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Sarcopenia is a skeletal muscle disease that often manifests with aging, characterized in part by low skeletal muscle strength and mass as well as low physical performance (i.e., exercise intolerance) compared to young adults, which contributes to increased frailty. There remains a need to develop novel and adherable interventions for mitigating these losses with aging. Increased flavonoid consumption is inversely related to frailty in mid-life/older adults. Late-life oral supplementation with the natural flavonoid apigenin can improve select age-related conditions; however, its effect on treating features of sarcopenia is unkown. Hypothesis. We hypothesized that late-life oral supplementation with apigenin would result in greater grip strength and skeletal muscle mass, increased exercise tolerance, and lower frailty with aging. Methods. Young (Y; 6 mo) and old (O; 27 mo) male C57BL/6N mice consumed drinking water containing vehicle (control [C]; 0.2% carboxymethylcellulose; YC, n=10; OC, n=7) or apigenin (Api; 0.5 mg/mL in vehicle; Y-Api, n=10; O-Api, n=9]) for 6 weeks. Skeletal muscle strength was measured by forelimb grip strength. Skeletal muscle mass was assessed as quadriceps weight relative to body mass. Exercise tolerance was determined using a rota-rod and quantified as time spent running while the rota-rod accelerated from 50-100% of maximal running speed. Frailty was evaluated using a validated frailty index. Exercise tolerance was assessed before and after the intervention, and skeletal muscle strength, mass, and frailty were quantified at the end of the intervention. Data are reported as mean±SEM. Results. Forelimb grip strength. Forelimb grip strength (g of force/bodyweight) was higher in YC vs. OC (6.7±0.3 vs 5.3±0.2, P=0.001) and in O-Api (6.1±0.2, P=0.006 vs OC). However, O-Api was still lower than Y-Api (YApi, 6.9±0.3, P=0.025 vs O-Api). There were no differences between Y-Api and YC ( P=0.520). Quadriceps mass. Quadriceps mass (mg/bodyweight) was higher in YC vs OC (9.5±0.6 vs 7.8±0.4, P=0.051), in O-Api vs OC (O-Api, 9.9±0.6; P=0.005 vs OC), and in Y-Api vs YC (11.1±0.5, P =0.064 vs YC). O-Api was not different than Y-Api ( P=0.111). Exercise tolerance. Prior to the intervention we observed differences with aging (YC, 1233±141 sec vs OC, 614±128 sec; P=0.010), but no differences between treatment groups (YC vs Y-Api, P=0.115; OC vs O-Api, P=0.177). Following the intervention, exercise tolerance was increased in the O-Api group (pre: 443±50 sec vs post: 1118±113 sec, P NIH AG055822-02S1, K99 HL159241, H2020-MSCA-IF-2019: 892267, T32 DK007135 This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

BACKGROUND: Cardiovascular disease (CVD) risk is elevated in patients with advanced chronic kidney disease (CKD). Vascular dysfunction, including vascular endothelial dysfunction and large elastic artery stiffening, is a key contributor to increased CVD risk in advanced CKD. Individuals with mild to moderately reduced renal function may also be at increased risk for CVD, but endothelial function and arterial stiffness are not well characterized in this group. Further, women with CKD have lower risk for CVD than men. Whether women are protected from renal dysfunction-related decreases in vascular function is unknown. PURPOSE: We sought to determine: i) whether individuals with mild to moderate renal dysfunction (MRD) have lower endothelial function and greater arterial (aortic) stiffness compared to healthy controls (HC) and ii) if the presence of vascular dysfunction in individuals with MRD is influenced by sex. METHODS & RESULTS: Fifty-nine adults were split into two groups based on renal function. Thirty-two adults (mean±SEM: 66±2 years; 16 women) were identified with MRD, characterized as an estimated glomerular filtration rate (eGFR) of 45-89 (CKD-EPI; 75.0±2.0 mL/min/1.73m2) and/or the presence of microalbuminuria (albumin: creatinine ratio between 30-300 mg/g). Twenty-seven adults (63±2 years; 13 women) were identified as HC with an eGFR >90 (CKD-EPI; 98.7±0.9 mL/min/1.73m2). Subject characteristics known to influence vascular function (e.g., blood pressure, cholesterol, blood glucose, etc.) were not different between groups (all p>0.05). Endothelial function, assessed by brachial artery flow mediated dilation, was not different between groups (MRD: 4.4±0.5%, HC: 4.6±0.5%, p=0.75). Aortic stiffness, assessed via carotid-femoral pulse wave velocity, also was not different (MRD: 950.7±50.6 cm/s, HC: 864.3±33.1 cm/s, p=0.18). When analyzed by sex, men with MRD had lower endothelial function (MRD: 2.8±0.4%, HC: 4.4±0.6%, p=0.04) and tended to have stiffer arteries (MRD: 879.0±42.7 cm/s, HC: 787.7±35.5 cm/s, p=0.11) compared to HC men. In women with MRD, there were no differences in endothelial function (MRD: 5.9±0.8%, HC: 4.8±0.8%, p=0.36) or aortic stiffness (MRD: 1022.3±89.9 cm/s, HC: 953.6±49.4 cm/s, p=0.55) versus HC women. CONCLUSIONS: Men with MRD exhibit vascular dysfunction, while women appear to be protected from MRD-related vascular dysfunction. The lower risk of CVD in women versus men with CKD may, in part, be due to resistance against renal dysfunction-related vascular dysfunction. NIH K01DK115524 to MJR; 5T32DK007135-46 to KOM; K01HL153326 to DHC This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

The aorta stiffens with aging in both men and women and predicts cardiovascular mortality. Stiffening occurs largely due to aortic wall structural remodeling induced, in part, by chronic low-grade inflammation. Use of animal models in biomedical research is essential for screening interventions for safety/efficacy and providing mechanistic insight that is difficult, or impossible, to obtain in humans. Male C57Bl/6N mice are an established model of aortic aging and have predominantly been used to determine mechanisms of and investigate interventions for mitigating age-related vascular changes. Although some studies have investigated mechanisms of artery stiffening in female mice in midlife, there is still a paucity of information regarding whether female mice are an appropriate model of age-related aortic stiffening/structural remodeling, especially in late life. Purpose: To determine if female C57Bl/6N mice are an appropriate model (i.e., reflective of the human condition) of age-related aortic stiffening, associated structural remodeling and systemic inflammation. Methods and Results: Data are mean ± SE. Aortic stiffness (pulse wave velocity) was higher in old (O; 27-29 mo) vs. young adult (Y; 6-7 mo) male (M) and female (F) C67Bl/6N mice (cm/sec: YM: 347±27, YF: 345±23, OM: 401±32, OF: 413±45; p0.15). Aortic elasticity was inversely related to IL-6 (r2=0.49, p NIH T32 AG000279, F31 FHL160173A, K99 HL159241 This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Doxorubicin (DOXO) chemotherapy induces vascular dysfunction (aortic stiffening and endothelial dysfunction). Cellular senescence and the related increase in oxidative stress, notably mitochondrial reactive oxygen species (mtROS), are implicated in DOXO-induced vascular dysfunction. Hypothesis. Targeting cellular senescence, via administration of a senolytic (senescent cell clearing drug), following exposure to DOXO chemotherapy will prevent aortic stiffening and endothelial dysfunction by suppressing excess mtROS bioactivity and preserving nitric oxide (NO) bioavailability. Methods/Results Animals. Young (6 mo) male and female C57BL/6 mice received a single intraperitoneal injection of Sham (saline) or DOXO (10 mg/kg in Sham). One week later, mice received the vehicle (V; 10% EtOH, 30% PEG400, 60% Phosal 50 PG) or the senolytic ABT263 (ABT; 50 mg/kg/day in V) by oral gavage (1 week on; 2 weeks off; 1 week on). There were 4 groups/sex (Sham-V; Sham-ABT; DOXO-V; DOXO-ABT; n=10-12/group). There were no sex differences, so results were combined. Data are mean±SEM. Aortic Stiffness. Aortic stiffness (aortic pulse wave velocity [aPWV, cm/sec]) was assessed pre and post intervention. There were no group differences at baseline ( P=.63). After the dosing period, aPWV increased by 15% in the DOXO-V group (pre: 353±2 vs post: 414±6, P NIH K99 HL159241 and NIH R21 AG078408 This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Aortic stiffnessincreases with aging and is an independent risk factor for the development of cardiovascular diseases. Cellular senescence, a state of cell cycle arrest that accumulates in arteries with aging, contributes to age-related aortic stiffening. 25-Hydroxycholesterol (25HC) is a novel senolytic — a compound that eliminates senescent cells by modulating senescent cell anti-apoptotic pathways (SCAPs). However, the senolytic effects of 25HC in the vasculature and its influence on age-related aortic stiffness and associated arterial wall structural remodeling are unknown. Hypothesis: 25HC treatment in old mice treated would favorably modulate the vascular SCAP profile, suppress vascular cell senescence, and reduce aortic stiffness relative to old control mice. Methods & Results. Animals. Two groups (n=10-15/group) of old (24-26mo) female p16-3MR mice were used: 1) untreated control; and 2) 25HC (50 mg/kg/day in 22.5% 2-hydroxypropyl-β-cyclodextrin administered intraperitoneally for 5 consecutive days). SCAPs. Aortic abundance of CRYAB, the therapeutic target of 25HC and a SCAP regulator, was 38% lower in 25HC-treated v. control mice (chemiluminescence units [CU]; PINK4A, a protein that confers cell cycle arrest in senescent cells (CU, P R01 AG055822, K99 HL159241, F31 HL165885 This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Background: Individuals infected with SARS-CoV-2, the virus that causes coronavirus disease 2019 (COVID-19), have increased risk of cardiovascular diseases (CVD). Endothelial dysfunction, characterized in part by a reduction in the vasodilatory molecule nitric oxide (NO), is a primary mechanism of CVD and has been observed in people with COVID-19. However, it is not well known if alterations in the circulating milieu (i.e., plasma) contribute to COVID-19-associated endothelial dysfunction. Moreover, the mechanisms by which circulating factors may contribute to endothelial dysfunction in COVID-19, including increased reactive oxygen species (ROS)-related oxidative stress and depletion of the key metabolic co-substrate nicotinamide adenine dinucleotide (NAD+), remain to be determined. Purpose: Here, we tested the hypotheses that circulating factors in plasma from patients with COVID-19 would cause ex vivo endothelial cell dysfunction (reduced acetylcholine-stimulated NO production), increase ROS bioactivity and decrease NAD+ levels. We also assessed whether the NAD+ precursor nicotinamide riboside (NR) would prevent endothelial cell dysfunction and oxidative stress induced by circulating factors in plasma from patients with COVID-19. Methods: Plasma samples were obtained from the University of Colorado Anschutz Medical Campus COVID-19 Biobank Specimen Repository. Samples were from men and women (age 18+ years) who were hospitalized and tested positive (+; n=34) or negative (–; n=13) for COVID-19. Human aortic endothelial cells (HAECs) in culture were exposed to 10% subject plasma, then stained with florescent probes to detect NO production (DAR-4M AM; before and 6 minutes after addition of 100μM acetylcholine), as an ex vivo model of endothelial function, and basal ROS bioactivity (CellROX), as a mechanism modulating endothelial cell function. NAD+ and NADH were measured in cells after plasma exposure using luminescence-based detection. HAECs exposed to COVID-19+ plasma also were co-incubated with 1mM NR. Results: Acetylcholine-stimulated NO production was lower (-27±12%; mean±SD; p+ (12.4±1.8 vs. 17.7±8.0au; p=0.0013) and NAD+/NADH (4.2±1.6 vs. 5.6±1.8au; p=0.0167) were lower in HAECs exposed to plasma from COVID-19+ vs. COVID-19– individuals. However, when HAECs exposed to plasma from COVID-19+ individuals were treated with NR, acetylcholine-stimulated NO production and ROS bioactivity were not different from that of HAECs exposed to plasma from COVID-19– individuals (all p>0.05). Conclusions: These findings suggest that circulating factors promote COVID-19-related endothelial cell dysfunction, increase ROS bioactivity and lead to NAD+ depletion. Supplementation with NAD+ precursors, such as NR, may be an effective strategy to prevent endothelial dysfunction and reduce the risk of CVD with COVID-19. F31HL154782, 3R01AG061514-02S2, K01DK115524 This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Aging is the main risk factor for cardiovascular diseases (CVD) due in part to stiffening of large elastic arteries (e.g., aorta). High-fiber diets (H-FIB) are associated with reduced risk of CVD, but most people do not meet guidelines for adequate fiber intake. Short-chain fatty acids (SCFA), which are produced by gut microbiome-dependent fermentation of soluble fiber, are thought to mediate many of the benefits of a H-FIB. Acetate, the most prevalent SCFA in circulation, may be an adherable intervention as an oral supplement for improving age-related arterial stiffening and reducing the risk of CVD. PURPOSE: To determine if oral acetate supplementation can reduce age-related aortic stiffness comparably to a H-FIB. METHODS: Young (Y: 3mo; N=11-12/group) and old (O: 24mo; N=24-27/group) C57Bl/6N mice were split into 3 groups per age: 1) controls (YC/OC): standard chow (15% total, 2.5% soluble fiber) and normal drinking water; 2) acetate supplemented (YA/OA): standard chow and 100mM of calcium acetate in drinking water; and 3) H-FIB (YF/OF): chow supplemented with fiber (20% total, 8% soluble fiber) and normal drinking water. Aortic pulse wave velocity (aPWV; standard reference method of in vivo aortic stiffness) and blood pressure (BP; tail cuff) were measured before and after 8 weeks of intervention. Thoracic aortas were harvested to measure: elastic modulus (aEM) of aortic rings (structural measurement of stiffness dependent on engagement of collagen fibers); aortic wall thickness (structural contributor to arterial stiffening); and collagen abundance by immunofluorescence (pro-stiffening structural protein). RESULTS: Data are mean ± SE. aPWV was higher in old vs. young mice at baseline (O: 404±10 vs. Y: 335±4 cm/sec, p FUNDING: K99/R00 HL151818, CU Boulder BSI Scholarship This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Aging is the primary risk factor for cardiovascular diseases (CVD), the leading cause of death worldwide. Vascular endothelial dysfunction, a key antecedent to CVD, is primarily mediated by excess superoxide-related oxidative stress and decreased nitric oxide (NO) bioavailability. High-fiber diets (H-FIB) are associated with lower CVD-related morbidity/mortality, but adherence to guidelines for recommended dietary fiber intake is poor. Benefits of H-FIB are likely mediated by fermentation of soluble fiber into short-chain fatty acids (SCFAs) in the gut, which may improve endothelial function. Thus, supplementing SCFAs may be a more practical/effective intervention to reduce CVD risk with aging. PURPOSE: To determine if oral supplementation with acetate, the most prevalent SCFA in circulation, reverses vascular endothelial dysfunction in old mice as effectively as a H-FIB. METHODS: Old (O: 24mo) and young (Y: 3mo) male C57BL/6 mice (N=10-17/group) were randomized into 3 groups per age: 1) standard chow (15% total & 2.5% soluble fiber) + normal drinking water (YC/OC); 2) standard chow + 100mM calcium acetate supplemented in drinking water (YA/OA); or 3) H-FIB (20% total & 8% soluble fiber [inulin added to the standard chow]) + normal drinking water (YF/OF). Endothelium-dependent dilation (EDD; ex vivo carotid artery dilation to increasing doses of acetylcholine) and associated mechanisms were assessed following 8 weeks of intervention. RESULTS: Data are mean ± SE. Age-related impairments in peak EDD (YC: 92±1%; OC: 81±2%, p0.8 vs. YC), suggesting that the interventions effects are specific to aging. There were no differences in smooth muscle sensitivity to NO (peak dilation to sodium nitroprusside; OC: 98±1%; OA: 99±0%; OF: 99±0%, p=0.1), indicating that differences in EDD were endothelium-dependent. Instead, these changes were likely NO-mediated, as addition of the endothelial NO synthase inhibitor L-NAME abolished differences in peak EDD (OC: 51±4%; OA: 47±8%; OF: 54±6%, p=0.8). Aortic superoxide production (electron paramagnetic resonance spectroscopy) was lower in old acetate-treated mice (OC: 50±7; OA: 29±3 AU, p=0.04), and tended to be lower in H-FIB mice (OC vs. OF: 33 AU, p=0.18). Incubation with the superoxide scavenger TEMPOL improved peak EDD in OC mice (OC: 81±2%; OC+TEMPOL: 93±2%, p K99/R00 HL151818, F31 HL164004 This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Age-related vascular endothelial dysfunction, characterized by reduced nitric oxide (NO) bioavailability, is mediated in part by excess production of mitochondrial reactive oxygen species (mtROS). Excess mtROS also oxidizes circulating lipids (i.e., low-density lipoprotein [LDL]) to promote endothelial dysfunction with aging. We recently showed that 6-weeks of treatment with the mitochondria-targeted antioxidant MitoQ (MQ) (20 mg/day) improved endothelial function in vivo in older adults. The improvements in endothelial function with MQ were mediated by reduced mtROS and associated with lower plasma levels of oxidized LDL (oxLDL). However, the complete mechanisms by which MQ improves endothelial function, and if MQ-related decreases in oxLDL are mechanistically involved, remain unknown. PURPOSE. To determine if chronic MQ supplementation-mediated changes in the circulating milieu contribute to improvements in endothelial cell (EC) function and the mechanistic role of reductions in oxLDL. METHODS. Plasma samples were obtained 24 h after the last dose from 19 older adults (mean±SEM: 67±1 yrs; 11 women) who completed our crossover design pilot trial to assess the efficacy of 6-weeks of MQ vs. placebo (PLA) treatment for improving endothelial function. Using an ex vivo model of endothelial function, acetylcholine (Ach)-stimulated NO bioavailability (DAR-4M-AM) was quantified in human aortic ECs (HAECs) following exposure to plasma collected after each phase of the study (MQ and PLA). The role of mtROS production (MitoSOX) in plasma-mediated changes in HAEC function was also assessed. To elucidate the role of lower oxLDL with MQ treatment on HAEC function, physiological levels of oxLDL were ‘added back’ to plasma collected from subjects after MQ treatment at a concentration corresponding to the average reduction after MQ (14 ng/mL). To determine if lower oxLDL with MQ treatment improves HAEC function through a reduction in binding to the oxLDL receptor LOX-1, HAECs were subjected to LOX-1 blocking antibodies prior to plasma exposure under the 3 experimental conditions (PLA; MQ; MQ + oxLDL [‘add back’]) in a subset of subjects (N=9; 67±1 yrs; 5 women). RESULTS. Ach-stimulated NO bioavailability was higher (PLA: 1.0±0.05; MQ: 1.24±0.08; P=0.0002) and mtROS was lower (PLA: 1.0±0.09; MQ: 0.76±0.08; P=0.003) in HAECs exposed to plasma collected from subjects after MQ treatment compared to PLA. Normalizing oxLDL (‘add back’) abolished the effects of MitoQ treatment. LOX-1 blockade modulated Ach-stimulated NO and mtROS production to levels comparable to (P>0.98) or lower than (P 1R21AG049451-01A1 (DRS); 5T32DK007135-46 (KOM); K01DK115524-01A1 (MJR) This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Age-related increases in aortic stiffness contribute to the development of cardiovascular diseases (CVD). To determine whether the gut microbiome (GM) modulates age-related aortic stiffening, we performed fecal microbiota transplants (FMT) between young (Y; 3 month) and older (O; 25 month) male C57BL/6N mice. Following antibiotic treatment (to suppress endogenous microbiota), mice received weekly FMT (fecal samples collected at baseline) via oral gavage for 8-16 weeks from their own (i.e., sham condition: Y-y, O-o [RECIPIENT-donor]) or opposite age group (Y-o, O-y) (N=8-12/group). In vivo aortic stiffness (pulse wave velocity [PWV]) was higher in older vs. young mice at baseline (382±8 vs. 328±7cm/sec, mean±SE, P

Age-related declines in cognitive function increase the risk of developing mild cognitive impairment and dementia, but select nutraceuticals (bioactive plant compounds) may hold promise for protecting the brain and improving cognitive function with age. Apigenin is a flavonoid nutraceutical found in chamomile and reported to inhibit multiple hallmarks of aging; however, it has not been studied in the context of brain aging specifically. We treated young (6 mo) and old (27 mo) C57BL/6N mice with apigenin (0.5 mg/mL in 0.2% carboxymethylcellulose) or control (0.2% carboxymethylcellulose) drinking water for 6 weeks. Then, we assessed cognitive function and performed RNA-seq to characterize global transcriptomic changes and potential mechanisms of action in the brain. We observed impaired novel object recognition (NOR) test performance (an index of learning/memory) in old vs. young control mice (P300 genes were differentially expressed in old apigenin-treated mice vs. old controls, and the biological processes linked with these differences were related to innate and adaptive immune function, and cytokine and chemokine regulation. We are performing protein/signaling pathway analyses to elucidate downstream cellular changes associated with apigenin treatment, but our current results suggest apigenin may be a promising nutraceutical candidate for preventing brain aging.