Clonal Hematopoiesis of Indeterminate Potential in Chronic Thromboembolic Pulmonary Hypertension: A Multicenter Study.

BACKGROUND: The pathogenesis of chronic thromboembolic pulmonary hypertension (CTEPH) is multifactorial and growing evidence has indicated that hematological disorders are involved. Clonal hematopoiesis of indeterminate potential (CHIP) has recently been associated with an increased risk of both hematological malignancies and cardiovascular diseases. However, the prevalence and clinical relevance of CHIP in patients with CTEPH remains unclear. METHODS: Using stepwise calling on next-generation sequencing data from 499 patients with CTEPH referred to 3 centers between October 2006 and December 2021, CHIP mutations were identified. We associated CHIP with all-cause mortality in patients with CTEPH. To provide insights into potential mechanisms, the associations between CHIP and inflammatory markers were also determined. RESULTS: In total, 47 (9.4%) patients with CTEPH carried at least 1 CHIP mutation at a variant allele frequency of ≥2%. The most common mutations were in DNMT3A, TET2, RUNX1, and ASXL1. During follow-up (mean, 55 months), deaths occurred in 22 (46.8%) and 104 (23.0%) patients in the CHIP and non-CHIP groups, respectively (P<0.001, log-rank test). The association of CHIP with mortality remained robust in the fully adjusted model (hazard ratio, 2.190 [95% CI, 1.257-3.816]; P=0.006). Moreover, patients with CHIP mutations showed higher circulating interleukin-1ß and interleukin-6 and lower interleukin-4 and IgG galactosylation levels. CONCLUSIONS: This is the first study to show that CHIP mutations occurred in 9.4% of patients with CTEPH are associated with a severe inflammatory state and confer a poorer prognosis in long-term follow-up.

Heart Rate Response Predicts 6-Minutes Walking Distance in Pulmonary Arterial Hypertension.

Because the 6-minute walking test (6MWT) is a self-paced submaximal test, the 6-minute walking distance (6MWD) is substantially influenced by individual effort level and physical condition, which is difficult to quantify. We aimed to explore the optimal indicator reflecting the perceived effort level during 6MWT. We prospectively enrolled 76 patients with pulmonary arterial hypertension and 152 healthy participants; they performed 2 6MWTs at 2 different speeds: (1) at leisurely speed, as performed in daily life without extra effort (leisure 6MWT) and (2) an increased walking speed, walking as the guideline indicated (standard 6MWT). The factors associated with 6MWD during standard 6MWT were investigated using a multiple linear regression analysis. The heart rate (HR) and Borg score increased and oxygen saturation (SpO2) decreased after walking in 2 6MWTs in both groups (all p <0.001). The ratio of difference in HR before and after each test (ΔHR) to HR before walking (HRat rest) and the difference in SpO2 (ΔSpO2) and Borg (ΔBorg) before and after each test were all significantly higher in both groups after standard 6MWT than after leisure 6MWT (all p <0.001). Multiple linear regression analysis revealed that ΔHR/HRat rest was an independent predictor of 6MWD during standard 6MWT in both groups (both p <0.001, adjusted R2 = 0.737 and 0.49, respectively). 6MWD and ΔHR/HRat rest were significantly lower in patients than in healthy participants (both p <0.001) and in patients with cardiac functional class III than in patients with class I/II (both p <0.001). In conclusion, ΔHR/HRat rest is a good reflector of combined physical and effort factors. HR response should be incorporated into 6MWD to better assess a participant's exercise capacity.

PD-1 inhibitor induces myocarditis by reducing regulatory T cells, activating inflammatory responses, promoting myocardial apoptosis and autophagy.

The present study aimed to explore the pathogenesis of autoimmune myocarditis induced by PD-1 inhibitors and their potential therapeutic targets. Mouse models of autoimmune myocarditis induced by PD-1 inhibitor in mouse models of polymyositis were established. The expression level of PD-1 and regulatory T cells (Tregs), CD4, CD8 + T cells, inflammation, apoptosis and autophagy-related factors, including IL-6, TGF-ß, AMA-M2, Fas/FasL, LC3 and p62 were detected in peripheral blood, muscle or myocardium of mice in each group, using ELISA, RT-PCR, Western Blot and immunofluorescence. In addition, HE and TUNEL staining and ultrastructural scanning were performed on the myocardium of mice in each group. Results showed that the expression level of PD-1 in the two myositis groups was significantly lower than that in the control group, and the level of PD-1 was lower in the myocarditis group than that in the polymyositis group. In the myocardium, TGF-ß, p62, and Tregs proportion showed the same expression level trend as PD-1, while CD8, IL-6, IL-10 and LC3 showed the opposite trend. Levels of Fas/FasL were significantly higher in both myositis groups, but were slightly lower in the myocarditis group, as was AMA-M2. Inflammation, apoptosis, and autophagy were observed in both myositis groups, but were more severe in the myocarditis group. In summary, the decreased expression level of PD-1 leads to decreased Tregs level in the myocardium, aggravated inflammatory response, apoptosis and autophagy, which may be the pathological mechanism of myocarditis induced by PD-1 inhibitors.

Delayed diagnosis of X-linked agammaglobulinaemia in a boy with recurrent meningitis.

BACKGROUND: X-linked agammaglobulinaemia (XLA) is a rare inherited primary immunodeficiency disease characterized by the B cell developmental defect, caused by mutations in the gene coding for Bruton's tyrosine kinase (BTK), which may cause serious recurrent infections. The diagnosis of XLA is sometimes challenging because a few number of patients have higher levels of serum immunoglobulins than expected. In this study, we reported an atypical case with recurrent meningitis, delayed diagnosis with XLA by genetic analysis at the second episode of meningitis at the age of 8 years. CASE REPORT: An 8-year-old Chinese boy presented with fever, dizziness and recurrent vomiting for 3 days. The cerebrospinal fluid (CSF) and magnetic resonance imaging (MRI) results were suggestive of bacterial meningoencephalitis, despite the negative gram staining and cultures of the CSF. The patient was treated with broad-spectrum antibiotics and responded well to the treatment. He had history of another episode of acute pneumococci meningitis 4 years before. The respective level of Immunoglobulin G (IgG), Immunoglobulin A (IgA) and Immunoglobulin M (IgM) was 4.85 g/L, 0.93 g/L and 0.1 g/L at 1st episode, whereas 1.9 g/L, 0.27 g/L and 0 g/L at second episode. The B lymphocytes were 0.21 and 0.06% of peripheral blood lymphocytes at first and second episode respectively. Sequencing of the BTK coding regions showed that the patient had a point mutation in the intron 14, hemizyous c.1349 + 5G > A, while his mother had a heterozygous mutation. It was a splice site mutation predicted to lead to exon skipping and cause a truncated BTK protein. CONCLUSION: Immunity function should be routinely checked in patients with severe intracranial bacterial infection. Absence of B cells even with normal level of serum immunoglobulin suggests the possibility of XLA, although this happens only in rare instances. Mutational analysis of BTK gene is crucial for accurate diagnosis to atypical patients with XLA.

High Sodium Intake Impairs Small Artery Vasoreactivity in vivo in Dahl Salt-Sensitive Rats.

The effects of high sodium intake on the functionality of resistance arteries have been repeatedly studied in vitro, but no study has focused on salt-sensitive hypertension in vivo. We studied the in vivo reactivity of mesenteric small arteries (MSAs) to vasoactive agents in Dahl salt-sensitive (DS) rats with various sodium diets. Twenty-four male DS rats were randomized into 3 groups: LS (0.3% NaCl diet), NS (0.6% NaCl diet), and HS (8% NaCl diet). After a 12-week intervention, the diameter changes of the MSAs after noradrenaline (NA) and acetylcholine (ACh) exposure were detected by a microscope, and changes in blood perfusion through the MSAs were measured by full-field laser perfusion imaging. HS enhanced the constrictive response of the MSAs to NA and attenuated the relaxing response to ACh. Low sodium intake reduced the response of the MSAs to NA and promoted ACh-induced vasodilatation. HS also aggravated NA-induced blood perfusion reduction and impaired ACh-induced hyperperfusion of the MSAs. Pathologically, HS was associated with arteriolar structural damage and fibrosis of the MSAs. We conclude that sodium intake affects the responsiveness of the MSAs to vasoactive agents in DS rats and might play important roles in modulating blood pressure in hypertensive individuals.

The effects of cigarette smoking and smoking cessation on high-density lipoprotein functions: implications for coronary artery disease.

BACKGROUND: Smoking cessation was associated with improved prognosis of coronary artery disease. This study was designed to investigate the effect of smoking cessation on high-density lipoprotein functionality in coronary artery disease patients. METHODS: In this prospective, randomized and parallel controlled study, coronary artery disease smokers ( n = 28) and healthy smokers ( n = 30) were divided into smoking cessation group and continuous smoking group, respectively. Blood samples were collected before and after three-month smoking cessation. Plasma high-density lipoprotein was isolated by density gradient centrifugation. The ability of high-density lipoprotein against copper-induced oxidation of lipoprotein was determined to evaluate the antioxidative property of high-density lipoprotein, and the macrophage migration inhibited by high-density lipoprotein was tested to identify the antichemotactic property of high-density lipoprotein. High-density lipoprotein-induced macrophage cholesterol efflux was measured by fluorescence spectrometry using NBD cholesterol analogue. Healthy non-smoking volunteers were enrolled as the baseline control. RESULTS: The baseline antioxidative, antichemotactic ability of high-density lipoprotein and high-density lipoprotein-induced cellular cholesterol efflux in coronary artery disease smokers and healthy smokers were significantly attenuated when compared with those in healthy non-smokers. After three-month smoking cessation, both the antioxidative ability and antichemotactic ability of high-density lipoprotein were improved significantly in coronary artery disease smokers. However, high-density lipoprotein-induced cellular cholesterol efflux was not increased by smoking cessation. In in vitro experiments, carbon monoxide reduced the antioxidative ability and nicotine enhanced the antichemotactic ability of high-density lipoprotein. CONCLUSIONS: Smoking cessation is an effective measure to improve high-density lipoprotein functions in coronary artery disease smokers. Our study re-emphasizes the importance of smoking cessation in the secondary prevention of coronary artery disease.

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.

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 AND 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). CONCLUSIONS 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.

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.

Effect of Right Heart Systolic Function on Outcomes in Patients with Constrictive Pericarditis Undergoing Pericardiectomy.

BACKGROUND: To determine the influence of right ventricular function in patients with constrictive pericarditis (CP) undergoing surgery and to compare the outcomes of patients who received surgery with those managed medically. METHODS: Patients with the diagnosis of CP and healthy volunteers were recruited from January 2006 to November 2011. Patients with CP chose to either receive pericardiectomy or medical management. Echocardiographic measurements were performed to evaluate heart function, and survival was recorded. RESULTS: A total of 58 patients with CP (36 received pericardiectomy, 22 managed medically), and 43 healthy volunteers were included. CP patients who received surgery had a higher survival rate than those managed medically (P = 0.003), and higher survival was also seen in the subgroup of CP patients with severely impaired right systolic function. Albumin level, left ventricular end-diastolic dimension, and tricuspid regurgitation velocity were associated with survival in CP patients who received surgery. CONCLUSIONS: Preoperative right heart function does not affect surgical outcomes. Patients with severely impaired preoperative right systolic function obtain a greater survival advantage with surgery than with medical treatment.

[Growth Differentiation Factor 15 Inhibits Elevated Expression of Matrix Metalloproteinase 1 Induced by Pooled Serum in Patients with Coroanry Artery Ectasia].

OBJECTIVE: To observe the effects of coroanry artery ectasia (CAE) patients' pooled serum on the main proteinases and extracellular matrix (ECM) synthesis and explore whether the growth differentiation factor 15(GDF 15) can regulate the characteristic changes induced by CAE patients' pooled serum. METHODS: Serum samples were collected from 32 CAE patients, 30 patients with coronary heart disease (CHD), and 31 subjects with normal coronary arteries (CON) and then mixed in the same volumes by groups. Then human umbilical vein smooth muscle cells were cultured with the media containing 25% pooled serum. After having been disposed, proteinase system and ECM synthesis system were detected in the cell and culture media samples. GDF15 or GDF15 antibodies was added into the 25% pooled serum in each group to observe if GDF 15 could impact the characteristic changes induced by CAE patients' pooled serum. RESULTS: The expression of matrix metalloproteinases (MMP) 1 mRNA in CAE group was significantly higher than CON group (P=0.002) and CHD group (P=0.000), the secretory MMP1 protein and total MMPs activity in culture media were also upregulated in CAE group (both P<0.01). After adding GDF 15 into the culture media (GDF15+CAE group), the MMP1 mRNA ,secretory MMP1 protein, and total MMPs activity were significantly lower than CAE group (all P<0.01), while in the GDF15 antibody+CAE group, the MMP1 mRNA and total MMPs activities were significantly higher than in GDF15+CAE group (both P<0.01), but the secretory MMP1 protein was not different from GDF 15+CAE group (P>0.05). CONCLUSION: The vascular smooth muscle cells may participate in the CAE process mainly by regulating MMPs system but not the elastase 2 or ECM synthesis system, and GDF15 may be an compensatory factor to prohibit the over-destruction of coronary ECM induced by MMPs.

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 implication of cigarette smoking and cessation on macrophage cholesterol efflux in coronary artery disease patients.

We investigated ATP-binding cassette transporters A1/G1 expression and function in mediating cholesterol efflux by examining the macrophages of cigarette-smoking patients with coronary artery disease (CAD) before and after smoking abstinence. Peripheral blood monocyte cells were collected from nonsmokers (n = 17), non-CAD (NCAD) smokers (n = 35), and CAD smokers (n = 32) before and after 3 months of smoking cessation. We found that the ABCA1 expression level was lower in macrophages from NCAD and CAD smokers than from nonsmokers at baseline. The ABCA1 function of mediating cholesterol efflux was reduced in NCAD and CAD smokers as compared with nonsmokers. After 3 months of smoking cessation, ABCA1 expression and function were improved in CAD smokers. However, ABCG1 expression and function did not change after smoking cessation. Furthermore, ABCA1 expression was inhibited by tar in human acute monocytic leukemia cell line THP-1-derived macrophages through the inhibition of liver X receptors. Nicotine and carbon monoxide did not inhibit ABCA1 expression. Our results indicate that chronic cigarette smoking impaired ABCA1-mediated cholesterol efflux in macrophages and that tobacco abstinence reversed the function and expression of ABCA1, especially in CAD patients. It was tobacco tar, rather than nicotine or carbon monoxide, that played a major role in the tobacco-induced disturbance of cellular cholesterol homeostasis.

[Effects of Rapamycin and Rapamycin-loaded Poly(lactic-co-glycolic)Acid Nanoparticles on Apoptosis and Expression of bcl-2 and p27(kip1) Proteins of Human Umbilical Arterial Vascular Smooth Muscle Cell].

OBJECTIVE: To investgate the effects of rapamycin(RPM)and RPM-loaded poly(lactic-co-glycolic)acid(PLGA)nanoparticles(NPs)on the apoptosis of human umbilical arterial vascular smooth muscle cells(HUASMCs)in vitro and expression of bcl-2 and p27(kip1) protein. METHODS: HUASMCs were cultured in vitro and divided to RPM and RPM-PLGA-NPs groups treated at 3 different concentration by 12 and 24 hours,with M231-smooth muscle growth supplements medium and null-PLGA-NPs treated groups as controlled. The apoptosis of HUASMCs was determined by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling staining and flow cytometry. The expressions of bcl-2 and p27(kip1) were detected by streptacidin/peroxidase immunohistochemical method. The effect on cellular proliferation was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromidecolorimetry. RESULTS: The proliferation of HUASMCs was inhibited by RPM and RPM-PLGA-NPs in a dose-dependent manner. DNA electrophoresis showed DNA ladder in RPM and RPM-PLGA-NPs groups and classical scalar strips in control groups. The apoptotic indexes of RPM 100 ng/ml group and RPM-PLGA-NPs 500 ng/ml group detected by flow cytometry were(45.45<2.36)% and(35.04<5.64)%,respectively,which were significantly higher than that of M231-smooth muscle growth supplements control group [(2.60<0.95)%,all P<0.01]. The apoptotic indexes of groups incubated with RPM and RPM-PLGA-NPs for 24 hours were significantly higher than those of groups which incubated for 12 hours(P<0.05,P<0.01). The positive expression indexes(PEI)of p27(kip1) and bcl-2 protein were higher in RPM and RPM-PLGA-NPs groups than that of control groups. The Spearman's rank correlation coefficient test showed that there was no significant correlation between the PEI of p27(kip1) and the apoptotic indexes in the RPM group and RPM-PLGA-NPs group(P>0.05). CONCLUSIONS: Rapamycin-loaded PLGA nanoparticles and rapamycin have similar effects in inhibiting proliferation and inducing apoptosis;meanwhile,they upregulate the expression of p27(kip1) protein without downregulating the expression of bcl-2 protein in HUASMCs in vitro. RPM-PLGA-NPs has more potent pro-apoptotic effect than equivalent dose of RPM but is not linearly correlated with the p27(kip1) expression level.

Attenuated macrophage cholesterol efflux function in patients with obstructive sleep apnea-hypopnea syndrome.

BACKGROUND: Obstructive sleep apnea-hypopnea syndrome (OSAHS) is associated with premature atherosclerosis. However, the associated mechanism remains unknown. This study investigates the expression of adenosine triphosphate (ATP)-binding cassette transporter protein A1 (ABCA1) and cellular cholesterol efflux in cultured macrophages from OSAHS patients. METHODS: Of the 18 subjects enrolled in this study, six subjects with apnea-hypopnea index (AHI) <5 were placed into the control group, and 12 subjects with AHI ≥5 were placed into the OSAHS group. Peripheral blood mononuclear cells (PBMCs) from each subject were isolated, purified, cultured, and differentiated into macrophages in vitro. ABCA1 mRNA and protein expression were evaluated by reverse transcription PCR and Western Blot, respectively. Both ABCA1-mediated and autologous serum induced cholesterol efflux were measured by isotopic cholesterol efflux assays. RESULTS: The levels of AHI and high sensitivity C-reactive protein (hsCRP) were significantly higher in the OSAHS group than in the control group. ABCA1 mRNA and protein expressions in PBMCs-derived macrophages were significantly reduced in patients with OSAHS compared to that in controls (p < 0.05). Both ABCA1-mediated and autologous serum-induced cholesterol efflux were significantly lower in the OSAHS group than that in the control group (p = 0.033 and p = 0.01, respectively). Pearson's correlation analysis revealed a negative correlation between AHI and the mRNA (r = -0.7726, p = 0.0007) and protein (r = -0.8112, p = 0.0044) expression of ABCA1, a positive correlation between ABCA1-mediated cholesterol efflux and the minimum oxygen saturation (r = 0.7954, p < 0.0001), and a negative correlation between AHI and autologous serum induced cholesterol efflux (r = -0.7756, p = 0.0002). CONCLUSION: ABCA1 expression and cellular cholesterol efflux in macrophages were significantly decreased in OSAHS patients, which closely correlated with the severity of disease. Our findings provide meaningful insights into the mechanism of atherogenesis in OSAHS patients.

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.

Enhanced differentiation potential of human amniotic mesenchymal stromal cells by using three-dimensional culturing.

The therapeutic potential of human amniotic mesenchymal stromal cells (hAMSCs) remains limited because of their differentiation towards mesenchymal stem cells (MSCs) following adherence. The aim of this study was to develop a three-dimensional (3-D) culture system that would permit hAMSCs to differentiate into cardiomyocyte-like cells. hAMSCs were isolated from human amnions of full-term births collected after Cesarean section. Immunocytochemistry, immunofluorescence and flow cytometry analyses were undertaken to examine hAMSC marker expression for differentiation status after adherence. Membrane currents were determined by patch clamp analysis of hAMSCs grown with or without cardiac lysates. Freshly isolated hAMSCs were positive for human embryonic stem-cell-related markers but their marker profile significantly shifted towards that of MSCs following adherence. hAMSCs cultured in the 3-D culture system in the presence of cardiac lysate expressed cardiomyocyte-specific markers, in contrast to those maintained in standard adherent cultures or those in 3-D cultures without cardiac lysate. hAMSCs cultured in 3-D with cardiac lysate displayed a cardiomyocyte-like phenotype as observed by membrane currents, including a calcium-activated potassium current, a delayed rectifier potassium current and a Ca(2+)-resistant transient outward K(+) current. Thus, although adherence limits the potential of hAMSCs to differentiate into cardiomyocyte-like cells, the 3-D culture of hAMSCs represents a more effective method of their culture for use in regenerative medicine.

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

The possible pharmacological mechanism by which partial PPARγ-activating angiotensin II (Ang II) type 1 receptor blocker (ARB) telmisartan and non-PPARγ-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 PPARγ-activating ARB telmisartan, angiotensin II type 2 (AT2) receptor blocker PD123319, full PPARγ agonist pioglitazone, and PPARγ 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 PPARγ 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 PPARγ-dependent pathway.

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.

A polymorphism in the ABCG1 promoter is functionally associated with coronary artery disease in a Chinese Han population.

OBJECTIVE: In this study, we examine the association of single nucleotide polymorphisms (SNPs) of the human ATP binding cassette transporter G1 (ABCG1) gene with atherosclerotic coronary artery disease (CAD) in a Chinese Han population. METHODS: 1021 patients with CAD and 1013 unaffected control subjects were enrolled. PCR-based ligation detection reaction (PCR-LDR) method was used to genotype four SNPs of ABCG1, three (rs2234714, rs2234715 and rs57137919) in the promoter region and one (rs1044317) in the 3'-untranslated region (UTR). RESULTS: The human ABCG1 -367G>A polymorphism (rs57137919) showed a significantly decreased risk for CAD and myocardial infarction (MI) in a dominant model (adjusted OR = 0.73, p = 0.033 for CAD, and adjusted OR = 0.65, p = 0.014 for MI, respectively). The rs57137919 also showed an association with angiographic severity of CAD (multi-vessel vs. single-vessel CAD, adjusted OR = 0.40, p = 0.005). The findings were further supported by luciferase reporter assay, in which the polymorphism impaired reporter gene expression. The ABCG1 -768G>A polymorphism (rs2234714) showed an association with CAD in a recessive model (adjusted OR = 0.64, p = 0.015), but did not demonstrate a functional influence on reporter gene expression in the luciferase reporter assay. CONCLUSIONS: The SNP rs57137919 in the ABCG1 promoter region is functionally associated with a reduced risk of CAD in a Chinese Han population.