Proteomics identifies novel biomarkers of synovial joint disease in a canine model of mucopolysaccharidosis I

Abstract

Mucopolysaccharidosis I is a lysosomal storage disorder characterized by deficient alpha-L-iduronidase activity, leading to abnormal accumulation of glycosaminoglycans in cells and tissues. Synovial joint disease is prevalent and significantly reduces patient quality of life. There is a critical need for improved understanding of joint disease pathophysiology in MPS I, including specific biomarkers to predict and monitor joint disease progression, and response to treatment. The objective of this study was to leverage the naturally-occurring MPS I canine model and undertake an unbiased proteomic screen to identify systemic biomarkers predictive of local joint disease in MPS I. Synovial fluid and serum samples were collected from MPS I and healthy dogs at 12 months-of-age, and protein abundance characterized using liquid chromatography tandem mass spectrometry. Stifle joints were evaluated postmortem using magnetic resonance imaging (MRI) and histology. Proteomics identified 40 proteins for which abundance was significantly correlated between serum and synovial fluid, including markers of inflammatory joint disease and lysosomal dysfunction. Elevated expression of three biomarker candidates, matrix metalloproteinase 19, inter-alpha-trypsin inhibitor heavy-chain 3 and alpha-1-microglobulin, was confirmed in MPS I cartilage, and serum abundance of these molecules was found to correlate with MRI and histological degenerative grades. The candidate biomarkers identified have the potential to improve patient care by facilitating minimally-invasive, specific assessment of joint disease progression and response to therapeutic intervention.

Keywords: Cartilage; Dog; Inflammation; Lysosomal storage disease; Serum; Synovial fluid.

Figure 1.

Global analysis of proteomic screening of synovial fluid and serum from 12-month-old MPS I (n=6) and healthy control (n=5) dogs. A. Principal component analyses; B. Heat maps; and C. Volcano plots, showing clustering of samples, and relative up and down regulation of proteins as a function of disease state.

Figure 2.

Up and down regulation of selected proteins with significantly different abundance in MPS I dogs compared to controls. A. Top 10 proteins significantly upregulated and B. Downregulated in MPS I synovial fluid. C. Top 10 proteins significantly upregulated and D. Downregulated in MPS I serum. E. Selected proteins significantly up or down regulated in both MPS I synovial fluid and serum with previously described roles in inflammatory joint disease or lysosomal dysfunction. All p<0.05, N = 5–6.

Figure 3.

Pathway analysis of proteomic screening results for synovial fluid and serum from 12-month-old MPS I (n=6) and healthy control (n=5) dogs. A. Gene ontology (GO) analysis. B. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis.

Figure 4.

Representative immunostaining and quantification of cells immunopositive for A. ITIH3; B. MMP19; and C. A1M in the femoral condylar cartilage of control and MPS I animals at 12 months-of-age. Scale = 100μm (higher magnification = 30μm); *p<0.05 vs control; N=5–6; mean ± SD; unpaired t-test; N = 5–6.

Figure 5.

Magnetic resonance imaging (MRI) of stifle joints from 12-month-old control and MPS I dogs. A. Representative T2-weighted, mid-sagittal images showing fluid effusions cranial (yellow arrow) and caudal (teal arrow) to the joint in an MPS I dog. B. Representative proton density-weighted, dorsal plane images showing meniscal intrasubstance degeneration (red arrow) in an MPS I dog. C-L. Semi-quantitative MRI grades. *p<0.05 vs control; median and interquartile range; Mann-Whitney Test; N = 5–6. M-P. Spearman correlations between protein abundance (normalized log2 intensity) in synovial fluid and overall MRI grade. Q-T. Spearman correlations between protein abundance (normalized log2 intensity) in serum and overall MRI grade.

Figure 6.

Histological assessment of femoral condylar cartilage. A. Representative mid-sagittal sections of femoral medial condylar cartilage from control and MPS I animals at 12 months-ofage. Scale = 2 mm; Safranin O/fast green staining. B. Higher magnification views of the regions indicated in A. showing cell clustering, enlargement and increased density in MPS I cartilage. Scale = 100 μm (inset 30 μm). Quantification of: C. Chondrocyte pathology; D. Proteoglycan staining; E. Cartilage structure; and F. Overall cartilage grade. *p<0.05 vs control; median and interquartile range; Mann-Whitney Test; N = 5–6. G-J. Spearman correlations between protein abundance (normalized log2 intensity) in synovial fluid and overall cartilage grade. K-N. Spearman correlations between protein abundance (normalized log2 intensity) in serum and overall cartilage grade.