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Chia‐Chi Chen,1– 4 Wei-Hua Tang,5,6 Cheng-Ching Wu,2,7,8 Thung-Lip Lee,7,9 I-Ting Tsai,2,10 Chin-Feng Hsuan,2,7,11 Chao-Ping Wang,7,9 Fu-Mei Chung,7 Yau-Jiunn Lee,12 Teng-Hung Yu,2,7,* Ching-Ting Wei4,13,* 1Department of Pathology, E-Da Hospital, I-Shou University, Kaohsiung, 82445, Taiwan; 2School of Medicine, College of Medicine, I-Shou University, Kaohsiung, 82445, Taiwan; 3Department of Physical Therapy, I-Shou University, Kaohsiung, 82445, Taiwan; 4The School of Chinese Medicine for Post Baccalaureate, College of Medicine, I-Shou University, Kaohsiung, 82445, Taiwan; 5Division of Cardiology, Department of Internal Medicine, Taipei Veterans General Hospital, Yuli Branch, Hualien, 98142, Taiwan; 6Faculty of Medicine, School of Medicine, National Yang Ming Chiao Tung University, Taipei, 112304, Taiwan; 7Division of Cardiology, Department of Internal Medicine, E-Da Hospital, I-Shou University, Kaohsiung, 82445, Taiwan; 8Division of Cardiology, Department of Internal Medicine, E-Da Cancer Hospital, I-Shou, University, Kaohsiung, 82445, Taiwan; 9School of Medicine for International Students, College of Medicine, I-Shou University, Kaohsiung, 82445, Taiwan; 10Department of Emergency, E-Da Hospital, I-Shou University, Kaohsiung, 82445, Taiwan; 11Division of Cardiology, Department of Internal Medicine, E-Da Dachang Hospital, I-Shou University, Kaohsiung, Taiwan; 12Lee’s Endocrinologic Clinic, Pingtung, 90000, Taiwan; 13Division of General Surgery, Department of Surgery, E-Da Hospital, I-Shou University, Kaohsiung, 82445, Taiwan*These authors contributed equally to this workCorrespondence: Teng-Hung Yu; Ching-Ting Wei, E-Da Hospital, I-Shou University, No. 1, Yi-Da Road, Jiau-Shu Village, Yan-Chao District, Kaohsiung, 82445, Taiwan, Tel +886-7-615-1100 ext. 5914 or 5018, Email tenghungy@gmail.com; triplet0826@gmail.comObjective: Breast cancer is the second most common malignancy globally and a leading cause of cancer death in women. Analysis of factors related to disease-free survival (DFS) has improved understanding of the disease and characteristics related to recurrence. The aim of this study was to investigate the predictors of DFS in patients with breast cancer to enable the identification of patients at high risk who may benefit from prevention interventions.Methods: We retrospectively analyzed 559 women with breast cancer who underwent treatment between 2004 and 2022. The study endpoint was DFS. Recurrence was defined as local recurrence, regional recurrence, distant metastases, contralateral breast cancer, other second primary cancer, and death. Baseline tumor-related characteristics, treatment-related characteristics, sociodemographic and biochemical data were analyzed using Cox proportional hazards analysis.Results: The median DFS was 45 months (range, 2 to 225 months). Breast cancer recurred in 86 patients (15.4%), of whom 10 had local recurrence, 10 had regional recurrence, 17 had contralateral breast cancer, 29 had distant metastases, 10 had second primary cancer, and 10 patients died. Multivariate forward stepwise Cox regression analysis showed that AJCC stage III, Ki67 ≥ 14%, albumin, platelet, and red cell distribution width-standard deviation (RDW-SD) were predictors of worse DFS. In addition, the effects of albumin, platelet, and RDW-SD on disease recurrence were confirmed by structural equation model (SEM) analysis.Conclusion: In addition to the traditional predictors of worse DFS such as AJCC stage III and Ki67 ≥ 14%, lower pretreatment circulating albumin, higher pretreatment circulating platelet count and RDW-SD could significantly predict worse DFS in this study, and SEM delineated possible causal pathways and inter-relationships of albumin, platelet, and RDW-SD contributing to the disease recurrence among Chinese women with breast cancer.Keywords: breast cancer, albumin, platelet, RDW-SD, disease-free survival

Yau-Jiunn Lee,1 Chao-Ping Wang,2,3 Wei-Chin Hung,2,4 Wei-Hua Tang,1 Yu-Hung Chang,1 Der-Wei Hu,1 Yung-Chuan Lu,3,5 Teng-Hung Yu,2,4 Cheng-Ching Wu,2,4,6 Fu-Mei Chung,2 Chia-Chang Hsu4,7,8 1Lee’s Endocrinology Clinic, Pingtung 90000, Taiwan; 2Division of Cardiology, Department of Internal Medicine, E-Da Hospital, Kaohsiung 82445, Taiwan; 3School of Medicine for International Students, College of Medicine, I-Shou University, Kaohsiung 82445, Taiwan; 4The School of Chinese Medicine for Post Baccalaureate, College of Medicine, I-Shou University, Kaohsiung 82445, Taiwan; 5Division of Endocrinology and Metabolism, Department of Internal Medicine, E-Da Hospital, Kaohsiung 82445, Taiwan; 6Division of Cardiology, Department of Internal Medicine, E-Da Cancer Hospital, Kaohsiung 82445, Taiwan; 7Division of Gastroenterology and Hepatology, Department of Internal Medicine, E-Da Hospital, Kaohsiung 82445, Taiwan; 8Health Examination Center, E-Da Dachang Hospital, Kaohsiung 80794, TaiwanCorrespondence: Chia-Chang HsuDivision of Gastroenterology and Hepatology, Department of Internal Medicine, E-Da Hospital, Kaohsiung 82445, TaiwanTel +886-7-615-1100 ext. 5914 or 5018Email aladarhsu1107@gmail.comPurpose: This study aimed to investigate the common and unique risk factors and bidirectional relationship between chronic kidney disease (CKD) and nonalcoholic fatty liver disease (NAFLD) in patients with type 2 diabetes mellitus (T2DM).Patients and Methods: This was a cross-sectional study of patients with T2DM enrolled in a disease management program at two specialized diabetes outpatient clinics. Common and unique risk factors for CKD and NAFLD were examined using structural equation models (SEMs). SEMs were also used to examine direct and indirect effects of NAFLD on CKD and those of CKD on NAFLD.Results: A total of 1992 subjects with T2DM were enrolled in this study. In multivariate analysis, NAFLD was independently associated with the odds of CKD (adjusted odds ratio=1.59, 95% confidence interval=1.12– 2.25, P=0.009). SEMs showed that age, triglyceride, uric acid (UA), albumin, and HbA1c levels had statistically significant direct effects on CKD, and the final model could explain 22% of the variability in CKD. Age, triglycerides, body mass index (BMI), UA, white blood cell (WBC) count, serum glutamic pyruvic transaminase (SGPT) level, and smoking status had statistically significant direct effects on NAFLD, and the final model could explain 43% of the variability in NAFLD. The common risk factors contributing to both CKD and NAFLD were age, triglycerides, and UA. The unique risk factors were albumin and HbA1c for CKD, and BMI, WBC, SGPT, and smoking for NAFLD. In addition, SEM analysis also confirmed the bidirectional causal relationship between NAFLD and CKD.Conclusion: Common and unique risk factors and a bidirectional relationship existed between CKD and NAFLD in our patients with T2DM.Keywords: bidirectional relationship, chronic kidney disease, nonalcoholic fatty liver disease, risk factor, structural equation modeling, type 2 diabetes mellitus

The heightened cardiovascular disease (CVD) risk observed among omnivores is thought to be linked, in part, to gut microbiota-dependent generation of trimethylamine-N-oxide (TMAO) from L-carnitine, a nutrient abundant in red meat. Gut microbial transformation of L-carnitine into trimethylamine (TMA), the precursor of TMAO, occurs via the intermediate γ-butyrobetaine (γBB). However, the interrelationship of γBB, red meat ingestion and CVD risks, as well as the gut microbial genes responsible for the transformation of γBB to TMA, are unclear. In the present study, we show that plasma γBB levels in individuals from a clinical cohort (n = 2,918) are strongly associated with incident CVD event risks. Culture of human faecal samples and microbial transplantation studies in gnotobiotic mice with defined synthetic communities showed that the introduction of Emergencia timonensis, a human gut microbe that can metabolize γBB into TMA, is sufficient to complete the carnitine → γBB → TMA transformation, elevate TMAO levels and enhance thrombosis potential in recipients after arterial injury. RNA-sequencing analyses of E. timonensis identified a six-gene cluster, herein named the γBB utilization (gbu) gene cluster, which is upregulated in response to γBB. Combinatorial cloning and functional studies identified four genes (gbuA, gbuB, gbuC and gbuE) that are necessary and sufficient to recapitulate the conversion of γBB to TMA when coexpressed in Escherichia coli. Finally, reanalysis of samples (n = 113) from a clinical, randomized diet, intervention study showed that the abundance of faecal gbuA correlates with plasma TMAO and a red meat-rich diet. Our findings reveal a microbial gene cluster that is critical to dietary carnitine → γBB → TMA → TMAO transformation in hosts and contributes to CVD risk.

Background Trimethylamine-N-oxide (TMAO) is a small molecule derived from the metabolism of dietary nutrients by gut microbes and contributes to cardiovascular disease. Plasma TMAO increases following consumption of red meat. This metabolic change is thought to be partly because of the expansion of gut microbes able to use nutrients abundant in red meat. Methods and Results We used data from a randomized crossover study to estimate the degree to which TMAO can be estimated from fecal microbial composition. Healthy participants received a series of 3 diets that differed in protein source (red meat, white meat, and non-meat), and fecal, plasma, and urine samples were collected following 4 weeks of exposure to each diet. TMAO was quantitated in plasma and urine, while shotgun metagenomic sequencing was performed on fecal DNA. While the cai gene cluster was weakly correlated with plasma TMAO (rho=0.17, P=0.0007), elastic net models of TMAO were not improved by abundances of bacterial genes known to contribute to TMAO synthesis. A global analysis of all taxonomic groups, genes, and gene families found no meaningful predictors of TMAO. We postulated that abundances of known genes related to TMAO production do not predict bacterial metabolism, and we measured choline- and carnitine-trimethylamine lyase activity during fecal culture. Trimethylamine lyase genes were only weakly correlated with the activity of the enzymes they encode. Conclusions Fecal microbiome composition does not predict systemic TMAO because, in this case, gene copy number does not predict bacterial metabolic activity. Registration URL: https://www.clinicaltrials.gov; Unique identifier: NCT01427855.

Background Trimethylamine N-oxide (TMAO) is a gut microbiota-dependent metabolite of dietary choline, L-carnitine, and phosphatidylcholine-rich foods. On the basis of experimental studies and patients with prevalent disease, elevated plasma TMAO may increase risk of atherosclerotic cardiovascular disease (ASCVD). TMAO is also renally cleared and may interact with and causally contribute to renal dysfunction. Yet, how serial TMAO levels relate to incident and recurrent ASCVD in community-based populations and the potential mediating or modifying role of renal function are not established. Methods and Results We investigated associations of serial measures of plasma TMAO, assessed at baseline and 7 years, with incident and recurrent ASCVD in a community-based cohort of 4131 (incident) and 1449 (recurrent) older US adults. TMAO was measured using stable isotope dilution liquid chromatography-tandem mass spectrometry (laboratory coefficient of variation,

Abstract Background Reduced gas transfer in patients with pulmonary arterial hypertension (PAH) is traditionally attributed to remodeling and progressive loss of pulmonary arterial vasculature that results in decreased capillary blood volume available for gas exchange. Methods We tested this hypothesis by determination of lung diffusing capacity (DL) and its components, the alveolar capillary membrane diffusing capacity (Dm) and lung capillary blood volume (Vc) in 28 individuals with PAH in comparison to 41 healthy individuals, and in 19 PAH patients over time. Using single breath simultaneous measure of diffusion of carbon monoxide (DLCO) and nitric oxide (DLNO), DL and Dm were respectively determined, and Vc calculated. Dm and Vc were evaluated over time in relation to standard clinical indicators of disease severity, including brain natriuretic peptide (BNP), 6-minute walk distance (6MWD) and right ventricular systolic pressure (RVSP) by echocardiography. Results Both DLCO and DLNO were reduced in PAH as compared to controls and the lower DL in PAH was due to loss of both Dm and Vc (all p CO of PAH patients did not change over time, DLNO decreased by 24 ml/min/mmHg/year (p = 0.01). Consequently, Dm decreased and Vc tended to increase over time, which led to deterioration of the Dm/Vc ratio, a measure of alveolar-capillary membrane functional efficiency without changes in clinical markers. Conclusions The findings indicate that lower than normal gas transfer in PAH is due to loss of both Dm and Vc, but that deterioration of Dm/Vc over time is related to worsening membrane diffusion.

Right ventricular dysfunction is a hallmark of advanced pulmonary vascular, lung parenchymal, and left heart disease, yet the underlying mechanisms that govern (mal)adaptation remain incompletely characterized. Owing to the knowledge gaps in our understanding of the right ventricle (RV) in health and disease, the National Heart, Lung, and Blood Institute (NHLBI) commissioned a working group to identify current challenges in the field. These included a need to define and standardize normal RV structure and function in populations; access to RV tissue for research purposes and the development of complex experimental platforms that recapitulate the in vivo environment; and the advancement of imaging and invasive methodologies to study the RV within basic, translational, and clinical research programs. Specific recommendations were provided, including a call to incorporate precision medicine and innovations in prognosis, diagnosis, and novel RV therapeutics for patients with pulmonary vascular disease.

AimsWhile most patients with myocardial infarction (MI) have underlying coronary atherosclerosis, not all patients with coronary artery disease (CAD) develop MI. We sought to address the hypothesis that some of the genetic factors which establish atherosclerosis may be distinct from those that predispose to vulnerable plaques and thrombus formation.Methods and resultsWe carried out a genome-wide association study for MI in the UK Biobank (n∼472 000), followed by a meta-analysis with summary statistics from the CARDIoGRAMplusC4D Consortium (n∼167 000). Multiple independent replication analyses and functional approaches were used to prioritize loci and evaluate positional candidate genes. Eight novel regions were identified for MI at the genome wide significance level, of which effect sizes at six loci were more robust for MI than for CAD without the presence of MI. Confirmatory evidence for association of a locus on chromosome 1p21.3 harbouring choline-like transporter 3 (SLC44A3) with MI in the context of CAD, but not with coronary atherosclerosis itself, was obtained in Biobank Japan (n∼165 000) and 16 independent angiography-based cohorts (n∼27 000). Follow-up analyses did not reveal association of the SLC44A3 locus with CAD risk factors, biomarkers of coagulation, other thrombotic diseases, or plasma levels of a broad array of metabolites, including choline, trimethylamine N-oxide, and betaine. However, aortic expression of SLC44A3 was increased in carriers of the MI risk allele at chromosome 1p21.3, increased in ischaemic (vs. non-diseased) coronary arteries, up-regulated in human aortic endothelial cells treated with interleukin-1β (vs. vehicle), and associated with smooth muscle cell migration in vitro.ConclusionsA large-scale analysis comprising ∼831 000 subjects revealed novel genetic determinants of MI and implicated SLC44A3 in the pathophysiology of vulnerable plaques.

Abstract Background The perioperative period is characterized by an intense inflammatory response. Perioperative inflammation promotes postoperative morbidity and increases mortality. Blunting the inflammatory response to surgical trauma might thus improve perioperative outcomes. We are studying three interventions that potentially modulate perioperative inflammation: corticosteroids, tight glucose control, and light anesthesia. Methods/Design The DeLiT Trial is a factorial randomized single-center trial of dexamethasone vs placebo, intraoperative tight vs. conventional glucose control, and light vs deep anesthesia in patients undergoing major non-cardiac surgery. Anesthetic depth will be estimated with Bispectral Index (BIS) monitoring (Aspect medical, Newton, MA). The primary outcome is a composite of major postoperative morbidity including myocardial infarction, stroke, sepsis, and 30-day mortality. C-reactive protein, a measure of the inflammatory response, will be evaluated as a secondary outcome. One-year all-cause mortality as well as post-operative delirium will be additional secondary outcomes. We will enroll up to 970 patients which will provide 90% power to detect a 40% reduction in the primary outcome, including interim analyses for efficacy and futility at 25%, 50% and 75% enrollment. Discussion The DeLiT trial started in February 2007. We expect to reach our second interim analysis point in 2010. This large randomized controlled trial will provide a reliable assessment of the effects of corticosteroids, glucose control, and depth-of-anesthesia on perioperative inflammation and morbidity from major non-cardiac surgery. The factorial design will enable us to simultaneously study the effects of the three interventions in the same population, both individually and in different combinations. Such a design is an economically efficient way to study the three interventions in one clinical trial vs three. Trial registration This trial is registered at Clinicaltrials.gov #: NTC00433251

Using untargeted metabolomics (n = 1,162 subjects), the plasma metabolite (m/z = 265.1188) phenylacetylglutamine (PAGln) was discovered and then shown in an independent cohort (n = 4,000 subjects) to be associated with cardiovascular disease (CVD) and incident major adverse cardiovascular events (myocardial infarction, stroke, or death). A gut microbiota-derived metabolite, PAGln, was shown to enhance platelet activation-related phenotypes and thrombosis potential in whole blood, isolated platelets, and animal models of arterial injury. Functional and genetic engineering studies with human commensals, coupled with microbial colonization of germ-free mice, showed the microbial porA gene facilitates dietary phenylalanine conversion into phenylacetic acid, with subsequent host generation of PAGln and phenylacetylglycine (PAGly) fostering platelet responsiveness and thrombosis potential. Both gain- and loss-of-function studies employing genetic and pharmacological tools reveal PAGln mediates cellular events through G-protein coupled receptors, including α2A, α2B, and β2-adrenergic receptors. PAGln thus represents a new CVD-promoting gut microbiota-dependent metabolite that signals via adrenergic receptors.

Background Recent studies have revealed sexually dimorphic associations between the carbamoyl-phosphate synthase 1 locus, intermediates of the metabolic pathway leading from choline to urea, and risk of coronary artery disease ( CAD ) in women. Based on evidence from the literature, the atheroprotective association with carbamoyl-phosphate synthase 1 could be mediated by the strong genetic effect of this locus on increased circulating glycine levels. Methods and Results We sought to identify additional genetic determinants of circulating glycine levels by carrying out a meta-analysis of genome-wide association study data in up to 30 118 subjects of European ancestry. Mendelian randomization and other analytical approaches were used to determine whether glycine-associated variants were associated with CAD and traditional risk factors. Twelve loci were significantly associated with circulating glycine levels, 7 of which were not previously known to be involved in glycine metabolism ( ACADM , PHGDH , COX 18- ADAMTS 3, PSPH , TRIB 1, PTPRD , and ABO ). Glycine-raising alleles at several loci individually exhibited directionally consistent associations with decreased risk of CAD . However, these effects could not be attributed directly to glycine because of associations with other CAD -related traits. By comparison, genetic models that only included the 2 variants directly involved in glycine degradation and for which there were no other pleiotropic associations were not associated with risk of CAD or blood pressure, lipid levels, and obesity-related traits. Conclusions These results provide additional insight into the genetic architecture of glycine metabolism, but do not yield conclusive evidence for a causal relationship between circulating levels of this amino acid and risk of CAD in humans.

Large-scale untargeted lipidomics experiments involve the measurement of hundreds to thousands of samples. Such data sets are usually acquired on one instrument over days or weeks of analysis time. Such extensive data acquisition processes introduce a variety of systematic errors, including batch differences, longitudinal drifts, or even instrument-to-instrument variation. Technical data variance can obscure the true biological signal and hinder biological discoveries. To combat this issue, we present a novel normalization approach based on using quality control pool samples (QC). This method is called systematic error removal using random forest (SERRF) for eliminating the unwanted systematic variations in large sample sets. We compared SERRF with 15 other commonly used normalization methods using six lipidomics data sets from three large cohort studies (832, 1162, and 2696 samples). SERRF reduced the average technical errors for these data sets to 5% relative standard deviation. We conclude that SERRF outperforms other existing methods and can significantly reduce the unwanted systematic variation, revealing biological variance of interest.

AimsCarnitine and choline are major nutrient precursors for gut microbiota-dependent generation of the atherogenic metabolite, trimethylamine N-oxide (TMAO). We performed randomized-controlled dietary intervention studies to explore the impact of chronic dietary patterns on TMAO levels, metabolism and renal excretion.Methods and resultsVolunteers (N = 113) were enrolled in a randomized 2-arm (high- or low-saturated fat) crossover design study. Within each arm, three 4-week isocaloric diets (with washout period between each) were evaluated (all meals prepared in metabolic kitchen with 25% calories from protein) to examine the effects of red meat, white meat, or non-meat protein on TMAO metabolism. Trimethylamine N-oxide and other trimethylamine (TMA) related metabolites were quantified at the end of each diet period. A random subset (N = 13) of subjects also participated in heavy isotope tracer studies. Chronic red meat, but not white meat or non-meat ingestion, increased plasma and urine TMAO (each >two-fold; P

Heart failure is a leading cause of mortality, yet our understanding of the genetic interactions underlying this disease remains incomplete. Here, we harvest 1352 healthy and failing human hearts directly from transplant center operating rooms, and obtain genome-wide genotyping and gene expression measurements for a subset of 313. We build failing and non-failing cardiac regulatory gene networks, revealing important regulators and cardiac expression quantitative trait loci (eQTLs). PPP1R3A emerges as a regulator whose network connectivity changes significantly between health and disease. RNA sequencing after PPP1R3A knockdown validates network-based predictions, and highlights metabolic pathway regulation associated with increased cardiomyocyte size and perturbed respiratory metabolism. Mice lacking PPP1R3A are protected against pressure-overload heart failure. We present a global gene interaction map of the human heart failure transition, identify previously unreported cardiac eQTLs, and demonstrate the discovery potential of disease-specific networks through the description of PPP1R3A as a central regulator in heart failure.

Interindividual variation in the composition of the human gut microbiome was examined in relation to demographic and anthropometric traits, and to changes in dietary saturated fat intake and protein source. One hundred nine healthy men and women aged 21 to 65, with BMIs of 18 to 36, were randomized, after a two-week baseline diet, to high (15% total energy [E])- or low (7%E)-saturated-fat groups and randomly received three diets (four weeks each) in which the protein source (25%E) was mainly red meat (beef, pork) (12%E), white meat (chicken, turkey) (12%E), and nonmeat sources (nuts, beans, soy) (16%E). Taxonomic characterization using 16S ribosomal DNA was performed on fecal samples collected at each diet completion. Interindividual differences in age, body fat (%), height, ethnicity, sex, and alpha diversity (Shannon) were all significant factors, and most samples clustered by participant in the PCoA ordination. The dietary interventions did not significantly alter the overall microbiome community in ordination space, but there was an effect on taxon abundance levels. Saturated fat had a greater effect than protein source on taxon differential abundance, but protein source had a significant effect once the fat influence was removed. Higher alpha diversity predicted lower beta diversity between the experimental and baseline diets, indicating greater resistance to change in people with higher microbiome diversity. Our results suggest that interindividual differences outweighed the influence of these specific dietary changes on the microbiome and that moderate changes in saturated fat level and protein source correspond to modest changes in the microbiome.IMPORTANCE The microbiome has proven to influence health and disease, but how combinations of external factors affect the microbiome is relatively unknown. Diet can cause changes, but this is usually achieved by altering macronutrient ratios and has not focused on dietary protein source or saturated fat intake levels. In addition, each individual's unique microbiome profile can be an important factor during studies, and it has even been shown to affect therapeutic outcomes. We show here that the effects of individual differences outweighed the effect of experimental diets and that protein source is less influential than saturated fat level. This suggests that fat and protein composition, separate from macronutrient ratio and carbohydrate composition, is an important consideration in dietary studies.

BackgroundMitochondrial oxidation is a major source of reactive oxygen species (ROS) and mitochondrial dysfunction plays a central role in development of heart failure (HF). Paraoxonase 2 deficient (PON2-def) mitochondria are impaired in function. In this study, we tested whether PON2-def aggravates HF progression.Methods and resultsUsing qPCR, immunoblotting and lactonase activity assay, we demonstrate that PON2 activity was significantly decreased in failing hearts despite increased PON2 expression. To determine the cardiac-specific function of PON2, we performed heart transplantations in which PON2-def and wild type (WT) donor hearts were implanted into WT recipient mice. Beating scores of the donor hearts, assessed at 4 weeks post-transplantation, were significantly decreased in PON2-def hearts when compared to WT donor hearts. By using a transverse aortic constriction (TAC) model, we found PON2 deficiency significantly exacerbated left ventricular remodeling and cardiac fibrosis post-TAC. We further demonstrated PON2 deficiency significantly enhanced ROS generation in heart tissues post-TAC. ROS generation was measured through dihydroethidium (DHE) using high-pressure liquid chromatography (HPLC) with a fluorescent detector. By using neonatal cardiomyocytes treated with CoCl2 to mimic hypoxia, we found PON2 deficiency dramatically increased ROS generation in the cardiomyocytes upon CoCl2 treatment. In response to a short CoCl2 exposure, cell viability and succinate dehydrogenase (SDH) activity assessed by MTT assay were significantly diminished in PON2-def cardiomyocytes compared to those in WT cardiomyocytes. PON2-def cardiomyocytes also had lower baseline SDH activity. By using adult mouse cardiomyocytes and mitochondrial ToxGlo assay, we found impaired cellular ATP generation in PON2-def cells compared to that in WT cells, suggesting that PON2 is necessary for proper mitochondrial function.ConclusionOur study suggests a cardioprotective role for PON2 in both experimental and human heart failure, which may be associated with the ability of PON2 to improve mitochondrial function and diminish ROS generation.

Using an untargeted metabolomics approach in initial (N = 99 subjects) and replication cohorts (N = 1,162), we discovered and structurally identified a plasma metabolite associated with cardiovascular disease (CVD) risks, N6,N6,N6-trimethyl-L-lysine (trimethyllysine, TML). Stable-isotope-dilution tandem mass spectrometry analyses of an independent validation cohort (N = 2,140) confirmed TML levels are independently associated with incident (3-year) major adverse cardiovascular event risks (hazards ratio [HR], 2.4; 95% CI, 1.7-3.4) and incident (5-year) mortality risk (HR, 2.9; 95% CI, 2.0-4.2). Genome-wide association studies identified several suggestive loci for TML levels, but none reached genome-wide significance; and d9(trimethyl)-TML isotope tracer studies confirmed TML can serve as a nutrient precursor for gut microbiota-dependent generation of trimethylamine (TMA) and the atherogenic metabolite trimethylamine N-oxide (TMAO). Although TML was shown to be abundant in both plant- and animal-derived foods, mouse and human fecal cultures (omnivores and vegans) showed slow conversion of TML to TMA. Furthermore, unlike chronic dietary choline, TML supplementation in mice failed to elevate plasma TMAO or heighten thrombosis potential in vivo. Thus, TML is identified as a strong predictor of incident CVD risks in subjects and to serve as a dietary precursor for gut microbiota-dependent generation of TMAO; however, TML does not appear to be a major microbial source for TMAO generation in vivo.

Elevated hepatic ceramide levels have been implicated in both insulin resistance (IR) and hepatic steatosis. To understand the factors contributing to hepatic ceramide levels in mice of both sexes, we have quantitated ceramides in a reference population of mice, the Hybrid Mouse Diversity Panel that has been previously characterized for a variety of metabolic syndrome traits. We observed significant positive correlations between Cer(d18:1/16:0) and IR/hepatic steatosis, consistent with previous findings, although the relationship broke down between sexes, as females were less insulin resistant, but had higher Cer(d18:1/16:0) levels than males. The sex difference was due in part to testosterone-mediated repression of ceramide synthase 6. One ceramide species, Cer(d18:1/20:0), was present at higher levels in males and was associated with IR only in males. Clear evidence of gene-by-sex and gene-by-diet interactions was observed, including sex-specific genome-wide association study results. Thus, our studies show clear differences in how hepatic ceramides are regulated between the sexes, which again suggests that the physiological roles of certain hepatic ceramides differ between the sexes.

More than 1 million heart failure hospitalizations occur annually, and congestion is the predominant cause. Rehospitalizations for recurrent congestion portend poor outcomes independently of age and renal function. Persistent congestion trumps serum creatinine increases in predicting adverse heart failure outcomes. No decongestive pharmacological therapy has reduced these harmful consequences. Simplified ultrafiltration devices permit fluid removal in lower-acuity hospital settings, but with conflicting results regarding safety and efficacy. Ultrafiltration performed at fixed rates after onset of therapy-induced increased serum creatinine was not superior to standard care and resulted in more complications. In contrast, compared with diuretic agents, some data suggest that adjustment of ultrafiltration rates to patients' vital signs and renal function may be associated with more effective decongestion and fewer heart failure events. Essential aspects of ultrafiltration remain poorly defined. Further research is urgently needed, given the burden of congestion and data suggesting sustained benefits of early and adjustable ultrafiltration.

Corticotropin-releasing factor (CRF) and the CRF-related peptides, urocortin (Ucn)-1, Ucn2, and Ucn3 signal through receptors CRFR1 and CRFR2 to restore homeostasis in response to stress. The Ucns exert potent cardioprotective effects and may have clinical utility in heart failure. To explore the activity of this system in the heart, we measured the levels of myocardial gene expression of the CRF/Ucn family of ligands/receptors and investigated genetic variation and alternative splicing of CRFR1 in 110 heart failure patients and 108 heart donors. Using quantitative real-time PCR, we detected CRFR1, CRFR2, CRF, Ucn1, Ucn2, and Ucn3 in all samples. CRFR2α was the most abundant receptor and Ucn3 the most abundant ligand, both in patients and donors. Compared with donors, cardiac expression of CRFR1, CRF, and Ucn3 was higher (P < .001) and CRFR2α lower (P = .012) in patients. In patients and donors, genetic variation within CRFR1, represented by the chromosome 17q21.31 inversion polymorphism, was associated with markedly higher CRFR1 expression (P < .001), making CRFR1 and CRFR2α expression almost equivalent in some patients. A novel, truncated splice variant of CRFR1, designated CRFR1j, was identified and shown to exert a dominant-negative effect on CRFR1 signaling in vitro. The novel variant was expressed in a greater proportion of patients (60%) than donors (3%, P < .001). In summary, cardiac expression of CRFR1, CRF, and Ucn3 genes is elevated in heart failure and may contribute to the activation of the CRF/Ucn system in these patients. A common variant within the CRFR1 gene and a novel CRFR1 splice variant may modulate CRFR1 expression and signaling.

Objective: To systematically evaluate the risk factors for developing diabetic foot ulcers (DFU). Methods: The Cochrane Library, PubMed, Web of Science, SinoMed, CNKI, VIP, and Wanfang Data were searched for relevant studies on DFU risk factors, and the search time frame was from database creation to March 2022. Meta-analysis was performed using RevMan 5.3. Results: A total of 26 publications were included, including 3 cohort studies, 22 case–control studies, and one cross-sectional study. Meta-analysis showed that advanced age [MD = 6.04, 95% CI (3.92, 8.16)], male [OR = 1.84, 95% CI (1.48, 2.29)], elevated body mass index [MD = 1.58, 95% CI (0.47, 2.69)], prolonged duration of diabetes [MD = 2.72, 95% CI (2.33, 3.11)], comorbid nephropathy [OR = 3.15, 95% CI (2.68, 4.60)], comorbid neuropathy [OR = 4.80, 95% CI (2.79, 8.27)], comorbid retinopathy [OR = 3.37, 95% CI (2.26, 5.02)], elevated systolic blood pressure [OR = 8.19, 95% CI (6.33, 10.05)], elevated fasting glucose [MD = 1.60, 95% CI (0.21, 3.00)], elevated glycated hemoglobin [MD = 1.24, 95% CI (0.94, 1.54)], elevated triglycerides [MD = 0.44 (95% CI (0.15, 0.73)], elevated fibrinogen [MD = 1.35, 95% CI (0.62, 2.08)], elevated white blood cell count [MD = 2.56, 95% CI (2.24, 2.88)], elevated C-reactive protein [MD = 15.08, 95% CI (8.03, 22.13)], decreased ankle-brachial ratio [MD = −0.26, 95% CI (−0.36, −0.16)], and decreased total protein levels [MD = −4.58, 95% CI (−6.91, −2.25)] were risk factors for developing DFU (p < 0.05), and increased HDL cholesterol levels [MD = −0.22, 95% CI (−0.36, −0.08)] were protective factor for developing DFU (p < 0.05). Conclusion: There are many risk factors for developing of ulcers in the diabetic feet, and early prevention and intervention should be performed as early as possible to assess the risk of developing diabetic foot patients. [ABSTRACT FROM AUTHOR]

Intestinal microbiota metabolism of choline and phosphatidylcholine produces trimethylamine (TMA), which is further metabolized to a proatherogenic species, trimethylamine-N-oxide (TMAO). We demonstrate here that metabolism by intestinal microbiota of dietary L-carnitine, a trimethylamine abundant in red meat, also produces TMAO and accelerates atherosclerosis in mice. Omnivorous human subjects produced more TMAO than did vegans or vegetarians following ingestion of L-carnitine through a microbiota-dependent mechanism. The presence of specific bacterial taxa in human feces was associated with both plasma TMAO concentration and dietary status. Plasma L-carnitine levels in subjects undergoing cardiac evaluation (n = 2,595) predicted increased risks for both prevalent cardiovascular disease (CVD) and incident major adverse cardiac events (myocardial infarction, stroke or death), but only among subjects with concurrently high TMAO levels. Chronic dietary L-carnitine supplementation in mice altered cecal microbial composition, markedly enhanced synthesis of TMA and TMAO, and increased atherosclerosis, but this did not occur if intestinal microbiota was concurrently suppressed. In mice with an intact intestinal microbiota, dietary supplementation with TMAO or either carnitine or choline reduced in vivo reverse cholesterol transport. Intestinal microbiota may thus contribute to the well-established link between high levels of red meat consumption and CVD risk.

Traditional risk factors fail to explain the increased risk for cardiovascular morbidity and mortality in ESRD. Cyanate, a reactive electrophilic species in equilibrium with urea, posttranslationally modifies proteins through a process called carbamylation, which promotes atherosclerosis. The plasma level of protein-bound homocitrulline (PBHCit), which results from carbamylation, predicts major adverse cardiac events in patients with normal renal function, but whether this relationship is similar in ESRD is unknown. We quantified serum PBHCit in a cohort of 347 patients undergoing maintenance hemodialysis with 5 years of follow-up. Kaplan-Meier analyses revealed a significant association between elevated PBHCit and death (log-rank P

BackgroundThe development of tools to monitor the right ventricle in pulmonary arterial hypertension (PAH) is of clinical importance. PAH is associated with pathologic expression of the transcription factor hypoxia-inducible factor (HIF)-1α, which induces glycolytic metabolism and mobilization of proangiogenic progenitor (CD34(+)CD133(+)) cells. We hypothesized that PAH cardiac myocytes have a HIF-related switch to glycolytic metabolism that can be detected with fasting 2-deoxy-2-[(18)F]fluoro-d-glucose positron emission tomography (FDG-PET) and that glucose uptake is informative for cardiac function.MethodsSix healthy control subjects and 14 patients with PAH underwent fasting FDG-PET and echocardiogram. Blood CD34(+)CD133(+) cells and erythropoietin were measured as indicators of HIF activation. Twelve subjects in the PAH cohort underwent repeat studies 1 year later to determine if changes in FDG uptake were related to changes in echocardiographic parameters or to measures of HIF activation.Measurements and resultsFDG uptake in the right ventricle was higher in patients with PAH than in healthy control subjects and correlated with echocardiographic measures of cardiac dysfunction and circulating CD34(+)CD133(+) cells but not erythropoietin. Among patients with PAH, FDG uptake was lower in those receiving β-adrenergic receptor blockers. Changes in FDG uptake over time were related to changes in echocardiographic parameters and CD34(+)CD133(+) cell numbers. Immunohistochemistry of explanted PAH hearts of patients undergoing transplantation revealed that HIF-1α was present in myocyte nuclei but was weakly detectable in control hearts.ConclusionsPAH hearts have pathologic glycolytic metabolism that is quantitatively related to cardiac dysfunction over time, suggesting that metabolic imaging may be useful in therapeutic monitoring of patients.

Background: Diabetic cardiomyopathy (DbCM) is a specific form of heart muscle disease that may result in substantial morbidity and mortality in individuals with type 2 diabetes mellitus (T2DM). Hyperactivation of the polyol pathway is one of the primary mechanisms in the pathogenesis of diabetic complications, including development of DbCM. There is an unmet need for therapies targeting the underlying metabolic abnormalities that drive this form of Stage B heart failure (HF).Methods: Aldose reductase (AR) catalyzes the first and rate-limiting step in the polyol pathway, and AR inhibition has been shown to reduce diabetic complications, including DbCM in animal models and in patients with DbCM. Previous AR inhibitors (ARIs) were limited by poor specificity resulting in unacceptable tolerability and safety profile. AT-001 is a novel investigational highly specific ARI with higher binding affinity and greater selectivity than previously studied ARIs. ARISE-HF (NCT04083339) is an ongoing Phase 3 randomized, placebo-controlled, double blind, global clinical study to investigate the efficacy of AT-001 (1000 mg twice daily [BID] and 1500 mg BID) in 675 T2DM patients with DbCM at high risk of progression to overt HF. ARISE-HF assesses the ability of AT-001 to improve or prevent decline in exercise capacity as measured by functional capacity (changes in peak oxygen uptake [peak VO2]) over 15 (and possibly 27) months of treatment. Additional endpoints include percentage of patients progressing to overt HF, health status metrics, echocardiographic measurements, and changes in cardiacbiomarkers.Results: The ARISE-HF Trial is fully enrolled.Conclusions: This report describes the rationale and study design of ARISE-HF. [ABSTRACT FROM AUTHOR]