Measurement of mouse vascular smooth muscle and atheroma cell proliferation by 2H2O incorporation into DNA

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Abstract
Vascular smooth muscle cell (VSMC) and leukocyte proliferation are central features of atherosclerosis. Using 2H2O to label the deoxyribose moiety of newly synthesized DNA in VSMC and atheroma cells from mouse aorta, we developed a method to measure DNA replication and, hence, cell division. Cell turnover/proliferation in aortae from normal and apolipoprotein E (ApoE)-knockout ( ApoE−/−) mice was measured. Mice were injected with 2H2O to achieve 2% body water enrichments and then maintained on 4% 2H2O in drinking water for weeks to months. DNA from the intimal-medial layer of the aorta was extracted and hydrolyzed to deoxyribonucleosides. Purified deoxyadenosine was derivatized to pentane tetraacetate for analysis of 2H enrichment by gas chromatography-mass spectrometry. VSMC proliferation was measurable but slow in adult mice (0.12 ± 0.08%/day) and higher in young mice (0.25 ± 0.08%/day). VSMC delabeling revealed that 2H died away slowly in VSMC DNA, confirming the low turnover rate. Atheroma cell proliferation was elevated in ApoE−/− mice fed low- or high-fat diets for 15 wk, concurrent with histological appearance of atherosclerosis. Validation of the method for VSMC was confirmed by comparison of in vitro rat VSMC proliferation rates using 2H2O with cell counts and bromodeoxyuridine proliferative index. In summary, proliferation of VSMC and atheroma cells can be quantified reliably and sensitively without radioactivity and may be an informative biomarker in vascular hyperplastic diseases, including atherosclerosis.

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