Upregulation of elastase activity in aorta in mucopolysaccharidosis I and VII dogs may be due to increased cytokine expression

Abstract

Mucopolysaccharidosis I (MPS I) and MPS VII are due to loss-of-function mutations within the genes that encode the lysosomal enzymes alpha-l-iduronidase and beta-glucuronidase, respectively, and result in accumulation of glycosaminoglycans and multisystemic disease. Both disorders are associated with elastin fragmentation and dilatation of the aorta. Here, the pathogenesis and effect of gene therapy on aortic disease in canine models of MPS was evaluated. We found that cathepsin S is upregulated at the mRNA and enzyme activity level, while matrix metalloproteinase 12 (MMP-12) is upregulated at the mRNA level, in aortas from untreated MPS I and MPS VII dogs. Both of these proteases can degrade elastin. In addition, mRNA levels for the interleukin 6-like cytokine oncostatin M were increased in MPS I and MPS VII dog aortas, while mRNA for tumor necrosis factor alpha and toll-like receptor 4 were increased in MPS VII dog aortas. These cytokines could contribute to upregulation of the elastases. Neonatal intravenous injection of a retroviral vector expressing beta-glucuronidase to MPS VII dogs reduced RNA levels of cathepsin S and MMP-12 and aortic dilatation was delayed, albeit dilatation developed at late times after gene therapy. A post-mortem aorta from a patient with MPS VII also exhibited elastin fragmentation. We conclude that aortic dilatation in MPS I and MPS VII dogs is likely due to degradation of elastin by cathepsin S and/or MMP-12. Inhibitors of these enzymes or these cytokine-induced signal transduction pathways might reduce aortic disease in patients with MPS.

Keywords: Mucopolysaccharidosis; aorta; canine; cathepsin S; elastin; gene therapy; lysosomal storage disease.

Fig. 1. Aorta diameters in MPS VII dogs

Echocardiograms were performed for normal dogs, untreated MPS VII dogs, and MPS VII dogs that received IV injection of 0.3 to 1×10¹⁰ TU/kg of the RV designated hAAT-cGUSB-WPRE at 2 to 3 days after birth (RV-treated). The latter achieved stable expression of GUSB activity in serum for the duration of evaluation. A. Representative images of aortas. Images are shown for one normal dog at 6 months (aortic dilatation score 0), one of 5 untreated MPS VII dog at 6 months (score +2), one of 13 RV-treated MPS VII dog at 6 months (score 0; dog M2420 with 317 U/ml of serum GUSB activity), and one of three RV-treated MPS VII dog that was evaluated at 8 years (score +2; M1332 with 111 U/ml of serum GUSB activity). The double-headed arrows indicate the inner diameters of the aorta at the position where dilatation was evaluated during diastole (top) and systole (bottom), and the left ventricle (LV) is indicated. For some images, the vertical single-headed arrows identify the narrowing of the aorta seen at the sinotubular junction in diastole, which is a normal finding. For other images, the horizontal single-headed arrow identifies a thickened base of the aortic valve that shows little movement during systole. The size marker represents 1 cm. B. Average aortic dilatation scores. Aortas were scored from 0 (normal) to +4 (severe dilatation) for the indicated (N) number of untreated MPS VII and RV-treated MPS VII dogs as shown in panel A, and the average +/− standard error of the mean (SEM) plotted. Normal dogs are not shown but generally have scores of 0. Untreated MPS VII dogs do not survive beyond 2 years. The Student’s t-test was used to compare values in the 2 groups at each time point where MPS VII dogs were evaluated, and * indicates a p value of 0.01 to 0.05, and ** indicates a p value <0.01.

Fig. 2. Elastin stain in MPS VII dog aortas

Aortas were obtained from normal, untreated MPS VII, or RV-treated MPS VII dogs. Sections were stained with VVG, which stains elastin dark, and representative examples are shown here, which are quantified for all animals that were evaluated in Fig. 3. The left panels show low power images, where the intima is located at the lower right and the adventitia is at the upper left, and the scale bar is 200 µm. The boxes identify Region 1 (inner media), Region 2 (middle media), and Region 3 (outer media) where high power images were obtained, which are shown in the 3 columns to the right, where the scale bar represents 25 µm. In some panels, the black arrows identify fragmented elastin fibers and black arrowheads identify the clear-appearing lysosomal storage. For RV-treated dogs, M2420 (317 U/ml of serum GUSB activity), M1653 (88 U/ml), and M1337 (281 U/ml) were evaluated at 6 months, years, and 8 years, respectively.

Fig. 3. Quantitation of elastin in MPS VII dog aortas

Computer software was used to determine the percent of the inner region of the aorta that consisted of elastin , as detailed in the methods section, and as shown for representative examples in Fig. 2. For normal dogs, 5 animals were evaluated at 3 months and are shown as the black circle that shows the average percentage elastin +/− SEM , while 6 adult dogs are shown as individual black circles, with the linear regression line shown for those values. The indicated number of MPS VII dogs were evaluated at the indicated age; values for two 3 month-old MPS VII dogs compared with those from five 3 month-old normal dogs, and values from the nine 6 month-old, four 12 month-old, and four 24 month-old MPS VII dogs were compared with the values in six adult normal dogs using the Student’s t test. Four RV-treated MPS VII dogs were evaluated at 6 months (M1312, M2420, M2427, and M2428 with 281, 317, 1227, and 752 U/ml of serum GUSB activity, respectively) and values were compared with 6 month-old MPS VII dogs and with adult normal dogs. Three additional RV-treated MPS VII dogs were evaluated at older ages (M1653 at 4 years with 88 U/ml, M1328 at 7.7 years with 503 U/ml, and M1337 at 8.3 years with 281 U/ml) and are shown as individual data points.

Fig. 4. mRNA levels of genes involved in extracellular matrix and signal transduction in MPS VII dog aortas

Real-time RT-PCR was used to determine the amount of each mRNA in the aorta for samples from 4 normal and 9 untreated MPS VII dogs that were isolated at 6 months of age, and the ratio of expression to that in normal dogs was determined. For genes whose expression was elevated in MPS VII dogs and was sufficiently abundant for accurate evaluation, levels were also evaluated in 7 MPS VII dogs that received neonatal gene therapy with an RV; 3 were sacrificed at 6 months of age (M2420, M2427, and M2428 with 317, 1227, and 752 U/ml of serum GUS activity, respectively), and 1 dog each was sacrificed at 24 months (M2065 with 2089 U/ml), 52 months (M1653 with 88 U/ml), 92 months (M1328 with 503 U/ml), and 99 months (M1337 with 281 U/ml). Since there were no major differences for samples isolated at different ages, these values were pooled. When only 2 groups were evaluated, statistical comparisons were between values from normal and untreated MPS VII dogs using the student’s T test and are indicated with the number of asterisks about the bar for normal mice, where * indicates a p value of 0.01 to 0.05, and ** indicates a p value <0.01. When 3 groups were evaluated, ANOVA with Tukey post-hoc analysis was used to compare values between normal dogs and RV-treated dogs with those in untreated MPS VII dogs. Abbreviations are collagen 1α2 (Col1α2), collagen III (Col III), cathepsin (Cath), and a disintegrin and metalloproteinase with a thrombospondin type 1 motif (ADAMTS).

Fig. 5. Cathepsin and MMP activities in MPS VII dogs

Ascending aortas were isolated at 6 months after birth from 6 normal and 5 untreated MPS VII dogs, and from 2 RV-treated MPS VII dogs at 25 months (M2065; 2090 U/ml of serum GUSB activity) and at 52 months (M1653; 88 U/ml) of age and homogenenates were prepared. A. Cathepsin activity. Cathepsin assays were performed with Z-Phe-Arg-AMC (Phe-Arg) or Z-Arg-Arg-AMC (Arg-Arg) at the indicated pH to measure primarily activities for cathepsin S (Cath S), cathepsin B (Cath B), or several cathepsins (All Cath) as indicated, and the mean activity +/− SEM is shown. Statistics were performed with ANOVA with Tukey post-hoc analysis, with ** indicating a p value <0.01 for comparison of values from the indicated group with those in untreated MPS VII dogs. B. Effect of cathepsin inhibitors. Only samples from 3 untreated MPS VII dogs were analyzed for the effect of specific cathepsin inhibitors at the indicated final concentrations upon enzyme activity using the substrate Z-Phe-Arg-AMC at pH 7.5, which primarily measures cathepsin S activity. Values with the inhibitor were compared with values without the inhibitor using the Student’s t-test. C. MMP-12 activity. Average MMP-12 activity ± SEM was determined.

Fig. 6. Evaluation of MPS I dog aortas

Some MPS I dogs received neonatal IV injection of 1×10¹⁰ TU/kg of the RV hAAT-cIDUA-WPRE (RV-treated) as previously described [14], while other MPS I and normal dogs were untreated. Aortas were evaluated at 1 to 1.8 years of age. A. Elastin fragmentation. Elastin fragmentation was quantified in the inner region of the aorta (see Region 1 in Fig. 2) for the indicated number (N) of animals, as described in Fig. 3, and the average +/− SEM is shown. RV-treated MPS I dogs that were evaluated included two dogs with relatively low serum IDUA activity [I-99 with 23 U/ml (2-fold normal) serum IDUA activity and I-101 with 27 U/ml (2-fold normal)], and two dogs with relatively high serum IDUA activity [I-107 with 553 U/ml (43-fold normal), and I-140 with 888 U/ml (68-fold normal)]. The p value determined using ANOVA with Tukey post-hoc analysis for comparison of the groups joined with a bracket are shown. B. mRNA levels. mRNA levels for the indicated genes in aortas of 8 normal, 3 untreated MPS I, and 3 RV-treated MPS I dogs are shown as a ratio to the values found in normal dogs. The RV-treated dogs that were analyzed included two dogs with relatively low expression (I-99 and I-101) and one dog with relatively high expression (I-107). C and D. Cathepsin and MMP-12 enzyme activity. Cathepsin and MMP-12 assays were performed as described in Fig. 5A and 5C for the number of animals described in panel A.

Fig. 7. Evaluation of aorta from a patient with MPS VII

This patient was the first to be diagnosed with MPS VII [19, 49]. A. Chest radiograph. At 18 years-of-age, a posterior-anterior radiograph of this patient demonstrates dilatation of the ascending aorta, as indicated by the black arrows. B to D. Elastin stain. The patient died at 19 years of age and an autopsy was performed. Sections of the aorta were stained with VVG. B. Atheroma. A low power view demonstrated a large atheroma (arrow) on the inner surface of the aorta. Scale bar = 400 µm. C. Lysosomal storage at low power. The white arrow identifies a region in the inner third of the aorta with lysosomal storage and elastin fragmentation. The intima is at the upper right and the adventitia is at the lower left. Scale bar = 200 µm. D. Lysosomal storage and elastin fragmentation at high power. A high power image was obtained from the inner aortic media. The black arrow identifies fragmented elastin while the white arrowhead identifies the clear-appearing lysosomal storage. Scale bar = 25 µm.