Faculty Bibliography

INTRODUCTION: An increase in the concentration of loose cartilage debris in the joint space has been associated with aging, overuse and obesity resulting in osteoarthritis (OA) [1]. Cartilage and bone particulates have been observed bound to the surface or embedded within the synovium of OA patients who also present with synovial hyperplasia and metaplasia [2]. While the involvement of cartilage debris in synovial inflammation has been long suspected in the clinic and demonstrated in animal models [3], the mechanism of action behind the interaction remains to be elucidated. In the current study, we hypothesize that synovial fibroblasts (SF) have an inflammatory response to sub-lOum cartilage debris that can be further modulated by the presence of pro-inflammatory cytokines and is also mediated by phagocytosis. Using similar sized latex beads as a non-biologic control, we also sought to examine and characterize differences in biosynthetic activity of SF to co-culture with biologic and non-biologic wear particulates in the presence or absence of cytokines. METHODS: Tissue Harvest: Synovial tissue and articular cartilage explants were harvested from the knee joints of bovine calf knees (n=3). Synovial tissue was digested with collagenase IV, and the isolated SF were plated and expanded for two passages with 5 ng/mL bFGF-2. Cartilage explants were maintained in serum free media [4]. Particulate Generation: Cartilage particles were sterilely generated from the superficial and middle zones by manually abrading explants (n=2) with 120 grit sandpaper. The particle solution was filtered with lOum nylon mesh filter. Latex and cartilage wear particles were counted and sized as in [5] (Figure 1 A,B). Imaging: SF were cultured with FITC labeled latex particles or cartilage particles labeled with dichlorotriazinyl aminofluorescein and counterstained with Calcein Red-Orange AM after 48 hours. Dosage Optimization: SF were plated overnight prior to treatment. The optimal particle dosage was defined as the lowest dosage of particles that yielded a significant increase in MTT (metabolism) absorbance normalized to the 0 particle dosage group after 5 days of culture (Figure 1C). Dense Monolayer Co-Culture: SF were plated at a high density overnight and co-cultured with +/- 10 ng/mL IL-la or TNF-a and +/- latex or cartilage particles for 5 days (n=6-8). Particles alone were cultured +/-10 ng/mL IL-la (not shown) or TNF-a. Biochemistry: DNA glycosaminoglycan (GAG), collagen (COL) and nitric oxide (NO) were determined using the Picogreen dsDNA, DMMB dye binding, OHP and Griess assays, respectively. Biochemical content of wear particles alone was subtracted from the measured values of the resulting SF sheet with particles to determine the contribution of SF alone. Statistics: Comparisons were analyzed using ANOVA with Fisher's LSD post-hoc test (p<0.05). RESULTS: Latex and cartilage wear particles had average diameters of 1.05 +/- 0.28 um and 0.85 +/- 0.45 um, respectively. For both latex and cartilage wear particles the lowest concentration that increased cellular metabolism was 250 particles per cell or <0.002%v/v for both particle types. Latex particles were readily phagocytosed by the majority of SF, with 5-20 particles internalized per cell (Figure 2A). Meanwhile, cartilage wear particles were selectively phagocytosed by SF, with positive cells only internalizing 1-5 particles (Figure 2A). Cartilage wear particles were also observed on the surface of the SF (not shown). With cartilage particle co-culture, cellular proliferation was significantly increased compared to non-treated controls or latex treated cells regardless of cytokine treatment (Figure 3A). The DNA content of the cartilage wear particles themselves constituted <1% that of the DNA of the final cell monolayer (not shown). Cell monolayer GAG and GAG synthesis decreased with cytokine treatment and was increased after cartilage wear particle treatment without cytokines only (Figure 3B). Cellular COL was significantly elevated in cartilage particle treated groups. However, COL synthesis was decreased compared to the control for latex and cartilage particle treated groups. Cell monolayer COL and COL synthesis per cell decreased with cytokine treatment across all groups. However, with TNF treatment, only cartilage particle synthesis was increased compared to the control (Figure 3C). Media GAG increased with cartilage treatment and cytokine treatment. However, in TNF treated groups, media GAG per cell was decreased in latex and cartilage treated groups (Figure 3D). NO in the media increased with cytokine and cartilage particle treated groups (Figure 3E). Across all cell and media biochemical measurements, cartilage particle treated groups had a muted response to cytokine treatment, compared to control and latex treated groups. DISCUSSION: Based on the results, we reject our hypothesis that the inflammatory response caused by cartilage wear particles is synergistic with cytokine insult, implicating perhaps a shift in the anabolic-catabolic balance of treated SF. Notably, the proliferative response of SF to cartilage wear particles resulted in an overall increase in extracellular matrix content, which may be suggestive of synovial hyperplasia often reported in OA. Treatment of SF with cartilage wear particles had an inflammatory effect, resulting in decreased anabolic synthesis (less overall matrix normalized by DNA) and increased catabolic activity (increased media GAG/DNA and NO/DNA). This observation is consistent with previous work that reported co-culture with cartilage particles results in increased TNF-a gene expression [1] and the release of proteinases [6]. This effect was not observed following phagocytosis of latex particles, suggesting that the inflammatory response of SF to cartilage wear particles is mediated by a mechanism beyond non-specific phagocytosis. Further characterization of the inflammatory response will investigate other factors upregulated in OA including prostaglandin E2, IL-6, and matrix metalloproteinases. Efforts to better understand the cartilage particle-induced proliferative response is ongoing, including study of the cadherin-11, which regulates synovial architecture [8]. SIGNIFICANCE: Cartilage wear debris play a role in the onset of synovium-mediated inflammation in OA. An in vitro model that can explore the interaction between cartilage particles and the synovium will foster the development of effective strategies to mitigate their negative interactions

The disabling and painful disease osteoarthritis (OA) is the most common form of arthritis. Strong evidence suggests that a subpopulation of OA patients has a form of OA driven by inflammation. Consequently, understanding when inflammation is the driver of disease progression and which OA patients might benefit from anti-inflammatory treatment is a topic of intense research in the OA field. We have reviewed the current literature on OA, with an emphasis on inflammation in OA, biochemical markers of structural damage, and anti-inflammatory treatments for OA. The literature suggests that the OA patient population is diverse, consisting of several subpopulations, including one associated with inflammation. This inflammatory subpopulation may be identified by a combination of novel serological inflammatory biomarkers. Preliminary evidence from small clinical studies suggests that this subpopulation may benefit from anti-inflammatory treatment currently reserved for other inflammatory arthritides.

BACKGROUND: During osteoarthritis and following surgical procedures, the environment of the knee is rich in proinflammatory cytokines such as IL-1. Introduction of tissue-engineered cartilage constructs to a chemically harsh milieu may limit the functionality of the implanted tissue over long periods. A chemical preconditioning scheme (application of low doses of IL-1) was tested as a method to prepare developing engineered tissue to withstand exposure to a higher concentration of the cytokine, known to elicit proteolysis as well as rapid degeneration of cartilage. METHODS: Using an established juvenile bovine model system, engineered cartilage was preconditioned with low doses of IL-1alpha (0.1 ng/mL, 0.5 ng/mL, and 1.0 ng/mL) for 7 days before exposure to an insult dose (10 ng/mL). The time frame over which this protection is afforded was investigated by altering the amount of time between preconditioning and insult as well as the time following insult. To explore a potential mechanism for this protection, one set of constructs was preconditioned with CoCl2, a chemical inducer of hypoxia, before exposure to the IL-1alpha insult. Finally, we examined the translation of this preconditioning method to extend to clinically relevant adult, passaged chondrocytes from a preclinical canine model. RESULTS: Low doses of IL-1alpha (0.1 ng/mL and 0.5 ng/mL) protected against subsequent catabolic degradation by cytokine insult, preserving mechanical stiffness and biochemical composition. Regardless of amount of time between preconditioning scheme and insult, protection was afforded. In a similar manner, preconditioning with CoCl2 similarly allowed for mediation of catabolic damage by IL-1alpha. The effects of preconditioning on clinically relevant adult, passaged chondrocytes from a preclinical canine model followed the same trends with low-dose IL-1beta offering variable protection against insult. CONCLUSIONS: Chemical preconditioning schemes have the ability to protect engineered cartilage constructs from IL-1-induced catabolic degradation, offering potential modalities for therapeutic treatments.

OBJECTIVE: Inflammatory mediators, such as PGE2 and IL-1beta, are produced by osteoarthritic joint tissues, where they may contribute to disease pathogenesis. We examined whether inflammation, reflected in plasma and peripheral blood leukocytes (PBLs) reflected presence of osteoarthritis (OA), progression or symptoms in patients with symptomatic knee osteoarthritis (SKOA). METHODS: SKOA patients were enrolled in a 24-month prospective study of radiographic progression. Standardized knee radiographs were obtained at baseline and 24 months. Biomarkers assessed at baseline included plasma lipids PGE2 and 15-HETE, and transcriptome analysis of PBLs by microarray and qPCR. RESULTS: Baseline PGE synthases (PGES) by PBL microarray gene expression, and plasma PGE2 distinguished SKOA patients from non-OA controls (AUCs 0.87 and 0.89 respectively, p<0.0001). Baseline plasma 15-HETE was significantly elevated in SKOA versus non-OA controls (p<0.019). In the 146 patients who completed the 24-month study, elevated baseline expression of IL-1beta, TNFalpha and COX-2 mRNA in PBLs predicted higher risk for radiographic progression by joint space narrowing (JSN). In a multivariate model, AUC point estimates of models containing COX-2 in combination with demographic traits overlap the confidence interval of the base model in two out of the three JSN outcome measures (JSN >0.0mm, >0.2mm and >0.5mm, AUC=0.62-0.67). CONCLUSION: Inflammatory plasma lipid biomarkers PGE2 and 15-HETE identify patients with SKOA. PBL inflammatory transcriptome identifies a subset of SKOA patients at higher risk for radiographic progression. These findings may reflect low-grade inflammation in OA and may be useful as diagnostic and prognostic biomarkers in clinical development of disease-modifying OA drugs

OBJECTIVE: Pro- and anti-inflammatory mediators, such as IL-1beta and IL1Ra, are produced by joint tissues in osteoarthritis (OA), where they may contribute to pathogenesis. We examined whether inflammatory events occurring within joints are reflected in plasma of patients with symptomatic knee osteoarthritis (SKOA). DESIGN: 111 SKOA subjects with medial disease completed a 24-month prospective study of clinical and radiographic progression, with clinical assessment and specimen collection at 6-month intervals. The plasma biochemical marker IL1Ra was assessed at baseline and 18 months; other plasma biochemical markers were assessed only at 18 months, including IL-1beta, TNFalpha, VEGF, IL-6, IL-6Ralpha, IL-17A, IL-17A/F, IL-17F, CRP, sTNF-RII, and MMP-2. RESULTS: In cross-sectional studies, WOMAC (total, pain, function) and plasma IL1Ra were modestly associated with radiographic severity after adjustment for age, gender and body mass index (BMI). In addition, elevation of plasma IL1Ra predicted joint space narrowing (JSN) at 24 months. BMI did associate with progression in some but not all analyses. Causal graph analysis indicated a positive association of IL1Ra with JSN; an interaction between IL1Ra and BMI suggested either that BMI influences IL1Ra or that a hidden confounder influences both BMI and IL1Ra. Other protein biomarkers examined in this study did not associate with radiographic progression or severity. CONCLUSIONS: Plasma levels of IL1Ra were modestly associated with the severity and progression of SKOA in a causal fashion, independent of other risk factors. The findings may be useful in the search for prognostic biomarkers and development of disease-modifying OA drugs.

We investigated the role of periostin, an extracellular matrix protein, in the pathophysiology of osteoarthritis (OA). In OA, dysregulated gene expression and phenotypic changes in articular chondrocytes culminate in progressive loss of cartilage from the joint surface. The molecular mechanisms underlying this process are poorly understood. We examined periostin expression by immunohistochemical analysis of lesional and nonlesional cartilage from human and rodent OA knee cartilage. In addition, we used small interfering (si)RNA and adenovirus transduction of chondrocytes to knock down and up-regulate periostin levels, respectively, and analyzed its effect on matrix metalloproteinase (MMP)-13, a disintegrin and MMP with thrombospondin motifs (ADAMTS)-4, and type II collagen expression. We found high periostin levels in human and rodent OA cartilage. Periostin increased MMP-13 expression dose [1-10 microg/ml (EC50 0.5-1 mug/ml)] and time (24-72 h) dependently, significantly enhanced expression of ADAMTS4 mRNA, and promoted cartilage degeneration through collagen and proteoglycan degradation. Periostin induction of MMP-13 expression was inhibited by CCT031374 hydrobromide, an inhibitor of the canonical Wnt/beta-catenin signaling pathway. In addition, siRNA-mediated knockdown of endogenous periostin blocked constitutive MMP-13 expression. These findings implicate periostin as a catabolic protein that promotes cartilage degeneration in OA by up-regulating MMP-13 through canonical Wnt signaling.-Attur, M., Yang, Q., Shimada, K., Tachida, Y., Nagase, H., Mignatti, P., Statman, L., Palmer, G., Thorsten, K., Beier, F., Abramson, A. B. Elevated expression of periostin in human osteoarthritic cartilage and its potential role in matrix degradation via matrix metalloproteinase-13.

Osteoarthritis (OA) is a highly prevalent, disabling joint disease with no existing therapies to slow or halt its progression. Cartilage degeneration hallmarks OA pathogenesis, and pannexin 3 (Panx3), a member of a novel family of channel proteins, is upregulated during this process. The function of Panx3 remains poorly understood, but we consistently observed a strong increase in Panx3 immunostaining in OA lesions in both mice and humans. Here, we developed and characterized the first global and conditional Panx3 knockout mice to investigate the role of Panx3 in OA. Interestingly, global Panx3 deletion produced no overt phenotype and had no obvious effect on early skeletal development. Mice lacking Panx3 specifically in the cartilage and global Panx3 knockout mice were markedly resistant to the development of OA following destabilization of medial meniscus surgery. These data indicate a specific catabolic role of Panx3 in articular cartilage and identify Panx3 as a potential therapeutic target for OA. Lastly, while Panx1 has been linked to over a dozen human pathologies, this is the first in vivo evidence for a role of Panx3 in disease. KEY MESSAGE: Panx3 is localized to cartilage lesions in mice and humans. Global Panx3 deletion does not result in any developmental abnormalities. Mice lacking Panx3 are resistant to the development of osteoarthritis. Panx3 is a novel therapeutic target for the treatment of osteoarthritis.

Purpose: We and others have demonstrated low grade inflammation exists in OA joint tissues, where it may contribute to disease pathogenesis. In the current studies we assessed whether inflammatory events occurring within joint tissues were reported in the peripheral blood leukocytes (PBLs) of patients with symptomatic knee OA (SKOA). Methods: PBL inflammatory gene expression (IL-1, TNFalpha, COX-2) was assessed in two independent cohorts of patients with SKOA, and a cohort of healthy control subjects: 1) 111 patients with tibiofemoral medial OA and 21 healthy volunteers from the NYUHJD Cohort, and 2) 200 patients from the OAI progression cohort who had "high quality radiographs", at both baseline and 24 months, and had KL2 or 3 in the signal knee at baseline. Radiographic progression was defined as narrowing of medial joint space width (JSW) in the signal knee between baseline and 24-months in each cohort. Radiographic progressors were defined as subjects who had JSN >0.0, 0.2 and 0.5mm over 24 months. For measuring predictive performance, we used the area under the curve (AUC) of a receiver operating characteristics (ROC). OAI SKOA subjects were dichotomized as radiographic non-progressors (JSN <0.0 mm) and progressors (JSN>0.0mm) for association studies. Results: Elevated PBL expression of IL-1, TNFalpha or COX-2 identified SKOA patients who were "fast progressors" (mean JSN 0= 0.71, 0.75 and 0.71 mm / 24 months, respectively) compared to patients with levels below the median. In a multivariable model, anthropometric traits alone (BMI, gender, age) did not predict progression, whereas addition of PBL gene expressions improved prediction of fast progressors (JSN>0.5mm). We next examined inflammatory gene expression in PBLs of radiographic progressors in the OAI cohort. Similar to the NYUHJD cohort, elevated expression of IL-1beta, TNFalpha and COX-2 mRNA distinguished radiographic progressors from non-progressors (Table 1). PBL IL-1beta expression found to be strongest predictor of all three radiographic progressors. In multivariate models that combine all three markers did not improve upon IL-1beta predictivity. We thus conclude that either the signal in TNFalpha and Cox-2 is already subsumed by IL-1beta and/or that it is not easy to capture the non-overlapping signals without increasing the sample size (i.e., fitting a stronger multivariate predictor will require more sample size). Conclusions: We identified, and confirmed in two cohorts, increased inflammatory gene expression (IL-1, TNFalpha or COX-2) by PBLs that predict radiographic progression in patients with SKOA. The data indicate that inflammatory events within joint tissues of patients with SKOA are reported in the peripheral blood. These PBL transcriptome signals of local joint inflammation merit further study as potential biomarkers for OA disease progression. (Table Presented)