P. Segui

Mitochondrial dysfunction in antiphospholipid syndrome: implications in the pathogenesis of the disease and effects of coenzyme Q(10) treatment

The exact mechanisms underlying the role of oxidative stress in the pathogenesis and the prothrombotic or proinflammatory status of antiphospholipid syndrome (APS) remain unknown. Here, we investigate the role of oxidative stress and mitochondrial dysfunction in the proatherothrombotic status of APS patients induced by IgG-antiphospholipid antibodies and the beneficial effects of supplementing cells with coenzyme Q(10) (CoQ(10)). A significant increase in relevant prothrombotic and inflammatory parameters in 43 APS patients was found compared with 38 healthy donors. Increased peroxide production, nuclear abundance of Nrf2, antioxidant enzymatic activity, decreased intracellular glutathione, and altered mitochondrial membrane potential were found in monocytes and neutrophils from APS patients. Accelerated atherosclerosis in APS patients was found associated with their inflammatory or oxidative status. CoQ(10) preincubation of healthy monocytes before IgG-antiphospholipid antibody treatment decreased oxidative stress, the percentage of cells with altered mitochondrial membrane potential, and the induced expression of tissue factor, VEGF, and Flt1. In addition, CoQ(10) significantly improved the ultrastructural preservation of mitochondria and prevented IgG-APS-induced fission mediated by Drp-1 and Fis-1 proteins. In conclusion, the oxidative perturbation in APS patient leukocytes, which is directly related to an inflammatory and pro-atherothrombotic status, relies on alterations in mitochondrial dynamics and metabolism that may be prevented, reverted, or both by treatment with CoQ(10).

Anticyclic Citrullinated Protein Antibodies Are Implicated in the Development of Cardiovascular Disease in Rheumatoid Arthritis

Objective— Previous studies have suggested a relationship between anticyclic citrullinated protein (CCP) levels and development of cardiovascular disease in rheumatoid arthritis (RA). However, a limited number of studies have demonstrated an involvement of anti-CCPs in those processes. This study was aimed to define the specific role of these auto-antibodies in the pro-oxidative, inflammatory, and proatherogenic profile observed in leukocytes from RA patients. Approach and Results— Seventy-five RA patients and 31 healthy donors were enrolled. Carotid intima media thickness was evaluated as atherosclerosis marker. Several procoagulant and inflammatory factors, leukocyte activation, and oxidative stress markers were analyzed in plasma and leukocyte subsets. Anti-CCPs were purified from plasma of RA patients, and in vitro treatment of healthy leukocytes was conducted. High titers of anti-CCPs were associated to altered expression of prothrombotic and inflammatory markers, high oxidative stress, and pathological carotid intima media thickness in RA patients. Notably, gene expression analysis showed that lymphocytes were major players in altered inflammatory profile, monocytes were responsible for the protrombotic and atherogenic status, and neutrophils mainly displayed a pro-oxidative feature. In vitro treatment with purified anti-CCPs fully recapitulated that pathogenic profile, promoting the activation of leukocytes. Conclusions— Anti-CCPs are key players in the inflammatory and proatherogenic status of RA patients. The effects are specific of the immune cell targeted, promoting overexpression of thrombotic, inflammatory, and pro-oxidative markers in monocytes; pro-oxidative status in neutrophils; and proinflammatory profile in lymphocytes. Targeting these autoantibodies would be an excellent strategy to prevent the development of cardiovascular disease in RA.

Atherosclerosis and cardiovascular disease in systemic lupus erythematosus: effects of in vivo statin treatment.

Objective Statins may have beneficial vascular effects in systemic lupus erythematosus (SLE) beyond their cholesterol-lowering action, although the mechanisms involved are not completely understood. We investigated potential mechanisms involved in the efficacy of fluvastatin in preventing atherothrombosis in SLE. Methods Eighty-five patients with SLE and 62 healthy donors were included in the study. Selected patients (n=27) received 20 mg/day fluvastatin for 1 month. Blood samples were obtained before the start and at the end of treatment. Monocytes from five patients were treated in vitro with fluvastatin. Results Increased prothrombotic and inflammatory variables were found in patients with SLE. SLE monocytes displayed altered mitochondrial membrane potential and increased oxidative stress. Correlation and association analyses demonstrated a complex interplay among autoimmunity, oxidative stress, inflammation and increased risk of atherothrombosis in SLE. Fluvastatin treatment of patients for 1 month reduced the SLE Disease Activity Index and lipid levels, oxidative status and vascular inflammation. Array studies on monocytes demonstrated differential expression in 799 genes after fluvastatin treatment. Novel target genes and pathways modulated by fluvastatin were uncovered, including gene networks involved in cholesterol and lipid metabolism, inflammation, oxidative stress and mitochondrial activity. Electron microscopy analysis showed increased density volume of mitochondria in monocytes from fluvastatin-treated patients, who also displayed higher expression of genes involved in mitochondrial biogenesis. In vitro treatment of SLE monocytes confirmed the results obtained in the in vivo study. Conclusions Our overall data suggest that fluvastatin improves the impairment of a redox-sensitive pathway involved in processes that collectively orchestrate the pathophysiology of atherothrombosis in SLE.

‘Atherothrombosis-associated microRNAs in Antiphospholipid syndrome and Systemic Lupus Erythematosus patients’

MicroRNAs markedly affect the immune system, and have a relevant role in CVD and autoimmune diseases. Yet, no study has analyzed their involvement in atherothrombosis related to APS and SLE patients. This study intended to: 1) identify and characterize microRNAs linked to CVD in APS and SLE; 2) assess the effects of specific autoantibodies. Six microRNAs, involved in atherothrombosis development, were quantified in purified leukocytes from 23 APS and 64 SLE patients, and 56 healthy donors. Levels of microRNAs in neutrophils were lower in APS and SLE than in healthy donors. Gene and protein expression of miRNA biogenesis-related molecules were also reduced. Accordingly, more than 75% of identified miRNAs by miRNA profiling were underexpressed. In monocytes, miR124a and -125a were low, while miR-146a and miR-155 appeared elevated. Altered microRNAs’ expression was linked to autoimmunity, thrombosis, early atherosclerosis, and oxidative stress in both pathologies. In vitro treatment of neutrophils, monocytes, and ECs with aPL-IgG or anti-dsDNA-IgG antibodies deregulated microRNAs expression, and decreased miRNA biogenesis-related proteins. Monocyte transfections with pre-miR-124a and/or -125a caused reduction in atherothrombosis-related target molecules. In conclusion, microRNA biogenesis, significantly altered in neutrophils of APS and SLE patients, is associated to their atherothrombotic status, further modulated by specific autoantibodies.

Ubiquinol Effects on Antiphospholipid Syndrome Prothrombotic Profile: A Randomized, Placebo-Controlled Trial

Objective— Antiphospholipid syndrome (APS) leukocytes exhibit an oxidative perturbation, directly linked to alterations in mitochondrial dynamics and metabolism. This disturbance is related to the patients’ prothrombotic status and can be prevented by in vitro treatment with coenzyme Q10. Our aim was to investigate short-term effects of in vivo ubiquinol (reduced coenzyme Q10 [Qred]) supplementation on markers related to inflammation and thrombosis in APS through a prospective, randomized, crossover, placebo-controlled trial. Approach and Results— Thirty-six patients with APS were randomized to receive Qred (200 mg/d) or placebo for 1 month. Thirty-three patients with APS completed the intervention, which increased plasma coenzyme Q10. Qred improved endothelial function and decreased monocyte expression of prothrombotic and proinflammatory mediators, inhibited phosphorylation of thrombosis-related protein kinases, and decreased peroxides and percentage of monocytes with depolarized mitochondria; mitochondrial size was increased, and mitochondrial biogenesis–related genes were upregulated. Qred ameliorated extruded neutrophil extracellular traps in neutrophils and downregulated peroxides, intracellular elastase, and myeloperoxidase. Nanostring microRNA profiling revealed 20 microRNAs reduced in APS monocytes, and 16 of them, with a preponderance of cardiovascular disease–related target mRNAs, were upregulated. Monocytes gene profiling showed differential expression of 29 atherosclerosis-related genes, 23 of them changed by Qred. Interaction networks of genes and microRNAs were identified. Correlation studies demonstrated co-ordinated effects of Qred on thrombosis and endothelial function–associated molecules. Conclusions— Our results highlight the potential of Qred to modulate the overexpression of inflammatory and thrombotic risk markers in APS. Because of the absence of clinically significant side effects and its potential therapeutic benefits, Qred might act as safe adjunct to standard therapies in APS. Clinical Trial Registration— URL: http://www.clinicaltrials.gov. Unique identifier: NCT02218476

THU0398 Beneficial Effects of in Vivo Ubiquinol Supplementation on Athero-Thrombosis Prevention in Antiphospholipid Syndrome Patients. Preliminary Results of a Clinical Trial

Background Previous studies highlighted the beneficial effects of CoQ10 supplementation in clinical conditions such as cardiovascular disease. Objectives To investigate the beneficial effects of in vivo ubiquinol (Q, reduced form of CoQ10) supplementation on athero-thrombosis prevention in APS patients. Methods The study was conducted on 20 APS patients randomized to receive either Q (200 mg/day) or placebo for one month. Blood was drawn at time 0 and at the end of the treatment. Studies were performed in plasma and purified leukocytes subsets. Plasma Q levels, various prothrombotic/proinflammatory parameters, and oxidative stress biomarkers were evaluated. Endothelial activity analysis was performed by Laser-Doppler measurement of post ischemic reactive hyperemia. Carotid intimae media thickness (CIMT) was measured as an early atherosclerosis marker. Results Seventeen out of 20 patients completed the intervention, which increased significantly plasma Q levels. Endothelial function improved notably, as shown by the amelioration in the highest perfusion value after occlusion was released, expressed as a percentage of change vs rest flow value (RF-PF). CD14highCD16- classical monocyte count was not changed, but CD14highCD16+ intermediate monocytes and CD14dimCD16+ non-classical monocytes were decreased. Q treatment decreased Tissue Factor (TF) expression levels in total monocytes, and more notably in intermediate monocytes, which also displayed a more robust reduction of intracellular IKK levels. Only this monocyte subset exhibited IL-8 reduction after intervention. Q supplementation produced a reduction in both the levels of peroxides and the percentage of monocytes with altered mitochondrial membrane potential (ΔΨm). Correlation studies showed that reduced monocyte TF expression after Q treatment were related to decreased peroxides levels, increased plasma Q levels and improved endothelial function. Improved endothelial function further correlated with IL8 levels. The decrease of non-classical monocytes count was related to reduction in the percentage of cells with altered ΔΨm as well as with the increase in RF-PF value. Q effects were particularly relevant in APS patients suffering from arterial thrombosis (AT) or showing pathologic CIMT since TF, IKK and peroxide levels, being particularly higher at baseline in that patients in comparison to those with venous thrombosis (VT) or obstetrical manifestations (OM), showed a more pronounced decline and endothelial function was better improved. Conversely, IL-8 levels, higher at baseline in patients suffering VT or OM, were found reduced after Q administration in those patients. Conclusions Q supplementation at 200 mg/d significantly improved endothelial function, and reduced mitochondrial dysfunction, oxidative stress, and the expression of prothrombotic/ proinflammatory proteins. Our results support the potential impact of Q in the prevention of atherothrombosis in APS patients. Acknowledgements Supported by: CTS-7940, PI12/01511, Spanish Rheumatology Society, KANEKA. Disclosure of Interest None declared

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.

AB0127 ANTI-DS-DNA antibodies regulate atherothrombosis in systemic lupus erythematosus through the induction of netosis, inflammation and endothelial activation

Background The role of anti-dsDNA in the pathogenesis of the systemic lupus erythematosus (SLE) has been clearly established. However, the influence of these autoantibodies in the atherothrombotic status of SLE patients has not yet been evaluated Objectives 1. To analyse in vivo the involvement of anti-dsDNA antibodies in the development of CVD in SLE patients. 2. To evaluate in vitro the mechanisms underlying the effects of anti-dsDNA antibodies in these processes Methods The study was conducted in 50 SLE patients and 38 healthy donors. Endothelial function was assessed by measuring the post-occlusive hyperaemia using Laser-Doppler. Various markers of oxidative stress, inflammatory cytokines, prothrombotic mediators and NETosis, were quantified in purified leukocytes and plasma from SLE patients and controls. Activation of intracellular pathways was analyzed in monocytes using pathscan intracellular signaling array. In vitro, purified neutrophils, monocytes and lymphocytes from healthy donors and endothelial cells (ECs) were treated separately and in a trans-well co-culture system with anti-dsDNA antibodies isolated from the serum of SLE patients. Then, markers of inflammation, thrombosis, oxidative stress and NETosis were evaluated by flow cytometry (protein), RT-PCR (mRNA) and electron microscopy Results SLE patients showed impaired micro-vascular endothelial function (reduction of hyperaemia post occlusion area) and altered expression levels of pro-inflammatory proteins (IL6, IL8, MCP-1 and PCR), prothrombotic molecules (TF), oxidative stress markers (peroxides and mitochondrial membrane potential) and netosis-related molecules (NE, MPO and cell free-DNA). Monocytes from anti-dsDNA-positive SLE patients showed activation of various pathways related to inflammation, thrombosis and apoptosis (ErK, STAT-3, p38, JNK, GSK, Bad and Caspase-3). Association studies demonstrated that molecules related to inflammation and thrombosis, endothelial dysfunction, oxidative status and netosis were linked to the occurrence of thrombotic events, as well as to the presence of anti-dsDNA antibodies. In vitro treatment of purified leukocytes with anti-dsDNA antibodies promoted an increase in the production of NETosis, levels of peroxides and percentage of cells with altered mitochondrial membrane potential, as well as enlarged expression of a number of proinflammatory and prothrombotic molecules. In vitro treatment of HUVEC with anti-dsDNA antibodies promoted an increase in endothelial activation molecules (ICAM-1, VCAM-1 and E-selectin). Conclusions 1. Positivity for anti-dsDNA antibodies is linked to an increased pro-atherothrombotic status in SLE patients. 2. Anti-dsDNA antibodies, in vitro, promote NETosis on neutrophils, apoptosis on monocytes, modulate the expression of molecules related to inflammation and thrombosis, and induce endothelial activation. Together, that data suggest the involvement of such autoantibodies on atherothrombosis development in SLE Acknowledgements CTS-794 and ISCIII (FIS15/1333;RIER RD16/0012/0015) Disclosure of Interest None declared

AB0128 Alterations of the splicing machinery components in leukocytes from patients with systemic lupus erythematosus influences its development and atherothrombotic profile and drives the therapeutic response

Background Recent studies emphasize the relevance of alternative splicing in the development of genetic and autoimmune diseases and suggest therapeutic possibilities based on the modulation of this process. Objectives To identify alterations in the leukocyte splicing machinery of patients with systemic lupus erythematosus (SLE) and to evaluate its influence on the development and activity of the disease, its atherothrombotic profile, and the response to specific therapies. Methods An array of selected components of the major-(n=12) and minor-spliceosome (n=4) and associated splicing factors (n=28) was developed, and their expression levels were evaluated using a Fluidigm methodology, in purified leukocytes from 36 SLE patients and 29 healthy donors (HD). In parallel, an extensive clinical/serological evaluation was performed. Carotid intimate media thickness (CIMT) was used as atherosclerosis marker. Endothelial activity was monitored by laser-doppler, and pro-inflammatory and oxidative stress markers were quantified by flow cytometry and RT-PCR. In a parallel cohort of SLE patients (n=12), the effects of in vivo treatment with ubiquinol on spliceosome components was evaluated. Results As a general feature, a significant reduction in splicing factors and spliceosome components was found in all the leukocytes of SLE patients. Interestingly, we found a specific altered profile of splicing factors and spliceosome components when compared monocytes (U2AF1, FBP11, SRSF9), lymphocytes (RBM22, PRP8, SRSF5) and neutrophils (RNU4, CA150). The reduced levels of some components of spliceosome in both monocytes and neutrophils were linked to the occurrence of thrombotic events, foetal loss and arterial hypertension. In lymphocytes, those reduced levels were strongly related to the positivity for anti-dsDNA antibodies in SLE patients, thus suggesting that reduced spliceosome machinery would contribute to increase in altered autoantigen assembly, inducing increased autoantibody production. Correlation studies demonstrated an inverse relationship among reduced levels of spliceosome components/splicing factors and high activity of the disease (measured as SLEDAI), endothelial dysfunction, and increased expression levels of peroxides and peroxynitrites, as well as of altered mitochondrial membrane potential in monocytes and neutrophils. In vitro treatment of leukocytes from HDs with anti-dsDNA promoted a reduction in spliceosome components associated with the expression of proinflammatory and oxidative mediators. Finally, in vivo treatment with ubiquinol reversed reduced expression in SLE of spliceosome components related to their proaterothrombotic profile. Conclusions These results reveal the existence of SLE- associated spliceosome alterations -promoted by anti-dsDNA antibodies-which could be related to the development and activity of this autoimmune condition and have influence on the induction of mechanisms that drive atherothrombosis as well as the therapeutic response. Acknowledgements Funded by CTS7940 and ISCIII (PI15/01333 and RIER RD16/0012/0015). Disclosure of Interest None declared

OP0310 Association of Neutrophil Extracellular Traps with Atherosclerosis in Rheumatoid Arthritis

Background Neutrophil extracellular traps (NETs) have recently been implicated in vascular damage and atherothrombosis. Extruded DNA fibers containing multiple proinflammatory and thrombotic molecules induce the activation and recruitment of monocytes and dendritic cells to the vessel wall. Enhanced NETosis occurs in rheumatoid arthritis (RA), which has been shown related to the pathogenesis of this disorder. However, the relevance of this abnormality in the development of atherosclerosis has not been elucidated yet. Objectives To evaluate the association of netosis-derived products with the development of atherosclerosis in RA. Methods One hundred RA patients and 30 healthy donors were included. Carotid intima media thickness (CIMT) was used as atherosclerosis marker. Inflammatory and prothrombotic molecules, nitric oxide (NO), total antioxidant capacity (TAC) and protein nitration (NTyr) were analyzed in plasma. Neutrophils were isolated from RA patients and controls. Spontaneous and induced NETs formation from RA and control neutrophils was assessed in vitro through fluorescence microscopy. Oxidative stress status (JC1 and DCFH) and myeloperoxidase (MPO), neutrophil elastase (NE) protein expression were measured in neutrophils through flow cytometry. Cell-free DNA plasma levels were analyzed using Sytox staining. mRNA expression of peptidyl arginine deiminase 4 (PAD4) was analyzed in neutrophils by RT-PCR. Receiver operator characteristic (ROC) curves were calculated. Results Induced NETosis was increased in RA patients alongside MPO and NE protein expression in neutrophils. Cell-free DNA plasma levels were found significantly increased in RA patients. ROC curve analysis showed an area under the curve (AUC): 84, with a high sensitivity (69%) and specificity (86%). Those DNA levels strongly correlated with clinical parameters such as evolution time, DAS28, CRP and ESR and autoimmunity state (RF and anti-CCPs levels). In addition, high levels of cell-free DNA were associated with elevated levels of IL-6, MCP-1, spSelectin and tPA in plasma. Moreover, high levels of cell-free DNA correlated with increased NTyr and TAC and decreased NO. At cellular level, cell-free DNA plasma levels correlated with increased oxidative status in RA neutrophils (JC1 and DCFH) and mRNA expression of PADI4. Those patients having pathologic CIMT showed increased cell-free DNA plasma levels (ROC curve analysis: AUC 77, sensitivity 60%, specifity 72%). Conclusions 1) Elements associated with the extrusion of NETs are significantly enhanced in RA compared with healthy controls. 2) NETosis-derived products, such as cell-free DNA strongly correlated with clinical parameters, inflammatory and oxidative status in RA patients. Thus, NETosis-derived products demonstrated diagnostic potential for atherosclerosis, which might be a complementary tool to discriminate between normal and pathologic CIMT in RA patients. Acknowledgement Funded by CTS7940, PI2013–0191, PI15/01333, CP15/00158. Disclosure of Interest None declared