Nwachukwu, Nneka

Background While recent work from our laboratory showed that DNA (cytosine‐5)‐methyltransferase 3 beta (DNMT3b) is significantly increased in tissue from humans with calcific aortic valve stenosis and is associated with increases in global genomic methylation, it is unknown whether the net effects of these changes are protective or deleterious. The objective of this study was to test the hypothesis that genetic reduction of DNMT3b allows for increased anti‐osteogenic gene expression, and consequently results in suppression of osteogenic signaling and slower progression of valvular stenosis. Methods We used Ldlr −/− /ApoB 100/100 (LA) mice that were wild‐type for DNMT3b (LA‐DNMT3b +/+ ) or deficient in DNMT3b (LA‐DNMT3b +/− ) and fed a western diet for six months. Results Using quantitative real‐time RT‐PCR, we confirmed that expression of DNMT3b was reduced by ~50% in valves from haploinsufficient mice. In line with our hypothesis, reductions in DNMT3b were associated with increases in expression of FABP4 (which opposes osteogenesis) and Smad6 (which opposes canonical BMP signaling). Similarly, expression of the osteogenic genes Msx2 and Spp1 were substantially reduced in DNMT3b‐deficient mice. Critically, aortic valve cusp separation distance was significantly greater in DNMT3b‐deficient mice, demonstrating slower progression of valvular dysfunction. No other gross abnormalities were noted in the mice. Conclusions Collectively, these data suggest that selectively reducing DNMT3b activity may be a viable therapeutic approach to slowing progression of calcific aortic valve stenosis in humans. Support or Funding Information Grant Funding Source: Supported by NHLBI

Aging is a major risk factor for calcific aortic valve disease (CAVD). While recent work suggests that DNA methylation patterns are dramatically altered with aging, changes in DNA methylation patterns in age‐related cardiovascular diseases remain largely uncharacterized. Thus, we tested the working hypothesis that de novo DNA methyltransferase levels (DNMT3b in particular) are significantly increased in stenotic human aortic valves and would serve to repress expression of protective genes. We used human aortic valves that were either normal (NORM) or stenotic (STEN). qRT‐PCR and Western blotting were used to examine analyze changes in DNMT3b levels and markers of osteogenesis. DNA methylation was examined using ELISA‐based methods for total methylation and Illumina 450k Beadchip assays for site‐specific methylation between NORM and STEN valves. The pro‐osteogenic marker expression, Sp7, was increased by 4±1 fold in STEN valves compared to NORM tissue. While mRNA levels of DNMT3b only tended to be increased in STEN valves, protein levels of DNMT3b were dramatically increased in STEN valves compared to NORM valves (13.9±3‐fold increase). Increases in DNMT3b levels were associated with an increase in global DNA methylation, and 450k beadchip analysis identified more than 6,000 differentially‐methylated sites between NORM and STEN tissue. Collectively, these data identify DNA methylation as a potentially novel and major regulator of gene expression in end‐stage calcific aortic valve stenosis. NHLBI Grant Funding Source : NHLBI