Search

Efficient stent implantation among others depends on avoiding the aggregation of platelets in the blood vessels and appropriate proliferation of endothelial cells and controlled proliferation of smooth muscle cells, which reduces the development of pathology, such as neointimal hyperplasia, thrombosis, and restenosis. The current article provides an elegant solution for prevention of platelet and smooth muscle cell adhesion and activation on stent surfaces while obtaining surface conditions to support the growth of human coronary artery endothelial cells. This was achieved by surface nanostructuring and chemical activation of the surface. Specific nanotopographies of titanium were obtained by electrochemical anodization, while appropriate chemical properties were attained by treatment of titanium oxide nanotubes by highly reactive oxygen plasma. Surface properties were studied by scanning electron microscopy, atomic force microscopy, and X-ray photoelectron spectroscopy. Wettability was evaluated by measuring the water contact angle. The influence of nanostructured morphology and plasma modification on in vitro biological response with human coronary artery endothelia and smooth muscle cells as well as whole blood was studied. Our results show that a combination of nanostructuring and plasma modification of the surfaces is an effective way to achieve desired biological responses necessary for implantable materials such as stents.

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.

The research presented herein follows an urgent global need for the development of novel surface engineering techniques that would allow the fabrication of next-generation cardiovascular stents, which would drastically reduce cardiovascular diseases (CVD). The combination of hydrothermal treatment (HT) and treatment with highly reactive oxygen plasma (P) allowed for the formation of an oxygen-rich nanostructured surface. The morphology, surface roughness, chemical composition and wettability of the newly prepared oxide layer on the Ti substrate were characterized by scanning electron microscopy (SEM) with energy-dispersive X-ray analysis (EDX), atomic force microscopy (AFM), X-ray photoelectron spectroscopy (XPS) and water contact angle (WCA) analysis. The alteration of surface characteristics influenced the material’s bio-performance, platelet aggregation and activation was reduced on surfaces treated by hydrothermal treatment, as well as after plasma treatment. Moreover, it was shown that surfaces treated by both treatment procedures (HT and P) promoted the adhesion and proliferation of vascular endothelial cells, while at the same time inhibiting the adhesion and proliferation of vascular smooth muscle cells. The combination of both techniques presents a novel approach for the fabrication of vascular implants, with superior characteristics.

Antiphospholipid syndrome (APS) is a systemic autoimmune disease characterized by thrombosis, obstetric complications and the presence of antiphospholipid antibodies (aPL) that cause endothelial injury and thrombophilia. Extracellular vesicles are involved in endothelial and thrombotic pathologies and may therefore have an influence on the prothrombotic status of APS patients. Intercellular communication and connectivity are important mechanisms of interaction between healthy and pathologically altered cells. Despite well-characterized in vitro and in vivo models of APS pathology, the field of extracellular vesicles is still largely unexplored and could therefore provide an insight into the APS mechanism and possibly serve as a biomarker to identify patients at increased risk. The analysis of EVs poses a challenge due to the lack of standardized technology for their isolation and characterization. Recent findings in the field of EVs offer promising aspects that may explain their role in the pathogenesis of various diseases, including APS.

Background:Myofibroblasts are the orchestrators of aberrant extracellular matrix (ECM) remodelling in fibrosis. Actin cytoskeleton is a central hub that integrates mechanical signals to promote myofibroblast differentiation and ECM remodelling. Targeting these pathways could represent a novel antifibrotic strategy. We have recently shown that metabolic intermediate dimethyl-α-ketoglutarate (dm-αKG) blocks TGFβ-driven myofibroblast differentiation in dermal fibroblasts (DF).Objectives:To investigate the mechanisms by which dm-αKG regulates TGFβ-driven myofibroblast differentiation and inflammatory responses in DF.Methods:DF from healthy controls and patients with systemic sclerosis (SSc) were treated with TGFβ (10 ng/ml) and/or dm-αKG (6 mM) for 24h, 48h and 72h. RNA sequencing (Ilumina 2000, n=3 per experimental group) was followed by the analysis of differentially expressed genes (DeSEQ2, log2 fold ≥ |0.5|, padj< 0.01), pathway enrichment analysis (GO terms) and supervised PCA analysis (ClustVis). Protein amounts (fibronectin, αSMA, IL-6), cell contraction and apoptosis were measured with Western blot (n=6), ELISA (n=4), collagen gel contraction assay (n=4) and real time Annexin V assay (n=6). Significance (pResults:TGFβ (24h) altered the expression of 4076 genes in DF as determined by RNA-seq, among which 1864 genes were upregulated. The upregulated genes were enriched in GO biological processes/molecular functions/cellular compartments related to ECM organization (p=1e-07), Wnt signalling (p=5e-06), actin binding (p=3e-07), focal adhesion (p=1e-10), stress fibers (p=3e-07) and actin cytoskeleton (p= 3e-06). Dm-αKG altered the expression of 589 genes in TGFβ-treated DF compared to TGFβ only. The most downregulated pathways in DF treated with dm-αKG + TGFβ compared to TGFβ only included actin binding (p=5e-05), muscle contraction (p=0.001), ECM organization (p=0.008), focal adhesion (p=0.01), Z disk (p=0.01) and stress fibers (p=0.03). Specifically, dm-aKG significantly (p-0.5) decreased the expression of many TGFβ-induced genes involved in actin organization and focal adhesion (NEXN, FRMD5, ANTXR1, ACTC1, LIMCH1, SORBS2, TGM2, CSRP2, CAP2, LMO7, FZD2), muscle contraction (SNTB1, LMOD1, ANKRD1, SULF1, JPH2, CAVIN4, OXTR, DYSF, FBXO32) and ECM organization (COL10A1, COL11A1, HAPLN1, MMP14, MMP3, SPINT2, GREM1, MATN3, ADAMTS4). The PCA analysis revealed that the experimental treatment (PC1, Fig 1A) accounted for 61% variability in the expression of these genes, while 19% was attributed to interdonor variability (PC2). Dm-αKG diminished TGFβ-induced production of αSMA protein (72h, p=0.02, mean O.D. ± SD in TGFβ + dm-αKG vs. TGFβ: 0.34 ± 0.38 vs. 3.1 ± 2.3) and repressed TGFβ-driven secretion of fibronectin protein (72h, p=0.047, 0.5 ± 0.1 vs. 1.2 ± 0.6). Dm-αKG reduced the contractile capacity of TGFβ-stimulated DF in collagen gel contraction assay (p=0.003, 0 vs. 67.1 ± 5.4%). Additionally, dm-αKG decreased TGFβ-driven production of IL-6 transcripts (24h, p=0.05, 2.9 ± 0.6 vs 1.9 ± 0.3) and protein (24h, p=0.0005, 5.9 ± 1.2 vs 3 ± 0.7, Fig 1B), but did not increase the apoptosis of DF (24h, 48h, 72h).Fig 1.A Supervised PCA analysis of RNA-seq data. B. IL-6 secretion (ELISA).Conclusion:Dm-αKG counteracted TGFβ-induced myofibroblast differentiation by regulating the cytoskeleton organization and ECM dynamics in DF and blocked the TGFβ-induced IL-6 production. This closely links metabolism to inflammatory and pro-fibrotic responses in DF. Therefore, regulating intracellular αKG might offer a novel strategy in combating the inflammatory and fibrotic stages of skin fibrosis in SSc.Acknowledgments:This work was supported by a research grant from FOREUM Foundation for Research in Rheumatology.Disclosure of Interests:Blaž Burja: None declared, Gabriela Kania: None declared, Matija Tomsic: None declared, Snežna Sodin-Šemrl: None declared, Oliver Distler Grant/research support from: Grants/Research support from Actelion, Bayer, Boehringer Ingelheim, Competitive Drug Development International Ltd. and Mitsubishi Tanabe; he also holds the issued Patent on mir-29 for the treatment of systemic sclerosis (US8247389, EP2331143)., Consultant of: Consultancy fees from Actelion, Acceleron Pharma, AnaMar, Bayer, Baecon Discovery, Blade Therapeutics, Boehringer, CSL Behring, Catenion, ChemomAb, Curzion Pharmaceuticals, Ergonex, Galapagos NV, GSK, Glenmark Pharmaceuticals, Inventiva, Italfarmaco, iQvia, medac, Medscape, Mitsubishi Tanabe Pharma, MSD, Roche, Sanofi and UCB, Speakers bureau: Speaker fees from Actelion, Bayer, Boehringer Ingelheim, Medscape, Pfizer and Roche, Katja Lakota: None declared, Mojca Frank-Bertoncelj: None declared

It was the aim of this work to determine whether the plasma concentration of extracellular vesicles (EVs) in active diabetic Charcot neuroarthropathy (CN) is connected to the inflammatory markers, temperature elevation in the affected foot and concentration of soluble receptor for advanced glycation end products (RAGE). EVs were isolated from peripheral blood of 35 patients with active CN. EVs were counted after repetitive centrifugation and washing of samples, by flow cytometry. Foot temperature was measured by infrared thermometer. Concentration of soluble receptor for advanced glycation end products (RAGE) was determined by enzyme-linked immunosorbent assay (ELISA). We found statistically significant correlations of EV concentration (but not soluble RAGE concentration) with C-Reactive Protein (CRP) and with temperature difference between the affected and the contralateral foot (r=0.40, p=0.032; r=0.89, p

Heparins represent an efficient treatment of acute thrombosis and obstetric complications in antiphospholipid syndrome (APS). Enhanced microvesiculation of cell membranes, as detected by reduced membrane adhesion, can contribute to hypercoagulability in APS. Healthy donor IgG antibodies significantly increased beta2-glycoprotein I (beta2-GPI)-induced membrane adhesion, indicating that IgG antibodies might supplement the role of beta2-GPI in the regulation of membrane microvesiculation in healthy individuals. Anti-beta2-GPI IgG antibodies significantly reduced beta2-GPI-induced membrane adhesion, suggesting a direct role of anti-beta2-GPI antibodies in enhancing membrane microvesiculation in APS. Therapeutic concentration of nadroparin completely restored beta2-GPI-induced membrane adhesion in the presence of anti-beta2-GPI IgG antibodies. A novel anticoagulant mechanism of nadroparin in APS is suggested that supplements its direct effect on the coagulation cascade. Restoration of adhesion between negatively charged membranes in the presence of nadroparin might decrease shedding of microvesicles into the surrounding solution and could thus contribute to the efficacy of heparin treatment in APS.

Lipoxin A4 (LXA4) and scrum amyloid A (SAA) are endogenous negative and positive modulators of inflammation, respectively. Both molecules bind the shared lipoxin A4 receptor (ALX) and elicit opposing effects on the production of inflammatory cytokines and matrix metalloproteinases. The aim of these studies is to examine the divergence of the intracellular signaling pathways triggered by lipid LXA4 (1 nM) and protein SAA (200 nM) ligands of ALX. Phospholipase D (PLD) is a phosphohydrolase enzyme that catalyzes the generation of phosphatidic acid (PA) from membrane phospholipids. Our results showed that in fibroblast-like synoviocytes, activation of PLD occurred only in response to LXA4, and not SAA. PA (30 μM) mimicked LXA4 and demonstrated inhibition of IL-8 production induced by SAA or interleukin-1β. In sharp contrast to LXA4, SAA confirmed the stimulation of IL-8 release as determined previously. Taken together, these findings suggest that two physiologic ligands sharing the common ALX receptor, LXA4 and SAA, differentially regulate the level of PLD activation and differentially modulate IL-8. These results may have important implications for understanding the regulation of inflammatory responses under physiologic and pathological conditions.

Microvesicles (MVs) found in peripheral blood are derived from the budding of cell membranes and are associated with a higher risk of thrombosis. Recently, a hypothesis has been suggested that certain plasma proteins could suppress microvesiculation by mediating adhesion of the buds to the mother cell membrane. In a pilot study, we have tested this hypothesis by considering the relation between the amount of MVs in peripheral blood and the ability of plasma to induce adhesion between giant phospholipid vesicles (GPVs). MVs were isolated from human plasma and counted by flow cytometry. The adhesion between GPVs was measured by assessing the average angle of contact between the adhered vesicles. It was found that greater ability of plasma to induce adhesion relates to smaller concentration of MVs in plasma. The ratio between the concentration of MVs and the concentration of platelets proved the most efficient parameter to predict the propensity of the membrane to shed vesicles. Our results indicate that a stronger attractive interaction between GPVs mediated by plasma is associated with a smaller amount of MVs per platelets. Plasma that mediates stronger attractive interaction between GPVs might potentially be associated with a smaller risk of thrombosis.

Antiprothrombin antibodies can be measured by ELISA using either a prothrombin/phosphatidylserine complex (aPS/PT) or prothrombin alone (aPT) as antigen. We aimed to compare the clinical features of autoimmune patients with avidity of aPS/PT and determine the diagnostic efficiency of aPS/PT and aPT for assessing antiphospholipid syndrome (APS). aPS/PT were of low ( n = 9), heterogeneous ( n = 31) and high ( n = 8) avidity out of 48 cases. None of the samples with low avidity were positive in aPT ELISA. Among patients with heterogeneous or high avidity aPS/PT, there was a significantly greater number of patients with APS as compared to patients with low avidity (38/39 vs. 7/9; p

Aims Oxidation reactions can modify protein activity or specificity. Recently, a novel redox-reactive family of autoantibodies was described, which indicated involvement of altered antibodies (beside altered antigens) into autoimmune reactions. The aim of our study was to determine the binding capacity alterations of electro-oxidized blood donors' IgGs, and to evaluate their effects on released proinflammatory interleukin 6 in HUVEC. Results We found out that 1.) Isolated blood donor IgGs bound after electro-oxidation to β2-glycoprotein I, cardiolipin, citrullinated cyclic peptide and protein 3 by enzyme-linked immunosorbent assay, extractable nuclear antigens by counterimmuno-electrophoresis, and cell antigens by indirect immunofluorescence; 2.) Alterations in immunoreactivity of IgGs due to oxidation highly depend on electric current, time of exposure and the presence of antioxidants, 3.) Treatment of HUVEC with oxidized IgGs resulted in changed cell morphology, accompanied by an increase in released interleukin-6. Conclusions Our data suggest repeatable transformation of antibodies present in the blood of healthy persons and patients. Inter-individual differences in chemical stability of antibodies, patient's antioxidant status, and the microenvironmental changes at the cellular level may influence the range of antibody alterations and their involvement in pathophysiological autoimmune processes.

The way proteins associate with the lipid bilayer defines not only their function, but in autoimmune diseases, such as antiphospholipid syndrome (APS), also represents the conformational change of an antigen necessary for the exposure of critical epitopes recognized by potential autoantibodies. These interactions between the antigen (bound to phospholipid bilayers) and its antibodies could lead to clinical implications. Our aim was to review and characterize the requirements of the plasma protein β2‐glycoprotein I (β2‐GPI), one of the major proteins implicated in autoantibody development in APS for interactions with phospholipids, to provide primary sequence and secondary structure information of its Vth phospholipid binding domain, and to perform a NCBI search for comparison between homology of the Vth β2‐GPI domain with other known proteins. The second part addresses the effects of phospholipid membrane composition, charge, and specifically, the mechanism of interaction with artificial negatively charged membranes as in vitro model systems (vesicle and other studies). The third part focuses on the influence of environmental factors on β2‐GPI–phospholipid binding, and the fourth part addresses antibody binding to membrane‐bound β2‐GPI, exposure of its epitopes, recognition by its autoantibodies, and the functional (clinical) implications (specifically in APS). The development of antigenicity is addressed, with an emphasis on human studies and pathogenicity of antibodies. In the future, we plan to concentrate also on the numerous, important and critical reports centered on cell‐specific and receptor‐mediated studies, which we have omitted in the present compilation, since they are beyond the scope of this chapter.

The loss of membrane asymmetry is an important feature in mechanisms involved in maintaining immunity. Negatively charged phospholipids are present only in small amounts in the outer leaflet of the membranes of viable cells. In some pathological conditions and in apoptosis, negatively charged phospholipids become exposed on the outer membrane surface facing among other surroundings, the blood plasma. There, they can contribute to the catalytic potential for chemical reactions leading to blood clot formation. Also, they serve, in connection with protein cofactors, as antigens in the immune system leading to autoantibody formation and autoimmune diseases, among them of special emphasis, antiphospholipid syndrome (APS). APS is manifested clinically by venous and arterial thromboses and fetal loss. Laboratory analysis reveals a presence of antiphospholipid antibodies (aPL) in the patients' blood and research analysis indicates an increased amount of cell-derived microvesicles (MVs). MVs often have an impaired membrane asymmetry and can be procoagulant. Elevation of MV quantity in patient sera from different cells has been shown to be associated with thromboses. It is indicated that MVs can also be antigenic, depending on the properties and content of the blood plasma, that is, the presence in plasma of certain proteins called protein cofactors. Although numerous methods have been used in attempt to understand the antigen—phospholipid—antibody interactions and their role in APS, knowledge of the underlying mechanisms remains fragmentary. In this chapter, we study complex interactions between protein cofactors, phospholipid membranes, and aPL in giant phospholipid vesicles and in erythrocytes. We consider the process of budding and vesiculation in giant phospholipid vesicles and in erythrocytes, plasma protein-mediated attraction of negatively charged phospholipid membranes, and effects of annexin A5 (ANXA5) on interaction of protein cofactors and aPL with the phospholipid membranes. Our results indicate that phospholipid vesicles and erythrocytes can be useful not only in elucidating basic mechanisms involved in APS but can serve as models of antigen—phospholipid—antibody interactions with potential for improving clinical diagnosis and/or prognosis of this debilitating and sometimes lethal autoimmune disorder. Further studies in this direction should be undertaken.

Background Methotrexate (MTX) may reduce the risk of developing type 2 diabetes in rheumatoid (RA) and psoriatic arthritis (PsA). Animal studies suggest that MTX could reduce this risk by inducing intracellular accumulation of 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranosyl-59-monophosphate (ZMP). However, whether MTX therapy leads to chronic increase in ZMP concentration in RA or PsA patients has not been determined. Objectives We explored whether improvements in glucose homeostasis during MTX therapy in RA or PsA are associated with changes in concentration of ZMP or its metabolite 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside (AICAR). Methods Newly diagnosed nondiabetic, MTX-naive, RA (2010 ACR/EULAR criteria) and PsA (CASPAR criteria) patients were enrolled between December 2012 and June 2013. Patients were treated according to the treat to target approach. DAS28ESR and the metabolic parameters were followed at baseline and at 1, 2, 3 and 6 months after MTX initiation. Concentration of glycated hemoglobin HbA 1c and HOMA-IR (fasting glucose (mmol/l) x fasting insulin (μU/ml)/22.5) were determined to assess glucose homeostasis. Erythrocyte ZMP concentration and urinary AICAR concentration were measured with liquid chromatography-mass spectrometry at baseline and at 1 and 6 months. Data were analyzed using repeated measures ANOVA or Friedman test, followed by an appropriate post hoc test. Results 16 RA and 10 PsA patients completed the study (16 female, 10 male). Mean age was 53±12, BMI 28.5±4.6 kg/m 2 and the initial DAS28ESR 5.3±1.3. Remission or low disease activity (DAS28ESR 1c concentration was reduced from 5.80%±0.29% (39.0±3.1 mmol/mol) at baseline to 5.52%±0.31% (36.8±3.5 mmol/mol) at 6 months ( P 1c concentration was observed in 19 out of 26 patients. The number of patients with prediabetes (5.7%≤HbA 1c concentration at 6 months were not associated with changes in ZMP or AICAR concentration. Conclusions Here we show that MTX therapy reduces HbA 1c concentration in RA or PsA patients. However, MTX does not produce a sustained increase in erythrocyte ZMP or urinary AICAR concentration, suggesting ZMP did not accumulate during MTX therapy. Collectively, our data do not support the notion that MTX improves glucose homeostasis via chronic accumulation of ZMP. Disclosure of Interest None declared