Lian-Feng Chen

Danon disease as a cause of concentric left ventricular hypertrophy in patients who underwent endomyocardial biopsy

BACKGROUND Danon disease is an X-linked dominant disorder; concentric left ventricular hypertrophy (LVH) is one of its manifestations. In this study, we investigated the prevalence of Danon disease in patients with concentric LVH who underwent endomyocardial biopsy (EMB). METHODS AND RESULTS A total of 50 patients with concentric LVH underwent EMB from January 2008 to December 2010. Cardiac amyloidosis was diagnosed in 14 patients; genetic analysis of lysosome-associated membrane protein 2 (LAMP2) was done in the remaining 36 patients. Three novel LAMP2 frameshift mutations were found. They were c.808_809 insG in exon 6, c.320_321 insCATC in exon 3, and c.257_258delCC in exon 3, leading to a premature stop codon on cDNA analysis. The prevalence of Danon disease was seen in 6% (3 of 50) of unselected concentric LVH patients who underwent EMB, or 8% (3 of 36) after excluding cardiac amyloidosis through EMB. All the three patients were male teenagers with a mean age of 15 ± 1 years, and had mild mental retardation, two of the three with Wolff-Parkinson-White (WPW) syndrome and markedly increased left ventricular voltage. All the three patients had increased serum hepatic enzymes and creatine kinase (CK) concentrations. There was no death or cardiovascular hospitalization during 20 ± 15 months of follow-up. CONCLUSIONS Danon disease may account for a number of patients with concentric LVH who underwent EMB. Danon disease should be suspected in the male teenager with concentric LVH, especially with elevated serum hepatic enzymes and CK concentrations, and/or WPW syndrome with markedly increased voltage of the left ventricle. Genetic analysis of LAMP2 can help make the diagnosis.

Urokinase receptor surface expression regulates monocyte migration and is associated with accelerated atherosclerosis

BACKGROUND The urokinase receptor (uPAR) is a key regulator of pericellular proteolysis, cell adhesion and migration, all of which are fundamental processes in atherogenesis. We hypothesized that increased monocytic uPAR expression in circulation is associated with the formation and development of atherosclerosis. METHODS A total of 42 male apoE-/- mice were ramdonly divided into high-fat (HF) diet and normal diet (n=21 per group). The percentage of uPAR expressing monocytes (PUEM) and the expression of uPAR within different types of atherosclerotic plaques were measured at an interval of 3 weeks from week 10 to week 16. In vitro, uPAR expression upon ox-LDL stimulation and the migration of monocytes were examined. RESULTS PUEM in circulation was significantly higher in animals with HF diet compared with those having normal diet (p<0.03). The augmented levels of PUEM were associated with body weight, visceral fat weight and numbers of uPAR+macrophages within atherosclerotic lesions. Accumulation of uPAR+macrophages increased with the progression of atherosclerosis. Monocytes upon ox-LDL stimulation exhibited an increased uPAR expression and uPAR antibody markedly suppressed monocyte migration induced by monocyte chemotactic protein-1. uPAR modulated monocyte migration was accelerated by uPA and was suppressed by amino terminal fragment of uPA dependent. CONCLUSIONS Over-expression of uPAR both in circulating monocytes and in atherosclerotic lesions is associated with the development of atherosclerotic plaques. uPAR and its interaction with uPA may contribute to enhanced monocyte migration. Thus, uPAR may be a novel target for prevention of uncontrolled monocyte recruitment in inflammatory atherogenic process.

ABCG1 rs57137919G>A Polymorphism Is Functionally Associated with Varying Gene Expression and Apoptosis of Macrophages

ATP-binding cassette transporter G1 (ABCG1) is a transmembrane cholesterol transporter involved in macrophage sterol homeostasis, reverse cholesterol transport (RCT), and atherosclerosis. The role of ABCG1 in atherosclerosis remains controversial, especially in animal models. Our previous study showed that single nucleotide polymorphism rs57137919 (-367G>A) in the ABCG1 promoter region was associated with reduced risk for atherosclerotic coronary artery disease (CAD). This study was designed to provide functional evidence for the role of rs57137919G>A in atherosclerosis in humans. We combined in vitro and ex vivo studies using cell lines and human monocyte-derived macrophages to investigate the functional consequences of the promoter polymorphism by observing the effects of the rs57137919A allele on promoter activity, transcription factor binding, gene expression, cholesterol efflux, and apoptosis levels. The results showed that the rs57137919A allele was significantly associated with decreased ABCG1 gene expression possibly due to the impaired ability of protein-DNA binding. ABCG1-mediated cholesterol efflux decreased by 23% with rs57137919 A/A versus the G/G genotype. Cholesterol-loaded macrophage apoptosis was induced 2-fold with the A/A genotype compared with the G/G genotype. Proapoptotic genes Bok and Bid mRNA levels were significantly increased in macrophages from the A/A genotype compared with those from the G/G genotype. These findings demonstrated that the ABCG1 promoter rs57137919G>A variant had an allele-specific effect on ABCG1 expression and was associated with an increased apoptosis in cholesterol-loaded macrophages, providing functional evidence to explain the reduced risk for atherosclerosis in subjects with the ABCG1 promoter rs57137919A allele as reported in our previous study.

Characterization of fluorescent NBD‑cholesterol efflux in THP‑1‑derived macrophages.

Macrophage cholesterol efflux is important in maintaining cellular lipid homeostasis and preventing the formation of lipid‑laden foam cells. Although radioactive [3H]‑cholesterol is widely used as a tracer in cholesterol efflux assays, the lengthy and labor‑intensive assay procedure, and the radioactivity disposal procedure limit the use of this assay for high‑throughput screening. In the present study, a novel procedure using fluorescent N‑(7‑nitrobenz‑2‑oxa‑1,3‑diazol‑4‑yl)amino)‑23,24‑bisnor‑5‑xholen‑3β‑ol (NBD)‑cholesterol was developed as a substitute for [3H]‑cholesterol for the measurement of cholesterol efflux in THP‑1‑derived macrophages. NBD‑cholesterol uptake and metabolism in the THP‑1 cells were characterized using fluorescent microscopy and spectrophotometry. NBD‑cholesterol distributed rapidly into the cell organelles, with the exception of the nucleus. The uptake of NBD‑cholesterol in the THP‑1 macrophages was concentration‑ and time‑dependent, and reached a plateau following 4 h incubation. The present study subsequently investigated whether NBD‑cholesterol efflux was correlated with [3H]‑cholesterol efflux in THP‑1 derived macrophages and in human peripheral blood mononuclear cells (PBMCs). The results demonstrated that the percentage of efflux of NBD‑cholesterol in the THP‑1 cells was significantly correlated with that of [3H]‑cholesterol, at various concentrations of HDL or apoA‑1 as lipid acceptors (R2=0.876 for HDL; R2=0.837 for apoA‑1; P<0.001). In the PBMCs, NBD‑cholesterol efflux also correlated significantly with [3H]‑cholesterol efflux (R2=0.887 for HDL; R2=0.872 for apoA‑1; P<0.001). Furthermore, NBD‑cholesterol efflux in the THP‑1 cells exhibited a similar trend to that obseved in the PBMCs. In conclusion, the results of the present study suggested that fluorescent NBD‑cholesterol can be used as a sensitive and specific probe in cholesterol efflux assays in THP‑1‑derived macrophages.

Association Between Oxidative Stress and Peripheral Leukocyte Telomere Length in Patients with Premature Coronary Artery Disease

Background Leukocyte telomere length (LTL) is regarded as a potential marker of biological aging. Oxidative stress plays a major role in the rate of telomeric DNA loss. The aim of this study was to explore whether the LTL was shorter in Chinese patients with premature coronary artery disease (PCAD) than in non-CAD controls and to determine the relationship between oxidative stress and LTL shortening in this population. Material/Methods Patients for coronary angiography were recruited. In total, 128 patients with PCAD and 128 non-CAD controls were enrolled. Samples of circulating leukocytes and plasma were collected. The mean LTL was measured using a polymerase chain reaction-based assay and expressed as the ratio of telomere repeat copies to single-copy gene (SCG) copies (T/S ratio). Reactive oxygen species (ROS) levels and total antioxidant capacity (T-AOC) were determined in plasma. Results Both the T/S ratio (0.88±0.86 vs. 1.10±0.57, P=0.015) and telomere base pairs (4.97±1.37 kb vs. 5.32±0.91 kb, P=0.015) were significantly shorter in the PCAD group than in non-CAD controls. The T-AOC levels of the PCAD group were significantly lower than those of the non-CAD controls (0.482 mM [0.279, 0.603 mM]) vs. 0.778 mM [0.421, 0.924 mM], P=0.000). The ratio of T-AOC to ROS in the PCAD patients was significantly decreased compared to that of the non-CAD controls (0.1026±0. 1587 [Mm*ml/ng] vs. 0.1435±0.1946 [Mm*ml/ng], P=0.013). Conclusion The results point to a potential link between reduced LTLs in patients with PCAD and early onset of atherosclerosis. The decline in antioxidant capacity may play an important role in accelerating the attrition of telomeres in PCAD patients.

Valsartan and telmisartan abrogate angiotensin II-induced downregulation of ABCA1 expression via AT1 receptor, rather than AT2 receptor or PPARγ activation.

Abstract: The possible pharmacological mechanism by which partial PPAR&ggr;-activating angiotensin II (Ang II) type 1 receptor blocker (ARB) telmisartan and non–PPAR&ggr;-activating ARB valsartan reverse Ang II–suppressed ABCA1 expression is still unclear. In this study, human monocyte-derived THP-1 cells were differentiated into macrophages. Cells were treated with various concentrations of Ang II alone or with Ang II and various drugs including highly selective ARB valsartan, partial PPAR&ggr;-activating ARB telmisartan, angiotensin II type 2 (AT2) receptor blocker PD123319, full PPAR&ggr; agonist pioglitazone, and PPAR&ggr; antagonist GW9662, respectively. After treatment, messenger RNA and protein expressions of ABCA1 and ABCG1 were analyzed by real-time polymerase chain reaction and Western blotting, respectively. ABCA1 expression was remarkably suppressed by Ang II at both messenger RNA and protein levels in a dose-dependent manner in THP-1–derived macrophages, whereas ABCG1 expression was not affected. Valsartan and telmisartan could both reverse the downregulation of Ang II on ABCA1 expression. Such effects were not affected by either AT2 receptor blocker PD123319 or PPAR&ggr; antagonist GW9662. Our findings suggest that the effect of Ang II on ABCA1 expression should be mediated by the AT1 receptor. Both valsartan and telmisartan abrogate Ang II–induced downregulation of ABCA1 expression mainly through AT1 receptor rather than through AT2 receptor or PPAR&ggr;-dependent pathway.

Association between serum chemokine CC-motif ligand 17 and coronary artery disease

Chemokines, a family of small chemotactic cytokines, have been recognized as important contributors to the pathogenesis of atherosclerosis and related cardiovascular disease [1]. Chemokine CC ligand 17 (CCL 17), also known as thymus activation and regulated chemokine (TARC), is a member of the chemokine family. Recently, Weber et al. found that dendritic cell-derived CCL17 accumulated in advanced human and mouse atherosclerotic lesions. Furthermore, deficiency or blockade of dendritic cell-derived CCL17 reduced atherosclerosis by expanding regulatory T cells (Tregs), whereas CCL17 expression in dendritic cells limited Treg retention to promote atherosclerosis, suggesting CCL17 as an important regulator of atherosclerosis [2]. To date, however, there has been no report exploring the relationship between serum CCL 17 levels and CAD. In the current study, we measured the serum CCL17 levels in patients with andwithout CAD and tried to determine the association between serum CCL17 levels and different forms of CAD or severity of CAD. The study population consisted of a cohort of 119 patients who underwent coronary angiography from January 2012 to December 2012 in a single academic center. Patients were divided into 4 groups according to diagnosis: 30 consecutive patients with angiographically normal coronary arteries (b20% stenosis), 30 consecutive patients with stable angina pectoris (SAP) and N50% stenosis in at least one major coronary artery branch, 29 consecutive patients with nonST elevation myocardial infarction (NSTEMI) and 30 consecutive patients with ST elevation myocardial infarction (STEMI). Exclusion criteria included dermatitis, current infection, malignancy, auto-immune disease, vasculitis, asthma, cirrhosis, severe renal failure (eGFR b 30 ml/min) and shock. This study has been approved by the local Ethics Committees and all participants have given their informed consent. Coronary angiography (CA) was performed with a conventional angiography unit (Integris H; Philips Medical Systems). The severity of atherosclerosis was determined using the Gensini score (GSS) [6]. Blood for determining serum levels of CCL 17 and high sensitive-C reactive protein (hs-CRP) was collected from the radial or femoral artery after the insertion of sheathe prior to any anticoagulation and coronary angiography. Serum CCL 17 concentrations weremeasured with the Quantikine ELISA development kit for human CCL17/TARC (RD NSTEMI 316.17 ± 162.26 pg/ml; STEMI 339.54 ± 130.26 pg/ml; p for ANOVA= 0.682). There was a linear relationship between serum CCL 17 levels and the Gensini Scores (R = 0.24, p = 0.024). No associationwas found between serum CCL 17 levels and age, BMI, lipid profile, creatinine or Hs-CRP levels.

Increased Urokinase-Type Plasminogen Activator Receptor Expression on Circulating Monocytes Is Correlated with Clinical Instability and Long-Term Adverse Cardiac Events in Patients with Coronary Artery Disease.

Objectives: This study sought to investigate the clinical correlates and prognostic roles of urokinase-type plasminogen activator receptor (uPAR) on circulating monocytes in patients with coronary artery disease (CAD). Methods: 263 angina patients were included in this study. The percentage of uPAR expressing monocytes (PUEM) and the mean fluorescence intensity (MFI) index of uPAR were measured using flow cytometry. Patient follow-up was on average 604 days. Major adverse cardiac events (MACE) were defined as a composite of cardiac death, reinfarction, acute heart failure and hospitalization for revascularization. Results: The PUEM and MFI index levels were significantly more elevated in acute coronary syndrome patients than in stable ones. uPAR expressions on circulating monocytes at admission were correlated to inflammatory biomarkers and myocardial necrosis. Logistic regression analysis revealed that PUEM ≥15% (OR 21.96, 95% CI 7.31-65.98, p < 0.001) and uPAR MFI index ≥3.00 (OR 3.54, 95% CI 1.18-10.59, p = 0.024) were independent determinants of clinical instability in patients with CAD. When followed up, a high PUEM level at admission was an independent prognostic parameter for long-term MACE (HR 3.99, 95% CI 1.31-12.11, p = 0.015). Conclusions: uPAR expression on circulating monocytes is associated with clinical instability and myocardial necrosis and independently predicts the risk of MACE in patients with CAD.

Association between the ABCA1-565C/T gene promoter polymorphism and coronary heart disease severity and cholesterol efflux in the Chinese Han population.

BACKGROUND ABCA1 -565C/T gene promoter variants have been associated with the severity of coronary artery disease in Western populations. The purpose of our study was to investigate the association between the -565C/T gene polymorphism and coronary artery disease severity and cholesterol efflux in the Chinese Han population. METHODS A cohort of 298 acute coronary syndrome (ACS) patients and 541 healthy controls was genotyped using the highly sensitive ligase detection reaction. ABCA1 -565C/T genotype was correlated with the clinical features of 164 acute myocardial infarction (AMI) patients. Monocytes from patients with various -565C/T gene polymorphisms were isolated and differentiated into foam cells by coincubation with [(3)H]-labeled acetyl-low-density lipoprotein cholesterol. ABCA1 mRNA and protein expression levels were evaluated, as well as cellular cholesterol efflux. RESULTS The frequency of the TT genotype in the -565C/T polymorphism of ACS patients was significantly increased when compared with controls (0.211 vs. 0.162, p<0.05). The TT genotype, but not the CT or CC genotypes, in the -565C/T gene polymorphism correlated with the severity of the coronary lesion observed in AMI patients. Patients with the TT homozygote genotype also exhibited significantly lower cellular cholesterol efflux (TT [6.37%±0.554%]) levels than controls and also had the lowest levels of ABCA1 mRNA and protein expression among the group of variants. In contrast, cholesterol efflux levels in AMI patients with CT [11.35%±3.975%] and CC ([15.32%±6.293%]) genotypes were not significantly different from controls. CONCLUSIONS Impaired ABCA1-mediated cholesterol efflux in macrophages may be associated with the severity of the coronary lesions in AMI patients with the TT genotype at the -565C/T gene polymorphism.

The Acute effects of cigarette smoking on the functional state of high density lipoprotein

Background: Cigarette smoking disturbs plasma lipid level and lipoprotein metabolism; however, the effects of smoking on the functional state of high density lipoprotein (HDL) are still not clear. This study aimed to determine the antioxidant and antichemotactic properties of HDL and HDL‐mediated cholesterol efflux in healthy subjects after cigarette smoking. Materials and Methods: Healthy male subjects, including nonsmokers (n = 16) and chronic smokers (n = 8), were enrolled. After smoking 8 cigarettes within 2 hours, plasma HDL was isolated and tested. Copper‐induced low density lipoprotein (LDL) oxidation was used to determine the antioxidant ability of HDL. The concentration of serum amyloid A was measured by Enzyme Linked Immunosorbent Assay. Chemotaxis was detected by transwell assay. HDL‐mediated cholesterol efflux was measured using fluorescent cholesterol analog. Results: LDL baseline oxidation state was higher in chronic smokers than that in nonsmokers. Meanwhile, HDL‐induced cholesterol efflux in macrophages in chronic smokers was significantly enhanced compared with that in nonsmokers. After acute smoking, both the antioxidant and antichemotactic ability of HDL declined in nonsmokers. However, in healthy chronic smokers, the effect of HDL on the susceptibility of LDL to oxidation was compensatorily enhanced. Nevertheless, their bodies were still in a higher oxidation state. Also, acute smoking did not affect HDL‐mediated cholesterol efflux significantly in both nonsmokers and chronic smokers. Conclusions: Our data suggest that acute smoking attenuates the antioxidant and antichemotactic abilities of HDL in nonsmokers. Chronic smokers are in a higher oxidative state, although the antioxidant function of their HDL is compensatorily enhanced.