Faculty Bibliography

MicroRNAs (miRNAs) represent a class of non-coding RNA molecules playing pivotal roles in cellular and developmental processes. miRNAs modulate the expression of multiple target genes at the post-transcriptional level and are predicted to affect up to one-third of all human protein-encoding genes. Recently, miRNA involvement in the adaptive and innate immune systems has been recognized. Rheumatoid arthritis serves an example of a chronic inflammatory disorder in which miRNAs modulate the inflammatory process in the joints, with the potential to serve as biomarkers for both the inflammatory process and the potential for therapeutic response. This review discusses the investigations that led to miRNA discovery, miRNA biogenesis and mode of action, and the diverse roles of miRNAs in modulating the immune and inflammatory responses. We conclude with a discussion of the implications of miRNA biology in rheumatoid arthritis and other autoimmune disorders

BACKGROUND: A lack of biomarkers that identify patients at risk for severe osteoarthritis (OA) complicates development of disease-modifying OA drugs. OBJECTIVE: To determine whether inflammatory genetic markers could stratify patients with knee OA into high and low risk for destructive disease. METHODS: Genotype associations with knee OA severity were assessed in two Caucasian populations. Fifteen single nucleotide polymorphisms (SNPs) in six inflammatory genes were evaluated for association with radiographic severity and with synovial fluid mediators in a subset of the patients. RESULTS: Interleukin 1 receptor antagonist (IL1RN) SNPs (rs419598, rs315952 and rs9005) predicted Kellgren-Lawrence scores independently in each population. One IL1RN haplotype was associated with lower odds of radiographic severity (OR=0.15; 95% CI 0.065 to 0.349; p<0.0001), greater joint space width and lower synovial fluid cytokine levels. Carriage of the IL1RN haplotype influenced the age relationship with severity. CONCLUSION: IL1RN polymorphisms reproducibly contribute to disease severity in knee OA and may be useful biomarkers for patient selection in disease-modifying OA drug trials

T cell receptor (TCR)-dependent regulatory T cell (Treg) activity controls effector T cell (Teff) function and is inhibited by the inflammatory cytokine tumor necrosis factor-alpha (TNF-alpha). Protein kinase C-theta (PKC-theta) recruitment to the immunological synapse is required for full Teff activation. In contrast, PKC-theta was sequestered away from the Treg immunological synapse. Furthermore, PKC-theta blockade enhanced Treg function, demonstrating PKC-theta inhibits Treg-mediated suppression. Inhibition of PKC-theta protected Treg from inactivation by TNF-alpha, restored activity of defective Treg from rheumatoid arthritis patients, and enhanced protection of mice from inflammatory colitis. Treg freed of PKC-theta-mediated inhibition can function in the presence of inflammatory cytokines and thus have therapeutic potential in control of inflammatory diseases

Osteoarthritis (OA) is often a progressive and disabling disease, which occurs in the setting of a variety of risk factors--such as advancing age, obesity and trauma--that collude to incite a cascade of pathophysiological events within joint tissues. An important emerging theme in OA is a broadening of focus from a disease of cartilage to one of the 'whole joint.' The synovium, bone and cartilage are each involved in pathological processes that lead to progressive joint degeneration. Additional themes that have emerged over the past decade are novel mechanisms of cartilage degradation and repair, the relationship between biomechanics and biochemical pathways, the importance of inflammation and the role of genetics. In this article, we review the molecular, clinical and imaging evidence that synovitis is not an 'incidental finding of OA', but plays a significant role in disease pathogenesis, and could therefore represent a target for future treatments

Background: The role of nitric oxide (NO) in osteoarthritis (OA) is controversial. NO, particularly when metabolized to peroxynitrite, is a recognized mediator of inflammation, which induces catabolic effects and promotes chondrocyte apoptosis. Conversely, NO delivered at low concentrations by NO donors exerts anti-inflammatory effects. Cyclooxygenase-Inhibiting Nitric Oxide Donators (CINODs) are anti-inflammatory compounds designed to inhibit both COX-1 and COX-2 while releasing nitric oxide, an important modulator of vascular tone. We studied the effect of naproxen and the CINOD NCX 429 in human chondrocytes and cartilage tissues from OA patients, to understand whether NO donation from CINODs may modulate the inflammatory/metabolic response. Methods: The experiments were performed with human cartilage and chondrocytes isolated from OA cartilage. Isolated chondrocytes stimulated with IL-1beta to induce inflammation, followed by measurement of inflammatory and matrix parameters, such as iNOS, COX-2, MMP1 and 13, PGE2, NO, matrix metalloproteinases, collagen degradation and NF-B nuclear translocation. Reference NSAIDs naproxen and celecoxib were used in comparison to NCX 429 (10 and 50 muM), and incubated 16 h before IL-1beta challenge. Cartilage explants culture supernatants (untreated and treated with modulators) were harvested at 24 and 72 hours post-treatment. RNA was extracted from chondrocytes and estimated by qPCR. NF-B binding was assessed using Marligen Bioscience kit and both cytoplasm and nuclear p65 subunit of NF-B was also assessed by Western blot. Results: The reference NSAIDs and the CINOD similarly upregulated MMP1 but inhibited MMP13, showing comparable net effect on collagen degradation as assayed by CTX-II ELISA. They similarly inhibited ADAMTS4, but only 50 muM NCX 429 downregulated COX-2 expression. In isolated OA chondrocytes, NCX 429 (10 and 50 muM) significantly and completely inhibited IL-1beta-induced PGE2 production, and both the constitutive and IL-1beta-stimulated induction of NF-B activity. Western blot analysis of p65 subunit isolated from nuclear fraction showed that the cells treated with CINOD (both in unstimulated and IL-1beta stimulated cells) had increased p65 accumulation although decreased NF-B binding and also inhibited NF-B promoter mediated luciferase reporter assay. Conclusions: Reference NSAIDs and the CINOD NCX 429 appear to similarly modulate human OA chondrocytes, both in unstimulated and stimulated conditions. NO has been considered detrimental for chondrocytes; however, donation of low concentrations of NO is recognized to be beneficial in a variety of conditions. Here, the CINOD NCX 429 does not differ from reference NSAIDs, indicating that CINODs do not adversely affect chondrocytes via NO donation. In addition, NCX 429 showed inhibition of the NF-B signalling, therefore presenting interesting anti-inflammatory features to be further explored

Purpose: There is insufficient understanding regarding how generalized OA involving the hand and knee differs from isolated knee OA, which may result from other factors such as obesity or trauma. The purpose of these studies is to determine whether the presence of hand OA involving interphalangeal (IP) and first carpometacarpal (CMC) joints, alone or in combination, predicts progression of patients with symptomatic knee OA. Methods: Hand radiographs were obtained on 94 patients at NYUHJD who met ACR criteria for symptomatic knee OA, and who were enrolled in a two-year NIH-sponsored prospective study. The patients completed standardized fixed-flexion knee radiographs at baseline and 24 months, with progression the signal (more painful) knee OA determined by change in joint space width (JSW) and KL score. For these analyses, the patients were separated into two groups by results on their signal knee: 17 progressors, defined by at least 30% decreased JSW over 24 months, and 77 non-progressors. For each set of hand x-rays, 2 radiologists evaluated 18 IP joints and 2 CMC joints for joint space narrowing and/or osteophytes, and whether or not there was erosive change at the IP joints; we averaged the scores from the two readers. Results: Kappa scores between the two scoring radiologists for the IP and CMC joints, and for the presence of erosive IP disease, were 0.79, 0.87 and 0.96, respectively. The overall mean IP score was 5.6 and 1st CMC score was 0.9, while medians were 5 and 1.0, respectively. The 17 progressors had a higher average IP (but not CMC) score than the non-progressors, 7.2+/- 5.4 vs. 5.0+/-4.6, p=0.13. Since the IP scores were not normally distributed, we further analyzed data by dichotomizing the study populations into two groups using the median IP total (5) as the cutoff point. When so analyzed, the presence of hand OA increased the odds ratio of knee OA progression to 2.8 (p=0.096). Of interest, the severity of knee OA correlated with hand OA scores: the average total hand OA scores (out of 20 joints) increased with baseline KL score, with mean scores of 3.8+/-5.5, 6.1+/-6.1 and 7.2+/-5.6 for KL 1 to 3 (p=0.06). There is also an increasing trend of total hand OA joint scores by KL score (p=0.042) when dichotomized around the median (5 joints), and with IP scores alone (p=0.026). The 8 patients with radiographic evidence of erosive IP disease, as compared with the 31 non-erosive IP OA patients (>5 IP joints) and the 54 without IP OA, demonstrated faster knee OA progression over 2 years by average KL increases (1.00, 0.35, 0.30) and decreases in joint space width (0.65, 0.56, 0.36), although perhaps given small numbers, this was not statistically significant (p=0.839). Conclusions: In cross-sectional analysis, the quantitative burden of hand OA correlates with the radiographic severity of knee OA (KL). Moreover, radiographic hand OA at the IP joints, but not at the 1st CMC joint, predicts more rapid progression of knee OA. Erosive IP disease may be an even stronger predictor than non-erosive IP disease of accelerated progression of knee OA

Purpose: We have previously shown that carriage of an IL1RN haplotype (CTA) was associated with substantially lower odds of radiographic severity (KL score, joint space width [JSW]) (Ann Rheum Dis. 2010). In this 24 month prospective study we assessed whether IL1-RN haplotypes predicted disease progression in patients with symptomatic knee OA. Methods: Ninety-seven (N=97) patients from NYUHJD who met ACR criteria for symptomatic knee OA were genotyped for single nucleotide polymorphisms (SNPs) in the IL-1b and IL-1RN genes. Standardized fixed-flexion radiographs were taken on all patients at baseline and 24 months. Radiographic progression of signal (more painful) knee OA was determined by change in JSW over 24 months. To account for variations in baseline JSW, we defined progression as greater than 30% joint space narrowing (JSN) of the diseased compartment over 24 months, rather than in change in absolute JSW in millimeters. Results: Decreases in JSW ranged from zero to 3.7 mm over the 24 months; 19 of 97 patients exhibited < 30% JSN. (Figure presented) Patients with the IL-1RN (rs419598/rs315952/9005) TTG haplotype exhibited increased radiographic knee OA severity at baseline compared to those without TTG (p>0.08). These TTG patients exhibited increased risk for radiographic progression at 24 months that approached significance based on <=30% JSN [OR = 2.85; 95%CI=0.68-11.67; p>0.15]. In contrast, OA patients with IL-1RN CTA haplotype showed decreased risk for JSN over 24 months in the signal knee [OR= 0.33; 95%CI=0.170-1.014; p>0.05]. Differences in reported VAS pain between the CTA and TTG group were significant at 24 months (p> 0.01), indicating that while these patients were not distinguishable by radiograph or symptoms at onset, IL1RN haplotype predicted symptomatic differences at two years. Finally, the TTG haplotype group of patients expressed relatively increased IL-1b gene expression [15.683 +/- 9.407 (p>0.0001)] as assessed by TaqMan QPCR in peripheral blood leukocytes. The TTG patients also exhibited decreased sIL-1Ra [283.64 +/- 36.4 pg/ml (p>0.001) in plasma samples compared to IL-1RN CTA haplotype protective groups [IL-1b (fold change), 5.444 +/- 10.083; sIL-1Ra, 370.35 +/- 43.3pg/ml] of patients respectively. Conclusion: IL-1RN gene family polymorphisms, which appear to affect host production of IL-1Ra, merit evaluation as biomarkers that predict the risk of progression in patients with symptomatic knee OA

Purpose: Stem cell-based therapies aimed at introducing progenitor cells into cartilage lesions hold great promise for the restoration of damaged articular surfaces following joint injury or osteoarthritis. Key to the generation of a functional repair tissue is the controlled differentiation into the desired phenotype. To this end microRNAs (miRNAs) may be important molecules that regulate this process. By acting as transcriptional repressors, their modulation during differentiation may enable commitment to a specific lineage by suppressing the expression of other lineage markers. In this study we profiled MSCs for miRNA expression following induction into the chondrocyte (C), osteoblast (O) and smooth muscle (SM) lineages. Results: Human bone marrow-derived Mesenchymal Stem Cells (MSCs) were obtained from NIH, or from the discarded hips of patients undergoing joint replacement surgery. SM differentiation was induced by treating monolayer cultures with 1 mM thromboxane-A2 [DP1] in the presence of 0.25% serum. C differentiation was induced by seeding MSCs in aggregate cultures in the presence of dexamethasone and TGF-b1 (10 ng/ml). O differentiation was induced by treatment of monolayer cultures with dexamethasone, ascorbate and beta-glycerolphosphate. Profiling of miRNAs by microarray (Agilent) or QPCR (SA Biosciences) revealed differential regulation of miR-7 and miR-130b, among 376 probes. Following SM and C differentiation, miR-7 expression was down-regulated up to 6.9-fold and 3-fold respectively. Conversely, during O differentiation, its expression was induced approximately 7-fold. Analysis of theoretical mRNA targets using TargetScan online software (www.targetscan.org) identified conserved sites in several genes associated with chondrocyte and myoblast lineages. Putative chondrogenic targets were found to include COL2A1, IGFR1, and GDF5, while potential smooth muscle modulators included EGFR1, PIK3CD, IRS1/IRS2, KLF4, CNN3 and IGF1R. Following a similar trend to miR-7, miR-130b was down-regulated up to 3.2-fold and 3.1-fold in C and SM differentiation respectively, while O differentiation induced its expression 2-fold. TargetScan analysis identified putative chondrogenic targets, TGF-BRII, Sox5, BMP-2 and IGF1; Potential smooth muscle regulators included ESR1, TGF-BRII, MBLN1, TGFBR1 and IGF2BP1. Together these observations suggest that miR-7 and miR-130b act to negatively regulate myogenic and chondrogenic cell fates via regulation of lineage specific genes. Conclusion: Our findings suggest that miR-7 and miR-130b, via the targeting of lineage specific molecules, regulate cell fate in adult human MSCs by inhibiting smooth muscle and chondrocyte differentiation, thereby promoting 'default' differentiation into the osteoblast lineage. [DP1]0.25% FBS 24 hours prior to addition of 1.0mM of the TxA2 chemical analog U46619