; and Summa Health Systems, Akron, Ohio (IG) S U M M A R Y To test the hypothesis that a perturbation of endoplasmic reticulum (ER) function is involved in the pathogenesis of osteoarthritis (OA), articular cartilage was isolated from non-OA patients secondary to resection of osteo-or chondrosarcomas. Intra-joint samples of minimal and advanced osteoarthritic cartilage were isolated from patients undergoing total knee arthroplasty and scored for disease severity. Glucose-regulated protein-78 (grp78) and bcl-2-associated athanogene-1 (bag-1) were detected via immunofluorescence as markers of non-homeostatic ER function. Additionally, the expression of type VI collagen and its integrin receptor, NG2, was determined to examine cartilage matrix health and turnover. There was an upregulation of grp78 in advanced OA, and variable expression in minimal OA. Non-OA cartilage was consistently grp78 negative. The downstream regulator bag-1 was also upregulated in OA compared with normal cartilage. Collagen VI was mainly cellassociated in non-OA cartilage, with a more widespread distribution observed in OA cartilage along with increased intracellular staining intensity. The collagen VI integral membrane proteoglycan receptor NG2 was downregulated in advanced OA compared with its patientmatched minimally involved cartilage sample. These results suggest that chondrocytes exhibit ER stress during OA, in association with upregulation of a large secreted molecule, type VI collagen. (J Histochem Cytochem 57:923-931, 2009) K E Y W O R D S osteoarthritis cartilage ER stress bag-1 grp78 collagen VI NG2 OSTEOARTHRITIS (OA) IS A PROGRESSIVE DISEASE, with changes in chondrocyte gene expression and extracellular matrix (ECM) composition occurring before macroscopic structural damage is observed (Hamerman 1989). Articular chondrocytes in OA exhibit a hypermetabolic phenotype, with studies describing highly proliferative clonal chondrocytes, along with the upand downregulation of ECM molecules such as aggrecan

A consequence of mitochondrial (mt) electron transport and energy production is continual formation of reactive oxygen species (ROS). MtROS production is usually compensated by high activities of antioxidants, e.g., superoxide dismutases 1 and 2. However, under various physiological or pathological states, mtROS production exceeds antioxidant defenses and oxidative (Ox) modification of mt proteins and mtDNA occurs. Current techniques to detect mtOx stress include estimations of ROS, Southern blotting to measure mtDNA fragmentation, or immunostaining and HPLC to measure Ox stress biomarkers such as 8‐hydroxy‐2′‐deoxyguanosine (8OHdG). These techniques are laborious and require specialized procedures (use of radioisotopes) or equipment (HPLC). Our goal was to devise and evaluate a new, simple method of assessing mtOx stress. Total cellular or mtDNA was isolated from liver and heart from Zucker lean and obese rats. Equal amounts of DNA were fixed to a nitrocellulose membrane and an immunoblotting protocol was performed with an antibody against 8OHdG. Levels of 8OHdG in heart mtDNA were less in Zucker lean than those from obese rats, consistent with other measurements from our laboratories showing greater mtROS production in obese compared to lean rats. The proposed method is useful to measure ROS‐induced DNA damage because it is convenient, semi‐quantitative, and accurate even with small amounts of DNA.