Faculty Bibliography

Interaction of estrogen with iron at the systemic level is long suspected, but direct evidence linking the two is limited. In the present study, we examined the effects of 17beta-estradiol (E2) on hepcidin, a key negative regulator of iron absorption from the liver. We found that transcription of hepcidin was suppressed by E2 treatment in human liver HuH7 and HepG2 cells, and this down-regulation was blocked by E2 antagonist ICI 182780. Chromatin immunoprecipitation, deletion, and EMSA detected a functional estrogen responsive element half-site that is located between -2474 and -2462 upstream from the start of transcription of the hepcidin gene. After cloning the human hepcidin promoter into the pGL3Luc-Reporter vector, luciferase activity was also down-regulated by E2 treatment in HepG2 cells. E2 reduced hepcidin mRNA in wild-type mice as well as in hemochromatosis Fe gene knockout mice. In summary, our data suggest that hepcidin inhibition by E2 is to increase iron uptake, a mechanism to compensate iron loss during menstruation. This mechanism may also contribute to increased iron stores in oral contraceptive users.

OBJECTIVE: The study aimed determining whether assessment of cartilage oligomeric matrix protein (COMP) degradation products could serve as a serological disease course and therapeutic response predictor in arthritis. METHODS: We generated a panel of monoclonal antibodies against COMP fragments and developed a novel capture enzyme-linked immunosorbent assay (ELISA) for detecting COMP fragments in patients with osteoarthritis (OA) and rheumatoid arthritis (RA). This test was also used to monitor COMP fragments in surgically-induced OA, collagen-induced arthritis (CIA), and tumor necrosis factor (TNF) transgenic animal models. RESULTS: Compared with a commercial COMP ELISA kit that detected no significant difference in COMP levels between OA and control groups, a significant increase of the COMP fragments were noted in the serum of OA patients assayed by this newly established ELISA. In addition, serum COMP fragment levels were well correlated with severity in OA patients and the progression of surgically-induced OA in murine models. Furthermore, the serum levels of COMP fragments in RA patients, mice with CIA, and TNF transgenic mice were significantly higher when compared with their controls. Interestingly, treatment with TNFalpha inhibitors and methotrexate led to a significant decrease of serum COMP fragments in RA patients. Additionally, administration of Atsttrin [Tang, et al., Science 2011;332(6028):478] also resulted in a significant reduction in COMP fragments in arthritis mice models. CONCLUSION: A novel sandwich ELISA is capable of reproducibly measuring serum COMP fragments in both arthritic patients and rodent arthritis models. This test also provides a valuable means to utilize serum COMP fragments for monitoring the effects of interventions in arthritis.

OBJECTIVE: Mutations in LMNA encoding the A-type lamins cause several diseases, including those with features of premature aging and skeletal abnormalities. The aim of this study was to examine the expression of lamin A in cartilage from patients with osteoarthritis (OA) and the effects of its overexpression on chondrocyte senescence and apoptosis. METHODS: Human chondrocyte-like cells (SW-1353) were used. RNA isolated from human OA and non-OA cartilage was used for profiling messenger RNA expression, using Affymetrix microarray analysis. The effects of lamin A overexpression on mitochondrial function and apoptosis were examined by assessing mitochondrial membrane potential, ATP levels, and cytochrome c release, and with a TUNEL assay. Western blotting was performed to determine protein expression. RESULTS: Lamin A expression was markedly elevated in OA cartilage samples compared with non-OA control samples. Western blot analysis confirmed increased expression of lamin A in OA compared with non-OA cartilage. Interleukin-1beta treatment inhibited lamin A accumulation, whereas treatment with prostaglandin E(2) (PGE(2) ) caused a marked increase in lamin A accumulation. These effects of exogenous PGE(2) on lamin A expression were mediated via the EP(2) /EP(4) receptors. Transfected chondrocytes that expressed lamin A displayed markers of early senescence/apoptosis. CONCLUSION: The results of this study suggest that lamin A is up-regulated in OA chondrocytes, and that increased nuclear accumulation of lamin A in response to catabolic stress may account for the premature aging phenotype and apoptosis of OA chondrocytes.

Purpose: To determine whether inhibition of inducible nitric oxide synthase (iNOS) can slow progression of knee osteoarthritis (OA), using the rate of joint space narrowing (JSN) in the medial tibiofemoral compartment as the primary outcome measure. Methods: This was a 2-year multinational, multicenter, double-blind, parallel group trial enrolling subjects with symptomatic knee OA in which subjects were randomly assigned to receive once daily 50 mg or 200 mg of the selective iNOS inhibitor, SD-6010, or placebo. Subjects were required to have a body mass index (BMI) >=25 and <= 40 kg/m² and Kellgren and Lawrence Grade (KLG) 2 or 3 in the study knee. Randomization was stratified by KLG. Radiographs were acquired using the modified Lyon-schuss protocol, at baseline, 48 and 96 weeks for measuring JSN. Clinical benefit was recorded at 12-week then every 24 weeks; use of acetaminophen, NSAIDs and/or weak opioids was permitted throughout the trial. The primary analysis of the rate of JSN used a continuous time random coefficients MMRM model. The slope over the entire 96 week period was used to assess the rate of change in joint space width (JSW). Results: Of 1457 randomized subjects (SD-6010 50mg, n=485; SD-6010 200mg, n=486; placebo, n=486), 1048 (71.9%) completed the study. Subjects were predominantly female (76.5%) with mean age 61. 0 years and mean BMI of 31.8 kg/m². Fifty-six percent had KLG3. The primary analysis did not demonstrate the superiority of SD-6010 in either treatment group over placebo. In an exploratory discrete time MMRM analysis of KLG2 subjects, the loss of JSW after 48 weeks was significantly smaller with SD-6010 (50 mg or 200 mg) than placebo. Least squared (LS) mean +/- standard error (SE) losses in JSW for SD-6010 50mg (-0.048 +/- 0.028 mm) and 200mg (-0.062 +/- 0.028 mm) were 59.9% (95% CI: 6.8%, 106.9%) and 48.7% (95% CI: -8.4%, 93.9%) of placebo (-0.120 +/- 0.028 mm; P=0.032 and P=0.081, respectively). After 96 weeks, the LS mean losses in JSW for SD-6010 50mg (-0.132 +/- 0.036 mm) and 200mg (-0.156 +/- 0.037 mm) were 20.9% (95% CI: -50.5%, 64.0%) and 6.8% (95% CI: -72.7%, 51.7%) of the LS-mean JSW loss in the placebo group (-0.167+/-0.036 mm) (P=0.460 and P=0.812, respectively). In a similar analysis of KLG3 subjects, no improvement in JSN was observed. Although SD-6010 showed no efficacy with respect to improvement in joint pain or function, it was generally safe and well tolerated in this population. Conclusions: During the first 48 weeks of treatment, subjects with mild OA who were treated with an iNOS inhibitor had a lower rate of JSN; however, this improvement was not sustained at 96 weeks. iNOS inhibition did not slow OA progression in subjects with more severe radiographic OA. The observed early effect on JSN in subjects with mild OA supports the role of iNOS in OA pathogenetic mechanisms. However, the loss of efficacy over time and the lack of effect in subjects with more severe disease suggest that alternative biochemical catabolic pathways overcame the effects of nitric oxide inhibition alone or that in more severe radiographic OA, biomechanical factors may not be amenable to iNOS inhibition

Foxp3(+)CD4(+)CD25(high) regulatory T cell (Treg) suppression of inflammation depends on T-cell receptor-mediated Nuclear Factor of Activated T cells c1 (NFATc1) activation with reduced Akt activity. We investigated the role of the scaffold protein Disc large homolog 1 (Dlgh1) in linking the T-cell receptor to this unique signaling outcome. The Treg immunological synapse (IS) recruited fourfold more Dlgh1 than conventional CD4(+) T-cell IS. Tregs isolated from patients with active rheumatoid arthritis, or treated with tumor necrosis factor-alpha, displayed reduced function and diminished Dlgh1 recruitment to the IS. Furthermore, Dlgh1 silencing abrogated Treg function, impaired NFATc1 activation, reduced phosphatase and tensin homolog levels, and increased Akt activation. Dlgh1 operates independently of the negative feedback pathway mediated by the related adapter protein Carma1 and thus presents an array of unique targets to selectively manipulate Treg function.

OBJECTIVE: To assess and compare subregional and whole T1rho values (median+/-interquartile range) of femorotibial cartilage and menisci in patients with doubtful (Kellgren-Lawrence (KL) grade 1) to severe (KL4) osteoarthritis (OA) at 3T. MATERIALS AND METHODS: 30 subjects with varying degrees of OA (KL1-4, 13 females, 17 males, mean age+/-SD=63.9+/-13.1 years) were evaluated on a 3T MR scanner using a spin-lock-based 3D GRE sequence for T1rho mapping. Clinical proton density (PD)-weighted fast spin echo (FSE) images in sagittal (without fat saturation), axial, and coronal (fat-saturated) planes were acquired for cartilage and meniscus Whole-organ MR imaging score (WORMS) grading. Wilcoxon rank sum test was performed to determine whether there were any statistically significant differences between subregional and whole T1rho values of femorotibial cartilage and menisci in subjects with doubtful to severe OA. RESULTS: Lateral (72+/-10ms, median+/-interquartile range) and medial (65+/-10ms) femoral anterior cartilage subregions in moderate-severe OA subjects had significantly higher T1rho values (P<0.05) than cartilage subregions and whole femorotibial cartilage in doubtful-minimal OA subjects. There were statistically significant differences in meniscus T1rho values of the medial posterior subregion of subjects with moderate-severe OA and T1rho values of all subregions and the whole meniscus in subjects with doubtful-minimal OA. When evaluated based on WORMS, statistically significant differences were identified in T1rho values between the lateral femoral anterior cartilage subregion in patients with WORMS5-6 (advanced degeneration) and whole femorotibial cartilage and all cartilage subregions in patients with WORMS0-1 (normal). CONCLUSION: T1rho values are higher in specific meniscus and femorotibial cartilage subregions. These findings suggest that regional damage of both femorotibial hyaline cartilage and menisci may be associated with osteoarthritis

The compartment-specific lipid changes in femoral-tibial bone of healthy controls and mild osteoarthritis (OA) patients were quantified at 3.0 T. Healthy volunteers [Kellgren-Lawrence (KL) grade = 0; n = 15, 4 females, 11 males, mean age 39 +/- 16 years, age range = 24-78 years] and mild OA patients (KL = 1, 2; n = 26, 12 females, 14 males, mean age 61 +/- 14 years, age range = 27-80 years) were scanned on a 3 T scanner. Clinical proton density (PD)-weighted fast spin echo (FSE) images in the sagittal (without fat-saturation), axial and coronal (fat-saturation) planes were acquired for cartilage Whole-Organ MR Imaging Score (WORMS) grading. A voxel of 10 x 10 x 10 mm(3) was positioned in the medial and lateral compartments of the tibia [medial tibial (MT) and lateral tibial (LT)] and femur [medial femoral (MF) and lateral femoral (LF)] for MRS measurements using the single voxel-stimulated echo acquisition mode (STEAM) pulse sequence. All MRS data were processed with Java-based Magnetic Resonance User Interface (JMRUI). Wilcoxon's rank sum test and mixed model two-way analysis of variance (anova) were performed to determine significant differences between different compartments as well as examine the effect of OA grade and compartment, and their interactions. Generally, the MF compartment index of unsaturation was increased in healthy subjects compared with OA subjects (whether graded by KL or WORMS score). Differences between MF at KL0 and all other compartments at KL1 except LF approached statistical significance (p < 0.05). Differences in saturated lipids signals could be observed predominantly in the 2.03 p.p.m. frequency shift. Healthy controls in the MF compartment had the lowest saturated lipid signals, and mild OA patients with KL2 and WORMS5-6 in the MF compartment had the highest saturated lipid signals compared with other compartments at 2.03 p.p.m. (p < 0.05).