Search

Preclinical rodent models are essential research tools for improving understanding of physiological aging processes in humans. However, the translatability of findings obtained leveraging rodent models to humans is limited, likely due in part to differences in macronutrient composition of the diets. Here, we investigated the impact of a 3-month diet intervention in old male C57BL/6JN mice in which the macronutrient composition was aligned with that of a midlife/older adult in the United States, compared to a traditional rodent diet, and assessed various phenotypes that are typically altered with aging. Following the diet period, mice fed the human macronutrient-matched diet had greater quadricep and subcutaneous adipose and visceral adipose tissue masses compared to animals fed a traditional mouse diet. Frailty, assessed using a clinical frailty index, was lower, while grip strength was higher in mice fed the human-matched diet. Circulating metabolite and inflammatory cytokine profiles were altered in mice fed the human-matched diet. Notably, mortality rate (assessed in animals who died or were euthanized per veterinary recommendation before the pre-determined end of study euthanasia), tended to be lower in mice fed the human-matched diet. The present study underscores the importance of diet in rodent studies of aging, as differences in macronutrient composition can affect various physiological processes in old mice that are relevant to aging research., (© 2024. The Author(s), under exclusive licence to American Aging Association.)

Endothelial function declines with aging and independently predicts future cardiovascular disease (CVD) events. Diving also impairs endothelial function in humans. Yet, dolphins, being long-lived mammals adapted to diving, undergo repetitive cycles of tissue hypoxia-reoxygenation and disturbed shear stress without manifesting any apparent detrimental effects, as CVD is essentially nonexistent in these animals. Thus, dolphins may be a unique model of healthy arterial aging and may provide insights into strategies for clinical medicine. Emerging evidence shows that the circulating milieu (bioactive factors in the blood) is at least partially responsible for transducing reductions in age-related endothelial function. To assess whether dolphins have preserved endothelial function with aging because of a protected circulating milieu, we tested if the serum (pool of the circulating milieu) of bottlenose dolphins ( Tursiops truncatus ) induces the same arterial aging phenotype as the serum of age-equivalent humans. We incubated conduit arteries from young and old mice with dolphin and human serum and measured endothelial function ex vivo via endothelium-dependent dilation to acetylcholine. Although young arteries incubated with serum from midlife/older adult human serum had lower endothelial function, those incubated with dolphin serum consistently maintained high endothelial function regardless of the age of the donor. Thus, studying the arterial health of dolphins could lead to potential novel therapeutic strategies to improve age-related endothelial dysfunction in humans. NEW & NOTEWORTHY We demonstrate that, unlike serum of midlife/older adult humans, age-matched dolphin serum elicits higher endothelial function ex vivo in young mouse carotid arteries, suggesting that the circulating milieu of bottlenose dolphins may be geroprotective. We propose that dolphins are a novel model to investigate potential novel therapeutic strategies to mitigate age-related endothelial dysfunction in humans.

Cardiovascular diseases (CVDs) are the leading cause of death in the United States. However, disparities in CVD-related morbidity and mortality exist as marginalized racial and ethnic groups are generally at higher risk for CVDs (Black Americans, Indigenous People, South and Southeast Asians, Native Hawaiians, and Pacific Islanders) and/or development of traditional CVD risk factors (groups above plus Hispanics/Latinos) relative to non-Hispanic Whites (NHW). In this comprehensive review, we outline emerging evidence suggesting these groups experience accelerated arterial dysfunction, including vascular endothelial dysfunction and large elastic artery stiffening, a nontraditional CVD risk factor that may predict risk of CVDs in these groups with advancing age. Adverse exposures to social determinants of health (SDOH), specifically lower socioeconomic status (SES), are exacerbated in most of these groups (except South Asians-higher SES) and may be a potential mediator of accelerated arterial aging. SES negatively influences the ability of marginalized racial and ethnic groups to meet aerobic exercise guidelines, the first-line strategy to improve arterial function, due to increased barriers, such as time and financial constraints, lack of motivation, facility access, and health education, to performing conventional aerobic exercise. Thus, identifying alternative interventions to conventional aerobic exercise that 1 ) overcome these common barriers and 2 ) target the biological mechanisms of aging to improve arterial function may be an effective, alternative method to aerobic exercise to ameliorate accelerated arterial aging and reduce CVD risk. Importantly, dedicated efforts are needed to assess these strategies in randomized-controlled clinical trials in these marginalized racial and ethnic groups.

The circulating milieu, bioactive molecules in the bloodstream, is altered with aging and interfaces constantly with the vasculature. This anatomic juxtaposition suggests that circulating factors may actively modulate arterial function. Here, we developed a novel, translational experimental model that allows for direct interrogation of the influence of the circulating milieu on age-related arterial dysfunction (aortic stiffening and endothelial dysfunction). To do so, we exposed young and old mouse arteries to serum from young and old mice and young and midlife/older (ML/O) adult humans. We found that old mouse and ML/O adult human, but not young, serum stiffened young mouse aortic rings, assessed via elastic modulus (mouse and human serum, P = 0.003 vs. young serum control), and impaired carotid artery endothelial function, assessed by endothelium-dependent dilation (EDD) (mouse serum, P < 0.001; human serum, P = 0.006 vs. young serum control). Furthermore, young mouse and human, but not old, serum reduced aortic elastic modulus (mouse serum, P = 0.009; human serum, P < 0.001 vs. old/MLO serum control) and improved EDD (mouse and human serum, P = 0.015 vs. old/MLO serum control) in old arteries. In human serum-exposed arteries, in vivo arterial function assessed in the human donors correlated with circulating milieu-modulated arterial function in young mouse arteries (aortic stiffness, r = 0.634, P = 0.005; endothelial function, r = 0.609, P = 0.004) and old mouse arteries (aortic stiffness, r = 0.664, P = 0.001; endothelial function, r = 0.637, P = 0.003). This study establishes novel experimental approaches for directly assessing the effects of the circulating milieu on arterial function and implicates changes in the circulating milieu as a mechanism of in vivo arterial aging. NEW & NOTEWORTHY Changes in the circulating milieu with advancing age may be a mechanism underlying age-related arterial dysfunction. Ex vivo exposure of young mouse arteries to the circulating milieu from old mice or midlife/older adults impairs arterial function whereas exposure of old mouse arteries to the circulating milieu from young mice or young adults improves arterial function. These findings establish that the circulating milieu directly influences arterial function with aging.

Cellular senescence and the senescence-associated secretory phenotype (SASP) contribute to age-related arterial dysfunction, in part, by promoting oxidative stress and inflammation, which reduce the bioavailability of the vasodilatory molecule nitric oxide (NO). In the present study, we assessed the efficacy of fisetin, a natural compound, as a senolytic to reduce vascular cell senescence and SASP factors and improve arterial function in old mice. We found that fisetin decreased cellular senescence in human endothelial cell culture. In old mice, vascular cell senescence and SASP-related inflammation were lower 1 week after the final dose of oral intermittent (1 week on-2 weeks off-1 weeks on dosing) fisetin supplementation. Old fisetin-supplemented mice had higher endothelial function. Leveraging old p16-3MR mice, a transgenic model allowing genetic clearance of p16 INK4A -positive senescent cells, we found that ex vivo removal of senescent cells from arteries isolated from vehicle- but not fisetin-treated mice increased endothelium-dependent dilation, demonstrating that fisetin improved endothelial function through senolysis. Enhanced endothelial function with fisetin was mediated by increased NO bioavailability and reduced cellular- and mitochondrial-related oxidative stress. Arterial stiffness was lower in fisetin-treated mice. Ex vivo genetic senolysis in aorta rings from p16-3MR mice did not further reduce mechanical wall stiffness in fisetin-treated mice, demonstrating lower arterial stiffness after fisetin was due to senolysis. Lower arterial stiffness with fisetin was accompanied by favorable arterial wall remodeling. The findings from this study identify fisetin as promising therapy for clinical translation to target excess cell senescence to treat age-related arterial dysfunction., (© 2023 The Authors. Aging Cell published by Anatomical Society and John Wiley & Sons Ltd.)

Cardiovascular disease (CVD) is a leading cause of morbidity and mortality worldwide, and senescent cells have emerged as key contributors to its pathogenesis. Senescent cells exhibit cell cycle arrest and secrete a range of proinflammatory factors, termed the senescence-associated secretory phenotype (SASP), which promotes tissue dysfunction and exacerbates CVD progression. Omics technologies, specifically transcriptomics and proteomics, offer powerful tools to uncover and define the molecular signatures of senescent cells in cardiovascular tissue. By analyzing the comprehensive molecular profiles of senescent cells, omics approaches can identify specific genetic alterations, gene expression patterns, protein abundances, and metabolite levels associated with senescence in CVD. These omics-based discoveries provide insights into the mechanisms underlying senescence-induced cardiovascular damage, facilitating the development of novel diagnostic biomarkers and therapeutic targets. Furthermore, integration of multiple omics data sets enables a systems-level understanding of senescence in CVD, paving the way for precision medicine approaches to prevent or treat cardiovascular aging and its associated complications.

Background: Here, we assessed the role of cellular senescence and the senescence associated secretory phenotype (SASP) in age-related aortic stiffening and endothelial dysfunction., Methods: We studied young (6-8 mo) and old (27-29 mo) p16-3MR mice, which allows for genetic-based clearance of senescent cells with ganciclovir (GCV). We also treated old C57BL/6N mice with the senolytic ABT-263., Results: In old mice, GCV reduced aortic stiffness assessed by aortic pulse wave velocity (PWV; 477±10 vs. 382±7 cm/s, P <0.05) to young levels (old-GCV vs. young-vehicle, P =0.35); ABT-263 also reduced aortic PWV in old mice (446±9 to 356±11 cm/s, P <0.05). Aortic adventitial collagen was reduced by GCV ( P <0.05) and ABT-263 ( P =0.12) in old mice. To show an effect of the circulating SASP, we demonstrated that plasma exposure from Old-vehicle p16-3MR mice, but not from Old-GCV mice, induced aortic stiffening assessed ex vivo (elastic modulus; P <0.05). Plasma proteomics implicated glycolysis in circulating SASP-mediated aortic stiffening. In old p16-3MR mice, GCV increased endothelial function assessed via peak carotid artery endothelium-dependent dilation (EDD; Old-GCV, 94±1% vs. Old-vehicle, 84±2%, P <0.05) to young levels (Old-GCV vs. young-vehicle, P =0.98), and EDD was higher in old C57BL/6N mice treated with ABT-263 vs. vehicle (96±1% vs. 82±3%, P <0.05). Improvements in endothelial function were mediated by increased nitric oxide (NO) bioavailability ( P <0.05) and reduced oxidative stress ( P <0.05). Circulating SASP factors related to NO signaling were associated with greater NO-mediated EDD following senescent cell clearance., Conclusions: Cellular senescence and the SASP contribute to vascular aging and senolytics hold promise for improving age-related vascular function., Competing Interests: Disclosures None.

The aorta stiffens with aging in both men and women, which predicts cardiovascular mortality. Aortic wall structural and extracellular matrix (ECM) remodeling, induced in part by chronic low-grade inflammation, contribute to aortic stiffening. Male mice are an established model of aortic aging. However, there is little information regarding whether female mice are an appropriate model of aortic aging in women, which we aimed to elucidate in the present study. We assessed two strains of mice and found that in C57BL/6N mice, in vivo aortic stiffness (pulse wave velocity, PWV) was higher with aging in both sexes, whereas in B6D2F1 mice, PWV was higher in old versus young male mice, but not in old versus young female mice. Because the age-related stiffening that occurs in men and women was reflected in male and female C57BL/6N mice, we examined the mechanisms of stiffening in this strain. In both sexes, aortic modulus of elasticity (pin myography) was lower in old mice, occurred in conjunction with and was related to higher plasma levels of the elastin-degrading enzyme matrix metalloproteinase-9 (MMP-9), and was accompanied by higher numbers of aortic elastin breaks and higher abundance of adventitial collagen-1. Plasma levels of the inflammatory cytokines interferon-γ, interleukin 6, and monocyte chemoattractant protein-1 were higher in both sexes of old mice. In conclusion, female C57BL/6N mice exhibit aortic stiffening, reduced modulus of elasticity and structural/ECM remodeling, and associated increases in MMP-9 and systemic inflammation with aging, and thus are an appropriate model of aortic aging in women. NEW & NOTEWORTHY Our study demonstrates that with aging, female C57BL/6N mice exhibit higher in vivo aortic stiffness, reduced modulus of elasticity, aortic wall structural and extracellular matrix remodeling, and elevations in systemic inflammation. These changes are largely reflective of those that occur with aging in women. Thus, female C57BL/6N mice are a viable model of human aortic aging and the utility of these animals should be considered in future biomedical investigations.

Age-associated cardiovascular (CV) dysfunction, namely arterial dysfunction, is a key antecedent to the development of CV disease (CVD). Arterial dysfunction with aging is characterized by impaired vascular endothelial function and stiffening of the large elastic arteries, each of which is an independent predictor of CVD. These processes are largely mediated by an excess production of reactive oxygen species (ROS) and an increase in chronic, low-grade inflammation that ultimately leads to a reduction in bioavailability of the vasodilatory molecule nitric oxide. Additionally, there are other fundamental aging mechanisms that may contribute to excessive ROS and inflammation termed the "hallmarks of aging"; these additional mechanisms of arterial dysfunction may represent therapeutic targets for improving CV health with aging. Aerobic exercise is the most well-known and effective intervention to prevent and treat the effects of aging on CV dysfunction. However, the majority of mid-life and older (ML/O) adults do not meet recommended exercise guidelines due to traditional barriers to aerobic exercise, such as reduced leisure time, motivation, or access to fitness facilities. Therefore, it is a biomedical research priority to develop and implement time- and resource-efficient alternative strategies to aerobic exercise to reduce the burden of CVD in ML/O adults. Alternative strategies that mimic or are inspired by aerobic exercise, that target pathways specific to the fundamental mechanisms of aging, represent a promising approach to accomplish this goal., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Cardiovascular diseases (CVD) are the leading cause of death worldwide, and age is by far the greatest risk factor for developing CVD. Vascular dysfunction, including endothelial dysfunction and arterial stiffening, is responsible for much of the increase in CVD risk with aging. A key mechanism involved in vascular dysfunction with aging is oxidative stress, which reduces the bioavailability of nitric oxide (NO) and induces adverse changes to the extracellular matrix of the arterial wall (e.g., elastin fragmentation/degradation, collagen deposition) and an increase in advanced glycation end products, which form crosslinks in arterial wall structural proteins. Although vascular dysfunction and CVD are most prevalent in older adults, several conditions can "accelerate" these events at any age. One such factor is chemotherapy with anthracyclines, such as doxorubicin (DOXO), to combat common forms of cancer. Children, adolescents and young adults treated with these chemotherapeutic agents demonstrate impaired vascular function and an increased risk of future CVD development compared with healthy age-matched controls. Anthracycline treatment also worsens vascular dysfunction in mid-life (50-64 years of age) and older (65 and older) adults such that endothelial dysfunction and arterial stiffness are greater compared to age-matched controls. Collectively, these observations indicate that use of anthracycline chemotherapeutic agents induce a vascular aging-like phenotype and that the latter contributes to premature CVD in cancer survivors exposed to these agents. Here, we review the existing literature supporting these ideas, discuss potential mechanisms as well as interventions that may protect arteries from these adverse effects, identify research gaps and make recommendations for future research., Competing Interests: Conflict of Interest The authors declare no conflicts of interest.

The neuropeptide galanin is present at high levels within the dorsal root ganglia (DRG) and spinal cord during development and after peripheral nerve damage in the adult. This pattern of expression suggests that it may play a role in the adaptive response of the peripheral nervous system (PNS) to injury. Several experimental paradigms have demonstrated that galanin modulates pain transmission, particularly after nerve injury. In our laboratory we have used a transgenic approach to further elucidate the functions of galanin within the somatosensory system. We have generated mice which over-express galanin (either inducibly after nerve injury, or constitutively), and knock-out (KO) mice, in which galanin is absent in all cells, throughout development and in the adult. Analysis of the nociceptive behaviour of the galanin over-expressing animals, before and after nerve injury, supports the view that galanin is an inhibitory neuromodulator of spinal cord transmission. In apparent contradiction to these findings, galanin KO animals fail to develop allodynia and hyperalgesia after nerve injury. However, further studies have shown that galanin is critical for the developmental survival of a subset of small diameter, unmyelinated sensory neurons that are likely to be nociceptors. This finding may well explain the lack of neuropathic pain-like behaviour after injury in the KO animals. Furthermore, the developmental survival role played by galanin is recapitulated in the adult where the peptide is required for optimal neuronal regeneration after injury, and in the hippocampus where it plays a neuroprotective role after excitotoxic injury.

The chimeric peptide M35 (galanin(1-3)-bradykinin(2-9)amide) is a high-affinity galanin receptor ligand which acts as a galanin receptor antagonist in many experimental models such as the flexor reflex and chronic constriction injury in rat. However, more recently there have been conflicting reports that M35 may act as a galanin receptor agonist in certain systems. Here we demonstrate that in the absence of endogenous galanin M35 has an agonistic effect, significantly enhancing neurite outgrowth from cultured adult mouse dorsal root ganglion neurons, albeit at a lower potency than galanin peptide itself. However, in the presence of galanin its agonistic activity is masked and thus it appears to act as a galanin receptor antagonist.

Expression of the neuropeptide galanin is markedly upregulated within the adult dorsal root ganglion (DRG) after peripheral nerve injury. We demonstrated previously that the rate of peripheral nerve regeneration is reduced in galanin knock-out mice, with similar deficits observed in neurite outgrowth from cultured mutant DRG neurons. Here, we show that the addition of galanin peptide significantly enhanced neurite outgrowth from wild-type sensory neurons and fully rescued the observed deficits in mutant cultures. Furthermore, neurite outgrowth in wild-type cultures was reduced to levels observed in the mutants by the addition of the galanin antagonist M35 [galanin(1-13)bradykinin(2-9)]. Study of the first galanin receptor (GalR1) knock-out animals demonstrated no differences in neurite outgrowth compared with wild-type animals. Similarly, use of a GalR1-specific antagonist had no effect on neuritogenesis. In contrast, use of a GalR2-specific agonist had equipotent effects on neuritogenesis to galanin peptide, and inhibition of PKC reduced neurite outgrowth from wild-type sensory neurons to that observed in galanin knock-out cultures. These results demonstrate that adult sensory neurons are dependent, in part, on galanin for neurite extension and that this crucial physiological process is mediated by activation of the GalR2 receptor in a PKC-dependent manner.

The formation of covalent isopeptide cross-links between cell surface protein molecules by the enzyme transglutaminase C influences cell adhesion and morphology. Retinoid-inducible cross-linking activity associated with this enzyme is present in the developing rat cerebellar cortex [Perry M. J. M. et al. (1995) Neuroscience 65, 1063-1076]. A monoclonal antibody was used to localize transglutaminase C to granule neurons in the developing cerebellar cortex. The enzyme was inducible by retinoic acid both in granule neurons cultured from postnatal rat cerebellar cortex and in cells of the embryonic dorsal rhombic lip, which contain granule neuron precursors. A possible biological function for transglutaminase activity was investigated in living granule neurons, cultured on a biomatrix substratum, studied by time-lapse cinematographic analysis using the transglutaminase inactivator RS-48373-007. Inhibition of cross-linking activity did not influence the number of neurites formed by granule neurons, but caused the destabilization of neurites during the initial outgrowth period, seen as an increase in the number of growth cone retractions and the onset of premature axon collateral formation (bifurcation). Inactivation of cross-linking activity prevented the formation of fascicles between neurites only when cells were cultured on a biomatrix surface. Two glial proteins involved in cell-extracellular matrix interactions, midkine and galectin-3, were identified as putative substrates for granule neuron transglutaminase. The results suggest that covalent cross-link formation by transglutaminase C or a related enzyme generates multimeric molecular forms of glial-derived proteins, and plays a role in stabilizing newly formed neurites. A possible non-pathological role for transglutaminase in the control of axon collateral branching by developing granule neurons in the cerebellar cortex is discussed.

Midkine is a prominent acyl donor substrate for the protein cross-linking enzyme transglutaminase type 2 in rat brain neurons. Transglutaminase type 2 and midkine immunoreactivity are regionally colocalized in developing cerebellar cortex. Monomeric midkine is present in the embryonic dorsal rhombic lip which gives rise to the cerebellar cortex. A high-molecular weight (29-30 kDa) midkine appears during postnatal cerebellar development. The presence of the high-molecular weight midkine in cultured cerebellar cortical interneurons is dependent upon culture conditions. Transglutaminase catalyzes the calcium-dependent cross-linking of midkine predominantly into 29-30 kDa dimers. Dimer-formation of midkine in vitro and in cultured neurons is reduced in the presence of a transglutaminase inactivator. Neurons plated onto previously cross-linked midkine exhibit larger growth cones and enhanced neurite outgrowth compared to those plated onto monomeric midkine alone.

Covalent cross-linking of cell surface proteins by the calcium-dependent enzyme transglutaminase C may be implicated in cell-cell interactions and growth regulation. We demonstrate the presence of the enzyme in rat cerebellar cortex during postnatal development. Transglutaminase C was induced in cerebellar granule neurons in culture by retinoic acid, dibutyryl- and 8-bromo-cyclic AMP analogues and by cultivation on a biomatrix substratum. Cyclic AMP analogues stimulated transglutaminase activity in protein synthesis-dependent and -independent phases. The enzyme was distributed at focal adhesion sites on the axon. By calcium-dependent covalent incorporation of the primary amine acceptor substrate, 5-(biotinamido)pentylamine, an increase in the Ca(2+)-dependent cross-linking of at least 11 substrate proteins in the presence of retinoic acid and dibutyryl-cyclic AMP was detected. Of these substrates, a subset was labelled on the surface of living granule neurons. A low-molecular-weight substrate, p18, was tentatively identified as the retinoic acid-inducible neurite-promoting factor, midkine. Transglutaminase-mediated amine incorporation, midkine and isopeptide cross-links were co-localized to axonal adhesion sites. The results provide evidence of transglutaminase C-catalysed protein cross-linking activity in cerebellar granule neurons and its possible implication in cell-substratum interactions.

A young patas monkey was run on a treadmill for 15-20 min at speeds in excess of 15 km times h-1 over a range of laboratory air temperatures from 0 to 55 degrees C. Cost of locomotion for the monkey was 83% that predicted for a running mammal of similar weight, 4 kg. At the highest steady-state running of speeds, the patas monkey's energy expenditure was 11 times the resting rate. Heat storage and respiratory evaporation during running each accounted for less than 16% of the heat production. Cutaneous evaporation, mainly sweating, was the major means of heat loss at high ambient temperatures and during heavy exercise. Maximum sweat rates of 0.5 mg times cm-2 times min-1 found in these experiments approach the sweat rates of humans. Whole-body dry thermal conductance increased 2-3 times the rest value at a given temperature. Environmentally gained heat contributed about two-thirds of the total heat load on the patas monkey at rest at 53 degrees C. At these same temperatures, environmental and metabolic heat loads were about equal when the animal ran at moderately high speeds (11-13.4 km times h-1).