Identification and validation of mutual hub genes in idiopathic pulmonary fibrosis and rheumatoid arthritis-associated usual interstitial pneumonia.

Objectives: The study aims at exploring common hub genes and pathways in idiopathic pulmonary fibrosis (IPF) and rheumatoid arthritis-associated usual interstitial pneumonia (RA-UIP) through integrated bioinformatics analyses. Methods: The GSE199152 dataset containing lung tissue samples from IPF and RA-UIP patients was acquired from the Gene Expression Omnibus (GEO) database. The identification of overlapping differentially expressed genes (DEGs) in IPF and RA-UIP was carried out through R language. Protein-protein interaction (PPI) network analysis and module analysis were applied to filter mutual hub genes in the two diseases. Enrichment analyses were also conducted to analyze the possible biological functions and pathways of the overlapped DEGs and hub genes. The diagnostic value of key genes was assessed with R language, and the expressions of these genes in pulmonary cells of IPF and rheumatoid arthritis-associated interstitial lung disease (RA-ILD) patients were analyzed with single cell RNA-sequencing (scRNA-seq) datasets. The expression levels of hub genes were validated in blood samples from patients, specimens of human lung fibroblasts, lung tissue samples from mice, as well as external GEO datasets. Results: Four common hub genes (THBS2, TIMP1, POSTN, and CD19) were screened. Enrichment analyses showed that the abnormal expressions of DEGs and hub genes may be connected with the onset of IPF and RA-UIP by regulating the progression of fibrosis. ScRNA-seq analyses illustrated that for both IPF and RA-ILD patients, THBS2, TIMP1, and POSTN were mainly expressed in lung fibroblasts, while CD19 was uniquely high-expressed in B cells. The qRT-PCR and immunohistochemistry (IHC) results verified that the expression levels of hub genes were mostly in accordance with the findings obtained from the bioinformatics analyses. Conclusion: Though IPF and RA-UIP are distinct diseases, they may to some extent have mutual pathogenesis in the development of fibrosis. THBS2, TIMP1, POSTN, and CD19 may be the potential biomarkers of IPF and RA-UIP, and intervention on related pathways of these genes could offer new strategies for the precision treatment of IPF and RA-UIP.

Screening and validation of differentially expressed genes in polymyositis.

Background: Polymyositis (PM), a prevalent inflammatory myopathy, currently lacks defined pathogenic mechanism. To illuminate its pathogenesis, we integrated bioinformatics and clinical specimens to examine potential aberrant gene expression patterns and their localization. Methods: We obtained GSE128470 and GSE3112 dataset from the Gene Expression Omnibus, performed Gene Set Enrichment Analysis (GSEA) and immune infiltration analysis using CiberSort, identified differentially expressed genes with Limma, conducted functional annotation and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis, constructed a Protein-Protein Interaction network, and identified hub genes using Cytoscape. ROC analysis evaluated hub gene diagnostic accuracy for PM, validated their expression levels with clinical specimens. Results: DEG analysis revealed 51 upregulated and 779 downregulated genes. Gene Ontology (GO) analysis implicated Type I interferon (IFN-Ⅰ) signaling, while KEGG pointed to cell adhesion molecule activation and oxidative phosphorylation inhibition. Protein-Protein Interaction (PPI) analysis identified 8 diagnosffftic hub genes. Clinical samples confirmed their upregulation in PM, especially IRF1 and IRF9 between muscle fibers. Different immune cell infiltrations were observed in PM patients versus controls. Conclusions: Our study explores potential pathogenic factors, diagnostic markers, and immune cells in PM, with a focus on verifying IRF1 and IRF9 upregulation in the IFN-I signaling pathway. These findings bear significance for PM diagnosis and treatment.

CD4+LAG3+T cells are decreased in SSc-ILD and affect fibroblast mesenchymal transition by TGF-ß3.

Pulmonary fibrosis frequently occurs in rheumatic conditions, particularly systemic sclerosis-associated interstitial lung disease (SSc-ILD). The pathology involves cell transformation into interstitial structures and collagen accumulation. CD4+LAG3+T cells, known for immune inhibition, are relevant in autoimmunity. This study investigates CD4+LAG3+T cells in SSc-ILD. Clinical analysis revealed a correlation between CD4+LAG3+T cells and interleukin-6 (IL-6) and erythrocyte sedimentation rate (ESR). Using primary human lung fibroblasts (pHLFs) and murine bone marrow-derived macrophages (BMDMs), we showed that CD4+LAG3+T cells secreted TGF-ß3 inhibits TGF-ß1-induced mesenchymal transformation, modulates cellular function, and reduces collagen release. In mouse models, CD4+LAG3+T cells exhibited potential in alleviating bleomycin-induced pulmonary fibrosis. This study emphasizes CD4+LAG3+T cells' therapeutic promise against fibrosis and proposes their role as biomarkers.

Diagnostic efficacy of joint ultrasonography, dual-energy computed tomography and minimally invasive arthroscopy on knee gouty arthritis, a comparative study.

OBJECTIVE: This study aimed to investigate the diagnostic performance of minimally invasive arthroscopy for knee gout when comparing with joint ultrasonography and dual-energy computed tomography (DECT). METHODS: From January 2016 to December 2018, 121 inpatients with knee joint swelling and pain were prospectively enrolled, including 63 gout patients and 58 non-gout patients. All patients underwent pre-operative ultrasonography and DECT to evaluate knee joint monosodium urate (MSU) deposits, followed by minimally invasive arthroscopy. The gold-standard for gout diagnosis was defined as the detection of MSU crystals in the synovial fluid under polarizing microscopic or pathological analysis. RESULTS: The diagnostic results of ultrasonic double contour sign, hyperechogenic foci, MSU deposition (detected by DECT), MSU deposition (detected by arthroscopy) and MSU deposition in cartilage (detected by arthroscopy) were significantly associated with that of the gold-standard. Except for hyperechogenic foci, the other four indexes had high sensitivity and specificity (approximately or over 80%) and a large odds ratio (OR) (14.73 to 36.56), indicating good diagnostic performance. Detection of MSU deposition in cartilage by arthroscopy had a good diagnostic agreement with the ultrasonic double contour sign (κ = 0.711, p < 0.001). CONCLUSION: Joint ultrasonography, DECT, and minimally invasive arthroscopy had high sensitivity and specificity for the diagnosis of knee gouty arthritis. Minimally invasive arthroscopy was superior to joint ultrasonography and DECT, which can be a useful supplement for the diagnosis of gout. ADVANCES IN KNOWLEDGE: This is the first study comparing the diagnostic performance for knee gout among the joint ultrasonography, DECT, and minimally invasive arthroscopy.

The effects of oral treatment for systemic sclerosis related pulmonary arterial hypertension: A systematic review and meta-analysis.

OBJECTIVES: The usage of oral therapies, endothelin receptor antagonists (ERAs), phosphodiesterase type-5 (PDE-5) inhibitors and prostaglandin analogs has resulted in improved outcomes in patients with pulmonary arterial hypertension related to systemic sclerosis (SSc-PAH). However, the optimal therapeutics have not been determined. METHODS: A systematic searching in the databases of Medline (PubMed), Embase, the Cochrane Library (Central) and unpublished clinical trials (clinicaltrials.gov) was conducted to identify the clinical studies with oral treatment for SSc-PAH patients published before 1 June 2019. The data were extracted and the quality was assessed. The main outcomes are exercise capacity and hemodynamic parameters, which were synthesized and analyzed. RESULTS: In total, 27 clinical trials were enrolled for further analysis. It was demonstrated that bosentan treatment, the widely used drug for PAH, might improve the exercise capacity and pulmonary arterial pressure (PAP) and pulmonary vascular resistance (PVR) in this clinical setting, although without significant difference. Meanwhile, the usage of prostaglandin analogs could improve the parameters mentioned above. Furthermore, combined therapy with ambrisentan and tadalafil significantly increased the treatment efficacy of key parameters in SSc-PAH patients compared with basic treatment. CONCLUSION: This meta-analysis reveals that combination therapy might provide more benefits to exercise capacity and hemodynamic parameters in SSc-PAH patients. Still more RCTs are needed to provide more solid evidence.

Anti-fibrotic effect of iguratimod on pulmonary fibrosis by inhibiting the fibroblast-to-myofibroblast transition.

PURPOSE: Pulmonary fibrosis (PF) is a severe lung disease causing significant morbidity and mortality. PF pathogenesis is attributed to the fibroblast-to-myofibroblast transition (FMT) driven by the most potent pro-fibrogenic factor TGF-ß1 activating the Smad3-dependent TGF-ß1 canonical pathway. Iguratimod (IGU) is a novel anti-rheumatic drug that suppresses the secretion of inflammatory factors, but is also able to modulate the differentiation of multiple cells. Therefore, the aim of this work was to investigate the effect of IGU on FMT. MATERIALS/METHODS: PF mouse model was induced in C57BL/6 male mice by bleomycin. The effect of IGU was assessed through the evaluation of lung morphology by H&E and through the collagen accumulation in the lung by Masson staining. Primary human lung fibroblasts (pHLFs) were also used to evaluate the effect of IGU in vitro on TGF-ß1-stimulated cells, and proliferation, migration and invasion were measured, together with genes and proteins involved in FMT. RESULTS: IGU attenuated bleomycin-induced PF in mice and improved the pathological changes in their lungs. In addition, IGU significantly inhibited proliferation, migration and invasion in TGF-ß1-stimulated pHLFs without causing apoptosis. Moreover, IGU significantly reduced TGF-ß1-induced increase of collagen I and III mRNA expression, thus reducing lung function impairment, and α-SMA, Smad2 and Smad3 phosphorylation, fibronectin expression and F-actin microfilament formation, thus attenuating FMT through the inhibition of the Smad3 pathway. CONCLUSIONS: Our results collectively revealed the beneficial effect of IGU on the inhibition of FMT, thus suggesting that it might act as an effective anti-fibrotic agent in preventing the progression of PF.

Clinical Characteristics and Risk Factors of Diffuse Alveolar Hemorrhage in Systemic Lupus Erythematosus: a Systematic Review and Meta-Analysis Based on Observational Studies.

Diffuse alveolar hemorrhage (DAH) is a rare but potentially deadly manifestation of systemic lupus erythematosus (SLE). The aim of this study was to investigate the clinical characteristics and risk factors of DAH in SLE. A systematic review and meta-analysis of previous observational studies compared the clinical characteristics and risk factors between DAH-SLE and SLE patients without DAH. A total of 5 observational studies were included in this meta-analysis. Compared with the SLE patients without DAH, DAH-SLE patients had a significantly higher incidence of neuropsychiatric events (OR = 4.321, 95% CI (1.686-11.073), P = 0.002, I2 = 49.2%), nephritis (OR = 3.146, 95% CI (1.663-5.955,), P = 0.000, I2 = 0.0%), serositis (OR = 6.028, 95% CI (1.418-25.635), P = 0.015, I2 = 80.3%), dyspnea (OR = 31.241,95% CI (0.202-4833.203), P = 0.181, I2 = 94.6%), and a significantly lower level of C3 (SMD = - 1.358, 95% CI - 1.685, - 1.031), P = 0.000, I2 = 98.0%), C4 (SMD = - 1.251, 95% CI (- 1.648, - 0.855), P = 0.000, I2 = 87.7%), hemoglobin (SMD = - 2.074, 95% CI (- 2.433, - 1.715), P = 0.000, I2 = 94.2%), and a higher SLEDAI-2K score (SMD = 1.284, 95% CI (0.959, 1.608), P = 0.000, I2 = 98.2%). However, due to significant heterogeneity, some of these results should be interpreted cautiously. Nevertheless, when the above abnormal indicators are found, especially neuropsychiatric involvement and nephritis, besides the existed diagnostic criteria for DAH in SLE patients, a diagnosis for DAH should be considered and relevant treatment timely initiated. Further prospective multi-center SLE studies with a large cohort of patients and long-term follow-up are needed to clarify further or find out the specific clinical indexes for DAH in SLE patients.

Accumulation of cytosolic dsDNA contributes to fibroblast-like synoviocytes-mediated rheumatoid arthritis synovial inflammation.

The accumulation of cytosolic dsDNA plays important roles in the regulation of cellular processes. However, whether cytosolic dsDNA is involved in the pathogenesis of rheumatoid arthritis (RA) is not clear. Therefore, the present study investigated the roles of cytosolic dsDNA in the modulation of inflammatory responses of fibroblast-like synoviocytes (FLS) in patients with RA. FLS were obtained from active RA patients. dsDNA accumulation in the cytosol was detected by immunofluorescence staining and the Qubit® dsDNA HS Assay. Immunohistochemistry was employed to detect the dsDNA and cGMP-AMP synthase (cGAS) expression in the synovium. Short hairpin RNA (shRNA) was used to knockdown the expression of cGAS and stimulator of interferon genes (STING). Protein expression was detected by Western blotting and immunofluorescence staining. We observed increased cytosolic dsDNA and cGAS expression in FLS and synovium from RA patients. dsDNA and cGAS expression correlated with the severity of rheumatoid synovitis. Transfection of dsDNA into the cytosol of RA FLS promoted pro-inflammatory cytokines production. DNaseII overexpression downregulated cytosolic dsDNA expression and inhibited dsDNA-induced cytokines secretion. We also found that dsDNA and TNF-α enhanced cGAS and STING expression, and dsDNA-induced cytokine secretion was reduced by cGAS or STING knockdown. Furthermore, we determined that the dsDNA-induced phosphorylation of IRF3 and NF-κBp65 was decreased by DNaseII overexpression or cGAS/STING knockdown. Overall, our findings show that increased cytosolic dsDNA level promoted inflammatory responses via the cGAS/STING pathway in RA FLS, which suggests that cytosolic dsDNA accumulation is an important contributor to FLS-mediated rheumatoid synovial inflammation.

Inhibitory effects of pirfenidone on fibroblast to myofibroblast transition in rheumatoid arthritis-associated interstitial lung disease via the downregulation of activating transcription factor 3 (ATF3).

OBJECTIVE: Pirfenidone (PFD) is an oral anti-fibrotic drug used for idiopathic pulmonary fibrosis (IPF) therapy. We determined the role of activating transcription factor 3 (ATF3) and the effect of PFD on fibroblast to myofibroblast transition (FMT) in rheumatoid arthritis-associated interstitial lung disease (RA-ILD). METHODS: RA-ILD lung specimens were obtained by CT-guided percutaneous transthoracic biopsy. A pulmonary fibrosis mouse model was established by the intratracheal injection of bleomycin. Pathological variation and the expression of α-SMA and ATF3 were observed by H&E, Masson and immunofluorescence staining. Primary human lung fibroblasts (pHLFs) were isolated from lung tissues that were pathologically confirmed to be normal by pneumonectomy. Cell viability was detected using an MTT assay. Cell migration and invasion were detected using a Transwell chamber. The protein levels of α-SMA, ATF3, Smad3 and p-Smad3 were measured by Western blot. HLFs were infected with lentiviruses expressing ATF3 or scrambled shRNA. RESULTS: ATF3 was dramatically upregulated in lung tissues from both bleomycin-induced mice and patients with RA-ILD compared with controls. The upregulation of ATF3 and the accumulation of collagen in the lung tissues of mice with pulmonary fibrosis were reduced by PFD. PFD significantly inhibited increases in the proliferation, invasion and migration of pHLFs stimulated by TGF-ß1. Moreover, we observed the inhibitory effect of PFD on FMT via the downregulation of ATF3, which was further confirmed in ATF3 knockdown (KD) pHLFs. CONCLUSIONS: This work shows the inhibitory effect of PFD on FMT in pHLFs, which is mediated by the downregulation of ATF3. Our findings suggest that PFD might have therapeutic potential for the treatment of RA-ILD.

Increased serum levels of MIC1/GDF15 correlated with bone erosion in spondyloarthritis: A pilot study.

INTRODUCTION: To assess the association between growth differentiation factor-15 (GDF15) and radiographic features including bone marrow edema and bone erosion in Spondyloarthritis (SpA). METHODS: Patients with SpA (n = 120) receiving treatment in the Guangdong General Hospital, China, between August 2012 and December 2016 were retrospectively included. Serum of patients and healthy controls (n = 30) were collected and GDF15 levels were measured using ELISA. Inflammation was assessed by C-reactive protein (CRP), and magnetic resonance imaging (MRI) of the sacroiliac joint using Spondyloarthritis Research Consortium of Canada score and a method of dichotomy to assess fat metaplasia, bone erosion, and ankylosis. Radiographs of the pelvis were scored using the modified New York (mNY) score. RESULTS: Serum GDF15 levels were higher in SpA patients compared to controls (503.52 ±â€Š222.92 vs. 190.86 ±â€Š104.18 pg/mL, P < .0001). Patients who suffered from bone erosion on MRI had higher levels of GDF15 (525.72 [186.33, 801.62]vs. 428.06 [255.15, 670.98] pg/mL, P = .0375). There was a positive correlation between serum GDF15 and CRP (r = 0.5442, P < .0001). Moreover, GDF15 levels were related to CRP levels (r = 0.5658, P < .0001) in those X-ray scores were III, according to 1984mNY criteria. Receiver operating characteristic (ROC) analysis showed that GDF15 levels above 501.98pg/mL could predict presence of bone erosion on MRI. CONCLUSION: The present study suggested that serum GDF15 levels are higher in SpA patients than in healthy controls. The GDF15 level was correlated with CRP and may be a surrogate biomarker in bone erosion.

Design of uniformly collimating light-emitting diode lenses of multiple freeform and Fresnel surfaces with different inter-surface mapping orders.

In this work, we propose a design method of composite freeform collimating lenses that integrates freeform reflection, refraction, and Fresnel surfaces to realize highly collimated and uniformed light output in a single lens element. The algorithm is designed considering a consistent combination of different surfaces, so the light rays reaching the Fresnel surface through refraction and reflection should generate the same luminous intensity. The Fresnel rings can be designed in two different ways, depending on the sequence of light rays propagating between the reflection and the Fresnel surfaces. The lighting effects of the lenses are analyzed in a 3D simulation, and the light output of both schemes reaches a high level of collimation and uniformity, agreeing well with design expectations. The obtained lenses have the advantages of a compact volume, high light extraction efficiency, and variable geometry by changing the combination form of the freeform surfaces.

Attenuation of antimalarial agent hydroxychloroquine on TNF-α-induced endothelial inflammation.

OBJECTIVE: Hydroxychloroquine (HCQ) is an antimalarial drug that is widely used in the treatment of some autoimmune diseases. In the present study, we explore the role of HCQ in regulating endothelial inflammation and its underlying mechanism. METHODS: Human umbilical vein endothelial cells (HUVECs) were isolated from fresh umbilical cords. Protein expression was measured by Western blot or immunofluorescence staining. Endothelial adhesion ability was determined by leukocyte-endothelial monolayer adhesion assay. Transwell assay was used to measure the transendothelial-migration of PBMCs. RESULTS: TNF-α-induced endothelial-leukocyte adhesion and the leukocyte transmigration were profoundly reduced by HCQ treatment. HCQ treatment dramatically inhibited the expression of TNF-α-induced endothelial ICAM-1 and VCAM-1. Furthermore, treatment with HCQ prevented the TNF-α-induced translocation of NF-κB p65 into the nucleus and the phosphorylation of the p65 subunit in HUVECs. HCQ inhibited the expression of phosphorylated p38 and JNK protein but not ERK. Treatment with NF-κB, p38 and JNK inhibitor could also reduce TNF-α-induced endothelial-leukocyte adhesion and the endothelial expression of ICAM-1 and VCAM-1. HCQ administration also suppressed TNF-α induced lung injury in mice by reducing neutrophil infiltration in pulmonary interstitial tissue. CONCLUSIONS: This work shows the inhibitory effect of HCQ on endothelial inflammatory response through, at least in part, blocking NF-κB, p38 and JNK pathways. Our findings suggest that HCQ may be a promising approach for the treatment of inflammatory vascular disease beyond its immunomodulatory actions.

Anti-fibrotic effects of pirfenidone by interference with the hedgehog signalling pathway in patients with systemic sclerosis-associated interstitial lung disease.

AIM: To determine whether pirfenidone attenuates lung fibrosis by interfering with the hedgehog (Hh) signalling pathway in patients with systemic sclerosis-associated interstitial lung disease (SSc-ILD). METHODS: Twenty-five SSc-ILD patients (20 first visit, five who underwent pirfenidone treatment for 6 months) and 10 healthy controls were recruited. Lung tissues were obtained by open-chest surgery, and primary lung fibroblasts were isolated, cultured and stimulated with pirfenidone. The levels of the proteins glioma-associated oncogene 1 (GLI1), suppressor of fused (Sufu), α-smooth muscle actin, and fibronectin in lung tissues or fibroblasts were determined by Western blotting. The messenger RNA levels of GLI1, glioma-associated oncogene 2, protein patched homolog 1, and Sufu in lung tissues or fibroblasts were determined by quantitative reverse-transcription polymerase chain reaction. Meanwhile, the levels of phosphorylation glycogen synthase kinasep-3ß (pGSK-3ß), phosphorylation SMAD2 (pSMAD2), and phosphorylation c-Jun N-terminal kinase (pJNK) in fibroblasts were determined by Western blotting. RESULTS: Hh pathway activation was increased in the lung tissue of SSc-ILD patients and was decreased by pirfenidone, Sufu was upregulated in lung fibroblasts isolated from SSc-ILD patients after pirfenidone challenge, and pirfenidone inhibited the phosphorylation of GSK-3ß signalling. CONCLUSION: Pirfenidone has anti-fibrotic effects in SSc-ILD patients by interfering with both the Hh signalling pathway and the GSK-3ß signalling pathway via the regulation of Sufu expression. These results might promote its use in other Hh driven lung diseases such as idiopathic pulmonary fibrosis and especially the interstitial lung disease associated with connective tissue diseases.

Realization of uniform and collimated light distribution in a single freeform-Fresnel double surface LED lens.

In this work, a design method of a freeform-Fresnel composite surface lens is proposed, which can be applied on LED lighting optics to realize uniform and collimated light output in a single lens element. The lower lens surface is of freeform type, which transforms the light distribution from the LED sources to a uniform one, and the upper lens surface is of the Fresnel type, which further transforms the lights to a collimated beam. Through the optimization of lens parameters, the obtained lens has the advantages of compact volume, wide acceptance angle, high light extraction efficiency, and high design accuracy.

Inhibitory effects of simvastatin on migration and invasion of rheumatoid fibroblast-like synoviocytes by preventing geranylgeranylation of RhoA.

To investigate the effect of simvastatin on the migration and invasion of fibroblast-like synoviocytes (FLS) from patients with rheumatoid arthritis (RA) and its cellular signal mechanisms, FLS from active RA patients were stimulated with 3 % FBS or GM-CSF in the presence or absence of simvastatin. Cells migration and invasion in vitro were measured by the Boyden chamber method. RhoA activity was assessed by a pull-down assay. Matrix metalloproteinases-2 (MMP-2) activity was evaluated by zymography. Simvastatin inhibits FBS- or GM-CSF-induced migration in a dose-dependent manner by RA FLS, and this inhibitory effect is independent of cell apoptosis. We also found that simvastatin suppressed in vitro invasion, adhesion, MMP-2 activity, cytoskeletal reorganization and RhoA activation. Furthermore, mevalonate or GGPP treatment reversed the inhibitory effect of simvastatin not only on migration and invasion in vitro but also on RhoA activation, and inhibition of RhoA by specific siRNA transfection reduced migration, adhesion and invasion of RA FLS. This study shows that simvastatin reduces RA FLS migration and invasion through the prevention of protein geranylgeranylation and RhoA activation. These findings provide a novel evidence that statin may be benefit for preventing RA arthritic destruction, and also indicate that RhoA may be a new target for the modulation of RA FLS migration and invasion.

Role of p21-activated kinase 1 in regulating the migration and invasion of fibroblast-like synoviocytes from rheumatoid arthritis patients.

OBJECTIVE: To investigate the role of p21-activated kinase 1 (PAK1) in regulating migration, invasion and MMP expression in RA fibroblast-like synoviocytes (FLS). METHODS: RA FLS migration and invasion in vitro were measured by the Boyden chamber method. Invasion of RA FLS into cartilage was detected in the severe combined immunodeficiency (SCID) mouse co-implantation model of RA in vivo. PAK1 and MT1-MMP expression were examined by western blotting. ELISA was used to measure the production and activity of MMPs. RESULTS: Phosphorylated PAK1 (p-PAK1) protein expression was increased in ex vivo synovial membrane cells from RA patients. Stimulation with IL-1ß or TNF-α up-regulated p-PAK1 expression. Inhibition of PAK1 by transfection with dominant negative PAK1 mutant (dnPAK1) reduced in vitro migration and invasion of RA FLS. In the SCID mouse model, RA FLS invasion into cartilage was attenuated by transfection with dnPAK1 in vivo. PAK1 regulated IL-1ß-induced production and activity of MMP-13 and MT1-MMP. Inhibition of MMP-13 or MT1-MMP activity also reduced RA FLS invasion. Furthermore, dnPAK1 transfection inhibited c-Jun N-terminal kinase (JNK) activation, but did not affect the activities of extracellular signal-regulated kinases and p38. Inhibition of the JNK activity by chemical inhibitor significantly reduced the migration, invasion and production of MMP-13 and MT1-MMP. CONCLUSION: PAK1 plays an important role in regulating the migration, invasion and production and activity of MMPs in RA FLS, which is mediated by the JNK pathway. This suggests a novel strategy targeting PAK1 to prevent joint destruction of RA.

[Modulation of RhoA/ROCK pathway on TLR-2 ligand-induced chemokine secretion in fibroblast-like synoviocytes from patients with rheumatoid arthritis].

OBJECTIVE: To evaluate the modulation of RhoA/Rho kinase (ROCK) signaling pathway, a small Rho GTPase that is considered as an important modulator in inflammatory responses, on Toll-like receptor-2 mediated chemokine secretion in fibroblast-like synoviocytes (FLS) from rheumatoid arthritis (RA) patients. METHODS: The RhoA activity was measured by a pull-down assay. And the ROCK activity was assessed by Western blot. The secretion of chemokines was measured by ELISA (enzyme-linked immunosorbent assay). MTT test was used to detect the cellular viability. RESULTS: The stimulation of peptidoglycan (PG, 5 mg/L) increased the levels of IL-8 (interleukin-8), RANTES (regulated upon activation normal T cell expressed & secreted) and MCP-2 (monocyte chemotactic protein-2) and boosted the activities of RhoA and ROCK versus the unstimulated RA FLS. And these effects of PG were suppressed by anti-TLR-2 monoclonal antibody. Inhibition of RhoA and ROCK with a specific inhibitor inhibited the secretion of IL-8, RANTES and MCP-2 in PG-induced RA FLS. CONCLUSION: The present study provides novel evidence that the RhoA/ROCK signal pathway modulates the TLR-2-mediated secretion of chemokines in RA FLS. It suggests that the inhibition of RhoA/ROCK may be a new therapeutic approach for RA.

Increased phosphorylation of ezrin/radixin/moesin proteins contributes to proliferation of rheumatoid fibroblast-like synoviocytes.

OBJECTIVES: Increasing evidence indicates that ezrin/radixin/moesin (ERM) proteins may play a critical role in cell proliferation. This study examined the role of ERM proteins in proliferation of fibroblast-like synoviocytes (FLS) from patients with RA. METHODS: Synovial tissues (STs) were obtained from 18 RA and 6 OA patients. The expression of ERM and its phosphorylated proteins in cultured FLS and ST was assessed by western blots or IF staining. Small interference RNA (siRNA)-mediated ERM knockdown was used to inhibit phosphorylation of ERM. Proliferation of FLS was measured by bromodeoxyuridine (BrdU) incorporation into cell DNA and by PCNA immunoblotting. RESULTS: Our study showed that increased phosphorylation of ERM proteins was found in ST and FLS from patients with RA as compared with OA patients and non-arthritis controls. Treatment with TNF-α, IL-1ß or PDGF-induced phosphorylation of ERM proteins in dose- and time-dependent manner by RA FLS, but did not affect the expression of total ERM protein. Rho kinase and p38MAPK signal pathways were involved in TNF-α-induced ERM phosphorylation. We further showed that inhibition of ERM phosphorylation by siRNA-mediated ERM knockdown suppressed TNF-α- or IL-1ß-induced BrdU incorporation and PCNA expression in RA FLS. CONCLUSIONS: This study provides the novel evidence that increased phosphorylation of ERM proteins may contribute to proliferation of RA FLS, suggesting that specific inhibition of ERM phosphorylation may be a new therapeutic approach for RA.

The anti-malaria agent artesunate inhibits expression of vascular endothelial growth factor and hypoxia-inducible factor-1α in human rheumatoid arthritis fibroblast-like synoviocyte.

Increasing evidence indicates that the anti-malarial agent artemisinin and its derivatives may exert anti-angiogenic effect. In the present study, we explored the effect of artesunate, a artemisinin derivative, on TNFα- and hypoxia-induced expression of hypoxia inducible factor-1α (HIF-1α) and secretion of vascular endothelial growth factor (VEGF) and inteleukin-8 (IL-8) in human rheumatoid arthritis fibroblast-like synoviocytes (RA FLS), and further investigated the signal mechanism by which this compound modulates HIF-1α, VEGF and IL-8 expression. RA FLS obtained from patients with active rheumatoid arthritis were pretreated with artesunate, and then stimulated with TNFα and hypoxia. Production of VEGF and IL-8 was measured by ELISA. Nuclear location of HIF-1α was measured by confocal fluorescence microscopy. HIF-1α and other signal transduction proteins expression was measured by Western blot. Artesunate decreased the secretion of VEGF and IL-8 from TNFα- or hypoxia-stimulated RA FLS in a dose-dependent manner. Artesunate also inhibited TNFα- or hypoxia-induced nuclear expression and translocation of HIF-1α. We also showed that artesunate prevented Akt phosphorylation, but did not find evidence that phosphorylation of p38 and ERK was affected. TNFα- or hypoxia-induced secretion of VEGF and IL-8 and expression of HIF-1α were hampered by treatment with the PI3 kinase inhibitor LY294002, suggesting that inhibition of PI3 kinase/Akt activation might inhibit VEGF and IL-8 secretion and HIF-1α expression induced by TNFα or hypoxia. Our results suggest that artesunate inhibits angiogenic factor expression in RA FLS, and provide novel evidence that, as a low-cost agent, artesunate may have therapeutic potential for RA.

HMG-CoA reductase inhibitor simvastatin suppresses Toll-like receptor 2 ligand-induced activation of nuclear factor kappa B by preventing RhoA activation in monocytes from rheumatoid arthritis patients.

To investigate whether anti-inflammatory effects of HMG-CoA reductase inhibitor simvastatin (SMV) in rheumatoid arthritis (RA) is mediated by Toll-like receptor-2 (TLR-2) signal via inhibiting activation of RhoA, a small Rho GTPase that plays an important role in inflammatory responses. Peripheral blood monocytes from active RA patients were treated with Staphylococcus aureus peptidoglycan (PG), a ligand of TLR-2, in the presence or absence of SMV. RhoA activity was assessed by a pull-down assay. DNA-binding activity was measured by a sensitive multi-well colorimetric assay. Cytokine secretion was measured by ELISA. PG stimulation increased the level of active GTP-bound RhoA compared with unstimulated monocytes, and the effect of PG on RhoA activity was suppressed with anti-TLR-2 monoclonal antibody. RhoA inhibition either with a specific inhibitor or by siRNA transfection inhibited activation of NF-κB and secretion of TNFα and IL-1ß in PG-induced RA monocytes. SMV mitigated PG-induced increase in RhoA activity and NF-κB activation as well as secretion of TNFα and IL-1ß. The inhibitory effects of SMV were completely reversed by mevalonate and geranylgeranyl pyrophosphate. Our results indicate the modulation of RhoA on TLR-2-mediated inflammatory signaling in RA and provide a novel evidence for anti-inflammatory effects of statins through influencing TLR-2 signaling via RhoA in RA.