Comparative evaluation of different modalities for measuring in vivo carnosine levels.

Carnosine is an endogenous di-peptide (ß-alanine -L- histidine) involved in maintaining tissue homeostasis. It is most abundant in skeletal muscle where its concentration has been determined in biopsy samples using tandem mass spectrometry (MS-MS). Carnosine levels can also be assessed in intact leg muscles by proton magnetic resonance spectroscopy (1H-MRS) or in blood and urine samples using mass spectrometry. Nevertheless, it remains uncertain how carnosine levels from these distinct compartments are correlated with each other when measured in the same individual. Furthermore, it is unclear which measurement modality might be most suitable for large-scale clinical studies. Hence, in 31 healthy volunteers, we assessed carnosine levels in skeletal muscle, via 1H-MRS, and in erythrocytes and urine by MS-MS. While muscle carnosine levels were higher in males (C2 peak, p = 0.010; C4 peak, p = 0.018), there was no sex-associated difference in urinary (p = 0.433) or erythrocyte (p = 0.858) levels. In a linear regression model adjusted for age, sex, race, and diet, there was a positive association between erythrocyte and urinary carnosine. However, no association was observed between 1H-MRS and erythrocytes or urinary measures. In the relationship between muscle versus urinary and erythrocyte measures, females had a positive association, while males did not show any association. We also found that 1H-MRS measures were highly sensitive to location of measurement. Thus, it is uncertain whether 1H-MRS can accurately and reliably predict endogenous carnosine levels. In contrast, urinary and erythrocyte carnosine measures may be stable and in greater synchrony, and given financial and logistical concerns, may be a feasible alternative for large-scale clinical studies.

Obesogenic polystyrene microplastic exposures disrupt the gut-liver-adipose axis.

Microplastics (MP) derived from the weathering of polymers, or synthesized in this size range, have become widespread environmental contaminants and have found their way into water supplies and the food chain. Despite this awareness, little is known about the health consequences of MP ingestion. We have previously shown that the consumption of polystyrene (PS) beads was associated with intestinal dysbiosis and diabetes and obesity in mice. To further evaluate the systemic metabolic effects of PS on the gut-liver-adipose tissue axis, we supplied C57BL/6J mice with normal water or that containing 2 sizes of PS beads (0.5 and 5 µm) at a concentration of 1 µg/ml. After 13 weeks, we evaluated indices of metabolism and liver function. As observed previously, mice drinking the PS-containing water had a potentiated weight gain and adipose expansion. Here we found that this was associated with an increased abundance of adipose F4/80+ macrophages. These exposures did not cause nonalcoholic fatty liver disease but were associated with decreased liver:body weight ratios and an enrichment in hepatic farnesoid X receptor and liver X receptor signaling. PS also increased hepatic cholesterol and altered both hepatic and cecal bile acids. Mice consuming PS beads and treated with the berry anthocyanin, delphinidin, demonstrated an attenuated weight gain compared with those mice receiving a control intervention and also exhibited a downregulation of cyclic adenosine monophosphate (cAMP) and peroxisome proliferator-activated receptor (PPAR) signaling pathways. This study highlights the obesogenic role of PS in perturbing the gut-liver-adipose axis and altering nuclear receptor signaling and intermediary metabolism. Dietary interventions may limit the adverse metabolic effects of PS consumption.

Polystyrene bead ingestion promotes adiposity and cardiometabolic disease in mice.

Vast amounts of plastic materials are produced in the modern world and despite recycling efforts, large amounts are disposed in water systems and landfills. Under these storage conditions, physical weathering and photochemical processes break down these materials into smaller particles of the micro- and nano-scale. In addition, ecosystems can be contaminated with plastic particles which are manufactured in these size ranges for commercial purposes. Independent of source, microplastics are abundant in the environment and have found their way into water supplies and the food cycle where human exposure is inevitable. Nevertheless, the health consequences of microplastic ingestion, inhalation, or absorption are largely unknown. In this study we sought to determine if ingestion of microplastics promoted pre-clinical cardiovascular disease (CVD). To do this, we supplied mice with normal drinking water or that supplemented with polystyrene beads of two different sizes (0.5 µm and 5 µm) and two different doses (0.1 µg/ml and 1 µg/ml) each for 12 weeks and measured several indices of metabolism and glucose homeostasis. As early as 3 weeks of consumption, we observed an accelerated weight gain with a corresponding increase in body fat for some exposure groups versus the control mice. Some exposure groups demonstrated increased levels of fasting plasma glucose. Those mice consuming the smaller sized beads (0.5 µm) at the higher dose (1 µg/ml), had increased levels of fasting plasma insulin and higher homeostatic model assessment of insulin resistance (HOMA-IR) scores as well. This was accompanied by changes in the gut microbiome consistent with an obese phenotype. Using samples of perivascular adipose tissue collected from the same group, we observed changes in gene expression consistent with increased adipogenesis. These results suggest that ingestion of polystyrene beads promotes a cardiometabolic disease phenotype and thus may be an unrecognized risk factor for CVD.

Subclinical markers of cardiovascular toxicity of benzene inhalation in mice.

Benzene is a ubiquitous environmental pollutant. Recent population-based studies suggest that benzene exposure is associated with an increased risk for cardiovascular disease. However, it is unclear whether benzene exposure by itself is sufficient to induce cardiovascular toxicity. We examined the effects of benzene inhalation (50 ppm, 6 h/day, 5 days/week, 6 weeks) or HEPA-filtered air exposure on the biomarkers of cardiovascular toxicity in male C57BL/6J mice. Benzene inhalation significantly increased the biomarkers of endothelial activation and injury including endothelial microparticles, activated endothelial microparticles, endothelial progenitor cell microparticles, lung endothelial microparticles, and activated lung and endothelial microparticles while having no effect on circulating levels of endothelial adhesion molecules, endothelial selectins, and biomarkers of angiogenesis. To understand how benzene may induce endothelial injury, we exposed human aortic endothelial cells to benzene metabolites. Of the metabolites tested, trans,trans-mucondialdehyde (10 µM, 18h) was the most toxic. It induced caspases-3, -7 and -9 (intrinsic pathway) activation and enhanced microparticle formation by 2.4-fold. Levels of platelet-leukocyte aggregates, platelet macroparticles, and a proportion of CD4+ and CD8+ T-cells were also significantly elevated in the blood of the benzene-exposed mice. We also found that benzene exposure increased the transcription of genes associated with endothelial cell and platelet activation in the liver; and induced inflammatory genes and suppressed cytochrome P450s in the lungs and the liver. Together, these data suggest that benzene exposure induces endothelial injury, enhances platelet activation and inflammatory processes; and circulatory levels of endothelial cell and platelet-derived microparticles and platelet-leukocyte aggregates are excellent biomarkers of cardiovascular toxicity of benzene.

Exposure to Fine Particulate Matter Air Pollution Alters mRNA and miRNA Expression in Bone Marrow-Derived Endothelial Progenitor Cells from Mice.

Exposure to fine particulate matter (PM2.5) air pollution is associated with quantitative deficits of circulating endothelial progenitor cells (EPCs) in humans. Related exposures of mice to concentrated ambient PM2.5 (CAP) likewise reduces levels of circulating EPCs and induces defects in their proliferation and angiogenic potential as well. These changes in EPC number or function are predictive of larger cardiovascular dysfunction. To identify global, PM2.5-dependent mRNA and miRNA expression changes that may contribute to these defects, we performed a transcriptomic analysis of cells isolated from exposed mice. Compared with control samples, we identified 122 upregulated genes and 44 downregulated genes in EPCs derived from CAP-exposed animals. Functions most impacted by these gene expression changes included regulation of cell movement, cell and tissue development, and cellular assembly and organization. With respect to miRNA changes, we found that 55 were upregulated while 53 were downregulated in EPCs from CAP-exposed mice. The top functions impacted by these miRNA changes included cell movement, cell death and survival, cellular development, and cell growth and proliferation. A subset of these mRNA and miRNA changes were confirmed by qRT-PCR, including some reciprocal relationships. These results suggest that PM2.5-induced changes in gene expression may contribute to EPC dysfunction and that such changes may contribute to the adverse cardiovascular outcomes of air pollution exposure.

Fine particulate matter air pollution and aortic perivascular adipose tissue: Oxidative stress, leptin, and vascular dysfunction.

Exposure to fine particulate matter (PM2.5 ) air pollution increases blood pressure, induces vascular inflammation and dysfunction, and augments atherosclerosis in humans and rodents; however, the understanding of early changes that foster chronic vascular disease is incomplete. Because perivascular adipose tissue (PVAT) inflammation is implicated in chronic vascular diseases, we investigated changes in aortic PVAT following short-term air pollution exposure. Mice were exposed to HEPA-filtered or concentrated ambient PM2.5 (CAP) for 9 consecutive days, and the abundance of inflammatory, adipogenic, and adipokine gene mRNAs was measured by gene array and qRT-PCR in thoracic aortic PVAT. Responses of the isolated aorta with and without PVAT to contractile (phenylephrine, PE) and relaxant agonists (acetylcholine, ACh; sodium nitroprusside, SNP) were measured. Exposure to CAP significantly increased the urinary excretion of acrolein metabolite (3HPMA) as well as the abundance of protein-acrolein adducts (a marker of oxidative stress) in PVAT and aorta, upregulated PVAT leptin mRNA expression without changing mRNA levels of several proinflammatory genes, and induced PVAT insulin resistance. In control mice, PVAT significantly depressed PE-induced contractions-an effect that was dampened by CAP exposure. Pulmonary overexpression of extracellular dismutase (ecSOD-Tg) prevented CAP-induced effects on urinary 3HPMA levels, PVAT Lep mRNA, and alterations in PVAT and aortic function, reflecting a necessary role of pulmonary oxidative stress in all of these deleterious CAP-induced changes. More research is needed to address how exactly short-term exposure to PM2.5 perturbs PVAT and aortic function, and how these specific genes and functional changes in PVAT could lead over time to chronic inflammation, endothelial dysfunction, and atherosclerosis.

Endothelial progenitor cells as critical mediators of environmental air pollution-induced cardiovascular toxicity.

Environmental air pollution exposure is a leading cause of death worldwide, and with increasing industrialization and urbanization, its disease burden is expected to rise even further. The majority of air pollution exposure-associated deaths are linked to cardiovascular disease (CVD). Although ample research demonstrates a strong correlation between air pollution exposure and CVD risk, the mechanisms by which inhalation of polluted air affects cardiovascular health are not completely understood. Inhalation of environmental air pollution has been associated with endothelial dysfunction, which suggests that air pollution exposure impacts CVD health by inducing endothelial injury. Interestingly, recent studies demonstrate that air pollution exposure affects the number and function of endothelial progenitor cells (EPCs), subpopulations of bone marrow-derived proangiogenic cells that have been shown to play an essential role in maintaining cardiovascular health. In line with their beneficial function, chronically low levels of circulating EPCs and EPC dysfunction (e.g., in diabetic patients) have been associated with vascular dysfunction, poor cardiovascular health, and increases in the severity of cardiovascular outcomes. In contrast, treatments that improve EPC number and function (e.g., exercise) have been found to attenuate cardiovascular dysfunction. Considering the critical, nonredundant role of EPCs in maintaining vascular health, air pollution exposure-induced impairments in EPC number and function could lead to endothelial dysfunction, consequently increasing the risk for CVD. This review article covers novel aspects and new mechanistic insights of the adverse effects of air pollution exposure on cardiovascular health associated with changes in EPC number and function.

Urinary Levels of the Acrolein Conjugates of Carnosine Are Associated with Cardiovascular Disease Risk.

Carnosine is a naturally occurring dipeptide (ß-alanine-L-histidine) which supports physiological homeostasis by buffering intracellular pH, chelating metals, and conjugating with and neutralizing toxic aldehydes such as acrolein. However, it is not clear if carnosine can support cardiovascular function or modify cardiovascular disease (CVD) risk. To examine this, we measured urinary levels of nonconjugated carnosine and its acrolein conjugates (carnosine-propanal and carnosine-propanol) in participants of the Louisville Healthy Heart Study and examined associations with indices of CVD risk. We found that nonconjugated carnosine was significantly associated with hypertension (p = 0.011), heart failure (p = 0.015), those categorized with high CVD risk (p < 0.001), body mass index (BMI; p = 0.007), high sensitivity C-reactive protein (hsCRP; p = 0.026), high-density lipoprotein (HDL; p = 0.007) and certain medication uses. Levels of carnosine-propanal and carnosine-propanol demonstrated significant associations with BMI, blood glucose, HDL and diagnosis of diabetes. Carnosine-propanal was also associated with heart failure (p = 0.045) and hyperlipidemia (p = 0.002), but no associations with myocardial infarction or stroke were identified. We found that the positive associations of carnosine conjugates with diabetes and HDL remain statistically significant (p < 0.05) in an adjusted, linear regression model. These findings suggest that urinary levels of nonconjugated carnosine, carnosine-propanal and carnosine-propanol may be informative biomarkers for the assessment of CVD risk-and particularly reflective of skeletal muscle injury and carnosine depletion in diabetes.

Protocol to assess the efficacy of carnosine supplementation in mitigating the adverse cardiovascular responses to particulate matter (PM) exposure: the Nucleophilic Defense Against PM Toxicity (NEAT) trial.

INTRODUCTION: Exposure to airborne particulate matter (PM) is associated with cardiovascular disease. These outcomes are believed to originate from pulmonary oxidative stress and the systemic delivery of oxidised biomolecules (eg, aldehydes) generated in the lungs. Carnosine is an endogenous di-peptide (ß-alanine-L-histidine) which promotes physiological homeostasis in part by conjugating to and neutralising toxic aldehydes. We hypothesise that an increase of endogenous carnosine by dietary supplementation would mitigate the adverse cardiovascular outcomes associated with PM exposure in humans. METHODS AND ANALYSIS: To test this, we designed the Nucleophilic Defense Against PM Toxicity trial. This trial will enroll 240 participants over 2 years and determine if carnosine supplementation mitigates the adverse effects of PM inhalation. The participants will have low levels of endogenous carnosine to facilitate identification of supplementation-specific outcomes. At enrollment, we will measure several indices of inflammation, preclinical cardiovascular disease and physical function. Participants will be randomly allocated to carnosine or placebo groups and instructed to take their oral supplement for 12 weeks with two return clinical visits and repeated assessments during times of peak PM exposure (June-September) in Louisville, Kentucky, USA. Statistical modelling approaches will be used to assess the efficacy of carnosine supplementation in mitigating adverse outcomes. ETHICS AND DISSEMINATION: This study protocol has been approved by the Institutional Review Board at the University of Louisville. Results from this study will be disseminated at scientific conferences and in peer-reviewed publications.Trial registration: NCT03314987; Pre-results.

Urinary levels of the acrolein conjugates of carnosine are associated with inhaled toxicants.

OBJECTIVE: The inhalation of air-borne toxicants is associated with adverse health outcomes which can be somewhat mitigated by enhancing endogenous anti-oxidant capacity. Carnosine is a naturally occurring dipeptide (ß-alanine-L-histidine), present in high abundance in skeletal and cardiac muscle. This multi-functional dipeptide has anti-oxidant properties, can buffer intracellular pH, chelate metals, and sequester aldehydes such as acrolein. Due to these chemical properties, carnosine may be protective against inhaled pollutants which can contain metals and aldehydes and can stimulate the generation of electrophiles in exposed tissues. Thus, assessment of carnosine levels, or levels of its acrolein conjugates (carnosine-propanal and carnosine-propanol) may inform on level of exposure and risk assessment. METHODS: We used established mass spectroscopy methods to measure levels of urinary carnosine (n = 605) and its conjugates with acrolein (n = 561) in a subset of participants in the Louisville Healthy Heart Study (mean age = 51 ± 10; 52% male). We then determined associations between these measures and air pollution exposure and smoking behavior using statistical modeling approaches. RESULTS: We found that higher levels of non-conjugated carnosine, carnosine-propanal, and carnosine-propanol were significantly associated with males (p < 0.02) and those of Caucasian ethnicity (p < 0.02). Levels of carnosine-propanol were significantly higher in never-smokers (p = 0.001) but lower in current smokers (p = 0.037). This conjugate also demonstrated a negative association with mean-daily particulate air pollution (PM2.5) levels (p = 0.01). CONCLUSIONS: These findings suggest that urinary levels of carnosine-propanol may inform as to risk from inhaled pollutants.

Choice of library size normalization and statistical methods for differential gene expression analysis in balanced two-group comparisons for RNA-seq studies.

BACKGROUND: High-throughput RNA sequencing (RNA-seq) has evolved as an important analytical tool in molecular biology. Although the utility and importance of this technique have grown, uncertainties regarding the proper analysis of RNA-seq data remain. Of primary concern, there is no consensus regarding which normalization and statistical methods are the most appropriate for analyzing this data. The lack of standardized analytical methods leads to uncertainties in data interpretation and study reproducibility, especially with studies reporting high false discovery rates. In this study, we compared a recently developed normalization method, UQ-pgQ2, with three of the most frequently used alternatives including RLE (relative log estimate), TMM (Trimmed-mean M values) and UQ (upper quartile normalization) in the analysis of RNA-seq data. We evaluated the performance of these methods for gene-level differential expression analysis by considering the factors, including: 1) normalization combined with the choice of a Wald test from DESeq2 and an exact test/QL (Quasi-likelihood) F-Test from edgeR; 2) sample sizes in two balanced two-group comparisons; and 3) sequencing read depths. RESULTS: Using the MAQC RNA-seq datasets with small sample replicates, we found that UQ-pgQ2 normalization combined with an exact test can achieve better performance in term of power and specificity in differential gene expression analysis. However, using an intra-group analysis of false positives from real and simulated data, we found that a Wald test performs better than an exact test when the number of sample replicates is large and that a QL F-test performs the best given sample sizes of 5, 10 and 15 for any normalization. The RLE, TMM and UQ methods performed similarly given a desired sample size. CONCLUSION: We found the UQ-pgQ2 method combined with an exact test/QL F-test is the best choice in order to control false positives when the sample size is small. When the sample size is large, UQ-pgQ2 with a QL F-test is a better choice for the type I error control in an intra-group analysis. We observed read depths have a minimal impact for differential gene expression analysis based on the simulated data.

Exposure to airborne fine particulate matter is associated with impaired endothelial function and biomarkers of oxidative stress and inflammation.

Epidemiological evidence suggests that exposure to air pollution is a leading risk factor for cardiovascular disease (CVD). However, there is little direct evidence linking exposure to vascular dysfunction. We conducted a cross-sectional study of 100 participants, recruited from the University of Louisville Clinics. Endothelial function was assessed by calculating the reactive hyperemia index (RHI). Oxidative stress was indexed by measuring urinary levels of isoprostanes (n = 91). Inflammatory biomarkers were measured in the plasma (n = 80). Daily average PM2.5 levels were obtained from regional monitoring stations. Adjusted associations between PM2.5 levels and measured outcomes were tested using generalized linear models. The average age of participants was 48 years (44% male, 62% white); 52% had a diagnosis of hypertension, and 44% had type-2 diabetes. A 12.4% decrease in RHI was associated with 10 µg/m3 increase in PM2.5 (95% CI: 21.0, -2.7). The F-2 isoprostane metabolite showed a positive association of 28.4% (95% CI: 2.7, 60.3) per 10 µg/m3 increase in PM2.5. Positive associations were observed with angiopoietin 1 (17.4%; 95% CI: 2.8, 33.8), vascular endothelial growth factor (10.4%; 95% CI: 0.6, 21.0), placental growth factor (31.7%; 95% CI: 12.2, 54.5), intracellular adhesion molecule-1 (24.6%; 95% CI: 1.6, 52.8), and matrix metalloproteinase-9 (30.3%; 95% CI: 8.0, 57.5) per 10 µg/m3 increase in PM2.5. Additionally, a 10 µg/m3 increase in PM2.5 was associated with 15.9% decrease in vascular cell adhesion molecule-1 (95% CI: 28.3, -1.3). These findings suggest that exposure to PM2.5 is associated with impaired vascular function, which may result from oxidative stress and inflammation, thereby leading to a pro-atherogenic state.

Carnosine Supplementation Mitigates the Deleterious Effects of Particulate Matter Exposure in Mice.

Background Exposure to fine airborne particulate matter ( PM 2.5) induces quantitative and qualitative defects in bone marrow-derived endothelial progenitor cells of mice, and similar outcomes in humans may contribute to vascular dysfunction and the cardiovascular morbidity and mortality associated with PM 2.5 exposure. Nevertheless, mechanisms underlying the pervasive effects of PM 2.5 are unclear and effective interventional strategies to mitigate against PM 2.5 toxicity are lacking. Furthermore, whether PM 2.5 exposure affects other types of bone marrow stem cells leading to additional hematological or immunological dysfunction is not clear. Methods and Results Mice given normal drinking water or that supplemented with carnosine, a naturally occurring, nucleophilic di-peptide that binds reactive aldehydes, were exposed to filtered air or concentrated ambient particles. Mice drinking normal water and exposed to concentrated ambient particles demonstrated a depletion of bone marrow hematopoietic stem cells but no change in mesenchymal stem cells. However, HSC depletion was significantly attenuated when the mice were placed on drinking water containing carnosine. Carnosine supplementation also increased the levels of carnosine-propanal conjugates in the urine of CAPs-exposed mice and prevented the concentrated ambient particles-induced dysfunction of endothelial progenitor cells as assessed by in vitro and in vivo assays. Conclusions These results suggest that exposure to PM 2.5 has pervasive effects on different bone marrow stem cell populations and that PM 2.5-induced hematopoietic stem cells depletion, endothelial progenitor cell dysfunction, and defects in vascular repair can be mitigated by excess carnosine. Carnosine supplementation may be a viable approach for preventing PM 2.5-induced immune dysfunction and cardiovascular injury in humans.

Acute exposure to air pollution is associated with novel changes in blood levels of endothelin-1 and circulating angiogenic cells in young, healthy adults.

Acute and chronic exposures to particulate matter (PM2.5) air pollution increase the risk for cardiovascular disease (CVD). A hypothesized mechanism linking PM2.5 exposure and CVD is the induction of endothelial dysfunction - a key step to increased CVD risk. Although PM2.5 exposure is associated with endothelial dysfunction and the vasoconstrictor peptide endothelin-1 (ET-1) is upregulated in endothelial dysfunction, the effects of PM2.5 on ET-1 and whether or not ET-1 mediates the downstream effects of PM2.5 are unclear. In addition to examining associations between acute changes in ambient PM2.5 and circulating levels of ET-1, we also looked at whether changes in ET-1 were associated with changes in markers of vascular health and systemic injury. For example, endothelial function is maintained in part by circulating angiogenic cell (CAC)-mediated repair, and our recent studies show that CACs in humans and mice are decreased by ambient PM2.5 exposure. In the current study, we recruited young, healthy adults who were exposed to natural variations in PM2.5, and we analyzed associations between PM2.5 and circulating levels of ET-1, between ET-1 and CACs, and between ET-1 and other biomarkers of injury using linear regression analyses. Surprisingly, ET-1 levels were negatively associated with PM2.5 levels (ß = -0.773, P = 0.0005), yet, in contrast, positively associated with two CACs: CAC-2 (CD31+/CD34+/CD45+) and CAC-4 (CD31+/CD34+/CD45+/CD133+). Interestingly, ET-1 levels were negatively associated with some biomarkers (platelet factor 4, ß = -0.148, P = 0.0003; triglycerides, ß = -0.095, P = 0.041) and positively with other biomarkers: albumin (ß = 0.035, P = 0.006) and IL-lß (ß = 0.082, P = 0.012). These findings further reveal the insidious nature of PI2.5's anti-angiogenic effect including a novel relationship between ET-1 and CACs in young adults exposed to acute elevations of air pollution.

Benzene Exposure Induces Insulin Resistance in Mice.

Benzene is a ubiquitous pollutant associated with hematotoxicity but its metabolic effects are unknown. We sought to determine if and how exposure to volatile benzene impacted glucose handling. We exposed wild type C57BL/6 mice to volatile benzene (50 ppm × 6 h/day) or HEPA-filtered air for 2 or 6 weeks and measured indices of oxidative stress, inflammation, and insulin signaling. Compared with air controls, we found that mice inhaling benzene demonstrated increased plasma glucose (p = .05), insulin (p = .03), and HOMA-IR (p = .05), establishing a state of insulin and glucose intolerance. Moreover, insulin-stimulated Akt phosphorylation was diminished in the liver (p = .001) and skeletal muscle (p = .001) of benzene-exposed mice, accompanied by increases in oxidative stress and Nf-κb phosphorylation (p = .025). Benzene-exposed mice also demonstrated elevated levels of Mip1-α transcripts and Socs1 (p = .001), but lower levels of Irs-2 tyrosine phosphorylation (p = .0001). Treatment with the superoxide dismutase mimetic, TEMPOL, reversed benzene-induced effects on oxidative stress, Nf-κb phosphorylation, Socs1 expression, Irs-2 tyrosine phosphorylation, and systemic glucose intolerance. These findings suggest that exposure to benzene induces insulin resistance and that this may be a sensitive indicator of inhaled benzene toxicity. Persistent ambient benzene exposure may be a heretofore unrecognized contributor to the global human epidemics of diabetes and cardiovascular disease.

Association Between Residential Greenness and Cardiovascular Disease Risk.

Background Exposure to green vegetation has been linked to positive health, but the pathophysiological processes affected by exposure to vegetation remain unclear. To study the relationship between greenness and cardiovascular disease, we examined the association between residential greenness and biomarkers of cardiovascular injury and disease risk in susceptible individuals. Methods and Results In this cross-sectional study of 408 individuals recruited from a preventive cardiology clinic, we measured biomarkers of cardiovascular injury and risk in participant blood and urine. We estimated greenness from satellite-derived normalized difference vegetation index ( NDVI ) in zones with radii of 250 m and 1 km surrounding the participants' residences. We used generalized estimating equations to examine associations between greenness and cardiovascular disease biomarkers. We adjusted for residential clustering, demographic, clinical, and environmental variables. In fully adjusted models, contemporaneous NDVI within 250 m of participant residence was inversely associated with urinary levels of epinephrine (-6.9%; 95% confidence interval, -11.5, -2.0/0.1 NDVI ) and F2-isoprostane (-9.0%; 95% confidence interval, -15.1, -2.5/0.1 NDVI ). We found stronger associations between NDVI and urinary epinephrine in women, those not on ß-blockers, and those who had not previously experienced a myocardial infarction. Of the 15 subtypes of circulating angiogenic cells examined, 11 were inversely associated (8.0-15.6% decrease/0.1 NDVI ), whereas 2 were positively associated (37.6-45.8% increase/0.1 NDVI ) with contemporaneous NDVI . Conclusions Independent of age, sex, race, smoking status, neighborhood deprivation, statin use, and roadway exposure, residential greenness is associated with lower levels of sympathetic activation, reduced oxidative stress, and higher angiogenic capacity.

Circulating angiogenic stem cells in type 2 diabetes are associated with glycemic control and endothelial dysfunction.

Circulating angiogenic cells (CACs) of various described phenotypes participate in the regeneration of the damaged endothelium, but the abundance of these cells is highly influenced by external cues including diabetes. It is not entirely clear which CAC populations are most reflective of endothelial function nor which are impacted by diabetes. To answer these questions, we enrolled a human cohort with variable CVD risk and determined relationships between stratified levels of CACs and indices of diabetes and vascular function. We also determined associations between CAC functional markers and diabetes and identified pro-angiogenic molecules which are impacted by diabetes. We found that subjects with low levels of CD34+/AC133+/CD31+/CD45dim cells (CAC-3) had a significantly higher incidence of diabetes (p = 0.004), higher HbA1c levels (p = 0.049) and higher CVD risk scores. Furthermore, there was an association between low CAC-3 levels and impaired vascular function (p = 0.023). These cells from diabetics had reduced levels of CXCR4 and VEGFR2, while diabetics had higher levels of certain cytokines and pro-angiogenic molecules. These results suggest that quantitative and functional defects of CD34+/AC133+/CD31+/CD45dim cells are associated with diabetes and vascular impairment and that this cell type may be a prognostic indicator of CVD and vascular dysfunction.

A combined approach with gene-wise normalization improves the analysis of RNA-seq data in human breast cancer subtypes.

Breast cancer (BC) is increasing in incidence and resistance to treatment worldwide. The challenges in limited therapeutic options and poor survival outcomes in BC subtypes persist because of its molecular heterogeneity and resistance to standard endocrine therapy. Recently, high throughput RNA sequencing (RNA-seq) has been used to identify biomarkers of disease progression and signaling pathways that could be amenable to specific therapies according to the BC subtype. However, there is no single generally accepted pipeline for the analysis of RNA-seq data in biomarker discovery due, in part, to the needs of simultaneously satisfying constraints of sensitivity and specificity. We proposed a combined approach using gene-wise normalization, UQ-pgQ2, followed by a Wald test from DESeq2. Our approach improved the analysis based on within-group comparisons in terms of the specificity when applied to publicly available RNA-seq BC datasets. In terms of identifying differentially expressed genes (DEGs), we combined an optimized log2 fold change cutoff with a nominal false discovery rate of 0.05 to further minimize false positives. Using this method in the analysis of two GEO BC datasets, we identified 797 DEGs uniquely expressed in triple negative BC (TNBC) and significantly associated with T cell and immune-related signaling, contributing to the immunotherapeutic efficacy in TNBC patients. In contrast, we identified 1403 DEGs uniquely expressed in estrogen positive and HER2 negative BC (ER+HER2-BC) and significantly associated with eicosanoid, notching and FAK signaling while a common set of genes was associated with cellular growth and proliferation. Thus, our approach to control for false positives identified two distinct gene expression profiles associated with these two subtypes of BC which are distinguishable by their molecular and functional attributes.