The effectiveness of tumor necrosis factor-α blocker therapy in patients with axial spondyloarthritis who failed conventional treatment: a comparative study focused on improvement in ASAS Health Index.

Objective: The purpose of this study is to evaluate the impact of tumor necrosis factor (TNF)-α blocker therapy on the Assessment of SpondyloArthritis international Society Health Index (ASAS-HI) among patients who have failed conventional nonsteroidal anti-inflammatory drugs. Methods: A comparative study was conducted involving axial spondyloarthritis (axSpA) patients treated with either TNF-α blocker or conventional therapy. Patient data, including demographics, disease characteristics, and ASAS-HI scores, were collected before and after treatment. Statistical analysis was performed to compare changes in ASAS-HI scores between the TNF-α blocker and the conventional therapy group. Results: The study population consisted of patients with axSpA, with a mean age of 38.3 years in conventional treatment group and 29.3 years in TNF-α blocker group. Most variables, including C-reactive protein levels, other comorbidities, and disease assessment scores showed no significant difference between groups. Longitudinal analysis within each treatment group from Week 0 to 12 showed no significant change in the conventional treatment group, whereas the TNF-α blocker group experienced a significant reduction in ASAS-HI scores, demonstrating the effectiveness of the treatment. The TNF-α blocker group exhibited a significantly greater improvement in ASAS-HI scores compared to the conventional therapy group. The Bath Ankylosing Spondylitis Functional Index and the Bath Ankylosing Spondylitis Disease Activity Index demonstrated strong positive correlations with ASAS-HI scores, indicating higher disease activity and functional limitation are associated with worse health outcomes in patients. Conclusion: The research demonstrates that ASAS-HI scores significantly improve with TNF-α blocker therapy in axSpA patients, underscoring ASAS-HI's effectiveness as a tool for evaluating drug responses.

Exosome-mediated delivery of super-repressor IκBα alleviates inflammation and joint damages in rheumatoid arthritis.

BACKGROUND: This study aims to investigate the potential anti-inflammatory effects of exosomes engineered to carry super-repressor IκB (Exo-srIκB), an exosome-based NF-κB inhibitor, in the context of RA. METHODS: Peripheral blood mononuclear cells (PBMCs) and synovial fluid mononuclear cells (SFMCs) were collected from patients diagnosed with RA and treated with Exo-srIκB to test the therapeutic potential. Flow cytometry analysis was performed to assess the production of inflammatory cytokines (IL-17A and GM-CSF) by the cells. ELISA was utilized to measure the levels of TNF-α, IL-17A, IL-6, and GM-CSF. Arthritis was induced in SKG mice by intraperitoneal injection of curdlan. DBA/1 J mice were used in collagen-induced arthritis (CIA) experiments. After the development of arthritis, mice were injected with either Exo-Naïve (control exosome) or Exo-srIκB. Arthritis scores were recorded biweekly, and histological observations of the ankle joint were conducted using H&E and safranin-O staining. Additionally, bone erosion was evaluated using micro-CT imaging. RESULTS: In the ex vivo study involving human PBMCs and SFMCs, treatment with Exo-srIκB demonstrated a notable reduction in inflammatory cytokines. Furthermore, in both the SKG and CIA models, Exo-srIκB treatment exhibited significant reductions in inflammation, cartilage destruction, and bone erosion within the joint tissues when compared to the Exo-Naive control group. Additionally, the radiographic score assessed through microCT showed a significant decrease compared to the Exo-Naive control group. CONCLUSION: Overall, these findings suggest that Exo-srIκB possesses anti-inflammatory properties in human RA cells and animal models, making it a promising therapeutic candidate for the treatment of RA.

Breast Tumor Cell-Stimulated Bone Marrow-Derived Mesenchymal Stem Cells Promote the Sprouting Capacity of Endothelial Cells by Promoting VEGF Expression, Mediated in Part through HIF-1α Increase.

Breast tumor cells recruit bone marrow-derived mesenchymal stem cells (BM-MSCs) and alter their cellular characteristics to establish a tumor microenvironment. BM-MSCs enhance tumor angiogenesis through various mechanisms. We investigated the mechanisms by which BM-MSCs promote angiogenesis in response to breast tumor. Conditioned media from MDA-MB-231 (MDA CM) and MCF7 (MCF7 CM) breast tumor cells were used to mimic breast tumor conditions. An in vitro spheroid sprouting assay using human umbilical vein endothelial cells (HUVECs) was conducted to assess the angiogenesis-stimulating potential of BM-MSCs in response to breast tumors. The ROS inhibitor N-acetylcysteine (NAC) and JAK inhibitor ruxolitinib attenuated increased HIF-1α in BM-MSCs in response to MDA CM and MCF7 CM. HIF-1α knockdown or HIF-1ß only partially downregulated VEGF expression and, therefore, the sprouting capacity of HUVECs in response to conditioned media from BM-MSCs treated with MDA CM or MCF7 CM. Inactivation of the VEGF receptor using sorafenib completely inhibited the HUVECs' sprouting. Our results suggest that increased HIF-1α expression under normoxia in BM-MSCs in response to breast tumor cells is mediated by ROS and JAK/Stat3, and that both HIF-1α-dependent and -independent mechanisms increase VEGF expression in BM-MSCs to promote the angiogenic sprouting capacity of endothelial cells in a VEGF-dependent manner.

Bone Marrow-Derived Vasculogenic Mesenchymal Stem Cells Enhance In Vitro Angiogenic Sprouting of Human Umbilical Vein Endothelial Cells.

Vasculogenic properties of bone marrow-derived mesenchymal stem cells (MSCs) have been reported, but it is still unclear whether the vasculogenic properties are restricted to some populations of MSCs or whether the entire population of MSCs has these properties. We cultured two different populations of MSCs in different culture media and their vasculogenic properties were evaluated using In vitro spheroid sprouting assay. Neither population of MSCs expressed markers of endothelial progenitor cells (EPCs), but they were different in the profiling of angiogenic factor expression as well as vasculogenic properties. One population of MSCs expressed basic fibroblast growth factor (bFGF) and another expressed hepatocyte growth factor (HGF). MSCs expressing HGF exhibited In vitro angiogenic sprouting capacity in response to bFGF derived from other MSCs as well as to their autocrine HGF. The vasculogenic mesenchymal stem cells (vMSCs) derived from the bone marrow also enhanced In vitro angiogenic sprouting capacity of human umbilical vein endothelial cells (HUVECs) in an HGF-dependent manner. These results suggest that MSCs exhibit different vasculogenic properties, and vMSCs that are different from EPCs may contribute to neovascularization and could be a promising cellular therapy for cardiovascular diseases.

Effect of onion peel extract on endothelial function and endothelial progenitor cells in overweight and obese individuals.

OBJECTIVES: Acute or chronic intake of polyphenol-rich foods has been reported to improve endothelial function. Quercetin, found abundantly in onion, is a potent antioxidant flavonoid. The aim of this study was to investigate whether consumption of onion peel extract (OPE) improves endothelial function in healthy overweight and obese individuals. METHODS: This was a randomized double-blind, placebo-controlled study. Seventy-two healthy overweight and obese participants were randomly assigned to receive a red, soft capsule of OPE (100 mg quercetin/d, 50 mg quercetin twice daily; n = 36 participants) or an identical placebo capsule (n = 36) for 12 wk. Endothelial function, defined by flow-mediated dilation (FMD), circulating endothelial progenitor cells (EPCs) by flow cytometry, and laboratory test were determined at baseline and after treatment. RESULTS: Baseline characteristics and laboratory findings did not significantly differ between the two groups. Compared with baseline values, the OPE group showed significantly improved FMD at 12 wk (from 12.5 ± 5.2 to 15.2 ± 6.1; P = 0.002), whereas the placebo group showed no difference. Nitroglycerin-mediated dilation did not change in either group. EPC counts (44.2 ± 25.6 versus 52.3 ± 18.6; P = 0.005) and the percentage of EPCs were significantly increased in the OPE group. When FMD was divided into quartiles, rate of patients with endothelial dysfunction defined as lowest quartile (cutoff value, 8.6%) of FMD improved from 26% to 9% by OPE. CONCLUSION: Medium-term administration of OPE an improvement in FMD and circulating EPCs.

Structural and biophysical characterization of human cytochromes P450 2B6 and 2A6 bound to volatile hydrocarbons: analysis and comparison.

X-ray crystal structures of complexes of cytochromes CYP2B6 and CYP2A6 with the monoterpene sabinene revealed two distinct binding modes in the active sites. In CYP2B6, sabinene positioned itself with the putative oxidation site located closer to the heme iron. In contrast, sabinene was found in an alternate conformation in the more compact CYP2A6, where the larger hydrophobic side chains resulted in a significantly reduced active-site cavity. Furthermore, results from isothermal titration calorimetry indicated a much more substantial contribution of favorable enthalpy to sabinene binding to CYP2B6 as opposed to CYP2A6, consistent with the previous observations with (+)-α-pinene. Structural analysis of CYP2B6 complexes with sabinene and the structurally similar (3)-carene and comparison with previously solved structures revealed how the movement of the F206 side chain influences the volume of the binding pocket. In addition, retrospective analysis of prior structures revealed that ligands containing -Cl and -NH functional groups adopted a distinct orientation in the CYP2B active site compared with other ligands. This binding mode may reflect the formation of Cl-π or NH-π bonds with aromatic rings in the active site, which serve as important contributors to protein-ligand binding affinity and specificity. Overall, the findings from multiple techniques illustrate how drugs metabolizing CYP2B6 and CYP2A6 handle a common hydrocarbon found in the environment. The study also provides insight into the role of specific functional groups of the ligand that may influence the binding to CYP2B6.

Impact of lysophosphatidylcholine on survival and function of UEA-1(+)acLDL (+) endothelial progenitor cells in patients with coronary artery disease.

Lysophosphatidylcholine (LPC) generated from oxidized low-density lipoprotein by lipoprotein-associated phospholipase A2 plays a key role in plaque inflammation and vulnerability. Endothelial progenitor cells (EPCs) can repair injured endothelium and exert anti-inflammatory effects of vulnerable plaque. We study the impact and mechanisms of LPC on UEA-1 and acLDL binding EPCs (UEA-1(+)acLDL(+) EPCs). UEA-1(+)acLDL(+) EPCs from coronary artery disease (CAD) patients were cultured and exposed to LPC at different concentrations and different timepoints. We determined the significant concentration (40 µM). UEA-1(+)acLDL(+) EPCs were preincubated for 30 min with pravastatin (20 µM) with LY249002, a specific inhibitor of the Akt signaling pathway, and exposed for 24 h to LPC 40 µM. The survival, migration, adhesion, and proliferation of UEA-1(+)acLDL(+) EPCs were assessed. To examine the mechanisms of LPC toxicity and pravastatin effects, phosphorylated Akt and endothelial nitric oxide synthase (eNOS) levels and the ratio of Bcl-2/Bax protein expression were assessed. LPC induced apoptosis and impaired migration and adhesion of UEA-1(+)acLDL(+) EPCs significantly. The detrimental effects of LPC were attenuated by pravastatin. However, when UEA-1(+)acLDL(+) EPCs were pretreated with pravastatin and LY249002, a specific inhibitor of the Akt signaling pathway, simultaneously, the beneficial effects of pravastatin were abolished. Furthermore, LPC suppressed Akt and eNOS phosphorylation and increased Bcl-2/Bax expression. The effects of LPC on Akt/eNOS and Bcl-2/Bax activity were reversed by pravastatin. In conclusion, LPC inhibited UEA-1(+)acLDL(+) EPCs survival and impaired its functions, and these were attributable to inhibition of the Akt/eNOS and Bcl-2/Bax pathway. Pravastatin reversed the detrimental action of LPC. These findings suggest that LPC inhibition can be a possible strategy for CAD through EPC revitalization.

Altered miRNA expression profiles are involved in the protective effects of troxerutin against ultraviolet B radiation in normal human dermal fibroblasts.

The aim of this study was to investigate the mechanisms by which troxerutin protects cells against ultraviolet B (UVB) radiation. First, we demonstrate that pre-treatment with troxerutin protects normal human dermal fibroblasts (nHDFs) against UVB-induced cytotoxicity. As shown by migration assay and DNA repair analysis, troxerutin increased cell migration and DNA repair activity in the nHDFs. Subsequently, we analyzed microRNA (miRNA) expression profiles in the nHDFs. miRNAs are 19- to 24-nucleotide (nt) non-coding RNA molecules that regulate the translation of target genes through RNA interference. In UVB-exposed cells, miRNAs act on crucial functions, such as apoptosis and cellular senescence. miRNA expression is significantly altered during the protective process induced by phytochemicals. Therefore, understanding changes that occur in miRNA expression profiles may help to elucidate the protective mechanisms of troxerutin. We identified 11 miRNAs that were significantly (>2-fold) upregulated and 12 that were significantly downregulated (>2-fold) following treatment of the nHDFs with troxerutin. In addition, we investigated the biological functions of these miRNAs through the prediction of miRNA targets and Gene Ontology analysis of the putative targets. Overall, our findings indicate that pre-treatment with troxerutin increases the viability of UVB-exposed nHDFs through the alteration of the miRNA expression profiles.

Effect of platelet reactivity, endothelial function, and inflammatory status on outcomes in patients with stable angina pectoris on clopidogrel therapy.

Although high on-treatment platelet reactivity (HTPR) is an important predictor of clinical outcomes in patients undergoing coronary stenting, it is unknown whether endothelial dysfunction and HTPR are associated. We examined the platelet function, peripheral vascular function, endothelial progenitor cell (EPC) number, platelet activation markers, high-sensitivity C-reactive protein (hs-CRP) level, and clinical outcomes in patients receiving chronic clopidogrel therapy. We consecutively enrolled 91 patients who underwent follow-up angiography because of chest discomfort. All patients took aspirin and clopidogrel for an average of 498 ± 138 days. Platelet reactivity was assessed by light transmittance aggregometry (maximal platelet aggregation by 5 µmol/L of adenosine diphosphate ≤50% in group 1 [optimal response] and >50% as group 2 [HTPR]). Flow-mediated dilation of the brachial artery and brachial-ankle pulse wave velocity (PWV), numbers of EPCs isolated from peripheral blood, platelet activation markers (soluble CD40 ligand and soluble P-selectin), and hs-CRP levels were assessed before follow-up angiography. There were no significant differences in baseline characteristics and previous percutaneous coronary intervention (PCI) data between groups 1 (n = 59) and 2 (n = 32). Group 2 showed poorer flow-mediated dilation (6.1 ± 4.1% vs 12.9 ± 6.2%, p <0.001), pulse wave velocity (1925.4 ± 362.2 vs 1571.0 ± 306.5 ms, p <0.001), and lower circulating EPCs by flow cytometry (21.9 ± 14.7 vs 65.2 ± 30.1 per 10 fields, p <0.001) compared with group 1. Significantly higher levels of soluble CD40 ligand, soluble P-selectin, and hs-CRP were observed in group 2. In multivariate analysis, elevated hs-CRP level, but not HTPR, was independently associated with repeated PCI. In patients with angina, HTPR was associated endothelial dysfunction and elevated hs-CRP, although elevated hs-CRP level was significantly associated with poorer outcomes.

Estrogen-related receptor γ controls hepatic CB1 receptor-mediated CYP2E1 expression and oxidative liver injury by alcohol.

BACKGROUND: The hepatic endocannabinoid system and cytochrome P450 2E1 (CYP2E1), a key enzyme causing alcohol-induced reactive oxygen species (ROS) generation, are major contributors to the pathogenesis of alcoholic liver disease. The nuclear hormone receptor oestrogen-related receptor γ (ERRγ) is a constitutively active transcriptional activator regulating gene expression. OBJECTIVE: To investigate the role of ERRγ in the alcohol-mediated regulation of CYP2E1 and to examine the possibility to control alcohol-mediated oxidative stress and liver injury through an ERRγ inverse agonist. DESIGN: For chronic alcoholic hepatosteatosis study, C57BL/6J wild-type and CB1(-/-) mice were administered alcohol for 4 weeks. GSK5182 and chlormethiazole (CMZ) were given by oral gavage for the last 2 weeks of alcohol feeding. Gene expression profiles and biochemical assays were performed using the liver or blood of mice. RESULTS: Hepatic ERRγ gene expression induced by alcohol-mediated activation of CB1 receptor results in induction of CYP2E1, while liver-specific ablation of ERRγ gene expression blocks alcohol-induced expression of CYP2E1 in mouse liver. An ERRγ inverse agonist significantly ameliorates chronic alcohol-induced liver injury in mice through inhibition of CYP2E1-mediated generation of ROS, while inhibition of CYP2E1 by CMZ abrogates the beneficial effects of the inverse agonist. Finally, chronic alcohol-mediated ERRγ and CYP2E1 gene expression, ROS generation and liver injury in normal mice were nearly abolished in CB1(-/-) mice. CONCLUSIONS: ERRγ, as a previously unrecognised transcriptional regulator of hepatic CB1 receptor, controls alcohol-induced oxidative stress and liver injury through CYP2E1 induction, and its inverse agonist could ameliorate oxidative liver injury due to chronic alcohol exposure.

Titrated extract of Centella asiatica provides a UVB protective effect by altering microRNA expression profiles in human dermal fibroblasts.

The titrated extract of Centella asiatica (TECA) is a reconstituted mixture comprising of asiatic acid, madecassic acid, asiaticoside and madecassoside, and is used as a therapeutic agent in wound healing and also as an anti-microbial, anticancer and anti-aging agent. Although these properties and the associated cell signaling pathways have been elucidated, the cellular mechanism of anti-photoaging upon ultraviolet (UV) exposure in normal human dermal fibroblasts (NHDFs) remains unknown. In this study, we investigated the photoprotective role of TECA via microRNA (miRNA) expression profiling analysis. Low dose of TECA did not exhibit toxicity and showed a protective effect against UVB irradiation in NDHFs. miRNA microarray experiments revealed that specific miRNAs were altered by TECA stimulation in UVB-irradiated NHDFs. Functional bioinformatic analysis showed that the putative target genes of the altered miRNAs were associated with the positive regulation of cell proliferation, anti-apoptosis, small GTPase- and Ras-mediated signal transduction and activation of MAPKK. Therefore, these results suggest that TECA may serve as a potential natural chemoprotective agent against UVB-mediated damage in NHDFs through changes in the expression of specific miRNAs.

Increase of human CYP1B1 activities by acidic phospholipids and kinetic deuterium isotope effects on CYP1B1 substrate oxidation.

The effect of phospholipids on the kinetic parameters of three substrates, 7-ethoxy-4 -(trifluoromethyl)coumarin (7-EFC), 7-ethoxycoumarin (7-EC) and 17ß-estradiol (E(2)), of human CYP1B1 was studied. In general, anionic phospholipids, phosphatidic acid and cardiolipin increased catalytic efficiency by increasing k(cat) values or decreasing K(m) values. The advantages of using the 7-EFC as a substrate over 7-EC and E(2) include high k(cat), low K(m) and high catalytic efficiency. Spectral binding titrations indicated that the binding affinity of 7-EFC to CYP1B1 in the presence or absence of phospholipids is higher than that of 7-EC or E(2). Furthermore, phosphatidylcholine increased the binding affinity of the substrates to the CYP1B1. High non-competitive intermolecular kinetic deuterium isotope effects (values 5.4-12) were observed for O-deethylation of 7-EFC and 7-EC with deuterium substitution at the ethoxy group, indicating that the C-H bond-breaking step makes a major contribution to the rate of these CYP1B1-catalyzed reactions. However, the intermolecular kinetic deuterium isotope effect is ~2 for the E(2) 4-hydroxylation reaction, indicating that the C-H bond-breaking step contributes only partially to the rate of this CYP1B1-catalyzed reaction. These results indicate that the reaction mechanism of CYP1B1-catalyzed reactions is distinct for each substrate.

CYP1A1 is a target of miR-892a-mediated post-transcriptional repression.

Cytochrome P450 1A1 (CYP1A1) is a member of the cytochrome p450 enzyme family, which is involved in the metabolisms of carcinogenic metabolites, such as benzo(a)pyrene. In this study, we identified miR-892a as a negative regulator of CYP1A1 expression. Luciferase assays revealed a sequence in the 3'-untranslated region of CYP1A1 that displayed a perfect match with miR-892a, and revealed that this sequence was a specific miR-892a target site. The overexpression of miR­892a inhibited the expression of the CYP1A1 protein, and the miR­892a antagonist increased CYP1A1 expression. Of note, benzo(a)pyrene, a major inducer of CYP1A1 transcription, decreased the expression of miR-892a. Moreover, the miR-892a-induced CYP1A1 repression inhibited the benzo(a)pyrene-mediated decrease in cell viability. These data provide insight into the CYP1A1 regulatory network.

Functional and conformational modulation of human cytochrome P450 1B1 by anionic phospholipids.

We investigated the interaction of human P450 1B1 (CYP1B1) with various phospholipid bilayers using the N-terminally deleted (Delta2-4)CYP1B1 and (Delta2-26)CYP1B1 enzymes. Among anionic phospholipids, phosphatidic acid (PA) and cardiolipin specifically increased the catalytic activities, membrane binding affinities, and thermal stabilities of both CYP1B1 proteins when phosphatidylcholine matrix was gradually replaced with these anionic phospholipids. PA- or cardiolipin-dependent changes of CYP1B1 conformation were revealed by altered Trp fluorescence and CD spectra. However, both PA and cardiolipin exerted more significant effects with the (Delta2-4)CYP1B1 than the (Delta2-26)CYP1B1 implying the functional importance of N-terminal region for the interaction with the phospholipid membranes. In contrast, other anionic phospholipids such as phosphatidylserine and the neutral phospholipid phosphatidylethanolamine had no apparent effects on the catalytic activity or conformation of CYP1B1. These data suggest that the chemical and physical properties of membranes influenced by PA or cardiolipin composition are critical for the functional roles of CYP1B1.