query: Higher plasma levels of I-carnitine, when associated with trimethylamine N-oxide, are negatively correlated with cardiovascular events in humans.

title: Intestinal microbiota metabolism of L-carnitine, a nutrient in red meat, promotes atherosclerosis

text: 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.

title: Circulating trimethylamine N‐oxide and the risk of cardiovascular diseases: a systematic review and meta‐analysis of 11 prospective cohort studies

text: Circulating trimethylamine N-oxide (TMAO), a canonical metabolite from gut flora, has been related to the risk of cardiovascular disorders. However, the association between circulating TMAO and the risk of cardiovascular events has not been quantitatively evaluated. We performed a systematic review and meta-analysis of all available cohort studies regarding the association between baseline circulating TMAO and subsequent cardiovascular events. Embase and PubMed databases were searched for relevant cohort studies. The overall hazard ratios for the developing of cardiovascular events (CVEs) and mortality were extracted. Heterogeneity among the included studies was evaluated with Cochran's Q Test and I2 statistics. A random-effect model or a fixed-effect model was applied depending on the heterogeneity. Subgroup analysis and meta-regression were used to evaluate the source of heterogeneity. Among the 11 eligible studies, three reported both CVE and mortality outcome, one reported only CVEs and the other seven provided mortality data only. Higher circulating TMAO was associated with a 23% higher risk of CVEs (HR = 1.23, 95% CI: 1.07-1.42, I2 = 31.4%) and a 55% higher risk of all-cause mortality (HR = 1.55, 95% CI: 1.19-2.02, I2 = 80.8%). Notably, the latter association may be blunted by potential publication bias, although sensitivity analysis by omitting one study at a time did not significantly change the results. Further subgroup analysis and meta-regression did not support that the location of the study, follow-up duration, publication year, population characteristics or the samples of TMAO affect the results significantly. Higher circulating TMAO may independently predict the risk of subsequent cardiovascular events and mortality.

title: Transmission of atherosclerosis susceptibility with gut microbial transplantation.

text: Recent studies indicate both clinical and mechanistic links between atherosclerotic heart disease and intestinal microbial metabolism of certain dietary nutrients producing trimethylamine N-oxide (TMAO). Here we test the hypothesis that gut microbial transplantation can transmit choline diet-induced TMAO production and atherosclerosis susceptibility. First, a strong association was noted between atherosclerotic plaque and plasma TMAO levels in a mouse diversity panel (n = 22 strains, r = 0.38; p = 0.0001). An atherosclerosis-prone and high TMAO-producing strain, C57BL/6J, and an atherosclerosis-resistant and low TMAO-producing strain, NZW/LacJ, were selected as donors for cecal microbial transplantation into apolipoprotein e null mice in which resident intestinal microbes were first suppressed with antibiotics. Trimethylamine (TMA) and TMAO levels were initially higher in recipients on choline diet that received cecal microbes from C57BL/6J inbred mice; however, durability of choline diet-dependent differences in TMA/TMAO levels was not maintained to the end of the study. Mice receiving C57BL/6J cecal microbes demonstrated choline diet-dependent enhancement in atherosclerotic plaque burden as compared with recipients of NZW/LacJ microbes. Microbial DNA analyses in feces and cecum revealed transplantation of donor microbial community features into recipients with differences in taxa proportions between donor strains that were transmissible to recipients and that tended to show coincident proportions with TMAO levels. Proportions of specific taxa were also identified that correlated with plasma TMAO levels in donors and recipients and with atherosclerotic lesion area in recipients. Atherosclerosis susceptibility may be transmitted via transplantation of gut microbiota. Gut microbes may thus represent a novel therapeutic target for modulating atherosclerosis susceptibility.

title: Gut microbial diversity is associated with lower arterial stiffness in women

text: Aims The gut microbiome influences metabolic syndrome (MetS) and inflammation and is therapeutically modifiable. Arterial stiffness is poorly correlated with most traditional risk factors. Our aim was to examine whether gut microbial composition is associated with arterial stiffness.    Methods and results We assessed the correlation between carotid-femoral pulse wave velocity (PWV), a measure of arterial stiffness, and gut microbiome composition in 617 middle-aged women from the TwinsUK cohort with concurrent serum metabolomics data. Pulse wave velocity was negatively correlated with gut microbiome alpha diversity (Shannon index, Beta(SE)= -0.25(0.07), P = 1 × 10-4) after adjustment for covariates. We identified seven operational taxonomic units associated with PWV after adjusting for covariates and multiple testing-two belonging to the Ruminococcaceae family. Associations between microbe abundances, microbe diversity, and PWV remained significant after adjustment for levels of gut-derived metabolites (indolepropionate, trimethylamine oxide, and phenylacetylglutamine). We linearly combined the PWV-associated gut microbiome-derived variables and found that microbiome factors explained 8.3% (95% confidence interval 4.3-12.4%) of the variance in PWV. A formal mediation analysis revealed that only a small proportion (5.51%) of the total effect of the gut microbiome on PWV was mediated by insulin resistance and visceral fat, c-reactive protein, and cardiovascular risk factors after adjusting for age, body mass index, and mean arterial pressure.    Conclusions Gut microbiome diversity is inversely associated with arterial stiffness in women. The effect of gut microbiome composition on PWV is only minimally mediated by MetS. This first human observation linking the gut microbiome to arterial stiffness suggests that targeting the microbiome may be a way to treat arterial ageing.

title: Original Article

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UNLABELLED Fruit and vegetable consumption has been inversely associated with the risk of chronic diseases including cancer and cardiovascular disease, with the beneficial effects attributed to a variety of protective antioxidants, carotenoids and phytonutrients. The objective of the present study was to determine the effect of supplementation with dehydrated concentrates from mixed fruit and vegetable juices (Juice Plus+R) on serum antioxidant and folate status, plasma homocysteine levels and markers for oxidative stress and DNA damage. Japanese subjects (n=60; age 27.8 yrs; BMI 22.1) were recruited to participate in a double-blind placebo controlled study and were randomized into 2 groups of 30, matched for sex, age, BMI and smoking status (39 males, 22 smokers; 21 females, 13 smokers). Subjects were given encapsulated supplements containing mixed fruit and vegetable juice concentrates or a matching placebo for 28 days, with blood and urine samples collected at baseline, day 14 and day 28 for analytical testing. Compared with the placebo, 28 day supplementation significantly increased the concentration of serum beta-carotene 528% (p<0.0001), lycopene 80.2% (p<0.0005), and alpha tocopherol 39.5% (p<0.0001). Serum folate increased 174.3% (p<0.0001) and correlated with a decrease in plasma homocysteine of -19.9% (p<0.03). Compared with baseline, measures of oxidative stress decreased with serum lipid peroxides declining -10.5% (p<0.02) and urine 8OHdG decreasing -21.1% (p<0.02). Evaluation of data from smokers only (n=17) after 28 days of active supplementation showed comparable changes.
 CONCLUSION In the absence of dietary modification, supplementation with the fruit and vegetable juice concentrate capsules proved to be a highly bioavailable source of phytonutrients. Important antioxidants were elevated to desirable levels associated with decreased risk of disease while markers of oxidative stress were reduced, and folate status improved with a concomitant decrease in homocysteine, and these benefits occurred to a similar extent in smokers when compared to non-smokers.

title: Effects of exercise training of 8 weeks and detraining on plasma levels of endothelium-derived factors, endothelin-1 and nitric oxide, in healthy young humans.

text: Vascular endothelial cells produce nitric oxide (NO), which is a potent vasodilator substance and has been proposed as having antiatherosclerotic property. Vascular endothelial cells also produce endothelin-1 (ET-1), which is a potent vasoconstrictor peptide and has potent proliferating activity on vascular smooth muscle cells. Therefore, ET-1 has been implicated in the progression of atheromatous vascular disease. Because exercise training has been reported to produce an alteration in the function of vascular endothelial cells in animals, we hypothesized that exercise training influences the production of NO and ET-1 in humans. The purpose of the present study was to examine whether chronic exercise could influence the plasma levels of NO (measured as the stable end product of NO, i.e., nitrite/nitrate [NOx]) and ET-1 in humans. Eight healthy young subjects (20.3 +/- 0.5 yr old) participated in the study and exercised by cycling on a leg ergometer (70% VO2max for 1 hour, 3-4 days/week) for 8 weeks. Venous plasma concentrations of NOx and ET-1 were measured before and after (immediately before the end of 8-week exercise training) the exercise training, and also after the 4th and 8th week after the cessation of training. The VO2max significantly increased after exercise training. After the exercise training, the plasma concentration of NOx significantly increased (30.69 +/- 3.20 vs. 48.64 +/- 8.16 micromol/L, p < 0.05), and the plasma concentration of ET-1 significantly decreased (1.65 +/- 0.14 vs. 1.23 +/- 0.12 pg/mL, p < 0.05). The increase in NOx level and the decrease in ET-1 level lasted to the 4th week after the cessation of exercise training and these levels (levels of NOx and ET-1) returned to the basal levels (the levels before the exercise training) in the 8th week after the cessation of exercise training. There was a significant negative correlation between plasma NOx concentration and plasma ET-1 concentration. The present study suggests that chronic exercise causes an increase in production of NO and a decrease in production of ET-1 in humans, which may produce beneficial effects (i.e., vasodilative and antiatherosclerotic) on the cardiovascular system.

title: LPA Gene, Ethnicity, and Cardiovascular Events

text: Background: The relationship of LPA single nucleotide polymorphisms (SNPs), apolipoprotein(a) isoforms, and lipoprotein(a) [Lp(a)] levels with major adverse cardiovascular events (MACE) in different ethnic groups is not well known. Methods: LPA SNPs, apolipoprotein(a) isoforms, Lp(a), and oxidized phospholipids on apolipoprotein B-100 (OxPL-apoB) levels were measured in 1792 black, 1030 white, and 597 Hispanic subjects enrolled in the Dallas Heart Study. Their interdependent relationships and prospective association with MACE after median 9.5-year follow-up were determined. Results: LPA SNP rs3798220 was most prevalent in Hispanics (42.38%), rs10455872 in whites (14.27%), and rs9457951 in blacks (32.92%). The correlation of each of these SNPs with the major apolipoprotein(a) isoform size was highly variable and in different directions among ethnic groups. In the entire cohort, Cox regression analysis with multivariable adjustment revealed that quartiles 4 of Lp(a) and OxPL-apoB were associated with hazard ratios (95% confidence interval) for time to MACE of 2.35 (1.50–3.69, P<0.001) and 1.89 (1.26–2.84, P=0.003), respectively, versus quartile 1. Addition of the major apolipoprotein(a) isoform and the 3 LPA SNPs to these models attenuated the risk, but significance was maintained for both Lp(a) and OxPL-apoB. Evaluating time to MACE in specific ethnic groups, Lp(a) was a positive predictor and the size of the major apolipoprotein(a) isoform was an inverse predictor in blacks, the size of the major apolipoprotein(a) isoform was an inverse predictor in whites, and OxPL-apoB was a positive predictor in Hispanics. Conclusions: The prevalence and association of LPA SNPs with size of apolipoprotein(a) isoforms, Lp(a), and OxPL-apoB levels are highly variable and ethnicity-specific. The relationship to MACE is best explained by elevated plasma Lp(a) or OxPL-apoB levels, despite significant ethnic differences in LPA genetic markers.

title: Plasma Corin as a Predictor of Cardiovascular Events in Patients With Chronic Heart Failure.

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OBJECTIVES The aim of this study was to determine the prognostic value of plasma corin in patients with chronic heart failure (CHF).
 BACKGROUND In recent years, accumulating evidence has indicated that corin plays a critical role in regulating blood pressure and cardiac function.
 METHODS We enrolled 1,148 consecutive CHF patients in a prospective cohort study and explored the association between plasma corin levels and clinical prognosis using multivariate Cox regression analysis.
 RESULTS Patients with low corin levels (<458 pg/ml) were more likely to be women and to be hypertensive. Low corin was found to be associated with an increase in New York Heart Association (NYHA) functional class and N-terminal pro-B-type natriuretic peptide (NT-proBNP) levels, and a decrease in left ventricular ejection fraction (LVEF) and the estimated glomerular filtration rate (eGFR). Multivariate Cox regression analysis suggested that log corin was an independent predictor of major adverse cardiac event(s) (MACE) (hazard ratio: 0.62; 95% confidence interval: 0.39 to 0.95), together with age, diabetes, NYHA functional class, LVEF, eGFR, and log NT-proBNP. In addition, log corin was also a significant predictor for cardiovascular death (p = 0.041) and heart failure rehospitalization (p = 0.015) after adjustment for clinical variables and established biomarkers of adverse prognosis. The Kaplan-Meier survival curves showed that low corin was a significant predictor of MACE in patients with NT-proBNP levels above and below the median.
 CONCLUSIONS Our study demonstrates that plasma corin is a valuable prognostic marker of MACE in patients with CHF, independent of established conventional risk factors.

title: Preventing coronary heart disease: B vitamins and homocysteine.

text: The list of preventable and reversible risk factors for atherosclerotic cardiovascular disease continues to grow. Cigarette smoking, high blood pressure, physical inactivity, elevated cholesterol, underlying lipoprotein abnormalities, lipoprotein(a), diabetes, overweight, male gender, and age are well-established risk factors. During the 1990s, there have been many reports associating elevated plasma homocysteine levels with arteriosclerotic cardiovascular disease and consistent evidence that dietary and supplemental folic acid can reduce homocysteine levels.1 2   The article by Robinson and colleagues3 in this issue of Circulation presents further evidence of the importance of homocysteine and suggestive evidence that plasma folate and plasma pyrixodal-l-phosphate (vitamin B6) are protective factors. Their study is part of the European Concerted Action Project,4 which examined 750 patients younger than age 60 with diagnoses within the previous 12 months of coronary, cerebrovascular, or peripheral vascular disease and 800 healthy control subjects. The patient groups were young (47 years for cases and 44 years for control subjects) and heterogeneous, with nonfatal clinical events or symptoms of arteriosclerotic cardiovascular disease supported by ECG, angiographic, or Doppler evidence; the study involved 19 centers in nine European countries. Men in the highest quintile for fasting total homocysteine (tHcy), compared with the remainder of the population, had an estimated relative risk of 2.2 (95% confidence interval [CI], 1.6 to 2.9), with a striking dose-response relationship and a more-than-multiplicative interaction with cigarette smoking and high blood pressure on vascular disease risk4 ; the corresponding estimated relative risk for coronary heart disease was similar (2.0; 95% CI 1.6 to 2.8). (tHcy is the sum of homocysteine and homocysteinyl moieties of oxidized disulfides, homocystine, and cysteine- homocysteine. )   Robinson and colleagues3 examined three B vitamins in detail to determine their effects on fasting and post–methionine-loading tHcy levels and any independent effects on cardiovascular disease …

title: Hyperglycemia is associated with increased bone mineral density and decreased trabecular bone score in elderly Japanese men: The Fujiwara-kyo osteoporosis risk in men (FORMEN) study.

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PURPOSE Patients with type 2 diabetes mellitus (T2DM) have an increased fracture risk despite having higher areal bone mineral density (aBMD). This study aimed to clarify the association between glycemic and insulin resistance status and bone microarchitecture, and whether pentosidine and bone turnover markers play any roles in the association.
 METHODS A total of 2012 community-dwelling men aged ≥65years completed baseline measurements of spine aBMD, fasting plasma glucose (FPG) and serum insulin, hemoglobin A1c (HbA1c), osteocalcin, type I procollagen N-terminal propeptide, type I collagen C-terminal crosslinking telopeptide, tartrate-resistant acid phosphatase isoenzyme 5b, pentosidine, height and weight and an interview regarding past disease history. Homeostasis model assessment-insulin resistance (HOMA-IR) was also calculated. T2DM was defined as physician-diagnosed middle age or elderly-onset diabetes mellitus, or according to biochemical test results. To evaluate bone microarchitecture, trabecular bone score (TBS) was calculated at the same vertebrae as those used for aBMD measurement.
 RESULTS After excluding participants who had a disease history and/or were taking medications affecting bone metabolism, 1683 men (age, 72.9±5.2years) were analyzed. Men with T2DM had significantly higher aBMD compared to those without T2DM. There was no significant difference in TBS. However, FPG, HbA1c and HOMA-IR levels were significantly inversely correlated with TBS after adjusting for age, BMI and aBMD. Multivariate linear regression analyses revealed that glycemic indices (FPG and HbA1c) were significantly associated with increased aBMD and decreased TBS, and that HOMA-IR was associated only with TBS. These associations did not change after further adjusting for bone turnover makers and pentosidine levels.
 CONCLUSIONS Hyperglycemia and elevated insulin-resistance were associated with low TBS independently of bone turnover and pentosidine levels.

title: Exosomes in Plasma of Patients with Ovarian Carcinoma: Potential Biomarkers of Tumor Progression and Response to Therapy.

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BACKGROUND In patients with Ovarian Cancer (OvCa) exosomes released by tumor cells are present in the plasma and could be involved in tumor progression. This study examines the association between the exosome presence/protein content in plasma of OvCa patients and disease outcome, response to standard therapy and/or tumorresistance to therapies in patients studied at diagnosis and also serially during and after therapy.
 DESIGN AND METHODS Exosomes were purified from OvCa patients' plasma (n=22), patients with benign tumors (n=10) or (n=10) healthy controls (NC) using ultracentrifugation. Exosomes were visualized by scanning electron microscopy. Their protein content was measured. The presence of MAGE 3/6 and TGF-β1 in exosomes was evaluated in Western blots.
 RESULTS The OvCa patients' plasma contained higher levels of exosomal proteins (p<0.05) compared to those isolated from plasma of patients with benign tumors or NC. Exosomes isolated from OvCa patients's plasma carried TGF-β1 and MAGE3/6, which distinguished OvCa patients from those with benign tumors and NC. High protein levels of exosomes were seen in newly diagnosed patients; however in advanced stages of OvCa patients the protein content of isolated exosomes was significantly higher than that of early stages. The exosome levels variably changed during/after chemotherapy, and correlations between the changes in exosomal protein levels and clinical data suggested that the protein content of exosomes might be useful in predicting responses to therapy and prognosis in OvCa patients.
 CONCLUSION Analysis of plasma exosomes levels offers a novel approach to diagnosis and monitoring response to therapies in OvCa patients.

title: Impact of blood pressure variability on cardiovascular events in elderly patients with hypertension.

text: Blood pressure variability is one of the characteristic features of hypertension in the elderly. However, its clinical significance remains to be determined. We therefore examined the impact of blood pressure variability on the development of cardiovascular events in elderly hypertensive patients. A total of 106 consecutive hypertensive patients aged more than 60 years old (mean age, 73.9 +/- 8.1 years old; male, 54%), all of whom underwent 24-h ambulatory blood pressure monitoring, were followed up (median, 34 months; range, 3-60 months). During the follow-up period, 39 cardiovascular events were observed, including 14 cases of cerebral infarction and 7 cases of acute myocardial infarction. The coefficient of variation (CV) of 24-h systolic blood pressure (SBP) values was used as an index of blood pressure variability. The patients showed a mean CV value of 10.6%, and were divided into two groups according to this mean value as a cut-off point: a high CV group (n = 46) and a low CV group (n = 60). Although baseline clinical characteristics were similar in the two groups, Kaplan-Meier plots for event-free survival revealed that the rate of cardiovascular events was significantly higher in high CV group than in low CV group (p < 0.05). Cox's proportional hazards analysis showed that increased blood pressure variability (a high CV value of 24-h SBP) was an independent predictive variable for cardiovascular events. The CV value of daytime SBP and the SD value of both 24-h SBP and daytime SBP also had positive correlations with the onset of cardiovascular events. These results suggest that increased blood pressure variability may be an independent risk factor for cardiovascular events in elderly hypertensive patients.

title: Analysis of SiO2 nanoparticles binding proteins in rat blood and brain homogenate

text: A multitude of nanoparticles, such as titanium oxide (TiO2), zinc oxide, aluminum oxide, gold oxide, silver oxide, iron oxide, and silica oxide, are found in many chemical, cosmetic, pharmaceutical, and electronic products. Recently, SiO2 nanoparticles were shown to have an inert toxicity profile and no association with an irreversible toxicological change in animal models. Hence, exposure to SiO2 nanoparticles is on the increase. SiO2 nanoparticles are routinely used in numerous materials, from strengthening filler for concrete and other construction composites, to nontoxic platforms for biomedical application, such as drug delivery and theragnostics. On the other hand, recent in vitro experiments indicated that SiO2 nanoparticles were cytotoxic. Therefore, we investigated these nanoparticles to identify potentially toxic pathways by analyzing the adsorbed protein corona on the surface of SiO2 nanoparticles in the blood and brain of the rat. Four types of SiO2 nanoparticles were chosen for investigation, and the protein corona of each type was analyzed using liquid chromatography-tandem mass spectrometry technology. In total, 115 and 48 plasma proteins from the rat were identified as being bound to negatively charged 20 nm and 100 nm SiO2 nanoparticles, respectively, and 50 and 36 proteins were found for 20 nm and 100 nm arginine-coated SiO2 nanoparticles, respectively. Higher numbers of proteins were adsorbed onto the 20 nm sized SiO2 nanoparticles than onto the 100 nm sized nanoparticles regardless of charge. When proteins were compared between the two charges, higher numbers of proteins were found for arginine-coated positively charged SiO2 nanoparticles than for the negatively charged nanoparticles. The proteins identified as bound in the corona from SiO2 nanoparticles were further analyzed with ClueGO, a Cytoscape plugin used in protein ontology and for identifying biological interaction pathways. Proteins bound on the surface of nanoparticles may affect functional and conformational properties and distributions in complicated biological processes.

title: Mitochondrial respiration in subcutaneous and visceral adipose tissue from patients with morbid obesity.

text: Adipose tissue exerts important endocrine and metabolic functions in health and disease. Yet the bioenergetics of this tissue is not characterized in humans and possible regional differences are not elucidated. Using high resolution respirometry, mitochondrial respiration was quantified in human abdominal subcutaneous and intra-abdominal visceral (omentum majus) adipose tissue from biopsies obtained in 20 obese patients undergoing bariatric surgery. Mitochondrial DNA (mtDNA) and genomic DNA (gDNA) were determined by the PCR technique for estimation of mitochondrial density. Adipose tissue samples were permeabilized and respirometric measurements were performed in duplicate at 37 degrees C. Substrates (glutamate (G) + malate (M) + octanoyl carnitine (O) + succinate (S)) were added sequentially to provide electrons to complex I + II. ADP ((D)) for state 3 respiration was added after GM. Uncoupled respiration was measured after addition of FCCP. Visceral fat contained more mitochondria per milligram of tissue than subcutaneous fat, but the cells were smaller. Robust, stable oxygen fluxes were found in both tissues, and coupled state 3 (GMOS(D)) and uncoupled respiration were significantly (P < 0.05) higher in visceral (0.95 +/- 0.05 and 1.15 +/- 0.06 pmol O(2) s(1) mg(1), respectively) compared with subcutaneous (0.76 +/- 0.04 and 0.98 +/- 0.05 pmol O(2) s(1) mg(1), respectively) adipose tissue. Expressed per mtDNA, visceral adipose tissue had significantly (P < 0.05) lower mitochondrial respiration. Substrate control ratios were higher and uncoupling control ratio lower (P < 0.05) in visceral compared with subcutaneous adipose tissue. We conclude that visceral fat is bioenergetically more active and more sensitive to mitochondrial substrate supply than subcutaneous fat. Oxidative phosphorylation has a higher relative activity in visceral compared with subcutaneous adipose tissue.

title: Plasma soluble corin in patients with heart failure.

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BACKGROUND Corin is a transmembrane protease that processes natriuretic peptides in the heart. Like many membrane proteins, corin is shed from the cell surface.
 METHODS AND RESULTS In this study, we obtained plasma samples from healthy controls and patients with heart failure (HF) and acute myocardial infarction. Soluble corin levels in plasma were measured by an ELISA method. In healthy adults (n=198), plasma corin levels were 690 pg/mL (SD, 260 pg/mL). The corin levels did not differ significantly among different age groups. In patients with HF (n=291), plasma corin levels were significantly lower compared with that of healthy controls (365 pg/mL [SD, 259]; P<0.001). The reduction in plasma corin levels seemed to correlate with the severity of HF. In patients of New York Heart Association classes II, III, and IV, plasma corin levels were 450 pg/mL (SD, 281 pg/mL; n=69), 377 pg/mL (SD, 270 pg/mL; n=132), and 282 pg/mL (SD, 194 pg/mL; n=90), respectively (P<0.001 class II vs class IV; P<0.05 class III vs class IV). In contrast, plasma corin levels in patients with acute myocardial infarction (n=73) were similar to that of healthy controls (678 pg/mL [SD, 285 pg/mL]; P>0.05).
 CONCLUSIONS Soluble corin was detected in human plasma. Plasma corin levels were reduced significantly in patients with HF but not in those with acute myocardial infarction. Our results indicate that corin deficiency may contribute to the pathogenesis of HF and that plasma corin may be used as a biomarker in the diagnosis of HF.

title: Intestinal methane production in obese individuals is associated with a higher body mass index.

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BACKGROUND Obesity is an epidemic that affects 1 in 3 individuals in the United States, and recent evidence suggests that enteric microbiota may play a significant role in the development of obesity. This study evaluated the association between methanogenic archaea and obesity in human subjects.
 METHODS Subjects with a body mass index (BMI) of 30 kg/m² or higher were prospectively recruited from the weight loss program of a tertiary care medical center. Subjects who met the study's inclusion criteria were asked to complete a questionnaire that included a series of visual analogue scores for bowel symptom severities. Subjects then provided a single end-expiratory breath sample to quantitate methane levels. Bivariate and multivariate analyses were used to determine associations with BMI.
 RESULTS A total of 58 patients qualified for enrollment. Twenty percent of patients (n = 12) had breath test results that were positive for methane (>3 parts per million [ppm]), with a mean breath methane concentration of 12.2±3.1 ppm. BMI was significantly higher in methane-positive subjects (45.2±2.3 kg/m²) than in methane-negative subjects (38.5±0.8 kg/m²; P=.001). Methane-positive subjects also had a greater severity of constipation than methane-negative subjects (21.3±6.4 vs 9.5±2.4; P=.043). Multiple regression analysis illustrated a significant association between BMI and methane, constipation, and antidepressant use. However, methane remained an independent predictor of elevated BMI when controlling for antidepressant use (P<.001) and when controlling for both constipation and antidepressant use (6.55 kg/m² greater BMI; P=.003).
 CONCLUSION This is the first human study to demonstrate that a higher concentration of methane detected by breath testing is a predictor of significantly greater obesity in overweight subjects.

title: Prediction of coronary artery disease by a systemic atherosclerosis score index derived from whole-body MR angiography

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BACKGROUND Whole-body magnetic resonance angiography (WB-MRA) has shown its potential for the non-invasive assessment of nearly the entire arterial vasculature within one examination. Since the presence of extra-cardiac atherosclerosis is associated with an increased risk of coronary events, our goal was to establish the relationship between WB-MRA findings, including a systemic atherosclerosis score index, and the presence of significant coronary artery disease (CAD).
 METHODS WB-MRA was performed on a 1.5T scanner in 50 patients scheduled to undergo elective cardiac catheterization for suspected CAD. In each patient, 40 extra-cardiac vessel segments were evaluated and assigned scores according to their luminal narrowing. The atherosclerosis score index (ASI) was generated as the ratio of summed scores to analyzable segments.
 RESULTS ASI was higher in patients with significant (> 50% stenosis) CAD (n = 27) vs. patients without CAD (n = 22; 1.56 vs. 1.28, p = 0.004). ASI correlated with PROCAM (R = 0.57, p < 0.001) and Framingham (R = 0.36, p = 0.01) risk scores as estimates of the 10-year risk of coronary events. A ROC derived ASI of > 1.54 predicted significant CAD with a sensitivity of 59%, specificity of 86% and a positive predictive value of 84%. Logistic regression revealed ASI > 1.54 as the strongest independent predictor for CAD with a 11-fold increase in likelihood to suffer from significant coronary disease. On the contrary, while 15/27 (55%) of patients with CAD exhibited at least one extra-cardiac stenosis > 50%, only 3/22 (14%) of those patients without CAD did (p = 0.003). The likelihood for an extra-cardiac stenosis when CAD is present differed between vascular territories and ranged from 15% for a carotid stenosis to 44% for a stenosis in the lower extremities.
 CONCLUSION This study provides important new evidence for the close association of extra-cardiac and coronary atherosclerosis. The novel findings that a WB-MRA derived systemic atherosclerosis score index is not only associated with established cardiovascular risk scores but is also predictive of significant CAD suggest its potential prognostic implications and underline the importance to screen for coronary disease in patients with extra-cardiac manifestations of atherosclerosis.

title: Effects of soy isoflavones and phytate on homocysteine, C-reactive protein, and iron status in postmenopausal women.

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BACKGROUND Soy protein or its components may protect against the atherosclerotic cardiovascular disease (CVD) risk factors total homocysteine (tHcy), C-reactive protein (CRP), and excess body iron, which generally increase with menopause.
 OBJECTIVE The primary objective of this study was to determine the independent effect of the soy protein components isoflavones and phytate on CVD risk factors in postmenopausal women. The secondary objective was to identify factors [blood lipids, oxidative stress indexes, serum ferritin, plasma folate, plasma vitamin B-12, and body mass index (BMI)] contributing to tHcy and CRP concentrations.
 DESIGN In a double-blind, 6-wk study, 55 postmenopausal women aged 47-72 y were randomly assigned to 1 of 4 soy protein (40 g/d) isolate treatments: native phytate and native isoflavone (n = 14), native phytate and low isoflavone (n = 13), low phytate and native isoflavone (n = 14), or low phytate and low isoflavone (n = 14). We measured iron indexes, tHcy, CRP, and BMI.
 RESULTS Soy protein with native phytate significantly reduced tHcy (P = 0.017), transferrin saturation (P = 0.027), and ferritin (P = 0.029), whereas soy protein with native isoflavones had no effect on any variables. At baseline, BMI was highly correlated with tHcy (r = 0.39, P = 0.003) and CRP (r = 0.55, P < 0.0001), whereas HDL cholesterol was correlated with CRP (r = -0.30, P = 0.02). Multiple regression analysis showed that LDL cholesterol and BMI contributed significantly (R2= 19.9%, P = 0.003) to the overall variance in tHcy.
 CONCLUSION Consuming phytate-rich foods and maintaining a healthy weight may reduce atherosclerotic CVD risk factors in postmenopausal women.

title: Relation of plasma leptin concentrations to sex, body fat, dietary intake, and peak oxygen uptake in young adult women and men.

text: The purpose of this study was to examine the relation of leptin to metabolic and dietary factors in college-age adults. Young adult women and men (n = 32) were recruited and underwent testing for measurement of body mass index, body composition, peak oxygen consumption (VO2peak), dietary intake, and plasma levels of leptin and insulin. Ln leptin was significantly greater for women than for men (2.1 versus 1.2 ng/mL, respectively). This difference remained significant even after adjusting ln leptin for fat mass and fat-free mass as covariates in separate analyses. VO2peak was higher for men than for women and this remained significant after adjustment for differences in fat-free mass and total body mass. Significant correlations were found between ln leptin and indicators of fat mass in women and men, with higher correlations for similar variables observed in men (r = 0.548, 0.674, and 0.732 for body mass index, percentage of body fat, and fat mass [kg] for women, respectively, and r = 0.740, 0.888, 0.858 for body mass index, percentage of body fat, and fat mass [kg] for men, respectively). Ln leptin showed a significant inverse relationship with VO2peak (r = -0.751) in men only. After adjusting ln leptin for body fat mass using partial correlations, ln leptin was not significantly associated with any of the measured variables. Alternatively, after normalization of ln leptin using fat mass as the divisor, a less adequate statistical analysis method, men showed statistical significant correlations between ln leptin and dietary intake and VO2peak. Although plasma leptin values were higher in women, stronger associations were evident for men than for women between leptin and metabolic and dietary factors.

title: Effect of losartan on ambulatory short-term blood pressure variability and cardiovascular remodeling in hypertensive patients on hemodialysis.

text:
OBJECTIVE Previous studies have shown increases in ambulatory short-term blood pressure (BP) variability to be related to cardiovascular disease. In this study, we examined whether an angiotensin II type 1 receptor blocker losartan would improve ambulatory short-term BP variability in hypertensive patients on hemodialysis.
 METHODS Forty hypertensive patients on hemodialysis therapy were randomly assigned to the losartan treatment group (n=20) or the control treatment group (n=20). At baseline and 6 and 12 months after the treatment, 24-h ambulatory BP monitoring was performed. Echocardiography and measurements of brachial-ankle pulse wave velocity (baPWV) and biochemical parameters were also performed before and after therapy.
 RESULTS After 6- and 12-months of treatment, nighttime short-term BP variability, assessed on the basis of the coefficient of variation of ambulatory BP, was significantly decreased in the losartan group, but remained unchanged in the control group. Compared with the control group, losartan significantly decreased left ventricular mass index (LVMI), baPWV, and the plasma levels of brain natriuretic peptide and advanced glycation end products (AGE). Furthermore, multiple regression analysis showed significant correlations between changes in LVMI and changes in nighttime short-term BP variability, as well as between changes in LVMI and changes in the plasma levels of AGE.
 CONCLUSION These results suggest that losartan is beneficial for the suppression of pathological cardiovascular remodeling though its inhibitory effect on ambulatory short-term BP variability during nighttime.


query: Higher plasma levels of I-carnitine, when associated with trimethylamine N-oxide, are positively correlated with cardiovascular events in humans.

title: Intestinal microbiota metabolism of L-carnitine, a nutrient in red meat, promotes atherosclerosis

text: 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.

title: Circulating trimethylamine N‐oxide and the risk of cardiovascular diseases: a systematic review and meta‐analysis of 11 prospective cohort studies

text: Circulating trimethylamine N-oxide (TMAO), a canonical metabolite from gut flora, has been related to the risk of cardiovascular disorders. However, the association between circulating TMAO and the risk of cardiovascular events has not been quantitatively evaluated. We performed a systematic review and meta-analysis of all available cohort studies regarding the association between baseline circulating TMAO and subsequent cardiovascular events. Embase and PubMed databases were searched for relevant cohort studies. The overall hazard ratios for the developing of cardiovascular events (CVEs) and mortality were extracted. Heterogeneity among the included studies was evaluated with Cochran's Q Test and I2 statistics. A random-effect model or a fixed-effect model was applied depending on the heterogeneity. Subgroup analysis and meta-regression were used to evaluate the source of heterogeneity. Among the 11 eligible studies, three reported both CVE and mortality outcome, one reported only CVEs and the other seven provided mortality data only. Higher circulating TMAO was associated with a 23% higher risk of CVEs (HR = 1.23, 95% CI: 1.07-1.42, I2 = 31.4%) and a 55% higher risk of all-cause mortality (HR = 1.55, 95% CI: 1.19-2.02, I2 = 80.8%). Notably, the latter association may be blunted by potential publication bias, although sensitivity analysis by omitting one study at a time did not significantly change the results. Further subgroup analysis and meta-regression did not support that the location of the study, follow-up duration, publication year, population characteristics or the samples of TMAO affect the results significantly. Higher circulating TMAO may independently predict the risk of subsequent cardiovascular events and mortality.

title: Transmission of atherosclerosis susceptibility with gut microbial transplantation.

text: Recent studies indicate both clinical and mechanistic links between atherosclerotic heart disease and intestinal microbial metabolism of certain dietary nutrients producing trimethylamine N-oxide (TMAO). Here we test the hypothesis that gut microbial transplantation can transmit choline diet-induced TMAO production and atherosclerosis susceptibility. First, a strong association was noted between atherosclerotic plaque and plasma TMAO levels in a mouse diversity panel (n = 22 strains, r = 0.38; p = 0.0001). An atherosclerosis-prone and high TMAO-producing strain, C57BL/6J, and an atherosclerosis-resistant and low TMAO-producing strain, NZW/LacJ, were selected as donors for cecal microbial transplantation into apolipoprotein e null mice in which resident intestinal microbes were first suppressed with antibiotics. Trimethylamine (TMA) and TMAO levels were initially higher in recipients on choline diet that received cecal microbes from C57BL/6J inbred mice; however, durability of choline diet-dependent differences in TMA/TMAO levels was not maintained to the end of the study. Mice receiving C57BL/6J cecal microbes demonstrated choline diet-dependent enhancement in atherosclerotic plaque burden as compared with recipients of NZW/LacJ microbes. Microbial DNA analyses in feces and cecum revealed transplantation of donor microbial community features into recipients with differences in taxa proportions between donor strains that were transmissible to recipients and that tended to show coincident proportions with TMAO levels. Proportions of specific taxa were also identified that correlated with plasma TMAO levels in donors and recipients and with atherosclerotic lesion area in recipients. Atherosclerosis susceptibility may be transmitted via transplantation of gut microbiota. Gut microbes may thus represent a novel therapeutic target for modulating atherosclerosis susceptibility.

title: Original Article

text: Aims The gut microbiome influences metabolic syndrome (MetS) and inflammation and is therapeutically modifiable. Arterial stiffness is poorly correlated with most traditional risk factors. Our aim was to examine whether gut microbial composition is associated with arterial stiffness.    Methods and results We assessed the correlation between carotid-femoral pulse wave velocity (PWV), a measure of arterial stiffness, and gut microbiome composition in 617 middle-aged women from the TwinsUK cohort with concurrent serum metabolomics data. Pulse wave velocity was negatively correlated with gut microbiome alpha diversity (Shannon index, Beta(SE)= -0.25(0.07), P = 1 × 10-4) after adjustment for covariates. We identified seven operational taxonomic units associated with PWV after adjusting for covariates and multiple testing-two belonging to the Ruminococcaceae family. Associations between microbe abundances, microbe diversity, and PWV remained significant after adjustment for levels of gut-derived metabolites (indolepropionate, trimethylamine oxide, and phenylacetylglutamine). We linearly combined the PWV-associated gut microbiome-derived variables and found that microbiome factors explained 8.3% (95% confidence interval 4.3-12.4%) of the variance in PWV. A formal mediation analysis revealed that only a small proportion (5.51%) of the total effect of the gut microbiome on PWV was mediated by insulin resistance and visceral fat, c-reactive protein, and cardiovascular risk factors after adjusting for age, body mass index, and mean arterial pressure.    Conclusions Gut microbiome diversity is inversely associated with arterial stiffness in women. The effect of gut microbiome composition on PWV is only minimally mediated by MetS. This first human observation linking the gut microbiome to arterial stiffness suggests that targeting the microbiome may be a way to treat arterial ageing.

title: hClock gene expression in human colorectal carcinoma.

text: In this study, we aimed to investigate changes in the expression of human Clock (hClock), a gene at the core of the circadian gene family, in colorectal carcinomas (CRCs) and to discuss the possible effects. Previous studies have revealed that the disruption of circadian rhythms is one of the endogenous factors that contribute to the initiation and development of CRCs. However, the underlying molecular changes to the circadian genes associated with CRCs have not been explored. Immunofluorescence and quantitative polymerase chain reaction (qPCR) analysis of the hCLOCK protein and gene expression were performed in 30 cases of CRC. The hCLOCK protein was expressed in all specimens obtained from 30 CRC patients. Higher levels of hCLOCK expression were observed in human CRC tissues compared with the paired non-cancerous tissues. hCLOCK expression was significantly higher in poorly differentiated, or late-stage, Dukes' grade tumors and in 64.3% of tumor cases with lymph node metastasis. The hClock gene was expressed in all specimens. A significantly higher expression of hClock was found in human CRC cases compared with paired non-cancerous tissues. There was a strong positive linear correlation between hClock gene expression and protein expression in human CRCs. A strong positive linear correlation was also found between hClock gene expression and ARNT, HIF-1α and VEGF expression in human CRCs. There was no significant correlation between hClock and Bak, Bax, Bid, tumor necrosis factor receptor I (TNFR I) and TNFR II. The circadian gene hClock was stably expressed in human colorectal mucosa and was important in regulating the expression of downstream clock-controlled genes. hCLOCK may interact with HIF-1α/ARNT and activate VEGF to stimulate tumor angiogenesis and metastasis.

title: Chronic renal failure accelerates atherogenesis in apolipoprotein E-deficient mice.

text: Cardiovascular mortality is 10 to 20 times increased in patients with chronic renal failure (CRF). Risk factors for atherosclerosis are abundant in patients with CRF. However, the pathogenesis of cardiovascular disease in CRF remains to be elucidated. The effect of CRF on the development of atherosclerosis in apolipoprotein E-deficient male mice was examined. Seven-week-old mice underwent 5/6 nephrectomy (CRF, n = 28), unilateral nephrectomy (UNX, n = 24), or no surgery (n = 23). Twenty-two weeks later, CRF mice showed increased aortic plaque area fraction (0.266 +/- 0.033 versus 0.045 +/- 0.006; P < 0.001), aortic cholesterol content (535 +/- 62 versus 100 +/- 9 nmol/cm(2) intimal surface area; P < 0.001), and aortic root plaque area (205,296 +/- 22,098 versus 143,662 +/- 13,302 micro m(2); P < 0.05) as compared with no-surgery mice; UNX mice showed intermediate values. The plaques from uremic mice contained CD11b-positive macrophages and showed strong staining for nitrotyrosine. Systolic BP and plasma homocysteine concentrations were similar in uremic and nonuremic mice. Plasma urea and cholesterol concentrations were elevated 2.6-fold (P < 0.001) and 1.5-fold (P < 0.001) in CRF compared with no-surgery mice. Both variables correlated with aortic plaque area fraction (r(2) = 0.5, P < 0.001 and r(2) = 0.3, P < 0.001, respectively) and with each other (r(2) = 0.5, P < 0.001). On multiple linear regression analysis, only plasma urea was a significant predictor of aortic plaque area fraction. In conclusion, the present findings suggest that uremia markedly accelerates atherogenesis in apolipoprotein E-deficient mice. This effect could not be fully explained by changes in BP, plasma homocysteine levels, or total plasma cholesterol concentrations. Thus, the CRF apolipoprotein E-deficient mouse is a new model for studying the pathogenesis of accelerated atherosclerosis in uremia.

title: Relation between colonic proglucagon expression and metabolic response to oligofructose in high fat diet-fed mice.

text: Several data suggest that fermentable dietary fiber could play a role in the control of obesity and associated metabolic disorders. The aim of this study was to investigate the putative role of short chain fructo-oligosaccharide (OFS) - a non-digestible oligosaccharide - in mice fed a standard diet and in mice fed two distinct high fat diets inducing metabolic disorders associated to obesity. We confirmed, in mice, several effects previously shown in rats fed a standard diet enriched with OFS, namely an increase in total and empty caecum weight, a significant decrease in epididymal fat mass, and an increase in colonic and portal plasma glucagon-like peptide-1 (GLP-1), a phenomenon positively correlated with a higher colonic proglucagon mRNA level. Curiously, 4-week treatment with OFS added at the same dose induced different effects when added in the two different high fat diets. OFS decreased energy intake, body weight gain, glycemia, and epididymal fat mass only when added together with the high fat-carbohydrate free diet, in which OFS promoted colonic proglucagon expression and insulin secretion. Our results support an association between the increase in proglucagon expression in the proximal colon and OFS effects on glycemia, fat mass development, and/or body weight gain. In conclusion, dietary oligosaccharides would constitute an interesting class of dietary fibers promoting, in certain conditions, endogenous GLP-1 production, with beneficial physiological consequences. This remains to be proven in human studies.

title: Amylase, Lipase, and Acute Pancreatitis in People With Type 2 Diabetes Treated With Liraglutide: Results From the LEADER Randomized Trial.

text:
OBJECTIVE To evaluate serum amylase and lipase levels and the rate of acute pancreatitis in patients with type 2 diabetes and high cardiovascular risk randomized to liraglutide or placebo and observed for 3.5-5.0 years.    RESEARCH DESIGN AND METHODS A total of 9,340 patients with type 2 diabetes were randomized to either liraglutide or placebo (median observation time 3.84 years). Fasting serum lipase and amylase were monitored. Acute pancreatitis was adjudicated in a blinded manner.
 RESULTS Compared with the placebo group, liraglutide-treated patients had increases in serum lipase and amylase of 28.0% and 7.0%, respectively. Levels were increased at 6 months and then remained stable. During the study, 18 (0.4% [1.1 events/1,000 patient-years of observation] [PYO]) liraglutide-treated and 23 (0.5% [1.7 events/1,000 PYO]) placebo patients had acute pancreatitis confirmed by adjudication. Most acute pancreatitis cases occurred ≥12 months after randomization. Liraglutide-treated patients with prior history of pancreatitis (n = 147) were not more likely to develop acute pancreatitis than similar patients in the placebo group (n = 120). Elevations of amylase and lipase levels did not predict future risk of acute pancreatitis (positive predictive value <1.0%) in patients treated with liraglutide.
 CONCLUSIONS In a population with type 2 diabetes at high cardiovascular risk, there were numerically fewer events of acute pancreatitis among liraglutide-treated patients (regardless of previous history of pancreatitis) compared with the placebo group. Liraglutide was associated with increases in serum lipase and amylase, which were not predictive of an event of subsequent acute pancreatitis.


query: Homozygous deletion of murine Sbds gene from osterix-expressing mesenchymal stem and progenitor cells (MPCs) induces oxidative stress.

title: Mesenchymal Inflammation Drives Genotoxic Stress in Hematopoietic Stem Cells and Predicts Disease Evolution in Human Pre-leukemia.

text: Mesenchymal niche cells may drive tissue failure and malignant transformation in the hematopoietic system, but the underlying molecular mechanisms and relevance to human disease remain poorly defined. Here, we show that perturbation of mesenchymal cells in a mouse model of the pre-leukemic disorder Shwachman-Diamond syndrome (SDS) induces mitochondrial dysfunction, oxidative stress, and activation of DNA damage responses in hematopoietic stem and progenitor cells. Massive parallel RNA sequencing of highly purified mesenchymal cells in the SDS mouse model and a range of human pre-leukemic syndromes identified p53-S100A8/9-TLR inflammatory signaling as a common driving mechanism of genotoxic stress. Transcriptional activation of this signaling axis in the mesenchymal niche predicted leukemic evolution and progression-free survival in myelodysplastic syndrome (MDS), the principal leukemia predisposition syndrome. Collectively, our findings identify mesenchymal niche-induced genotoxic stress in heterotypic stem and progenitor cells through inflammatory signaling as a targetable determinant of disease outcome in human pre-leukemia.

title: Bone progenitor dysfunction induces myelodysplasia and secondary leukemia

text: Mesenchymal cells contribute to the 'stroma' of most normal and malignant tissues, with specific mesenchymal cells participating in the regulatory niches of stem cells. By examining how mesenchymal osteolineage cells modulate haematopoiesis, here we show that deletion of Dicer1 specifically in mouse osteoprogenitors, but not in mature osteoblasts, disrupts the integrity of haematopoiesis. Myelodysplasia resulted and acute myelogenous leukaemia emerged that had acquired several genetic abnormalities while having intact Dicer1. Examining gene expression altered in osteoprogenitors as a result of Dicer1 deletion showed reduced expression of Sbds, the gene mutated in Schwachman-Bodian-Diamond syndrome-a human bone marrow failure and leukaemia pre-disposition condition. Deletion of Sbds in mouse osteoprogenitors induced bone marrow dysfunction with myelodysplasia. Therefore, perturbation of specific mesenchymal subsets of stromal cells can disorder differentiation, proliferation and apoptosis of heterologous cells, and disrupt tissue homeostasis. Furthermore, primary stromal dysfunction can result in secondary neoplastic disease, supporting the concept of niche-induced oncogenesis.

title: MicroRNA-29 induces cellular senescence in aging muscle through multiple signaling pathways

text: The mechanisms underlying the development of aging-induced muscle atrophy are unclear. By microRNA array and individual qPCR analyses, we found significant up-regulation of miR-29 in muscles of aged rodents vs. results in young. With aging, p85α, IGF-1 and B-myb muscle levels were lower while the expression of certain cell arrest proteins (p53, p16 and pRB) increased. When miR-29 was expressed in muscle progenitor cells (MPC), their proliferation was impaired while SA-βgal expression increased signifying the development of senescence. Impaired MPC proliferation resulted from interactions between miR-29 and the 3'-UTR of p85a, IGF-1 and B-myb, suppressing the translation of these mediators of myoblast proliferation. In vivo, electroporation of miR-29 into muscles of young mice suppressed the proliferation and increased levels of cellular arrest proteins, recapitulating aging-induced responses in muscle. A potential stimulus of miR-29 expression is Wnt-3a since we found that exogenous Wnt-3a stimulated miR-29 expression 2.7-fold in primary cultures of MPCs. Thus, aging-induced muscle senescence results from activation of miR-29 by Wnt-3a leading to suppressed expression of several signaling proteins (p85α, IGF-1 and B-myb) that act coordinately to impair the proliferation of MPCs contributing to muscle atrophy. The increase in miR-29 provides a potential mechanism for aging-induced sarcopenia.

title: Epithelial and mesenchymal subpopulations within normal basal breast cell lines exhibit distinct stem cell/progenitor properties.

text: It has been proposed that epithelial-mesenchymal transition (EMT) in mammary epithelial cells and breast cancer cells generates stem cell features, and that the presence of EMT characteristics in claudin-low breast tumors reveals their origin in basal stem cells. It remains to be determined, however, whether EMT is an inherent property of normal basal stem cells, and if the presence of a mesenchymal-like phenotype is required for the maintenance of all their stem cell properties. We used nontumorigenic basal cell lines as models of normal stem cells/progenitors and demonstrate that these cell lines contain an epithelial subpopulation ("EpCAM+," epithelial cell adhesion molecule positive [EpCAM(pos)]/CD49f(high)) that spontaneously generates mesenchymal-like cells ("Fibros," EpCAM(neg)/CD49f(med/low)) through EMT. Importantly, stem cell/progenitor properties such as regenerative potential, high aldehyde dehydrogenase 1 activity, and formation of three-dimensional acini-like structures predominantly reside within EpCAM+ cells, while Fibros exhibit invasive behavior and mammosphere-forming ability. A gene expression profiling meta-analysis established that EpCAM+ cells show a luminal progenitor-like expression pattern, while Fibros most closely resemble stromal fibroblasts but not stem cells. Moreover, Fibros exhibit partial myoepithelial traits and strong similarities with claudin-low breast cancer cells. Finally, we demonstrate that Slug and Zeb1 EMT-inducers control the progenitor and mesenchymal-like phenotype in EpCAM+ cells and Fibros, respectively, by inhibiting luminal differentiation. In conclusion, nontumorigenic basal cell lines have intrinsic capacity for EMT, but a mesenchymal-like phenotype does not correlate with the acquisition of global stem cell/progenitor features. Based on our findings, we propose that EMT in normal basal cells and claudin-low breast cancers reflects aberrant/incomplete myoepithelial differentiation.

title: Tet and TDG mediate DNA demethylation essential for mesenchymal-to-epithelial transition in somatic cell reprogramming.

text: Tet-mediated DNA oxidation is a recently identified mammalian epigenetic modification, and its functional role in cell-fate transitions remains poorly understood. Here, we derive mouse embryonic fibroblasts (MEFs) deleted in all three Tet genes and examine their capacity for reprogramming into induced pluripotent stem cells (iPSCs). We show that Tet-deficient MEFs cannot be reprogrammed because of a block in the mesenchymal-to-epithelial transition (MET) step. Reprogramming of MEFs deficient in TDG is similarly impaired. The block in reprogramming is caused at least in part by defective activation of key miRNAs, which depends on oxidative demethylation promoted by Tet and TDG. Reintroduction of either the affected miRNAs or catalytically active Tet and TDG restores reprogramming in the knockout MEFs. Thus, oxidative demethylation to promote gene activation appears to be functionally required for reprogramming of fibroblasts to pluripotency. These findings provide mechanistic insight into the role of epigenetic barriers in cell-lineage conversion.

title: Murine embryonic stem cell differentiation is promoted by SOCS-3 and inhibited by the zinc finger transcription factor Klf4.

text: Embryonic stem (ES) cells homozygous for a Shp-2 mutation (Shp-2(Delta46-110)) demonstrate leukemia inhibitory factor (LIF) hypersensitivity and increased LIF-stimulated phosphorylation of signal transducer and activator of transcription (STAT3). We hypothesized that LIF-responsive genes in Shp-2(Delta46-110) cells would represent potential candidates for molecules vital for ES cell self-renewal. Using microarray analysis, we detected 41 genes whose expression was modified by LIF in Shp-2(Delta46-110) ES cells. Induction of 2 significantly up-regulated genes, suppressor of cytokine signaling-3 (SOCS-3) and Kruppel-like factor 4 (Klf4), was verified using Northern blotting. ES cells overexpressing SOCS-3 had an increased capacity to differentiate to hematopoietic progenitors, rather than to self-renew. In contrast, ES cells overexpressing Klf4 had a greater capacity to self-renew based on secondary embryoid body (EB) formation. Klf4-transduced d6 EBs expressed higher levels of Oct-4, consistent with the notion that Klf4 promotes ES cell self-renewal. These findings verify the negative role of SOCS-3 on LIF signaling and provide a novel role for Klf4 in ES cell function.

title: Reprogramming committed murine blood cells to induced hematopoietic stem cells with defined factors.

text: Hematopoietic stem cells (HSCs) sustain blood formation throughout life and are the functional units of bone marrow transplantation. We show that transient expression of six transcription factors Run1t1, Hlf, Lmo2, Prdm5, Pbx1, and Zfp37 imparts multilineage transplantation potential onto otherwise committed lymphoid and myeloid progenitors and myeloid effector cells. Inclusion of Mycn and Meis1 and use of polycistronic viruses increase reprogramming efficacy. The reprogrammed cells, designated induced-HSCs (iHSCs), possess clonal multilineage differentiation potential, reconstitute stem/progenitor compartments, and are serially transplantable. Single-cell analysis revealed that iHSCs derived under optimal conditions exhibit a gene expression profile that is highly similar to endogenous HSCs. These findings demonstrate that expression of a set of defined factors is sufficient to activate the gene networks governing HSC functional identity in committed blood cells. Our results raise the prospect that blood cell reprogramming may be a strategy for derivation of transplantable stem cells for clinical application.

title: Circadian mechanisms in murine and human bone marrow mesenchymal stem cells following dexamethasone exposure.

text: A core group of regulatory factors control circadian rhythms in mammalian cells. While the suprachiasmatic nucleus in the brain serves as the central core circadian oscillator, circadian clocks also exist within peripheral tissues and cells. A growing body of evidence has demonstrated that >20% of expressed mRNAs in bone and adipose tissues oscillate in a circadian manner. The current manuscript reports evidence of the core circadian transcriptional apparatus within primary cultures of murine and human bone marrow-derived mesenchymal stem cells (BMSCs). Exposure of confluent, quiescent BMSCs to dexamethasone synchronized the oscillating expression of the mRNAs encoding the albumin D binding protein (dbp), brain-muscle arnt-like 1 (bmal1), period 3 (per3), rev-erb alpha (Rev A), and rev-erb beta (Rev B). The genes displayed a mean oscillatory period of 22.2 to 24.3 h. The acrophase or peak expression of mRNAs encoding "positive" (bmal1) and "negative" (per3) components of the circadian regulatory apparatus were out of phase with each other by approximately 8-12 h, consistent with in vivo observations. In vivo, phosphyrylation by glycogen synthase kinase 3beta (GSK3beta) is known to regulate the turnover of per3 and components of the core circadian regulatory apparatus. In vitro addition of lithium chloride, a GSK3beta inhibitor, significantly shifted the acrophase of all genes by 4.2-4.7 h oscillation in BMSCs; however, only the male murine BMSCs displayed a significant increase in the length of the period of oscillation. We conclude that human and murine BMSCs represent a valid in vitro model for the analysis of circadian mechanisms in bone metabolism and stem cell biology.

title: Haematopoietic stem cells and early lymphoid progenitors occupy distinct bone marrow niches

text: Although haematopoietic stem cells (HSCs) are commonly assumed to reside within a specialized microenvironment, or niche, most published experimental manipulations of the HSC niche have affected the function of diverse restricted progenitors. This raises the fundamental question of whether HSCs and restricted progenitors reside within distinct, specialized niches or whether they share a common niche. Here we assess the physiological sources of the chemokine CXCL12 for HSC and restricted progenitor maintenance. Cxcl12(DsRed) knock-in mice (DsRed-Express2 recombined into the Cxcl12 locus) showed that Cxcl12 was primarily expressed by perivascular stromal cells and, at lower levels, by endothelial cells, osteoblasts and some haematopoietic cells. Conditional deletion of Cxcl12 from haematopoietic cells or nestin-cre-expressing cells had little or no effect on HSCs or restricted progenitors. Deletion of Cxcl12 from endothelial cells depleted HSCs but not myeloerythroid or lymphoid progenitors. Deletion of Cxcl12 from perivascular stromal cells depleted HSCs and certain restricted progenitors and mobilized these cells into circulation. Deletion of Cxcl12 from osteoblasts depleted certain early lymphoid progenitors but not HSCs or myeloerythroid progenitors, and did not mobilize these cells into circulation. Different stem and progenitor cells thus reside in distinct cellular niches in bone marrow: HSCs occupy a perivascular niche and early lymphoid progenitors occupy an endosteal niche.

title: Endothelial KLF2 links local arterial shear stress levels to the expression of vascular tone-regulating genes.

text: Lung Krüppel-like factor (LKLF/KLF2) is an endothelial transcription factor that is crucially involved in murine vasculogenesis and is specifically regulated by flow in vitro. We now show a relation to local flow variations in the adult human vasculature: decreased LKLF expression was noted at the aorta bifurcations to the iliac and carotid arteries, coinciding with neointima formation. The direct involvement of shear stress in the in vivo expression of LKLF was determined independently by in situ hybridization and laser microbeam microdissection/reverse transcriptase-polymerase chain reaction in a murine carotid artery collar model, in which a 4- to 30-fold induction of LKLF occurred at the high-shear sites. Dissection of the biomechanics of LKLF regulation in vitro demonstrated that steady flow and pulsatile flow induced basal LKLF expression 15- and 36-fold at shear stresses greater than approximately 5 dyne/cm2, whereas cyclic stretch had no effect. Prolonged LKLF induction in the absence of flow changed the expression of angiotensin-converting enzyme, endothelin-1, adrenomedullin, and endothelial nitric oxide synthase to levels similar to those observed under prolonged flow. LKLF repression by siRNA suppressed the flow response of endothelin-1, adrenomedullin, and endothelial nitric oxide synthase (P < 0.05). Thus, we demonstrate that endothelial LKLF is regulated by flow in vivo and is a transcriptional regulator of several endothelial genes that control vascular tone in response to flow.

title: Differential expression of bcl-2 in intestinal epithelia. Correlation with attenuation of apoptosis in colonic crypts and the incidence of colonic neoplasia.

text: The cell-positional incidence of both spontaneous and damage-induced apoptosis of epithelial cells was assessed in longitudinal sections of the crypts of small intestine and colon of BDF1 mice. This was compared, using immunohistochemistry, with the pattern of expression of bcl-2, a suppressor of apoptosis. In the small intestine, apoptosis was maximal around cell position 4 from the base of the crypt; this closely corresponds to the position considered to contain the stem cells. In the colon, however, apoptosis was not confined to the area considered to harbour the stem cells (position 1 and 2). Instead, apoptosis was attenuated and distributed along the length of the crypt. Some cells at the base of murine colonic crypts expressed bcl-2 protein, whereas bcl-2 was absent in the crypts of the small intestine. Most pertinently, bcl-2 was absent from small intestinal crypt cells at positions 4-5 (the stem cell region). The importance of the expression of bcl-2 to the attenuation of apoptosis in stem cells was confirmed by analysis of the levels of both spontaneous and induced apoptosis in homozygously bcl-2 null C57BL/6 mice: in colonic crypts the level of spontaneous apoptosis rose significantly, and selectively at the base of the crypt, in comparison with crypts from wild-type animals. In contrast, there was no rise in spontaneous apoptosis in the small intestinal crypts from the bcl-2 null animals. Analysis of sections of human colon and small intestine also showed that expression of bcl-2 was confined to the base of the colonic crypt. The attenuation of apoptosis by bcl-2 in the region of the stem cells of the colonic crypts may dispose these to neoplastic transformation. Indeed, analysis of human carcinomas revealed expression of bcl-2, which in some samples was reciprocal with the expression of p53.

title: Senescent growth arrest in mesenchymal stem cells is bypassed by Wip1-mediated downregulation of intrinsic stress signaling pathways.

text: Human mesenchymal stem cells (hMSCs) have been widely studied as a source of primary adult stem cells for cell therapy because of their multidifferentiation potential; however, the growth arrest (also known as "premature senescence") often found in hMSCs cultured in vitro has been a major obstacle to the in-depth characterization of these cells. In addition, the inability to maintain constant cell growth hampers the development of additional genetic modifications aimed at achieving desired levels of differentiation to specific tissues; however, the molecular mechanisms that govern this phenomenon remain unclear, with the exception of a few studies demonstrating that induction of p16INK4a is responsible for this senescence-like event. Here, we observed that the premature growth arrest in hMSCs occurs in parallel with the induction of p16INK4a, following abrogation of inhibitory phosphorylation of retinoblastoma protein. These stress responses were concurrent with increased formation of reactive oxygen species (ROSs) from mitochondria and increased p38 mitogen-activated protein kinase (MAPK) activity. The introduction of Wip1 (wild-type p53 inducible phosphatase-1), a well-studied stress modulator, significantly lowered p16INK4a expression and led to p38 MAPK inactivation, although it failed to affect the levels of ROSs. Moreover, the suppression of stress responses by Wip1 apparently extended the life span of hMSCs, compared with control conditions, while maintaining their multilineage differentiation potential. Based on these results, we suggest that senescent growth arrest in hMSCs may result from activation of stress signaling pathways and consequent onset of stress responses, due in part to ROS production during prolonged in vitro culture.

title: Disruption of the Hepcidin/Ferroportin Regulatory System Causes Pulmonary Iron Overload and Restrictive Lung Disease

text: Emerging evidence suggests that pulmonary iron accumulation is implicated in a spectrum of chronic lung diseases. However, the mechanism(s) involved in pulmonary iron deposition and its role in the in vivo pathogenesis of lung diseases remains unknown. Here we show that a point mutation in the murine ferroportin gene, which causes hereditary hemochromatosis type 4 (Slc40a1C326S), increases iron levels in alveolar macrophages, epithelial cells lining the conducting airways and lung parenchyma, and in vascular smooth muscle cells. Pulmonary iron overload is associated with oxidative stress, restrictive lung disease with decreased total lung capacity and reduced blood oxygen saturation in homozygous Slc40a1C326S/C326S mice compared to wild-type controls. These findings implicate iron in lung pathology, which is so far not considered a classical iron-related disorder.

title: Detection, characterization, and spontaneous differentiation in vitro of very small embryonic-like putative stem cells in adult mammalian ovary.

text: The present study was undertaken to detect, characterize, and study differentiation potential of stem cells in adult rabbit, sheep, monkey, and menopausal human ovarian surface epithelium (OSE). Two distinct populations of putative stem cells (PSCs) of variable size were detected in scraped OSE, one being smaller and other similar in size to the surrounding red blood cells in the scraped OSE. The smaller 1-3 μm very small embryonic-like PSCs were pluripotent in nature with nuclear Oct-4 and cell surface SSEA-4, whereas the bigger 4-7 μm cells with cytoplasmic localization of Oct-4 and minimal expression of SSEA-4 were possibly the tissue committed progenitor stem cells. Pluripotent gene transcripts of Oct-4, Oct-4A, Nanog, Sox-2, TERT, and Stat-3 in human and sheep OSE were detected by reverse transcriptase-polymerase chain reaction. The PSCs underwent spontaneous differentiation into oocyte-like structures, parthenote-like structures, embryoid body-like structures, cells with neuronal-like phenotype, and embryonic stem cell-like colonies, whereas the epithelial cells transformed into mesenchymal phenotype by epithelial-mesenchymal transition in 3 weeks of OSE culture. Germ cell markers like c-Kit, DAZL, GDF-9, VASA, and ZP4 were immuno-localized in oocyte-like structures. In conclusion, as opposed to the existing view of OSE being a bipotent source of oocytes and granulosa cells, mammalian ovaries harbor distinct very small embryonic-like PSCs and tissue committed progenitor stem cells population that have the potential to develop into oocyte-like structures in vitro, whereas mesenchymal fibroblasts appear to form supporting granulosa-like somatic cells. Research at the single-cell level, including complete gene expression profiling, is required to further confirm whether postnatal oogenesis is a conserved phenomenon in adult mammals.

title: Parathyroid Hormone Directs Bone Marrow Mesenchymal Cell Fate.

text: Intermittent PTH administration builds bone mass and prevents fractures, but its mechanism of action is unclear. We genetically deleted the PTH/PTHrP receptor (PTH1R) in mesenchymal stem cells using Prx1Cre and found low bone formation, increased bone resorption, and high bone marrow adipose tissue (BMAT). Bone marrow adipocytes traced to Prx1 and expressed classic adipogenic markers and high receptor activator of nuclear factor kappa B ligand (Rankl) expression. RANKL levels were also elevated in bone marrow supernatant and serum, but undetectable in other adipose depots. By cell sorting, Pref1+RANKL+ marrow progenitors were twice as great in mutant versus control marrow. Intermittent PTH administration to control mice reduced BMAT significantly. A similar finding was noted in male osteoporotic patients. Thus, marrow adipocytes exhibit osteogenic and adipogenic characteristics, are uniquely responsive to PTH, and secrete RANKL. These studies reveal an important mechanism for PTH's therapeutic action through its ability to direct mesenchymal cell fate.

title: Endogenous bone marrow MSCs are dynamic, fate-restricted participants in bone maintenance and regeneration.

text: Mesenchymal stem cells (MSCs) commonly defined by in vitro functions have entered clinical application despite little definition of their function in residence. Here, we report genetic pulse-chase experiments that define osteoblastic cells as short-lived and nonreplicative, requiring replenishment from bone-marrow-derived, Mx1(+) stromal cells with "MSC" features. These cells respond to tissue stress and migrate to sites of injury, supplying new osteoblasts during fracture healing. Single cell transplantation yielded progeny that both preserve progenitor function and differentiate into osteoblasts, producing new bone. They are capable of local and systemic translocation and serial transplantation. While these cells meet current definitions of MSCs in vitro, they are osteolineage restricted in vivo in growing and adult animals. Therefore, bone-marrow-derived MSCs may be a heterogeneous population with the Mx1(+) population, representing a highly dynamic and stress responsive stem/progenitor cell population of fate-restricted potential that feeds the high cell replacement demands of the adult skeleton.

title: Crypt stem cells as the cells-of-origin of intestinal cancer

text: Intestinal cancer is initiated by Wnt-pathway-activating mutations in genes such as adenomatous polyposis coli (APC). As in most cancers, the cell of origin has remained elusive. In a previously established Lgr5 (leucine-rich-repeat containing G-protein-coupled receptor 5) knockin mouse model, a tamoxifen-inducible Cre recombinase is expressed in long-lived intestinal stem cells. Here we show that deletion of Apc in these stem cells leads to their transformation within days. Transformed stem cells remain located at crypt bottoms, while fuelling a growing microadenoma. These microadenomas show unimpeded growth and develop into macroscopic adenomas within 3-5weeks. The distribution of Lgr5+ cells within stem-cell-derived adenomas indicates that a stem cell/progenitor cell hierarchy is maintained in early neoplastic lesions. When Apc is deleted in short-lived transit-amplifying cells using a different cre mouse, the growth of the induced microadenomas rapidly stalls. Even after 30weeks, large adenomas are very rare in these mice. We conclude that stem-cell-specific loss of Apc results in progressively growing neoplasia.

title: Islet1 derivatives in the heart are of both neural crest and second heart field origin.

text:
RATIONALE Islet1 (Isl1) has been proposed as a marker of cardiac progenitor cells derived from the second heart field and is utilized to identify and purify cardiac progenitors from murine and human specimens for ex vivo expansion. The use of Isl1 as a specific second heart field marker is dependent on its exclusion from other cardiac lineages such as neural crest.
 OBJECTIVE Determine whether Isl1 is expressed by cardiac neural crest.
 METHODS AND RESULTS We used an intersectional fate-mapping system using the RC::FrePe allele, which reports dual Flpe and Cre recombination. Combining Isl1(Cre/+), a SHF driver, and Wnt1::Flpe, a neural crest driver, with Rc::FrePe reveals that some Isl1 derivatives in the cardiac outflow tract derive from Wnt1-expressing neural crest progenitors. In contrast, no overlap was observed between Wnt1-derived neural crest and an alternative second heart field driver, Mef2c-AHF-Cre.
 CONCLUSIONS Isl1 is not restricted to second heart field progenitors in the developing heart but also labels cardiac neural crest. The intersection of Isl1 and Wnt1 lineages within the heart provides a caveat to using Isl1 as an exclusive second heart field cardiac progenitor marker and suggests that some Isl1-expressing progenitor cells derived from embryos, embryonic stem cultures, or induced pluripotent stem cultures may be of neural crest lineage.

title: Islet1 deletion causes kidney agenesis and hydroureter resembling CAKUT.

text: Islet1 (Isl1) is a transcription factor transiently expressed in a subset of heart and limb progenitors. During studies of limb development, conditional Isl1 deletion produced unexpected kidney abnormalities. Here, we studied the renal expression of Isl1 and whether it has a role in kidney development. In situ hybridization revealed Isl1 expression in the mesenchymal cells surrounding the base of the ureteric bud in mice. Conditional deletion of Isl1 caused kidney agenesis or hypoplasia and hydroureter, a phenotype resembling human congenital anomalies of the kidney and urinary tract (CAKUT). The absence of Isl1 led to ectopic branching of the ureteric bud out from the nephric duct or to the formation of accessory buds, both of which could lead to obstruction of the ureter-bladder junction and consequent hydroureter. The abnormal elongation and poor branching of the ureteric buds were the likely causes of the kidney agenesis or hypoplasia. Furthermore, the lack of Isl1 reduced the expression of Bmp4, a gene implicated in the CAKUT-like phenotype, in the metanephric region before ureteric budding. In conclusion, Isl1 is essential for proper development of the kidney and ureter by repressing the aberrant formation of the ureteric bud. These observations call for further studies to investigate whether Isl1 may be a causative gene for human CAKUT.

title: Mammalian RAD52 Functions in Break-Induced Replication Repair of Collapsed DNA Replication Forks

text: Human cancers are characterized by the presence of oncogene-induced DNA replication stress (DRS), making them dependent on repair pathways such as break-induced replication (BIR) for damaged DNA replication forks. To better understand BIR, we performed a targeted siRNA screen for genes whose depletion inhibited G1 to S phase progression when oncogenic cyclin E was overexpressed. RAD52, a gene dispensable for normal development in mice, was among the top hits. In cells in which fork collapse was induced by oncogenes or chemicals, the Rad52 protein localized to DRS foci. Depletion of Rad52 by siRNA or knockout of the gene by CRISPR/Cas9 compromised restart of collapsed forks and led to DNA damage in cells experiencing DRS. Furthermore, in cancer-prone, heterozygous APC mutant mice, homozygous deletion of the Rad52 gene suppressed tumor growth and prolonged lifespan. We therefore propose that mammalian RAD52 facilitates repair of collapsed DNA replication forks in cancer cells.