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.

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.

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.

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.

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.

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.

Inhalation of Fine Particulate Matter Impairs Endothelial Progenitor Cell Function Via Pulmonary Oxidative Stress.

OBJECTIVE: Exposure to fine particulate matter (PM2.5) air pollution is associated with the depletion of circulating endothelial progenitor cells (EPCs), as well as vascular injury and dysfunction. Nevertheless, it remains unclear whether PM2.5 exposure leads to significant impairments in EPC function. Hence, we studied the effects of PM2.5 on EPC-mediated recovery of vascular perfusion after hindlimb ischemia and examined the mechanisms whereby PM2.5 exposure affects EPC abundance and function. APPROACH AND RESULTS: In comparison with EPCs isolated from mice breathing filtered air, EPCs from mice exposed for 9 consecutive days (6 hours per day) to concentrated ambient PM2.5 (CAP) had defects in both proliferation and tube formation. However, CAP exposure of mice overexpressing extracellular superoxide dismutase (ecSOD-Tg) in the lungs did not affect EPC tube formation. Exposure to CAP also suppressed circulating EPC levels, VEGF (vascular endothelial growth factor)-stimulated aortic Akt phosphorylation, and plasma NO levels in wild-type but not in ecSOD-Tg mice. EPCs from CAP-exposed wild-type mice failed to augment basal recovery of hindlimb perfusion when injected into unexposed mice subjected to hindlimb ischemia; however, these deficits in recovery of hindlimb perfusion were absent when using EPCs derived from CAP-exposed ecSOD-Tg mice. The improved reparative function of EPCs from CAP-exposed ecSOD-Tg mice was also reflected by greater expression of Mmp-9 and Nos3 when compared with EPCs from CAP-exposed wild-type mice. CONCLUSIONS: Exposure to PM2.5 impairs EPC abundance and function and prevents EPC-mediated vascular recovery after hindlimb ischemia. This defect is attributed, in part, to pulmonary oxidative stress and was associated with vascular VEGF resistance and a decrement in NO bioavailability.

Residential Proximity to Major Roadways Is Associated With Increased Levels of AC133+ Circulating Angiogenic Cells.

OBJECTIVES: Previous studies have shown that residential proximity to a roadway is associated with increased cardiovascular disease risk. Yet, the nature of this association remains unclear, and its effect on individual cardiovascular disease risk factors has not been assessed. The objective of this study was to determine whether residential proximity to roadways influences systemic inflammation and the levels of circulating angiogenic cells. APPROACH AND RESULTS: In a cross-sectional study, cardiovascular disease risk factors, blood levels of C-reactive protein, and 15 antigenically defined circulating angiogenic cell populations were measured in participants (n=316) with moderate-to-high cardiovascular disease risk. Attributes of roadways surrounding residential locations were assessed using geographic information systems. Associations between road proximity and cardiovascular indices were analyzed using generalized linear models. Close proximity (<50 m) to a major roadway was associated with lower income and higher rates of smoking but not C-reactive protein levels. After adjustment for potential confounders, the levels of circulating angiogenic cells in peripheral blood were significantly elevated in people living in close proximity to a major roadway (CD31(+)/AC133(+), AC133(+), CD34(+)/AC133(+), and CD34(+)/45(dim)/AC133(+) cells) and positively associated with road segment distance (CD31(+)/AC133(+), AC133(+), and CD34(+)/AC133(+) cells), traffic intensity (CD31(+)/AC133(+) and AC133(+) cells), and distance-weighted traffic intensity (CD31(+)/34(+)/45(+)/AC133(+) cells). CONCLUSIONS: Living close to a major roadway is associated with elevated levels of circulating cells positive for the early stem marker AC133(+). This may reflect an increased need for vascular repair. Levels of these cells in peripheral blood may be a sensitive index of cardiovascular injury because of residential proximity to roadways.

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.

Episodic exposure to fine particulate air pollution decreases circulating levels of endothelial progenitor cells.

RATIONALE: Acute and chronic exposures to airborne particulate matter (PM) have been linked in epidemiological studies to a wide spectrum of cardiovascular disorders that are characterized by a dysfunctional endothelium. The pathophysiological mechanisms underlying these associations are unclear. OBJECTIVE: To examine whether exposure to fine PM with an aerodynamic diameter of <2.5 microm (PM(2.5)) affects the circulating levels of endothelial progenitor cell (EPC) populations, systemic inflammation and coagulation. METHODS AND RESULTS: Phenotypically distinct EPC populations were quantified by flow cytometry in young (18 to 25 years) adult humans exposed to episodic increases in PM(2.5) along the Wasatch Mountain Front in Utah. In addition, Sca-1+/Flk-1+ cells were measured in the peripheral blood of mice exposed to concentrated particles from ambient air in Louisville, Ky. In both studies, PM exposure was negatively correlated with circulating EPC levels. In humans, statistically significant associations between PM(2.5) exposure and the plasma levels of platelet-monocyte aggregates, high-density lipoprotein, and nonalbumin protein were also observed. Episodic increases in PM(2.5) did not change plasma levels of C-reactive protein, interleukin-1beta, interleukin-6, fibrinogen, or serum amyloid A. CONCLUSIONS: Episodic exposure to PM(2.5) induces reversible vascular injury, reflected in part by depletion of circulating EPC levels, and increases in platelet activation and the plasma level of high-density lipoprotein. These changes were also accompanied by an increase in nonalbumin protein and may be related to mechanisms by which exposure to particulate air pollution increases the risk of cardiovascular disease and adverse cardiovascular events.

Acrolein inhalation prevents vascular endothelial growth factor-induced mobilization of Flk-1+/Sca-1+ cells in mice.

OBJECTIVE: Acrolein is a toxic chemical present in tobacco, wood, and coal smoke, as well as automobile exhaust. Because exposure to these pollutants is associated with an increase in cardiovascular disease risk, we studied the effects of acrolein on Flk-1(+)/Sca-1(+) cells that are involved in vascular repair. METHODS AND RESULTS: In adult male C57BL/6 mice, inhalation of acrolein (1 part per million [ppm], 6 hours/day for 4 days or 5 ppm for 2 or 6 hours) led to the formation of protein-acrolein adducts in the bone marrow and diminished levels of plasma nitric oxide metabolites and circulating Flk-1(+)/Sca-1(+) but not Sca-1(+)-only cells. Acrolein exposure increased the number of apoptotic Flk-1(+)/Sca-1(+) cells in circulation and increased bone marrow-derived cells with endothelial characteristics (DiI-ac-low-density lipoprotein [DiI-acLDL]/UE-lectin and Flk-1(+)/Sca-1(+)) in culture. Deficits in the circulating levels of Flk-1(+)/Sca-1(+) cells were reversed after 7 days of recovery in acrolein-free air. Exposure to acrolein blocked vascular endothelial growth factor (VEGF)/AMD3100-stimulated mobilization of Flk-1(+)/Sca-1(+) but not Sca-1(+)-only cells and prevented VEGF-induced phosphorylation of Akt and endothelial nitric oxide synthase in the aorta. CONCLUSIONS: Inhalation of acrolein increases apoptosis and suppresses the circulating levels of Flk-1(+)/Sca-1(+) cells while increasing these cells in the bone marrow and preventing their mobilization by VEGF. Exposure to acrolein-rich pollutants could impair vascular repair capacity.

Tiam1 is recruited to ß1-integrin complexes by 14-3-3ζ where it mediates integrin-induced Rac1 activation and motility.

14-3-3 is an adaptor protein that localizes to the leading edge of spreading cells, returning to the cytoplasm as spreading ceases. Previously, we showed that integrin-induced Rac1 activation and spreading were inhibited by sequestration of 14-3-3ζ and restored by its overexpression. Here, we determined whether 14-3-3 mediates integrin signaling by localizing a guanine nucleotide exchange factor (GEF) to Rac1-activating integrin complexes. We showed that GST-14-3-3ζ recruited the Rac1-GEF, Tiam1, from cell lysates through Tiam1 residues 1-182 (N(1-182) Tiam1). The physiological relevance of this interaction was examined in serum-starved Hela cells plated on fibronectin. Both Tiam1 and N(1-182) Tiam1 were recruited to 14-3-3-containing ß1-integrin complexes, as shown by co-localization and co-immunoprecipitation. Integrin-induced Rac1 activation was inhibited when Tiam1 was depleted with siRNA or by overexpression of catalytically inactive N(1-182) Tiam1, which was incorporated into 14-3-3/ß1-integrin complexes and inhibited spreading in a manner that was overcome by constitutively active Rac1. Integrin-induced Rac1 activation, spreading, and migration were also inhibited by overexpression of 14-3-3ζ S58D, which was unable to recruit Tiam1 from lysates, co-immunoprecipitate with Tiam1, or mediate its incorporation into ß1-integrin complexes. Taken together, these findings suggest a previously unrecognized mechanism of integrin-induced Rac1 activation in which 14-3-3 dimers localize Tiam1 to integrin complexes, where it mediates integrin-dependent Rac1 activation, thus initiating motility-inducing pathways. Moreover, since Tiam1 is recruited to other sites of localized Rac1 activation through its PH-CC-EX domain, the present findings show that a mechanism involving its N-terminal 182 residues is utilized to recruit Tiam1 to motility-inducing integrin complexes.

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.

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.

Exposure to acrolein by inhalation causes platelet activation.

Acrolein is a common air pollutant that is present in high concentrations in wood, cotton, and tobacco smoke, automobile exhaust and industrial waste and emissions. Exposure to acrolein containing environmental pollutants such as tobacco smoke and automobile exhaust has been linked to the activation of the coagulation and hemostasis pathways and thereby to the predisposition of thrombotic events in human. To examine the effects of acrolein on platelets, adult male C57Bl/6 mice were subjected acute (5ppm for 6h) or sub-chronic (1ppm, 6h/day for 4days) acrolein inhalation exposures. The acute exposure to acrolein did not cause pulmonary inflammation and oxidative stress, dyslipidemia or induce liver damage or muscle injury. Platelet GSH levels in acrolein-exposed mice were comparable to controls, but acrolein-exposure increased the abundance of protein-acrolein adducts in platelets. Platelets isolated from mice, exposed to both acute and sub-chronic acrolein levels, showed increased ADP-induced platelet aggregation. Exposure to acrolein also led to an increase in the indices of platelet activation such as the formation of platelet-leukocyte aggregates in the blood, plasma PF4 levels, and increased platelet-fibrinogen binding. The bleeding time was decreased in acrolein exposed mice. Plasma levels of PF4 were also increased in mice exposed to environmental tobacco smoke. Similar to inhalation exposure, acrolein feeding to mice also increased platelet activation and established a pro-thrombotic state in mice. Together, our data suggest that acrolein is an important contributing factor to the pro-thrombotic risk in human exposure to pollutants such as tobacco smoke or automobile exhaust, or through dietary consumption.

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.