Characterization of the inflammatory proteome of synovial fluid from patients with psoriatic arthritis: Potential treatment targets.

Objectives: 1) To characterize the inflammatory proteome of synovial fluid (SF) from patients with Psoriatic Arthritis (PsA) using a high-quality throughput proteomic platform, and 2) to evaluate its potential to stratify patients according to clinical features. Methods: Inflammatory proteome profile of SF from thirteen PsA patients with active knee arthritis were analyzed using proximity extension assay (PEA) technology (Olink Target 96 Inflammation panel). Four patients with OA were included as control group. Results: Seventy-nine inflammation-related proteins were detected in SF from PsA patients (SF-PsA). Unsupervised analyzes of the molecular proteome profile in SF-PsA identified two specific phenotypes characterized by higher or lower levels of inflammation-related proteins. Clinically, SF-PsA with higher levels of inflammatory proteins also showed increased systemic inflammation and altered glucose and lipid metabolisms. Besides, SF from PsA patients showed 39 out of 79 proteins significantly altered compared to SF-OA specifically related to cell migration and inflammatory response. Among these, molecules such as TNFα, IL-17A, IL-6, IL-10, IL-8, ENRAGE, CCL20, TNFSF-14, OSM, IFNγ, MCP-3, CXCL-11, MCP4, CASP-8, CXCL-6, CD-6, ADA, CXCL-10, TNFß and IL-7 showed the most significantly change. Conclusion: This is the first study that characterizes the inflammatory landscape of synovial fluid of PsA patients by analyzing a panel of 92 inflammatory proteins using PEA technology. Novel SF proteins have been described as potential pathogenic molecules involved in the pathogenesis of PsA. Despite the flare, inflammatory proteome could distinguish two different phenotypes related to systemic inflammation and lipid and glucose alterations.

Preclinical Characterization of Pharmacologic NAD+ Boosting as a Promising Therapeutic Approach in Rheumatoid Arthritis.

OBJECTIVE: We analyzed NAD+ metabolism in patients with rheumatoid arthritis (RA), its association with disease activity and clinical outcomes of RA, and the therapeutic potential of pharmacologic NAD+ boosting. METHODS: Our study included 253 participants. In the first cohort, comprising 153 RA patients and 56 healthy donors, we assessed NAD+ levels and NAD+ -related gene pathways. We analyzed 92 inflammatory molecules by proximity extension assay. In the second cohort, comprising 44 RA patients starting anti-tumor necrosis factor (anti-TNF) drugs, we evaluated changes in NAD+ levels and their association with clinical response after 3 months. Mechanistic studies were performed ex vivo on peripheral blood mononuclear cells (PBMCs) from patients with RA to test the beneficial effects of NAD+ boosters, such as nicotinamide and nicotinamide riboside. RESULTS: Reduced NAD+ levels were found in RA samples, in line with altered activity and expression of genes involved in NAD+ consumption (sirtuins, poly[ADP-ribose] polymerase, CD38), transport (connexin 43), and biosynthesis (NAMPT, NMNATs). Unsupervised clustering analysis identified a group of RA patients with the highest inflammatory profile, the lowest NAD+ levels, and the highest disease activity (as shown by the Disease Activity Score in 28 joints). NAD+ levels were modulated by anti-TNF therapy in parallel with the clinical response. In vitro studies using PBMCs from RA patients showed that nicotinamide riboside and nicotinamide increased NAD+ levels via NAMPT and NMNAT and reduced their prooxidative, proapoptotic, and proinflammatory status. CONCLUSION: RA patients display altered NAD+ metabolism, directly linked to their inflammatory and disease activity status, which was reverted by anti-TNF therapy. The preclinical beneficial effects of NAD+ boosters, as shown in leukocytes from RA patients, along with their proven clinical safety, might pave the way for the development of clinical trials using these compounds.

Nonalcoholic fatty liver disease in inflammatory arthritis: Relationship with cardiovascular risk.

Liver disease is one of the most important causes of morbidity and mortality worldwide whose prevalence is dramatically increasing. The first sign of hepatic damage is inflammation which could be accompanied by the accumulation of fat called non-alcoholic fatty liver disease (NAFLD), causing damage in the hepatocytes. This stage can progress to fibrosis where the accumulation of fibrotic tissue replaces healthy tissue reducing liver function. The next stage is cirrhosis, a late phase of fibrosis where a high percentage of liver tissue has been replaced by fibrotic tissue and liver functionality is substantially impaired. There is a close interplay of cardiovascular disease (CVD) and hepatic alterations, where different mechanisms mediating this relation between the liver and systemic vasculature have been described. In chronic inflammatory diseases such as rheumatoid arthritis (RA) and psoriatic arthritis (PsA), in which the CVD risk is high, hepatic alterations seem to be more prevalent compared to the general population and other rheumatic disorders. The pathogenic mechanisms involved in the development of this comorbidity are still unraveled, although chronic inflammation, autoimmunity, treatments, and metabolic deregulation seem to have an important role. In this review, we will discuss the involvement of liver disease in the cardiovascular risk associated with inflammatory arthritis, the pathogenic mechanisms, and the recognized factors involved. Likewise, monitoring of the liver disease risk in routine clinical practice through both, classical and novel techniques and indexes will be exposed. Finally, we will examine the latest controversies that have been raised about the effects of the current therapies used to control the inflammation in RA and PsA, in the liver damage of those patients, such as methotrexate, leflunomide or biologics.

Pathogenic mechanisms involving the interplay between adipose tissue and auto-antibodies in rheumatoid arthritis.

We aimed to evaluate the association between adipose tissue (AT) dysfunction, autoimmunity, and disease activity in rheumatoid arthritis (RA). A cross-sectional study including 150 RA patients and 50 healthy donors and longitudinal study with 122 RA patients treated with anti-tumor necrosis factor (TNF)-α, anti-interleukin 6 receptor (IL6R) or anti-CD20 therapies for 6 months were carried out. In vitro experiments with human AT and adipocyte and macrophage cell lines were performed. A collagen-induced arthritis mouse model was developed. The insulin resistance and the altered adipocytokine profile were associated with disease activity, the presence of anti-citrullinated proteins anti-bodies (ACPAs), and worse response to therapy in RA. AT in the context of arthritis is characterized by an inflammatory state alongside the infiltration of macrophages and B/plasmatic cells, where ACPAs can have a direct impact, inducing inflammation and insulin resistance in macrophages and promoting a defective adipocyte differentiation, partially restored by biologicals.

The clinical and molecular cardiometabolic fingerprint of an exploratory psoriatic arthritis cohort is associated with the disease activity and differentially modulated by methotrexate and apremilast.

OBJECTIVES: (1) To evaluate clinical and molecular cardiovascular disease (CVD) signs and their relationship with psoriatic arthritis (PsA) features and (2) to identify a clinical patient profile susceptible to benefit from methotrexate (MTX) and/or apremilast regarding CVD risk. METHODS: This cross-sectional study included 100 patients with PsA and 100 age-matched healthy donors. In addition, an exploratory cohort of 45 biologically naïve patients treated for 6 months with apremilast, MTX or combined therapy according to routine clinical practice was recruited. Extensive clinical and metabolic profiles were obtained. Ninety-nine surrogate CVD-related molecules were analysed in plasma and peripheral blood mononuclear cells (PBMCs). Hard cluster analysis was performed to identify the clinical and molecular phenotypes. Mechanistic studies were performed on adipocytes. RESULTS: Cardiometabolic comorbidities were associated with disease activity and long-term inflammatory status. Thirty-five CVD-related proteins were altered in the plasma and PBMCs of PsA patients and were associated with the key clinical features of the disease. Plasma levels of some of the CVD-related molecules might distinguish insulin-resistant patients (MMP-3, CD163, FABP-4), high disease activity (GAL-3 and FABP-4) and poor therapy outcomes (CD-163, LTBR and CNTN-1). Hard cluster analysis identified two phenotypes of patients according to the rates of cardiometabolic comorbidities with distinctive clinical and molecular responses to each treatment. CONCLUSIONS: (1) Novel CVD-related proteins associated with clinical features could be emerging therapeutic targets in the context of PsA and (2) the pleiotropic action of apremilast could make it an excellent choice for the management of PsA patients with high CVD risk, targeting metabolic alterations and CVD-related molecules.

Splicing machinery is impaired in rheumatoid arthritis, associated with disease activity and modulated by anti-TNF therapy.

OBJECTIVES: To characterise splicing machinery (SM) alterations in leucocytes of patients with rheumatoid arthritis (RA), and to assess its influence on their clinical profile and therapeutic response. METHODS: Leucocyte subtypes from 129 patients with RA and 29 healthy donors (HD) were purified, and 45 selected SM elements (SME) were evaluated by quantitative PCR-array based on microfluidic technology (Fluidigm). Modulation by anti-tumour necrosis factor (TNF) therapy and underlying regulatory mechanisms were assessed. RESULTS: An altered expression of several SME was found in RA leucocytes. Eight elements (SNRNP70, SNRNP200, U2AF2, RNU4ATAC, RBM3, RBM17, KHDRBS1 and SRSF10) were equally altered in all leucocytes subtypes. Logistic regressions revealed that this signature might: discriminate RA and HD, and anti-citrullinated protein antibodies (ACPAs) positivity; classify high-disease activity (disease activity score-28 (DAS28) >5.1); recognise radiological involvement; and identify patients showing atheroma plaques. Furthermore, this signature was altered in RA synovial fluid and ankle joints of K/BxN-arthritic mice. An available RNA-seq data set enabled to validate data and identified distinctive splicing events and splicing variants among patients with RA expressing high and low SME levels. 3 and 6 months anti-TNF therapy reversed their expression in parallel to the reduction of the inflammatory profile. In vitro, ACPAs modulated SME, at least partially, by Fc Receptor (FcR)-dependent mechanisms. Key inflammatory cytokines further altered SME. Lastly, induced SNRNP70-overexpression and KHDRBS1-overexpression reversed inflammation in lymphocytes, NETosis in neutrophils and adhesion in RA monocytes and influenced activity of RA synovial fibroblasts. CONCLUSIONS: Overall, we have characterised for the first time a signature comprising eight dysregulated SME in RA leucocytes from both peripheral blood and synovial fluid, linked to disease pathophysiology, modulated by ACPAs and reversed by anti-TNF therapy.

Integrative Clinical, Molecular, and Computational Analysis Identify Novel Biomarkers and Differential Profiles of Anti-TNF Response in Rheumatoid Arthritis.

Background: This prospective multicenter study developed an integrative clinical and molecular longitudinal study in Rheumatoid Arthritis (RA) patients to explore changes in serologic parameters following anti-TNF therapy (TNF inhibitors, TNFi) and built on machine-learning algorithms aimed at the prediction of TNFi response, based on clinical and molecular profiles of RA patients. Methods: A total of 104 RA patients from two independent cohorts undergoing TNFi and 29 healthy donors (HD) were enrolled for the discovery and validation of prediction biomarkers. Serum samples were obtained at baseline and 6 months after treatment, and therapeutic efficacy was evaluated. Serum inflammatory profile, oxidative stress markers and NETosis-derived bioproducts were quantified and miRNomes were recognized by next-generation sequencing. Then, clinical and molecular changes induced by TNFi were delineated. Clinical and molecular signatures predictors of clinical response were assessed with supervised machine learning methods, using regularized logistic regressions. Results: Altered inflammatory, oxidative and NETosis-derived biomolecules were found in RA patients vs. HD, closely interconnected and associated with specific miRNA profiles. This altered molecular profile allowed the unsupervised division of three clusters of RA patients, showing distinctive clinical phenotypes, further linked to the TNFi effectiveness. Moreover, TNFi treatment reversed the molecular alterations in parallel to the clinical outcome. Machine-learning algorithms in the discovery cohort identified both, clinical and molecular signatures as potential predictors of response to TNFi treatment with high accuracy, which was further increased when both features were integrated in a mixed model (AUC: 0.91). These results were confirmed in the validation cohort. Conclusions: Our overall data suggest that: 1. RA patients undergoing anti-TNF-therapy conform distinctive clusters based on altered molecular profiles, which are directly linked to their clinical status at baseline. 2. Clinical effectiveness of anti-TNF therapy was divergent among these molecular clusters and associated with a specific modulation of the inflammatory response, the reestablishment of the altered oxidative status, the reduction of NETosis, and the reversion of related altered miRNAs. 3. The integrative analysis of the clinical and molecular profiles using machine learning allows the identification of novel signatures as potential predictors of therapeutic response to TNFi therapy.

Clinical Utility of microRNAs in Exhaled Breath Condensate as Biomarkers for Lung Cancer.

This study represents a novel proof of concept of the clinical utility of miRNAs from exhaled breath condensate (EBC) as biomarkers of lung cancer (LC). Genome-wide miRNA profiling and machine learning analysis were performed on EBC from 21 healthy volunteers and 21 LC patients. The levels of 12 miRNAs were significantly altered in EBC from LC patients where a specific signature of miR-4507, miR-6777-5p and miR-451a distinguished these patients with high accuracy. Besides, a distinctive miRNA profile between LC adenocarcinoma and squamous cell carcinoma was observed, where a combined panel of miR-4529-3p, miR-8075 and miR-7704 enabling discrimination between them. EBC levels of miR-6777-5p, 6780a-5p and miR-877-5p predicted clinical outcome at 500 days. Two additional miRNA signatures were also associated with other clinical features such as stage and invasion status. Dysregulated EBC miRNAs showed potential target genes related to LC pathogenesis, including CDKN2B, PTEN, TP53, BCL2, KRAS and EGFR. We conclude that EBC miRNAs might allow the identification, stratification and monitorization of LC, which could lead to the development of precision medicine in this and other respiratory diseases.

Clinical and laboratory characteristics of Brazilian versus non-Brazilian primary antiphospholipid syndrome patients in AntiPhospholipid Syndrome Alliance for Clinical Trials and InternatiOnal Networking (APS ACTION) clinical database and repository

Abstract Background: Antiphospholipid syndrome (APS) is characterized by episodes of thrombosis, obstetric morbidity or both, associated with persistently positive antiphospholipid antibodies (aPL). Studying the profile of a rare disease in an admixed population is important as it can provide new insights for understanding an autoimmune disease. In this sense of miscegenation, Brazil is characterized by one of the most heterogeneous populations in the world, which is the result of five centuries of interethnic crosses of people from three continents. The objective of this study was to compare the clinical and laboratory characteristics of Brazilian vs. non-Brazilian primary antiphospholipid syndrome (PAPS) patients. Methods: We classified PAPS patients into 2 groups: Brazilian PAPS patients (BPAPS) and PAPS patients from other countries (non-BPAPS). They were compared regarding demographic characteristics, criteria and non-criteria APS manifestations, antiphospholipid antibody (aPL) profile, and the adjusted Global Antiphospholipid Syndrome Score (aGAPSS). Results: We included 415 PAPS patients (88 [21%] BPAPS and 327 [79%] non-BPAPS). Brazilian patients were significantly younger, more frequently female, sedentary, obese, non-white, and had a higher frequency of livedo (25% vs. 10%, p < 0.001), cognitive dysfunction (21% vs. 8%, p = 0.001) and seizures (16% vs. 7%, p = 0.007), and a lower frequency of thrombocytopenia (9% vs. 18%, p = 0.037). Additionally, they were more frequently positive for lupus anticoagulant (87.5% vs. 74.6%, p = 0.01), and less frequently positive to anticardiolipin (46.6% vs. 73.7%, p < 0.001) and anti-ß2-glycoprotein-I (13.6% vs. 62.7%, p < 0.001) antibodies. Triple aPL positivity was also less frequent (8% vs. 41.6%, p < 0.001) in Brazilian patients. Median aGAPSS was lower in the Brazilian group (8 vs. 10, p < 0.0001). In the multivariate analysis, BPAPS patients still presented more frequently with livedo, cognitive dysfunction and sedentary lifestyle, and less frequently with thrombocytopenia and triple positivity to aPL. They were also less often white. Conclusions: Our study suggests a specific profile of PAPS in Brazil with higher frequency of selected non-criteria manifestations and lupus anticoagulant positivity. Lupus anticoagulant (not triple positivity) was the major aPL predictor of a classification criteria event.

Impaired microRNA processing in neutrophils from rheumatoid arthritis patients confers their pathogenic profile. Modulation by biological therapies.

The aim of this study was to investigate the microRNA (miRNA) expression pattern in neutrophils from rheumatoid arthritis (RA) patients and its contribution to their pathogenic profile and to analyze the effect of specific autoantibodies or inflammatory components in the regulation of miRNA in RA neutrophils and its modulation by biological therapies. Neutrophils were isolated from paired peripheral blood (PB) and synovial fluid samples of 40 patients with RA and from PB of 40 healthy donors. A miRNA array was performed using nCounter technology. Neutrophils from healthy donors were treated in vitrowith antibodies to citrullinated protein antigens isolated from RA patients and tumor necrosis factor-a (TNF-a) or interleukin-6. A number of cytokines and chemokines were analyzed. In vitro treatments of RA-neutrophils with tocilizumab or infliximab were carried out. Transfections with pre-miRNA and DICER downregulation experiments were further performed. RA-neutrophils showed a global downregulation of miRNA and genes involved in their biogenesis, alongside with an upregulation of various potential mRNA targets related to migration and inflammation. Decreased levels of miRNA and DICER correlated with autoimmunity, inflammation and disease activity. Citrullinated protein antigens and TNF-a decreased the expression of numerous miRNA and their biogenesis-related genes, increasing their potential mRNA targets. Infliximab reversed those effects. Transfections with pre-miRNA-223, -126 and -148a specifically modulated genes regulating inflammation, survival and migration whereas DICER depletion influenced the inflammatory profile of neutrophils. Taken together RA-neutrophils exhibited a global low abundance of miRNA induced by autoantibodies and inflammatory markers, which potentially contributed to their pathogenic activation. miRNA biogenesis was significantly impaired in RAneutrophils and further associated with a greater downregulation of miRNA mainly related to migration and inflammation in synovial fluid neutrophils. Finally, anti-TNF-a and anti-interleukin-6 receptor treatments can modulate miRNA levels in the neutrophils, minimizing their inflammatory profile.

Enhanced NETosis generation in radiographic axial spondyloarthritis: utility as biomarker for disease activity and anti-TNF-α therapy effectiveness.

BACKGROUND: Radiographic axial spondyloarthritis (r-axSpA) is a chronic inflammatory form of arthritis in which tumor necrosis factor (TNF)-α, a potent inducer of inflammatory response and a key regulator of innate immunity and of Th1 immune responses, plays a central role. NETosis is a mechanism of innate immune defense that is involved in diverse rheumatology diseases. Nevertheless, spontaneous NETosis generation in r-axSpA, its association to disease pathogenesis, and the NETosis involvement on anti-TNF-α therapy's effects has never been explored. METHODS: Thirty r-axSpA patients and 32 healthy donors (HDs) were evaluated. Neutrophil extracellular trap (NET) formation, mediators of signal-transduction cascade required for NETosis induction and cell-free NETosis-derived products were quantified. An additional cohort of 15 r-axSpA patients treated with infliximab (IFX) for six months were further analyzed. In vitro studies were designed to assess the effects of IFX in NETosis generation and the inflammatory profile triggered. RESULTS: Compared to HDs, neutrophils from r-axSpA patients displayed augmented spontaneous NET formation, elevated expression of NET-associated signaling components, nuclear peptidylarginine deiminase 4 translocation and increased citrullinated histone H3. Furthermore, patients exhibited altered circulating levels of cell-free NETosis-derived products (DNA, nucleosomes and elastase). Additional studies revealed that cell-free NETosis-derived products could be suitable biomarkers for distinguish r-axSpA patients from HDs. Correlation studies showed association between cell-free NETosis-derived products and clinical inflammatory parameters. Besides, nucleosomes displayed potential as a biomarker for discriminate patients according to disease activity. IFX therapy promoted a reduction in both NETosis generation and disease activity in r-axSpA patients. Mechanistic in vitro studies further unveiled the relevance of IFX in reducing NET release and normalizing the augmented inflammatory activities promoted by NETs in mononuclear cells. CONCLUSIONS: This study reveals that NETosis is enhanced in r-axSpA patients and identifies the NETosis-derived products as potential disease activity biomarkers. In addition, the data suggests the potential role of NET generation analysis for assessment of therapeutic effectiveness in r-axSpA.

Assessment of the relationship between estimated cardiovascular risk and structural damage in patients with axial spondyloarthritis.

AIMS: To evaluate the association of estimated cardiovascular (CV) risk and subclinical atherosclerosis with radiographic structural damage in patients with axial spondyloarthritis (axSpA). METHODS: Cross-sectional study including 114 patients axSpA from the SpA registry of Córdoba (CASTRO) and 132 age- and sex-matched healthy controls (HCs). Disease activity and the presence of traditional CV risk factors were recorded. The presence of atherosclerotic plaques and carotid intima media thickness (cIMT) were evaluated through carotid ultrasound and the SCORE index was calculated. Radiographic damage was measured though modified Stoke Ankylosing Spondylitis Spinal Score (mSASSS). The association between mSASSS and SCORE was tested using generalized linear models (GLM), and an age-adjusted cluster analysis was performed to identify different phenotypes dependent on the subclinical CV risk. RESULTS: Increased traditional CV risk factors, SCORE, and the presence of carotid plaques were found in axSpA patients compared with HCs. The presence of atherosclerotic plaques and SCORE were associated with radiographic structural damage. The GLM showed that the total mSASSS was associated independently with the SCORE [ß coefficient 0.24; 95% confidence interval (CI) 0.10-0.38] adjusted for disease duration, age, tobacco, C-reactive protein, and non-steroidal anti-inflammatory drugs (NSAID) intake. Hard cluster analysis identified two phenotypes of patients. Patients from cluster 1, characterized by the presence of plaques and increased cIMT, had a higher prevalence of CV risk factors and SCORE, and more structural damage than cluster two patients. CONCLUSION: Radiographic structural damage is associated closely with increased estimated CV risk: higher SCORE levels in axSpA patients were found to be associated independently with mSASSS after adjusting for age, disease duration, CRP, tobacco and NSAID intake.

Complement component 3 as biomarker of disease activity and cardiometabolic risk factor in rheumatoid arthritis and spondyloarthritis.

OBJECTIVE: To analyze the relationship between complement component 3 (C3) and the prevalence of cardiometabolic risk factors and disease activity in the rheumatic diseases having the highest rates of cardiovascular morbidity and mortality: rheumatoid arthritis (RA), psoriatic arthritis (PsA) and axial spondyloarthritis (axSpA). METHODS: This is a cross-sectional study including 200 RA, 80 PsA, 150 axSpA patients and 100 healthy donors. The prevalence of cardiometabolic risk factors [obesity, insulin resistance, type 2 diabetes mellitus, hyperlipidemia, apolipoprotein B/apolipoprotein A (apoB/apoA) and atherogenic risks and hypertension] was analyzed. Serum complement C3 levels, inflammatory markers and disease activity were evaluated. Cluster analysis was performed to identify different phenotypes. Receiver operating characteristic (ROC) curve analysis to assess the accuracy of complement C3 as biomarker of insulin resistance and disease activity was carried out. RESULTS: Levels of complement C3, significantly elevated in RA, axSpA and PsA patients, were associated with the prevalence of cardiometabolic risk factors. Hard clustering analysis identified two distinctive phenotypes of patients depending on the complement C3 levels and insulin sensitivity state. Patients from cluster 1, characterized by high levels of complement C3 displayed increased prevalence of cardiometabolic risk factors and high disease activity. ROC curve analysis showed that non-obesity related complement C3 levels allowed to identify insulin resistant patients. CONCLUSIONS: Complement C3 is associated with the concomitant increased prevalence of cardiometabolic risk factors in rheumatoid arthritis and spondyloarthritis. Thus, complement C3 should be considered a useful marker of insulin resistance and disease activity in these rheumatic disorders.

Circulating microRNAs as biomarkers of disease and typification of the atherothrombotic status in antiphospholipid syndrome.

We aimed to identify the plasma miRNA profile of antiphospholipid syndrome (APS) patients and to investigate the potential role of specific circulating miRNAs as non-invasive disease biomarkers. Ninety APS patients and 42 healthy donors were recruited. Profiling of miRNAs by PCR-array in plasma of APS patients identified a set of miRNAs differentially expressed and collectively involved in clinical features. Logistic regression and ROC analysis identified a signature of 10 miRNA ratios as biomarkers of disease. In addition, miRNA signature was related to fetal loss, atherosclerosis, and type of thrombosis, and correlated with parameters linked to inflammation, thrombosis, and autoimmunity. Hard clustering analysis differentiated 3 clusters representing different thrombotic risk profile groups. Significant differences between groups for several miRNA ratios were found. Moreover, miRNA signature remained stable over time, demonstrated by their analysis three months after the first sample collection. Parallel analysis in two additional cohorts of patients, including thrombosis without autoimmune disease, and systemic lupus erythematosus without antiphospholipid antibodies, each displayed specific miRNA profiles that were distinct from those of APS patients. In vitro, antiphospholipid antibodies of IgG isotype promoted deregulation in selected miRNAs and their potential atherothrombotic protein targets in monocytes and endothelial cells. Taken together, differentially expressed circulating miRNAs in APS patients, modulated at least partially by antiphospholipid antibodies of IgG isotype, might have the potential to serve as novel biomarkers of disease features and to typify patients' atherothrombotic status, thus constituting a useful tool in the management of the disease.

Circulating microRNAs as potential biomarkers of disease activity and structural damage in ankylosing spondylitis patients.

Ankylosing spondylitis (AS) remains difficult to diagnose before irreversible damage to sacroiliac joint is noticeable. Circulating microRNAs have demonstrated to serve as diagnostic tools for several human diseases. Here, we analysed plasma microRNAs to identify potential AS biomarkers. Higher expression levels of microRNA (miR)-146a-5p, miR-125a-5p, miR-151a-3p and miR-22-3p, and lower expression of miR-150-5p, and miR-451a were found in AS versus healthy donors. Interestingly, higher miR-146a-5p, miR-125a-5p, miR-151a-3p, miR-22-3p and miR-451a expression was also observed in AS than psoriatic arthritis patients. The areas under the curve, generated to assess the accuracy of microRNAs as diagnostic biomarkers for AS, ranged from 0.614 to 0.781; the six-microRNA signature reached 0.957. Bioinformatics analysis revealed that microRNAs targeted inflammatory and bone remodeling genes, underlying their potential role in this pathology. Indeed, additional studies revealed an association between these six microRNAs and potential target proteins related to AS pathophysiology. Furthermore, miR-146a-5p, miR-125a-5p and miR-22-3p expression was increased in active versus non-active patients. Moreover, miR-125a-5p, miR-151a-3p, miR-150-5p and miR-451a expression was related to the presence of syndesmophytes in AS patients. Overall, this study identified a six-plasma microRNA signature that could be attractive candidates as non-invasive biomarkers for the AS diagnosis, and may help to elucidate the disease pathogenesis.

Oxidative stress in the pathogenesis of atherothrombosis associated with anti-phospholipid syndrome and systemic lupus erythematosus: new therapeutic approaches.

Atherothrombosis is a recurrent complication in APS and SLE patients. Oxidative stress has been suggested as a key player underlying this process. Autoantibodies have been pointed to as the main contributors to abnormality in the oxidative status observed in APS and SLE patients, promoting the increased production of oxidant species and the reduction of antioxidant molecules. This imbalance causes vascular damage through the activation of immune cells, including monocytes, lymphocytes and neutrophils, causing the expression of pro-inflammatory and procoagulant molecules, the formation of neutrophil extracellular traps and the adhesion of these cells to the endothelium; the induction of cellular apoptosis and impaired cell clearance, which in turn enhances autoantibody neogeneration; and cytotoxicity of endothelial cells. This review describes the mechanisms underlying the role of oxidative stress in the pathogenesis of atherothrombosis associated with APS and SLE, focused on the effect of autoantibodies, the different cell types involved and the diverse effectors, including cytokines, procoagulant proteins and their main modulators, such as oxidant/antioxidant species and intracellular pathways in each pathology. We further discuss new therapies aimed at restoring the oxidative stress balance and subsequently to tackle atherothrombosis in APS and SLE.

Altered S-nitrosothiol homeostasis provides a survival advantage to breast cancer cells in HER2 tumors and reduces their sensitivity to trastuzumab.

The monoclonal antibody trastuzumab against HER2/neu, which is overexpressed in 15-20% of breast cancers, has clinical efficacy but many patients do not respond to initial treatment or develop resistance during treatment. Nitric oxide (NO) regulates cell signaling by targeting specific cysteine residues in proteins, forming S-nitrosothiols (SNO) in a process known as S-nitrosylation. We previously reported that molecular characteristics in breast cancer may dictate the tumor response to impaired SNO homeostasis. In the present study, we explored the role of SNO homeostasis in HER2 breast tumors. The antiproliferative action of trastuzumab in HER2-overexpressing BT-474 and SKBR-3 cells was suppressed when S-nitrosoglutathione reductase (GSNOR/ADH5) activity, which plays a key role in SNO homeostasis, was specifically inhibited with the pyrrole derivative compound N6022. Moreover, GSNOR inhibition restored the activation of survival signaling pathways involved in the resistance to anti-HER2 therapies (AKT, Src and c-Abl kinases and TrkA/NRTK1, TrkB/NRTK2, EphA1 and EphA3 receptors) and reduced the apoptotic effect of trastuzumab. Accordingly, GSNOR inhibition augmented the S-nitrosylation of apoptosis-related proteins, including Apaf-1, pSer73/63 c-Jun, calcineurin subunit α and HSF1. In agreement with in vitro data, immunohistochemical analyses of 51 breast tumors showed that HER2 expression was associated with lower expression of GSNOR protein. Moreover, gene expression analysis confirmed that high ADH5/GSNOR gene expression was associated with high patient survival rates in HER2 tumors. In conclusion, our data provide evidence of molecular mechanisms contributing to the progression of HER2+ breast cancers and could facilitate the development of therapeutic options to counteract resistance to anti-HER2 therapies.

Simultaneous inhibition of EGFR/VEGFR and cyclooxygenase-2 targets stemness-related pathways in colorectal cancer cells.

Despite the demonstrated benefits of anti-EGFR/VEGF targeted therapies in metastatic colorectal cancer (mCRC), many patients initially respond, but then show evidence of disease progression. New therapeutic strategies are needed to make the action of available drugs more efficient. Our study aimed to explore whether simultaneous targeting of EGFR/VEGF and cyclooxygenase-2 (COX-2) may aid the treatment and management of mCRC patients. The dual tyrosine kinase inhibitor AEE788 and celecoxib were used to inhibit EGFR/VEGFR and COX-2, respectively, in colorectal cancer cells. COX-2 inhibition with celecoxib augmented the antitumoral and antiangiogenic efficacy of AEE788, as indicated by the inhibition of cell proliferation, induction of apoptosis and G1 cell cycle arrest, down-regulation of VEGF production by cancer cells and reduction of cell migration. These effects were related with a blockade in the EGFR/VEGFR signaling axis. Notably, the combined AEE788/celecoxib treatment prevented ß-catenin nuclear accumulation in tumor cells. This effect was associated with a significant downregulation of FOXM1 protein levels and an impairment in the interaction of this transcription factor with ß-catenin, which is required for its nuclear localization. Furthermore, the combined treatment also reduced the expression of the stem cell markers Oct 3/4, Nanog, Sox-2 and Snail in cancer cells, and contributed to the diminution of the CSC subpopulation, as indicated by colonosphere formation assays. In conclusion, the combined treatment of AEE788 and celecoxib not only demonstrated enhanced anti-tumoral efficacy in colorectal cancer cells, but also reduced colon CSCs subpopulation by targeting stemness-related pathways. Therefore, the simultaneous targeting of EGFR/VEGF and COX-2 may aid in blocking mCRC progression and improve the efficacy of existing therapies in colorectal cancer.

Circulating miRNAs as potential biomarkers of therapy effectiveness in rheumatoid arthritis patients treated with anti-TNFα.

INTRODUCTION: The advent of anti-tumor necrosis factor alpha (anti-TNFα) drugs has considerably improved medical management in rheumatoid arthritis (RA) patients, although it has been reported to be ineffective in a fraction of them. MicroRNAs (miRNAs) are small, non-coding RNAs that act as fine-tuning regulators of gene expression. Targeting miRNAs by gain or loss of function approaches have brought therapeutic effects in various disease models. The aim of this study was to investigate serum miRNA levels as predictive biomarkers of response to anti-TNFα therapy in RA patients. METHODS: In total, 95 RA patients undergoing anti-TNFα/disease-modifying antirheumatic drugs (anti-TNFα/DMARDs) combined treatments were enrolled. Serum samples were obtained at 0 and 6 months and therapeutic efficacy was assessed. miRNAs were isolated from the serum of 10 patients before and after anti-TNFα/DMARDs combination therapy, cDNA transcribed and pooled, and human serum miRNA polymerase chain reaction (PCR) arrays were performed. Subsequently, selected miRNAs were analyzed in a validation cohort consisting of 85 RA patients. Correlation studies with clinical and serological variables were also performed. RESULTS: Ninety percent of RA patients responded to anti-TNFα/DMARDs combination therapy according to European League Against Rheumatism (EULAR) criteria. Array analysis showed that 91% of miRNAS were overexpressed and 9% downregulated after therapy. Functional classification revealed a preponderance of target mRNAs involved in reduction of cells maturation--especially on chondrocytes--as well as in immune and inflammatory response, cardiovascular disease, connective tissue and musculoskeletal system. Six out of ten miRNAs selected for validation were found significantly upregulated by anti-TNFα/DMARDs combination therapy (miR-16-5p, miR-23-3p, miR125b-5p, miR-126-3p, miRN-146a-5p, miR-223-3p). Only responder patients showed an increase in those miRNAs after therapy, and paralleled the reduction of TNFα, interleukin (IL)-6, IL-17, rheumatoid factor (RF), and C-reactive protein (CRP). Correlation studies demonstrated associations between validated miRNAs and clinical and inflammatory parameters. Further, we identified a specific plasma miRNA signature (miR-23 and miR-223) that may serve both as predictor and biomarker of response to anti-TNFα/DMARDs combination therapy. CONCLUSIONS: miRNA levels in the serum of RA patients before and after anti-TNFα/DMARDs combination therapy are potential novel biomarkers for predicting and monitoring therapy outcome.

CoCl2, a mimic of hypoxia, induces formation of polyploid giant cells with stem characteristics in colon cancer.

The induction of polyploidy is considered the reproductive end of cells, but there is evidence that polyploid giant cancer cells (PGCCs) contribute to cell repopulation during tumor relapse. However, the role of these cells in the development, progression and response to therapy in colon cancer remains undefined. Therefore, the main objective of this study was to investigate the generation of PGCCs in colon cancer cells and identify mechanisms of formation. Treatment of HCT-116 and Caco-2 colon cancer cells with the hypoxia mimic CoCl2 induced the formation of cells with larger cell and nuclear size (PGCCs), while the cells with normal morphology were selectively eliminated. Cytometric analysis showed that CoCl2 treatment induced G2 cell cycle arrest and the generation of a polyploid cell subpopulation with increased cellular DNA content. Polyploidy of hypoxia-induced PGCCs was confirmed by FISH analysis. Furthermore, CoCl2 treatment effectively induced the stabilization of HIF-1α, the differential expression of a truncated form of p53 (p47) and decreased levels of cyclin D1, indicating molecular mechanisms associated with cell cycle arrest at G2. Generation of PGCCs also contributed to expansion of a cell subpopulation with cancer stem cells (CSCs) characteristics, as indicated by colonosphere formation assays, and enhanced chemoresistance to 5-fluorouracil and oxaliplatin. In conclusion, the pharmacological induction of hypoxia in colon cancer cells causes the formation of PGCCs, the expansion of a cell subpopulation with CSC characteristics and chemoresistance. The molecular mechanisms involved, including the stabilization of HIF-1 α, the involvement of p53/p47 isoform and cell cycle arrest at G2, suggest novel targets to prevent tumor relapse and treatment failure in colon cancer.