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 (PM2.5) inhalation-induced alterations in the plasma lipidome as promoters of vascular inflammation and insulin resistance.

Fine particulate matter (PM2.5) air pollution exposure increases the risk of developing cardiovascular disease (CVD). Although the precise mechanisms by which air pollution exposure increases CVD risk remain uncertain, research indicates that PM2.5-induced endothelial dysfunction contributes to CVD risk. Previous studies demonstrate that concentrated ambient PM2.5 (CAP) exposure induces vascular inflammation and impairs insulin and vascular endothelial growth factor (VEGF) signaling dependent on pulmonary oxidative stress. To assess whether CAP exposure induces these vascular effects via plasmatic factors, we incubated aortas from naïve mice with plasma isolated from mice exposed to HEPA-filtered air or CAP (9 days) and examined vascular inflammation and insulin and VEGF signaling. We found that treatment of naïve aortas with plasma from CAP-exposed mice activates NF-κBα and induces insulin and VEGF resistance, indicating transmission by plasmatic factor(s). To identify putative factors, we exposed lung-specific ecSOD-transgenic (ecSOD-Tg) mice and wild-type (WT) littermates to CAP at concentrations of either ∼60 µg/m3 (CAP60) or ∼100 µg/m3 (CAP100) and measured the abundance of plasma metabolites by mass spectrometry. In WT mice, both CAP concentrations increased levels of fatty acids such as palmitate, myristate, and palmitoleate and decreased numerous phospholipid species; however, these CAP-induced changes in the plasma lipidome were prevented in ecSOD-Tg mice. Consistent with the literature, we found that fatty acids such as palmitate are sufficient to promote endothelial inflammation. Collectively, our findings suggest that PM2.5 exposure, by inducing pulmonary oxidative stress, promotes unique lipidomic changes characterized by high levels of circulating fatty acids, which are sufficient to trigger vascular pathology.NEW & NOTEWORTHY We found that circulating plasma constituents are responsible for air pollution-induced vascular pathologies. Inhalation of fine particulate matter (≤PM2.5) promotes a unique form of dyslipidemia that manifests in a manner dependent upon pulmonary oxidative stress. The air pollution-engendered dyslipidemic phenotype is characterized by elevated free fatty acid species and diminished phospholipid species, which could contribute to vascular inflammation and loss of insulin sensitivity.

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.

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.

Biomarkers of Chronic Acrolein Inhalation Exposure in Mice: Implications for Tobacco Product-Induced Toxicity.

Exposure to tobacco smoke, which contains several harmful and potentially harmful constituents such as acrolein increases cardiovascular disease (CVD) risk. Although high acrolein levels induce pervasive cardiovascular injury, the effects of low-level exposure remain unknown and sensitive biomarkers of acrolein toxicity have not been identified. Identification of such biomarkers is essential to assess the toxicity of acrolein present at low levels in the ambient air or in new tobacco products such as e-cigarettes. Hence, we examined the systemic effects of chronic (12 weeks) acrolein exposure at concentrations similar to those found in tobacco smoke (0.5 or 1 ppm). Acrolein exposure in mice led to a 2- to 3-fold increase in its urinary metabolite 3-hydroxypropyl mercapturic acid (3-HPMA) with an attendant increase in pulmonary levels of the acrolein-metabolizing enzymes, glutathione S-transferase P and aldose reductase, as well as several Nrf2-regulated antioxidant proteins. Markers of pulmonary endoplasmic reticulum stress and inflammation were unchanged. Exposure to acrolein suppressed circulating levels of endothelial progenitor cells (EPCs) and specific leukocyte subsets (eg, GR-1+ cells, CD19+ B-cells, CD4+ T-cells; CD11b+ monocytes) whilst other subsets (eg, CD8+ cells, NK1.1+ cells, Ly6C+ monocytes) were unchanged. Chronic acrolein exposure did not affect systemic glucose tolerance, platelet-leukocyte aggregates or microparticles in blood. These findings suggest that circulating levels of EPCs and specific leukocyte populations are sensitive biomarkers of inhaled acrolein injury and that low-level (<0.5 ppm) acrolein exposure (eg, in secondhand smoke, vehicle exhaust, e-cigarettes) could increase CVD risk by diminishing endothelium repair or by suppressing immune cells or both.

Role of TRPA1 in acute cardiopulmonary toxicity of inhaled acrolein.

Acrolein is a highly toxic, volatile, unsaturated aldehyde generated during incomplete combustion as in tobacco smoke and indoor fires. Because the transient receptor potential ankyrin 1 (TRPA1) channel mediates tobacco smoke-induced lung injury, we assessed its role in high-level acrolein-induced toxicity in mice. Acrolein (100-275ppm, 10-30min) caused upper airway epithelial sloughing, bradypnea and oral gasping, hypothermia, cardiac depression and mortality. Male wild-type mice (WT, C57BL/6; 5-52weeks) were significantly more sensitive to high-level acrolein than age-matched, female WT mice. Both male and female TRPA1-null mice were more sensitive to acrolein-induced mortality than age- and sex-matched WT mice. Acrolein exposure increased lung weight:body weight ratios and lung albumin and decreased plasma albumin to a greater extent in TRPA1-null than in WT mice. Lung and plasma protein-acrolein adducts were not increased in acrolein-exposed TRPA1-null mice compared with WT mice. To assess TRPA1-dependent protective mechanisms, respiratory parameters were monitored by telemetry. TRPA1-null mice had a slower onset of breathing rate suppression ('respiratory braking') than WT mice suggesting TRPA1 mediates this protective response. Surprisingly, WT male mice treated either with a TRPA1 antagonist (HC030031; 200mg/kg) alone or with combined TRPA1 (100mg/kg) and TRPV1 (capsazepine, 10mg/kg) antagonists at 30min post-acrolein exposure (i.e., "real world" delay in treatment) were significantly protected from acrolein-induced mortality. These data show TRPA1 protects against high-level acrolein-induced toxicity in a sex-dependent manner. Post-exposure TRPA1 antagonism also protected against acrolein-induced mortality attesting to a complex role of TRPA1 in cardiopulmonary injury.

Insulin sensitizers prevent fine particulate matter-induced vascular insulin resistance and changes in endothelial progenitor cell homeostasis.

Exposure to fine particular matter (PM2.5) increases the risk of developing cardiovascular disease and Type 2 diabetes. Because blood vessels are sensitive targets of air pollutant exposure, we examined the effects of concentrated ambient PM2.5 (CAP) on vascular insulin sensitivity and circulating levels of endothelial progenitor cells (EPCs), which reflect cardiovascular health. We found that CAP exposure for 9 days decreased insulin-stimulated Akt phosphorylation in the aorta of mice maintained on control diet. This change was accompanied by the induction of IL-1ß and increases in the abundance of cleaved IL-18 and p10 subunit of Casp-1, consistent with the activation of the inflammasome pathway. CAP exposure also suppressed circulating levels of EPCs (Flk-1(+)/Sca-1(+) cells), while enhancing the bone marrow abundance of these cells. Although similar changes in vascular insulin signaling and EPC levels were observed in mice fed high-fat diet, CAP exposure did not exacerbate diet-induced changes in vascular insulin resistance or EPC homeostasis. Treatment with an insulin sensitizer, metformin or rosiglitazone, prevented CAP-induced vascular insulin resistance and NF-κB and inflammasome activation and restored peripheral blood and bone marrow EPC levels. These findings suggest that PM2.5 exposure induces diet-independent vascular insulin resistance and inflammation and prevents EPC mobilization, and that this EPC mobilization defect could be mediated by vascular insulin resistance. Impaired vascular insulin sensitivity may be an important mechanism underlying PM2.5-induced vascular injury, and pharmacological sensitization to insulin action could potentially prevent deficits in vascular repair and mitigate vascular inflammation due to exposure to elevated levels of ambient air pollution.

Glutathione S-transferase P protects against cyclophosphamide-induced cardiotoxicity in mice.

High-dose chemotherapy regimens using cyclophosphamide (CY) are frequently associated with cardiotoxicity that could lead to myocyte damage and congestive heart failure. However, the mechanisms regulating the cardiotoxic effects of CY remain unclear. Because CY is converted to an unsaturated aldehyde acrolein, a toxic, reactive CY metabolite that induces extensive protein modification and myocardial injury, we examined the role of glutathione S-transferase P (GSTP), an acrolein-metabolizing enzyme, in CY cardiotoxicity in wild-type (WT) and GSTP-null mice. Treatment with CY (100-300 mg/kg) increased plasma levels of creatine kinase-MB isoform (CK · MB) and heart-to-body weight ratio to a significantly greater extent in GSTP-null than WT mice. In addition to modest yet significant echocardiographic changes following acute CY-treatment, GSTP insufficiency was associated with greater phosphorylation of c-Jun and p38 as well as greater accumulation of albumin and protein-acrolein adducts in the heart. Mass spectrometric analysis revealed likely prominent modification of albumin, kallikrein-1-related peptidase, myoglobin and transgelin-2 by acrolein in the hearts of CY-treated mice. Treatment with acrolein (low dose, 1-5 mg/kg) also led to increased heart-to-body weight ratio and myocardial contractility changes. Acrolein induced similar hypotension in GSTP-null and WT mice. GSTP-null mice also were more susceptible than WT mice to mortality associated with high-dose acrolein (10-20 mg/kg). Collectively, these results suggest that CY cardiotoxicity is regulated, in part, by GSTP, which prevents CY toxicity by detoxifying acrolein. Thus, humans with low cardiac GSTP levels or polymorphic forms of GSTP with low acrolein-metabolizing capacity may be more sensitive to CY toxicity.

Acrolein decreases endothelial cell migration and insulin sensitivity through induction of let-7a.

Acrolein is a major reactive component of vehicle exhaust, and cigarette and wood smoke. It is also present in several food substances and is generated endogenously during inflammation and lipid peroxidation. Although previous studies have shown that dietary or inhalation exposure to acrolein results in endothelial activation, platelet activation, and accelerated atherogenesis, the basis for these effects is unknown. Moreover, the effects of acrolein on microRNA (miRNA) have not been studied. Using AGILENT miRNA microarray high-throughput technology, we found that treatment of cultured human umbilical vein endothelial cells with acrolein led to a significant (>1.5-fold) upregulation of 12, and downregulation of 15, miRNAs. Among the miRNAs upregulated were members of the let-7 family and this upregulation was associated with decreased expression of their protein targets, ß3 integrin, Cdc34, and K-Ras. Exposure to acrolein attenuated ß3 integrin-dependent migration and reduced Akt phosphorylation in response to insulin. These effects of acrolein on endothelial cell migration and insulin signaling were reversed by expression of a let-7a inhibitor. Also, inhalation exposure of mice to acrolein (1 ppm x 6 h/day x 4 days) upregulated let-7a and led to a decrease in insulin-stimulated Akt phosphorylation in the aorta. These results suggest that acrolein exposure has broad effects on endothelial miRNA repertoire and that attenuation of endothelial cell migration and insulin signaling by acrolein is mediated in part by the upregulation of let-7a. This mechanism may be a significant feature of vascular injury caused by inflammation, oxidized lipids, and exposure to environmental pollutants.

Exposure to ambient air fine particulate matter prevents VEGF-induced mobilization of endothelial progenitor cells from the bone marrow.

BACKGROUND: Exposure to ambient fine particulate matter air pollution (PM(2.5); < 2.5 µm in aerodynamic diameter) induces endothelial dysfunction and increases the risk for cardiovascular disease. Endothelial progenitor cells (EPCs) contribute to postnatal endothelial repair and regeneration. In humans and mice, EPC levels are decreased upon exposure to elevated levels of PM(2.5). OBJECTIVE: We examined the mechanism by which PM(2.5) exposure suppresses circulating levels of EPCs. METHODS: Mice were exposed to HEPA-filtered air or concentrated ambient fine particulate matter (CAP, 30-100 µg/m³) from downtown Louisville (Kentucky) air, and progenitor cells from peripheral blood or bone marrow were analyzed by flow cytometry or by culture ex vivo. RESULTS: Exposure of the mice to CAP (6 hr/day) for 4-30 days progressively decreased circulating levels of EPCs positive for both Flk-1 and Sca-1 (Flk-1(+)/Sca-1(+)) without affecting stem cells positive for Sca-1 alone (Sca-1(+)). After 9 days of exposure, a 7-day exposure-free period led to complete recovery of the circulating levels of Flk-1(+)/Sca-1(+) cells. CAP exposure decreased circulating levels of EPCs independent of apoptosis while simultaneously increasing Flk-1(+)/Sca-1(+) cells in the bone marrow. We observed no change in tissue deposition of these cells. CAP exposure suppressed vascular endothelial growth factor (VEGF)-induced Akt and endothelial nitric oxide synthase (eNOS) phosphorylation in the aorta, and it prevented VEGF/AMD3100-induced mobilization of Flk-1(+)/Sca-1(+) cells into the peripheral blood. Treatment with stem cell factor/AMD3100 led to a greater increase in circulating Flk-1(+)/Sca-1(+) cells in CAP-exposed mice than in mice breathing filtered air. CONCLUSION: Exposure to PM(2.5) increases EPC levels in the bone marrow by preventing their mobilization to the peripheral blood via inhibition of signaling events triggered by VEGF-receptor stimulation that are upstream of c-kit activation. Suppression of EPC mobilization by PM(2.5) could induce deficits in vascular repair or regeneration.

Lipid peroxidation product 4-hydroxy-trans-2-nonenal causes endothelial activation by inducing endoplasmic reticulum stress.

Lipid peroxidation products, such as 4-hydroxy-trans-2-nonenal (HNE), cause endothelial activation, and they increase the adhesion of the endothelium to circulating leukocytes. Nevertheless, the mechanisms underlying these effects remain unclear. We observed that in HNE-treated human umbilical vein endothelial cells, some of the protein-HNE adducts colocalize with the endoplasmic reticulum (ER) and that HNE forms covalent adducts with several ER chaperones that assist in protein folding. We also found that at concentrations that did not induce apoptosis or necrosis, HNE activated the unfolded protein response, leading to an increase in XBP-1 splicing, phosphorylation of protein kinase-like ER kinase and eukaryotic translation initiation factor 2α, and the induction of ATF3 and ATF4. This increase in eukaryotic translation initiation factor 2α phosphorylation was prevented by transfection with protein kinase-like ER kinase siRNA. Treatment with HNE increased the expression of the ER chaperones, GRP78 and HERP. Exposure to HNE led to a depletion of reduced glutathione and an increase in the production of reactive oxygen species (ROS); however, glutathione depletion and ROS production by tert-butyl-hydroperoxide did not trigger the unfolded protein response. Pretreatment with a chemical chaperone, phenylbutyric acid, or adenoviral transfection with ATF6 attenuated HNE-induced monocyte adhesion and IL-8 induction. Moreover, phenylbutyric acid and taurine-conjugated ursodeoxycholic acid attenuated HNE-induced leukocyte rolling and their firm adhesion to the endothelium in rat cremaster muscle. These data suggest that endothelial activation by HNE is mediated in part by ER stress, induced by mechanisms independent of ROS production or glutathione depletion. The induction of ER stress may be a significant cause of vascular inflammation induced by products of oxidized lipids.

Contrasting macrophage activation by fine and ultrafine titanium dioxide particles is associated with different uptake mechanisms.

Inhalation of (nano)particles may lead to pulmonary inflammation. However, the precise mechanisms of particle uptake and generation of inflammatory mediators by alveolar macrophages (AM) are still poorly understood. The aim of this study was to investigate the interactions between particles and AM and their associated pro-inflammatory effects in relation to particle size and physico-chemical properties.NR8383 rat lung AM were treated with ultrafine (uf), fine (f) TiO2 or fine crystalline silica (DQ12 quartz). Physico-chemical particle properties were investigated by transmission electron microscopy, elemental analysis and thermogravimetry. Aggregation and agglomeration tendency of the particles were determined in assay-specific suspensions by means of dynamic light scattering.All three particle types were rapidly taken up by AM. DQ12 and ufTiO2 , but not fTiO2 , caused increased extracellular reactive oxygen species (ROS), heme oxygenase 1 (HO-1) mRNA expression and tumor necrosis factor (TNF)-α release. Inducible nitric oxide synthase (iNOS) mRNA expression was increased most strongly by ufTiO2 , while DQ12 exclusively triggered interleukin (IL) 1ß release. However, oscillations of intracellular calcium concentration and increased intracellular ROS were observed with all three samples. Uptake inhibition experiments with cytochalasin D, chlorpromazine and a Fcγ receptor II (FcγRII) antibody revealed that the endocytosis of fTiO2 by the macrophages involves actin-dependent phagocytosis and macropinocytosis as well as clathrin-coated pit formation, whereas the uptake of ufTiO2 was dominated by FcγIIR. The uptake of DQ12 was found to be significantly reduced by all three inhibitors. Our findings suggest that the contrasting AM responses to fTiO2 , ufTiO2 and DQ12 relate to differences in the involvement of specific uptake mechanisms.

Chronic oral exposure to the aldehyde pollutant acrolein induces dilated cardiomyopathy.

Environmental triggers of dilated cardiomyopathy are poorly understood. Acute exposure to acrolein, a ubiquitous aldehyde pollutant, impairs cardiac function and cardioprotective responses in mice. Here, we tested the hypothesis that chronic oral exposure to acrolein induces inflammation and cardiomyopathy. C57BL/6 mice were gavage-fed acrolein (1 mg/kg) or water (vehicle) daily for 48 days. The dose was chosen based on estimates of human daily unsaturated aldehyde consumption. Compared with vehicle-fed mice, acrolein-fed mice exhibited significant (P < 0.05) left ventricular (LV) dilatation (LV end-diastolic volume 36 ± 8 vs. 17 ± 5 µl), contractile dysfunction (dP/dt(max) 4,697 ± 1,498 vs. 7,016 ± 1,757 mmHg/s), and impaired relaxation (tau 15.4 ± 4.3 vs. 10.4 ± 2.2 ms). Histological and biochemical evaluation revealed myocardial oxidative stress (membrane-localized protein-4-hydroxy-trans-2-nonenal adducts) and nitrative stress (increased protein-nitrotyrosine) and varying degrees of plasma and myocardial protein-acrolein adduct formation indicative of physical translocation of ingested acrolein to the heart. Acrolein also induced myocyte hypertrophy (~2.2-fold increased myocyte area, P < 0.05), increased apoptosis (~7.5-fold), and disrupted endothelial nitric oxide synthase in the heart. DNA binding studies, immunohistochemistry, and PCR revealed significant (P < 0.05) activation of nuclear factor-κB in acrolein-exposed hearts, along with upregulated gene expression of proinflammatory cytokines tumor necrosis factor-α and interleukin-1ß. Long-term oral exposure to acrolein, at an amount within the range of human unsaturated aldehyde intake, induces a phenotype of dilated cardiomyopathy in the mouse. Human exposure to acrolein may have analogous effects and raise consideration of an environmental, aldehyde-mediated basis for heart failure.

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.

Oral exposure to acrolein exacerbates atherosclerosis in apoE-null mice.

BACKGROUND: Acrolein is a dietary aldehyde that is present in high concentrations in alcoholic beverages and foods including cheese, donuts and coffee. It is also abundant in tobacco smoke, automobile exhaust and industrial waste and is generated in vivo during inflammation and oxidative stress. OBJECTIVES: The goal of this study was to examine the effects of dietary acrolein on atherosclerosis. METHODS: Eight-week-old male apoE-null mice were gavage-fed acrolein (2.5mg/kg/day) for 8 weeks. Atherosclerotic lesion formation and composition and plasma lipids and platelet factor 4 (PF4) levels were measured. Effects of acrolein and PF4 on endothelial cell function was measured in vitro. RESULTS: Acrolein feeding increased the concentration of cholesterol in the plasma. NMR analysis of the lipoproteins showed that acrolein feeding increased the abundance of small and medium VLDL particles. Acrolein feeding also increased atherosclerotic lesion formation in the aortic valve and the aortic arch. Immunohistochemical analysis showed increased macrophage accumulation in the lesions of acrolein-fed mice. Plasma PF4 levels and accumulation of PF4 in atherosclerotic lesions was increased in the acrolein-fed mice. Incubation of endothelial cells with the plasma of acrolein-fed mice augmented transmigration of monocytic cells, which was abolished by anti-PF4 antibody treatment. CONCLUSIONS: Dietary exposure to acrolein exacerbates atherosclerosis in apoE-null mice. Consumption of foods and beverages rich in unsaturated aldehydes such as acrolein may be a contributing factor to the progression of atherosclerotic lesions.

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.

Arsenic exacerbates atherosclerotic lesion formation and inflammation in ApoE-/- mice.

Exposure to arsenic-contaminated water has been shown to be associated with cardiovascular disease, especially atherosclerosis. We examined the effect of arsenic exposure on atherosclerotic lesion formation, lesion composition and nature in ApoE-/- mice. Early post-natal exposure (3-week-old mice exposed to 49 ppm arsenic as NaAsO(2) in drinking water for 7 weeks) increased the atherosclerotic lesion formation by 3- to 5-fold in the aortic valve and the aortic arch, without affecting plasma cholesterol. Exposure to arsenic for 13 weeks (3-week-old mice exposed to 1, 4.9 and 49 ppm arsenic as NaAsO(2) in drinking water) increased the lesion formation and macrophage accumulation in a dose-dependent manner. Temporal studies showed that continuous arsenic exposure significantly exacerbated the lesion formation throughout the aortic tree at 16 and 36 weeks of age. Withdrawal of arsenic for 12 weeks after an initial exposure for 21 weeks (to 3-week-old mice) significantly decreased lesion formation as compared with mice continuously exposed to arsenic. Similarly, adult exposure to 49 ppm arsenic for 24 weeks, starting at 12 weeks of age increased lesion formation by 2- to 3.6-fold in the aortic valve, the aortic arch and the abdominal aorta. Lesions of arsenic-exposed mice displayed a 1.8-fold increase in macrophage accumulation whereas smooth muscle cell and T-lymphocyte contents were not changed. Expression of pro-inflammatory chemokine MCP-1 and cytokine IL-6 and markers of oxidative stress, protein-HNE and protein-MDA adducts were markedly increased in lesions of arsenic-exposed mice. Plasma concentrations of MCP-1, IL-6 and MDA were also significantly elevated in arsenic-exposed mice. These data suggest that arsenic exposure increases oxidative stress, inflammation and atherosclerotic lesion formation.

Investigation of the cytotoxic and proinflammatory effects of cement dusts in rat alveolar macrophages.

Exposure to cement dust, a specifically alkaline and irritant dust, is one of the most common occupational dust exposures worldwide. Although several adverse respiratory health effects have been associated with cement dust exposure, the evidence is not conclusive. In the current study, cytotoxic and pro-inflammatory effects as well as oxidative stress elicited by a number of cement dusts, including a limestone and cement clinker sample, were tested using the NR8383 rat alveolar macrophage cell line and primary rat alveolar macrophages. DQ12 quartz and TiO(2) were included as positive and negative controls, respectively. Cytotoxicity was determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide assay and the lactate dehydrogenase assay, oxidative stress was determined by measurement of the depletion of total cellular glutathione, and electron spin resonance was applied to determine reactive oxygen species (ROS) generation. The release of the cytokines tumor necrosis factor-alpha (TNFalpha), interleukin-1 beta (IL-1 beta), and macrophage inflammatory protein-2 (MIP-2) was determined by enzyme-linked immunosorbent assay. None of the dust samples were found to cause toxicity to the macrophages or notable glutathione depletion when compared to DQ12. The cement samples also failed to activate macrophages for the generation of ROS and the production of inflammatory cytokines IL-1 beta and MIP-2. In contrast, however, most of the cement dusts were found to activate macrophage TNFalpha production, and this was significantly associated with their content of CaO. Further research is needed to determine the relevance of these in vitro observations for occupational cement dust exposure settings.

Increased sensitivity of glutathione S-transferase P-null mice to cyclophosphamide-induced urinary bladder toxicity.

Hemorrhagic cystitis and diffuse inflammation of the bladder, common side effects of cyclophosphamide (CY) treatment, have been linked to the generation of acrolein derived from CY metabolism. Metabolic removal of acrolein involves multiple pathways, which include reduction, oxidation, and conjugation with glutathione. Herein, we tested the hypothesis that glutathione S-transferase P (GSTP), the GST isoform that displays high catalytic efficiency with acrolein, protects against CY-induced urotoxicity by detoxifying acrolein. Treatment of wild-type (WT) and mGstP1/P2 null (GSTP-null) mice with CY caused hemorrhagic cystitis, edema, albumin extravasation, and sloughing of bladder epithelium; however, CY-induced bladder ulcerations of the lamina propria were more numerous and more severe in GSTP-null mice. CY treatment also led to greater accumulation of myeloperoxidase-positive cells and specific protein-acrolein adducts in the bladder of GSTP-null than WT mice. There was no difference in hepatic microsomal production of acrolein from CY or urinary hydroxypropyl mercapturic acid output between WT and GSTP-null mice, but CY induced greater c-Jun NH(2)-terminal kinase (JNK) and c-Jun, but not extracellular signal-regulated kinase or p38, activation in GSTP-null than in WT mice. Pretreatment with mesna (2-mercaptoethane sulfonate sodium) abolished CY toxicity and JNK activation in GSTP-null mice. Taken together, these data support the view that GSTP prevents CY-induced bladder toxicity, in part by detoxifying acrolein. Because polymorphisms in human GSTP gene code for protein variants differing significantly in their catalytic efficiency toward acrolein, it is likely that GSTP polymorphisms influence CY urotoxicity. In addition, pretreatment with dietary or nutrient inducers of GSTP may be of use in minimizing bladder injury in patients undergoing CY therapy.

Glutathione-S-transferase P protects against endothelial dysfunction induced by exposure to tobacco smoke.

Exposure to tobacco smoke impairs endothelium-dependent arterial dilation. Reactive constituents of cigarette smoke are metabolized and detoxified by glutathione-S-transferases (GSTs). Although polymorphisms in GST genes are associated with the risk of cancer in smokers, the role of these enzymes in regulating the cardiovascular effects of smoking has not been studied. The P isoform of GST (GSTP), which catalyzes the conjugation of electrophilic molecules in cigarette smoke such as acrolein, was expressed in high abundance in the mouse lung and aorta. Exposure to tobacco smoke for 3 days (5 h/day) decreased total plasma protein. These changes were exaggerated in GSTP(-/-) mice. Aortic rings isolated from tobacco smoke-exposed GSTP(-/-) mice showed greater attenuation of ACh-evoked relaxation than those from GSTP(+/+) mice. The lung, plasma, and aorta of mice exposed to tobacco smoke or acrolein (for 5 h) accumulated more acrolein-adducted proteins than those tissues of mice exposed to air, indicating that exposure to tobacco smoke results in the systemic delivery of acrolein. Relative to GSTP(+/+) mice, modification of some proteins by acrolein was increased in the aorta of GSTP(-/-) mice. Aortic rings prepared from GSTP(-/-) mice that inhaled acrolein (1 ppm, 5 h/day for 3 days) or those exposed to acrolein in an organ bath showed diminished ACh-induced arterial relaxation more strongly than GSTP(+/+) mice. Acrolein-induced endothelial dysfunction was prevented by pretreatment of the aorta with N-acetylcysteine. These results indicate that GSTP protects against the endothelial dysfunction induced by tobacco smoke exposure and that this protection may be related to the detoxification of acrolein or other related cigarette smoke constituents.