Twelve-week-old male ApoE−/− mice from The Jackson Laboratory (Bar Harbor, ME) were housed at constant temperature (22 ± 2°C) on a 12-h light/dark cycle. They were fed ad libitum on standard laboratory mouse chow and had free access to water. The investigation conforms to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996), and the study protocols were approved by the Institutional Animal Care and Use Committee of New York University and the Ohio State University.
2.2. Scaffold Preparation and Implantation
To provide a biologically-compatible platform for cell growth, a system of scaffold with matrigel was used. The scaffolds consisted of a blend of NaCl and poly-L-lactic acid sponge (PLLA, Boehringer Ingelheim Inc., Winchester, VA). The sponges were leached out of salt by water, and cut into rectangular pieces of 6 × 6 × 6 mm. The physical characterization of the scaffold has been documented previously (Sun et al., 2005
). The scaffolds were sterilized overnight in 70% (vol/vol) ethanol and washed three times in PBS. Then, 1×106
murine endothelial cells (ATCC # CRL-2586) were seeded into each piece of the scaffold with matrigel (growth factor-reduced, Becton Dickinson, MA). After the mice were anesthetized by a mixture of ketamine (100 mg/kg) and xylazine (20 mg/kg) intraperitoneally, an incision on the dorsal region (between the scapulae) was performed, and one scaffold containing the cells was implanted subcutaneously into the dorsum of each mouse (near the scapula).
2.3. Hindlimb Ischemic Model
After anesthesia as above, another set of the mice were subjected to unilateral femoral artery and vein ligation, as described previously (Sun et al., 2005
). Briefly, severe unilateral hind limb ischemia was surgically created via ligation and division of the left superficial femora artery and vein, left external iliac artery and vein, and left deep femoral and circumflex arteries and veins.
2.4. Mouse Diesel Exhaust Exposure
Mice, with either scaffold implantation or hindlimb ischemia, were exposed to either diluted whole diesel exhaust (WDE, containing diesel exhaust particles [DEP] at a concentration of about 1 mg/m3
, as well as all of the gaseous pollutants in the exhaust) based on previous studies (McDonald et al., 2004a
; Reed et al., 2004
), or to filtered outdoor air (FA) for 6 hours/day, 5 days/week for 2, 5, or 8 weeks while within a whole body exposure chamber (n = 8) in a diesel exhaust exposure facility at New York University School of Medicine’s A.J. Lanza Laboratory in Sterling Forest at Tuxedo, NY. The diluted air was sufficient to maintain the carbon monoxide (CO) concentration in the exposure chamber at 5.0 ± 1.5 ppm.
The WDE was produced by a 5500-watt single-cylinder diesel engine generator (Model YDG 5500EE-6EI, Yanmar, Osaka, Japan) that contains a 418cc displacement air-cooled engine (Model LE100EE-DEGY6). Engine oil (SAE, 15W/40, Delo400, Chevron Products Company, San Ramon, CA) was changed every two weeks during the series of daily exposures. The diesel engine used #2 on-road diesel fuel from a local gas station (SOS fuels, Tuxedo, NY) and the diesel engine was run at a maximum engine load condition. The characterization and exposure concentrations of the WDE, including both the gaseous components and the DEP components when using #2 diesel fuel have been described previously (McDonald et al., 2004a
; McDonald et al., 2004b
). The FA control mice in the experiment were subjected to an identical protocol, with the exception that a High Efficiency Particulate Air filter (HEPA, Pall Life Sciences, East Hills, NY) was positioned in the inlet valve to the exposure system (a separate line from the diesel exposure system) to remove essentially all of the ambient air PM from the fresh air stream.
2.5. Mouse Sacrifice
Mice were euthanized immediately after each exposure period. The implanted scaffolds were retrieved and aortas dissected out. The scaffolds were fixed in 10% formalin, and embedded in paraffin, while the aorta and skeletal muscles distal to the ischemic surgery site were removed, snap-frozen in OCT, and stored at −80 °C.
2.6. Micro-CT Scanning
To investigate the functional vascular blood flow, micro-CT scanning was used. In brief, after exposure to WDE or FA for 8 weeks, the mice were euthanized and perfused with radiopaque polymer contrast Microfil® MV-122 (Flowtech, Carver, MA). The images were acquired on an Invion™ micro-CT scanner (Siemens Medical Solution, Knoxville, TN) with 4K × 4K detector using the following parameters: 80 kV voltage, 500 mA current, effective pixel size 20.30 mM, exposure time of 515 ms with high system magnification and binning of 2 giving image resolution of 37.20 mM with slice thickness of 0.010 mm. The images were analyzed with IRW software from Siemens.
2.7. Histology and Immunohistochemical Staining
Hematoxylin and eosin (H&E) staining was performed on the sections of thoracic aorta and retrieved scaffolds. The images were digitized with a digital camera, and analyzed under a research microscope (Zeiss 510 META, Jena, Germany) with Metamorph V.7.1.2 software (Universal Imaging, West Chester, PA). The percentage of total cells was calculated by the area of positive hematoxylin staining over the area of entire analyzed tissue.
For immunohistochemical staining, after quenching the endogenous peroxidase activity with 0.3% H2O2, the sections were incubated in 1% BSA/PBS for 10 minutes, followed by overnight incubation with primary antibodies (1:200 diluted in 1% BSA/PBS) at 4°C. After incubation with appropriate peroxidase-conjugated secondary antibodies, the stain was developed using Fast 3, 3′-diaminobenzidine tablet sets (D4293; Sigma). The sections were then counterstained with hematoxylin and examined by light microscopy. The primary antibodies were rat anti-CD31 (BD/pharmingen, San Diego, CA), mouse monoclonal anti-α-smooth muscle actin (α-SMA, 1:200; Sigma), rabbit anti-endothelial nitric oxide synthase (eNOS, Cell Signaling Technology, Danvers, MA), and rabbit anti-CD68 and rabbit anti-inducible NOS (iNOS, Santa Cruz Biotechnology Inc., Santa Cruz, CA).
2.8. DEP Extraction
The particles used in in vitro assays were collected from the filters of the diesel exhaust exposure system where the mice were exposed. The particles were extracted from the filters in 95% ethanol, and ultrasonicated for 20 min. The supernatant liquid was then decanted into evaporative concentrator tubes and concentrated under a stream of nitrogen gas in a Dry Bath Incubator (Fisher Scientific, Pittsburgh, PA). Thereafter, PBS was added to re-suspend the DEP to 0.5 mg/ml.
2.9. Cell Culture
Human umbilical vein endothelial cells (HUVECs) were purchased from ATCC (# CRL-1730) and cultured at 37 °C in a 5% CO2 atmosphere in M199 culture medium (Lonza, Williamsport, PA) supplemented with 10% fetal bovine serum (FBS; Lonza), 5 units/ml heparin, 2mM L-glutamine(Sigma, St. Louis, MO), 10 ng/ml β-Endothelial Cell Growth Factor (β-ECGF; Sigma), 100 U/ml penicillin, and 100μg/ml streptomycin (Invitrogen, Carlsbad, CA). The HUVECs at 6th to 8th passage were used in this study.
2.10. Cytotoxicity Evaluation
Cell viability was evaluated under the microscope with the trypan blue dye exclusion method. The HUVECs (1.5 ×105/well) were cultured in a 24-well plate coated with 0.2% gelatin (Sigma) followed the incubation with DEP for 24 hours. Live cell number was counted after 1, 3, 5, 7 and 9 days by trypan blue dye exclusion. In addition, the cells (1.5 ×105/well) were incubated with DEP for 10, 20, 30 and 40 hours, and then the numbers of live cells were counted. The total number of viable cells in the FA control group (DEP-free) was considered as 100% viability, while DEP-treated cells were compared with the FA control group for the determination of percentage viability.
2.11. Endothelial Capillary Tube Formation Assay in Vitro
Confluent HUVECs were treated with different concentration of DEP (0, 5, 15, and 30 μg/ml) for 4 or 24 hours, then resuspended in a density of 3 ×104 cells/ml with M199 supplemented with 10% FBS but without β-ECGF and seeded onto extracellular matrix (ECM) gel (Sigma). The cells were incubated at 37 °C for 24 hours in a humidified 5% CO2 incubator. Three random fields of view in three replicate wells in triplicate were visualized under 100×magnifications.
2.12. Capillary Sprouting From Aortic Rings ex Vivo
The aortas were harvested and 1–2 mm thick aortic rings were transversally cut after the dissection of all connective tissue. The rings were treated with different concentration of DEP (0, 5, 15, and 30 μg/ml) for 24 h and then seeded in a 24-well ECM gel-coated plate. DMEM supplemented with 10% FBS, 20 U/ml of heparin (Sigma), and penicillin/streptomycin was added to each well of gelled ECM. After 5 days of culture, the number and length of capillary sprouting from each aortic ring were imaged using an inverted microscope and assessed.
2.13. Quantitative Real-time Polymerase Chain Reaction
RNA was isolated using TRIzol Reagent (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions. Total RNA were converted into cDNA using the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA). The quantification of gene expression was determined by real-time polymerase chain reaction (RT-PCR). The primers for human and mouse HIF-1α, PHD2, VEGF and β–actin are showed in Table E1 in the online data supplement
2.14. Statistical Analysis
Data are expressed as mean ± SEM unless otherwise stated. All of the values were analyzed using one-way ANOVA and the Newman-Keuls-Student t test. A p value of less than 0.05 was considered statistically significant.