The transient receptor potential vanilloid 4 ion channel regulates the biosynthetic response of chondrocytes to dynamic loading

s / Osteoarthritis and Cartilage 21 (2013) S9–S62 S17 immuno-histological staining was analyzed using an arbitrary scale. Osteophyte formation was measured using digital image analysis. Bone marrow derived cells were differentiated into macrophages and preincubated with oxLDL or LDL for 24 hours, after which they were stimulatedwith the endogenous toll-like receptor 4 ligand S100A8. RNA was analyzed for gene expression. Data are depicted as mean SEM. Results: WT mice receiving a normal diet developed moderate cartilage destruction (6.1 1.5), synovial thickening (1.4 0.2) and osteophyte formation (32.4mm2 14.6). SerumLDL levelswere significantly higher in LDLr-/-micecompared toWTmice (7.33mmol/L 0.46and0.54mmol/L 0.04 respectively; p<0.05), which was additionally increased by a cholesterol-rich diet (38.73 mmol/L 3.11; p<0.0001). Despite differences in serum LDL levels, no significant differences between the four groups were found regarding synovial thickening and cartilage destruction. Expression of S100A8 by the synovial lining, however, was increased after receiving a cholesterol-rich diet, suggesting synovial activation. Furthermore, a cholesterol-rich diet increased ApoB accumulation in synovial lining macrophages of LDLr-/mice. Interestingly, at the tibial plateau, LDLr-/mice showed almost a fourfold increase of osteophyte formation compared to WT mice (206.3 mm2 36.3; p<0.05). When receivingacholesterol-richdiet, osteophyte formationat the lateral sideof the tibialplateau inLDLr-/-mice further increased from107.0mm2 49.3 to 309.4 mm2 41.7 (p<0.05). In vitro stimulation of oxLDL-laden macrophages with S100A8 showed a significant decrease of anti-inflammatory cytokine IL-10 expression and an increase of BMP6 expression compared to macrophages that were not pre-incubated with oxLDL. Conclusions: Increased serum cholesterol levels by either LDL receptor deficiency or as a result of a cholesterol-rich diet stimulate oxLDL uptake by synovial lining macrophages, resulting in synovial activation. In accordance with in vitro data, synovial activation elicited by oxLDL leads to an inflammatorymilieu reflected by increased S100A8 levels andmay stimulate osteophyte formation at the margins of the tibial plateau. 18 THE TRANSIENT RECEPTOR POTENTIAL VANILLOID 4 ION CHANNEL REGULATES THE BIOSYNTHETIC RESPONSE OF CHONDROCYTES TO DYNAMIC LOADING C.J. O'Conor y,z, H.C. Benefield y, W. Liedtke y, F. Guilak y. yDuke Univ., Durham, NC, USA; zUniv. of North Carolina, Chapel Hill, NC, USA Purpose: Mechanical factors play an important role in modulating the metabolic and functional properties of normal and engineered articular cartilage, but the mechanisms of mechanical signal transduction are not fully understood. TRPV4 (Transient Receptor Potential Vanilloid 4) is a Ca2+-permeable non-selective cation channel that functions in the transduction of osmotic changes in chondrocytes. Cartilage loading produces changes in local tissue osmolality due to fluid exudation and changes infixed charge density. Therefore, TRPV4maybe involved in the metabolic and functional response of articular cartilage to mechanical stimulation. The goal of this study was to determine the role of TRPV4 activation in the response of chondrocyte-laden agarose constructs to dynamic compressive loading (DL), as well as to determine if specific chemical activation of TRPV4 enhances cartilage matrix biosynthesis. Methods: Porcine chondrocytes were enzymatically isolated from skeletally immature pigs (w30kg) and mixed with agarose to form chondrocyte-laden disks (2% agarose, 4mm diameter x 2.25mm thick, 10M cells/mL). Constructswere cultured in 10% FBS containingmedia for 2 weeks, following which disks were loaded between impermeable platens in unconfined compression (10% peak-to-peak strain, 7% offset, 1.0Hz sine wave, 3 hrs/day, 5 days/wk for 4 wks), or left in free swelling conditions. To better understand the role of TRPV4-mediated signaling during DL, GSK205, a specific TRPV4 antagonist, or vehicle control (0.1% DMSO), was added prior to and washed off immediately after loading (n1⁄43-4). To determine the response of chondrocytes to selective chemical activation of TRPV4, day 3 constructs (20M cells/mL) were pulsedwith GSK101, a potent and specific TRPV4 agonist, or with vehicle control (0.1% DMSO), for 1 hr/day, 5 days/wk, for 4 wks (n1⁄46-8). Constructs were evaluated for sulfated glycosaminoglycan (s-GAG) and collagen content using the DMMB and OHP assays. To evaluate construct functional properties, disks were mechanically tested in unconfined compression between impermeable platens in PBS. After the application of a small (1.3gf) tare load for 5 min, constructs were equilibrated under a 10% strain for 20min, fromwhich the Young'smodulus was calculated. The dynamicmoduluswasmeasured by superimposing a 0.1Hz 2% peakto-peak sine wave upon this 10% strain, and dividing the amplitude of the resulting stress wave by the applied strain. One-way ANOVA was performed followed by Fisher's LSD post-hoc analysis. Results: DL increased both biochemical measures and the dynamic modulus of the constructs. Daily exposure to GSK205 alone had little effect on construct bulk biochemical content and functional properties. Underdynamic loadingconditions,GSK205blocked thedynamic loadinginduced increase in GAG and collagen content, and reduced both the Young's modulus and dynamic modulus compared to DL disks without GSK205. In unloaded constructs, daily chemical activation of TRPV4 with GSK101 significantly increased total s-GAG and collagen contents, as well as enhanced the Young's and dynamic moduli of the constructs. Conclusions: These findings suggest that the biosynthetic response of chondrocytes to dynamic compressive loading is mediated by TRPV4. Inhibition of TRPV4 with GSK205 largely blocked the anabolic response of chondrocyte-laden constructs to dynamic loading. Furthermore, periodic selective chemical activation of TRPV4 increasedmatrix content and functional properties of constructs in a similar manner to what was observed with dynamic loading. An improved understanding of TRPV4 in chondrocytemechanotransductionmay provide new insights into the development of novel therapies for osteoarthritis as well as the enhancement of tissue-engineering approaches for cartilage repair. Abstracts / Osteoarthritis and Cartilage 21 (2013) S9–S62 S18s / Osteoarthritis and Cartilage 21 (2013) S9–S62 S18 Ă19 PROTEOGLYCAN 4 EXPRESSION PROTECTS AGAINST THE DEVELOPMENT OF OSTEOARTHRITIS M.Z. Ruan, A. Erez, K. Guse, B. Dawson, T. Bertin, Y. Chen, M.-M. Jiang, J. Yustein, F. Gannon, B. Lee. Baylor Coll. of Med., Houston, TX, USA Purpose: Osteoarthritis (OA) is a common degenerative condition that afflicts more than 70% of the population between 55 and 77 years of age. Although its prevalence is rising globally with aging of the population, current therapy is limited to symptomatic relief and in severe cases, joint replacement surgery.We aim todevelop a novel therapeuticmodality by intra-articular over-expression of proteoglycan 4. In addition, we aim to investigate the related mechanism in the pathogenesis of OA. Methods: To study the effects of long-term expression of Prg4 on OA, we generated proteoglycan 4 (Prg4) transgenic mice. To translate localized expression of Prg4 into a therapeutic approach, we delivered Prg4 by intra-articular injection of helper-dependent adenoviral vectors (HDV). To investigate the related mechanism of OA pathogenesis, we performed gene expression profiling of mouse articular cartilage obtained by laser capture micro-dissection (LCM), in vitro cell studies and analysis of human OA gene profiling. Results: Long-term Prg4 expression under the type II collagen promoter (Col2a1) does not adversely affect skeletal development but protects from developing signs of age-related osteoarthritis. The protective effect is also shown in a model of post-traumatic osteoarthritis created by cruciate ligament transection (CLT). Moreover, intra-articular injection of HDV expressing Prg4 protected against the development of posttraumatic osteoarthritis when administered either before or after injury. Gene expression profiling of mouse articular cartilage obtained by LCM and in vitro cell studies show that Prg4 expression inhibits the transcriptional programs that promote cartilage catabolism and hypertrophy. Analyses of available human OA datasets are consistent with the predictions of this model. Conclusions: Our data offer long-term intra-articular expression of Prg4 as a potential chondro-protective approach to OA treatment and provide insight into the mechanisms for OA development. 20 EARLY CHONDROCYTE HYPERCELLULARITY AND APOPTOSIS MAY BE CORRELATED WITH OSTEOCHONDRAL JUNCTION CHANGE AT OSTEOARTHRITIS ONSET IN DUNKIN-HARTLEY STRAIN GUINEA PIGS T. Wang, C.-Y. Wen, W.-W. Lu, K.-Y. Chiu. The Univ. of Hong Kong, Hong