Kafa, Rami

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

Introduction While progression of calcific aortic valve stenosis (CAVS) is known to be strongly associated with activation of pro-inflammatory and pro-osteogenic pathways, effective treatments for CAVS have remained elusive. TNF-α is a pleiotropic inflammatory cytokine and is upregulated in patients with CAVS. Hence, we hypothesized that TNF-α inhibition, using the neutralizing antibody infliximab, can slow the progression of CAVS in vivo . Methods We used Ldlr -/- Apob 100/100 mice, which develop robust CAVS in an age-dependent manner, as a model. Mice were fed a Western-type diet for 6 months, then randomized into one of two groups: 1) twice-weekly infliximab injections (10 mg/kg intraperitoneally; n=10), and 2) saline injections (n=9). Aortic valve function was evaluated using echocardiography, and gene expression in aortic valves was measured using quantitative real-time PCR. Data are presented as mean ± SEM. Results During the 3 months preceding treatment initiation, aortic valve cusp separation distance decreased progressively in both groups (reduction by 0.082±0.011 mm in control group, 0.095±0.016 mm in infliximab group; p =0.27). Three months after initiation of treatment, cusp separation continued to decrease in control mice (by 0.108±0.021 mm), while progression of valve dysfunction was slower by 42% in the infliximab group (reduction of 0.063±0.009 mm; p =0.03 vs control). Expression of the pro-inflammatory genes Vcam1 and Nos2 was significantly reduced in valves of the infliximab group (by 37% and 60%, respectively). Moreover, endothelium-dependent vasodilation was improved by infliximab (improvement in maximum relaxation to acetylcholine in infliximab group 18±6% compared to control). Conclusions This study is the first to report that in vivo inhibition of TNF-α slows progression of CAVS in mice with established valve disease. This beneficial effect was accompanied by reduction in pro-inflammatory gene expression and improvement of systemic endothelial function, suggesting infliximab may exert its effects in part by increasing nitric oxide bioavailability. The findings warrant further investigation into the role of TNF-α in initiation and progression of CAVS, as well as its potential as a therapeutic target in human CAVS.

Endothelial dysfunction and vascular calcification are major problems associated with prolonged hypercholesterolemia and aging, and are strongly associated with increases in TNFα. We hypothesized that inhibition of TNF‐α would reduce inflammatory and pro‐osteogenic gene expression in aorta, leading to improved endothelial function and reductions in intimal plaque calcification. We used hypercholesteremic mice (Ldlr −/− , apoB 100/100 ) fed Western Diet for 6 months, at which point they received either intraperitoneal injections of saline (CTRL) or Infliximab (INX) for 3 additional months. Expression of the pro‐inflammatory genes TNF‐α, VCAM, and ICAM were all reduced in the INX group (qRT‐PCR). INX treatment improved endothelium‐dependent relaxation compared to the CTRL group (acetylcholine responses in isolated organ chamber; 50.7±3.4%, 43.7±3.7%; respectively), but treatment did not affect relaxation to nitroprusside. Furthermore, expression of the osteogenic gene MSX2 was reduced in INX mice, PPARγ target gene expression was increased, and intimal plaque calcification was reduced in aorta (Alizarin Red; CTRL = 13.7±2.9%, INX = 5.4±1.3%, respectively). In conclusion, blockade of TNF‐α may be a viable therapeutic target to reduce inflammation, improve endothelial dysfunction, and attenuate vascular calcification in the advanced atherosclerotic lesions commonly found in aging humans. 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