Shih-Yao Chen

Brief Report: Amelioration of collagen-induced arthritis in mice by lentivirus-mediated silencing of microRNA-223

OBJECTIVE MicroRNA (miRNA) plays a role in autoimmune diseases. MiRNA-223 (miR-223) is up-regulated in patients with rheumatoid arthritis (RA) and is involved in osteoclastogenesis, which contributes to erosive disease. The aim of this study was to test the feasibility of using lentiviral vectors expressing the miR-223 target sequence (miR-223T) to suppress miR-223 activity as a therapeutic strategy in a mouse model of collagen-induced arthritis (CIA). METHODS Levels of miR-223 in the synovial tissue of patients with RA or osteoarthritis (OA), as well as in the ankle joints of mice with CIA, were determined by quantitative reverse transcriptase-polymerase chain reaction (RT-PCR). Lentiviral vectors expressing miR-223T (LVmiR-223T) or luciferase short hairpin RNA (LVshLuc) as a control vector were injected intraperitoneally into mice with CIA. Treatment responses and disease-related bone mineral density were monitored. Levels of nuclear factor 1A (NF-1A), a direct target of miR-223, and macrophage colony-stimulating factor receptor (M-CSFR), which is critical for osteoclastogenesis, were measured by immunohistochemistry and quantitative RT-PCR. Osteoclasts were assessed by tartrate-resistant acid phosphatase staining. RESULTS MiR-223 expression was significantly higher in the synovium of RA patients and in the ankle joints of mice with CIA as compared to OA patients and normal mice. LVmiR-223T treatment reduced the arthritis score, histologic score, miR-223 expression, osteoclastogenesis, and bone erosion in mice with CIA. Down-regulation of miR-223 with concomitant increases in NF-1A levels and decreases in M-CSFR levels was detected in the synovium of LVmiR-223T-treated mice. CONCLUSION This study is the first to demonstrate that lentivirus-mediated silencing of miR-223 can reduce disease severity of experimental arthritis. Furthermore, our results indicate that inhibition of miR-223 activity should be further explored as a therapeutic strategy in RA.

Amelioration of collagen-induced arthritis in rats by adenovirus-mediated PTEN gene transfer.

OBJECTIVE The phosphatidylinositol 3-kinase (PI 3-kinase)/Akt pathway is known to be activated in rheumatoid arthritis (RA) synovial tissue, which impacts cell growth, proliferation, survival, and migration. Phosphatase and tensin homolog deleted from chromosome 10 (PTEN) functions as a negative regulator of PI 3-kinase signaling, thus blocking Akt activation. The aim of this study was to examine the effect of PTEN gene transfer in rats with collagen-induced arthritis (CIA). METHODS Adenoviral vectors encoding human PTEN (AdPTEN) or beta-galactosidase (AdLacZ) were injected intraarticularly into rats with CIA, and their treatment responses were monitored by measures of clinical, radiographic, and histologic changes. The expression of phosphorylated Akt, total Akt, vascular endothelial growth factor (VEGF), proinflammatory cytokines, and chemokines, as well as the extent of microvessel density in the ankle joints were determined. RESULTS AdPTEN treatment reduced Akt phosphorylation and decreased VEGF production in human RA synovial fibroblasts. Compared with AdLacZ treatment of the rats with CIA, AdPTEN treatment significantly reduced ankle circumference, articular index scores, radiography scores, and histology scores, and also decreased microvessel density and levels of VEGF and interleukin-1beta. Furthermore, PTEN gene transfer led to down-regulation of Akt activation and increased apoptosis in the ankle joints. CONCLUSION This study is the first to demonstrate the in vivo effect of intraarticular gene delivery of PTEN on amelioration of arthritis symptoms in rats with CIA, which involved antiangiogenic, antiproliferative, and antiinflammatory effects of PTEN via inhibition of the PI 3-kinase/Akt signaling pathway. Our findings also implicate the PI 3-kinase/Akt pathway as a therapeutic target for the treatment of RA or other inflammatory diseases.

Amelioration of Rat Collagen-Induced Arthritis Through CD4+ T Cells Apoptosis and Synovial Interleukin-17 Reduction by Indoleamine 2,3-Dioxygenase Gene Therapy

Indoleamine 2,3-dioxygenase (IDO) has been known as an emerging therapeutic target in autoimmunity-related arthritis. The treatment responses of adenoviral vectors encoding IDO (AdIDO) gene therapy in rat collagen-induced arthritis (CIA) were examined in this study. The therapeutic effects on ankle circumference, articular index, and radiographic and histological scores were evaluated in AdIDO-injected ankle joints. We further determined CD4+ T-cell numbers and their apoptotic status, CD68(+) macrophage numbers, kynurenine (a downstream tryptophan metabolite) concentrations, interleukin-17 (IL-17) levels, and retinoic acid-related orphan receptor γt (RORγt) expression in synovial tissues of CIA rats receiving AdIDO treatment. Reduction of ankle circumference, articular index, and radiographic and histological scores were noted in AdIDO-treated ankles, as compared with those receiving injection of control vectors. Furthermore, IDO gene transfer led to decreased infiltrating CD4+ T cells with enhanced apoptosis, reduced CD68+ macrophage numbers, increased kynurenine levels, lower IL-17 concentrations, and decreased RORγt expression within the ankle joints. In addition, such a therapy diminished type II collagen-specific IL-17 production and RORγt expression in CD4+ T cells from draining lymph nodes of CIA rats. Our results demonstrate for the first time that intra-articular delivery of IDO gene ameliorated ankle arthritis of CIA rats by induction of CD4+ T-cell apoptosis and reduction of synovial IL-17 production through the supplement of kynurenine. Taken together, these findings implicate the novel strategy of using IDO gene as a therapeutic approach in treating patients with rheumatoid arthritis.

Transcription Factor Snail Regulates Tumor Necrosis Factor α–Mediated Synovial Fibroblast Activation in the Rheumatoid Joint

The transcription factor Snail is involved in various biologic functions. We hypothesized that this molecule regulates tumor necrosis factor α (TNFα)–mediated synovial fibroblast activation in the rheumatoid joint. The aim of this study was to examine the role of Snail in the expression of cadherin‐11 (Cad‐11) and myofibroblast markers, interleukin‐6 (IL‐6) production, and the invasive ability of cells.

High-molecular-weight hyaluronic acid attenuated matrix metalloproteinase-1 and -3 expression via CD44 in tendinopathy

Evidence indicates that hyaluronic acid (HA) mitigates tendinopathy, but the effect of molecular weight is unclear. We investigated the effects of different concentrations and different molecular weights of HA (350 kDa, 1500 kDa, and 3000 kDa) on matrix metalloproteinase (MMP)-1 and -3 expression in IL-1β-stimulated rat tenocytes, and on their dynamic expression in peritendinous effusion from patients with long head of biceps (LHB) tendinopathy after high-molecular-weight (HMW)-HA treatments. Reverse transcription PCR, real-time PCR, and ELISA were used to determine MMP-1 and -3expression. Because CD44 was clearly expressed in the plasma membranes of cultured tenocytes, OX-50, a CD44 antagonist, was used to inhibit CD44 to evaluate the HA mechanism. HA (3000 kDa) significantly (p < 0.001) downregulated the mRNA and protein expression of MMP-1 and -3 in IL-1β-stimulated tenocytes. Its attenuating effects were dose-dependent (p < 0.01). In OX-50-pretreated cells, the mRNA expression of CD44 was not significantly altered, but the mRNA expression of MMP-1 and -3 was significantly upregulated. Visual analogue scale scores were significantly lower, and MMP-1 and -3 expression was significantly (p < 0.05) lower one month posttreatment. HMW-HA attenuated tendinopathy by downregulating MMP-1 and -3 expression. Inhibiting CD44 blocked the effects of HMW-HA.

Knockdown of toll-like receptor 4 signaling pathways ameliorate bone graft rejection in a mouse model of allograft transplantation

Non-union occurring in structural bone grafting is a major problem in allograft transplantation because of impaired interaction between the host and graft tissue. Activated toll-like receptor (TLR) induces inflammatory cytokines and chemokines and triggers cell-mediated immune responses. The TLR-mediated signal pathway is important for mediating allograft rejection. We evaluated the effects of local knockdown of the TLR4 signaling pathway in a mouse segmental femoral graft model. Allografts were coated with freeze-dried lentiviral vectors that encoded TLR4 and myeloid differentiation primary response gene 88 (MyD88) short-hairpin RNA (shRNA), which were individually transplanted into the mice. They were assessed morphologically, radiographically, and histologically for tissue remodeling. Union occurred in autografted but not in allografted mice at the graft and host junctions after 4 weeks. TLR4 and MyD88 expression was up-regulated in allografted mice. TLR4 and MyD88 shRNAs inhibited TLR4 and MyD88 expression, which led to better union in the grafted sites. More regulatory T-cells in the draining lymph nodes suggested inflammation suppression. Local inhibition of TLR4 and MyD88 might reduce immune responses and ameliorate allograft rejection.

Estrogen and mechanical loading-related regulation of estrogen receptor-β and apoptosis in tendinopathy

Female-dominant tendinopathies are musculoskeletal disorders caused by repetitive hand posture and motion; they are considered overuse syndromes. Both external mechanical stress and changes in hormone levels might affect disease progression. We have previously reported that estrogen receptor-β (ER)-β expression was associated with the pathogenesis of de Quervains disease. To study the underlying mechanisms, a cyclic stretching culture system was applied to tendon tissue from ovariectomized (OVX) rats. Furthermore, a collagenase I-induced rat tendinopathy model was established to examine the association of ER-β with disease progression. Our results showed that ER-β expression and the number of apoptotic cells were higher and associated with disease severity in rats with tendinopathy. Mechanical stress altered the morphology of primary tenocytes and collagen fiber alignment in tendons, and up-regulated the expression of matrix metalloproteinase-9, ER-β, and interleukin-1β, as well as induced apoptosis in tenocytes and tendon tissue from OVX rats. This is the first report on the effects of ER-β and mechanical stress in tendinopathy. We hope these findings contribute to new pharmacological therapies targeting ER-β signaling pathways to treat tendon-related diseases.

Dynamic weight bearing analysis is effective for evaluation of tendinopathy using a customized corridor with multi-directional force sensors in a rat model

Few studies discuss kinetic changes in tendinopathy models. We propose a customized corridor to evaluate dynamic weight bearing (DWB) and shearing forces. Sixty rats were randomly given ultrasound-assisted collagenase injections (Collagenase rats) or needle punctures (Control rats) in their left Achilles tendons, and then evaluated 1, 4, and 8 weeks later. The Collagenase rats always had significantly (p < 0.001) higher histopathological and ultrasound feature scores than did the Controls, significantly lower DWB values in the injured than in the right hindlimbs, and compensatorily higher (p < 0.05) DWB values in the contralateral than in the left forelimbs. The injured hindlimbs had lower outward shearing force 1 and 4 weeks later, and higher (p < 0.05) push-off shearing force 8 weeks later, than did the contralateral hindlimbs. Injured Control rat hindlimbs had lower DWB values than did the contralateral only at week 1. The Collagenase rats had only lower static weight bearing ratios (SWBRs) values than did the Controls at week 1 (p < 0.05). Our customized corridor showed changes in DWB compatible with histopathological and ultrasound feature changes in the rat tendinopathy model. The hindlimb SWBRs did not correspond with any tendinopathic changes.

P53-derived hybrid peptides induce apoptosis of synovial fibroblasts in the rheumatoid joint

Loss of p53-mediated suppression by its dominant-negative counterpart is commonly observed in human cancers, and activating p73 is a therapeutic strategy in p53-mutated oncological patients. In synovial fibroblasts (SFs) from rheumatoid arthritis (RA), mutant p53 can lead to the transformation-like features with resistance to the apoptosis induction. We examined whether intra-articular (i.a.) administration of p53-derived hybrid peptides to activate p73 can induce apoptosis of SFs by using adenoviral vectors encoding 37 amino acid (Ad37AA), a p53-derived hybrid peptide capable of activating p73, to transduce SFs in vitro and inject collagen-induced arthritis (CIA) joints in vivo. Increased p73 expression was found in synovial lining layers and SFs of RA patients and CIA rats. Higher expression of p53 up-regulated modulator of apoptosis (PUMA) and Bax with enhanced apoptosis were found in Ad37AA-transduced SFs, and silencing p73 abrogated the up-regulation of PUMA and Bax. Articular indexes and histologic scores were reduced in Ad37AA-injected joints with decreased SF densities, increased apoptotic cell numbers, and higher PUMA expression levels. We demonstrate that i.a. administration of p53-derived hybrid peptides can activate p73 to induce apoptosis of SFs and ameliorate the rheumatoid joint, implicating an enhancement of the p73-dependent apoptotic mechanism as a pharmacological strategy in the RA therapy.

Amelioration Of Collagen-Induced Arthritis By Modulation Of Inhibitory Apoptosis Stimulating Protein Of p53 To Activate Transcription Factor p73

SUPPLEMENTSex Bias In Autoimmune Diseases : Increased Risk Of 47,XXX In Systemic Lupus Erythematosus (SLE) and Sjogrens Syndrome (SS) Supports The Gene Dose HypothesisBackground/Purpose: Human FoxP3+ Th-cells are heterogeneous in function and include not only suppressive cells (TRegs) but also nonsuppressive cells that abundantly secrete proinflammatory cytokines. We have previously shown that FoxP3+ Th-cells were increased in GPA-patients during remission as compared to healthy controls (HCs). In this group of patients, however, we observed a defective suppressor function of TRegs, and an increase in the percentage of Th-17 cells. These observations make it tempting to investigate whether increased FoxP3+ Th-cells in GPA-patients are attributed to an increase in the cytokine-secreting non-suppressive FoxP3+Th-cells. Methods: Peripheral blood mononuclear cells (PBMCs) were isolated from 46 GPA-patients in remission and from 22 age- and sex-matched HCs. Expression of CD4, CD45RO, and FoxP3 were determined by flow cytometric analysis. The expression levels of FoxP3 and CD45RO were used for distinction between activated suppressor TRegs (FoxP3HighCD45RO+; ASTReg), resting suppressor TRegs (FoxP3LowCD45RO-; RSTReg), and cytokine-secreting non-suppressor TRegs (FoxP3LowCD45RO+; NONTReg) cells. Intracellular expression of IFNg, IL-17, and IL-21 were determined in the various FoxP3+ Th-cell subsets after in vitro activation of PBMCs by PMA and Ca-Ionophore. Results: A significant increase in the frequency of NONTReg cells was observed in GPA-patients as compared with HCs, whereas no differences were detected in RSTReg- and ASTReg cells between GPA-patients and HCs. The distribution of RSTReg- and NONTReg cells did not differ between ANCA-negative and ANCA-positive patients, whereas lower percentages of ASTReg cells were observed in ANCA-positive patients as compared to ANCA-negative patients and HCs. Importantly, a significant increase in the percentage of IL-17+ and IL-21+ cells was seen within the NONTRegcells from ANCA-positive patients (n= 9) when compared to ANCA-negative (n= 10) and HCs (n= 12), whereas no differences were found between ANCA-negative and HCs. Conclusion: Increased FoxP3 expression in Th-cells from GPA-patients is related to an increase in a subset of non-suppressive Th-cells. Increased production of IL-17 and IL-21 cytokines, in NONTReg cells from ANCApositive patients points towards FoxP3+ effector cells and decrease in suppressive TReg cells in relation to ANCA production.Complex Functional Effects Within The HLA Contribute To Sjogrens Syndrome Pathogenesis and May Influence Both Transcriptional Regulation and Peptide Binding