Faculty Bibliography

Background/Purpose: We aimed to understand the mechanism by which membrane-type 1 matrix metalloproteinase (MT1-MMP, MMP-14) controls bone and cartilage homeostasis. MT1-MMP, a cell-membrane-bound proteinase with an extracellular catalytic site and a 20-amino acid cytoplasmic tail, plays a key role in postnatal bone formation. The genetic deficiency of MT1-MMP in the mouse causes dwarfism, osteopenia and severe arthritis. Deletion of MT1-MMP in bone marrow-derived mesenchymal progenitor cells (BM-MSC) recapitulates this phenotype, showing that MT1-MMP controls osteogenic differentiation in MSC. The phenotype of MT1-MMP-/- mice has been proposed to result from lack of MT1-MMP proteolytic activity. However, mounting evidence shows a variety of proteolysis-independent signaling functions of MT1-MMP. The unique tyrosine (Y573) in the MT1-MMP cytoplasmic tail is fundamental for the control of intracellular signaling. Methods: We generated a mouse with the Y573D mutation in MT1-MMP (MT1-MMP Y573D) and characterized its skeletal phenotype by histological and microCT analyses. Isolated BM-MSC were induced to differentiate into osteoblasts, chondrocytes and adipocytes, using qRT-PCR to analyze gene expression. Mouse C3H10T1/2 MSC were transfected with MT1-MMP cDNA and analyzed for Wnt signaling by luciferase reporter assays. Results: MT1-MMP Y573D mice had increased trabecular bone relative to wt littermates, marked thinning of articular cartilage with disorganized tissue architecture, clustering and cloning of chondrocytes, and pronounced decrease in bone marrow-associated and total body fat. We induced BM-MSC from wt and MT1-MMP Y573D littermates to differentiate into osteoblast and chondrocytes, and myeloid precursors into osteoclasts. The Y573D mutation dramatically increased MSC expression of osteoblast markers and strongly downregulated chondrocyte and osteoclast markers. These findings indicated that Wnt signaling is upregulated in MT1-MMP Y573D-expressing MSC. Therefore, we analyzed Wnt signaling. We transiently transfected C3H10T1/2 MSC cells in osteoblast medium with the cDNAs for wt MT1-MMP and MT1-MMP Y573D. As controls the cells were transfected with the empty vector (pcDNA) or with MT1-MMP E240A, a mutant devoid of proteolytic activity. MT1-MMP Y573D dramatically upregulated Wnt signaling relative to wt MT1-MMP and MT1-MMP E240A. Conclusion: MT1-MMP controls Wnt signaling by a mechanism independent of extracellular proteolysis and mediated by its cytoplasmic tail. MT1-MMP is a bifunctional protein, with an extracellular proteolytic activity that promotes bone formation through ECM remodeling and a cytoplasmic tail that controls osteogenesis by interacting with a key pro-osteogenic signaling pathway

Background/Purpose: Despite significant advances in the therapeutics of inflammatory arthritides, methotrexate (MTX) remains the mainstay of treatment for rheumatoid arthritis (RA) and related conditions worldwide. However, it has a hihgly variable inter-individual bioavailabity (ranging from 20 to 80%) and there is currently no mechanism to predict efficacy. One of the fundamental roles of the intestinal microbiome is to metabolize xenobiotics and synthetic drugs. Here we characterize the effects of oral methotrexate on the gut community composition of patients with RA and the potential role of baseline human microbiota in predicting response to MTX therapy. Methods: Demographic characteristics, drug use and disease activity scores-28 (DAS28) were recorded from new-onset rheumatoid arthritis (NORA) patients (n=33). For each participant, fecal samples were collected at baseline and at pre-established intervals for at least 3 months after initiation of oral methotrexate (range 3-48 months). 16S rDNA was extracted per protocol (MoBio, USA) and amplicons targeting the hypervariable V4 region were sequenced using 454 and MiSeq (Illumina) platforms to define the microbiota composition. The obtained 16S rRNA sequences were analyzed using the Quantitative Insights into Microbial Ecology (QIIME) pipeline. Taxonomic relative abundance at all hierarchical levels was determined to establish baseline microbiota composition prior to MTX initiation. Two-tailed Wilcoxon non-parametric test was applied to identify significant microbiota taxonomic changes that occur after MTX therapy. The False Discovery Rate (FDR) approach was applied to adjust for multiple hypothesis testing. Changes with a P<0.05 and FDR<0.2 were considered significant.Spearman correlations between baseline relative composition of intestinal microbiota and clinical response to MTX at each time point were also applied. Results: To quantify microbiota similarities among fecal samples we used unweighted UniFrac and hierarchical clustering. Samples from MTX-treated patients clustered with their respective baseline samples, indicating that the gut microbiota is stable with inter-individual taxonomic differences maintained for at least 6 months. NORA patients receiving MTX developed minimal changes over time. Intriguingly, however, baseline microbiome signatures in these patients predicted clinical response to MTX at 3 and 6 months, including the overabundance of unclassified Coriobacteriaceae (r=-0.756; P <0.01) and a Coprococcus-related OTU (r=-0.755; P =0.022). Conclusion: Although oral methotrexate does not induce significant changes in the over all structure of the human intestinal microbiota of NORA patients, the abundance of several taxa at baseline correlate with a significant improvement in clinical disease activity 3 and 6 months into therapy. Whether specific intestinal commensals can modulate the pharmacokinetics and bioavailability of methotrexate (and other DMARDs), remains to be elucidated. Better understanding of MTX pharamcomicrobiomics will be necessary to achieve precision medicine strategies in RA and related conditions

Background/Purpose: Osteoarthritis (OA) etiopathogenesis includes an inflammatory component. Published reports indicate that synovial fluid urate levels, even in patients without gout, associate with OA prevalence/severity. Whether serum urate (sUA), the precursor for gout and a biomarker for cardiovascular and kidney disease, may serve as a biomarker to convey or predict OA risk is not known. We investigated whether sUA levels associate with knee OA radiographic severity and contrast MRI-measured quantitative synovial volume (SV), and whether sUA levels predict radiographic progression, in a gout-free knee OA cohort. Methods: We assessed sUA in 88 gout-free subjects who completed a 24-month prospective, natural history knee OA study. Subjects had symptomatic medial knee OA, met ACR knee OA criteria and had BMI <33 at study entry. sUA was measured (enzyme-colorimetry) in serum frozen and banked at baseline. At baseline and 24 months, patients underwent standardized weight-bearing fixed-flexion posteroanterior knee radiographs (SynaFlexerTM). Twenty-seven subjects additionally had a dynamic gadolinium-enhanced 3.0T knee MRI that was read for quantitative synovial volume (SV). A musculoskeletal radiologist, blinded to subject data, determined joint space width (JSW) and Kellgren-Lawrence (KL) grades at each time point. Joint space narrowing (JSN) was determined as JSW change from baseline to 24 months. Pearson's correlations, student's t-tests, one-way ANOVA with post hoc Tukey-Kramer tests, ROC and AUC curves were used in statistical analyses, as appropriate. Results: sUA correlated with JSN in both univariate (r=0.40, p<0.01) and multivariate analyses (adjusting for age, gender and BMI, r=0.28, p=0.010). There was a significant difference in mean JSN after dichotomization of sUA at 6.8mg/dL, the solubility point for serum urate, even after adjustment for age, gender and BMI (JSN [+/-SEM] of 0.90mm+/-0.20mm for sUA>6.8; JSN [+/-SEM] of 0.31mm+/-0.09mm for sUA<6.8, p<0.01). Baseline sUA distinguished progressors (JSN>0.2mm), and fast progressors (JSN>0.5mm), from non-progressors (JSN<0.0mm) in multivariate analyses (area under the receiver operating characteristic curve [AUC] 0.626, p=0.027; AUC 0.620, p=0.045, respectively). sUA also correlated with SV (r=0.44, p=0.0040), a possible marker of JSN, though this correlation did not persist after controlling for age, gender and BMI (r=0.13, p=0.562). Conclusion: In non-gout patients with knee OA, sUA levels predict JSN and may serve as a biomarker for OA progression. (Figure presented)

Background/Purpose: Osteoarthritis (OA) is a disease characterized by pain and tissue destruction, in some cases concomitant with inflammation. The link between pain and tissue destruction is yet unknown, and there is a lack objective quantifiable parameters. Collagens are the main structural proteins of the joint extracellular matrix. The degradation of especially type I (connective tissue), II (cartilage), III (synovium) and IV (basement membrane) collagens have been shown to be elevated in OA. So we investigated whether biomarkers reflecting collagen degradation were associated with knee OA representing with different pain and inflammatory phenotypes. Methods: 111 knee OA patients, 62% women, from NYUHJD progression cohort study with varying degree of OA were included: mean (SD) age, 62 (10); mean(SD) BMI, 27(4); NSAID users, 23%; radiographic OA (KL>2) 68%; and bilateral knee OA; 87%. Pain was assessed by VASpain and WOMAC at baseline (BL) at a 2- year follow-up (FU) visit. Median (IQR) were 39 (13-69) and 37 (13-52) for BL VASpain and WOMACpain. 4 BL serum biomarkers of type I, II, III and IV collagen degradation (C1M, C2M, C3M, C4M), and the 2 inflammatory biomarkers CRPM and hsCRP, were assessed. Data were log2 transformed. Associations between BL biomarkers, BL pain and change (CHG) pain scores were assessed by multivariate linear model including gender, age, BMI, KLsignalknee, bilateral knee OA and NSAID use. Patients with cont. mild/moderate pain had a BL VASpain<54 and FU VASpain<30, cont. moderate/severe pain had VASpain>30 at baseline and FU, and transitional severe pain had either VASpain-BL<30 and VASpain-FU>54 or VASpain-BL>54 and VASpain-FU<30 (ref). Patients with; low biochemical disease activity index (bDAI) low in CRPM (<12nM) moderate bDAI were high in CRPM but low in hsCRP (<5), and high bDAI (flare) were high in CRPM and hsCRP. Results: BL association between pain and biomarkers C2M (beta -17.9, p<0.0001) and KLsignalknee (beta -5.4, p=0.0031) were significantly associated with WOMAC pain. C2M (beta -12.4, p=0.0033), C3M (beta -19.9, p=0.059), age (beta -0.84, p<0.0018), KLsignalknee (beta 8.9, p=0.0021) and bilateral knee OA (beta -12.2, p=0.087) were associated with VASpain. Association between BL biomarkers and CHG pain C2M (beta 13.3, p=0.0016), age (beta 0.5, p=0.029) and bilateral OA (beta -12.0, p=0.043) were significantly associated with delta WOMACpain. Only age, BMI and NSAID use was associated with CHG VASpain. Association between pain phenotypes and BL biomarkers Patients with cont. mild/moderate pain had significantly higher C2M compared patients with transitional severe pain (p=0.0014) and cont. moderate/severe pain (p=0.04). Biomarker, BL pain and CHG pain in patients w. inflammatory OA Patient with low bDAI had lower WOMACpain (p<0.05) and VASpain(p<0.1). C1M was higher (p<0.05) in the flare group compared to the low and moderate bDAI groups. C3M was higher (p<0.05) in the moderate bDAI group than the low DAI group. Conclusion: Different collagen degradation products are linked differentially to different phenotypes. Cartilage degradation (C2M) was consistently linked to CHG pain phenotypes, whereas it was not associated with an inflammatory phenotype. In contrast, C1M and C3M were linked to inflammatory and flared OA

Background/Purpose: Growing numbers of studies show increased expression in Osteoarthritis (OA) of inflammatory cytokines, such as IL-1beta and TNFalpha, in joint tissues and peripheral blood mononuclear (PBM) cells. The IL1 receptor antagonist (IL1RN) gene cluster region has been associated with susceptibility to knee OA, thereby further implicating inflammation in OA pathogenesis. In these studies, we examined the association of IL-1RN haplotype with the radiographic severity of symptomatic knee OA (SKOA). Methods: Genomic DNA from SKOA patients from three cohorts (NYU I, NYU-II and O

Background/Purpose: Persistent inflammation is a characteristic of several joint diseases, including OA. It is nowadays appreciated that this could be a result of a failure to (optimally) activate inflammation resolution pathways. Therefore, we investigated the presence of specialized pro-resolving lipid mediators (SPM) and their precursors as pathway markers of the resolution process in the joint of OA patients and controls. Methods: SF was obtained from knee OA (2 populations) and rheumatoid arthritis (RA) patients fulfilling the ACR criteria for OA and RA, respectively, and healthy controls. Lipid mediators (LMs) were determined by targeted lipidomics using liquid-chromatography mass spectrometry. Sixty different lipids including pro-inflammatory (e.g. prostaglandins, leukotrienes) and anti-inflammatory/pro-resolving LM (e.g. SPM), as well as their precursors can be detected with our technique. Results: SF from 24 OA and 12 RA patients were first studied. Thirty-seven lipids were detected in the soluble fraction of SF, including polyunsaturated fatty acids (PUFA) and their lipoxygenase (LOX) and cyclooxygenase (COX) pathway markers in both OA and RA patients. Among these, pro-inflammatory LM such as PGE2 and thromboxane B2, as well as the pathway markers of resolution and precursors of SPM, 17-HDHA and 18-HEPE, were detected. Except for the LOX products of arachidonic acid: 15-HETE, 6-trans-LTB4 and 20-OH- LTB4, which were lower in OA than in RA SF, all other lipid mediators and PUFA were comparable between OA and RA samples. Ratios of metabolites to their precursors indicated that both pro- (e.g. LTB4) and anti-inflammatory LOX products (e.g. 17- HDHA) are more efficiently generated in RA than in OA patients, while no differences were observed in COX products. Interestingly, the SPM resolvin D2 (RvD2) could also be detected, but only in the insoluble fraction (cells and undigested matrix), indicating that the resolution pathways are activated in OA. This expands our previous publication showing activation of resolution in RA patients. To assess the efficiency of activation of resolution in OA, we have performed targeted lipidomics on total SF in an additional study with 32 OA patients and 10 healthy controls. Confirming earlier data, most LMs were also detected in this study, including the pro-inflammatory PGE2, the SPM precursors 17-HDHA and 18-HEPE, and the SPM RvD2. Additionally, we detected 18S-resolvin E3 (18S-RvE3). Remarkably, both the absolute concentrations of the SPM RvD2 and 18S-RvE3, and the ratio to their precursors, 17-HDHA and 18-HEPE, were lower in OA compared to healthy SF, indicating less efficient generation of SPM in OA compared to healthy joints. In contrast, the proinflammatory lipid PGE2was higher in OA than in healthy SF, indicating that the lower activation of resolution is paired by a higher inflammatory load in OA compared with healthy individuals. Conclusion: By using a state-of-the-art technique, we show for the first time that resolution pathways are activated in OA patients. Importantly, resolution seems to be less efficiently activated than in healthy individuals, which could account for the persistent inflammation observed in OA and RA patients

BACKGROUND: The purpose of this study was to use MRI to determine if a loss of meniscal intra-substance integrity, as determined by T2* relaxation time, is associated with an increase of Kellgren-Lawrence (KL) grade, and if this was correlated with risk factors for cartilage degeneration, namely meniscal extrusion, contact area and anterior-posterior (AP) displacement. METHODS: Eleven symptomatic knees with a KL 2 to 4 and 11 control knees with a KL 0 to 1 were studied. A 3 Tesla MRI scanner was used to scan all knees at 15 degrees of flexion. With a 222N compression applied, a 3D SPACE sequence was obtained, followed by a spin echo 3D T2* mapping sequence. Next, an internal tibial torque of 5Nm was added and a second 3D SPACE sequence obtained. The MRI scans were post-processed to evaluate meniscal extrusion, contact area, AP displacement and T2* relaxation time. RESULTS: KL grade was correlated with T2* relaxation time for both the anterior medial meniscus (r=0.79, p<0.001) and the posterior lateral meniscus (r=0.55, p=0.009). In addition, T2* relaxation time was found to be correlated with risk factors for cartilage degeneration. The largest increases in meniscal extrusion and decreases in contact area were noted for those with meniscal tears (KL 3 to 4). All patients with KL 3 to 4 indicated evidence of meniscal tears. CONCLUSIONS: This suggests that a loss of meniscal integrity, in the form of intra-substance degeneration, is correlated with risk factors for cartilage degeneration.

The role of the gut microbiome in animal models of inflammatory and autoimmune disease is now well established. The human gut microbiome is currently being studied as a potential modulator of the immune response in rheumatic disorders. However, the vastness and complexity of this host-microorganism interaction is likely to go well beyond taxonomic, correlative observations. In fact, most advances in the field relate to the functional and metabolic capabilities of these microorganisms and their influence on mucosal immunity and systemic inflammation. An intricate relationship between the microbiome and the diet of the host is now fully recognized, with the microbiota having an important role in the degradation of polysaccharides into active metabolites. This Review summarizes the current knowledge on the metabolic role of the microbiota in health and rheumatic disease, including the advances in pharmacomicrobiomics and its potential use in diagnostics, therapeutics and personalized medicine.

Background: Osteoarthritis (OA) is heterogeneous disease characterized by pain and tissue destruction which is in some case concomitant with inflammation. However, the link between pain and tissue destruction is yet unknown, and there is a lack objective quantifiable parameters. Collagens are the main structural proteins of the joint extracellular matrix. The degradation type I (connective tissue), II (cartilage), III (synovium) and IV (basement membrane) collagens have been shown to be elevated in joint degenerative diseases. Objectives: To investigate whether biomarkers reflecting collagen degradation were associated with symptomatic knee OA representing with different pain and inflammatory phenotypes. Methods: 111 knee OA patients, 62% women, from NYUHJD progression cohort study with varying degree of OA were included: mean (SD) age, 32 (10); mean (SD) BMI, 27 (4); NSAID users, 23%; radiographic OA (KL>2) 68%; and bilateral knee OA; 87%. Pain was assessed by VASpain and WOMAC at baseline (BL) at a 2-year follow-up (FU) visit. Median (IQR) were 39 (13-69) and 37 (13-52) for BL VASpain and WOMACpain. 4 BL serum biomarkers of type I, II, III and IV collagen degradation (C1M, C2M, C3M, C4M), and the 2 inflammatory biomarkers CRPM and hsCRP, were assessed. Data were log2 transformed. Associations between BL biomarkers, BL pain and change (CHG) pain scores were assessed by multivariate linear model including gender, age, BMI, KLsignal knee, bilateral knee OA and NSAID use. Patients with cont. mild/moderate pain had a BL VASpain<54 and FU VASpain<30, cont. moderate/severe pain had VASpain>30 at baseline and FU, and transitional severe pain had either VASpain-BL<30 and VASpain-FU>54 or VASpain-BL>54 and VASpain-FU<30 (ref). Patients with; low biochemical disease activity index (bDAI) low in CRPM (<12nM) moderate bDAI were high in CRPM but low in hsCRP (<5), and high bDAI (flare) were high in CRPM and hsCRP. Results: BL association between pain and biomarkers. C2M (beta -17.9, p<0.0001) and KLsignal knee (beta -5.4, p=0.0031) were significantly associated with WOMAC pain. C2M (beta -12.4, p=0.0033), C3M (beta -19.9, p=0.059), age (beta -0.84, p<0.0018), KLsignal knee (beta 8.9, p=0.0021) and bilateral knee OA (beta -12.2, p=0.087) were associated with VASpain. Association between BL biomarkers and CHG pain. C2M (beta 13.3, p=0.0016), age (beta 0.5, p=0.029) and bilateral OA (beta -12.0, p=0.043) were significantly associated with delta WOMACpain. Only age, BMI and NSAID use was associated with CHG VASpain. Association between pain phenotypes and BL biomarkers. Patients with cont. mild/moderate pain had significantly higher C2M compared patients with transitional severe pain (p=0.0014) and cont. moderate/severe pain (p=0.04). Biomarker, BL pain and CHG pain in patients w. inflammatory OA. Patient with low bDAI had lower WOMACpain (p<0.05) and VASpain (p<0.1). C1M was higher (p<0.05) in the flare group compared to the low and moderate bDAI groups. C3M was higher (p<0.05) in the moderate bDAI group than the low DAI group. Conclusions: Different collagen degradation products are linked differentially to different phenotypes. Cartilage degradation (C2M) was consistently linked to pain CHG and phenotypes, whereas it was not associated with an inflammatory phenotype. In contrast, C1M and C3M were linked to inflammatory OA and flared OA