Roos, Carolyn M

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

Asymmetric dimethylarginine (ADMA), an endogenous inhibitor of nitric oxide synthases, is primarily degraded by DDAH1. We tested the hypotheses that over expression DDAH1 reduces endothelial dysfunction and slows progression of fibrocalcific aortic valve disease (FCAVD) in hypercholesterolemic mice. We used low density lipoprotein receptor‐deficient, apolipoprotein B100‐only (LA) mice that were either wild‐type (D 0/0 ) or overexpressed human DDAH1 (D Tg/0 ). Systolic blood pressure (SBP, tail cuff), vasomotor function (isolated organ bath), and aortic valve function (echocardiography), were measured at 3 and 6 month time points. Compared to LA‐D 0/0 mice, SBP was reduced by ~15 mmHg in LA‐D Tg/0 mice at both 3 and 6 months (p < 0.05). Although aortic endothelial function progressively declined with prolonged hypercholesterolemia, there were no differences between LA‐D 0/0 and LA‐D Tg/0 mice. While aortic valve function was similar between LA‐D 0/0 and LA‐D Tg/0 mice at 3 months (peak transvalvular velocity: 1.95±0.09 m/sec versus 2.03±0.07 m/sec), overexpression of DDAH1 slowed progression of valvular dysfunction at 6 months (2.07±0.10 m/sec versus 2.30±0.05 m/sec). In conclusion, our data suggest that increasing DDAH1 may be an effective therapeutic strategy to slow progression of FCAVD, and is likely to exert protective effects independent of changes in systemic endothelial function.

We have previously shown that global reductions in mitochondrial antioxidant capacity and induction of reactive oxygen species (ROS)‐induced ROS‐release (RI‐RR) do not impair endothelial function in young mice due to compensatory increases in an apparent NAD(P)H oxidase‐derived endothelium‐derived hyperpolarizing factor (EDHF). In the present study, we hypothesized that endothelial MnSOD is a major determinant of the functional consequences of RI‐RR, and NAD(P)H oxidase‐derived H 2 O 2 is the major compensatory EDHF released from endothelium when MnSOD is reduced. MnSOD was reduced in endothelium by crossing MnSOD FL/FL mice with mice carrying cre‐recombinase driven by a Tie‐2 promoter (Cre + ). Aorta from Cre + /MnSOD +/+ and Cre + /MnSOD FL/+ mice were used for studies of endothelial function. Maximum relaxation to acetylcholine (M R , ACH ) was not affected between Cre +/ / MnSOD +/+ and Cre + /MnSOD FL/+ mice (61%±1.9, 61%±2.6; respectively). Incubation with apocynin did not change M R , ACH in Cre + / MnSOD +/+ mice (63%±2.0), but significantly impaired M R , ACH in Cre +/ MnSOD FL/+ (42%±4.7). Incubation with catalase did not effect M R , ACH in Cre + /MnSOD +/+ mice, but substantially impaired M R , ACH in Cre + /MnSOD FL/+ mice (61%±1.2, 48%±1.7). We conclude that inducing RI‐RR in endothelium by reducing MnSOD elicits a compensatory increase in EDHF production, most likely from an increase in NOX2‐derived H 2 O 2 . 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

Reduced antioxidant enzyme activity and subsequent impairments in endothelial function are major contributors to progression of atherosclerosis. We previously reported that endothelium‐dependent relaxation is well‐preserved in manganese superoxide dismutase‐deficient mice (MnSOD +/‐ ) due to production of a compensatory, H 2 O 2 ‐derived endothelial hyperpolarizing factor (EDHF). It is unclear, however, whether similar phenomena occur in hypercholesterolemic mice, and whether reconstitution of mitochondrial antioxidant capacity with mitochondria‐targeted antioxidants (mito‐Tempo) can reverse this phenomena. We used Ldlr ‐/‐ ApoB 100/100 (LA) mice that were either wild‐type (LA‐MnSOD +/+ ) or heterozygous (LA‐MnSOD +/‐ ) for MnSOD. After 6 months of Western diet, mice were treated with either saline (SAL) or MT (0.7mg/kg/day) using osmotic minipumps for 2 weeks. Endothelial function in aorta was assessed using isolated organ chamber baths in the presence or absence of apocynin (an NAD(P)H oxidase inhibitor, APO) . Osmotic minipump implantation significantly worsened baseline endothelial function compared to non‐implanted mice. Overall, relaxation to acetylcholine (MR ACH ) was unchanged between LA‐MnSOD +/+ and LA‐MnSOD +/‐ treated with SAL (3.8±4.5% and 13.6±14.5%, respectively). Treatment with MT significant improved endothelial function in LA‐MnSOD +/+ mice (21.6±7.1%), and MR ACH was further improved after acute incubation with APO (25±6.7%). In LA‐MnSOD +/‐ , MT greatly improved MR ACH (38.6±3.4%). In contrast to LA‐MnSOD +/+ , however, acute incubation of vessels with APO impaired MR ACH (27.3±10%). Collectively, our data suggest that increasing mitochondrial antioxidant capacity substantially improves endothelial function in hypercholesterolemic mice, but does not completely reverse compensatory molecular mechanisms that preserve endothelial function during prolonged mitochondrial antioxidant deficiency. NHLBI Grant Funding Source : Supported by NHLBI

Endothelial cell senescence promotes inflammation and impairs endothelium‐dependent vasodilation. While aging, hypercholesterolemia, and caloric excess are known to drive cellular senescence through activation of mTOR, it is unknown whether reduction of the mTORC1 adapter protein RapTOR can improve vasomotor function. Thus, we used hypercholesterolemic mice (Ldlr ‐/‐ /apoB 100/100 , or LA) carrying intact copies of Raptor (LA‐Raptor +/+ ) or were deficient in one copy of Raptor (LA‐ Raptor +/‐ ). Mice were fed Western Diet for 6 or 12 months, and vasomotor function in aorta was evaluated using isolated organ chamber baths. Maximum relaxation to acetylcholine (MR ACH ) was progressively impaired from 6 months to 12 months in LA‐Raptor +/+ mice (69±4% and 42±7%, respectively). In contrast to our hypothesis, MR ACH was not significantly improved in LA‐Raptor +/‐ mice at either time point (72±3% and 50±8%, respectively). Relaxation to acetylcholine was abrogated by L‐NAME in both strains at both time points and relaxation to nitroprusside was identical between strains at each time point, suggesting that changes in endothelial function were not masked by compensatory mechanisms (e.g., endothelium‐derived hyperpolarizing factors). Furthermore, reduction of RapTOR did not elicit changes in maximal tension generated in response to Prostaglandin F 2 α at either time point. Collectively, despite strong previous evidence from in vitro model systems, genetic reduction of mTORC1 signaling in vivo does not appear to be sufficient to improve vasomotor function in aging hypercholesterolemic mice . Grant Funding Source : Supported by NIH/NHLBI