Treatment with the BCL-2/BCL-xL inhibitor senolytic drug ABT263/Navitoclax improves functional hyperemia in aged mice

Abstract

Moment-to-moment adjustment of regional cerebral blood flow to neuronal activity via neurovascular coupling (NVC or “functional hyperemia”) has a critical role in maintenance of healthy cognitive function. Aging-induced impairment of NVC responses importantly contributes to age-related cognitive decline. Advanced aging is associated with increased prevalence of senescent cells in the cerebral microcirculation, but their role in impaired NVC responses remains unexplored. The present study was designed to test the hypothesis that a validated senolytic treatment can improve NVC responses and cognitive performance in aged mice. To achieve this goal, aged (24-month-old) C57BL/6 mice were treated with ABT263/Navitoclax, a potent senolytic agent known to eliminate senescent cells in the aged mouse brain. Mice were behaviorally evaluated (radial arms water maze) and NVC was assessed by measuring CBF responses (laser speckle contrast imaging) in the somatosensory whisker barrel cortex evoked by contralateral whisker stimulation. We found that NVC responses were significantly impaired in aged mice. ABT263/Navitoclax treatment improved NVC response, which was associated with significantly improved hippocampal-encoded functions of learning and memory. ABT263/Navitoclax treatment did not significantly affect endothelium-dependent acetylcholine-induced relaxation of aorta rings. Thus, increased presence of senescent cells in the aged brain likely contributes to age-related neurovascular uncoupling, exacerbating cognitive decline. The neurovascular protective effects of ABT263/Navitoclax treatment highlight the preventive and therapeutic potential of senolytic treatments (as monotherapy or as part of combination treatment regimens) as effective interventions in patients at risk for vascular cognitive impairment (VCI).

Introduction

The prevalence of age-related cognitive impairment is becoming a critical public health concern as proportions of older individuals in populations of many developed countries rapidly grow. Over 20% of people in Japan and many countries in the European Union (e.g. Germany and Italy) are already 65 or older. By 2050, 27% of the population of the European Union will be at least 65 years old, as will 22% of that of the USA. Many of these older individuals will experience cognitive decline resulting in a progressive reduction in functional independence and social relationships and imposing a substantial financial burden on society. The identification of potentially reversible age-related pathophysiological mechanisms contributing to cognitive decline is expected to lead to the development of clinically relevant treatments for prevention.

There is strong evidence that aging-induced dysregulation of cerebral blood flow critically contributes to the genesis of cognitive impairment in older adults [1, 2]. The brain has a high metabolic demand (20% of total oxygen consumption), but it has no energy or oxygen reserves. Maintenance of cerebral homeostasis requires adequate supply of oxygen and glucose at all time. During periods of intense neuronal activity, there is a need for prompt adjustment of oxygen and glucose delivery to the increased metabolic demand of the firing neurons as well as for effective washout of harmful metabolites to maintain the humoral microenvironment in the brain tissue. These goals are achieved by spatially localized adaptive increases in cerebral blood flow through a critical homeostatic mechanism termed neurovascular coupling (NVC) or functional hyperemia [3]. Multiple lines of evidence support the concept that aging-induced impairment of NVC responses contributes to the genesis of age-related cognitive decline [4–7]. First, NVC responses are decreased in older adults [2, 8–13], which predict impaired cognitive performance. Second, pharmacological disruption of NVC in young mice alters cognitive performance, mimicking aspects of the cognitive aging phenotype [14, 15]. Third, rescue of NVC responses by pharmacological treatments or dietary interventions targeting cellular and molecular mechanisms of aging has been reported to improve cognitive performance in experimental rodent models [4–6, 16–18].

The cellular mechanisms of NVC responses involve release of vasodilator nitric oxide (NO) from the cerebromicrovascular endothelial cells, in response to mediators released from activated neurons and astrocytes [19, 20]. Endothelium-derived NO relaxes the smooth muscle cells and, likely, the contractile pericytes at the resistance arterioles and precapillary sphincters. Additionally, endothelial cells also mediate the upstream propagation of conducted vasodilation, which further amplifies functional hyperemia [21]. Translational studies show that endothelial function is compromised in older adults [22] and aged laboratory animals [5, 6, 23], which importantly contributes to the age-related decline in NVC responses. Understanding the cellular mechanisms contributing to cerebromicrovascular endothelial aging will enable the development of clinically translatable interventions for neurovascular rejuvenation [24] and improvement of cerebral blood flow to prevent development and delay progression of vascular cognitive impairment.

Increased cellular oxidative stress and macromolecular damage associated with advanced age induce a complex stress response termed cellular senescence, which has been shown to cause or exacerbate aging and promote the genesis of a wide range of age-related pathologies, including vascular alterations [25–53]. Accordingly, pharmacological or genetic depletion of senescent cells in mice prolongs median lifespan and improves overall health [25, 44, 48, 49, 54–59], supporting a critical role for cellular senescence in organismal aging [52, 56, 60–64]. Endothelial cells exhibit increased sensitivity to molecular stressors causing DNA damage [39, 49, 50, 65, 66], including oxygen free radicals [67, 68] and readily develop a senescent phenotype [62, 63]. It is significant [69, 70] that approximately 10% of the cerebromicrovascular endothelial cells in the aging mouse brain are senescent [71]. Importantly, endothelial senescence has been causally linked to endothelial dysfunction in large arteries [44, 47]. Pharmacological treatments that preferentially eliminate senescent cells (termed “senolytics”) were shown to restore endothelial function in aged mice [44, 47]. Importantly, recent studies demonstrate that senolytic treatments also restore NVC responses and prevent cognitive decline that develops after whole brain irradiation [69, 72]. Despite these advances, the effects of senolytic treatments on age-related impairment of NVC responses remain elusive.

The present study was designed to test the hypothesis that senolytic treatments can improve NVC responses and cognitive performance in aged mice. To achieve this goal, aged C57BL/6 mice were treated with ABT263/Navitoclax, a potent senolytic agent [73]. ABT263/Navitoclax is an experimental orally active anti-cancer drug which inhibits the apoptosis regulator proteins Bcl-2 and Bcl-XL and thereby induces apoptosis in senescent cells, but not in non-senescent cells [74]. Senescent endothelial cells are particularly susceptible to apoptosis induced by ABT263/Navitoclax due to a reduction in Bcl-2 and increased BAX expression [75]. Mice were behaviorally evaluated and functional tests for NO-mediated NVC responses were performed. To substantiate the in vivo findings, the effects of ABT263/Navitoclax on endothelium-dependent vasorelaxation were obtained ex vivo in isolated aortic ring preparations.

Material and methods

Animals, ABT263/Navitoclax treatment

Young (3-month, n = 20) and aged (24-month, n = 40) male C57BL/6 mice were purchased from the aging colony maintained by the National Institute on Aging at Charles River Laboratories (Wilmington, MA). Animals were housed under specific pathogen-free barrier conditions in the Rodent Barrier Facility at University of Oklahoma Health Sciences Center under a controlled photoperiod (12 h light; 12 h dark) with unlimited access to water and were fed a standard AIN-93G diet (ad libitum). Mice in the aged cohort were assigned to two groups (n = 20 each group). One group of the aged mice received a daily dose of the BCL-2/BCL-xL inhibitor senolytic drug ABT263/Navitoclax (intraperitoneal (i.p.) injection, 1.5 mg/kg/daily) [73, 76, 77]. For maximum solubility in aqueous buffer, ABT-263 was first dissolved in DMSO plus 70% ethanol (4:1) and then diluted with PBS. All procedures were approved by the Institutional Animal Use and Care Committees of the University of Oklahoma Health Sciences Center.

Behavioral studies

Previous studies demonstrate that changes in NVC responses result in changes in cognitive performance [78]. Thus, after the treatment period, spatial memory and long-term memory were tested using the radial arms water maze as reported [5, 6, 72]. In brief, the maze consisted of eight arms 9 cm wide that radiated out from an open central area, with a submerged escape platform located at the end of one of the arms. Paint was added into the water to make it opaque. The maze was surrounded by privacy blinds with extramaze visual cues. Intramaze visual cues were placed at the end of the arms. The mice were monitored by a video tracking system directly above the maze as they waded and parameters were measured using Ethovision software Noldus Information Technology Inc., Leesburg, VA, USA). Experimenters were unaware of the experimental conditions of the mice at the time of testing. During the learning period each day, mice were given the opportunity to learn the location of the submerged platform during two sessions each consisting of four consecutive acquisition trials. On each trial, the mouse was started in one arm not containing the platform and allowed to wade for up to 1 min to find the escape platform. All mice spent 30 s on the platform following each trial before beginning the next trial. The platform was located in the same arm on each trial. Over the 3 days of training, mice in the young control group gradually improved performance as they learned the procedural aspects of the task. Upon entering an incorrect arm (all four paws within the distal half of the arm) or failing to select an arm after 15 s, the mouse was charged an error. Learning capability was assessed by comparing performance on days 2 and 3 of the learning period. A fourth trial, the probe trial, was conducted 7 days after the last learning trial to assess long-term memory retention. The reversal trials were carried out the following day by replacing the hidden platform in another arm while the errors were continuously assessed as during the learning phase. The reversal trials are a shorter version of the learning task which is performed after the animals have become proficient with the task and aims to evaluate the ability of each mouse to extinguish and re-learn the correct escape platform location.

Measurement of neurovascular coupling responses

After behavioral testing, mice in each group were anesthetized with isoflurane (4% induction and 1% maintenance), endotracheally intubated and ventilated (MousVent G500; Kent Scientific Co, Torrington, CT). A thermostatic heating pad (Kent Scientific Co, Torrington, CT) was used to maintain rectal temperature at 37 °C [20]. End-tidal CO₂ was controlled between 3.2 and 3.7% to keep blood gas values within the physiological range, as described [78, 79]. The right femoral artery was canulated for arterial blood pressure measurement (Living Systems Instrumentations, Burlington, VT) [20]. The blood pressure was within the physiological range throughout the experiments (90–110 mmHg). Mice were immobilized and placed on a stereotaxic frame (Leica Microsystems, Buffalo Grove, IL), the scalp and periosteum were pulled aside, and the skull was gently thinned using a dental drill while cooled with dripping buffer. A laser speckle contrast imager (Perimed, Järfälla, Sweden) was placed 10 cm above the thinned skull, and to achieve the highest CBF response, the right whiskers were stimulated for 30 s at 5 Hz from side to side [6]. Differential perfusion maps of the brain surface were captured. Changes in CBF were assessed above the left barrel cortex in six trials in each group, separated by 5–10-min intervals. To assess the role of NO mediation, CBF responses to whisker stimulation were repeated 15 min after intravenous administration of the nitric oxide synthase inhibitor N^ω-Nitro-L-arginine methyl ester (L-NAME). Changes in CBF were averaged and expressed as percent (%) increase from the baseline value [80]. All drugs used in this study were purchased from Sigma-Aldrich (St. Louis, MO) unless otherwise indicated.

Assessment of endothelial function in the aorta

To assess the specific effect of ABT263/Navitoclax treatment on endothelial function, endothelium-dependent vasorelaxation was assessed in isolated aorta ring preparations as described previously [81–85]. In brief, aortas were cut into ring segments 1.5 mm in length and mounted in myograph chambers (Danish Myo Technology A/S, Inc., Denmark) for measurement of isometric tension. The chambers were filled with Krebs buffer solution (118 mM NaCl, 4.7 mM KCl, 1.5 mM CaCl₂, 25 mM NaHCO₃, 1.1 mM MgSO₄, 1.2 mM KH₂PO₄, and 5.6 mM glucose; at 37 °C; gassed with 95% air and 5% CO₂). After an equilibration period of 1 h during which an optimal passive tension was applied to the rings (as determined from the vascular length-tension relationship), they were pre-contracted with 10⁻⁶ M phenylephrine and relaxation in response to acetylcholine was measured in the absence and presence of the NO synthase inhibitor L-NAME (3 × 10⁻⁴ mol/L).

Statistical analysis

Statistical analysis was carried out by one-way ANOVA followed by Tukey’s post hoc test, as appropriate. Dose–response curves for vascular relaxations were analyzed by two-way ANOVA for repeated measures followed by Bonferroni multiple comparison test. A p value less than 0.05 was considered statistically significant. Data are expressed as mean ± S.E.M.

Results

ABT263/Navitoclax treatment improves NVC responses in aged mice by restoring endothelial NO mediation

Representative pseudocolour baseline CBF maps were obtained using laser speckle contrast imaging to provide better anatomical and orientation information (Fig. 1A). NVC responses measured in the somatosensory whisker barrel cortex elicited by contralateral whisker stimulation were significantly decreased in 24-month-old mice compared to young animals indicating impaired functional hyperemia in old age (Fig. 1) [23]. Treatment with ABT263/Navitoclax significantly increased NVC responses in aged mice (Fig. 1). Our previous studies, using both pharmacological inhibitors and genetically modified animals, provide strong evidence that endothelial NO production plays an important role in mediation of NVC responses and that cerebromicrovascular endothelial dysfunction significantly contributes to age-related impairment of functional hyperemia [19, 20]. In accord with our previous findings in the present study, we found that in untreated aged animals, administration of the NO synthase inhibitor L-NAME was without effect, whereas in young mice, it significantly decreased NVC responses (Fig. 1C). In ABT263/Navitoclax-treated aged mice, L-NAME significantly decreased NVC responses (Fig. 1C), suggesting that senolytic treatment improves NO mediation of NVC responses in aged animals. There are also other cellular mechanisms contributing to functional hyperemia (e.g. astrocytic release of eicosanoid vasodilators, activation of potassium channels [86–89], but the non-NO-dependent component(s) of NVC responses appear to be unaffected by either aging or ABT263/Navitoclax treatment.

Fig. 1.

Treatment with the BCL-2/BCL-xL inhibitor senolytic drug ABT263/Navitoclax improves neurovascular coupling responses in aged mice. A Representative pseudocolour laser speckle flowmetry maps of baseline CBF (upper row; shown for orientation purposes) and CBF changes in the whisker barrel field relative to baseline during contralateral whisker stimulation (bottom row, right oval, 30 s, 5 Hz) in young (3-month-old), aged (24-month-old), and ABT263/Navitoclax-treated aged mice. Color bar represents CBF as percent change from baseline. The NO synthase inhibitor L-NAME was administered to test NO mediation of functional hyperemia. Panel B shows the time-course of CBF changes after the start of contralateral whisker stimulation (horizontal bars). Summary data are shown in panel C. Data are mean ± S.E.M. (n = 8–9 in each group), *P < 0.05 vs. young; ^#P < 0.05 vs. aged. (one-way ANOVA with post hoc Tukey’s tests). n.s.: not significant

Our results show that treatment with ABT263/Navitoclax treatment did not restore acetylcholine-induced, endothelium-dependent relaxation of aged mouse aortas (Fig. 2). To assess the role of endothelium-derived NO, L-NAME was applied. L-NAME abolished acetylcholine-induced vasorelaxation, eliminating the differences between the three groups (data not shown).

Fig. 2.

Effect of treatment with the BCL-2/BCL-xL inhibitor senolytic drug ABT263/Navitoclax on acetylcholine-induced vasorelaxation in aged mice. Shown are acetylcholine (ACh)-induced relaxations in aortic ring preparations isolated from young (3-month-old), aged (24-month-old), and ABT263/Navitoclax-treated aged mice. The trend for improvement in endothelial function in aged mice treated with ABT263/Navitoclax treatment did not reach statistical significance. Data are mean ± S.E.M. (n = 5–8 for each data point). *P < 0.05 vs. young

Improved functional hyperemia is associated with improved cognitive function in aged mice treated with ABT263/Navitoclax

Our previous studies provide proof-of-concept that pharmacologically induced neurovascular dysfunction results in detectable cognitive impairment [78]. To determine how improvement of neurovascular function by ABT263/Navitoclax treatment impacts cognitive performance in aged mice, each cohort of animals was tested in the radial arms water maze (Fig. 3). We compared the learning performance of mice in each experimental group by analyzing the day-to-day changes in the combined error rate and successful escape rate. During acquisition, or learning phase, mice from all groups showed a decrease in the combined error rate (Fig. 3B) across days, indicating improved proficiency at the task. After the 3 days of learning, young mice consistently had lower combined error rate than aged mice or ABT263/Navitoclax-treated aged mice (Fig. 3B). In the probe trial, error rates did not differ from that of the respective last trial of the learning phase in any of the groups. During the reversal trials, young and ABT263/Navitoclax-treated aged mice performed significantly better at re-learning the task compared to the untreated aged control animals.

Fig. 3.

Treatment of aged mice with the BCL-2/BCL-xL inhibitor senolytic drug ABT263/Navitoclax associates with improved radial-arm water maze (RAWM) performance. Young (3-month-old), aged (24-month-old), and ABT263/Navitoclax-treated aged mice were tested in the RAWM. A Heatmap representing the percentage of time spent in different locations in the maze for a randomly selected animal from each group during experimental day 10. Note that the untreated aged mouse required a greater amount of time and a longer path length in order to find the hidden escape platform. The older mice also re-enter a previously visited arm multiple time, accruing working memory errors as compared to the young mouse. B Older animals have higher combined error rates throughout the learning phase and probe day 10. Older animals also make significantly more errors as compared to young mice during the reversal trial. In contrast, aged mice treated with ABT263/Navitoclax perform this task significantly better than untreated aged mice during the reversal phase (p < 0.01). Combined error rate is calculated by adding 1 error for each incorrect arm entry as well as for every 15 s spent not exploring the arms. C The ratio of successful escapes, averaged across trial blocks, is shown for each group. Note day-to-day improvement in the performance of young mice, which was significantly delayed in aged mice. Aged mice treated with ABT263/Navitoclax were significantly more successful at finding the hidden escape platform in comparison to untreated age-matched controls both during the probe trial and the reversal trial. D There was no significant difference in swimming speed with ABT263/Navitoclax treatment. n = 20 in each group. All data are shown as mean ± SEM. Statistical significance was calculated using one-way ANOVA with Tukey’s post hoc test to determine differences among groups. *P < 0.05 vs. young; ^#P < 0.05 vs. aged

Successful escape rate from the maze was assessed by measuring the percent of animals that could find the hidden platform within the 60 s allowed for each trial. During acquisition, mice from all groups showed an increase in successful escape rate consistent with the learning of the task. Young mice exhibited significantly better escape success than both ABT263/Navitoclax-treated aged mice and untreated aged control mice (Fig. 3C). In the probe trial, ABT263/Navitoclax-treated aged mice exhibited significantly better successful escape rate than untreated aged control mice (Fig. 3C). Performance of aged mice treated with ABT263/Navitoclax was statistically identical to young mice in successful escape rate during the retrieval (day 10) and reversal phase (day 11). ABT263/Navitoclax treatment did not affect swimming speed of aged mice (Fig. 3D).

Discussion

The key finding of this study is that treatment with the BCL-2/BCL-xL inhibitor senolytic drug ABT263/Navitoclax improves NVC responses and cognitive function in a mouse model of aging that recapitulates key aspects of cerebromicrovascular dysfunction and deficits of higher brain function manifested in older adults.

Our findings that ABT263/Navitoclax improves endothelium-mediated NVC responses suggest that increased presence of senescent cells contributes to neurovascular dysfunction in aged mice. Results from studies using single-cell RNA sequencing show that in addition to senescent endothelial cells, senescent microglia and other cells types are also abundantly present in the aged mouse brain [71]. Further studies are needed to determine which senescent cell types exert negative effects on neurovascular function. Using the novel senescence reporter 3-MR mouse model, we have obtained preliminary data to show that ABT263/Navitoclax effectively removes senescent endothelial cells from the brains of aged mice (Nyul-Toth, Csiszar and Ungvari, 2021, unpublished observation). Thus, it is logical to assume that presence of senescent endothelial cells in the cerebromicrovascular network may adversely affect NVC responses. Possible mechanisms include disruption of conducted vasodilation, adverse paracrine effects, non-cell autonomous propagation of cellular senescence, and endothelial dysfunction (e.g. via tight junctions along the endothelial syncytium). It has been proposed that, in addition to the induction of neurovascular dysfunction, endothelial senescence may also contribute to the genesis of a spectrum of cerebromicrovascular aging phenotypes, including microvascular rarefaction, pro-inflammatory alterations, and blood–brain barrier disruption. Future studies should determine how ABT263/Navitoclax affects these age-related microvascular alterations.

Interestingly, contrary to our expectation, we found that ABT263/Navitoclax did not improve significantly endothelium-mediated vasorelaxation in the aged aorta. This observation warrants further investigations to determine the prevalence of senescent endothelial cells in different vascular beds in aged mice. It is possible that greater presence of senescent endothelial cells in the aged cerebral microvasculature as compared to that in the conduit arteries is responsible for the differential functional effects of the senolytic treatment used in the brain and the aorta [90]. It is also possible that increased presence of perivascular senescent microglia also plays an important role in the aged brain [91] by altering endothelial function in a paracrine fashion, whereas this effect is not present in the conduit arteries. Additionally, it has been reported that significant morphological and functional heterogeneity exists between endothelial cells from different anatomic sites [92].

Our previous studies have shown that endothelial oxidative stress and endothelial NAD depletion [93] play a critical role in age-related cerebromicrovascular endothelial dysfunction and neurovascular uncoupling, as chronic treatment with a mitochondria-targeted antioxidative peptide (SS-31) [6] or a NAD precursor (nicotinamide mononucleotide) [5] or a PARP-1 inhibitor (that is thought to restore cellular NAD levels by inhibiting age-related overactivation of the NAD-utilizing enzyme PARP-1) [4] can each improve both endothelium-mediated vasodilation and NVC responses in aged mice. We posit that increased presence of senescent cells in the aged mouse brain may affect endothelial redox status and/or cellular NAD homeostasis directly or indirectly [94]; however, the underlying mechanisms remain unexplored. Senescent cells readily acquire a pro-inflammatory phenotype, termed senescence-associated secretory phenotype (“SASP”) [69]. It is possible that secretion of inflammatory cytokines by senescent cells may exacerbate age-related mitochondrial oxidative stress, thereby exacerbating cerebromicrocrovascular endothelial dysfunction and neurovascular impairment. There are also reports that the NAD-degrading enzyme CD38/NADase increases during aging, in part, due to effects mediated by senescent cells [93], and this contributes to cellular NAD decline and subsequent mitochondrial dysfunction [95]. These hypotheses linking age-related cellular senescence to generalized decline in cellular NAD in the neurovascular unit should be tested experimentally in future studies.

There is a growing evidence from clinical [2, 10] and experimental [78] studies that impairment of NVC responses contributes to the age-related decline in higher cortical functions. Thus, development of novel interventions that restore cerebromicrovascular endothelial function and NVC responses is critical for the prevention of vascular cognitive impairment. Improvement of this key homeostatic mechanism matching oxygen and energy supply with the increased needs of active neurons is expected to confer beneficial effects on brain function in aged organisms. The present study is the first to show that partial restoration of NVC responses in ABT263/Navitoclax-treated aged mice associates with improvement of hippocampal encoded functions of learning and memory. Similar improvement of cognitive function has been recently documented in aged INK-ATTAC mice treated with the drug AP20187 (which eliminates p16^Ink4a-positive senescent cells in this genetically engineered mouse model) or with the senolytic drug combination dasatinib and quercetin [91]. Importantly, NVC is compromised both in patients with Alzheimer’s disease (AD) and in mouse models of AD, which is believed to accelerate clinical deterioration [1, 96]. Thus, it is significant that similar trends for improved cognitive function have been observed recently in animal models of Alzheimer’s disease [97, 98] after treatment with senolytic compounds. NVC is also reduced in obese mice, which contributes to the impairment of cognitive function [99–101]. Importantly, senolytic treatments were also shown to be protective in mice with obesity-related cognitive decline [102].

In conclusion, our findings show that ABT263/Navitoclax treatment exerts significant neurovascular protective effects in aged mice, which likely contribute to the observed significant cognitive benefit. Our findings, taken together with the results of recent studies, point to benefits at several levels of cerebrovascular and systemic pathology of aging [36, 59, 73, 91] and to the potential use of senolytic treatments as therapy for prevention of aging-induced vascular cognitive impairment. ABT263/Navitoclax is an orally active, synthetic small molecule that exerts antineoplastic activity in humans in addition to its senolytic activity proven in experimental studies. However, it also has important side effects, including induction of thrombocytopenia, liver toxicity, and gastrointestinal bleeding, which would limit its use in otherwise healthy older adults for prevention of cognitive impairment and other chronic age-related diseases [103, 104]. There is an acute need for novel senolytic drugs devoid of such side effects for eventual translation into clinical interventions. Potential candidates to be tested in preclinical models include fisetin, a naturally occurring flavone with low toxicity [105]. Future studies should test the effects of these senolytic drugs on senescent cell burden in the brain, NVC responses, and cognitive function in preclinical models of aging.

Funding

This work was supported by grants from the Oklahoma Center for the Advancement of Science and Technology, the National Institute on Aging (R01-AG047879; R01-AG055395; R01-AG068295: K01AG073614 to ST), the National Institute of Neurological Disorders and Stroke (NINDS; R01-NS100782), the National Cancer Institute (NCI; R01-CA255840-01), the National Institute of General Medical Sciences Oklahoma Shared Clinical and Translational Resources (OSCTR) (GM104938, to AY), the Presbyterian Health Foundation, the NIA-supported Geroscience Training Program in Oklahoma (T32AG052363), the Oklahoma Nathan Shock Center (P30AG050911), the Cellular and Molecular GeroScience CoBRE (1P20GM125528, sub#5337), the American Federation for Aging Research (Irene/Diamond Postdoctoral Transition Award to PB), and the NKFIH (Nemzeti Szivlabor).

Declarations

Conflict of interest

The authors declare no competing interests.

Disclaimer

The funding sources had no role in the study design; in the collection, analysis, and interpretation of data; in the writing of the report; and in the decision to submit the article for publication.