Proteomic and functional analysis of HDL subclasses in humans and rats: a proof-of-concept study.

BACKGROUND: The previous study investigated whether the functions of small, medium, and large high density lipoprotein (S/M/L-HDL) are correlated with protein changes in mice. Herein, the proteomic and functional analyses of high density lipoprotein (HDL) subclasses were performed in humans and rats. METHODS: After purifying S/M/L-HDL subclasses from healthy humans (n = 6) and rats (n = 3) using fast protein liquid chromatography (FPLC) with calcium silica hydrate (CSH) resin, the proteomic analysis by mass spectrometry was conducted, as well as the capacities of cholesterol efflux and antioxidation was measured. RESULTS: Of the 120 and 106 HDL proteins identified, 85 and 68 proteins were significantly changed in concentration among the S/M/L-HDL subclasses in humans and rats, respectively. Interestingly, it was found that the relatively abundant proteins in the small HDL (S-HDL) and large HDL (L-HDL) subclasses did not overlap, both in humans and in rats. Next, by searching for the biological functions of the relatively abundant proteins in the HDL subclasses via Gene Ontology, it was displayed that the relatively abundant proteins involved in lipid metabolism and antioxidation were enriched more in the medium HDL (M-HDL) subclass than in the S/L-HDL subclasses in humans, whereas in rats, the relatively abundant proteins associated with lipid metabolism and anti-oxidation were enriched in M/L-HDL and S/M-HDL, respectively. Finally, it was confirmed that M-HDL and L-HDL had the highest cholesterol efflux capacity among the three HDL subclasses in humans and rats, respectively; moreover, M-HDL exhibited higher antioxidative capacity than S-HDL in both humans and rats. CONCLUSIONS: The S-HDL and L-HDL subclasses are likely to have different proteomic components during HDL maturation, and results from the proteomics-based comparison of the HDL subclasses may explain the associated differences in function.

Insulin Rescued MCP-1-Suppressed Cholesterol Efflux to Large HDL2 Particles via ABCA1, ABCG1, SR-BI and PI3K/Akt Activation in Adipocytes.

PURPOSE: Intracellular cholesterol imbalance plays an important role in adipocyte dysfunction of obesity. However, it is unclear whether obesity induced monocyte chemoattractant protein-1 (MCP-1) causes the adipocyte cholesterol imbalance. In this study, we hypothesize that MCP-1 impairs cholesterol efflux of adipocytes to HDL2 and insulin rescues this process. METHODS: We recruited coronary artery disease (CAD) patients with obesity and overweight to analyze the association between MCP-1 and HDL2-C by Pearson correlation coefficients. We performed [3H]-cholesterol efflux assay to demonstrate the effect of MCP-1 and insulin on cholesterol efflux from 3T3-L1 adipocytes to large HDL2 particles. Western blot, RT-qPCR, cell-surface protein assay, and confocal microscopy were performed to determine the regulatory mechanism. RESULTS: Plasma MCP-1 concentrations were negatively correlated with HDL2-C in CAD patients with obesity and overweight (r = -0.60, p < 0.001). In differentiated 3T3-L1 adipocytes, MCP-1 reduced cholesterol efflux to large HDL2 particles by 55.4% via decreasing ATP-binding cassette A1 (ABCA1), ABCG1, and scavenger receptor class B type I (SR-BI) expression. Intriguingly, insulin rescued MCP-1 mediated-inhibition of cholesterol efflux to HDL2 in an Akt phosphorylation-dependent manner. The rescue efficacy of insulin was 138.2% for HDL2. Moreover, insulin increased mRNA and protein expression of ABCA1, ABCG1, and SR-BI at both transcriptional and translational levels via the PI3K/Akt activation. CONCLUSIONS: These findings indicate that MCP-1 impairs cholesterol efflux to large HDL2 particles in adipocytes, which is reversed by insulin via the upregulation of ABCA1, ABCG1, and SR-BI. Therefore, insulin might improve cholesterol imbalance by an anti-inflammatory effect in adipocytes. CLINICAL TRIAL REGISTRATION NUMBER: ChiCTR2000033297; Date of registration: 2020/05/ 27; Retrospectively registered.

CC-Chemokine Ligand 2 (CCL2) Suppresses High Density Lipoprotein (HDL) Internalization and Cholesterol Efflux via CC-Chemokine Receptor 2 (CCR2) Induction and p42/44 Mitogen-activated Protein Kinase (MAPK) Activation in Human Endothelial Cells.

High density lipoprotein (HDL) has been proposed to be internalized and to promote reverse cholesterol transport in endothelial cells (ECs). However, the mechanism underlying these processes has not been studied. In this study, we aim to characterize HDL internalization and cholesterol efflux in ECs and regulatory mechanisms. We found mature HDL particles were reduced in patients with coronary artery disease (CAD), which was associated with an increase in CC-chemokine ligand 2 (CCL2). In cultured primary human coronary artery endothelial cells and human umbilical vein endothelial cells, we determined that CCL2 suppressed the binding (4 °C) and association (37 °C) of HDL to/with ECs and HDL cellular internalization. Furthermore, CCL2 inhibited [(3)H]cholesterol efflux to HDL/apoA1 in ECs. We further found that CCL2 induced CC-chemokine receptor 2 (CCR2) expression and siRNA-CCR2 reversed CCL2 suppression on HDL binding, association, internalization, and on cholesterol efflux in ECs. Moreover, CCL2 induced p42/44 mitogen-activated protein kinase (MAPK) phosphorylation via CCR2, and p42/44 MAPK inhibition reversed the suppression of CCL2 on HDL metabolism in ECs. Our study suggests that CCL2 was elevated in CAD patients. CCL2 suppressed HDL internalization and cholesterol efflux via CCR2 induction and p42/44 MAPK activation in ECs. CCL2 induction may contribute to impair HDL function and form atherosclerosis in CAD.

MCP-1 impacts RCT by repressing ABCA1, ABCG1, and SR-BI through PI3K/Akt posttranslational regulation in HepG2 cells.

Monocyte chemoattractant protein-1 (MCP-1) plays crucial roles at multiple stages of atherosclerosis. We hypothesized that MCP-1 might impair the reverse cholesterol transport (RCT) capacity of HepG2 cells by decreasing the cell-surface protein expression of ATP binding cassette A1 (ABCA1), ATP binding cassette G1 (ABCG1), and scavenger receptor class B type I (SR-BI). MCP-1 reduced the total protein and mRNA levels of ABCA1 and SR-BI, but not of ABCG1. MCP-1 decreased the cell-surface protein expression of ABCA1, ABCG1, and SR-BI in dose-dependent and time-dependent manners, as measured using cell-surface biotinylation. We further studied the phosphoinositide 3-kinase (PI3K)/serine/threonine protein kinase Akt pathway in regulating receptor trafficking. Both the translation and transcription of ABCA1, ABCG1, and SR-BI were not found to be regulated by the PI3K/Akt pathway. However, the cell-surface protein expression of ABCA1, ABCG1, and SR-BI could be regulated by PI3K activity, and PI3K activation corrected the MCP-1-induced decreases in the cell-surface protein expression of ABCA1, ABCG1, and SR-BI. Moreover, we found that MCP-1 decreased the lipid uptake by HepG2 cells and the ABCA1-mediated cholesterol efflux to apoA-I, which could be reversed by PI3K activation. Our data suggest that MCP-1 impairs RCT activity in HepG2 cells by a PI3K/Akt-mediated posttranslational regulation of ABCA1, ABCG1, and SR-BI cell-surface expression.

Mental health is correlated with lipoprotein(a) levels in male patients with premature coronary heart disease.

BACKGROUND: High levels of lipoprotein(a) (Lp(a)) is an independent risk factor for premature coronary heart disease (PCHD). It is also considered a residual risk for controlled low density lipoprotein cholesterol (LDL-C). Dietary control, exercise, and drugs have limited effects on the levels of Lp(a). Recently, mental health was found to be associated with lipid levels and increased risk of PCHD. However, the relationship between mental health and Lp(a) is still unknown. This study explored the association between mental health and Lp(a) levels in men with PCHD. METHODS: A retrospective, observational study was conducted. A total of 226 male patients with PCHD, aged 49.65±3.68 years, was included in this study. The control group consisted of 230 age-matched healthy male volunteers. Serum Lp(a) levels ≥30 mg/dL, as measured by the immunoturbidimetry method, were considered high. All participants received health related quality of life (HRQoL) scores using the self-assessed 36-Item Short Form Health Survey (SF-36). The HRQoL includes both a physical component summary (PCS) and a mental component summary (MCS). RESULTS: Patients with PCHD were found to have higher levels of Lp(a) (51.61±33.39 vs. 26.42±21.93, P<0.001), and lower MCS (35.83±4.21 vs. 39.85±4.12) and PCS scores (38.02±3.73 vs. 39.63±3.21) compared to healthy volunteers. The MCS score was negatively correlated with Lp(a) levels in the PCHD group (R=-0.295, P<0.001), but no correlation was detected in the control group. There was no relationship between the PCS score and Lp(a) levels in neither the PCHD group nor the healthy control group. Multivariate logistic regression analysis indicated that the MCS and PCS scores were negatively correlated with the risk of PCHD. CONCLUSIONS: These findings suggested that poor mental health may be associated with high levels of Lp(a) and increased risk of PCHD in men. Therefore, improving the mental state in men with PCHD may be crucial.

Efficacy and Safety of High-Density Lipoprotein/Apolipoprotein A1 Replacement Therapy in Humans and Mice With Atherosclerosis: A Systematic Review and Meta-Analysis.

Background: Although elevation of HDL-C levels by pharmaceutical drugs have no benefit of cardiovascular endpoint, the effect of high-density lipoprotein/apolipoprotein A1 (HDL/apoA-1) replacement therapy on atherosclerosis is controversial. The current meta-analysis analyzed the effects of HDL/apoA-1 replacement therapies on atherosclerotic lesions both in humans and mice. Methods: The PubMed, Cochrane Library, Web of Science, and EMBASE databases were searched through June 6, 2020. The methodological quality of the human studies was assessed using Review Manager (RevMan, version 5.3.). The methodological quality of the mouse studies was assessed using a stair list. STATA (version 14.0) was used to perform all statistical analyses. Results: Fifteen randomized controlled human trials and 17 animal studies were included. The pooled results showed that HDL/apoA-1 replacement therapy use did not significantly decrease the percent atheroma volume (p = 0.766) or total atheroma volume (p = 0.510) in acute coronary syndrome (ACS) patients (N = 754). However, HDL/apoA-1 replacement therapies were significantly associated with the final percent lesion area, final lesion area, and changes in lesion area (SMD, -1.75; 95% CI: -2.21~-1.29, p = 0.000; SMD, -0.78; 95% CI: -1.18~-0.38, p = 0.000; SMD: -2.06; 95% CI, -3.92~-0.2, p = 0.03, respectively) in mice. Conclusions: HDL/apoA-1 replacement therapies are safe but do not significantly improve arterial atheroma volume in humans. The results in animals suggest that HDL/apoA-1 replacement therapies decrease the lesion area. Additional studies are needed to investigate and explain the differences in HDL/apoA-1 replacement therapy efficacies between humans and animals. Trial registration number: Human pooled analysis: PROSPERO, CRD42020210772. prospectively registered.

Large HDL2 combined with inflammatory factors as superior predictors for coronary artery disease than small HDL3.

BACKGROUND: This study investigated whether combinations of high-density lipoprotein (HDL) subfractions and inflammatory markers would add value to coronary artery disease (CAD) prediction. METHODS: Non-CAD subjects (n=245) were stratified into low/moderate/high-Framingham risk (L/M/H-FR) groups and 180 CAD patients were enrolled. Levels of HDL-C, HDL2, HDL3, monocyte chemoattractant protein-1 (MCP-1), and high-sensitivity C-reactive protein (hsCRP) were measured. Multivariable logistic models for CAD were estimated with a single parameter or all parameters together after adjustment for conventional risk factors (CRFs), and Z statistics, net reclassification improvement (NRI), and integrated discrimination improvement (IDI) were used to compare discrimination among different models. RESULTS: The results show that HDL-C, HDL2, and HDL3 gradually decreased, while MCP-1 and hsCRP gradually increased from L/M/H-FR to the CAD group. When applying a single factor in the CRFs-adjusted models, HDL-C (OR 0.011, 95% CI, 0.002-0.071, P<0.05) and HDL2 (OR 0.000072, 95% CI, 0.000001-0.004, P<0.05), but not HDL3, were significantly related to CAD risk. Only HDL2 (OR 0.000072, 95% CI, 0.000001-0.004, P<0.001) remained significant when applying all HDL parameters. In the model including all HDL and inflammatory parameters, HDL2 (OR 0.001, 95% CI, 0.000027-0.051), MCP-1 (OR 1.066, 95% CI, 1.039-1.094), and hsCRP (OR 1.130, 95% CI, 1.041-1.227) showed significant differences (all P<0.05). This combined model showed improved discrimination over the models with a single factor (P<0.05) or all HDL parameters (Z=3.299, NRI =0.179, IDI =0.081, P<0.001). CONCLUSIONS: Large HDL2 is superior to small HDL3 in the inverse association with CAD. The combination of HDL2, MCP-1, and hsCRP with CRFs provides an optimal prediction for CAD.

Association of renal cyst and type A acute aortic dissection with hypertension.

BACKGROUND: Type A acute aortic dissection (TA-AAD) has high mortality, with 50% of patients dying before hospital admission. Hypertension is the most common comorbidity for acute aortic dissection, and effective antihypertensive therapy is still unable to predict the risk of aortic rupture at the medium- and long-term stages. While the presence of renal cyst has been found to increases the risk of thoracic aortic disease, the correlation between renal cyst and TA-AAD with hypertension remains poorly understood. Thus, this study aimed to determine the relationship of renal cyst and TA-AAD with hypertension. METHODS: A retrospective analysis was performed in 464 hypertension patients from August 2014 to August 2019. A total of 230 TA-AAD patients were enrolled in the AD with hypertension group (age 53.79±11.31 years, male 90.87%), and matched by age, sex, and hypertension control to 234 patients without TA-AAD who were enrolled in the non-AD with hypertension group. Patients were divided into three subgroups according to the numbers of renal cysts: no renal cyst, single renal cyst, and multiple renal cysts. RESULTS: In this study, the AD with hypertension group had significantly more single renal cyst and multiple renal cyst cases than did the non-AD with hypertension group. The mean age of the multiple renal cyst subgroup was significantly older than that of the single renal cyst subgroup (57.25±13.00 vs. 51.57±10.75 years) in the AD with hypertension group. There was significantly different distribution of dissection starting points and dissection ending points across three renal cyst subgroups. Multivariate logistic regression analysis indicated that having no renal cyst significantly decreased the risk of TA-AAD in middle-aged and elderly patents, but showed no correlations with those of younger ages. Single renal cyst status also significantly decreased the risk of TA-AAD in elderly patients [odds ratio (OR) =0.129, 95% confidence interval (CI): 0.029-0.575, P=0.007]. CONCLUSIONS: Renal cyst status correlates with the risk of TA-AAD with hypertension in middle-aged and elderly patients, and exhibits different degrees of vascular lesion in aortic dissection. We therefore suggest that different antihypertensive standards should be adopted in different renal cyst status to more effectively prevent aortic dissection.

Comprehensive Construction of a Circular RNA-Associated Competing Endogenous RNA Network Identified Novel Circular RNAs in Hypertrophic Cardiomyopathy by Integrated Analysis.

Hypertrophic cardiomyopathy (HCM), the most common heritable cardiomyopathy, is associated with a high risk of sudden cardiac death. The complexity and behavior of the circular RNA (circRNA)-associated competing endogenous RNA (ceRNA) network in HCM have not been thoroughly elucidated. Plasma circRNA and messenger RNA (mRNA) expression profiles were acquired by using a microarray. Weighted correlation network analysis (WGCNA) and linear models for microarray data (Limma) were used to analyze microarray data. Gene modules, consisting of genes with high correlations, were detected and represented by a designated color. The ceRNA network, including circRNA, microRNA (miRNA), and mRNA, was constructed based on the "ceRNA hypothesis" using an integrated systems biology method. By WGCNA, two modules, namely magenta and red modules, were identified as being positively correlated with HCM. In the combined analysis of WGCNA and Limma, 36 hub circRNAs in the magenta module and 83 hub circRNAs in the red module were significantly upregulated compared with the controls. By coexpression analysis, 270 circRNA-mRNA pairs were identified with a coefficient ≥0.9 and p < 0.05. With Starbase and miRWalk tools, circRNA-miRNA pairs and miRNA-mRNA pairs were predicted. Once these pairs were combined, the ceRNA network with 6 circRNAs, 29 miRNAs, and 6 mRNAs was constructed. Functional analysis demonstrated that these circRNAs in the ceRNA network were associated with calcium-release channel activity and muscle filament sliding. Our study provided a global perspective and systematic analysis of the circRNA-associated ceRNA network in HCM. The identified circRNAs hsa_circ_0043762, hsa_circ_0036248, and hsa_circ_0071269 may be key regulators involved in HCM pathogenesis.