Hepatic Cdkal1 deletion regulates HDL catabolism and promotes reverse cholesterol transport.

BACKGROUND AND AIMS: Associations between CDKAL1 variants and cholesterol efflux capacity (CEC) have been reported. This study aimed to investigate the effects of Cdkal1 deficiency on high-density lipoprotein (HDL) metabolism, atherosclerosis, and related pathways. METHODS: Lipid and glucose metabolic profiles, CEC, and in vivo reverse cholesterol transport (RCT) were compared in liver-specific Alb-Cre:Cdkal1fl/fl and Cdkal1fl/fl mice. Aortic atherosclerosis was compared in Apoe-/-Alb-Cre:Cdkal1fl/fl and Apoe-/- mice fed high-fat diets. HDL subclasses and mediators of HDL metabolism from Alb-Cre:Cdkal1fl/fl mice were examined. RESULTS: HDL-cholesterol level tended to be higher in the Alb-Cre:Cdkal1fl/fl mice (p = 0.050). Glucose and other lipid profiles were similar in the two groups of mice, irrespective of diet. The mean CEC was 27% higher (p = 0.007) in the Alb-Cre:Cdkal1fl/fl mice, as were the radioactivities of bile acids (mean difference 17%; p = 0.035) and cholesterol (mean difference 42%; p = 0.036) from faeces. The radioactivity tendency was largely similar in mice fed a high-fat diet. Atherosclerotic lesion area tended to be smaller in the Apoe-/-Alb-Cre:Cdkal1fl/fl mice than in the Apoe-/- mice (p = 0.067). Cholesterol concentrations in large HDLs were higher in the Alb-Cre:Cdkal1fl/fl mice (p = 0.024), whereas in small HDLs, they were lower (p = 0.024). Endothelial lipase (mean difference 39%; p = 0.002) and hepatic lipase expression levels (mean difference 34%; p < 0.001) were reduced in the Alb-Cre:Cdkal1fl/fl mice, whereas SR-B1 expression was elevated (mean difference 35%; p = 0.007). CONCLUSIONS: The promotion of CEC and RCT in Alb-Cre:Cdkal1fl/fl mice verified the effect of CDKAL1 seen in human genetic data. These phenotypes were related to regulation of HDL catabolism. This study suggests that CDKAL1 and associated molecules could be targets for improving RCT and vascular pathology.

Single-cell transcriptomics reveal cellular diversity of aortic valve and the immunomodulation by PPARγ during hyperlipidemia.

Valvular inflammation triggered by hyperlipidemia has been considered as an important initial process of aortic valve disease; however, cellular and molecular evidence remains unclear. Here, we assess the relationship between plasma lipids and valvular inflammation, and identify association of low-density lipoprotein with increased valvular lipid and macrophage accumulation. Single-cell RNA sequencing analysis reveals the cellular heterogeneity of leukocytes, valvular interstitial cells, and valvular endothelial cells, and their phenotypic changes during hyperlipidemia leading to recruitment of monocyte-derived MHC-IIhi macrophages. Interestingly, we find activated PPARγ pathway in Cd36+ valvular endothelial cells increased in hyperlipidemic mice, and the conservation of PPARγ activation in non-calcified human aortic valves. While the PPARγ inhibition promotes inflammation, PPARγ activation using pioglitazone reduces valvular inflammation in hyperlipidemic mice. These results show that low-density lipoprotein is the main lipoprotein accumulated in the aortic valve during hyperlipidemia, leading to early-stage aortic valve disease, and PPARγ activation protects the aortic valve against inflammation.

LncRNA HSPA7 in human atherosclerotic plaques sponges miR-223 and promotes the proinflammatory vascular smooth muscle cell transition.

Although there are many genetic loci in noncoding regions associated with vascular disease, studies on long noncoding RNAs (lncRNAs) discovered from human plaques that affect atherosclerosis have been highly limited. We aimed to identify and functionally validate a lncRNA using human atherosclerotic plaques. Human aortic samples were obtained from patients who underwent aortic surgery, and tissues were classified according to atherosclerotic plaques. RNA was extracted and analyzed for differentially expressed lncRNAs in plaques. Human aortic smooth muscle cells (HASMCs) were stimulated with oxidized low-density lipoprotein (oxLDL) to evaluate the effect of the identified lncRNA on the inflammatory transition of the cells. Among 380 RNAs differentially expressed between the plaque and control tissues, lncRNA HSPA7 was selected and confirmed to show upregulated expression upon oxLDL treatment. HSPA7 knockdown inhibited the migration of HASMCs and the secretion and expression of IL-1ß and IL-6; however, HSPA7 knockdown recovered the oxLDL-induced reduction in the expression of contractile markers. Although miR-223 inhibition promoted the activity of Nf-κB and the secretion of inflammatory proteins such as IL-1ß and IL-6, HSPA7 knockdown diminished these effects. The effects of miR-223 inhibition and HSPA7 knockdown were also found in THP-1 cell-derived macrophages. The impact of HSPA7 on miR-223 was mediated in an AGO2-dependent manner. HSPA7 is differentially increased in human atheroma and promotes the inflammatory transition of vascular smooth muscle cells by sponging miR-223. For the first time, this study elucidated the molecular mechanism of action of HSPA7, a lncRNA of previously unknown function, in humans.

Erratum to: Common and differential effects of docosahexaenoic acid and eicosapentaenoic acid on helper T-cell responses and associated pathways.

[Erratum to: BMB Reports 2021; 54(5): 278-283, PMID: 33972011] In the originally published version of this article, there was an error in the Supplementary information. Fig. 1 as following image was missing in the Supplementary Information. The Supplementary file in the original version has now been updated to include the corrected. We apologize for any inconvenience that this may have caused.

Common and differential effects of docosahexaenoic acid and eicosapentaenoic acid on helper T-cell responses and associated pathways.

Our understanding of the differential effects between specific omega-3 fatty acids is incomplete. Here, we aimed to evaluate the effects of docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) on T-helper type 1 (Th1) cell responses and identify the pathways associated with these responses. Naïve CD4+ T cells were co-cultured with bone marrow-derived dendritic cells (DCs) in the presence or absence of palmitate (PA), DHA, or EPA. DHA or EPA treatment lowered the number of differentiated IFN-γ-positive cells and inhibited the secretion of IFN-γ, whereas only DHA increased IL-2 and reduced TNF-α secretion. There was reduced expression of MHC II on DCs after DHA or EPA treatment. In the DC-independent model, DHA and EPA reduced Th1 cell differentiation and lowered the cell number. DHA and EPA markedly inhibited IFN-γ secretion, while only EPA reduced TNF-α secretion. Microarray analysis identified pathways involved in inflammation, immunity, metabolism, and cell proliferation. Moreover, DHA and EPA inhibited Th1 cells through the regulation of diverse pathways and genes, including Igf1 and Cpt1a. Our results showed that DHA and EPA had largely comparable inhibitory effects on Th1 cell differentiation. However, each of the fatty acids also had distinct effects on specific cytokine secretion, particularly according to the presence of DCs. [BMB Reports 2021; 54(5): 278-283].

Cholesterol Efflux and Collateral Circulation in Chronic Total Coronary Occlusion: Effect-Circ Study.

Background The mechanism through which high-density lipoprotein (HDL) induces cardioprotection is not completely understood. We evaluated the correlation between cholesterol efflux capacity (CEC), a functional parameter of HDL, and coronary collateral circulation (CCC). We additionally investigated whether A1BP (apoA1-binding protein) concentration correlates with CEC and CCC. Methods and Results In this case-control study, clinical and angiographic data were collected from 226 patients (mean age, 58 years; male, 72%) with chronic total coronary occlusion. CEC was assessed using a radioisotope and J774 cells, and human A1BP concentration was measured using enzyme-linked immunosorbent assay. Differences between the good and poor CCC groups were compared, and associations between CEC, A1BP, and other variables were evaluated. Predictors of CCC were identified by multivariable logistic regression analysis. The CEC was higher in the good than in the poor CCC group (22.0±4.6% versus 20.2±4.7%; P=0.009). In multivariable analyses including age, sex, HDL-cholesterol levels, age (odds ratio [OR], 0.96; P=0.003), and CEC (OR, 1.10; P=0.004) were identified as the independent predictors of good CCC. These relationships remained significant after additional adjustment for diabetes mellitus, acute coronary syndrome, and Gensini score. The A1BP levels were not significantly correlated with CCC (300 pg/mL and 283 pg/mL in the good CCC and poor CCC groups, respectively, P=0.25) or CEC. Conclusions The relationship between higher CEC and good CCC indicates that well-functioning HDL may contribute to CCC and may be cardioprotective; this suggests that a specific function of HDL can have biological and clinical consequences.

Nlrp3, Csf3, and Edn1 in Macrophage Response to Saturated Fatty Acids and Modified Low-Density Lipoprotein.

BACKGROUND AND OBJECTIVES: The relationship between metabolic stress, inflammation, and cardiovascular disease is being studied steadily. The aim of this study was to evaluate the effect of palmitate (PA) and minimally modified low-density lipoprotein (mmLDL) on macrophages and to identify the associated pathways. METHODS: J774 macrophages were incubated with PA or mmLDL and lipopolysaccharide (LPS). Secretion of inflammatory chemokines and the expression of corresponding genes were determined. The phosphorylation of extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase was also assessed. RNA sequencing of macrophages was performed to identify the genes regulated by PA or mmLDL. Some of the genes regulated by the 2 agents were validated by knocking down the cells using small interfering RNA. RESULTS: PA or mmLDL promoted the secretion of interleukin (IL)-6 and IL-1ß in LPS-stimulated macrophages, and this was accompanied by higher phosphorylation of ERK. RNA sequencing revealed dozens of genes that were regulated in this process, such as Csf3 and Edn1, which were affected by PA and mmLDL, respectively. These agents also increased Nlrp3 expression. The effect of Csf3 or Edn1 silencing on inflammation was modest, whereas toll-like receptor (TLR) 4 inhibition reduced a large proportion of macrophage activation. CONCLUSIONS: We demonstrated that the proinflammatory milieu with high levels of PA or mmLDL promoted macrophage activation and the expression of associated genes such as Nlrp3, Csf3, and Edn1. Although the TLR4 pathway appeared to be most relevant, additional role of other genes in this process provided insights regarding the potential targets for intervention.

Novel Associations between Related Proteins and Cellular Effects of High-Density Lipoprotein.

BACKGROUND AND OBJECTIVES: Recent studies have examined the structure-function relationship of high-density lipoprotein (HDL). This study aimed to identify and rank HDL-associated proteins involved in several biological function of HDL. METHODS: HDLs isolated from 48 participants were analyzed. Cholesterol efflux capacity, effect of HDL on nitric oxide production, and vascular cell adhesion molecule-1 expression were assessed. The relative abundance of identified proteins in the highest vs. lowest quartile was expressed using the normalized spectral abundance factor ratio. RESULTS: After adjustment by multiple testing, six proteins, thyroxine-binding globulin, alpha-1B-glycoprotein, plasma serine protease inhibitor, vitronectin, angiotensinogen, and serum amyloid A-4, were more abundant (relative abundance ratio ≥2) in HDLs with the highest cholesterol efflux capacity. In contrast, three proteins, complement C4-A, alpha-2-macroglobulin, and immunoglobulin mu chain C region, were less abundant (relative abundance ratio <0.5). In terms of nitric oxide production and vascular cell adhesion molecule-1 expression, no proteins showed abundance ratios ≥2 or <0.5 after adjustment. Proteins correlated with the functional parameters of HDL belonged to diverse biological categories. CONCLUSIONS: In summary, this study ranked proteins showing higher or lower abundance in HDLs with high functional capacities and newly identified multiple proteins linked to cholesterol efflux capacity.

CETP, LIPC, and SCARB1 variants in individuals with extremely high high-density lipoprotein-cholesterol levels.

The concentration of high-density lipoprotein-cholesterol (HDL-C) in humans is partially determined by genetic factors; however, the role of these factors is incompletely understood. The aim of this study was to examine the prevalence and characteristics of CETP, LIPC, and SCARB1 variants in Korean individuals with extremely high HDL-C levels. We also analysed associations between these variants and cholesterol efflux capacity (CEC), reactive oxygen species (ROS) generation, and vascular cell adhesion molecule-1 (VCAM-1) expression. Of 13,545 participants in the cardiovascular genome cohort, 42 subjects with HDL-C levels >100 mg/dL were analysed. The three target genes were sequenced by targeted next-generation sequencing, the functional effects of detected variants were predicted, and CEC was assessed using a radioisotope and apolipoprotein B-depleted sera. We observed two rare variants of CETP in 13 individuals (rare variant c.A1196G [p.D399G] of CETP was discovered in 12 subjects) and one rare variant of SCARB1 in one individual. Furthermore, all subjects had at least one of four common variants (one CETP and three LIPC variants). Two additional novel CETP variants of unknown frequency were found in two subjects. However, the identified variants did not show significant associations with CEC, ROS generation, or VCAM-1 expression. Our study provides additional insights into the role of genetics in individuals with extremely high HDL-C.

Palmitate and minimally-modified low-density lipoprotein cooperatively promote inflammatory responses in macrophages.

Increased consumption of Western-type diets and environmental insults lead to wide-spread increases in the plasma levels of saturated fatty acids and lipoprotein oxidation. The aim of this study is to examine whether palmitate and minimally modified low-density lipoprotein (mmLDL) exert an additive effect on macrophage activation. We found that CXCL2 and TNF-α secretion as well as ERK and p38 phosphorylation were additively increased by co-treatment of J774 macrophages with palmitate and mmLDL in the presence of lipopolysaccharide (LPS). Furthermore, the analysis of differentially expressed genes using the KEGG database revealed that several pathways, including cytokine-cytokine receptor interaction, and genes were significantly altered. These results were validated with real-time PCR, showing upregulation of Il-6, Csf3, Il-1ß, and Clec4d. The present study demonstrated that palmitate and mmLDL additively potentiate the LPS-induced activation of macrophages. These results suggest the existence of synergistic mechanisms by which saturated fatty acids and oxidized lipoproteins activate immune cells.

Metabolic phenotyping of human atherosclerotic plaques: Metabolic alterations and their biological relevance in plaque-containing aorta.

BACKGROUND AND AIMS: Atherosclerosis is a chronic inflammatory disease characterized by thickening of the arterial wall. However, a limited number of studies have been conducted on metabolic profiling of human aortic tissue. METHODS: We applied liquid chromatography/mass spectrometry to perform global and targeted profiling of plaque-containing aortic tissue. The aorta samples included plaque-containing (n = 18) and control plaque-free (n = 24) aortic tissue from patients undergoing aortic surgery. RESULTS: The metabolic patterns of atherosclerotic and control vessels were significantly different. Metabolites in the purine and glutathione pathways showed dysregulation of oxidative stress in plaques, and levels of glucosylceramide, tryptophan, and kynurenine, which are related to inflammation, were also altered. Interestingly, an increased level of quinic acid was observed in plaques (p < 0.000), and we demonstrated an inhibitory effect of quinic acid on inflammatory activation and oxidative stress in macrophages. CONCLUSIONS: Our study provides insight into the disease mechanism and potential markers of atherosclerosis through comprehensive metabolic profiling of human aortic tissue samples containing plaque.

Effect of two lipid-lowering strategies on high-density lipoprotein function and some HDL-related proteins: a randomized clinical trial.

BACKGROUND: The influence of lipid-lowering therapy on high-density lipoprotein (HDL) is incompletely understood. We compared the effect of two lipid-lowering strategies on HDL functions and identified some HDL-related proteins. METHODS: Thirty two patients were initially screened and HDLs of 21 patients were finally analyzed. Patients were randomized to receive atorvastatin 20 mg (n = 11) or atorvastatin 5 mg/ezetimibe 10 mg combination (n = 10) for 8 weeks. The cholesterol efflux capacity and other anti-inflammatory functions were assessed based on HDLs of the participants before and after treatment. Pre-specified HDL proteins of the same HDL samples were measured. RESULTS: The post-treatment increase in cholesterol efflux capacities was similar between the groups (35.6% and 34.6% for mono-therapy and combination, respectively, p = 0.60). Changes in nitric oxide (NO) production, vascular cell adhesion molecule-1 (VCAM-1) expression, and reactive oxygen species (ROS) production were similar between the groups. The baseline cholesterol efflux capacity correlated positively with apolipoprotein (apo)A1 and C3, whereas apoA1 and apoC1 showed inverse associations with VCAM-1 expression. The changes in the cholesterol efflux capacity were positively correlated with multiple HDL proteins, especially apoA2. CONCLUSIONS: Two regimens increased the cholesterol efflux capacity of HDL comparably. Multiple HDL proteins, not limited to apoA1, showed a correlation with HDL functions. These results indicate that conventional lipid therapy may have additional effects on HDL functions with changes in HDL proteins. TRIAL REGISTRATION: ClinicalTrials.gov, number NCT02942602 .

PPARα agonists inhibit inflammatory activation of macrophages through upregulation of ß-defensin 1.

BACKGROUND: Effects of peroxisome proliferator-activated receptor alpha (PPARα) agonists on cardiovascular outcome have been controversial. Although these agents primarily affect lipoprotein metabolism, their pleiotropic anti-inflammatory effect is one of the potential anti-atherosclerotic mechanisms. This study aimed to evaluate the effect of fenofibrate and gemfibrozil on inflammation in macrophages and reveal pathways these agents may affect. METHODS AND RESULTS: The two PPARα agonists inhibited secretion of CXCL2, TNF-α, IL-6, activation of p65 of NF-κB, ERK, and TLR4 expression. These changes occurred simultaneously with upregulation and secretion of ß-defensin 1, an inflammation-modulating peptide. To demonstrate the role of ß-defensin 1, it was knocked-down by target-specific siRNA. The effects of PPARα agonists on TLR4 expression and chemokine secretion were obviously abrogated with this treatment. In experiments investigating whether ß-defensin 1 acts extracellularly, inflammatory chemokines decreased significantly after the addition of recombinant ß-defensin 1 or conditioned media to cells. In experiments designed to clarify if the effects of the two agents are PPARα-dependent, induction of mRNA and secretion ß-defensin 1 and inhibition of chemokine release were clearly reduced with GW6471, a PPARα blocker. CONCLUSIONS: Our results reveal the pathways by which fenofibrate and gemfibrozil inhibit LPS-induced inflammatory activation of macrophages. This study elucidated a novel anti-inflammatory mechanism that acts through PPARα, ß-defensin 1, and TLR4 pathways.