Citations

Angiotensin II produces oxidative stress and endothelial dysfunction. ACE2 converts angiotensin II to angiotensin 1–7 and may protect against effects of angiotensin II. We tested the hypothesis that ACE2 deficiency increases oxidative stress and vasomotor dysfunction in cerebral arteries, and examined the role of ACE2 in vascular aging. ACE2 was present at relatively low levels in cerebral arteries. Systolic arterial pressure was similar in adult and old ACE2 knockout (KO) and wild‐type (WT) mice. Maximal dilatation to acetylcholine (ACh) was impaired in the basilar artery from adult ACE2 KO mice compared to adult WT. In old mice, maximal dilatation to ACh was impaired in WT mice but severely impaired in ACE2 KO mice (P<0.05). The antioxidant tempol (1 mM) improved responses to ACh in adult and old ACE2 KO and WT mice. Nitrotyrosine staining in the basilar artery was increased in adult ACE2 KO mice and in old ACE2 KO and WT mice. Expression of NAD(P)H oxidase subunits Nox2 and p47phox, and of pro‐inflammatory molecules Rcan1 and TNFα, was increased in cerebral arteries from old ACE2 KO and WT animals. In summary, ACE2 deficiency impaired vasomotor function in cerebral arteries from adult mice and augments endothelial dysfunction during aging. Oxidative stress plays a critical role in cerebrovascular dysfunction induced by ACE2 deficiency and aging. NIH grants NS‐24621, HL‐38091, HL‐62984; AHA 0815525G, 10PRE3780044.

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

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

Nutrient excess and physical inactivity are primary drivers of numerous age‐related chronic diseases. A growing body of evidence has implicated cellular senescence, a process in which cells lose the ability to divide and concurrently damage neighboring cells by the factors they secrete, as an underlying mechanism of aging and its associated conditions. However, the impact of lifestyle choices on the age‐associated accumulation of senescent cells remains poorly understood. Thus, we examined the effects of a fast food diet (FFD) and a voluntary exercise intervention on multiple parameters of healthspan and cellular senescence using a transgenic mouse model that expresses green fluorescent protein (GFP) in p16 Ink4a ‐positive senescent cells. As expected, 12‐month old male mice that had received the FFD diet for 5 months demonstrated significantly greater gains in body weight and fat mass compared to mice fed a standard chow diet. Moreover, mice fed the FFD exhibited hyperinsulinemia, impaired glucose tolerance, reduced exercise capacity, and diastolic dysfunction. Impressively, providing an exercise wheel (26.9 ± 6.9 km/wk) in parallel with the FFD nullified the deleterious effects of the FFD on body weight and composition, whole‐body metabolism, physical performance and cardiac function. Strikingly, our data further suggest that exercise markedly lowers the number of cells in visceral fat depots that express GFP and stain positively for senescent‐associated β‐galactosidase; markers of senescence, that are highly abundant in sedentary mice fed the FFD. Correspondingly, FFD‐induced expression of senescence‐associated inflammatory factors was also diminished by the exercise intervention. Collectively, our data suggest that lifestyle choices may affect the abundance of senescence cells with advancing age, and exercise may partly mediate its salutary effects on multiple organ systems by preventing their accumulation and/or mediating their removal.

Ankyrin B (AnkB) plays an important role in normal cardiac and neuronal function by linking membrane proteins to the cytoskeleton. Little is known, however, about the role of AnkB in the vasculature. Thus, we studied vasomotor function in aortas from wild‐type mice (AnkB +/+ ) and AnkB‐deficient littermates (AnkB +/− ). Maximum relaxation (M R ) to acetylcholine (Ach) was significantly reduced in AnkB +/− mice relative to AnkB +/+ mice (68 ± 5% vs 80 ± 1%), while responses to sodium nitroprusside were unaffected. While constrictor responses to PGF 2 α (1.5 ± 0.1g vs 1.4 ± 0.1g) were unchanged in AnkB +/− mice, constriction to phenylephrine was reduced by >35% in AnkB +/− mice compared to their AnkB +/+ littermates (p < 0.05). Incubation of AnkB +/+ vessels with the IP 3 ‐receptor inhibitor 2‐APB impaired M R to Ach in both AnkB +/+ and AnkB +/− mice (28 ± 6% and 29 ± 3%, respectively). Vasoconstrictor responses to phenylephrine were also dramatically attenuated by 2‐APB in AnkB +/+ mice (0.43 ± 0.05g vs 0.18 ± 0.02g) but not in AnkB +/− mice (0.28 ± 0.05g vs 0.27 ± 0.05g). These data suggest that Ankyrin B plays a significant role in determining both endothelial and vascular smooth muscle function, in part through the modulation of the IP 3 receptor. Future studies examining the molecular mechanisms whereby Ankyrin B affects vasomotor function may lend critical insights into vasomotor dysfunction in health and disease.

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

Senescence is the consequence of a signaling mechanism activated in stressed cells to prevent proliferation of cells with damage. Senescent cells (Sncs) often develop a senescence-associated secretory phenotype to prompt immune clearance, which drives chronic sterile inflammation and plays a causal role in aging and age-related diseases. Sncs accumulate with age and at anatomical sites of disease. Thus, they are regarded as a logical therapeutic target. Senotherapeutics are a new class of drugs that selectively kill Sncs (senolytics) or suppress their disease-causing phenotypes (senomorphics/senostatics). Since 2015, several senolytics went from identification to clinical trial. Preclinical data indicate that senolytics alleviate disease in numerous organs, improve physical function and resilience, and suppress all causes of mortality, even if administered to the aged. Here, we review the evidence that Sncs drive aging and disease, the approaches to identify and optimize senotherapeutics, and the current status of preclinical and clinical testing of senolytics.