Hagler, Michael A

Activation of NADPH oxidase is a major contributor to hypertension, reactive oxygen species generation, endothelial dysfunction and increased vascular distensibility in angiotensin II (AngII)‐induced hypertension. Since exercise has been associated with increased antioxidant enzyme expression in blood vessels, we hypothesized that exercise training would improve endothelial function and vascular distensibility in mice with AngII‐induced hypertension. C57BL/6J mice received saline or AngII (1000 ng/kg/min) via osmotic minipumps, and were assigned to sedentary or treadmill exercise groups (13 m/min for 30 minutes, 5 days/week) for 14 days. Endothelium‐dependent relaxation in response to acetylcholine and arterial distensibility were assessed in carotid arteries using a pressurized arteriograph system. Gene expression was examined by qRT‐PCR. In AngII‐treated sedentary mice, endothelium‐dependent relaxation was impaired, while distensibility was higher than in saline‐infused sedentary mice. Compared to AngII‐infused sedentary mice, however, endothelium‐dependent relaxation was partially improved, and distensibility normalized in AngII‐infused exercised mice. In response to exercise, mRNA levels of SOD1 and SOD2 in aorta were increased, while mRNA levels of NOX2 were reduced. We conclude that exercise training can dramatically improve endothelial dysfunction and reverse derangements in carotid distensibility following prolonged AngII‐induced hypertension. Additional work examining mechanisms underlying our observed transcriptional changes will be critical to development of novel therapeutic targets.

We previously reported that MnSOD‐deficiency does not cause overt vasomotor dysfunction in aged or hypercholesterolemic mice. To determine whether increasing MnSOD protects against vasomotor dysfunction and deleterious molecular changes in atherosclerosis, we crossed hypercholesterolemic mice with mice overexpressing MnSOD to test the hypothesis that lifelong increases in MnSOD protect against vasomotor dysfunction. We used Ldlr ‐/‐ /ApoB 100/100 mice that were wild‐type (MnSOD 0/0 ) or transgenic (MnSOD Tg/0 ) and fed a western diet for 6 months. We used organ bath chambers to measure aortic endothelium‐dependent relaxation (relaxation to acetylcholine, Ach), and mRNA from aortic arch was used for qRT‐PCR. Surprisingly, relaxation to Ach was unchanged between MnSOD 0/0 and MnSOD Tg/0 mice, as was relaxation to sodium nitroprusside. As expected, gene expression of MnSOD was significantly increased in the MnSOD Tg/0 mice compared to MnSOD 0/0 mice. Unlike our findings in MnSOD +/‐ mice, however (where Nox2 was increased), Nox2 expression was unchanged between MnSOD 0/0 and MnSOD Tg/0 mice. Importantly, markers of osteogenesis such as Runx2 and BMP2 were unchanged between genotypes. Furthermore, MnSOD overexpression did not alter cellular senescence markers. Collectively, our data support the surprising conclusion that physiologically relevant increases in MnSOD do not significantly affect endothelial function or molecular regulators of histopathological changes in advanced atherosclerosis in hypercholesterolemic mice.

Sirtuin 3 (SIRT3)—a deacetylase localized to the mitochondria—deacetylates a number of mitochondria‐specific proteins, including the mitochondrial antioxidant enzyme MnSOD. We previously reported that genetic inactivation of SIRT3 impairs endothelial function and increases pro‐osteogenic markers in early stages of atherosclerosis. While recent data suggest that the molecular and phenotypic effects of SIRT enzyme inactivation can act in a highly sex‐dependent manner, we have not systematically studied sex differences in our unique mouse strains. Here, we report our first effort to explore the presence of molecular and phenotypic sex differences following inactivation of SIRT3 in hypercholesterolemic mice. We used female and male Ldlr −/− /ApoB 100/100 mice that were either wild‐type (LA‐SIRT3 +/+ ) or null for SIRT3 (LA‐SIRT3 −/− ). Following 3 months of western diet feeding, we assessed endothelial function of aorta using isolated organ bath chambers and measured changes in gene expression using qRT‐PCR. We did not observe sex differences at baseline in hypercholesterolemic mice, as endothelium‐dependent relaxation to acetylcholine (ACH) was similar between female LA‐SIRT3 +/+ and their male LA‐SIRT3 +/+ littermates (61.1%±2.2, 57.9%±2.7; respectively). Interestingly, genetic inactivation of SIRT3 did not impair responses to ACH in female LA‐SIRT3 −/− mice (61.1%±4.2), whereas responses to ACH were modestly impaired in male LA‐SIRT3 −/− mice (53.9%±1.9). Consistent with SIRT3's role as a post‐translational protein modifier, expression of MnSOD was unchanged across all sexes and genotypes. Expression of genes related to pathophysiological changes in atherosclerotic plaques did change in a highly sex‐dependent manner, however, as osterix gene expression levels (related to plaque calcification) were markedly increased in male LA‐SIRT3 −/− compared to their sex‐matched wild‐type littermates and to their female counterparts. Surprisingly, while female LA‐SIRT3 −/− mice showed dramatic increases in expression of interleukin 6 (related to inflammation) compared to their wild‐type littermates, male LA‐SIRT3 −/− had robust reductions in expression in interleukin 6 compared to their LA‐SIRT3 +/+ littermates. Collectively, these data suggest there are significant, sex‐dependent molecular and phenotypic effects of SIRT3 inactivation in hypercholesterolemic mice, which may also drive a paradoxical dissociation between pro‐osteogenic and pro‐inflammatory signaling under conditions of mitochondrial dysfunction. Support or Funding Information NHLBI R01‐HL111121 This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .

Increased oxidative stress is associated with initiation and progression of atherosclerosis. While catalase catalyzes the breakdown of hydrogen peroxide (H 2 O 2 ) to water, it is unclear whether overexpression of catalase is sufficient to protect against cardiovascular dysfunction and advanced plaque formation. Therefore, we hypothesized overexpression of catalase in hypercholesterolemic mice following 3 months or 12 months of Western diet feeding will have preserved endothelial function, reduced H 2 O 2 levels, reduced levels of osteogenic markers, and reduced calcium burden in atherosclerotic plaques. We used Ldlr − / − ApoB 100/100 (LA) mice (hypercholesterolemic mice with a more “humanized” lipid profile) that were either expressed endogenous levels of murine catalase (LA‐Cat 0/0 ) or overexpressed one copy of a human catalase construct (LA‐Cat Tg/0 ). These mice were fed a Western Diet (TD88137) for either 3 months (early disease) or 12 months (late disease). Following 3 months or 12 months on diet, we assessed endothelial function using isolated organ chambers, quantified H 2 O 2 production (staining of frozen cross‐sections of aorta with CM‐H2DCFDA), measured changes of gene expression in aortic arch (qRT‐PCR), and performed histopathological analyses of intimal plaque calcium burden (Alizarin Red). Following 3 months on diet, endothelium‐dependent relaxation to acetylcholine (ACH) was unchanged between LA‐Cat 0/0 and LA‐Cat Tg/0 mice, and we did not find substantive molecular or histopathological differences in aortic tissue between the two genotypes. Interestingly, and contrast to our hypothesis, endothelium‐dependent relaxation was modestly but not significantly impaired in LA‐Cat Tg/0 mice compared to LA‐Cat 0/0 mice after 12 months of western diet feeding. Evaluation of female mice alone, however, revealed profoundly impaired endothelium‐dependent relaxation in LA‐Cat Tg/0 . While H 2 O 2 levels were slightly reduced in LA‐Cat Tg/0 at 3 months, H 2 O 2 levels were not reduced by catalase overexpression following 12 months of Western diet feeding. In further contrast to our original hypothesis, expression of the osteogenic genes osterix and osteopontin were modestly increased in LA‐Cat Tg/0 mice following 3 months on diet compared to their wild‐type littermates, and further increased at the 12 month time point. Functionally, the molecular changes in 12 month animals were associated with increases in intimal plaque calcium burden in LA‐Cat Tg/0 (12.3% ± 2.5%) compared to their wild‐type littermates (7.8% ±1.2%). In conclusion, these data are in line with previous negative clinical trials of antioxidant treatment strategies, and suggest targeting a single antioxidant enzyme/reactive oxygen species to delay initiation or slow progression of atherosclerosis is not a viable therapeutic target, and may in fact paradoxically lead to deleterious changes in plaque composition and endothelial dysfunction. Support or Funding Information NHLBI HL111121 This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .

Myxomatous mitral valve disease (MMVD) is associated with increases in valvular fibrosis, matrix remodeling, and transforming growth factor beta (TGF β) signaling. While interleukin 10 (IL‐10) can suppress tissue fibrosis and matrix metalloproteinase (MMP) expression, its role in MMVD is unknown. We tested the hypotheses that reduced IL‐10 expression contributes to increased tissue fibrosis in MMVD, and that IL‐10 tonically suppresses TGF‐β signaling in normal mitral valves. We used qRT‐PCR to measure expression of IL10, TGF‐β1, MMP 2 and 9, and connective tissue growth factor (CTGF) in normal and myxomatous human mitral valves and in mitral and tricuspid valves from wild‐type and IL‐10 knockout mice. IL‐10 expression was significantly reduced in human myxomatous valves compared to normal valves (53± 12%), and was associated with increases in TGFβ, CTGF, and MMP2. Mitral valve gene expression patterns in IL‐10 −/− mice favored fibrosis and matrix remodeling compared to wild‐type littermates, with increases in TGF β (132±7%), CTGF (154±3%), and MMP9 (259±7%) (p< 0.05 for all), but not MMP2. Interestingly, expression of these genes was not altered in tricuspid valves from IL‐10 −/− mice. Collectively, these data are the first to show that IL‐10 tonically suppresses TGF β signaling in normal mitral valves and that deficiency of IL‐10 is likely to contribute to fibrosis and matrix remodeling in MMVD. NHLBI

Myxomatous mitral valve disease is characterized by fibrosis and matrix degradation and increases in transforming growth factor beta‐1 (TGFbeta‐1). Little is known, however, about changes in molecules that regulate TGFbeta‐1 signaling in myxomatous valves. Thus, we tested the hypothesis that TGFbeta‐1 promotes valvular fibrosis and matrix remodeling in myxomatous valves through canonical Smad2 signaling and permissive reductions in Smad inhibitors. Expression of TGFbeta‐1, TGFbeta‐1 target genes (connective tissue growth factor, matrix metalloproteinases 2/9), E3 ubiquitin‐protein ligases (Smurf 1/2, which degrade Smad2), and NAD(P)H oxidase catalytic subunits (Nox 2, 4, and 5) was measured using quantitative real‐time RT‐PCR. Phospho‐Smad2 levels were measured using western blotting. TGFbeta‐1 expression was higher in myxomatous valves (138.3 ± 12.5% of control, n = 24 per group; p < 0.05), while CTGF and MMP2 were increased by 5‐ and 2‐fold, respectively. Surprisingly, phospho‐Smad2 levels were unchanged between myxomatous and control tissue due to selective increases in Smurf2 expression in myxomatous valve tissue (165.2 ± 17.7% of control, p < 0.05), as Smurf1 expression was unchanged. Expression of Nox2 and Nox4—which can drive pro‐fibrotic and matrix remodeling gene expression in other tissues—were significantly elevated in myxomatous valves (211 ± 44% and 224 ± 35 % of control, respectively; p < 0.05). In conclusion, our data suggest that canonical TGFbeta‐1/Smad2 signaling is not required for progression of myxomatous valve disease, and that reducing Nox2‐and Nox4‐derived reactive oxygen species may be a novel intervention to slow progression of fibrosis and matrix remodeling in myxomatous valves.

Myxomatous mitral valve disease (MMVD), if untreated, progresses to valve prolapse, mitral regurgitation, and heart failure. Molecular mechanisms contributing to MMVD are poorly understood, and surgery remains the primary treatment for this disease. In the current study, we used whole‐genome expression microarrays in normal (n = 12) and myxomatous (n = 11) mitral valves as a nonbiased approach to identify novel pathways contributing to MMVD. In addition to identifying known contributors to fibrosis in MMVD (e.g. TGFβ signaling), we found robust evidence of immune cell infiltration and activation in myxomatous valves. Specifically, toll‐like receptors (TLRs) 3 and 7, which play a key role in antigen recognition, alongside cell surface receptors CD14 and CD83, characteristic of antigen presenting cells of monocytic lineage, were overexpressed in myxomatous tissue. Interleukin‐7 and the chemokine receptor CX3CR1, implicated in monocyte activation and recruitment, were also upregulated. Interestingly, immune localization appeared independent of “classic” inflammatory cytokines such as IL‐6. Differential expression of a subset of genes was confirmed with qRT‐PCR and immunohistochemistry. In conclusion, these data are the first to demonstrate infiltration of immune cells in MMVD, which may represent a critical process in the initiation and/or progression of this disease. NHLBI

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

In angiotensin II‐induced hypertension, activation of NADPH oxidase is a major contributor to reactive oxygen species generation and endothelial dysfunction. As exercise has been associated with the augmentation of antioxidant enzyme expression in endothelial cells, we hypothesized that exercise would improve endothelial function in mice angiotensin II‐induced hypertension. Three‐month old received either saline or a pressor dose of angiotensin II (1000 ng/kg/min) using subcutaneous osmotic minipumps, and were housed in cages with or without a running wheel. Endothelium‐dependent relaxation in response to acetylcholine was studied in carotid arteries using isolated organ chamber baths. Gene expression was examined using qRT‐PCR. In angiotensin II‐infused sedentary mice, endothelium‐dependent relaxation was significantly impaired, and was partially improved when NADPH oxidase was inhibited by apocynin. mRNA levels of SOD1, SOD2, SOD3, and NOX2 were increased in aorta, and mRNA levels of NOX4 and NOS3 were substantially decreased. In contrast to our hypothesis, endothelium‐dependent relaxation was not improved in angiotensin II‐infused exercised mice housed with running wheels, despite marked increases in physical activity over the two week time period. We conclude that angiotensin II‐induced hypertension results in profound endothelial dysfunction that is not attenuated by voluntary wheel running exercise. Ongoing work is examining molecular mechanisms underlying these observations, as well as evaluation of the protective effects of regimented exercise of varying intensities.

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