Upregulation of elastase proteins results in aortic dilatation in mucopolysaccharidosis I mice

Abstract

Mucopolysaccharidosis I (MPS I), known as Hurler syndrome in the severe form, is a lysosomal storage disease due to alpha-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.

Fig. 1

Aortic diameter and elastin histochemistry. (A) Outer diameter of aortas. The average outer diameter of ascending aortas ± standard deviation (SD) at the indicated age after birth was determined at the indicated pressures for 5–6 animals in each group. Untreated MPS I and normal mice were evaluated at all times. Retroviral vector-treated MPS I mice were evaluated at 6 months and appear as the triangles that are almost indistinguishable from the values in normal mice. * indicates a p value of 0.01–0.05 and ** indicates a p value <0.01 when values in other groups were compared with those in normal mice. (B and C) Internal aortic diameter. The internal diameter of aortas from normal and untreated MPS I mice was determined by echocardiography at 8 months. (B) Representative examples of enddiastolic images, while (C) the average end-diastolic diameter for 5–7 animals in each group at the aortic valve (AV), sinus of Valsalva (SV), sinotubular junction (STJ), ascending aorta (AA), aortic arch (Arch), and descending aorta (DA). (D) Elastin breaks in the aorta. The number of elastin breaks throughout the thickness of the aorta was determined as described in Materials and methods. (E–J) Elastin stain of ascending aorta. A VVG stain for elastin was performed on an ascending aorta from a normal mouse at 3 months (E), on aortas from untreated MPS I mice at 1.5–8 months (F–I), or on an aorta from a retroviral vector (RV)-treated MPS I mouse at 8 months. The adventitia is at the bottom right, and the intima is at the top left. Fragmented elastin fibers are identified with white arrows, and lysosomal storage with black arrows.

Fig. 2

mRNA levels of genes involved in extracellular matrix biogenesis and degradation. Real-time RT-PCR was used to determine the amount of each mRNA in the aorta for 3–6 mice in each group. (A) mRNA relative to normal at 6–8 months. The average ratio to normal ± SD was determined for each gene in aortas isolated at 6–8 months. Abbreviations are elastin binding protein (EBP), microfibril-associated glycoprotein (MAGP), matrix metalloproteinase (MMP), urokinase plasminogen activator (uPA), tissue inhibitor of metalloproteinase (TIMP), and neutrophil elastase (NE). (B) Time course of mRNA levels relative to β-actin. RNA from untreated MPS I and normal mice was evaluated at all times. RNA from retroviral vector (RV)-treated MPS I mice was only evaluated at 6–8 months, and the values were artifacturally plotted at a slightly later time to distinguish them from values in normal mice. The ratio of the signal for each mRNA to β-actin was determined by a comparison of the C_T values. * indicates a p value of 0.01–0.05 and ** indicates a p value <0.01 when values in other groups were compared with those in normal mice.

Fig. 3

Evaluation of MMP-12 and cathepsin S activity. Ascending aortas were isolated at 6–8 months after birth from untreated MPS I or normal mice. (A and B) MMP-12 activity. In (A), average MMP-12 activity ± SD was determined for 6 mice for each group, and * indicates a p value of 0.01–0.05 and ** indicates a p value <0.01 when values in MPS I mice were compared with those in normal mice. In (B), samples from MPS I mice were incubated with protease inhibitors, and activity with the inhibitor was compared with the activity without the inhibitor for samples from 4 different mice, with the same criterion for statistically significance as shown in (A). Abbreviations include cathepsin inhibitor 1 (CI-1), cathepsin S inhibitor (CSI; 1 nM was used), calpain inhibitor I (Cal1), iodoacetamide (IA), phenylmethylsulfonyl fluoride (PMSF), pepstatin A (PepA), and bestatin (Bes). (C and D) Cathepsin S activity. In (C), cathepsin S activity was determined for 4 mice from each group using Z-Phe-Arg-AMC at pH 7.5, while (D) tests the effect of inhibitors upon activity in MPS I mice.

Fig. 4

Immunostaining for phosphorylated transcription factors. Ascending aortas from normal or untreated MPS I mice collected at 8 months were immunostained with antibodies specific for transcription factors that were activated by phosphorylation at the indicated amino acid. (A and B) Hematoxylin and eosin stain (H and E). The black arrows indicate smooth muscle cell nuclei (blue). (C and D) Negative control. Negative controls that received a non-specific rabbit IgG antibody as the first antibody. (E and F) STAT-1 Ser727. The black arrow indicates a nucleus with STAT-1 that is phosphorylated at serine 727, which stains brown. (G and H) STAT-1 Tyr701. (I and J) STAT-3 Ser727. (K and L) STAT-3 Tyr705. (M and N) MAPK Thr202/Tyr204.

Fig. 5

Evaluation of transcription factor mRNA in aorta. Aortas obtained at 6–8 months after birth were evaluated for levels of mRNAs that are known to regulate MMP-12 or cathepsin S transcriptionally, and shown as the average relative to normal ± SD for 4–6 animals in each group. IL-1β represents interleukin 1β. * indicates a p value of 0.01–0.05 and ** indicates a p value <0.01 when values in other groups were compared with those in normal mice.