Defective Base Excision Repair of Oxidative DNA Damage in Vascular Smooth Muscle Cells Promotes Atherosclerosis

Supplemental Digital Content is available in the text.

8oxoG and TUNEL. Plaque extent and composition was measured using ImageJ analysis software with an observer blind to experimental group. SMA content of fibrous caps was defined using Masson's Trichrome to identify the fibrous cap region. Fibrous caps were defined as the area rich in VSMCs and proteoglycan overlying the cholesterol-rich, matrixpoor, acellular regions of the necrotic cores. Very small plaques (<90,000µm 2 ) were not included in the above analysis since it was impossible to demarcate a fibrous cap.
Human VSMCs were cultured from explants. The endothelial layer was removed using a scalpel and tissue cut into 2-3mm² pieces, placed into 6-well plates containing 1ml media and grown for 1-2 weeks to allow cells to emerge. Human VSMCs were studied at passages 2-5; VSMC cultures from individual patients were not pooled. Rat and mouse aortic VSMCs were prepared by removing surrounding tissues and enzymatic dispersion using Type I collagenase (1 mg/ml, Sigma) and elastase (0.5 mg/ml, Worthington Biochemical) in serumfree medium for 1h at 37 °C.

Transfections and virus infections
Retrovirus infection was used to produce stable expression of wild type OGG1 or the acetylation mutant OGG1 K-R in rat aortic VSMCs. hOGG1 was subcloned from pCMV-myc-Nuc-hOGG1 (Addgene) and site-directed mutagenesis performed using the QuikChange Lightning Multi Site-Directed Mutagenesis Kit (Stratagene) following the manufacturer's instructions.

CRISPR-mediated gene silencing
For gene silencing experiments, rat aortic VSMCs were transfected with a U6-gRNA/CMV-Cas9-GFP CRISPR plasmid targeting OGG1 (Sigma). To generate rat OGG1 -/-VSMCs, cells were trypsinized 3 days after transfection and resuspended in PBS with 0.1% BSA and 2 mM EDTA. Single GFP-positive cells were sorted by FACS into 96-well plates, and clonal cell populations expanded. OGG1 protein expression of each clone was examined by immunoblotting, and clones with efficient OGG1 knockout used for further studies. Genomic DNA was extracted from 1 × 10 6 cells, and locus-specific cleavage was analyzed using the GeneArt Genomic Cleavage Detection Kit (Life Technologies).

Real-time polymerase chain reaction
RNA extraction from mouse tissues and primary VSMCs was performed using the RNeasy kit (Qiagen). First strand cDNA synthesis was performed using Superscript III Reverse Transcriptase (Invitrogen). Quantitative real-time PCR was performed using Rotor-Gene SYBR Green PCR Kit (Qiagen) on a Rotor-Gene 6000 QPCR thermocycler (Corbett Research). Oligonucleotide sequences used are listed in Supplementary Table S1.

Oligonucleotide incision assay
8oxoG BER activity in nuclear lysates was determined using a 40-mer oligonucleotide containing an 8oxoG at position 19 and labelled at the 3' end with indodicarbocyanine (5'-AGAGAAGAAGAAGAA(G*)AGATGGGTTATTCGAA-CTAGC-Cy5Sp-3'). This oligonucleotide was hybridized to its complementary sequence containing a cytosine opposite the 8oxoG lesion (G*). Nuclear extracts were added to a 10μL standard incision reaction mixture containing 200 fmoles of the 8oxoG:C-labeled duplex in 20 mmol/L Tris-HCl (pH 7.1), 1 mmol/L EDTA, 200 mmol/L NaCl, 1 mg/mL bovine serum albumin (BSA), and 5% glycerol. After 15 min at 37°C, the incision reaction was stopped by adding 4 μL of formamide dye and heating for 5 min at 95°C. The cleaved product was separated from the intact substrate in a 20% polyacrylamide gel containing 8 M urea in Tris-borate-EDTA buffer, pH 8.4. Fluorescence in the separated DNA bands was visualized using a LI-COR Odyssey CLx system.

8oxoG ELISA
Cell lysates were assessed for 8oxoG expression using a competitive ELISA assay (Abcam) as per the manufacturer's instructions. Total DNA was purified from VSMC lysates by using the DNeasy Blood & Tissue Kit (Qiagen). DNA was digested using nuclease P1 following the manufacturer's instructions. The pH was adjusted to 7.5-8.5 using 1M Tris and 1 unit alkaline phosphatase added per 100 μg DNA and incubated at 37°C for 30 minutes. Samples were boiled for 10 min and placed on ice until use. Absorbance was measured on a Synergy HT Plate Reader (Biotek) with standard curves generated from known concentrations of 8oxoG standards.

Intracellular ROS measurement
Intracellular ROS was measured by using the oxidant-sensitive fluorescent probe 5-(and-6)chloromethyl-2',7'-dichlorodihydrofluorescein diacetate acetyl ester (CM-H2DCFDA,10 mM; Molecular Probes). Cells were incubated continuously with CM-H2DCFDA during experimental conditions. Subsequently, cells were washed and lysed in 0.1% Triton X-100. Fluorescence was measured in the Biotek synergy HT plate reader with excitation of 488 nm and emission of 530 nm.

Annexin V/PI flow cytometry
Cell death was determined using an Apoptosis Detection kit according to the manufacturer's instructions (BD BioSciences). Briefly, cells (1 × 10 5 cells/sample) were washed twice in cold PBS and suspended in binding buffer containing fluorescein isothiocyanate-conjugated annexin V (10 μg/ml) and PI (10 μg/ml). The cell suspension was incubated in the dark for 15 min and fluorescence measured using an Accuri C6 flow cytometer (BD Biosciences). A total of 10,000 events were analyzed for each sample with Cell Quest software (BD Biosciences).
Comet assay 5 × 10 4 VSMCs were washed with PBS, resuspended in 150 µl 1% low melting point agarose (LMPA) and left to cool on Gel-bond films (Trevigen) at 4˚C for 10 min to set the agarose. Gel-bond films were incubated overnight in lysis buffer at 4˚C, washed briefly with water, and placed in alkaline electrophoresis buffer for 30 min prior to electrophoresis at 26V for 30 min at 4˚C. Gel-bond films were washed in neutralization buffer (100 mM Tris pH 7.6) and subsequently immersed in ice-cold ethanol for 10 min. Films were dried overnight, rehydrated in distilled H 2 O for 10 min and stained with 2µg/ml ethidium bromide (Sigma) solution in the dark at RT for 2 h. Multiple images per slide were analysed using a Zeiss microscope and comet tail length/moment measured using Comet Assay IV software (Perception Instruments).

ChIP-qPCR
ChIP was performed using the ChIP-IT express kit (Active Motif), VSMCs were crosslinked for 10 min with formaldehyde (to a final concentration of 1 %). Chromatin was sheared using a Bioruptor UCD-200 ultrasound sonicator (Diagenode), resulting in DNA fragments of 500-1000 bp in size. Chromatin was immunoprecipitated with 2 μg mouse anti-8oxoG antibody (MAB3560, Millipore) or negative control mouse IgG (PP64, Millipore). Immunoprecipitated DNA was then used as a template for quantitative PCR using primers specific for genomic loci. Oligonucleotide sequences used are listed in Supplementary Table S1.