Faculty Bibliography

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)

INTRODUCTION: The synovial membrane encapsulates diarthrodial joints to create a fluid-filled cavity that provides a lubricating environment for the articulating cartilage surfaces within. Fibroblast-like synoviocytes (FLS) regulate the composition of this synovial fluid, maintaining its viscous and lubricating properties. Fluid shear arising during joint articulation is recognized as a relevant stimulus for FLS in both healthy and pathologic conditions1. Gentle oscillatory shear has been shown to influence the response of FLS to inflammatory cytokines overexpressed in osteoarthritis (OA)2. Cartilage wear particles (CWP) are also recognized as a key factor in OA3,4, but their influence on FLS mechanosensing sensing is unknown. The specific mechanisms of FLS shear sensing and its downstream consequences, as well as the influence of the OA environment on this sensing are still unknown. This work seeks to characterize the real-time intracellular calcium ([Ca2+]i) response of FLS to physiologic fluid shear and determine how this response is modulated by both chemical and physical OA factors, Interleukin-1alpha (IL1) and CWP. Intracellular calcium is an important cell signaling molecule in cell mechanotransduction. The aim of the current study is to test the hypothesis that these OA factors heighten FLS shear sensitivity as determined using an in vitro model system. METHODS: Cell Culture. FLS were isolated from juvenile bovine synovium, expanded in alphaMEM containing 10% fetal bovine serum (FBS), 100 U/mL penicillin, 100 mg/mL streptomycin, and 5 ng/mL bovine FGF for two passages to obtain a pure population, and plated into silicone wells on 5 mug/cm2 collagen type 1 coated glass slides at high density to obtain a confluent monolayer after 24hr attachment. To mitigate artifacts due to cellular starvation the media was switched to 0.5% FBS for 12hr prior to imaging, with 10 ng/mL IL1 or CWP at 50 particles/cell for preconditioned groups. CWP were generated by mechanical abrasion of cartilage explants and sorting via Coulter counter to obtain a size of 0.9 mum5. Calcium Signaling. FLS were loaded with 5muM Fura Red to track relative [Ca2+]i in real time, where increasing [Ca2+]i results in decreasing fluorescence. A parallel plate flow chamber was employed to expose cells to 1 dyne/cm2 shear stress. Hank's Buffered Salt Solution with 0.1% FBS was flowed, with 10 ng/mL IL1 or 1 mM octanol for indicated groups. Fluorescence intensity over time was determined for individual cells and normalized as relative [Ca2+]i with respect to average intensity during equilibrium period prior to flow. Cells were considered responders if relative [Ca2+]i rose 20% above equilibrium level, which was found to be sufficient to exclude any false responses due to natural variation in equilibrium levels. Percentile response, peak magnitude, area, and latency were determined for 100 cells/slide across 3 slides/group. Dye Transfer Assay. 'Parachute' FLS were loaded with Calcein and DiI and plated at low density onto a non-dyed FLS monolayer, where Calcein transfer to neighboring cells was quantified after 4hr as a measure of gap junction communication, for 40 parachute cells (identified by non-transferable DiI) across triplicate slides per group. Preconditioned monolayers received 10 ng/mL IL1, or 50 particle/cell CWP for 24hr prior, while 1 mM octanol was applied during imaging for the negative control. Statistical Analysis. Parametric statistics were performed via ANOVA with Tukey HSD post-hoc testing (alpha<0.05), and nonparametric statistics via Fischer Exact Test with Holms-Sidak correction (alpha<0.05). RESULTS: FLS display a single Ca2+ peak in response to fluid shear (Figure 1a) coordinated among a percentage of the population, dependent upon a minimum level of serum. IL1 showed differing effects on shear response depending on timing of exposure (Figure 1b). Exposure during flow alone (CTL/IL1) significantly decreased percentile response, while preconditioning had no significant effect alone (IL1/CTL) but abolished the decreasing effect of IL exposure during flow when combined (IL1/IL1). A separate vehicle-controlled experiment showed that FLS do not respond to IL exposure itself. CWP attachment to FLS (Figure 2b,c) also significantly increased percentile response (Figure 2a) as well as peak magnitude, area, and latency. The proposed mechanism for both IL1 and CWP preconditioning enhancement of shear sensing is increase gap junction formation and communication. Gap junction blocking via octanol significantly reduced percentile response by 22%, indicating that cell-cell passing of Ca2+ signals makes up a percentage of responding cells in normal conditions, while dye transfer experiments showed that IL1 and CWP both increased gap junction communication compared to control (Figure 3). DISCUSSION: FLS demonstrate a robust Ca2+ signaling response to fluid shear which is modulated by chemical and physical factors of the OA environment, namely by increasing percentile response and thus confirming the hypothesis of heightened sensitivity. While mechanisms for the suppressing influence of IL1 exposure alone are still being explored, the influence of IL1 and CWP preconditioning may be explained by an increase in gap junction communication leading to more cell-cell signaling and higher percentile response, which supports previous implications of increased gap junction communication between FLS in the pathology of OA6. Future work will focus on the mechanisms for individual cell shear sensing, and how they are influenced by OA factors, thus contributing to the phenomenon observed in the present study. The primary cilium is of particular interest as it is both involved in mechanosensing in other cell types7, and implicated in OA pathology in its modulation by the inflammatory environment8. (Figure Presented)