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.

A Fluorescence Cross-Correlation-Spectroscopy-Based Immunoassay for Rapid, Selective, and Accurate Protein Sizing in Human Plasma, Applied to the von Willebrand Factor.

Multimeric abnormalities in plasma von Willebrand factor (VWF) cause bleeding or clotting disorders. Electrophoretic analysis of multimers is used to detect such abnormalities but is qualitative, slow, and difficult to standardize. Fluorescence correlation spectroscopy (FCS) is a good alternative but is affected by low selectivity and concentration bias. Here, we report the development of a homogeneous immunoassay based on dual-color fluorescence cross-correlation spectroscopy (FCCS) that overcomes these challenges. By performing a mild denaturation treatment followed by reacting with polyclonal antibodies, the concentration bias was drastically reduced. The use of a dual antibody assay improved selectivity. Diffusion times of immunolabeled VWF were measured with FCCS and standardized relative to calibrator measurements. The assay measures size changes in VWF using 1 µL of plasma and less than 10 ng of antibody per measurement and was validated over a 16-fold range of VWF antigen concentration (VWF:Ag), with a sensitivity of VWF:Ag 0.8%. Concentration bias and imprecision were less than 10%. Measurements were unaffected by hemolytic, icteric, or lipemic interference. Strong correlations were obtained with reference densitometric readouts (0.97 for calibrators, 0.85 for clinical samples), and significant differences were found between normal (n = 10), type 2A (n = 5), and type 2B (n = 5) von Willebrand's disease and acquired thrombotic thrombocytopenic purpura (n = 10) samples (p < 0.01). This FCCS based immunoassay accurately and selectively determines changes in the multimeric status of plasma VWF and may be used as a simpler, faster, and a standardizable alternative for multimer analysis, following further clinical validation in larger cohorts.

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.

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.

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.

Impaired Cholesterol-Uptake Capacity of HDL Might Promote Target-Lesion Revascularization by Inducing Neoatherosclerosis After Stent Implantation.

Background We evaluated the importance of high-density lipoprotein (HDL) functionality for target-lesion revascularization in patients treated with coronary stents using a rapid cell-free assay system to evaluate the functional capacity of HDL to accept additional cholesterol (cholesterol-uptake capacity; CUC). Methods and Results From an optical coherence tomography (OCT) registry of patients treated with coronary stents, 207 patients were enrolled and their HDL was functionally evaluated by measuring the CUC. Follow-up OCT was performed (median duration, 24.5 months after stenting) to evaluate the presence of neoatherosclerosis. Clinical follow-up was performed to assess target-lesion revascularization for a median duration of 42.3 months after stent implantation. Neoatherosclerosis was identified in 37 patients (17.9%). Multivariate logistic regression analysis revealed that a decreased CUC was independently associated with neoatherosclerosis (odds ratio, 0.799; P<0.001). The CUC showed a significant inverse correlation with incidence of target-lesion revascularization (odds ratio, 0.887; P=0.003) and with lipid accumulation inside stents, suggesting that neoatherosclerosis contributes to the association between CUC and target-lesion revascularization. Conclusions Impaired HDL functionality, detected as decreased CUC, might lead to future stent failure by provoking atherogenic changes of the neointima within stents. Both quantitative and qualitative assessments of HDL might enable the improved prediction of clinical outcomes after stent implantation.

Cholesterol Uptake Capacity: A New Measure of HDL Functionality for Coronary Risk Assessment.

BACKGROUND: Recent studies have shown that the cholesterol efflux capacity of HDL is a better predictor of cardiovascular disease (CVD) than HDL cholesterol. However, the standard procedures used for measuring cholesterol efflux capacity involve radioisotope-labeled cholesterol and cultured macrophages. Thus, a simpler method to measure HDL functionality is needed for clinical application. METHODS: We established a cell-free assay system to evaluate the capacity of HDL to accept additional cholesterol, which we named cholesterol "uptake capacity," using fluorescently labeled cholesterol and an anti-apolipoprotein A1 antibody. We quantified cholesterol uptake capacity of apolipoprotein B (apoB)-depleted serum samples from patients with coronary artery disease who had previously undergone revascularization. RESULTS: This assay system exhibited high reproducibility (CV <10%) and a short processing time (<6 h). The myeloperoxidase-mediated oxidation of apoB-depleted serum impaired cholesterol uptake capacity. Cholesterol uptake capacity correlated significantly with cholesterol efflux capacity (r2 = 0.47, n = 30). Furthermore, cholesterol uptake capacity correlated inversely with the requirement for revascularization because of recurrence of coronary lesions in patients with optimal control of LDL cholesterol (P < 0.01, n = 156). A multivariate analysis adjusted for traditional coronary risk factors showed that only cholesterol uptake capacity remained significant (odds ratio, 0.48; 95% CI, 0.29-0.80; P = 0.0048). CONCLUSIONS: Cholesterol uptake capacity assay evaluates the functionality of HDL in a sensitive and high-throughput manner without using radioisotope label and cells. This assay system could be used for the assessment of CVD risk in the clinical settings.

Direct interaction of Rnd1 with FRS2 beta regulates Rnd1-induced down-regulation of RhoA activity and is involved in fibroblast growth factor-induced neurite outgrowth in PC12 cells.

The Rho family of small GTPases has been implicated in the reorganization of the actin cytoskeleton and subsequent morphological changes in various cells. Rnd1, a member of this family, has a low intrinsic GTPase activity and exerts antagonistic effects on RhoA signaling. However, how the activity of Rnd1 is regulated has not yet been elucidated. Here we have demonstrated that Rnd1 directly associates with FRS2alpha and FRS2beta, which are docking proteins of fibroblast growth factor (FGF) receptors and play important roles in the intracellular signals induced by FGFs. The interaction of FRS2beta with Rnd1 suppresses the inhibitory effect of Rnd1 on RhoA. Rnd1 binds to the COOH-terminal region of FRS2beta including tyrosine residues essential for the interaction with Shp2. When FGF receptor 1 is activated, it phosphorylates FRS2beta, recruits Shp2, and releases Rnd1 from FRS2beta. The liberated Rnd1 then inhibits RhoA activity. Furthermore, knockdown of Rnd1 by Rnd1-specific short interfering RNAs suppress the FGF-induced neurite outgrowth in PC12 cells. These results suggest that the activity of Rnd1 is regulated by FGF receptor through FRS2beta and that Rnd1 plays an important role in the FGF signaling during neurite outgrowth.

Direct interaction of Rnd1 with Plexin-B1 regulates PDZ-RhoGEF-mediated Rho activation by Plexin-B1 and induces cell contraction in COS-7 cells.

Plexins are receptors for the axon guidance molecule semaphorins, and several lines of evidence suggest that Rho family small GTPases are implicated in the downstream signaling of Plexins. Recent studies have demonstrated that Plexin-B1 activates RhoA and induces growth cone collapse through Rho-specific guanine nucleotide exchange factor PDZ-RhoGEF. Here we show that Rnd1, a member of Rho family GTPases, directly interacted with the cytoplasmic domain of Plexin-B1. In COS-7 cells, coexpression of Rnd1 and Plexin-B1 induced cell contraction in response to semaphorin 4D (Sema4D), a ligand for Plexin-B1, whereas expression of Plexin-B1 alone or coexpression of Rnd1 and a Rnd1 interaction-defective mutant of Plexin-B1 did not. The Sema4D-induced contraction in Plexin-B1/Rnd1-expressing COS-7 cells was suppressed by dominant negative RhoA, a Rho-associated kinase inhibitor, a dominant negative form of PDZ-RhoGEF, or deletion of the carboxyl-terminal PDZ-RhoGEF-binding region of Plexin-B1, indicating that the PDZ-RhoGEF/RhoA/Rho-associated kinase pathway is involved in this morphological effect. We also found that Rnd1 promoted the interaction between Plexin-B1 and PDZ-RhoGEF and thereby dramatically potentiated the Plexin-B1-mediated RhoA activation. We propose that Rnd1 plays an important role in the regulation of Plexin-B1 signaling, leading to Rho activation during axon guidance and cell migration.

Socius is a novel Rnd GTPase-interacting protein involved in disassembly of actin stress fibers.

Rho family small GTPases are key regulators of the actin cytoskeleton in various cell types. The Rnd proteins, Rnd1, Rnd2, and Rnd3/RhoE, have been recently identified as new members of the Rho family of GTPases, and expression of Rnd1 or Rnd3 in fibroblasts causes the disassembly of actin stress fibers and the retraction of the cell body to produce extensively branching cellular processes. Here we have performed a yeast two-hybrid screening by using Rnd1 as bait and identified a novel protein that specifically binds to Rnd GTPases. We named this protein Socius. Socius directly binds to Rnd GTPases through its COOH-terminal region. When transfected into COS-7 cells, Socius is translocated to the cell periphery in response to Rnd1 and Rnd3 and colocalized with the GTPases. While expression of wild-type Socius in Swiss 3T3 fibroblasts has little effect on the actin cytoskeleton, the expression of a membrane-targeted form of Socius, containing a COOH-terminal farnesylation motif (Socius-CAAX), induces a dramatic loss of stress fibers. The inhibitory effect of Socius-CAAX on stress fiber formation is enhanced by truncation of its NH(2) terminus. On the other hand, the expression of Socius-CAAX or its NH(2) terminus-truncated form suppresses the Rnd-induced retraction of the cell body and the production of extensively branching cellular processes, although the disassembly of stress fibers is observed. We propose that Socius participates in the Rnd GTPase-induced signal transduction pathways, leading to reorganization of the actin cytoskeleton.