56 research outputs found
Epigenetically-driven anatomical diversity of synovial fibroblasts guides joint-specific fibroblast functions
Joint-level responses to tofacitinib and methotrexate: a post hoc analysis of data from ORAL Start
BACKGROUND: Rheumatoid arthritis (RA) has a variable impact on different synovial joints, with inflammation being more commonly observed in some joints than others. Emerging evidence suggests that the anatomical variation in pathophysiology could result in differential responses to treatments across the joints, both within and between modes of action. This analysis aimed to characterize joint-specific responses to tofacitinib and methotrexate monotherapy in patients with RA.
METHODS: This was a post hoc analysis of data from the phase III trial ORAL Start (NCT01039688), in methotrexate-naïve patients with RA. A paired joint pathology score (PJPS), derived from bilateral tender/swollen joint counts, was calculated. The percentage change from baseline in PJPS (%∆PJPS) and treatment-specific responses (tofacitinib 5 and 10 mg twice daily [BID] vs methotrexate; tofacitinib 5 vs 10 mg BID) for each patient joint pair, except for those with baseline/post-baseline PJPS = 0, were calculated at month 3, month 6, and month 12. Radiographic progression was similarly assessed using the Modified Total Sharp Score at month 6 and month 12.
RESULTS: In methotrexate-naïve patients, differences in %∆PJPS demonstrated greater responses with tofacitinib vs methotrexate in most joint locations. Lesser responses with tofacitinib vs methotrexate were observed in most joints of the feet, particularly at month 12. Despite this, radiographic progression at month 12 was significantly worse in the foot (and metacarpophalangeal) joints of patients receiving methotrexate vs tofacitinib.
CONCLUSION: We observed variation in joint-specific responses with tofacitinib and methotrexate monotherapy. Despite a proximal-distal efficacy gradient, with better clinical responses in the feet, patients receiving methotrexate monotherapy demonstrated more radiographic progression in the foot joints compared with those receiving tofacitinib. These findings suggest that body site- and therapy-specific characteristics may interact to produce differential treatment responses
Laboratory Methodology Important in the Diagnosis and Prognosis of Antiphospholipid Syndrome
Antiphospholipid syndrome (APS) is an autoimmune disease, characterized by thrombosis and pregnancy complications with persistently elevated levels of antiphospholipid antibodies (aPL). Recently, a unique mathematical calculation has been presented to assess the risk of thrombosis in patients with APS called antiphospholipid score or global antiphospholipid syndrome score (GAPSS). This new approach in the diagnosis of APS leads to the assessment of the risk of thrombosis considering the results of different aPL (lupus anticoagulants (LA), anticardiolipin antibodies (aCL), antibodies against β2GPI (anti-β2GPI), and phosphatidylserine-dependent antiprothrombin antibodies (aPS/PT) (isotypes IgG and IgM). This chapter provides an overview of the algorithm strategy for APS diagnosis with the aims of characterizing in detail the laboratory methodology of criteria aPL (LA, aCL, and anti-β2GPI) and noncriteria aPL, such as IgA aCL and IgA anti-β2GPI, anti-domain I β2GPI, and antiprothrombin antibodies. In order to improve APS diagnosis, several new approaches in aPL detection have recently been suggested, such as multiline immunodot assay, detection of aPL by flow cytometry using beads with particular surface properties, and the newly developed automated BioPlex system technology for parallel detection of aCL and anti-β2GPI antibodies of IgG, IgA, and IgM isotypes. A completely different and promising approach in future research lies in the potential of microRNAs as biomarkers for risk of thrombosis and/or obstetric complication
Regulation and function of SIRT1 in rheumatoid arthritis synovial fibroblasts
Rheumatoid arthritis (RA) is a chronic autoimmune disease characterized by inflammation and destruction of synovial joints. The function of sirtuin (SIRT)1 in RA is inconclusive. In human synovial cells, SIRT1 was shown to promote cytokine production and apoptosis resistance. However, deletion of SIRT1 aggravated inflammatory arthritis in mice and increased production of pro-inflammatory cytokines in murine macrophages. In the current study, we investigated the regulation, expression, and function of SIRT1 in RA, in particular its role in adhesion and proliferation of human RA synovial fibroblasts (RASF). We found that expression of SIRT1 was increased in vivo in synovial tissues of RA smokers and in vitro by stimulation of RASF with TNFα, but decreased upon treatment with cigarette smoke extract. Synovial tissues of RA smokers showed higher leukocytic infiltration that positively correlated with enhanced levels of SIRT1. Global transcriptome analysis revealed that SIRT1 modulates expression of genes involved in the regulation of inflammatory response and cell adhesion. In functional studies, silencing of SIRT1 reduced proliferation and leukocytic adhesion to RASF but showed inconsistent results in the regulation of adhesion to plastic. In conclusion, SIRT1 modulates the proliferative and potentially also adhesive properties of RASF and can therefore promote progression of RA.
KEY MESSAGES: SIRT1 is upregulated by TNFα but decreased upon CSE treatment of RASF. Upregulation of SIRT1 in RA smokers correlates with increased leukocytic infiltration. SIRT1 modulates expression of genes regulating cell adhesion and inflammation. SIRT1 regulates proliferation of RASF
Dysregulated Expression of Arterial MicroRNAs and Their Target Gene Networks in Temporal Arteries of Treatment-Naïve Patients with Giant Cell Arteritis
In this study, we explored expression of microRNA (miR), miR-target genes and matrix remodelling molecules in temporal artery biopsies (TABs) from treatment-naïve patients with giant cell arteritis (GCA, n = 41) and integrated these analyses with clinical, laboratory, ultrasound and histological manifestations of GCA. NonGCA patients (n = 4) served as controls. GCA TABs exhibited deregulated expression of several miRs (miR-21-5p, -145-5p, -146a-5p, -146b-5p, -155-5p, 424-3p, -424-5p, -503-5p), putative miR-target genes (YAP1, PELI1, FGF2, VEGFA, KLF4) and matrix remodelling factors (MMP2, MMP9, TIMP1, TIPM2) with key roles in Toll-like receptor signaling, mechanotransduction and extracellular matrix biology. MiR-424-3p, -503-5p, KLF4, PELI1 and YAP1 were identified as new deregulated molecular factors in GCA TABs. Quantities of miR-146a-5p, YAP1, PELI1, FGF2, TIMP2 and MMP9 were particularly high in histologically positive GCA TABs with occluded temporal artery lumen. MiR-424-5p expression in TABs and the presence of facial or carotid arteritis on ultrasound were associated with vision disturbances in GCA patients. Correlative analysis of miR-mRNA quantities demonstrated a highly interrelated expression network of deregulated miRs and mRNAs in temporal arteries and identified KLF4 as a candidate target gene of deregulated miR-21-5p, -146a-5p and -155-5p network in GCA TABs. Meanwhile, arterial miR and mRNA expression did not correlate with constitutive symptoms and signs of GCA, elevated markers of systemic inflammation nor sonographic characteristics of GCA. Our study provides new insights into GCA pathophysiology and uncovers new candidate biomarkers of vision impairment in GCA
P38 Mediates Tumor Suppression through Reduced Autophagy and Actin Cytoskeleton Changes in NRAS-Mutant Melanoma
Hotspot mutations in the NRAS gene are causative genetic events associated with the development of melanoma. Currently, there are no FDA-approved drugs directly targeting NRAS mutations. Previously, we showed that p38 acts as a tumor suppressor in vitro and in vivo with respect to NRAS-mutant melanoma. We observed that because of p38 activation through treatment with the protein synthesis inhibitor, anisomycin leads to a transient upregulation of several targets of the cAMP pathway, representing a stressed cancer cell state that is often observed by therapeutic doses of MAPK inhibitors in melanoma patients. Meanwhile, genetically induced p38 or its stable transduction leads to a distinct cellular transcriptional state. Contrary to previous work showing an association of invasiveness with high p38 levels in BRAF-mutated melanoma, there was no correlation of p38 expression with NRAS-mutant melanoma invasion, highlighting the difference in BRAF and NRAS-driven melanomas. Although the role of p38 has been reported to be that of both tumor suppressor and oncogene, we show here that p38 specifically plays the role of a tumor suppressor in NRAS-mutant melanoma. Both the transient and stable activation of p38 elicits phosphorylation of mTOR, reported to be a master switch in regulating autophagy. Indeed, we observed a correlation between elevated levels of phosphorylated mTOR and a reduction in LC3 conversion (LCII/LCI), indicative of suppressed autophagy. Furthermore, a reduction in actin intensity in p38–high cells strongly suggests a role of mTOR in regulating actin and a remodeling in the NRAS-mutant melanoma cells. Therefore, p38 plays a tumor suppressive role in NRAS-mutant melanomas at least partially through the mechanism of mTOR upregulation, suppressed autophagy, and reduced actin polymerization. One or more combinations of MEK inhibitors with either anisomycin, rapamycin, chloroquine/bafilomycin, and cytochalasin modulate p38 activation, mTOR phosphorylation, autophagy, and actin polymerization, respectively, and they may provide an alternate route to targeting NRAS-mutant melanoma
Liposomal aggregates sustain the release of rapamycin and protect cartilage from friction
Liposomes show promise as biolubricants for damaged cartilage, but their small size results in low joint and cartilage retention. We developed a zinc ion-based liposomal drug delivery system for local osteoarthritis therapy, focusing on sustained release and tribological protection from phospholipid lubrication properties. Our strategy involved inducing aggregation of negatively charged liposomes with zinc ions to extend rapamycin (RAPA) release and improve cartilage lubrication. Liposomal aggregation occurred within 10 min and was irreversible, facilitating excess cation removal. The aggregates extended RAPA release beyond free liposomes and displayed irregular morphology influenced by RAPA. At nearly 100 µm, the aggregates were large enough to exceed the previously reported size threshold for increased joint retention. Tribological assessment on silicon surfaces and ex vivo porcine cartilage revealed the system's excellent protective ability against friction at both nano- and macro-scales. Moreover, RAPA was shown to attenuate the fibrotic response in human OA synovial fibroblasts. Our findings suggest the zinc ion-based liposomal drug delivery system has potential to enhance OA therapy through extended release and cartilage tribological protection, while also illustrating the impact of a hydrophobic drug like RAPA on liposome aggregation and morphology
Liposomal aggregates sustain the release of rapamycin and protect cartilage from friction.
Liposomes show promise as biolubricants for damaged cartilage, but their small size results in low joint and cartilage retention. We developed a zinc ion-based liposomal drug delivery system for local osteoarthritis therapy, focusing on sustained release and tribological protection from phospholipid lubrication properties. Our strategy involved inducing aggregation of negatively charged liposomes with zinc ions to extend rapamycin (RAPA) release and improve cartilage lubrication. Liposomal aggregation occurred within 10 min and was irreversible, facilitating excess cation removal. The aggregates extended RAPA release beyond free liposomes and displayed irregular morphology influenced by RAPA. At nearly 100 µm, the aggregates were large enough to exceed the previously reported size threshold for increased joint retention. Tribological assessment on silicon surfaces and ex vivo porcine cartilage revealed the system's excellent protective ability against friction at both nano- and macro-scales. Moreover, RAPA was shown to attenuate the fibrotic response in human OA synovial fibroblasts. Our findings suggest the zinc ion-based liposomal drug delivery system has potential to enhance OA therapy through extended release and cartilage tribological protection, while also illustrating the impact of a hydrophobic drug like RAPA on liposome aggregation and morphology
Olive Leaf Extract Attenuates Inflammatory Activation and DNA Damage in Human Arterial Endothelial Cells
Olive leaf extract (OLE) is used in traditional medicine as a food supplement and as an over-the-counter drug for a variety of its effects, including anti-inflammatory and anti-atherosclerotic ones. Mechanisms through which OLE could modulate these pathways in human vasculature remain largely unknown. Serum amyloid A (SAA) plays a causal role in atherosclerosis and cardiovascular diseases and induces pro-inflammatory and pro-adhesive responses in human coronary artery endothelial cells (HCAEC). Within this study we explored whether OLE can attenuate SAA-driven responses in HCAEC. HCAEC were treated with SAA (1,000 nM) and/or OLE (0.5 and 1 mg/ml). The expression of adhesion molecules VCAM-1 and E-selectin, matrix metalloproteinases (MMP2 and MMP9) and microRNA 146a, let-7e, and let-7g (involved in the regulation of inflammation) was determined by qPCR. The amount of secreted IL-6, IL-8, MIF, and GRO-alpha in cell culture supernatants was quantified by ELISA. Phosphorylation of NF-kappa B was assessed by Western blot and DNA damage was measured using the COMET assay. OLE decreased significantly released protein levels of IL-6 and IL-8, as well as mRNA expression of E-selectin in SAA-stimulated HCAEC and reduced MMP2 levels in unstimulated cells. Phosphorylation of NF-kappa B (p65) was upregulated in the presence of SAA, with OLE significantly attenuating this SAA-induced effect. OLE stabilized SAA-induced upregulation of microRNA-146a and let-7e in HCAEC, suggesting that OLE could fine-tune the SAA-driven activity of NF-kappa B by changing the microRNA networks in HCAEC. SAA induced DNA damage and worsened the oxidative DNA damage in HCAEC, whereas OLE protected HCAEC from SAA- and H2O2-driven DNA damage. OLE significantly attenuated certain pro-inflammatory and pro-adhesive responses and decreased DNA damage in HCAEC upon stimulation with SAA. The reversal of SAA-driven endothelial activation by OLE might contribute to its anti-inflammatory and anti-atherogenic effects in HCAEC
OTUD6B-AS1 Might Be a Novel Regulator of Apoptosis in Systemic Sclerosis
Antisense long non-coding RNAs (AS lncRNAs) have increasingly been recognized as important regulators of gene expression and they have been found to play key roles in several diseases. However, very little is known about the role of AS lncRNAs in fibrotic diseases such as systemic sclerosis (SSc). Our recent screening experiments by RNA sequencing showed that ovarian tumor domain containing 6B antisense RNA1 (OTUD6B-AS1) and its sense gene OTUD6B were significantly downregulated in SSc skin biopsies. Therefore, we aimed to identify key regulators of OTUD6B-AS1 and to analyze the functional relevance of OTUD6B-AS1 in SSc. OTUD6B-AS1 and OTUD6B expression in SSc and healthy control (HC) dermal fibroblasts (Fb) after stimulation with transforming growth factor-β (TGFβ), Interleukin (IL)-4, IL-13, and platelet-derived growth factor (PDGF) was analyzed by qPCR. To identify the functional role of OTUD6B-AS1, dermal Fb or human pulmonary artery smooth muscle cells (HPASMC) were transfected with a locked nucleic acid antisense oligonucleotide (ASO) targeting OTUD6B-AS1. Proliferation was measured by BrdU and real-time proliferation assay. Apoptosis was measured by Caspase 3/7 assay and Western blot for cleaved caspase 3. While no difference was recorded at the basal level between HC and SSc dermal Fb, the expression of OTUD6B-AS1 and OTUD6B was significantly downregulated in both SSc and HC dermal Fb after PDGF stimulation in a time-dependent manner. Only mild and inconsistent effects were observed with TGFβ, IL-4, and IL-13. OTUD6B-AS1 knockdown in Fb and HPASMC did not affect extracellular matrix or pro-fibrotic/proinflammatory cytokine production. However, OTUD6B-AS1 knockdown significantly increased Cyclin D1 expression at the mRNA and protein level. Moreover, silencing of OTUD6B-AS1 significantly reduced proliferation and suppressed apoptosis in both dermal Fb and HPASMC. OTUD6B-AS1 knockdown did not affect OTUD6B expression at the mRNA level and protein level. Our data suggest that OTUD6B-AS1 regulates proliferation and apoptosis via cyclin D1 expression in a sense gene independent manner. This is the first report investigating the function of OTUD6B-AS1. Our data shed light on a novel apoptosis resistance mechanism in Fb and vascular smooth muscle cells that might be relevant for pathogenesis of SSc
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