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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.HDL function, assessed by serum CUC level, rather than HDL-C level, adds risk stratification information among FH patients.

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.

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.

Atherosclerotic plaques consist mostly of smooth muscle cells (SMC), and genes that influence SMC phenotype can modulate coronary artery disease (CAD) risk. Allelic variation at 15q22.33 has been identified by genome-wide association studies to modify the risk of CAD and is associated with the expression of

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.

Atherosclerotic plaques consist mostly of smooth muscle cells (SMC), and genes that influence SMC phenotype can modulate coronary artery disease (CAD) risk. Allelic variation at 15q22.33 has been identified by genome-wide association studies to modify the risk of CAD, and is associated with expression of SMAD3 in SMC , however the mechanism by which this gene modifies CAD risk remains poorly understood. SMC-specific deletion of Smad3 in a murine atherosclerosis model resulted in greater plaque burden, more positive remodeling, and increased vascular calcification. Single-cell transcriptomic analyses revealed that loss of Smad3 altered SMC transition cell state toward two fates: a novel SMC phenotype that governs both vascular remodeling and recruitment of inflammatory cells, as well as a chondromyocyte fate. The remodeling population was marked by uniquely high Mmp3 and Cxcl12 expression, and its appearance correlated with higher risk plaque features such as increased positive remodeling and macrophage content. Further, investigation of transcriptional mechanisms by which Smad3 alters SMC cell fate revealed novel roles for Hox and Sox transcription factors whose direct interaction with Smad3 regulate an extensive transcriptional program balancing remodeling and vascular extracellular matrix. These findings have significant implications for atherosclerotic and Mendelian aortic aneurysmal diseases. Together, these data suggest that Smad3 expression in SMC inhibits the emergence of specific SMC phenotypic transition cells that mediate adverse plaque features, including positive remodeling, monocyte recruitment, and vascular calcification.

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.

Rationale: The gene encoding TCF21 (transcription factor 21) has been linked to coronary artery disease risk by human genome-wide association studies in multiple racial ethnic groups. In murine models, Tcf21 is required for phenotypic modulation of smooth muscle cells (SMCs) in atherosclerotic tissues and promotes a fibroblast phenotype in these cells. In humans, TCF21 expression inhibits risk for coronary artery disease. The molecular mechanism by which TCF21 regulates SMC phenotype is not known. Objective: To better understand how TCF21 affects the SMC phenotype, we sought to investigate the possible mechanisms by which it regulates the lineage determining MYOCD (myocardin)-SRF (serum response factor) pathway. Methods and Results: Modulation of TCF21 expression in human coronary artery SMC revealed that TCF21 suppresses a broad range of SMC markers, as well as key SMC transcription factors MYOCD and SRF, at the RNA and protein level. We conducted chromatin immunoprecipitation-sequencing to map SRF-binding sites in human coronary artery SMC, showing that binding is colocalized in the genome with TCF21, including at a novel enhancer in the SRF gene, and at the MYOCD gene promoter. In vitro genome editing indicated that the SRF enhancer CArG box regulates transcription of the SRF gene, and mutation of this conserved motif in the orthologous mouse SRF enhancer revealed decreased SRF expression in aorta and heart tissues. Direct TCF21 binding and transcriptional inhibition at colocalized sites were established by reporter gene transfection assays. Chromatin immunoprecipitation and protein coimmunoprecipitation studies provided evidence that TCF21 blocks MYOCD and SRF association by direct TCF21-MYOCD interaction. Conclusions: These data indicate that TCF21 antagonizes the MYOCD-SRF pathway through multiple mechanisms, further establishing a role for this coronary artery disease-associated gene in fundamental SMC processes and indicating the importance of smooth muscle response to vascular stress and phenotypic modulation of this cell type in coronary artery disease risk.

Atherosclerotic plaques consist mostly of smooth muscle cells (SMC), and genes that influence SMC biology can modulate coronary artery disease (CAD) risk. Allelic variation at 15q22.33 has been identified by genome-wide association studies to modify the risk of CAD, and is associated with expression of SMAD3 in SMC, but the mechanism by which this gene modifies CAD risk remains poorly understood. SMC-specific deletion of Smad3 in a murine atherosclerosis model resulted in greater plaque burden, more positive remodeling, and increased vascular calcification. Single-cell transcriptomic analyses revealed that loss of Smad3 altered SMC transition cell state toward two fates: a novel SMC phenotype that governs both vascular remodeling and recruitment of inflammatory cells, as well as a chondromyocyte fate. The remodeling population was marked by uniquely high Mmp3 and Cxcl12 expression, and its appearance correlated with higher risk plaque features such as increased positive remodeling and macrophage content. Further, investigation of transcriptional mechanisms by which Smad3 alters SMC cell fate revealed novel roles for Hox and Sox transcription factors whose direct interaction with Smad3 regulate an extensive transcriptional program balancing remodeling and vascular extracellular matrix with significant implications for atherosclerotic and Mendelian aortic aneurysmal diseases. Together, these data suggest that Smad3 expression in SMC inhibits the emergence of specific SMC phenotypic transition cells that mediate adverse plaque features, including positive remodeling, monocyte recruitment, and vascular calcification.

Background: Endothelial lipase (EL), a regulator of plasma high-density lipoprotein cholesterol (HDL-C), is secreted as a 68-kDa mature glycoprotein, and then cleaved by proprotein convertases. However, the clinical significance of the circulating EL fragments remains unclear. Objective: The objective of this study was to analyze the impact of serum EL fragments on HDL-C levels and major adverse cardiovascular events (MACE). Methods: Using novel monoclonal antibodies (RC3A6) against carboxy-terminal EL protein, we have established a new enzyme-linked immunosorbent assay (ELISA) system, which can detect both full-length EL protein (full EL) and carboxy-terminal truncated fragments (total EL) in serum. The previous sandwich ELISA detected only full EL. The full and total EL mass were measured in 556 patients with coronary artery disease. Among them, 272 patients who underwent coronary intervention were monitored for 2 years for MACE. Results: There was a significant correlation between serum full and total EL mass (R = 0.45, P < .0001). However, the total EL mass showed a stronger inverse correlation with serum HDL-cholesterol concentration than the full EL mass (R = −0.17 vs −0.02). Kaplan-Meier analysis documented an association of serum total EL mass and MACE (log-rank P = .037). When an optimal cutoff value was set at 96.23 ng/mL, total EL mass was an independent prognostic factor for MACE in the Cox proportional hazard model (HR; 1.75, 95% CI; 1.10–2.79, P = .018). Conclusion: Serum total EL mass could be a predictor for MACE in patients with coronary artery disease. This novel ELISA will be useful for further clarifying the impact of EL on HDL metabolism and atherosclerosis.

In response to various stimuli, vascular smooth muscle cells (SMCs) can de-differentiate, proliferate and migrate in a process known as phenotypic modulation. However, the phenotype of modulated SMCs in vivo during atherosclerosis and the influence of this process on coronary artery disease (CAD) risk have not been clearly established. Using single-cell RNA sequencing, we comprehensively characterized the transcriptomic phenotype of modulated SMCs in vivo in atherosclerotic lesions of both mouse and human arteries and found that these cells transform into unique fibroblast-like cells, termed 'fibromyocytes', rather than into a classical macrophage phenotype. SMC-specific knockout of TCF21-a causal CAD gene-markedly inhibited SMC phenotypic modulation in mice, leading to the presence of fewer fibromyocytes within lesions as well as within the protective fibrous cap of the lesions. Moreover, TCF21 expression was strongly associated with SMC phenotypic modulation in diseased human coronary arteries, and higher levels of TCF21 expression were associated with decreased CAD risk in human CAD-relevant tissues. These results establish a protective role for both TCF21 and SMC phenotypic modulation in this disease.

Background: Trans-fatty acid (TFA) intake increases the risk of coronary artery disease (CAD). Our previous cross-sectional survey showed that middle-aged patients with CAD in Japan have elevated serum TFA. In this study, we longitudinally investigated whether elevated TFA is a risk factor in the secondary prevention of CAD for the same-age patients. Methods and Results: A total of 112 patients (age, 21–66 years) who underwent percutaneous coronary intervention were followed up for up to 2 years. Serum elaidic acid was measured using gas chromatography/mass spectrometry as a marker of TFA intake and divided into quartiles. The primary endpoint was ischemia-driven target lesion revascularization (TLR). The hazard ratio (HR) for TLR increased significantly with higher serum elaidic acid (P

Background Recent studies have demonstrated the importance of high-density lipoprotein (HDL) functionality in the development of de novo coronary artery disease by using the cholesterol-efflux capacity, a measure of the ability of HDL to promote cholesterol removal from lipid-laden macrophages. Recently, we developed a rapid cell-free assay system to directly evaluate the capacity of HDL to accept additional cholesterol; the measurement of the cholesterol-uptake capacity (CUC) enables HDL functionality to be readily evaluated in our daily practice. However, prognostic implication of CUC measurement at the timing of percutaneous coronary intervention (PCI) remains unclear. Purpose We aimed to evaluate the association between baseline CUC and revascularization during follow-up in the patients who underwent PCI. Methods We retrospectively reviewed the patients who underwent PCI with follow-up coronary angiography (CAG) or ischemic-driven revascularization. The patients who had the frozen blood samples of which CUC were measurable at the index PCI and follow-up CAG or revascularization were enrolled. We excluded the patients under hemodialysis. Results Among a total of 703 consecutive patients who underwent PCI between Dec 2014 and Mar 2019, we finally enrolled 74 patients who underwent ischemic-driven revascularization (revascularization group) and 183 patients who underwent follow-up CAG without revascularization (non-revascularization group).There were no significant difference in baseline traditional cardiovascular risk factors between the groups. However, the presence of diabetes was significantly more frequent in the revascularization group (63.5% vs 41.0%; P=0.001) than in the non-revascularization group. CUC at the index PCI was significantly lower in the revascularization group than in the non-revascularization group (87.0±19.5 vs 93.9±19.2; P=0.004). Multivariate logistic regression analysis revealed that impaired HDL functionality assessed by decreased CUC level at the index PCI (odds ratio; 0.984, 95% confidence interval; 0.969–1.000) was independently associated with subsequent revascularization after PCI. Indeed, there was a graded inverse association between increasing tertiles of CUC levels and the incidence of revascularization during a median follow-up of 881 days (Figure). Especially in the subgroup analysis of non-diabetic patients, decreased CUC level at the index PCI was independently associated with subsequent revascularization (odds ratio; 0.947, 95% confidence interval; 0.915–0.981), while not in diabetic population. Conclusion Serum CUC level at the index procedure was associated with subsequent revascularization especially in non-diabetic patients who underwent PCI. Funding Acknowledgement Type of funding source: None

Atherosclerotic plaques consist mostly of smooth muscle cells (SMC), and genes that influence SMC biology can modulate coronary artery disease (CAD) risk. Allelic variation at 15q22.33 has been identified by genome-wide association studies to modify the risk of CAD, and is associated with expression of SMAD3 in SMC, but the mechanism by which this gene modifies CAD risk remains poorly understood. SMC-specific deletion of Smad3 in a murine atherosclerosis model resulted in greater plaque burden, positive remodeling, and increased vascular calcification. Single-cell transcriptomic analyses revealed that loss of Smad3 altered SMC progeny phenotype toward the previously described chondromyocyte fate, but importantly also promoted transition to a novel cell-state that governs remodeling and recruitment of inflammatory cells. This new remodeling population was marked by uniquely high Mmp3 and Cxcl12 expression, and its appearance correlated with higher-risk plaque features such as increased positive remodeling and macrophage content. Further, investigation of transcriptional mechanisms by which Smad3 alters SMC cell-fate revealed novel roles for Hox and Sox transcription factors whose direct interaction with Smad3 regulate an extensive transcriptional program balancing remodeling and vascular ECM with significant implications for human Mendelian aortic aneurysmal diseases. Together, these data suggest that Smad3 expression in SMC inhibits the emergence of specific SMC phenotypic transition cells that mediate adverse plaque features, including positive remodeling, monocyte recruitment, and vascular calcification.

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.

Background: Smooth muscle cells (SMC) play a critical role in atherosclerosis. The Aryl hydrocarbon receptor (AHR) is an environment-sensing transcription factor that contributes to vascular development, and has been implicated in coronary artery disease risk. We hypothesized that AHR can affect atherosclerosis by regulating phenotypic modulation of SMC. Methods: We combined RNA-sequencing, chromatin immunoprecipitation followed by sequencing, assay for transposase-accessible chromatin using sequencing, and in vitro assays in human coronary artery SMCs, with single-cell RNA-sequencing, histology, and RNAscope in an SMC-specific lineage-tracing Ahr knockout mouse model of atherosclerosis to better understand the role of AHR in vascular disease. Results: Genomic studies coupled with functional assays in cultured human coronary artery SMCs revealed that AHR modulates the human coronary artery SMC phenotype and suppresses ossification in these cells. Lineage-tracing and activity-tracing studies in the mouse aortic sinus showed that the Ahr pathway is active in modulated SMCs in the atherosclerotic lesion cap. Furthermore, single-cell RNA-sequencing studies of the SMC-specific Ahr knockout mice showed a significant increase in the proportion of modulated SMCs expressing chondrocyte markers such as Col2a1 and Alpl , which localized to the lesion neointima. These cells, which we term “chondromyocytes,” were also identified in the neointima of human coronary arteries. In histological analyses, these changes manifested as larger lesion size, increased lineage-traced SMC participation in the lesion, decreased lineage-traced SMCs in the lesion cap, and increased alkaline phosphatase activity in lesions in the Ahr knockout in comparison with wild-type mice. We propose that AHR is likely protective based on these data and inference from human genetic analyses. Conclusions: Overall, we conclude that AHR promotes the maintenance of lesion cap integrity and diminishes the disease-related SMC-to-chondromyocyte transition in atherosclerotic tissues.

IntroductionSmooth muscle cells (SMC) play a critical role in atherosclerosis. The Aryl hydrocarbon receptor (AHR) is an environment-sensing transcription factor that contributes to vascular development, and has been implicated in coronary artery disease (CAD) risk. We hypothesized that AHR can affect atherosclerosis by regulating phenotypic modulation of SMC.MethodsWe combined RNA-Seq, ChIP-Seq, ATAC-Seq and in-vitro assays in human coronary artery SMC (HCASMC), with single-cell RNA-Seq (scRNA-Seq), histology, and RNAscope in an SMC-specific lineage-tracing Ahr knockout mouse model of atherosclerosis to better understand the role of AHR in vascular disease.ResultsGenomic studies coupled with functional assays in cultured HCASMC revealed that AHR modulates HCASMC phenotype and suppresses ossification in these cells. Lineage tracing and activity tracing studies in the mouse aortic sinus showed that the Ahr pathway is active in modulated SMC in the atherosclerotic lesion cap. Furthermore, scRNA-Seq studies of the SMC-specific Ahr knockout mice showed a significant increase in the proportion of modulated SMC expressing chondrocyte markers such as Col2a1 and Alpl, which localized to the lesion neointima. These cells, which we term “chondromyocytes” (CMC), were also identified in the neointima of human coronary arteries. In histological analyses, these changes manifested as larger lesion size, increased lineage-traced SMC participation in the lesion, decreased lineage-traced SMC in the lesion cap, and increased alkaline phosphatase activity in lesions in the Ahr knockout compared to wild-type mice. We propose that AHR is likely protective based on these data and inference from human genetic analyses.ConclusionOverall, we conclude that AHR promotes maintenance of lesion cap integrity and diminishes the disease related SMC-to-CMC transition in atherosclerotic tissues.

High-density lipoprotein cholesterol (HDL-C) has been identified as a powerful independent negative predictor of cardiovascular disease. The beneficial effect of HDL is largely attributable to its key role in reverse cholesterol transport, whereby excess cholesterol in the peripheral tissues is transported to the liver, reducing the atherosclerotic burden. However, mounting evidence indicates that HDL also has pleiotropic properties, such as anti-inflammatory, anti-oxidative, and vasodilatory properties, which may contribute in reducing the incidence of heart failure. Actually, previous data from clinical and experimental studies have suggested that HDL exerts cardioprotective effects irrespective of the presence/absence of coronary artery disease. This review summarizes the currently available evidence regarding beneficial effects of HDL on the heart beyond its anti-atherogenic property. Understanding the mechanisms of cardiac protection by HDL will provide new insight into the underlying mechanism and therapeutic strategy for heart failure.

Background It has previously been reported that oral administration of purified eicosapentaenoic acid (EPA) generates EPA-rich high-density lipoprotein (HDL) particles with a variety of anti-inflammatory properties. In this study, the mechanism underlying the anti-atherogenic effects of EPA-rich HDL using reconstituted HDL (rHDL) was investigated.Methods and Results:rHDL was generated by the sodium cholate dialysis method, using apolipoprotein A-1 protein, cholesterol, and various concentrations of EPA-phosphatidylcholine (PC) or egg-PC. Increased EPA-PC contents in rHDL resulted in decreased particle size. Next, the effects of rHDL containing various amounts (0-100% of total PC) of EPA-PC on vascular cell adhesion molecule-1 (VCAM-1) expression in human umbilical vein endothelial cells (HUVECs) was examined. Cytokine-stimulated VCAM-1 expression was inhibited in a dose-dependent manner based on the amount of EPA-PC in rHDL. Surprisingly, the incubation of HUVECs with EPA-rich rHDL resulted in the production of resolvin E3 (RvE3), an anti-inflammatory metabolite derived from EPA. Incubation with EPA-PC alone did not adequately induce RvE3 production, suggesting that RvE3 production requires an endothelial cell-HDL interaction. The increased anti-inflammatory effects of EPA-rich HDL may be explained by EPA itself and RvE3 production. Furthermore, the increase in EPA-PC content enhanced cholesterol efflux. Conclusions The EPA-enriched HDL particles exhibit cardioprotective properties via the production of anti-inflammatory lipid metabolites and the increase in cholesterol efflux.

Low levels of plasma high-density lipoprotein (HDL) cholesterol are associated with an increased risk of heart failure, regardless of the presence or absence of coronary artery disease. However, the direct effects of HDL on failing myocardium have not been fully elucidated. We found that HDL treatment resulted in improved cell viability in H9c2 cardiomyocytes under oxidative stress. This cardioprotective effect of HDL was regulated via the phosphatidylinositol 3-kinase (PI3K)/mammalian target of rapamycin (mTOR) pathway. mTOR signaling promotes cell survival through the inactivation of the BCL2-associated agonist of cell death via phosphorylation of ribosomal protein S6 kinase. Modulation of cardiac PI3K/mTOR signaling by HDL could represent a novel therapeutic strategy for heart failure.

The gene encoding TCF21 (transcription factor 21) has been linked to coronary artery disease risk by human genome-wide association studies in multiple racial ethnic groups. In murine models, Tcf21 is required for phenotypic modulation of smooth muscle cells (SMCs) in atherosclerotic tissues and promotes a fibroblast phenotype in these cells. In humans, TCF21 expression inhibits risk for coronary artery disease. The molecular mechanism by which TCF21 regulates SMC phenotype is not known.To better understand how TCF21 affects the SMC phenotype, we sought to investigate the possible mechanisms by which it regulates the lineage determining MYOCD (myocardin)-SRF (serum response factor) pathway.Modulation ofThese data indicate that

Background Genome-wide association studies have identified over 160 loci that are associated with coronary artery disease. As with other complex human diseases, risk in coronary disease loci is determined primarily by altered expression of the causal gene, due to variation in binding of transcription factors and chromatin-modifying proteins that directly regulate the transcriptional apparatus. We have previously identified a coronary disease network downstream of the disease-associated transcription factor TCF21, and in work reported here extends these studies to investigate the mechanisms by which it interacts with the AP-1 transcription complex to regulate local epigenetic effects in these downstream coronary disease loci. Methods Genomic studies, including chromatin immunoprecipitation sequencing, RNA sequencing, and protein-protein interaction studies, were performed in human coronary artery smooth muscle cells. Results We show here that TCF21 and JUN regulate expression of two presumptive causal coronary disease genes, SMAD3 and CDKN2B-AS1, in part by interactions with histone deacetylases and acetyltransferases. Genome-wide TCF21 and JUN binding is jointly localized and particularly enriched in coronary disease loci where they broadly modulate H3K27Ac and chromatin state changes linked to disease-related processes in vascular cells. Heterozygosity at coronary disease causal variation, or genome editing of these variants, is associated with decreased binding of both JUN and TCF21 and loss of expression in cis, supporting a transcriptional mechanism for disease risk. Conclusions These data show that the known chromatin remodeling and pioneer functions of AP-1 are a pervasive aspect of epigenetic control of transcription, and thus, the risk in coronary disease-associated loci, and that interaction of AP-1 with TCF21 to control epigenetic features, contributes to the genetic risk in loci where they co-localize. Electronic supplementary material The online version of this article (10.1186/s13073-019-0635-9) contains supplementary material, which is available to authorized users.

Recent studies have shown that the ketone body β-hydroxybutyrate (βOHB) acts not only as a carrier of energy but also as a signaling molecule that has a role in diverse cellular functions. Circulating levels of ketone bodies have been previously reported to be increased in patients with congestive heart failure (HF). In this study, we investigated regulatory mechanism and pathophysiological role of βOHB in HF. First, we revealed that βOHB level was elevated in failing hearts, but not in blood, using pressure-overloaded mice. We also measured cellular βOHB levels in both cardiomyocytes and non-cardiomyocytes stimulated with or without H2O2 and revealed that increased myocardial βOHB was derived from cardiomyocytes but not non-cardiomyocytes under pathological states. Next, we sought to elucidate the mechanisms of myocardial βOHB elevation and its implication under pathological states. The gene and protein expression levels of CoA transferase (SCOT), a key enzyme involved in ketone body oxidation, was decreased in failing hearts. In cardiomyocytes, H2O2 stimulation caused βOHB accumulation concomitantly with SCOT downregulation, implying that the accumulation of myocardial βOHB occurs because of the decline in its utilization. Finally, we checked the effects of βOHB on cardiomyocytes under oxidative stress. We found that βOHB induced FOXO3a, an oxidative stress resistance gene, and its target enzyme, SOD2 and catalase. Consequently, βOHB attenuated reactive oxygen species production and alleviated apoptosis induced by oxidative stress. It has been reported that hyperadrenergic state in HF boost lipolysis and result in elevation of circulating free fatty acids, which can lead hepatic ketogenesis for energy metabolism alteration. The present findings suggest that the accumulation of βOHB also occurs as a compensatory response against oxidative stress in failing hearts.

Although numerous genetic loci have been associated with coronary artery disease (CAD) with genome wide association studies, efforts are needed to identify the causal genes in these loci and link them into fundamental signaling pathways. Recent studies have investigated the disease mechanism of CAD associated gene SMAD3, a central transcription factor (TF) in the TGFβ pathway, investigating its role in smooth muscle biology. In vitro studies in human coronary artery smooth muscle cells (HCASMC) revealed that SMAD3 modulates cellular phenotype, promoting expression of differentiation marker genes while inhibiting proliferation. RNA sequencing and chromatin immunoprecipitation sequencing studies in HCASMC identified downstream genes that reside in pathways which mediate vascular development and atherosclerosis processes in this cell type. HCASMC phenotype, and gene expression patterns promoted by SMAD3 were noted to have opposing direction of effect compared to another CAD associated TF, TCF21. At sites of SMAD3 and TCF21 colocalization on DNA, SMAD3 binding was inversely correlated with TCF21 binding, due in part to TCF21 locally blocking chromatin accessibility at the SMAD3 binding site. Further, TCF21 was able to directly inhibit SMAD3 activation of gene expression in transfection reporter gene studies. In contrast to TCF21 which is protective toward CAD, SMAD3 expression in HCASMC was shown to be directly correlated with disease risk. We propose that the pro-differentiation action of SMAD3 inhibits dedifferentiation that is required for HCASMC to expand and stabilize disease plaque as they respond to vascular stresses, counteracting the protective dedifferentiating activity of TCF21 and promoting disease risk., Author summary Coronary artery disease (CAD) is the worldwide leading cause of death. The majority of risk for CAD is genetic in nature, i.e., a feature of the genetic information that is transmitted to each individual from both parents, and primarily affects the disease processes in the blood vessel wall that regulate the disease molecular pathways. Modern genetic approaches have allowed mapping of the regions of the human genome that encode information that mediates this risk. The SMAD3 gene has been identified through these studies, a known master regulatory of other genes and molecular pathways, and we have investigated the functions of this gene that are important for disease risk. SMAD3 affects basic functions of a cellular component of the vessel wall, the smooth muscle cell (SMC), that is responsible for responding to vascular stresses to heal the lesions that are produced in conjunction with elevated lipids and other classic risk factors. Studies reported here show that SMAD3 actually inhibits the cellular processes that allow SMC to repair the vascular lesions, and its expression is promoted by the disease related variable sequences in the disease associated regions of the genome. SMAD3 is opposed by another CAD gene, TCF21, that functions to block the effects of SMAD3 expression, and these studies identify genetic mechanisms by which this is done. Thus, these studies identify an interactive pathway that directly contributes to disease risk, and the ability to block SMAD3 or promote TCF21 function could be exploited to inhibit vascular events such as myocardial infarction.

This paper proposes a new approach to simultaneous speech recognition and acoustic event detection of spontaneous speech based on one-pass decoding without rescoring. In this approach, an LSTM (long short-term memory) acoustic model outputs probabilities of acoustic event symbols, such as filler symbols and word fragment symbols, as well as probabilities of phonetic symbols. Then a WFST (weighted finite state transducer) decoder detects fillers based on a filler confidence score calculated by the ratio of the number of filler symbols to the number of phonetic symbols in each word. The WFST decoder also detects word fragments using a phonetic symbol loop followed by a path accepting the word fragment symbol, which is attached to a WFST of a lexicon. Experimental results show that precision and recall rates of filler detection can be controlled by the filler confidence score, and word fragments can be detected without registering all possible word fragments to the lexicon.

Glycosylphosphatidylinositol-anchored high-density lipoprotein-binding protein 1 (GPIHBP1), a glycosylphosphatidylinositol (GPI)-anchored protein of capillary endothelial cells, transports lipoprotein lipase to the capillary lumen and is essential for the lipolytic processing of triglyceride-rich lipoproteins.Because some GPI-anchored proteins have been detected in plasma, we tested whether GPIHBP1 is present in human blood and whether GPIHBP1 deficiency or a history of cardiovascular disease affected GPIHBP1 circulating levels.We developed 2 monoclonal antibodies against GPIHBP1 and used the antibodies to establish a sandwich enzyme-linked immunosorbent assay (ELISA) to measure GPIHBP1 levels in human blood.The GPIHBP1 ELISA was linear in the 8 to 500 pg/mL range and allowed the quantification of GPIHBP1 in serum and in pre- and post-heparin plasma (including lipemic samples). GPIHBP1 was undetectable in the plasma of subjects with null mutations in GPIHBP1. Serum GPIHBP1 median levels were 849 pg/mL (range: 740-1014) in healthy volunteers (n = 28) and 1087 pg/mL (range: 877-1371) in patients with a history of cardiovascular or metabolic disease (n = 415). There was an extremely small inverse correlation between GPIHBP1 and triglyceride levels (r = 0.109; P .0275). GPIHBP1 levels tended to be slightly higher in patients who had a major cardiovascular event after revascularization.We developed an ELISA for quantifying GPIHBP1 in human blood. This assay will be useful to identify patients with GPIHBP1 deficiency and patients with GPIHBP1 autoantibodies. The potential of plasma GPIHBP1 as a biomarker for metabolic or cardiovascular disease is yet questionable but needs additional testing.

Objective : It has been reported that high-density lipoprotein (HDL) loses anti-inflammatory function and promotes atherosclerosis under pathological conditions. However, no pharmacological therapy to improve HDL function is currently available. We aimed to evaluate the effect of oral administration of eicosapentaenoic acid (EPA) on HDL function. Methods : Japanese patients with dyslipidemia were treated with EPA (1800 mg/day, 4 weeks), and anti-inflammatory functions of HDL were assessed utilizing in vitro cell-based assays. Results : The EPA treatment did not change serum cholesterol and triglyceride levels, but it significantly increased EPA concentrations in the serum and HDL fraction. The EPA/arachidonic acid ratio in the HDL was in proportion to that in the serum, suggesting that the orally administered EPA was efficiently incorporated into the HDL particles. The HDL after EPA treatment showed significantly increased activity of anti-oxidative enzyme, paraoxonase-1. In addition, the EPA-rich HDL significantly improved endothelial cell migration, and markedly inhibited cytokine-induced expression of vascular cell adhesion molecule-1, in human umbilical vein endothelial cells, compared to HDL before the EPA treatment. Moreover, the EPA-rich HDL augmented cholesterol efflux capacity from macrophages. Conclusion : Oral administration of EPA regenerated anti-oxidative and anti-inflammatory functions of HDL, and promoted cholesterol efflux from macrophages. Therefore, EPA may transform “dysfunctional HDL” to “functional”, in patients with coronary risk factors.

Sugar concentrations in tomato (Solanum lycopersicum L.) fruits increase under water or salt stress. Water stress can be induced by root chilling, but the relationship with fruit sugars has not been reported. To elucidate this relationship, we compared fruit sugar concentrations and other characteristics between tomato plants cultivated at two root temperatures (20 and 12 °C) using a nutrient-film technique during the spring-to-summer season (SS) and during two summer-to-winter seasons (SW) in a greenhouse in Morioka, Japan. The soluble solids, glucose, and fructose concentrations in tomato fruits increased similarly under root chilling at 12 °C without a significant effect on acidity in all experiments. However, the increase was greater in the SW treatment, where water stress was smaller than in the SS treatment based on the vapour-pressure deficit. Root chilling also increased the starch, sucrose, glucose, and fructose concentrations in the leaves. The photosynthetic CO2 exchange rate and stomatal conductance per unit leaf area were not significantly affected by root chilling, despite a marked decrease in leaf area. Root chilling increased fruit dry weight but decreased the dry weight of vegetative organs, especially leaves and roots. The root chilling-induced increase in soluble sugar concentrations was likely caused by sink limitations in the vegetative organs. Water stress did not contribute directly to this result, although it might have contributed to the decreased dry weights of leaves and roots. Root chilling appears to be an effective way to improve tomato fruit quality and avoids the disadvantages of other methods of increasing fruit sugar content.

Objective: Myeloperoxidase (MPO) is known as major leukocyte enzyme that oxidizes lipoproteins. High density lipoprotein (HDL) contains paraoxonase 1 (PON1), which hydrolyzes oxidized phospholipids. HDL requires PON1 to attenuate accumulation of lipid peroxides in LDL. We recently reported that serum MPO/PON1 ratio could be used as a useful marker for dysfunctional HDL and showed elevated ratios in patients undergoing recurrent percutaneous coronary intervention (PCI). However, it remains obscure whether serum MPO/PON1 ratio can predict relapsing coronary atherosclerotic lesions after PCI. Methods and Results: Total 111 patients who had a history of successful PCI were enrolled. Their serum MPO mass and PON1 activities were measured at the time point of enrollment, and they had angiographical follow-up evaluation. Fourteen patients needed repeat-PCI due to restenosis and/or de novo lesions during the follow up period (143±730 days). With the established cut off value of 1.59 based on our previous work, Kaplan-Meier analysis showed significantly higher recurrence rate of coronary lesions which required PCI treatment in patients with higher MPO/PON1 ratio at enrollment than that in patients with lower MPO/PON1 ratio (66.7% vs. 6.0%, p Conclusions: This study demonstrated that higher MPO/PON1 ratio (>1.59) could predict future recurrence of coronary lesions after PCI. This ratio could be useful marker for secondary prevention of coronary artery disease.

Mosses are known to have the ability to develop high degrees of resistance to desiccation and freezing stress at cellular levels. However, underlying cellular mechanisms leading to the development of stress resistance in mosses are not understood. We previously showed that freezing tolerance in protonema cells of the moss Physcomitrella patens was rapidly increased by exogenous application of the stress hormone abscisic acid (ABA) [Minami, A., Nagao, M., Arakawa, K., Fujikawa, S., Takezawa, D., 2003a. Abscisic acid-induced freezing tolerance in the moss Physcomitrella patens is accompanied by increased expression of stress-related genes. J. Plant Physiol. 160, 475–483]. Herein it is shown that protonema cells with acquired freezing tolerance specifically accumulate low-molecular-weight soluble sugars. Analysis of the most abundant trisaccharide revealed that the cells accumulated theanderose (G 6 -α-glucosyl sucrose) in close association with enhancement of freezing tolerance by ABA treatment. The accumulation of theanderose was inhibited by cycloheximide, an inhibitor of nuclear-encoded protein synthesis, coinciding with a remarkable decrease in freezing tolerance. Furthermore, theanderose accumulation was promoted by cold acclimation and treatment with hyperosmotic solutes, both of which had been shown to enhance cellular freezing tolerance. These results reveal a novel role for theanderose, whose biological function has been obscure, in high freezing tolerance in moss cells.

Acid snow might be an environmental stress factor for wintering plants since acid precipitates are locally concentrated in snow and the period in which ice crystals are in contact with shoots might be longer than that of acid precipitates in rain. In this study, 'equilibrium' and 'prolonged' freezing tests with sulfuric acid, which simulate situations of temperature depression and chronic freezing at a subzero temperature with acid precipitate as acid snow stress, respectively, were carried out using leaf segments of cold-acclimated winter wheat. When leaf segments were frozen in the presence of sulfuric acid solution (pH 4.0, 3.0 or 2.0) by equilibrium freezing with ice seeding, the survival rate of leaf samples treated with sulfuric acid solution of pH 2.0 decreased markedly. Leaf samples after supercooling to -4 and -8 degrees C in the presence of sulfuric acid solution (pH 2.0) without ice seeding were less damaged. When leaf samples were subjected to prolonged freezing at -4 and -8 degrees C for 7 d with sulfuric acid (pH 2.0), the survival rates of leaf samples exposed to sulfuric acid decreased more than those of leaf samples treated with water. On the other hand, leaf samples were less damaged by prolonged supercooling at -4 and -8 degrees C for 7 d with sulfuric acid (pH 2.0). The results suggest that an acid condition (pH 2.0) in the process of extracellular freezing and/or thawing promotes freezing injury of wheat leaves.

High-density lipoprotein (HDL) interacts with various cells, particularly macrophages, in functional cell-HDL interactions. Here, we found that HDL protein quality and lipid quality play critical roles in HDL functions. HDL fractions from healthy volunteers (HDLHealthy) and patients with recurrent coronary atherosclerotic disease (HDLCAD) were prepared. To analyse functional HDL-macrophage interactions, macrophages were co-incubated with each HDL, and lipid mediator production was assessed by liquid chromatography/mass spectrometry-based metabololipidomics. HDLHealthy treatment attenuated the pro-inflammatory lipid mediator production, particularly that of leukotriene (LT) B4, and this treatment enhanced lipoxin (LX) B4 and resolvin (Rv) E2 production. HDLHealthy treatment enhanced the proteasome-mediated degradation of the LTB4-producing enzyme 5-lipoxygenase (LO) in activated macrophages; however, HDLCAD did not show these anti-inflammatory effects. HDLHealthy was engulfed by macrophages via clathrin-mediated endocytosis, which was a critical step in 5-LO/LTB4 regulation. We also found that HDLCAD showed higher levels of the LTB4-producing enzymes and thus promoted LTB4 production from HDLCAD. In addition, LTB4 attenuated HDL endocytosis, HDL-mediated 5-LO degradation in macrophages, and HDL-derived augmentation of macrophage phagocytosis. These results indicated that local LTB4 produced de novo from HDLCAD regulates HDL-macrophage functional interactions and plays critical roles in dysfunctional, inflammatory HDL characteristics.

Heart failure (HF) is a major cause of morbidity and mortality worldwide. However, conventional biomarkers are insufficient to detect early stage of HF and identification of more sensitive markers is an urgent task. We conducted the comprehensive quantification of water-soluble metabolites in blood of atrial septal defect (ASD) patients using a gas chromatograph-mass spectrometry. We found the mean level of 2-Aminobutyric acid (2-AB) in ASD patients was significantly higher than that in healthy volunteers, and 1 month after transcatherter closure of ASD using the Amplatzer septal occluder, serum 2-AB levels in patients decreased to almost the same levels as healthy volunteers (healthy-volunteers: 15.74±1.75 μM, ASD (pre-closure): 22.38±1.99 μM, ASD (post-closure):15.50±1.50 μM, P=0.017). In contrast, serum levels of brain natriuretic peptide (BNP), frequently used as the biomarker in heart disease, did not show such relationships. In addition, 2-AB concentration had a significant correlation with tricuspid regurgitation peak gradient (TRPG) (R2=0.517, P=0.029). Because TRPG reflects right ventricular loading conditions, we examined whether mechanical stress to cardiomyocyte induces 2-AB. Mechanical stretch induced an accumulation of 2-AB, suggesting that circulating 2-AB increased in ASD patients were derived from their hearts. Next, we tried to identify the 2-AB synthesis pathway. 2-AB is known as a key molecule in the biosynthesis of ophthalmic acid that is an analogue of glutathione (GSH) and indicates GSH depletion under oxidative stress. We revealed that 2-AB was produced as the byproduct of GSH synthesis reactions. We also demonstrated that oxidative stress lead to 2-AB production by H2O2 stimulation in cardiomyocyte. Mechanical stress-induced 2-AB accumulation might be mediated by oxidative stress. In summary, circulating 2-AB levels changed before and after closure of ASD and indicated heart condition more efficiently than BNP. We also demonstrated that 2-AB reflects myocardial redox state. Thus, 2-AB monitoring may facilitate the early diagnosis strategy for HF.

Objective: Previous studies have reported that inflammation converts high-density lipoprotein (HDL) to dysfunctional form lacking its anti-inflammatory properties. However, little is known about pharmacological therapy to improve HDL function. It has been recently postulated that fatty acid component in HDL-phospholipids modulates HDL functions. We investigated the effect of oral administration of high dose eicosapentaenoic acid (EPA) on HDL functions. Methods: Patients with dyslipidemia were treated with 1800 mg/day EPA for 4 weeks, and HDL fractions were isolated by ultracentrifuge before and after the EPA treatment. HDL functions were evaluated by multiple in vitro cell-based assays. Results: The EPA treatment did not affect the quantity in the serum lipid profile including serum cholesterol, triglyceride and apolipoprotein levels, but significantly increased EPA concentrations both in the serum and HDL fraction. The EPA/arachidonic acid ratio in the HDL was in proportion to that in the serum, suggesting that EPA was incorporated in HDL particles. The HDL after EPA treatment possessed high activity of paraoxonase-1, an HDL-associated anti-oxidative enzyme, compared to HDL before EPA treatment. In addition, the EPA-rich HDL significantly improved endothelial cell migration, and markedly inhibited cytokine-induced expression of vascular cell adhesion molecule-1 in human umbilical vein endothelial cells, compared to HDL before the EPA treatment. Moreover, the EPA-rich HDL had high capacity of cholesterol efflux capacity from macrophages. Conclusion: Administration of high dose EPA improves anti-inflammatory, anti-oxidative, and cholesterol efflux capacity of HDL in dyslipidemic patients. Thus, EPA may transform “dysfunctional HDL” to “functional”, in patients with coronary risk factors.

(Xanthophyceae), was examined. T. bombycinum shifted from vegetative cells to akinetes with starving by a prolonged batch culture, by culture with a diluted medium, or by culture with a single nutrient-deficient medium. In addition, akinetes developed by desiccation, but cold treatment at 4 C did not facilitate akinete formation. During starving, the vegetative cells, which had a large central vacuole in the protoplasm and thin cell walls, finally changed to akinetes, which had many small vacuoles and oil droplets in the protoplasm and thick cell walls. During akinete formation by starving, the freezing tolerance (LT50) increased gradually from −3 C in vegetative cells to far below −30 C in akinetes. When vegetative cells were subjected to equilibrium freezing, their size shrank greatly and aparticulate domains accompanied by fracture-jump lesions developed in the plasma membranes. Akinetes subjected to equilibrium freezing showed little shrinkage, and freezing-induced ultrastructural changes did not occur in the plasma membranes. The morphological changes in the process of akinete formation and the responses to equilibrium freezing resembled those of cold-acclimated terrestrial plants.