Citations

Physical resilience, defined as the ability to resist and respond to challenges and return to homeostasis, has been postulated to be a determinant of healthspan and lifespan. To test this premise, we have begun to optimize a standardized, clinically relevant battery of resilience challenges, including anesthesia and cisplatin chemotherapy, and recovery outcome measures in younger (2–3 month) and older (26–29 month) mice. We have also started to test whether interventions shown to increase murine lifespan‐acarbose, rapamycin, and 17a‐estradiol‐impact the resilience of older mice. Our preliminary data demonstrate that younger mice (n = 10) have faster dorsal to sternal recumbence recovery time compared to older mice (n = 8) (39 ± 1 sec vs. 67 ± 6 sec, p < 0.001) following a 5‐minute 4% isoflurane anesthesia challenge. In response to a 5‐day cisplatin (2 mg/kg/day) challenge, both younger (n=5) and older (n=4) mice lost a significant percent of their baseline body weight (younger mice −10.6 ± 0.8% and older mice −13.2 ± 2.1%, p = 0.26), however, older mice failed to regain body weight 11 days after the challenge (younger mice −0.9 ± 0.8% and older mice −9.8 ± 0.8%, p < 0.01). Changes in body weight in younger and older mice were largely attributable to the loss/regain of fat mass. Interestingly, 8 weeks of either acarbose (1,000 ppm) or 17α‐estradiol (14.4 ppm) administration to older female mice improved body weight recovery after the cisplatin challenge, while rapamycin (14 ppm) had no appreciable effect. Our preliminary results demonstrate that younger mice, as expected, have greater resilience than older mice to clinically relevant physical challenges. The age at which physical resilience starts to decline requires further investigation. Our early observations suggest interventions that extend health‐ and lifespan may impact resilience in older mice. Ultimately, a standardized battery of resilience challenges may provide a new framework for testing and translating interventions that target the fundamental biology of aging. Support or Funding Information This work was supported by grant AG52958 from the National Institutes of Health. This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .

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 .

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.

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

Real-time RT-PCR is considered the gold standard confirmatory test for coronavirus disease 2019 (COVID-19). However, many scientists disagree, and it is essential to understand that several factors and variables can cause a false-negative test. In this context, cycle threshold (Ct) values are being utilized to diagnose or predict SARS-CoV-2 infection. This practice has a significant clinical utility as Ct values can be correlated with the viral load. In addition, Ct values have a strong correlation with multiple haematological and biochemical markers. However, it is essential to consider that Ct values might be affected by pre-analytic, analytic, and post-analytical variables such as collection technique, specimen type, sampling time, viral kinetics, transport and storage conditions, nucleic acid extraction, viral RNA load, primer designing, real-time PCR efficiency, and Ct value determination method. Therefore, understanding the interpretation of Ct values and other influential factors could play a crucial role in interpreting viral load and disease severity. In several clinical studies consisting of small or large sample sizes, several discrepancies exist regarding a significant positive correlation between the Ct value and disease severity in COVID-19. In this context, a revised review of the literature has been conducted to fill the knowledge gaps regarding the correlations between Ct values and severity/fatality rates of patients with COVID-19. Various databases such as PubMed, Science Direct, Medline, Scopus, and Google Scholar were searched up to April 2021 by using keywords including “RT-PCR or viral load”, “SARS-CoV-2 and RT-PCR”, “Ct value and viral load”, “Ct value or COVID-19”. Research articles were extracted and selected independently by the authors and included in the present review based on their relevance to the study. The current narrative review explores the correlation of Ct values with mortality, disease progression, severity, and infectivity. We also discuss the factors that can affect these values, such as collection technique, type of swab, sampling method, etc.