where normal-appearing cartilage was present for all patients analyzed (Fig. 2A and insert), suggesting the hypermetabolic activity next to degenerated tissue. In contrast, our results indicated that the rate of sulfated-GAG synthesis was significantly reduced (1.2- to 3.9-fold) in late OA cartilage (Fig. 2A and insert), suggesting that it was less active in terms of sulfated-GAG synthesis. As illustrated in Fig. 1, staining for toluidine blue was also less evident in late OA cartilage section (compare a to b and c). Noteworthy, variations in the rate of GAG anabolism was observed in cartilage from normal areas among the patients studied indicating inter-individual variations in cartilage metabo- lism (Fig. 2A). Gene expression patterns of XT-I and aggrecan define the pathologic state of the disease in OA cartilage To determine if the differences observed in sulfated-GAG synthesis in human OA cartilage were associated with differences in the expression of XT-I responsible for initiation of chondroitin sulfate and heparan sulfate GAG synthesis, we performed qPCR analysis in cartilage samples obtained from areas of ‘‘normal’’, early disease, and late disease. Depending upon the severity of the lesions, alterations in expression pattern of XT-I gene was observed between diseased and normal areas. Striking increases (1.74- to 8-fold) in the expression level of XT-I gene in OA cartilage were seen in the regions next to lesion compared to normal (Fig. 2B and insert). However, in late-stage OA regions, levels of XT-I expression were significantly lower (1.5- to 7-fold) compared to normal (Fig. 2B and insert). Thus, XT-I downreg- ulation in diseased cartilage may be one of the major determinants of sulfated GAG depletion and disease progression. Furthermore, our results also showed that the alterations in aggrecan gene expression paralleled that of XT-I expression with an increase of 2- to 8-fold in regions next to lesion and a decrease of 1.5- to 7-fold in late-stage OA areas (Fig. 2C and insert). Disease-associated alterations in XT activity in human OA cartilage As shown in Fig. 3A, the xylosyltransferase activity in cartilage from three representative OA patients was increased by 1.2- to 2.5-fold in samples from regions next to lesion when compared with samples from normal regions. In contrast, the activity was decreased by 1.5- to 4-fold in late-stage OA cartilage samples compared with cartilage from normal areas. These results clearly demonstrate that changes in sulfated-GAG synthesis are related to the expression and enzymatic activity of XT-I and to the severity of cartilage lesion. FACE analyses of chondroitin sulfate in human OA cartilage Analysis of the fine structure and disaccharide composition of CS-GAG chains in human cartilage by FACE demonstrated the presence of unsaturated monosulfated (Ddi6S and Ddi4S) and nonsulfated (DDi0S) disaccharides from CS/DS chains in normal cartilage. Next, FACE results suggested that 6-sulfated disaccha- rides were abundant in adult human cartilage. While it is likely that disulfated and higher sulfated products might have been present in the CS-GAG chains, we were unable to profile such units under our assay conditions. Furthermore this study revealed a disease-related quantitative variation of CS disaccharides (Fig. 3B). Notably, a strong increase (80%) in disaccharides content occurs in cartilage from regions next to lesion, when compared to normal regions. In contrast, disaccharides content was dramatically reduced (2.5-fold) in tissue obtained from late- XT-I and Proteoglycan Synthesis in Osteoarthritis Figure 2. Analysis of GAG synthesis and expression of XT-I and aggrecan genes. (A) GAG synthesis analysis in cartilage from ‘‘normal’’, early OA and late-stage OA areas for each patient studied by 35S-sulfate incorporation. Insert shows the mean of GAG synthesis rate values corresponding to different areas of cartilage sample from all patients. Values are mean 6 SEM of 3 experiments per parameter, per joint, per patient. (B) and (C) Analyses of the expression of XT-I and aggrecan genes in cartilage from normal, early OA and late-stage OA areas for each patient. The expression level of the genes was analyzed by qPCR. Measurements were normalized to the control ‘‘normal’’. Inserts in (B) and (C) show the mean of XT-I and aggrecan gene expression level values corresponding to different areas of cartilage sample from all patients. Values are mean 6 SD of 3 experiments per parameter, per joint, per patient. **significantly (P,0.01) higher than normal and late groups; 1significantly (P,0.05) higher than normal and late groups; *significantly (P,0.05) lower than normal. doi:10.1371/journal.pone.0034020.g002 stage OA areas (Fig. 3B). Thus, it seems very likely that altered gene expression and activity of XT-I might explain the changes observed in CS content in cartilage from areas next to lesion and PLoS ONE | www.plosone.org 4 March 2012 | Volume 7 | Issue 3 | e34020