Impaired Cholesterol Uptake Capacity in Patients with Hypertriglyceridemia and Diabetes Mellitus.

BACKGROUND: Although low high-density lipoprotein cholesterol (HDL-C) levels are a common metabolic abnormality associated with insulin resistance, their role in cardiovascular risk stratification remains controversial. Recently, we developed a simple, high-throughput, cell-free assay system to evaluate the "cholesterol uptake capacity (CUC)" as a novel concept for HDL functionality. In this study, we assessed the CUC in patients with hypertriglyceridemia and diabetes mellitus. METHODS: The CUC was measured using cryopreserved serum samples from 285 patients who underwent coronary angiography or percutaneous coronary intervention between December 2014 and May 2019 at Kobe University Hospital. RESULTS: The CUC was significantly lower in diabetic patients (n = 125) than in nondiabetic patients (93.0 vs 100.7 arbitrary units (A.U.), P = 0.002). Patients with serum triglyceride (TG) levels >150 mg/dL (n = 94) also had a significantly lower CUC (91.8 vs 100.0 A.U., P = 0.004). Furthermore, the CUC showed a significant inverse correlation with TG, hemoglobin A1c (Hb A1c), homeostasis model assessment of insulin resistance (HOMA-IR), and body mass index (BMI). Finally, the HDL-C/Apolipoprotein A1 (ApoA1) ratio, calculated as a surrogate index of HDL particle size, was significantly positively correlated with the CUC (r2 = 0.49, P < 0.001), but inversely correlated with TG levels (r2 = -0.30, P < 0.001). CONCLUSIONS: The CUC decreased in patients with hypertriglyceridemia and diabetes mellitus, and HDL particle size was a factor defining the CUC and inversely correlated with TG levels, suggesting that impaired CUC in insulin-resistant states was partially due to the shift in HDL towards smaller particles. These findings provide a better understanding of the mechanisms underlying impaired HDL functionality.

Plasma cystine/methionine ratio is associated with left ventricular diastolic function in patients with heart disease.

Elevated circulating homocysteine (Hcy) is a well-known risk factor for cardiovascular diseases (CVDs), including coronary artery disease (CAD) and heart failure (HF). It remains unclear how Hcy and its derivatives relate to left ventricular (LV) diastolic function. The aim of the present study was to investigate the relationship between plasma Hcy-related metabolites and diastolic dysfunction (DD) in patients with heart disease (HD). A total of 62 HD patients with preserved LV ejection fraction (LVEF ≥ 50%) were enrolled. Plasma Hcy and its derivatives were measured by liquid chromatography‒mass spectrometry (LC-MS/MS). Spearman's correlation test and multiple linear regression models were used to analyze the associations between metabolite levels and LV diastolic function. The cystine/methionine (CySS/Met) ratio was positively correlated with LV diastolic function, which was defined from the ratio of mitral inflow E and mitral e' annular velocities (E/e') (Spearman's r = 0.43, p < 0.001). When the subjects were categorized into two groups by E/e', the high-E/e' group had a significantly higher CySS/Met ratio than the low-E/e' group (p = 0.002). Multiple linear regression models revealed that the CySS/Met ratio was independently associated with E/e' after adjustment for age, sex, body mass index (BMI), diabetes mellitus, hypertension, chronic kidney disease (CKD),　hemoglobin, and lipid peroxide (LPO) {standardized ß (95% CI); 0.14 (0.04-0.23); p = 0.005}. Hcy, CySS, and Met did not show a significant association with E/e' in the same models. A high plasma CySS/Met ratio reflected DD in patients with HD.

Fully automated immunoassay for cholesterol uptake capacity to assess high-density lipoprotein function and cardiovascular disease risk.

High-density lipoprotein (HDL) cholesterol efflux capacity (CEC), which is a conventional metric of HDL function, has been associated with coronary heart disease risk. However, the CEC assay requires cultured cells and takes several days to perform. We previously established a cell-free assay to evaluate cholesterol uptake capacity (CUC) as a novel measure of HDL functionality and demonstrated its utility in coronary risk stratification. To apply this concept clinically, we developed a rapid and sensitive assay system based on a chemiluminescent magnetic particle immunoassay. The system is fully automated, providing high reproducibility. Measurement of CUC in serum is completed within 20 min per sample without HDL isolation, a notably higher throughput than that of the conventional CEC assay. CUC decreased with myeloperoxidase-mediated oxidation of HDL or in the presence of N-ethylmaleimide, an inhibitor of lecithin: cholesterol acyltransferase (LCAT), whereas CUC was enhanced by the addition of recombinant LCAT. Furthermore, CUC correlated with CEC even after being normalized by ApoA1 concentration and was significantly associated with the requirement for revascularization due to the recurrence of coronary lesions. Therefore, our new assay system shows potential for the accurate measurement of CUC in serum and permits assessing cardiovascular health.

Impact of High-Density Lipoprotein Function, Rather Than High-Density Lipoprotein Cholesterol Level, on Cardiovascular Disease Among Patients With Familial Hypercholesterolemia.

BACKGROUND: Recently, the function of high-density lipoprotein (HDL), rather than the HDL cholesterol (HDL-C) level, has been attracting more attention in risk prediction for coronary artery disease (CAD).Methods and Results: Patients with clinically diagnosed familial hypercholesterolemia (FH; n=108; male/female, 51/57) were assessed cross-sectionally. Serum cholesterol uptake capacity (CUC) levels were determined using our original cell-free assay. Linear regression was used to determine associations between CUC and clinical variables, including low-density lipoprotein cholesterol and the carotid plaque score. Multivariable logistic regression analysis was used to test factors associated with the presence of CAD. Among the 108 FH patients, 30 had CAD. CUC levels were significantly lower among patients with than without CAD (median [interquartile range] 119 [92-139] vs. 142 [121-165] arbitrary units [AU]; P=0.0004). In addition, CUC was significantly lower in patients with Achilles tendon thickness ≥9.0 mm than in those without Achilles tendon thickening (133 [110-157] vs. 142 [123-174] AU; P=0.047). Serum CUC levels were negatively correlated with the carotid plaque score (Spearman's r=0.37; P=0.00018). Serum CUC levels were significantly associated with CAD, after adjusting for other clinical variables (odds ratio=0.86, 95% CI=0.76-0.96, P=0.033), whereas HDL-C was not. CONCLUSIONS: HDL function, assessed by serum CUC level, rather than HDL-C level, adds risk stratification information among FH patients.

Inhibition of glutaminase 1-mediated glutaminolysis improves pathological cardiac remodeling.

Alterations in cardiac metabolism are strongly associated with the pathogenesis of heart failure (HF). We recently reported that glutamine-dependent anaplerosis, termed glutaminolysis, was activated by H2O2 stimulation in rat cardiomyocytes, which seemed to be an adaptive response by which cardiomyocytes survive acute stress. However, the molecular mechanisms and fundamental roles of glutaminolysis in the pathophysiology of the failing heart are still unknown. Here, we treated wild-type mice (C57BL/6J) and rat neonatal cardiomyocytes (RNCMs) and fibroblasts (RNCFs) with angiotensin II (ANG II) to induce pathological cardiac remodeling. Glutaminase 1 (GLS1), a rate-limiting glutaminolysis enzyme, was significantly increased in ANG II-induced mouse hearts, RNCMs and RNCFs. Unexpectedly, a GLS1 inhibitor attenuated ANG II-induced left ventricular hypertrophy and fibrosis in the mice, and gene knockdown and pharmacological perturbation of GLS1 suppressed hypertrophy and the proliferation of RNCMs and RNCFs, respectively. Using mass spectrometry (MS)-based stable isotope tracing with 13C-labeled glutamine, we observed glutamine metabolic flux in ANG II-treated RNCMs and RNCFs. The incorporation of 13C atoms into tricarboxylic acid (TCA) cycle intermediates and their derivatives was markedly enhanced in both cell types, indicating the activation of glutaminolysis in hypertrophied hearts. Notably, GLS1 inhibition reduced the production of glutamine-derived aspartate and citrate, which are required for the biosynthesis of nucleic acids and lipids, possibly contributing to the suppression of cardiac hypertrophy and fibrosis. The findings of the present study reveal that GLS1-mediated upregulation of glutaminolysis leads to maladaptive cardiac remodeling. Inhibition of this anaplerotic pathway could be a novel therapeutic approach for HF.NEW & NOTEWORTHY To our knowledge, this study is the first to demonstrate that increased GLS1 expression and subsequent activation of glutaminolysis are associated with exacerbation of cardiac hypertrophy and fibrosis. Inhibiting GLS1 antagonized the adverse cardiac remodeling in vitro and in vivo, partly due to reduction of glutamine-derived metabolites, which are necessary for cellular growth and proliferation. Increased glutamine utilization for anabolic reactions in cardiac cells may be related to the pathogenesis and development of HF.

Cholesterol uptake capacity: A new measure of high-density lipoprotein functionality as a predictor of subsequent revascularization in patients undergoing percutaneous coronary intervention.

BACKGROUND AND AIMS: High-density lipoprotein (HDL) functionality is an important determinant of coronary artery disease (CAD) development. We recently developed cholesterol-uptake capacity (CUC), a rapid cell-free assay system that directly evaluates the capacity of HDL to accept additional cholesterol. We aimed to evaluate the association between CUC and revascularization in patients who have undergone percutaneous coronary intervention (PCI). METHODS: We retrospectively reviewed patients who underwent PCI with subsequent revascularization or coronary angiography (CAG) without revascularization. The patients who had frozen blood samples for which CUC were measurable at the index PCI and follow-up were enrolled. RESULTS: We finally enrolled 74 patients who underwent subsequent revascularization and 183 patients who underwent follow-up CAG without revascularization. The serum CUC level at the index PCI was significantly lower in the revascularization group than that in the non-revascularization group (84.3 [75.2-98.9] vs. 92.0 [81.6-103.3 A U.]; p = 0.004). Multivariate logistic regression analysis revealed that decreased serum CUC level at the index PCI was independently associated with subsequent revascularization (odds ratio, 0.98; 95% confidence interval, 0.969-1.000). After adjusting for 16 cardiovascular risk factors, the serum CUC level at the index PCI and follow-up and the absolute change in serum CUC level from the index PCI to follow-up were significantly lower in the revascularization group than those in the non-revascularization group. CONCLUSIONS: Serum CUC level at index PCI was independently associated with subsequent revascularization after PCI. Continuous assessment of HDL functionality by CUC might help predict subsequent revascularization after PCI.

Effects of Elaidic Acid on HDL Cholesterol Uptake Capacity.

Recently we established a cell-free assay to evaluate "cholesterol uptake capacity (CUC)" as a novel concept for high-density lipoprotein (HDL) functionality and demonstrated the feasibility of CUC for coronary risk stratification, although its regulatory mechanism remains unclear. HDL fluidity affects cholesterol efflux, and trans fatty acids (TFA) reduce lipid membrane fluidity when incorporated into phospholipids (PL). This study aimed to clarify the effect of TFA in HDL-PL on CUC. Serum was collected from 264 patients after coronary angiography or percutaneous coronary intervention to measure CUC and elaidic acid levels in HDL-PL, and in vitro analysis using reconstituted HDL (rHDL) was used to determine the HDL-PL mechanism affecting CUC. CUC was positively associated with HDL-PL levels but negatively associated with the proportion of elaidic acid in HDL-PL (elaidic acid in HDL-PL/HDL-PL ratio). Increased elaidic acid-phosphatidylcholine (PC) content in rHDL exhibited no change in particle size or CUC compared to rHDL containing oleic acid in PC. Recombinant human lecithin-cholesterol acyltransferase (LCAT) enhanced CUC, and LCAT-dependent enhancement of CUC and LCAT-dependent cholesterol esterification were suppressed in rHDL containing elaidic acid in PC. Therefore, CUC is affected by HDL-PL concentration, HDL-PL acyl group composition, and LCAT-dependent cholesterol esterification. Elaidic acid precipitated an inhibition of cholesterol uptake and maturation of HDL; therefore, modulation of HDL-PL acyl groups could improve CUC.

Quantification of Amyloid-ß in Plasma by Simple and Highly Sensitive Immunoaffinity Enrichment and LC-MS/MS Assay.

BACKGROUND: Numerous immunoassays have been developed to quantify amyloid ß1-40 (Aß40) and amyloid ß1-42 (Aß42). Nevertheless, given the low concentration of Aß and the high levels of interfering factors in plasma, quantification of plasma Aß is still challenging. To overcome the problems related to the specificity of Aß immunoassays, this study aimed to develop an immunoaffinity enrichment and LC-MS/MS (IA-MS) assay. METHODS: We developed an IA-MS assay using antibody-labeled magnetic beads for purification and LC-MS/MS for Aß quantification. To avoid the loss of Aß due to aggregation in acidic buffer, we used alkaline elution buffer for immunoaffinity enrichment. The concentrations of the Aßs in plasma samples were measured, and the correlation between the plasma and cerebrospinal fluid (CSF) Aß42/Aß40 ratio was also evaluated. RESULTS: The intensities of the Aß mass peaks were significantly higher with the alkaline elution buffer than with the acidic elution buffer (Aß40: 3.6-fold, Aß42: 5.4-fold). This assay exhibited high reproducibility (intra-assay and inter-assay precision, %CV <15), and the working ranges of Aß40 and Aß42 were determined to be 21.7 to 692.8 pg/mL and 5.6 to 180.6 pg/mL, respectively. The concentrations of Aß40 and Aß42 in plasma were measured by IA-MS, and the plasma Aß42/Aß40 ratio was correlated with the CSF Aß42/Aß40 ratio (rs = 0.439, P < 0.01). CONCLUSIONS: The IA-MS assay has sufficient analytic performance for measuring endogenous Aß40 and Aß42 in plasma. This assay can lead to new lines of clinical discovery related to amyloid pathology.

Critical role of glutamine metabolism in cardiomyocytes under oxidative stress.

BACKGROUND: Metabolic remodeling in cardiomyocytes is deeply associated with the pathogenesis of heart failure (HF). Glutaminolysis is an anaplerotic pathway that incorporates α-ketoglutarate (αKG) derived from glutamine into the tricarboxylic acid (TCA) cycle. It is well known that cancer cells depend on glutamine for their increased energy demand and proliferation; however, the physiological roles of glutamine metabolism in failing hearts remain unclear. OBJECTIVE: To investigate the regulatory mechanisms and biological effects of glutamine metabolism in oxidative stress-induced failing myocardium. METHODS AND RESULTS: The intracellular levels of glutamine, glutamate, and αKG were significantly decreased by H2O2 stimulation in rat neonatal cardiomyocytes (RNCMs). To better understand the metabolic flux in failing myocardium, we performed a stable isotope tracing study and found that glutaminolysis was upregulated in RNCMs under oxidative stress. Consistent with this, the enzymatic activity of glutaminase (Gls), which converts glutamine to glutamate, was augmented in RNCMs treated with H2O2. These findings suggest that glutamine anaplerosis is enhanced in cardiomyocytes under oxidative stress to compensate for the reduction of αKG. Furthermore, the inhibition of Gls reduced cardiac cell viability, ATP production, and glutathione (GSH) synthesis in RNCMs with H2O2 stimulation. Finally, we evaluated the effects of αKG on failing myocardium and observed that dimethyl α-ketoglutarate (DMKG) suppressed oxidative stress-induced cell death likely due to the enhancement of intracellular ATP and GSH levels. CONCLUSION: Our study demonstrates that under oxidative stress, glutaminolysis is upregulated to compensate for the loss of αKG and its replenishment into the TCA cycle, thereby exerting cardioprotective effects by maintaining ATP and GSH levels. Modulation of glutamine metabolism in failing hearts might provide a new therapeutic strategy for HF.

Fibronectin-containing High-Density Lipoprotein is Associated with Cancer Cell Adhesion and Proliferation.

A large amount of evidence suggests that high-density lipoprotein (HDL) has anti-atherosclerotic properties. HDL-cholesterol (HDL-C) has also been widely used as a marker of cardiovascular disease. Recently, it was reported that plasma HDL-C levels are inversely correlated with cancer risk. However, the relationship between HDL and cancer pathophysiology remains unknown. Here, we sought to investigate the effect of HDL on cancer progression. First, we focused on fibronectin-an essential extracellular matrix glycoprotein-as an HDL-associated protein and found that only 7.4% of subjects in this study had fibronectin in HDL isolated from their plasma. The fibronectin-containing HDL (FN-HDL) increased the phosphorylation of focal adhesion kinase (FAK) in HeLa cells compared to HDL without fibronectin, further inducing the phosphorylation in a dose-dependent manner. Second, we found that fibronectin-treated HDL activated the phosphorylation of FAK, and its upstream effector blocked the phosphorylation induced by FN-HDL. Finally, we demonstrated that FN-HDL promoted cancer cell proliferation and adhesion compared to HDL without fibronectin. Our study showed the possible mechanism by which FN-HDL enhanced cancer cell proliferation and adhesion via the FAK signaling pathway. Further investigation of the roles of HDL components in tumorigenesis might provide novel insight into cancer pathophysiology.

Association of cholesterol uptake capacity, a novel indicator for HDL functionality, and coronary plaque properties: An optical coherence tomography-based observational study.

BACKGROUND: Cholesterol efflux from atherosclerotic lesion is a key function of high-density lipoprotein (HDL). Recently, we established a simple, high-throughput, cell-free assay to evaluate the capacity of HDL to accept additional cholesterol, which is herein referred to as "cholesterol uptake capacity (CUC)". OBJECTIVE: To clarify the cross-sectional relationship between CUC and coronary plaque properties. METHODS: We enrolled 135 patients to measure CUC and assess the morphological features of angiographic stenosis by optical coherence tomography (OCT). We estimated the extent of the lipid-rich plaque by multiplying the mean lipid arc by lipid length (lipid index). The extent of the OCT-detected macrophage accumulation in the target plaque was semi-quantitatively estimated using a grading system. RESULTS: Lipid-rich plaque lesions were identified in 125 patients (92.6%). CUC was inversely associated with the lipid index (R = -0.348, P < 0.0001). In addition, CUC was also inversely associated with macrophage score (R = -0.327, P < 0.0001). Conversely, neither circulating levels of HDL cholesterol nor apoA1 showed a similar relationship. CONCLUSIONS: We demonstrated that CUC was inversely related to lipid-rich plaque burden and the extent of macrophage accumulation, suggesting that CUC could be useful for cardiovascular risk stratification.

Analytical performance of a new automated chemiluminescent magnetic immunoassays for soluble PD-1, PD-L1, and CTLA-4 in human plasma.

Current clinically approved biomarkers for the PD-1 blockade cancer immunotherapy are based entirely on the properties of tumour cells. With increasing awareness of clinical responses, more precise biomarkers for the efficacy are required based on immune properties. In particular, expression levels of immune checkpoint-associated molecules such as PD-1, PD-L1, and CTLA-4 would be critical to evaluate the immune state of individuals. Although quantification of their soluble form leased from the membrane will provide quick evaluation of patients' immune status, available methods such as enzyme-linked immunosorbent assays to measure these soluble factors have limitations in sensitivity and reproducibility for clinical use. To overcome these problems, we developed a rapid and sensitive immunoassay system based on chemiluminescent magnetic technology. The system is fully automated, providing high reproducibility. Application of this system to plasma of patients with several types of tumours demonstrated that soluble PD-1, PD-L1, and CTLA-4 levels were increased compared to those of healthy controls and varied among tumour types. The sensitivity and detection range were sufficient for evaluating plasma concentrations before and after the surgical ablation of cancers. Therefore, our newly developed system shows potential for accurate detection of soluble PD-1, PD-L1, and CTLA-4 levels in the clinical practice.

Transforming potential of Src family kinases is limited by the cholesterol-enriched membrane microdomain.

The upregulation of Src family kinases (SFKs) has been implicated in cancer progression, but the molecular mechanisms regulating their transforming potentials remain unclear. Here we show that the transforming ability of all SFK members is suppressed by being distributed to the cholesterol-enriched membrane microdomain. All SFKs could induce cell transformation when overexpressed in C-terminal Src kinase (Csk)-deficient fibroblasts. However, their transforming abilities varied depending on their affinity for the microdomain. c-Src and Blk, with a weak affinity for the microdomain due to a single myristate modification at the N terminus, could efficiently induce cell transformation, whereas SFKs with both myristate and palmitate modifications were preferentially distributed to the microdomain and required higher doses of protein expression to induce transformation. In contrast, disruption of the microdomain by depleting cholesterol could induce a robust transformation in Csk-deficient fibroblasts in which only a limited amount of activated SFKs was expressed. Conversely, the addition of cholesterol or recruitment of activated SFKs to the microdomain via a transmembrane adaptor, Cbp/PAG1, efficiently suppressed SFK-induced cell transformation. These findings suggest that the membrane microdomain spatially limits the transforming potential of SFKs by sequestering them away from the transforming pathways.

START-GAP2/DLC2 is localized in focal adhesions via its N-terminal region.

START-GAP2, also termed as DLC2, is a START domain-containing RhoGAP and a negative regulator of RhoA and Cdc42. Although it was reported as a tumor suppresser gene product, the molecular basis for function of START-GAP2 remains to be clarified. Here, we demonstrate that START-GAP2 is localized in focal adhesions through a "FAT (focal adhesion targeting)" region in the N-terminal half. START-GAP2 competes with START-GAP1/DLC1, another START domain-containing RhoGAP, in focal adhesion targeting. Moreover, the C-terminus of tensin2, one of focal adhesion components and reported to bind START-GAP1, also directly interacts with START-GAP2. These results suggest that START-GAP2 and START-GAP1 share the same molecular mechanism in targeting to focal adhesions.