M Liu

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.

Novel UMOD mutations in familial juvenile hyperuricemic nephropathy lead to abnormal uromodulin intracellular trafficking

BACKGROUND Familial juvenile hyperuricemic nephropathy (FJHN) is an autosomal dominant disorder characterized by hyperuricemia and progressive chronic kidney disease. Uromodulin gene (UMOD) mutations, leading to abnormalities of uromodulin intracellular trafficking contribute to the progress of the disease. METHODS We did UMOD screening in three Chinese FJHN families. We thus constructed mutant uromodulin express plasmids by site-mutagenesis from wild type uromodulin vector and transfected them into HEK293 (human embryonic kidney) cells. And then we detected uromodulin expression by western blot and observed intracellular distribution by immunofluorescence. RESULTS We found three heterozygous mutations. Mutation Val109Glu (c.326T/A; p.Val109Glu) and mutation Pro236Gln (c.707C/A; p.Pro236Gln) were newly indentified mutations in two distinct families (family F1 and family F3). Another previously reported UMOD mutation Cys248Trp (c.744C/G; p.Cys248Trp) was detected in family F2. Phenotypes varied both within the same family and between different families. Uromodulin expression is abnormal in the patient biopsy. Functional analysis of mutation showed that mutant types of uromodulin were secreted into the supernatant medium much less when compared with wild type. In mutant type uromodulin transfected cells, intracellular uromodulin localized less in the Golgi apparatus and more in endoplasmic reticulum(ER). CONCLUSIONS Our results suggested that the novel uromodulin mutations found in the Chinese families lead to misfolded protein, which was retained in the endoplasmic reticulum, finally contributed to the phenotype of FJHN.

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.

Interaction of uromodulin and complement factor H enhances C3b inactivation

Recent studies suggest that uromodulin plays an important role in chronic kidney diseases. It can interact with several complement components, various cytokines and immune system cells. Complement factor H (CFH), as a regulator of the complement alternative pathway, is also associated with various renal diseases. Thus, we have been suggested that uromodulin regulates complement activation by interacting with CFH during tubulointerstitial injury. We detected co‐localization of uromodulin and CFH in the renal tubules by using immunofluorescence. Next, we confirmed the binding of uromodulin with CFH in vitro and found that the affinity constant (KD) of uromodulin binding to CFH was 4.07 × 10−6M based on surface plasmon resonance results. The binding sites on CFH were defined as the short consensus repeat (SCR) units SCR1–4, SCR7 and SCR19–20. The uromodulin‐CFH interaction enhanced the cofactor activity of CFH for factor I‐mediated cleavage of C3b to iC3b. These results indicate that uromodulin plays a role via binding and enhancing the function of CFH.

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). Whats 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.

Implication of Urinary Complement Factor H in the Progression of Immunoglobulin A Nephropathy

Background After activation, the complement system is involved in the pathogenesis of Immunoglobulin A nephropathy (IgAN). Complement factor H (CFH) is a crucial inhibitory factor of the alternative pathway of the complement system. The study investigated the effects of urinary CFH levels on IgAN progression. Methods A total of 351patients with IgAN participated in this study. They were followed up for an average of 51.8±26.6 months. Renal outcome was defined as a composite endpoint, that included instances of end-stage renal disease (ESRD),≥ 50% decline in estimated glomerular filtration rate (eGFR) or doubling of plasma creatinine levels. Urinary CFH levels were measured by enzyme-linked immunosorbent assay and calculated as the ratio of urinary CFH over creatinine (uCFH/uCr). Results In the whole cohort, uCFH/uCr values were associated with disease progression either as continuous [log(uCFH/uCr)] or categorical traits (dichotomous and quartile variables) after adjusting for eGFR, proteinuria, mean arterial blood pressure, histological grading and immunosuppressive therapy in the Cox proportional hazard model. Kaplan-Meier analysis showed that higher uCFH/uCr values at baseline predicted worse renal outcome during follow-up (log-rank, P<0.001). Receiver operating characteristic curve (ROC) analysis showed that log(uCFH/uCr) had predictive value for renal outcome (area under curve [AUC]=0.745), and the AUC increased to 0.805 after being incorporated into baseline eGFR and proteinuria. In subgroup analysis with eGFR≥60 mL/min/1.73m2, log(uCFH/uCr) had better predictive value (AUC= 0.724, P=0.002) for renal outcome compared to eGFR (AUC = 0.582, P=0.259) and proteinuria (AUC = 0.615, P=0.114). Conclusions Urinary CFH levels are associated with renal function decline and increased urinary CFH levels are a risk factor for progression of IgA nephropathy.

ASSA14-03-41 Platelet thromboxane 11-dehydro-Thromboxane B2 (11dhTxB2) and aspirin response and its correlation with clinical outcome in patients with Coronary artery disease and Diabetes

Aims Many studies revealed that patients with diabetes (DM) had higher risk in developing atherosclerosis and its complicated clincal outcome. Aspirin, a widely prescribed antiplatelet drug, was beneficial in the primary and secondary of cardiovascular diseases. However, its efficacy varies. The aim of our studywas to determine if there is a correlation betweenAspirin Resistance (AR) and clinical outcomes in patients with DM and coronary artery disease (CAD). Methods 108 aged CAD patients were enrolled in this study,among who 27 were with DM, and 81 were without DM. Aspirin response was assessed by urinary11-DehydrothromboxaneB2 (11dhTxB2) measurement before and after 100 mg/d aspirin administration. Aspirin resistance (AR) was defined as urinary11dhTxB2 >1500 pg/mg. The measured clinical outcomes were defined as the occurrence of cardiovascular events and death. Results The mean baseline urinary 11dhTxB2 of CADpatients was 3463 ± 2465 pg/mg. In subgroup analysis, patients withDM (3887 ± 1857 pg/mg) had higher baseline urinary 11dhTxB2 than CAD patients (3210 ± 934 pg/mg) (p < 0.001). After aspirin administration, the urinary 11dhTxB2 of CAD patients with and without DM were significantly lower than their baseline value (856 ± 748 pg/mg, 1054 ± 859 pg/mg, P < 0.001). The prevalence of AR in CAD patients with or without DM were 18.3% and 6.4% according to the defined criterion Urinary11dhTxB2 >1500 pg/mg. Within a mean follow-up time of 12 months, the outcomesoccurred more frequently in AR patients withDM than AR patients without DM (p < 0.05). Conclusion The baseline and after administration urinary 11dhTxB2 levels in CAD patients with DM were significantly higher than CAD patients without DM. Incidence of 1-year clinical outcome in CAD patients with DM was significantly higher than those without DM. Further studies are needed to confirm our results.

ASSA14-03-40 Association between platelet thromboxane (11-dehydro-Thromboxane B2) and aspirin response in aged patients with coronary artery disease

Aims Aspirin, a widely used antiplatelet agent, irreversibly inhibits platelet cyclooxygenase-1 and reduce thromboxane-mediated platelet activation. Although it has been proved that aspirin played an important role in the primary and secondary prevention of cardiovascular diseases, its efficacy varies. The aim of our studywas to investigate the relationship betweenurinary 11-dehydro-thromboxane B2 (11dhTxB2) and clinical outcome in aged patients with coronaryartery disease (CAD). Methods 108 aged CAD patients on 100 mg/d aspirin administration at least 12 month were enrolled in this study. Aspirin response was assessed using urinary11-DehydrothromboxaneB2 (11dhTxB2) measurement. Urinary11dhTxB2 >1500 pg/mg was defined as Aspirin Resistance (AR). The measured clinical outcomes were defined as the occurrence of cardiovascular events and death. Results The mean age of all enrolled patients was 83.3 ± 10.5 years old. Baseline and post-treatment urinary 11-dhTXB2 levels are 3463 ± 2465 pg/mg and 1386 ± 1895 pg/mg, respectively. Prevalence of AR was 27.7% according to the defined criterion Urinary11dhTxB2 >1500 pg/mg. Within a mean follow-up time of 12 months, the outcomesoccurred more frequently in AR group as compared with no-AR patients (p < 0.05). After adjustment for the conventional risk factors for CAD, there still exists a significant association between AR based on urinary 11dhTxB2 level and clinical outcome (p < 0.05). Conclusion Our research showed that 27.7% enrolled CAD patients meet the criterion of AR using urinary 11dhTxB2measurement. It’s noteworthy that AR status was significantly associated with higher incidence of cardiovascular events and death than no-AR patients (p < 0.05).

ASSA14-03-39 Identification of Aspirin response related gene profiles in the elderly population with coronary artery disease

Aims Aspirin is widely used in the primary and secondary prevention of cardiovascular diseases. Nevertheless, the responses to aspirin administration vary from one patient to another. A small proportion of patients experience thrombo-embolic events during their daily aspirin therapy, defined as clinical aspirin resistance (AR). Recent studies showed that aspirin exposure might have an influence on the expression of various genes, thus responsible for the variability in aspirin effects. The aim of our studywas to identify aspirin responses related gene profiles, and investigate the correlation between target genes expression and clinical outcomes in the elderly population with coronary artery disease (CAD). Methods A total of 152 aged patients with CAD treated with low-dose aspirin (100 mg/d) were enrolled in this study. All enrolled patients were distributed according to quartile of 0.5 mmol/l AA-induced platelet aggregation, and AR was defined as the upper quartile of AA-induced platelet aggregation. Total blood RNA were extracted within 4 h and reversely transcribed immediately. Expression of fourteen genes (CLU, CMTM5, CTTN, MPL, TMEM64, SELP, HLA-DQA1, HLA-DRB4, ITGA2B, ITGB3, THBS1, CXCL5, PPBP, and SPARC) was measuredusing real-time quantitative PCR method. Clinical outcomes were defined as the occurrence of cardiovascular events and death during regular aspirin administration. Results The quartile cut points of AA-induced platelet aggregation for the 25th, 50th, and 75th percentiles of the enrolled population were 9.7%, 12.0%, and 14.3%, respectively. AR was defined as AA-induced platelet aggregation ≥14.3%. Expressions of six genes (CTTN CXCL5 HLA-DQA1 HLA-DRB4 THBS1 SPARC)were significantly higher in AR group than no-AR group (p < 0.01). What’s more, with a mean follow-up of 6 months, clinical outcomesoccurred more frequently in AR group as compared tono-AR group (p < 0.05). Conclusion Six genes (CTTN CXCL5 HLA-DQA1 HLA-DRB4 THBS1 SPARC) were up-regulated in AR group than no-AR group (p < 0.01). What’s more, AR patients have higher risks for cardiovascular events and death than non-AR group (p < 0.05)

ASSA14-03-38 Comparison of Urinary11-DehydrothromboxaneB2 detection and Plasma LTA Assay for evaluating aspirin response in the Aged Patients with Coronary Artery Disease

Aims Aspirin is widely used in the primary and secondary prevention of cardiovascular diseases. Nevertheless, the responses to aspirin administration vary from one patient to another, and there is a lack of efficient and platelet-specific method correlated well with clinical outcomes. The aim of our study was to make comparisons between 2 established methods of aspirin response evaluation)—measurement of urinary 11-dehydrothromboxane B2 (11dhTxB2) and plasma Light Transmission Aggregometry (LTA) assay—in aged patients with coronary artery disease (CAD), and examinethe relationship between AR and clinical outcomes. Methods A total of 145 aged patients with CAD on low-dose aspirin (100 mg/d) therapy were enrolled in this study. All patients were measuredby urinary11dhTxB2 and platelet aggregation. Aspirin response was assessed via urinary 11dhTxB2 measurement and plasma LTAassay. AspirinResistance (AR) was defined as urinary11dhTxB2 >1500 pg/mg or 0.5 mg/ml AA-induced platelet aggregation ≥ 15.07%. The Spearman correlation coefficients andkappa statistics were calculated to assess correlation and agreement between the2 tests. The measured clinical outcome was defined as the occurrence of cardiovascular events and death. Results Prevalence of aspirin non-responders was 32.1% and 13.2%, according to urinary 11dhTxB2 measurement and LTA assay, respectively. Poor correlation in the results between the 2 tests was observed (r = 0.135, p = 0.151). Besides, the correlation between the 2 methods and AR was weak (kappa = 0.113, p = 0.113). With a mean follow-up time of 12 months, the clinical outcomes occurred more frequently in AR patients based on urinary 11dhTxB2 measurement as compared with normal aspirin response patients (18% vs 2%, OR: 8.8, 95% CI: 1.18~65.4, p = 0.037). Conclusions Our research observed poor correlation and lack of agreement between the 2 tests. Urinary 11dhTxB2 measurement might be a better predictor in the risk of cardiovascular events and death as compared to LTA assay. Nevertheless, development of efficient and inexpensive platelet function tests, which should also have a good correlation with clinical outcomes was necessary.