Influence of the TLR4-mediated p38MAPK signaling pathway on chronic intermittent hypoxic-induced rat's oxidative stress and inflammatory cytokines in rats.

OBJECTIVE: To investigate the influence of the TOLL-like receptor 4 (TLR4)-mediated p38MAPK signaling pathway on chronic intermittent hypoxic (CIH)-induced oxidative stress and inflammatory cytokines in rats. MATERIALS AND METHODS: A total of 120 healthy male Sprague Dawley (SD) rats, aged between 8-10 weeks, were randomly divided into 9 groups (normoxia control group, CIH 2 weeks group, CIH 4 weeks group, CIH 6 weeks group, CIH 8 weeks group, CIH 6 weeks + p38MAPK receptor inhibit group, CIH 6 weeks + Tempol group, CIH 8 weeks + p38MAPK receptor inhibitor group and CIH 8 weeks + Tempol group). The expression of TLR4 and p38MAPK in the adipose tissue was evaluated, as well as the level of serum oxidative stress markers (SOD, TRx-1, MDA) and inflammatory cytokines (adiponectin, TNF-α, hsCRP and IL-6). RT-PCR and Western-blot were conducted to detect the expression of TLR4 and p38MAPK mRNA. RESULTS: With increased hypoxia, the levels of SOD and adiponectin in the serum of the CIH group decreased significantly, and the levels of TNF-α, hsCRP, IL-8 and IL-6 in serum increased significantly. After the intervention of antioxidant Tempol and p38MAPK inhibitor SB203580, SOD increased significantly but with significant MDA reduction. The levels of TNF-α, hsCRP, IL-8 and IL-6 in serum significantly decreased. The results of RT-PCR and Western-Blot indicated that the P-p38 and TLR4 proteins related to the MAPK pathway were expressed in rat adipose tissue. With the hypoxia intensity, expression of P-p38 decreased after initially increasing. The expression of TLR4 showed a continuously growing trend. After Tempol treatment, the expression of p38MAPK protein decreased, and the expression of TLR4 did not change significantly, indicating the inhibiting effect of Tempol on p38MAPK, without a significant inhibiting effect on TLR4. CONCLUSIONS: The TLR4-mediated p38MAPK signaling pathway was active in adipose tissue and the expression of the corresponding molecules increased as the duration of intermittent hypoxia increased. The expression of TLR4 and p38MAPK components regulated the level of oxidative stress and inflammatory cytokines; the application of p38MAPK inhibitors and antioxidant free radical scavengers improved the levels of oxidative stress and inflammatory cytokines.

Predictors of high on-aspirin platelet reactivity in elderly patients with coronary artery disease.

OBJECTIVES: Previous studies have illustrated the link between high on-aspirin platelet reactivity (HAPR) with increasing thrombotic risks. The aim of our study was to investigate relative risk factors of HAPR in elderly patients with coronary artery disease. METHODS: Elderly, hospitalized coronary artery disease patients on regular aspirin treatment were enrolled from January 2014 to September 2016. Medical records of each patient were collected, including demographic information, cardiovascular risk factors, concomitant drugs and routine biological parameters. Arachidonic acid (AA, 0.5 mg/mL) and adenosine diphosphate (ADP, 5 µmol/L) induced platelet aggregation were measured via light transmission assay (LTA) to evaluate antiplatelet responses, referred as LTA-AA and LTA-ADP. RESULTS: A total of 275 elderly patients were included, with mean age of 77.2±8.1 years, and males accounted for 81.8%. HAPR was defined as LTA-AA in the upper quartile of the enrolled population. HAPR patients tended to have lower renal function (P=0.052). Higher serum uric acid (SUA) level, as well as lower platelet count, hemoglobin and hematocrit were observed in HAPR patients, with a higher proportion of diuretics use (P<0.05). Multivariate analysis revealed that SUA (OR: 1.004, 95% CI: 1.000-1.007, P=0.048), platelet count (OR: 0.994, 95% CI: 0.989-1.000, P=0.045), hematocrit (OR: 0.921, 95% CI: 0.864-0.981, P=0.011) and concomitant P2Y12 receptor inhibitors use (OR: 1.965, 95% CI: 1.075-3.592, P=0.028) were correlated with HAPR. Spearman's correlation analysis demonstrated an inverse association of LTA-AA with hematocrit (r=-0.234, P<0.001), hemoglobin (r=-0.209, P<0.001) and estimated glomerular filtration rate (r=-0.132, P=0.031). CONCLUSION: SUA, platelet count, hematocrit and P2Y12 receptor inhibitors use were independently correlated with HAPR. These parameters might provide novel therapeutic targets for optimizing antiplatelet therapy.

Validation of aspirin response-related transcripts in patients with coronary artery disease and preliminary investigation on CMTM5 function.

Aspirin is widely used in the prevention of cardiovascular diseases, but the antiplatelet responses vary from one patient to another. To validate aspirin response related transcripts and illustrate their roles in predicting cardiovascular events, we have quantified the relative expression of 14 transcripts previously identified as related to high on-aspirin platelet reactivity (HAPR) in 223 patients with coronary artery disease (CAD) on regular aspirin treatment. All patients were followed up regularly for cardiovascular events (CVE). The mean age of our enrolled population was 75.80±8.57years. HAPR patients showed no significant differences in terms of co-morbidities and combined drugs. Besides, the relative expression of HLA-DQA1 was significantly lower in low on-aspirin platelet reactivity (LAPR) patients, when compared with HAPR and high normal (HN) group (p=0.028). What's more, the number of arteries involved, HAPR status and the relative expression of CLU, CMTM5 and SPARC were independent risk factors for CVE during follow up (p<0.05). In addition, overexpression of CMTM5 attenuated endothelial cells (ECs) migration and proliferation, with significantly decreased phosphorylated-Akt levels, while its inhibition promoted these processes in vitro (p<0.05).Our study provides evidence that circulating transcripts might be potential biomarkers in predicting cardiovascular events. CMTM5 might exert anti-atherosclerotic effects via suppressing migration and proliferation in the vessel wall. Nevertheless, larger-scale and long-term studies are still needed.

[Dose-response of aspirin on platelet function in very elderly patients].

OBJECTIVE: To assess the consequences of switching aspirin dosage from 100 mg/d to 40 mg/d on cardiovascular benefit, bleeding risk and platelet aggregation in very elderly patients. METHODS: Arachidonic acid induced platelet aggregation(AA-Ag) was measured in 537 patients aged 80 or older treated with aspirin (100 mg/d). In the study, 100 patients with low on-treatment platelet aggregation and at high risk of bleeding and low risk of cardiovascular events, were switched to aspirin (40 mg/d) and their platelet aggregation was measured again 7 days later.Their bleeding and upper gastrointestinal symptoms were also recorded in following 3 months. RESULTS: The study observed a heterogeneous distributed aspirin 100 mg/d AA-Ag (range: 0.42% to 28.78%)in the 537 very elderly patients.Aspirin 100 mg/d AA-Ag before the switch in aspirin 40 mg/d group was 5.00%±2.32% and the rate of the patients with low on-treatment platelet aggregation was 71.00%. The rates of melena or occult blood positive, other minimal bleeding,upper gastrointestinal symptoms and a history of gastrointestinal bleeding in 40 mg/d group were higher than those in 100 mg/d group. On a regimen of aspirin 40 mg/d, AA-Ag increased to 11.21%±4.95%(range: 2.12% to 28.84%) with 95.00%of the patients with AA-Ag<20% and the rate of the patients with low on-treatment platelet aggregation was 15.00%. Multiple variable analysis revealed that aspirin 40 mg/d AA-Ag was significantly influenced by aspirin 100 mg/d AA-Ag, BMI and platelet counts. The rate of gastrointestinal bleeding decreased from 12.00% to 5.00%,and upper gastrointestinal symptoms decreased from 59.00% to 21.00% after the switch in 40 mg/d group. CONCLUSION: Switching aspirin dosage from 100 mg/d to 40 mg/d reduces the bleeding events and improves upper gastrointestinal symptoms, thus inhibiting platelet aggregation effectively in very elderly patients.

Serum uric acid level and left ventricular hypertrophy in elderly male patients with nonvalvular atrial fibrillation.

BACKGROUND AND AIMS: Recent studies have suggested that serum uric acid (SUA) induces oxidative stress and inflammation, which are involved in the mechanism of cardiac hypertrophy. In patients with atrial fibrillation (AF), comorbidity of left ventricular hypertrophy (LVH) exacerbates cardiac function. In this study, we investigated the association between SUA and cardiac hypertrophy in AF patients. METHODS AND RESULTS: Initially, 1296 consecutive elderly patients (age >60) with nonvalvular AF were retrospectively selected from the inpatient clinic between January 2012 and April 2015. Demographic, clinical, and echocardiographic characteristics were carefully recorded. The final study population was 577 patients. The mean SUA level was significantly higher in patients with LVH than those without LVH. Compared with the non-LVH group, the LVH group was older, had a higher percentage of female patients, and had lower hemoglobin levels and estimated glomerular filtration rates. Patients in the LVH group also had a higher rate of coronary heart disease and fewer had history of radiofrequency ablation compared with the non-LVH group. In the hyperuricemia group, B-type natriuretic peptide levels, left atrial diameter, left ventricular mass index, and percentage of NYHA (New York Heart Association) class III/IV were significantly higher than the SUA normal group. Multivariate logistic regression analysis indicated the independent risk factors for LVH in elderly AF patients included SUA, age, male sex, the presence of coronary heart disease, and diuretic therapy. Subgroup analysis identified SUA as a significant risk factor associated with LVH in men. CONCLUSIONS: SUA was independently associated with LVH in elderly male patients with nonvalvular AF.

Uric acid promotes chemokine and adhesion molecule production in vascular endothelium via nuclear factor-kappa B signaling.

BACKGROUND AND AIMS: Hyperuricemia is an important risk factor for atherosclerosis, yet the potential mechanisms are not well understood. Migration and adhesion of leukocytes to endothelial cells play key roles in initiation and development of atherosclerosis. We investigated monocyte-endothelial cell interactions and potential signaling pathways under uric acid (UA)-stimulated conditions. METHODS AND RESULTS: Primary human umbilical vein endothelial cells (HUVECs) were cultured and exposed to different concentrations of UA for various periods. Experimental hyperuricemia rat models were established. Expression of chemoattractant protein-1 (MCP-1), interleukin 8 (IL-8), vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1) were evaluated. Monocyte-endothelial cell interactions were elucidated by chemotaxis and adhesion assays, and nuclear factor-kappa B (NF-κB) pathway was studied using fluorescent microscopy and electrophoretic mobility shift assay. Results showed that high concentration of UA stimulated generation of chemokines and adhesion molecules in ex vivo and in vivo experiments. Migration and adhesion of human monocytic leukemia cell line THP-1 cells to HUVECs were promoted and activated NF-κB was significantly increased. UA-induced responses were ameliorated by organic anion transporter inhibitor probenecid and NF-κB inhibitor BAY11-7082. It was also observed that human endothelial cells expressed urate transporter-1, which was not regulated by UA. CONCLUSION: High concentration of UA exerts unfavorable effects directly on vascular endothelium via the NF-κB signaling pathway, the process of which requires intracellular uptake of UA.

Development of haploid asparagus embryos from liquid cultures of anther-derived calli is enhanced by ancymidol.

The effect of ancymidol concentration on the development of haploid asparagus embryos was determined. Liquid cultures from anther-derived calli were grown for three weeks in MS medium plus 1.0 mg l(-1) 2,4-D, 0.1 mg l(-1) NAA, 0.2 mg l(-1) kinetin, 800 mg l(-1) glutamine, 500 mg l(-1) casein hydrolysate, 2% sucrose and 0.0-1.0 mg l(-1) ancymidol. Cell clumps (224-500 µm) were plated on solid embryo maturation medium (MS medium plus 3% sucrose, 0.1 mg l(-1) NAA, 0.5 mg l(-1) kinetin and 0.0-1.0 mg l(-1) ancymidol) and grown for eight weeks. Ancymidol enhanced embryo maturation and germination and was more critical in the solid than liquid medium. Total embryo number did not vary among most treatments. The best response was observed when ancymidol concentrations were 0.1 and 0.5 mg l(-1) in the liquid and solid media, respectively; two-thirds of the embryos produced were bipolar and 35% of bipolar embryos germinated. Seven to 82% of plants recovered from different ancymidol treatments were haploid; the others were diploid, triploid or chimeric for ploidy level.

High frequency production of haploid embryos in asparagus anther culture.

A method for obtaining a high frequency of haploid asparagus embryos through anther culture was developed. Flowers collected from plants in the field in July, August and September 1990, for the genotype G203, were stored at 5°C for 24 h. Anthers were placed on Murashige and Skoog medium (MS) containing 500 mg l (-1) casein hydrolysate, 800 mg l(-1) glutamine, 2 mg l (-1) NAA, 1 mg l (-1) BA and 5 % sucrose at 32 °C in the dark for three to four weeks to induce calli. Calli were then grown at 25 °C with a 16 h photoperiod for three to four weeks. Developing embryos and calli were transferred to embryo maturation medium, MS containing 6% sucrose, 0.1 mg l (-1) NAA, 0.1 mg l (-1) kinetin and 0.65 mg l (-1) ancymidol, for four weeks. More than 50% of the recovered mature embryos germinated on MS containing l mg l (-1) GA3. Anthers with microspores at the late-uninucleate stage had the highest frequency of total and embryogenic calli formation, 40% and 15%, respectively. Each embryogenic callus usually produced 10-15 embryos. Aproximately 75 plants per 100 anthers cultured were recovered: 76% haploid, 22% diploid and 2% triploid. High temperature was critical for the induction of embryogenic callus.