Faculty Bibliography

Purpose: The unique properties of articular cartilage are defined by quiescent, differentiated articular chondrocytes whose homeostasis is maintained through a strict balance between anabolic and catabolic processes. This balance is shifted toward catabolic activities in osteoarthritis (OA), a disease that involves all joint tissues and disrupts the structural integrity of articular cartilage. In OA, articular chondrocytes become more hypertrophic-ike, and abnormally increase not only extracellular matrix (ECM) production but also the expression of ECM-degrading enzymes. While chondrocyte hypertrophy is indispensable for endochondral bone formation, in OA it mediates detrimental changes that lead to cartilage destruction. Our previous work established that translational control of gene expression is critical for dedifferentiation of articular chondrocytes in OA. We showed that fine-tuned control of protein synthesis by translational repressor 4E-BP (eIF4E-Binding Protein) is required for proper chondrocyte homeostasis. By binding to the m7-GTP cap-binding protein eIF4E, 4E-BP inhibits cap-dependent mRNA translation by restricting the incorporation of eIF4E into a multi-subunit initiation factor called eIF4F, which is needed to recruit 40S ribosomes to mRNA. We hypothesized that proper translational control is crucial for maintaining healthy cartilage phenotype. We therefore investigated expression of which genes is controlled (completely or partially) at the translational level. Method(s): To identify the pool of mRNAs that are translationally control in OA cartilage we first compared translation efficiencies of mRNAs by analyzing polysome profiles of rat articular chondrocytes (RAC) untreated and treated with Interleukin 1beta (IL-1beta). Expression of several translationally regulated mRNAs was then compared to their expression in human and rodent OA cartilage. Additionally, the eIF4F inhibitor was used in vivo to determine if restriction of eIF4F activity affects OA progression in ACLT rat model. Result(s): We identified 617 mRNAs that are translationally regulated by IL-1beta. One of them is the orphan receptor Nr4a1, whose protein but not mRNA level is significantly increased in OA cartilage in vivo, highlighting the exceptional potential of our approach for discovering new possible targets in OA cartilage. Modulating Nr4a1 expression in IL-1beta-treated articular chondrocytes effects expression of MMP13, the metalloproteinase that has a predominant role in OA. Intra-articular injection of 4E1RCat, an inhibitor of cap-dependent translation not only precludes Fn and Nr4a upregulation in a rodent OA model, but also significantly delays cartilage degeneration in the ACLT rat model. Conclusion(s): We identified 617 mRNAs that are translationally regulated by IL-1beta. One of them is the orphan receptor Nr4a1, whose protein but not mRNA level is significantly increased in OA cartilage in vivo, highlighting the exceptional potential of our approach for discovering new possible targets in OA cartilage. Modulating Nr4a1 expression in IL-1beta-treated articular chondrocytes effects expression of MMP13, the metalloproteinase that has a predominant role in OA. Intra-articular injection of 4E1RCat, an inhibitor of cap-dependent translation not only precludes Fn and Nr4a upregulation in a rodent OA model, but also significantly delays cartilage degeneration in the ACLT rat model.

OBJECTIVE:Phenotypic changes of chondrocytes toward hypertrophy might be fundamental in the pathogenesis of OA, of which type X collagen (Col10) is a well-known marker. The purpose was to develop a specific immunoassay for blood quantification of a newly identified neo-epitope of type X collagen to assess its diagnostic value for radiographic knee osteoarthritis (OA). METHODS:A neo-epitope of Col10 was identified in urine samples from OA patients. A monoclonal antibody against the neo-epitope was produced in Balb/C mice. The enzyme responsible for the cleavage was identified. Immunohistochemical detection of this neo-epitope was performed on human OA cartilage. An immunoassay (Col10neo) was developed and quantified in two clinical studies: the C4Pain-003 and the NYU OA progression study. ROC curve analysis was carried out to evaluate the discriminative power of Col10neo between OA and RA. RESULTS:A neo-epitope specific mAb was produced. The Cathepsin K-generated neo-epitope was localized to the pericellular matrix of chondrocytes, while its presence was extended and more prominent in superficial fibrillation in the cartilage with advanced degradation. In the C4Pain study, a higher level of Col10neo was seen in subjects with greater KL grade. The group of the highest tertile of Col10neo included more subjects with KL3-4. In the NYU study, Col10neo was statistically higher in OA than control or RA. ROC curve analysis revealed area under the curve was 0.88 (95% CI 0.81-0.94). CONCLUSION/CONCLUSIONS:Our findings indicate that Col10neo linked to hypertrophic chondrocytes could be used as a diagnostic biochemical marker for knee OA.

Background/Purpose: Psoriatic Arthritis (PsA) affects up to 30% patients with psoriasis and is characterized by wide spread synovio-entheseal inflammation. Physiologically, the human gut microbiota metabolizes dietary fiber into shortchain fatty acids (FA)- which exert anti-inflammatory effects by increasing activity of regulatory T cells (Tregs).Moreover, we have previously shown decreased abundance of Akkermansia and Ruminococcus and concomitant decrease in mediumchain FA (MCFA) levels in stool of PsA patients. We therefore hypothesized that FA supplementation may have favorable effects on gut microbiome and lead to increase in tolerance, potentially serving as therapeutic target in psoriatic disease.
Method(s): Wild type (WT)animals were fed SCFA-rich diet for 14 days followed by 16S rRNA sequencing and microbiota analysis of pellet specimens.We then evaluated effects ofMCFA-rich diet in healthy subjects. Peripheral blood and stool samples were collected at days 0, 7 and 14 for 16s rRNA sequencing and FACS. Finally, we conducted a small, prospective, proof-ofprinciple study in new-onset, drug-naive psoriatic disease patients (with or without PsA). Each participant received MCFA (1 gm 4 times a day for 6 weeks). Clinical history was obtained at baseline. Skin and joint exam were performed at baseline and follow up. Serum and stool samples were collected at baseline, weeks 3, and 6 for 16S rRNA sequencing and FACS, respectively. Wilcoxon signed-rank test was used to compare differences in Tregs before and after MCFA-rich administration.
Result(s): SCFA rich diet in WT mice led to statistically significant perturbations in gut bacterial composition 14 days into intervention, with a dramatic increase in commensals (Fig 1A; p<0.001), most notably in Akkermansia(Fig 1B). MCFA administration to healthy subjects (n=7) also led to significant changes in community structure (Fig 2A; p=0.03) and associated increases in circulating Treg cells (Fig 2B; p<0.001). These findings were also observed in psoriatic disease patients (n=4) showing a significant alteration in specific taxa, including Actinobacteria (Fig 2 C; p<0.05) and Mollicutes (p=0.09) and concomitant increase in circulatory Treg cells (Fig 2D)
Conclusion(s): In both health and psoriatic disease, MCFA supplementation is associated with distinct changes in human gut microbiota composition and peripheral Treg cells. These findings rationalize the need for a larger placebo controlled, prospective trial to study the effects of MCFA in patients with psoriasis and PsA as a potential therapy alone or in combination with DMARDs. (Figure Presented)

Background/Purpose: To investigate the expression of vascular adhesion protein -1 (VAP-1) in joint tissues and serum in knee osteoarthritis (OA) patients and examine whether VAP-1 levels predict increased risk of disease severity or progression of knee OA.
Method(s): Baseline serum and synovial fluid VAP-1/semicarbazide-sensitive amine oxidase (SSAO) levels were assessed in cohorts of patients with tibiofemoral medial knee OA and healthy subjects. Standardized fixed-flexion posteroanterior knee radiographs were scored for Kellgren Lawrence (KL) grade (0-4) and medial joint space width (JSW) at the mid-portion of the joint space. Radiographic severity was defined by KL2/3 vs. KL4. Biochemical markers assessed comprised VAP-1/ SSAO, IL-1Ra, IL-6, sRAGE, CCL2, CCL4, CD163, hsCRP and MMPs-1,-3,-9. Associations between biomarkers and radiographic severity (logistic regression controlling for covariates) and pain (Spearman correlation) were evaluated.
Result(s): VAP-1 was locally overexpressed at least 2 fold in the OA synovium based on immunohistochemical, microarray and qRT-PCR analyses compared to controls. Synovial fluid SSAO levels was also significantly higher in OA (107.94+41.42) compared to normals (38.12 + 22.98 ng/ml; p=0.0001) and inversely associated with radiographic severity. We observed a positive correlation with the levels of SSAO in the synovial fluid and serum of OA patients (r=0.47; p=0.014). However, serum SSAO levels in OA patients were lower than in controls, and inversely correlated with pain and inflammation markers (CRP and soluble RAGE). Serum SSAO levels were also lower in radiographically severe (KL4) OA patients compared to KL2/3. Serum SSAO did not correlate with other markers of inflammation or radiographic joint space narrowing (JSN) over 24 months.
Conclusion(s): Synovial fluid VAP-1/SSAO levels were elevated in OA and correlate with radiographic severity. However, serum or circulating SSAO levels are lower in OA patients and inversely correlate with pain and inflammation. Serum VAP-1 levels could identify patients at increased risk for knee radiographic severity

OBJECTIVE: To investigate the safety, tolerability, pharmacokinetics, and pharmacodynamics of ABT-981, a human dual variable domain immunoglobulin simultaneously targeting interleukin (IL)-1alpha and IL-1beta, in patients with knee osteoarthritis. METHOD: This was a randomized, double-blind, placebo-controlled, single-center study of multiple subcutaneous (SC) injections of ABT-981 in patients with mild-to-moderate osteoarthritis of the knee (NCT01668511). Three cohorts received ABT-981 (0.3, 1, or 3 mg/kg) or placebo every other week for a total of 4 SC injections, and one cohort received ABT-981 (3 mg/kg) or placebo every 4 weeks for a total of 3 SC injections. Assessment of safety and tolerability were the primary objectives. A panel of serum and urine biomarkers of inflammation and joint degradation were evaluated. RESULTS: A total of 36 patients were randomized (ABT-981, n=28; placebo, n=8); 31 (86%) completed the study. Adverse event (AE) rates were comparable between ABT-981 and placebo (54% vs 63%). The most common AE reported with ABT-981 versus placebo was injection site erythema (14% vs 0%). ABT-981 significantly reduced absolute neutrophil count and serum concentrations of IL-1alpha/IL-1beta, high-sensitivity C-reactive protein, and matrix metalloproteinase (MMP)-derived type 1 collagen. Serum concentrations of MMP-derived type 3 collagen and MMP-degraded C-reactive protein demonstrated decreasing trends with ABT-981. Antidrug antibodies were found in 37% of patients but were not associated with the incidence or severity of AEs. CONCLUSION: ABT-981 was generally well tolerated in patients with knee osteoarthritis and engaged relevant tissue targets, eliciting an anti-inflammatory response. Consequently, ABT-981 may provide clinical benefit to patients with inflammation-driven osteoarthritis.

INTRODUCTION: The synovium of patients with osteoarthritis (OA), presenting with capsular synovial hyperplasia, has cartilage and bone debris bound to the surface or embedded within the tissue [1]. Arthroscopy with lavage and synovectomy can remove tissue debris from the joint space and the synovial lining to provide pain relief to patients with OA. Despite the results of six decades of preliminary animal model and in vitro studies, which have confirmed the deleterious interaction of cartilage wear particles with the synovium, the mechanisms that mediate this interaction remain poorly understood [2-4]. Cartilage wear particles are inherently more complex to study than their non-biologic counterparts, as they can interact with the cells through phagocytosis or integrin binding as well as undergo degradation by cells. In the current study, we hypothesize that the physical interaction between fibroblast-like synoviocytes (FLS) and cartilage wear particles is mediated by phagocytosis and surface binding. METHODS: Tissue Harvest: Synovial tissue and articular cartilage explants were harvested from bovine calf knees (n=3). Synovial tissue was digested with collagenase IV, and the isolated FLS were plated and expanded for two passages. Cartilage explants were maintained in serum free media until use [5]. Particulate Generation: Cartilage particles were generated by manual abrasion with 120 grit sandpaper. The cartilage particle solution was filtered with a 10mum nylon mesh filter to achieve desired particle size. Latex (~ 1mum) and cartilage wear particles (~0.9mum) were counted and sized as in [6]. Imaging: FLS were cultured with FITC labeled latex particles or cartilage particles labeled with dichlorotriazinyl aminofluorescein, fixed and counterstained with TRITC phalloidin (actin) and DAPI (nuclei) after 48 hours. Immunohistochemistry: High density FLS monolayers were cultured for 5 days, fixed and stained for type I collagen and lubricin and counterstained with DAPI. Monolayer Culture: FLS were plated at high density overnight and co-cultured with 250 latex or cartilage particles per cell for 5 days (n=6-8) [7]. Particles alone were also cultured. Conditioned Media: Dense monolayers were cultured +/- 250 cartilage particles per cell for 5 days. In parallel, acellular media only and particle only groups were cultured under the same conditions. Conditioned media was removed from the four original culture groups (media only, particles only, FLS only, FLS + particles). After filtering out the remaining particles, the conditioned media was supplemented with fresh media before application to recipient FLS monolayers for 5 days. Biochemistry: DNA, collagen (COL), nitric oxide (NO) and prostaglandin E2 (PGE2) were determined using the Picogreen, DMMB, OHP, Griess and PGE2 Parameter assays, respectively. Biochemical content of cartilage particles alone was subtracted from the measured sample values to determine the contribution of SF alone. Statistics: Comparisons were analyzed using ANOVA with Tukey's post-hoc test (p<0.05). RESULTS: Similar to the native synovium, FLS organized into a multi-layer cell sheets with positive staining for collagen type I and lubricin (Figure 1A,B,C). Both latex and cartilage particles were capable of being phagocytosed by FLS. Cartilage particles, not only appeared internalized, but attached to the surface of both dense monolayers and individual cells. Staining for the actin cytoskeleton revealed nuclear (internal) and cortical (external) remodeling of the actin cytoskeleton around cartilage wear particles (Figure 1D), compared to only internal remodeling of latex particulates (Figure 1E). Co-culture of FLS with cartilage particulates for 5 days resulted in a significant increase in cell sheet DNA and collagen content compared to control and latex treated groups (Figure 2A,B). Co-culture with cartilage wear particles induced an inflammatory response as measured by the up-regulation of both NO and PGE2 synthesis compared to both control and latex treated samples. Latex particles only had a deleterious effect on collagen synthesis (Figure 2B). No differences in proliferation or extracellular matrix (ECM) synthesis were observed between media only and particle + media groups and FLS + media only and particles + media + FLS. As such, the response of FLS to cartilage particles appears to require particulate surface binding and/or phagocytosis (Figure 3). DISCUSSION: In this study, we developed a 2D model of the synovium to study the interaction of cartilage wear particles with FLS. Based on our findings, we accept our hypothesis that the physical interaction between FLS and cartilage wear particles is mediated by phagocytosis and surface binding. Both latex and cartilage wear particles showed re-arrangement of the actin cytoskeleton around the wear particulate (Figure1 D,E) as has been observed previously with titanium particles [8]. Here, the actin cytoskeleton remodeled at the surface to form a U-shaped organization around the cartilage particle contact site (Figure 1D). Cartilage particles induced more proliferation and secreted more inflammatory factors than latex particles alone. The proliferative response of FLS to cartilage wear particles resulted in an overall increase in ECM content, representative of the thickening of the synovial lining observed in OA patients. Additional insight into the underlying mechanisms that mediate cartilage particulate-FLS interactions may be gleaned from literature studies that have looked at cellular activation through ECM binding to FLS integrins using ECM (e.g. collagen, fibronectin, etc.)-coated beads [9]. These studies have shown that binding to ECM-coated beads increases cellular proliferation and decreases collagenase synthesis, which supports the present results for FLS co-culture with cartilage wear particles (Figure 2). Parallel studies pursued by our laboratory are investigating how the interaction of cartilage wear particles and FLS are affected by physical loading, such as fluid shear that arises in situ from synovial fluid-synovium interactions during joint articulation. (Figure Presented)