"Materials

Ultra performance liquid chromatography (UPLC) grade acetonitrile, N-hexane, acetone, and pentafluorobenzyl bromide (PFBBr) were purchased from Fisher Scientific (Fairlawn, NJ). NAcetyl-S-(3-hydroxypropyl)cysteine, Dicyclohexylammonium Salt (3HPMA); N-Acetyl-d3-S-(3-hydroxypropyl)cysteine, and Dicyclohexylammonium Salt 3HPMA-D3 (3HPMA-d3) were purchased from Toronto Research Chemicals (Downsview, Ontario, CAN). alonaldehyde-1,3-d2 Bis(diethyl acetal) was purchased from CDN Isotopes (Pointe-Claire, Quebec, CAN). Antibodies for flow cytometric analyses: FITC-anti-Sca-1 (Ly-6A/E), APC-anti-Flk1 (CD309), APC-eFluor780-anti-CD41, PE-Cyanine7-anti-Sca-1, FITC-anti-Nk1.1, PE-anti-Ly6C, PerCPe710-anti-CD8, PECy7-anti- CD62, APC-anti-CD19, Alexa700-anti-Gr-1, APCe780-anti-CD3, eVolve605-CD11b, e650-anti-CD4, and OneComp eBeads, proprietary solution, and propidium iodide were purchased from eBioscience (San Diego, CA). Annexin V Buffer and Zenon Alexa Fluor488 labeling kit were obtained from Invitrogen (Waltham, MA). Sources for other reagents are: CD62E-PE (BD Bioscience; San Jose, CA), ACE ectodomain (R&D Systems, Minneapolis, MN), Fc Block (CD32/CD16) (Leinco Technologies; Fenton, MO), counting beads (Spherotech; Lake Forest, IL), PBS (Corning; Manassas, VA), BSA (Rockland Immunochemicals Inc.; Pottstown, PA). Sources for the ELISA kits are: insulin (Mercodia AB, Uppsala, SWE), endothelin-1 (Enzo Life Sciences Inc., Farmingdale, NY), vWF (My Biosource, San Diego, CA), PF4, MMP3, and MMP9 (R&D Systems), fibrinogen (Innovative Research, Novi, MI).

Primers for PCR Analyses

Primers for B2m, TNFa, IL-6, IL-8, GRP78, GRP94, and HERP were obtained from Qiagen, (Gaithersburgh, MD). Sources of antibodies for Western lotting: Anti-GSTP-1 (610719; 1:2500) was purchased from BD Bioscience (San Jose, CA); and anti-ALDH2 (LSB7924; 1:1500) was bought from ifeSpan BioSciences (Seattle, WA). Other antibodies anti-AR (Sc-271007; 1:1500), anti-b-actin (Sc-47778; 1:1000), anti-Nrf2 (Sc-722; 1:1500), anti-COX-2 (Sc-376861;1:1500), anti-HO-1 (Sc-10789; 1:1500), anti-SOD-1 (Sc-11407; 1:2000), anti-SOD-2 (Sc-137254; 1:2000), anti-SOD-3

(Sc-101338; 1:2000), anti-Catalase (Sc271803; 1:2000), and anti-GAPDH (Sc-47724; 1:1000) were obtained from Santa Cruz Biotechnology (Dallas, TX).

Mice and Acrolein Exposure

Male C57BL/6J mice were obtained from the Jackson Laboratories (Bar Harbor, ME). Mice were treated according to American Physiological Society Guiding Principles in the Care and Use of Animals, and all protocols were approved by University of Louisville Institutional Animal Care and Use committee. Mice were housed under pathogen-free conditions in the University of Louisville vivarium under controlled temperature and 12h light: 12h dark cycle. Mice were provided a standard chow diet (Rodent Diet 5010, 4.5% fat by weight, LabDiet; St. Louis, MO). Mice (8 weeks old) were exposed to either HEPA- and charcoal-filtered air or acrolein (0.5 or 1 ppm; 6 h/day, 5 days/week) for 12 weeks using a custom exposure system and certified

permeation tubes (Kin-Tek; LaMarque, TX) as described (O’Toole et al., 2014). Immediately after the end of the last exposure, mice were anesthetized with sodium pentobarbital (150 mg/kg) followed by ventral thoracotomy and exsanguination via cardiac puncture for blood collection in EDTA-coated

syringes. Organs were removed, weighed, snap frozen in N2, and stored at 80C.

Urine Collection and Acrolein Metabolism

Urine collection. On day 1, mice were weighed and briefly exposed to D-glucose/saccharin solution (w/v; 3.0%/0.125%; Sigma) immediately prior to HEPA- and charcoal-filtered air exposure (6 h), and then placed singly per metabolic cage (Harvard Apparatus) with glucose/saccharin solution in drinking water without food to collect urine (in graduated cylinders in 4C water-jacketed organ baths) for continuous 3h collection postexposure (Conklin et al., 2009a; Wood et al., 2001). After urine collection, mice were placed in home cages overnight with food and water per normal housing arrangements. On day 2, the same mice were exposed to acrolein (0.5 or 1 ppm, 6h) or KY Reference cigarette mainstream smoke (3R4F cigarettes: 12 cigarettes over 6h) and then placed singly per metabolic cage with glucose/saccharin solution to collect urine (without food) in 1h increments up to 3h post-exposure followed by an overnight (O/N; with food) urine collection. Urine samples were centrifuged (600 g, 5 min; to pellet any feces and food particles) in cold immediately after collection, decanted, and stored at 80C. Acrolein metabolite analysis. The major metabolite of acrolein, 3- hydroxypropylmercapturic acid (3-HPMA), was quantified in urine by mass spectrometry (negative ion mode). The following mass spectrometer settings were used: capillary voltage 0.8 kV, extractor lens 3 V, entrance 2. The interchannel delay was 5ms and interscan delay was 6 ms. Urine (25 ml) was mixed with 15mM ammonium acetate (975 ml) containing 3HPMA-d3 (400 ng) and filtered through a 0.2mm PTFE membrane. Two microliters of the samples were applied on UPLC and the eluate was analyzed for the MRM traces. To account for urinary dilution, all values for urinary metabolites were normalized to urinary creatinine (mg/dl).

Pre-diabetes evaluation

A glucose tolerance test (GTT) was performed after a 6h exposure (ie, fast) in the 10th week by injecting D-glucose (1 g/kg; i.p., saline) (Sansbury et al., 2012).

Complete blood counts

CBC were measured (25 ll blood) with a hematology analyzer calibrated with multispecies hematological reference controls (Hemavet 950FS, Drew scientific, Inc.).

Flow cytometry

Leukocyte subpopulations. To analyze circulating immune cell populations, 0.1 ml lysed whole blood was centrifuged and washed twice with PBS containing 1% BSA (PBS/BSA). The cell pellets were re-suspended in the same buffer and incubated with CD32/CD16 (0.25 lg) for 10 min at 4C to prevent nonspecific binding. The cells were then incubated with an antibody cocktail consisting of FITC-anti-Nk1.1 (0.5 lg), PE-anti-Ly6C (0.125 lg), PerCPe710-anti-CD8 (0.125 lg), PECy7-anti-CD62 (0.25 lg), APC-anti-CD19 (0.125 lg), Alexa 700-antiGr-1 (0.125 lg), APCe780-anti-CD3 (1 lg), Volve605-CD11b (1 lg), and e650-anti-CD4 (1 lg). After 30 min on ice, the cells were washed, resuspended, and analyzed on an LSR II flow cytometer (high speed, 90 s). Cell numbers were analyzed using the FlowJo software and normalized to sample volume. Monocytes double positive for Ly6C and CD62L were defined as Ly6high subpopulation.

Endothelial progenitor cells (EPCs)

Blood EPCs (Flk-1þ/Sca-1þ cells) were identified by flow cytometry as described (Wheat et al., 2011). Blood (0.3 ml) was mixed with 3ml proprietary solution to lyse RBCs. Cells were pelleted by centrifugation and washed twice with PBS containing 1% BSA (PBS/BSA). Pelleted cells were re-suspended in PBS/FCS incubated with Fc block for 10 min and then stained with a FITC-labeled Sca-1 antibody and an APC-labeled Flk-1 antibody

for 30 min on ice. Cells were rinsed with 1% BSA/PBS and the pellets were re-suspended in 250 ll of 1% BSA/PBS containing propidium iodide (PI, 5 ll). Sample analysis was performed using an LSRII. EPCs were defined as the number of events double positive for Flk-1 and Sca-1 and counts were normalized to the volume used. Data were analyzed using FlowJo software. For the measurement of EPCs in bone marrow, the bone marrow was

aspirated from tibia of right leg, and mononuclear cells were separated by Ficoll gradient centrifugation and analyzed as described (Haberzettl et al., 2012).

Microparticles (MP)

Plasma (110 ml) was centrifuged for 2min (11 000 g; 4 C) to pellet residual cells and debris. Supernatant (100 ml) was aspirated and centrifuged for 45min (17 000 g; 4 C). The resulting MP pellet was re-suspended in 70 ml 1.45 Annexin V Buffer pre-filtered through syringe filter (0.22 mm). Sample (35 ml) was mixed with Annexin-V Pacific Blue (2.5 ll), ACE ectodomain antibody (0.5 lg)—labeled with Zenon Alexa Fluor488 labeling kit, CD41 APCeFluor780 (0.625 ll), CD309 APC (1.25 ll), CD62E-PE (2.5 lg), and Ly-6A/E (1.25 lg; Sca-1) PE-Cyanine7 for 30min. The reaction was stoppedwith 235 mlAnnexinV buffer (pre-filtered). Counting beads (10 mm, 15 ml) were added to the samples were analyzed on LSR II flow cytometer for 5min. An identical sample without antibodies was used as a gating control.MPnumberswere quantified in gated populations<1 mm in size and positive for Annexin V staining using the FlowJo software. MP subpopulations were analyzed as the percentage of CD41- events in theMP gate (Pope et al., 2016).

Platelet-leukocyte aggregates

Platelet–leukocyte aggregates were identified by flow cytometry and quantified as events double positive for CD41 (platelets) and CD45 (leukocytes) as described (DeJarnett et al., 2014; Sithu et al., 2010).

Plasma biomarkers

Plasma lipoproteins, total protein, albumin, creatinine, aspartate transaminase (AST), alanine transaminase (ALT), and lactate dehydrogenase were measured on a Cobas Mira Plus Clinical chemistry autoanalyzer (Roche) as described previously (Conklin et al., 2009a; Srivastava et al., 2011). Levels of cytokines and chemokines in the plasma were measured by multiplex arrays (Eve Technologies; Calgary, Alberta, CAN) or by using commercially available ELISA kits according to manufacturer’s recommendations.

Pulmonary Oxidative Stress, Endoplasmic Reticulum Stress and Cytokines

MDA in lungs. Pulmonary malonaldialdehyde (MDA) levels were measured by gas chromatography-mass spectrometry (GC-MS). Lungs were pulverized and homogenized in phosphate buffered saline (1, pH 7.4; 5% wt/vol). MDA-d2 (20 pmoles) obtained from hydrolysis of malonaldehyde-1,3-d2 bis(diethyl acetal) was used as an internal standard. Samples and standard were derivatized with 2,3,4,5-pentafluorobenzyle bromide (PFBBr; 130 ml) for 1h at 80C, extracted with hexane and analyzed by GC-MS in chemical ionization (CI) mode. A six-point calibration curve was used to quantify levels of MDA in lung homogenates. Real time quantitative PCR analyses and Western blotting were performed on lung tissues as described (Vladykovskaya et al., 2012; Wheat et al., 2011). Expression of cytokine mRNA in the lungs of air and acrolein (1 ppm) exposed mice were measured

by Qiagen RT2 profiler inflammatory cytokines and receptors PCR array. Fold regulation cut off and statistical significance were set at 2.0 and 0.05, respectively, as per Qiagen’s standard protocol.

Statistics

Data are presented as mean6standard error of mean (SEM). Statistical significance was accepted at p<.05. Statistical analyses were performed on log transformed data using SAS 9.4 software. Student’s t-test was used for the data comparison between two groups, and one-way analysis of variance with

Tukey’s correction was used when comparing more than two experimental groups."

Male C57BK/Bj Mice