlying aortic disease in MPS I is enhanced elastin degradation.
Animal studies were approved by our Institutional Review Board. MPS I mice  were on a C57BL/6 background and normal controls were heterozygous littermates. Some MPS I mice were injected IV with 1010 transducing unit (U) U/kg of the retroviral vector hAAT-cIDUA-WPRE at 2–3 days after birth . For compliance curves, periadventitial fat was removed from ascending aortas and the outer diameters determined halfway between the sinotubular junction and the innominate artery . Echocardiography was performed with a Vevo 770 echocardiography machine (VisualSonics, Toronto ON) using inhaled isofluorane anaesthesia.
Ascending aortas were fixed as described  and a region 3 mm from the aortic valve was obtained. Six micrometer sections were stained with Verhoeff's Van Gieson (VVG) stain. A micrometer was used to measure a length of 0.4 mm that was parallel to the elastic fibers, and the number of fragmented elastin fibers throughout all layers of the aorta was determined at >5 different regions. The number of breaks per millimeter aorta length was calculated.
For RNA and enzyme analysis, the ascending aorta from the sinotubular junction to the innominate artery was stripped of periadventitial tissue. A 5–10 mg piece was homogenized for 30 s in 150 μl of RNeasy micro kit buffer (Qiagen Inc., Valencia, CA) with a hand-held pellet pestle (Kimble-Kontes; Vineland, NJ) and incubated for 10 min with proteinase K and purified according to the manufacturer's instructions. Reverse transcription (RT) was performed on DNase I-treated RNA with an oligo (dT) 20 primer using a Superscript III kit from Invitrogen Corp. (Carlsbad, CA) in a 20 μl volume, followed by real-time PCR on 0.4 μl of each cDNA sample per well using SYBR green reagents from Applied Biosystems (Foster City, CA) unless otherwise stated. The primers are listed in Table 1 of the supplementary methods section. The percent of an RNA to that of β-actin was calculated by subtracting the cycle to reach the threshold (CT) for a gene from the β-actin CT to determine the ΔCT, and the formula: Percent β-actin = (100) × 2ΔCT.
An MMP-12 assay kit (EnzolyteTM 490 MMP-12) was obtained from Anaspec (San Jose, CA). According to the manufacturer, the substrate is also cleaved by MMP-1, 2, 3, 8, and 13. Ascending aortas were homogenized in 100 μl of assay buffer, centrifuged at 10,000g for 5 min at 4 °C, and the protein concentration determined with the Bradford assay (Bio-Rad Laboratories, Hercules CA). Approximately 3 μg of extract was assayed in a 100 μl volume using substrate concentrations recommended by the manufacturer, and the fluorescent units were measured every 5 min with a Fluoroskan Ascent microplate fluorometer from Thermo Electron Corporation (Milford, MA) with excitation at 355 nm and emission at 460 nm. Standards were 5-[(2-aminoethyl)amino]naphthalene-1-sulfonic acid in assay buffer. One unit of enzyme releases 1 nmol of product per hour at 37 °C. All inhibitors were from Calbiochem (San Diego CA) and were used at the following final concentration. EDTA (10 mM) and EGTA (10 mM) chelate metals; cathepsin inhibitor I (Z-Phe-Gly-NHO-Bz; 200 μM) inhibits cathepsins B, L, and S; cathepsin S inhibitor Z-FL-COCHO (10–1000 pM) inhibits cathepsin S; calpain inhibitor I (20 μg/ml) inhibits cathepsin B and L and calpain I and II; the sulfhydryl inhibitor iodoacetamide inhibits cathepsins and other cysteine proteases; phenylmethylsulfonyl fluoride (PMSF; 1 mM) inhibits serine proteases; pepstatin A (1 μM) inhibits cathepsin D, pepsin, and renin; and bestatin (10 μM) inhibits aminopeptidase B and leucine aminopeptidase. Samples were incubated with the inhibitor for 10 min at 4 °C prior to starting the assay.
For the cathepsin S assay, aortas were homogenized in 100 mM Na–Acetate pH 5.5 containing 2.5 mM EDTA, 0.01% Triton X-100, and 2.5 mM dithiothreitol (DTT). Assays used 20 μM benzyloxycarbonyl-l-phenylalanyl-l-arginine-7-amido-4-methylcoumarin (Z-Phe-Arg-AMC; Anaspec) at pH 7.5 in 100 mM Na–Acetate with 2.5 mM EDTA, 0.01% Triton X-100, and 2.5 mM DTT in a microtiter plate  with ~0.3 μg of extract at 37 °C. The amount of product was determined by excitation at 355 nm and emission at 460 nm using a kinetic reading for a microtiter plate and comparison with 7-amino-4-methylcoumarin standards from Anaspec. One unit of enzyme releases 1 nmol of the product per hour at 37 °C.
Aortas were embedded in OCT from Akura Finetek (Torrance, CA) and 8 μm-thick sections were mounted on Superfrost adhesive slides (Fisher Scientific, USA) and fixed with 4% paraformaldehyde in PBS for 20 min at 4 °C. Endogenous peroxidase activity was inhibited using 0.6% hydrogen peroxidase in Tris Buffered Saline (TBS; 40 mM Tris pH 7.4 with 150 mM NaCl). Staining was performed using polyclonal rabbit antibodies from Cell Signaling Technology (Danvers MA) that were diluted 1:100. After overnight incubation at 4 °C with the first antibody in blocking buffer (TBS with 10% normal goat serum), samples were incubated with a 1:200 dilution of biotinylated anti-rabbit antibody (VECTOR Laboratories, Inc., Burlingame CA) for 1 h at room temperature, and developed with the ABC reagents provided in the kit. Negative controls used non-specific rabbit IgG as the primary antibody.
The Student's t test compared values between 2 groups, and ANOVA with Tukey post hoc analysis compared values between 3 groups using Sigma Stat software (Systat Software, Inc., Point Richmond, CA).
The time course of development of ascending aortic dilatation and elastin fragmentation in MPS I mice was evaluated by studying the mechanical properties and pathology of aortas at different ages at a point halfway between the sinotubular junction and the innominate artery (Fig. 1). The outer diameters of ascending aortas from MPS I mice were modestly dilated at 1.5 and 3 months when 75 mm of Hg of pressure was applied at 1.6 ± 0.1 and 1.7 ± 0.2 mm [error bars represent standard deviation (SD)], respectively, which were statistically higher than the values of 1.4 ± 0.08 and 1.4 ± 0.03 mm in age-matched heterozygous normal littermates, respectively (p = 0.005 and 0.027, respectively). Aortas from MPS I mice were massively dilated at 3.3 ± 0.5 and 3.2 ± 0.6 mm at 6 and 8 months, respectively, which was >2-fold the normal value of 1.5 ± 0.05 mm in 6-month-old and 1.5 ± 0.1 in 8-month-old normal mice (A).
Echocardiography at 8 months of age demonstrated that the internal diameter at a similar region of the aorta was 2.3 ± 0.5 mm in MPS I mice, which was 1.5-fold the value of 1.6 ± 0.1 mm in normal mice (B and C; p = 0.01). In addition, the aorta of MPS I mice was dilated at the aortic valve (1.2-fold; 1.17 ± 0.13 mm for normal and 1.37 ± 0.06 mm for MPS I; p = 0.01), sinus of Valsalva (1.2-fold; 2.12 ± 0.20 mm for normal and 2.61 ± 0.37 for MPS I; p = 0.02), sinotubular junction (1.4-fold; 1.50 ± 0.19 mm for normal and 2.19 ± 0.48 mm for MPS I; p = 0.02), arch (1.2-fold; 1.48 ± 0.07 mm for normal and 1.76 ± 0.27 mm for MPS I; p = 0.05), and the descending aorta (1.2-fold; 1.18 ± 0.08 mm for normal and 1.37 ± 0.14 mm for MPS I; p = 0.02). A video of the aortas can be viewed in the online version.
Panel D quantifies the number of elastin breaks in the ascending aorta, and E–J show representative examples of the elastin stains that were used to obtain these data. There were very few fragmented elastin fibers at early times in MPS I mice, but there were 5- and 10-fold as many elastin fiber breaks in MPS I mice as in normal mice at 6 and 8 months, respectively. Lysosomal storage material was minimal at early times and apparent at 6 and 8 months, although this was harder to appreciate on the relatively thick paraffin sections used here than in our previous study that used thin plastic sections .
Previously, we reported that neonatal intravenous (IV) injection of a retroviral vector expressing canine IDUA resulted in high IDUA activity in liver, serum, and aorta, and this prevented the accumulation of lysosomal storage, elastin fragmentation, and aortic dilatation in MPS I mice . The mechanism of correction was probably secretion of mannose 6-phosphate (M6P)-modified IDUA by transduced hepatocytes or other cells into blood, diffusion of enzyme to the aorta, and uptake of enzyme by cells via the M6P receptor. Retroviral vector-treated MPS I mice had normal compliance curves at 6 months (Fig. 1A), and there were only 8.3 ± 2.4 and 7.5 ± 0.8 elastin fiber breaks per mm of aorta at 6 and 8 months, respectively (Fig. 1D).
Genes that play a role in elastin or collagen biogenesis were expressed at normal levels in MPS I aortas at 6–8 months after birth, as determined by the real-time reverse transcriptase (RT) PCR shown in Fig. 2A. However, there were striking and statistically significant elevations in mRNA for two elastin-degrading proteins, matrix metalloprotease-12 (MMP-12; 23 ± 6-fold normal) and cathepsin S (11 ± 2-fold normal). In addition, there were elevations in mRNA for a protein that can degrade collagen but not elastin (MMP-3 at 7-fold normal), proteins that can activate MMPs (osteopontin at 16-fold normal and uPA at 3-fold normal), proteins that can associate with MMPs and affect their activity (TIMP-1 at 4-fold normal and TIMP-2 at 2-fold normal), and cathepsins that have low activity at neutral pH (cathepsin B at 2-fold normal, cathepsin D at 3-fold normal, and cathepsin L at 2-fold normal). In general, mRNA levels for genes that were elevated at 6 to 8 months were relatively normal at 1.5 months and modestly elevated at 3 months (Fig. 2B), which correlates well with the time course of lysosomal storage accumulation, elastin fiber fragmentation, and aortic dilatation. mRNA levels were normal for MMP-12 and cathepsin S in retroviral vector-treated MPS I mice at 6–8 months of age. However, osteopontin and TIMP-1 mRNA remained elevated in retroviral vector-treated mice at 7- and 3-fold normal, respectively, and values were not significantly different from those in untreated MPS I mice.
Ascending aorta extracts were tested for MMP activity. Cleavage of a peptide that is recognized by MMP-12 was 8-fold normal in MPS I mice at 6 months (p < 0.001; Fig. 3A), although it is possible that some other MMP such as MMP-3 contributed to this enzyme activity, as detailed in the methods. This activity was inhibited by the metal chelators EDTA and EGTA, but was not affected by other protease inhibitors (Fig. 3B).
Although the substrate Z-Phe-Arg-AMC is cleaved by cathepsins B and L in addition to cathepsin S, only cathepsin S is stable at pH 7.5  that was used for the reaction. Cathepsin S activity was markedly elevated at 65-fold normal (p < 0.001) at 6 months in MPS I mice (Fig. 3C). The response to inhibitors confirmed that this activity was due to cathepsin S, as it was inhibited with very low concentrations of a cathepsin S-specific inhibitor in addition to being inhibited by a non-specific cathepsin inhibitor and by iodoacetamide (Fig. 3D).
The MMP-12 promoter contains binding sites for STAT-5, N-myc, CCAAT, PEA3, AP-1 (fos/jun dimers), and ets-1  and sequence analysis suggests that it should also bind STAT-1 and STAT-3 . Phosphorylation of STATs at tyrosine residues results in dimerization, translocation to the nucleus, DNA binding, and activation of responsive genes. In addition, the activity of STATs is increased by phosphorylation of serines by mitogen-activated protein kinase (MAPK) . Immunostaining of ascending aortas isolated from MPS I mice at 8 months showed a marked increase in nuclear levels of STAT-1 that was phosphorylated at Ser727 and Tyr701, and in STAT-3 that was phosphorylated at Ser727, and Tyr705 (Fig. 4). In addition, MAPK was activated by phosphorylation at Thr202/Tyr204. In contrast, there were very low levels of STAT-5 that was phosphorylated at Tyr694 (data not shown). mRNA levels for transcription factors that are regulated transcriptionally and can influence MMP-12 expression were not altered in ascending aortas from MPS I (Fig. 5).
Although the cathepsin S promoter does not contain STAT binding sequences, it binds interferon-regulated factor 1 (IRF-1), which is regulated transcriptionally by activated STATs . Indeed, although the mRNA for IRF-1 was not elevated in MPS I mouse ascending aorta (Fig. 5), mRNA for the related proteins IRF-8 and IRF-3 were 2.3 ± 0.5-fold (p = 0.04 vs. normal) and 1.6 ± 0.2-fold normal (p = 0.02 vs. normal), respectively, at 6 to 8 months. In addition, mRNA for PU.1, a transcription factor that can increase expression of cathepsin S  was 3.0 ± 0.5-fold normal in MPS I mice (p = 0.01 vs. normal). Activation of SMAD2 via the TGF-β receptor has been implicated in the pathophysiology of Marfan Syndrome, which is also associated with progressive fragmentation of elastin fibers . Indeed, aortas from MPS I mice had a modest increase in the RNA levels of TGF-β1 to 1.9-fold normal, although there was no increase in RNA levels for TGF-β2, and there was no increase with immunostaining in the level of SMAD2 that was phosphorylated at Ser465 and Ser467 in the aorta of MPS I mice (date not shown).
The etiology of elastin fragmentation in the aorta and heart in MPS I is important because it is a major cause of morbidity, and is relatively refractory to treatments. For example, aortic insufficiency developed de novo or worsened in 89% of patients at 12 years after performing hematopoietic stem cell transplantation at ~2 years  and in 60% of patients at 4–6 years after starting enzyme replacement therapy [31,32]. Although pathological data were not available, progressive fragmentation of the elastic fibers likely contributed to worsening aortic valve function. In addition, gene therapy in mice  and dogs  only fully reduced aortic elastin fragmentation if serum IDUA activity was very high. The dense structure of the aorta likely reduces diffusion of enzyme, which may explain why the middle region is most refractory to treatment after gene therapy [5,7].
Elastin fiber fragmentation could be due to defects in elastin assembly or to elastin degradation. Our findings of relatively normal elastic fiber structure and vessel compliance in young adult MPS I mice suggest that elastin synthesis and elastic fiber assembly are normal during the formative period of the vessel wall. This finding is in contrast to the abnormal elastic fiber assembly reported in vitro for fibroblasts from MPS I patients, which was associated with low EBP levels . EBP is an enzymatically-inactive splice variant of β-galactosidase that binds to galactose moieties and potentiates elastin assembly  and it was proposed that the accumulation of the N-acetylgalactosamine-containing dermatan sulfate reduced EBP levels and prevented EBP from functioning as a chaperone for elastin biogenesis . It remains possible that a defect in EBP-mediated elastin assembly contributes to elastin fragmentation in ascending aorta in MPS I mice. However, at 8 months, immunostaining of aorta with an anti-EBP antibody  that recognizes the mouse protein (A. Hinek, personal communication) was negative for both normal and MPS I mouse aortas (data not shown), which is consistent with the low EBP mRNA levels at this age (Fig. 2).
Our data strongly favor the hypothesis that elastin degradation was the major cause of the fragmented fibers in MPS I mice at late times. Since RNA for MMP-12 and cathepsin S were 23- and 11-fold normal, respectively, these elastin-degrading proteins might be responsible. Indeed, enzyme activity against an MMP-12 substrate was 8-fold normal in MPS I mice, although it remains possible that another metalloproteinase such as MMP-3 was responsible. In addition, cathepsin activity was 65-fold normal in MPS I mice with an assay that was performed at a neutral pH at which cathepsin S is active but most other cathepsins are inactive  and this activity was extremely sensitive to a cathepsin S-specific inhibitor. Although it is likely that each enzyme can act independently to cleave elastin, we plan to breed MPS I mice with MMP-12  and/or cathepsin S  deficient mice to directly test the role of each protein in the pathogenesis of disease in the aorta and other sites. Since both MMP-12  and cathepsin S  are expressed by vascular smooth muscle cells (VSMC) in vitro, and there was no evidence of migration of macrophages into the aorta as assessed by immunostaining for Mac I (data not shown), VSMC were probably the source of MMP-12 and cathepsin S expression.
We propose that activation of STAT-1 and/or STAT-3 in MPS I mice by phosphorylation resulted in upregulation of MMP-12 and cathepsin S. As noted above, the MMP-12 promoter has a sequence that is predicted to bind to STAT-1 and STAT-3. Although the cathepsin S promoter does not have a respectable STAT binding site, cathepsin S expression in VSMC was activated by IL-6 in a STAT-3-dependent process , which probably involved upregulation of IRF proteins. Indeed, mRNA for IRF-8 and IRF-3 were increased in the ascending aorta of MPS I mice, and these proteins may function in a similar fashion on the cathepsin S promoter as IRF-1 . In addition, TIMP-1 is regulated by STAT-3 , and its mRNA was 4-fold normal (Fig. 2). It is interesting that mechanical stress to cultured VSMC resulted in phosphorylation of STAT-3 in a src-dependent fashion . We propose that the stress on the cell from accumulation of lysosomal storage material may activate STAT-1 and STAT-3, although alternative explanations are possible.
The MMP-12 promoter also contains a binding site for PEA3, which is activated by MAPK  and we showed here that MAPK was phosphorylated at sites that increase its activity. In addition, the osteopontin promoter contains a PEA3 binding site , and osteopontin mRNA was 16-fold normal in MPS I mice. PU.1 is an ets family transcription factor that acts synergistically with IRF-8 to induce expression of genes , and is expressed in VSMC  and regulates cathepsin S and osteopontin expression. PU.1 mRNA levels were 3-fold normal at 6 months in MPS I mice.
This study suggests that up-regulation of MMP-12 and/or cathepsin S may be pivotal to the development of aortic dilatation and valvular degeneration in MPS I and related diseases. In addition, these proteins may contribute to manifestations of disease in other organs, such as skin, ligaments, and tendons. Indeed, MMPs contributed to joint disease in other MPS models  while cathepsin S was upregulated in the brain of MPS I mice . If the genetic cross that is in progress shows that deficiency of one or both of these elastase proteins can reduce aortic disease in MPS I, it might be possible to treat MPS I patients with inhibitors of MMP-12  and/or cathepsin S .
We thank Alexander Hinek for the anti-EBP antibody, Xu Zhang for performing immunostaining, Elizabeth Neufeld for MPS I mice, Robert Thompson and John Curci for helpful discussions, and Clay Semenkovich and Trey Coleman for help with real-time PCR.
Sources of funding
This work was supported by the Ryan Foundation, the National MPS Society, and the National Institutes of Health (DK66448). Histology was supported by P30 DK52574 and real-time PCR was supported by DK20579 awarded to Clay Semenkovich.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.ymgme.2008.03.018.