Colocalization of Serum Amyloid A with Microtubules in Human Coronary Artery Endothelial Cells

Serum amyloid A (SAA) acts as a major acute phase protein and represents a sensitive and accurate marker of inflammation. Besides its hepatic origin, as the main source of serum SAA, this protein is also produced extrahepatically. The mRNA levels of SAA become significantly elevated following proinflammatory stimuli, as well as, are induced through their own positive feedback in human primary coronary artery endothelial cells. However, the intracellular functions of SAA are so far unknown. Colocalization of SAA with cytoskeletal filaments has previously been proposed, so we analyzed the colocalization of SAA with all three cytoskeletal elements: actin filaments, vimentin filaments, and microtubules. Immunofluorescent double-labeling analyses confirmed by PLA method revealed a strict colocalization of SAA with microtubules and a very infrequent attachment to vimentin while the distribution of actin filaments appeared clearly separated from SAA staining. Also, no significant colocalization was found between SAA and endomembranes labeled with the fluorescent lipid stain DiO6. However, SAA appears to be located also unbound in the cytosol, as well as inside the nucleus and within nanotubes extending from the cells or bridging neighboring cells. These different locations of SAA in endothelial cells strongly indicate multiple potential functions of this protein

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-α in cell culture supernatants was quantified by ELISA. Phosphorylation of NF-κB 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-κB (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-κB 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

Analysis of gene expression using public data and bionformatic tools in systemic sclerosis

Izhodišča: Sistemska skleroza (SSc) je kronična sistemska avtoimunska bolezen vezivnega tkiva. Patogeneza bolezni še ni popolnoma poznana. Pogost vzrok smrti pri bolnikih s SSc je intersticijska pljučna bolezen. Naš namen je bil z uporabo javno dostopnih podatkovnih baz in bioinformatičnih orodij ugotoviti značilen profil izražanja genov in vpletenost različnih celičnih tipov v razvoju fibroze pri bolnikih s SSc s pridruženo intersticijsko pljučno boleznijo (SSc-ILD). Raziskovalne metode: Za analizo podatkov genskega izražanja fibroblastov, izoliranih iz pljučnega tkiva, in celic celotnega pljučnega tkiva bolnikov smo uporabili program BRB-ArrayTools. V drugem delu smo s pomočjo bioinformatičnih orodij String in Reactome raziskali proteinske interakcije genov s spremenjenim izražanjem in jih analizirali glede na vpletenost v signalne poti. Rezultati: V analizi genskega izražanja pljučnih fibroblastov smo dokazali moteno regulacijo genov, vpletenih v procese fibroze. Z analizo pljučnega tkiva smo dokazali moteno regulacijo genov izvenceličnega matriksa, poti TGF-β in znižano izražanje citokinov, kemokinov in njihovih receptorjev. Ugotovili smo možne vplive različnih miRNA in transkripcijskih faktorjev družine NFkB, STAT in JUN na transkriptom fibroblastov ter vpliv PPAR in RAR na transkriptom celic pljučnega tkiva. Ugotovili smo epigenetski vpliv na celično proliferacijo, angiogenezo in izražanje signalne poti TGF-β prek hsa-miR-132 miRNA v celicah pljučnega tkiva. Diskusija in zaključek: Dokazali smo pomembno spremenjeno izražanje imunoglobulinov, kar kaže na pomembno vlogo celic B v pljučnem tkivu bolnikov v patogenezi bolezni, in ugotovili, da so geni, pomembno spremenjeni v fibroblastih, predvsem vpleteni v celično proliferacijo, geni drugih celic pljučnega tkiva pa so v veliki meri vključeni v mehanizme imunskega sistema in celične signalizacije, kar ponuja potencialne nove tarče za zdravljenje.Starting points: Systemic sclerosis (SSc) is chronic, autoimmune connective tissue disease. Its pathogenesis is not completely understood. Major cause for mortality in this disease is interstitial lung disease (ILD). Our aim was to use publicly available databases and bioinformatic tools to discern gene expression and cell type involvement in development of fibrosis in SSc-ILD patients. Methods: We used BRB-Array tools for analysis of gene expression in lung tissue and fibroblasts isolated from lung tissue of SSc patient and control. Furthermore, String and Reactome bioinformatic tools were used to analyze protein interactions between genes with changed expression and analyze pathways involving these genes, to better understand major drivers of disease. Results: In analysis of gene expression we found perturbations in expression of genes leading to fibrosis such as extracellular matrix proteins, TGF-β signaling pathway, decreased levels of cytokines, chemokines and their receptors expression. Based on gene expression profile we found possible miRNA and NFkB family STAT and JUN transcription factor involvement to/on transcriptome of fibroblasts and PPAR and RAR transcription factors to/on transcriptome of lung tissue. We found hsa-miR-132 miRNA epigenetic influence/signature on cell proliferation, angiogenesis and TGF-β signaling pathway expression in lung tissue withouth fibroblasts. We found profound changes in imunoglobulin gene expression, which is implying important role of B cells in lung tissue of these patients. Discussion and conclusion: The genes with significantly changed expression in fibroblast were those involved in cell proliferation, while differentially expressed genes of other cell types in lung tissue were those belonging to immune system and cell signalization, which implicates possible new therapeutic approaches

Verification, implementation and harmonization of automated chemiluminescent immunoassays for MPO- and PR3-ANCA detection

Objectives: Antineutrophil cytoplasmic antibody (ANCA) testing assists clinicians diagnose ANCA-associated vasculitis (AAV). We aimed to verify and harmonize chemiluminescent immunoassays for the detection of myeloperoxidase (MPO)- and proteinase 3 (PR3)-ANCA. Methods: An in-house ELISA, a capture ELISA, and a chemiluminescent assay QUANTA Flash on a BIO-FLASH analyzer were used to detect MPO- and PR3-ANCA in sera from 39 patients with AAV, 55 patients with various non-AAV, and 66 patients with connective tissue diseases. The results of the assays were evaluated, and their clinical performance was assessed. The precision and linearity of the QUANTA Flash assays were determined, and likelihood ratios (LRs) for AAV at diagnosis were calculated. Results: The precision and linearity of the QUANTA Flash assays were confirmed. Overall agreement between 97.5 and 98.8 % and Cohen’s kappa coefficients between 0.861 and 0.947 were observed for the results of the QUANTA Flash assays and ELISAs. The diagnostic sensitivity, specificity, and ROC analysis of the assays for AAV were statistically similar (in-house ELISA 89.7 %, 95.0 %, and 0.937capture ELISA 92.3 %, 98.3 %, and 0.939and QUANTA Flash 89.7 %, 95.9 %, and 0.972). For the QUANTA Flash assay results, the interval-specific LRs for AAV at diagnosis were: 0–8 CU had LR 0.08, 8–29 CU had LR 1.03, 29–121 CU had LR 7.76, 121–191 CU had LR 12.4, and >191 CU had LR ∞. Conclusions: The QUANTA Flash MPO and PR3 assays provide precise and consistent results and have comparable clinical utility for AAV. The calculated LRs were consistent with published LRs, confirming the utility of LRs for harmonization of ANCA results

Insights into the immunological description of cryoglobulins with regard to detection and characterization in Slovenian rheumatological patients

The detection of cryoglobulins (CG) used to diagnose cryoglobulemic vasculitis requires strict adherence to protocol, with emphasis on the preanalytical part. Our main objectives were to introduce a more sensitive and specific protocol for the detection of CG and to characterize CG in Slovenian patients diagnosed with cryoglobulinemic vasculitis, other vasculitides, connective tissue diseases or non-rheumatic diseases examined at the Department of Rheumatology (University Medical Centre Ljubljana). Samples were routinely analyzed for the presence of CG with the protocol using the Folin-Ciocalteu reagent. In the newly introduced protocol, the type of CG was determined by immunofixation on visually observed positive samples, and the concentration of CG in the cryoprecipitate and rheumatoid factor (RF) activity were measured by nephelometry. RF, C3c and C4 were measured in the patients` serum, and decision tree analysis was performed using all results. The agreement between negative and positive results between the two protocols was 86%. Of the 258 patient samples tested, we found 56 patients (21.7%) with positive CG (37.5% - type II, 62.5% - type III). The RF activity was observed in 21.4% of CG positive subjects. The median concentration of type II CG was significantly higher than that of type III CG (67.4 mg/L vs. 45.0 mg/L, p = 0.037). Patients with type II had lower C4 concentrations and higher RF activity compared to patients with type III CG. In the decision tree, C4 was the strongest predictor of cryoglobulinemia in patients. With the newly implemented protocol, we were able to improve the detection and quantification of CG in the samples of our rheumatology patients and report the results to adequately support clinicians

Protective Effects Of Olive Leaf Extract On Inflammatory Activation Of Endothelial Cells

Background and Aims: Olive leaf extract (OLE) has been traditionally used due to its anti-inflammatory and anti-atherogenic effects. ...87th Congress of the European-Atherosclerosis-Society (EAS), May 26-29, 2019, Maastricht, Netherland

Improved Protective Effect of Umbilical Cord Stem Cell Transplantation on Cisplatin-Induced Kidney Injury in Mice Pretreated with Antithymocyte Globulin

Mesenchymal stem cells (MSCs) are recognised as a promising tool to improve renal recovery in experimental models of cisplatin-induced acute kidney injury. However, these preclinical studies were performed on severely immunodeficient animals. Here, we investigated whether human umbilical cord derived MSC treatment could equally ameliorate acute kidney injury induced by cisplatin and prolong survival in mice with a normal immune system and those with a suppressed immune system by polyclonal antithymocyte globulin (ATG). We demonstrated that ATG pretreatment, when followed by MSC transplantation, significantly improved injured renal function parameters, as evidenced by decreased blood urea nitrogen and serum creatinine concentration, as well as improved renal morphology. This tissue restoration was also supported by increased survival of mice. The beneficial effects of ATG were associated with reduced level of inflammatory protein serum amyloid A3 and induced antioxidative expression of superoxide dismutase-1 (SOD-1), glutathione peroxidase (GPx), and hem oxygenase-1 (HO-1). Infused MSCs became localised predominantly in peritubular areas and acted to reduce renal cell death. In conclusion, these results show that ATG diminished in situ inflammation and oxidative stress associated with cisplatin-induced acute kidney injury, the effects that may provide more favourable microenvironment for MSC action, with consequential synergistic improvements in renal injury and animal survival as compared to MSC treatment alone

Could titanium dioxide nanotubes represent a viable support system for appropriate cells in vascular implants?

Nanoscale topography on various titanium surfaces has already been shown to improve vascular response in vitro. To propose a novel strategy for translation into clinically used vascular implants, it is imperative that the surface should also be properly conditioned to provide a better environment for adhesion and proliferation of cells. Electrochemical anodization process is one of the well-established strategies to produce controlled nanotopographic features on the surface of titanium. By combining electrochemical anodization process and gaseous plasma surface modification, it would be possible to fine-tune surface properties to enable improved biological response for specific application. The key surface properties that may influence biological responses, such as surface topography, surface chemistry, and surface wettability were studied in detail and their influences on in vitro biological responses were evaluated. Performance of platelets, human coronary artery endothelial cells (HCAEC), and stem cells on those surfaces was studied. It was shown that altering nanotube diameter (electrochemical anodization) and changing surface chemistry and wettability (gaseous plasma modification) significantly influenced platelet adhesion and activation as well as proliferation of HCAEC. The results provide evidence that by combining specific nanotopographic features and surface chemical modification by gaseous oxygen plasma, the optimized surface features necessary for improved performance of vascular implants in coronary arteries could be achieved. © 2017 Elsevier Inc