Age-related Vascular Dysfunction Research Articles

Age-Related Vascular Mitochondrial Dysfunction Is Ameliorated by Time-Restricted Feeding Intervention

Endothelial dysfunction constitutes a hallmark of vascular aging that can have serious implications for disease risk. In addition, aging is associated with mitochondrial dysfunction, which can lead to increased oxidative stress and create associated damages. Time-restricted feeding (TRF) is a dietary intervention known to have many benefits on age-related disease risk. The objective of this study is to elucidate the effects of TRF upon endothelial mitochondrial function of aorta during aging. We hypothesized that TRF protects the aorta from age-related endothelial mitochondrial dysfunction. Adult C57BL/6 mice underwent TRF regimen, including six hours of ad libitum feeding daily. Young and age-matched control mice were fed ad libitum. After 12 months of TRF, wire myography was performed on aorta tissue as a measure of endothelial function. The aorta received pharmacological treatments to induce contraction, and dose-dependent dilation was quantified. For the first time, simultaneous measurement of reactive oxygen species (ROS) and oxygen consumption were completed in aorta tissue using Oroboros O2K Fluorespirometry. Dihydroethidium (DHE) fluorescent staining of ROS in aorta tissue was used to validate ROS measurements from O2K. Serum ketone body levels related to metabolic switching and relevant to mitochondrial biogenesis were determined. TRF improved endothelial-dependent dilation of aorta tissue, representing the intervention’s protection of endothelial function in aging. Aged TRF mice had greater oxygen consumption and less ROS in aorta tissue than age-matched control mice, indicating improved vascular mitochondrial function after TRF intervention. Aged TRF mice had elevated serum levels of ketone bodies involved in mitochondrial biogenesis. This supports the hypothesis that age-related changes in vascular mitochondrial health are alleviated by TRF. The knowledge gained from this study is relevant to understanding the mechanisms of age-related vascular dysfunction. Stephenson Cancer Center, National Institute of Aging R03 AG070479, American Heart Association. This is the full abstract presented at the American Physiology Summit 2024 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.

Functional, Structural and Proteomic Effects of Ageing in Resistance Arteries.

The normal ageing process affects resistance arteries, leading to various functional and structural changes. Systolic hypertension is a common occurrence in human ageing, and it is associated with large artery stiffening, heightened pulsatility, small artery remodeling, and damage to critical microvascular structures. Starting from young adulthood, a progressive elevation in the mean arterial pressure is evidenced by clinical and epidemiological data as well as findings from animal models. The myogenic response, a protective mechanism for the microcirculation, may face disruptions during ageing. The dysregulation of calcium entry channels (L-type, T-type, and TRP channels), dysfunction in intracellular calcium storage and extrusion mechanisms, altered expression of potassium channels, and a change in smooth muscle calcium sensitization may contribute to the age-related dysregulation of myogenic tone. Flow-mediated vasodilation, a hallmark of endothelial function, is compromised in ageing. This endothelial dysfunction is related to increased oxidative stress, lower nitric oxide bioavailability, and a low-grade inflammatory response, further exacerbating vascular dysfunction. Resistance artery remodeling in ageing emerges as a hypertrophic response of the vessel wall that is typically observed in conjunction with outward remodeling (in normotension), or as inward hypertrophic remodeling (in hypertension). The remodeling process involves oxidative stress, inflammation, reorganization of actin cytoskeletal components, and extracellular matrix fiber proteins. Reactive oxygen species (ROS) signaling and chronic low-grade inflammation play substantial roles in age-related vascular dysfunction. Due to its role in the regulation of vascular tone and structural proteins, the RhoA/Rho-kinase pathway is an important target in age-related vascular dysfunction and diseases. Understanding the intricate interplay of these factors is crucial for developing targeted interventions to mitigate the consequences of ageing on resistance arteries and enhance the overall vascular health.

Chronic mitochondria-targeted antioxidant supplementation in older adults alters circulating factors to improve endothelial cell function

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<0.047) MQ in all 3 groups, respectively. CONCLUSIONS. Chronic MQ supplementation in older adults alters circulating factors by decreasing plasma oxLDL to reduce oxLDL-LOX-1 signaling-mediated mtROS production and improve EC function. These findings extend insight into the mechanisms by which mitochondria-targeted antioxidant treatment improves endothelial function with aging. 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.

Effect of home-based heat therapy on skeletal muscle blood flow during exercise in older adults

The arterial vasculature undergoes profound changes across the lifespan. This vascular aging manifests as structural (e.g., vascular rarefaction) and functional (e.g., endothelial dysfunction) maladaptations. These age-related maladaptations contribute to the hypo-perfusion of skeletal muscle during conditions that require an elevated blood supply and substrate delivery (e.g., exercise) by tipping the vasoactive balance in favor of vasoconstriction. Acute heat exposure increases body and tissue temperature, increases blood flow and shear stress throughout the arterial tree, and initiates a humoral response. Importantly, chronic exposure to these temperature-dependent stimuli may reverse age-related vascular dysfunction, thereby improving perfusion of active skeletal muscle (i.e., exercise hyperemia). However, the effect of chronic heat therapy on exercise hyperemia has never been explored. Therefore, the purpose of this pilot study was to test the hypothesis that home-based heat therapy would increase skeletal muscle blood flow in older adults performing plantar flexion exercise. Six older adults (5 women; 67 ± 4 years) performed graded (5, 9, and 14 kg) unilateral dynamic plantar flexion exercise before (pre) and after (post) 8 weeks of home-based heat therapy. Each session of heat therapy required participants to immerse their lower legs ~33 cm into a heated (~ 42°C) and circulated water bath 4 days per week, for 45 min per session. Dynamic plantar flexion exercise was performed using a duty cycle of 1 s contraction and 2 s relaxation. Superficial femoral artery blood velocity and diameter were measured via Duplex ultrasonography and used to calculate the change in blood flow measured from baseline to each stage of exercise. Vascular conductance was calculated as flow/pressure. The change in leg blood flow during exercise was increased following heat therapy ( P = 0.08 for main effect of time), an effect that was mediated by augmented vascular conductance (5 kg: pre, Δ143 ± 89 ml/min/mmHg vs post, Δ188 ± 89 ml/min/mmHg; 9 kg: pre, Δ225 ± 89 ml/min/mmHg vs post, Δ234 ± 89 ml/min/mmHg; 14 kg: pre, Δ264 ± 89 ml/min/mmHg vs post, Δ290 ± 89 ml/min/mmHg; P = 0.07). Our preliminary data suggest that home-based heat therapy increases skeletal muscle blood flow during exercise in older adults, and may serve as a pragmatic intervention to improve exercise and functional capacity in this population. Supported by: American Heart Association TPA958179 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.

Abstract 13: Aptamer-Based Plasma Proteomics Nominates Biomarkers Of Neurological Severity, Stroke Diagnosis And Age

Background: Plasma protein biomarkers measured in the hyperacute setting that are associated with stroke diagnosis or with clinical neurological severity, can be diagnostically and prognostically meaningful in patient care. We hypothesized that high-throughput proteomics can identify panels of plasma proteins that can differentiate stroke mimics from ischemic and hemorrhagic stroke, and are associated with neurological severity. Methods: From a clinically-characterized biorepository of 320 plasma samples collected at time of patient presentation to the Emergency Room at Grady Memorial Hospital (Atlanta), 40 samples (10 stroke mimics, 10 intracerebral hemorrhage [ICH], 20 acute ischemic stroke [AIS]) were included (Fig A). 7,000 proteins were measured per sample using aptamer-based proteomics (SomaLogic). A combination of differential-expression and variance partitioning analyses (VPA) identified proteins associated with diagnosis (mimic vs ICH vs AIS), neurological severity (NIHSS), age and sex. Results: Cohort characteristics are shown (Fig B). VPA identified top proteins that accounted for majority of variance in several traits (Fig C). One-way ANOVA identified >400 proteins showing group-wise differences, of which a panel of 56 proteins collectively distinguished mimics from both AIS and ICH, ICH from both mimics and AIS, as well as distinguished between AIS based on NIHSS (Fig D). The pattern of expression of these 56 plasma proteins across diagnostic groups is shown in Fig E. We also identified plasma biomarkers associated with increasing age, indicative of age-related vascular dysfunction. Conclusions: This pilot study of AIS, ICH and stroke mimics demonstrates the applicability of aptamer-based proteomics as a promising approach for plasma protein biomarker discovery in stroke. We identified acute plasma protein biomarkers of neurological severity, diagnostic group and age-related vascular dysfunction that need further validation.

Initiation of 3,3-dimethyl-1-butanol at midlife prevents endothelial dysfunction and attenuates in vivo aortic stiffening with ageing in mice.

Vascular dysfunction: develops progressively with ageing; increases the risk of cardiovascular diseases (CVD); and is characterized by endothelial dysfunction and arterial stiffening, which are primarily mediated by superoxide-driven oxidative stress and consequently reduced nitric oxide (NO) bioavailability and arterial structural changes. Interventions initiated before vascular dysfunction manifests may have more promise for reducing CVD risk than interventions targeting established dysfunction. Gut microbiome-derived trimethylamine N-oxide (TMAO) induces vascular dysfunction, is associated with higher CV risk, and can be suppressed by 3,3-dimethyl-1-butanol (DMB). We investigated whether DMB supplementation could prevent age-related vascular dysfunction in C57BL/6N mice when initiated prior to development of dysfunction. Mice received drinking water with 1% DMB or normal drinking water (control) from midlife (18 months) until being studied at 21, 24 or 27 months of age, and were compared to young adult (5 month) mice. Endothelial function [carotid artery endothelium-dependent dilatation (EDD) to acetylcholine; pressure myography] progressively declined with age in control mice, which was fully prevented by DMB via higher NO-mediated EDD and lower superoxide-related suppression of EDD (normalization of EDD with the superoxide dismutase mimetic TEMPOL). In vivo aortic stiffness (pulse wave velocity) increased progressively with age in controls, but DMB attenuated stiffening by ∼ 70%, probably due to preservation of endothelial function, as DMB did not affect aortic intrinsic mechanical (structural) stiffness (stress-strain testing) nor adventitial abundance of the arterial structural protein collagen. Our findings indicate that long-term DMB supplementation prevents/attenuates age-related vascular dysfunction, and therefore has potential for translation to humans for reducing CV risk with ageing. KEY POINTS: Vascular dysfunction, characterized by endothelial dysfunction and arterial stiffening, develops progressively with ageing and increases the risk of cardiovascular diseases (CVD). Interventions aimed at preventing the development of CV risk factors have more potential for preventing CVD relative to those aimed at reversing established dysfunction. The gut microbiome-derived metabolite trimethylamine N-oxide (TMAO) induces vascular dysfunction, is associated with higher CV risk and can be suppressed by supplementation with 3,3-dimethyl-1-butanol (DMB). In mice, DMB prevented the development of endothelial dysfunction and delayed and attenuated in vivo arterial stiffening with ageing when supplementation was initiated in midlife, prior to the development of dysfunction. DMB supplementation or other TMAO-suppressing interventions have potential for translation to humans for reducing CV risk with ageing.

CircCRIM1 Ameliorates Endothelial Cell Angiogenesis in Aging through the miR-455-3p/Twist1/VEGFR2 Signaling Axis.

Background Aging leads to vascular endothelial cell senescence. Decreased expression of VEGFA and VEGFR2 plays a crucial role in impairing angiogenesis in senescent endothelial cells. Noncoding RNAs, including circular RNAs (circRNAs) and microRNAs (miRNAs), regulate endothelial cell proliferation, differentiation, apoptosis, and migration and participate in the occurrence and development of vascular diseases. However, the mechanism of noncoding RNAs in age-related vascular endothelial dysfunction remains unclear. Here, we aimed to identify the circRNA that is associated with VEGF/VEGFR2 signaling pathway activation in angiogenesis. Methods Immunoblotting, quantitative reverse transcription-polymerase chain reaction (qRT–PCR), in vitro and in vivo experiments, luciferase assays, and chromatin immunoprecipitation followed by qRT–PCR (ChIP–qPCR) assays were performed to clarify the roles played by circCRIM1 in mouse aortic endothelial cell (MAEC) angiogenesis. Results CircCRIM1 expression was downregulated in both an aging mouse model of lower limb ischemia in vivo and aging MAECs in vitro. Overexpressing circCRIM1 mediated through a plasmid or adeno-associated virus (AAV) reversed the downregulation of angiogenesis-related phenotype acquisition during aging. MiR-455-3p was confirmed to be a potential target of circCRIM1 through luciferase assays followed by RNA fluorescence in situ hybridization (FISH), which revealed the colocalization of circCRIM1 and miR-455-3p. CircCRIM1 was found to be a competitive endogenous RNA that sponged miR-455-3p and regulated angiogenesis-related phenotypes in MAECs. Furthermore, Twist1 was found to be downstream of miR-455-3p. A ChIP–qPCR assay showed that Twist1 promoted VEGFR2 expression by binding to the promoter region, playing a vital role in angiogenesis. Conclusions Decreased expression of circCRIM1 impaired angiogenesis in aging via the miR-455-3p/Twist1/VEGFR2 axis. Our findings suggest that overexpression of circCRIM1 may be an effective therapeutic strategy for promoting ischemic lower limb blood flow recovery.

Mitochondrial-targeted antioxidant supplementation for improving age-related vascular dysfunction in humans: A study protocol.

Background: Cardiovascular disease (CVD) is the leading cause of death worldwide and aging is the primary risk factor for the development of CVD. The increased risk of CVD with aging is largely mediated by the development of vascular dysfunction. Excessive production of mitochondrial reactive oxygen species (mtROS) is a key mechanism of age-related vascular dysfunction. Therefore, establishing the efficacy of therapies to reduce mtROS to improve vascular function with aging is of high biomedical importance. Previously, in a small, randomized, crossover-design pilot clinical trial, our laboratory obtained initial evidence that chronic oral supplementation with the mitochondrial-targeted antioxidant MitoQ improves vascular function in healthy older adults. Here, we describe the protocol for an ongoing R01-funded phase IIa clinical trial to establish the efficacy of MitoQ as a therapy to improve vascular function in older adults (ClinicalTrials.gov Identifier: NCT04851288). Outcomes: The primary outcome of the study is nitric oxide (NO)-mediated endothelium-dependent dilation (EDD) as assessed by brachial artery flow-mediated dilation (FMDBA). Secondary outcomes include mtROS-mediated suppression of EDD, aortic stiffness as measured by carotid-femoral pulse wave velocity, carotid compliance and β-stiffness index, and intima media thickness. Other outcomes include the assessment of endothelial mitochondrial health and oxidative stress in endothelial cells obtained by endovascular biopsy; the effect of altered circulating factors following MitoQ treatment on endothelial cell NO bioavailability and whole cell and mitochondrial reactive oxygen species production ex vivo; and circulating markers of oxidative stress, antioxidant status, and inflammation. Methods: We are conducting a randomized, placebo-controlled, double-blind, parallel group, phase IIa clinical trial in 90 (45/group) healthy older men and women 60 years of age or older. Participants complete baseline testing and are then randomized to either 3 months of oral MitoQ (20 mg; once daily) or placebo supplementation. Outcome measures are assessed at the midpoint of treatment, i.e., 6 weeks, and again at the conclusion of treatment. Discussion: This study is designed to establish the efficacy of chronic supplementation with the mitochondrial-targeted antioxidant MitoQ for improving vascular endothelial function and reducing large elastic artery stiffness in older adults, and to investigate the mechanisms by which MitoQ supplementation improves endothelial function.

Preserved Endothelium-independent Function With Aging: Evidence From The Peripheral And Cerebral Vasculature

There is growing evidence that vascular endothelial dysfunction is not only antecedent to cardiovascular disease, but also cognitive dysfunction and Alzheimer’s disease. Vascular smooth muscle cells (VSMCs) translate the endothelial-derived signal to vasodilate into action. PURPOSE: However, in the peripheral, and especially the cerebral, vasculature, the impact of aging on the functional capacity of VSMCs, and any functional link between these vascular beds, is not well understood. METHODS: Therefore, endothelium-independent dilation, elicited by sublingual nitroglycerin (NTG, 2 tabs, 0.4 mg Nitrostat each), compared to sham-delivery (control), was assessed using Doppler ultrasound in both the popliteal and middle cerebral (MCA) arteries of 12 young (22 ± 4 yrs, 6 male/6 female) and 11 old (67 ± 3 yrs, 6 male/5 female) healthy adults. RESULTS: In the popliteal artery, when compared to control (∆ young: 0.00 ± 0.03 mm, ∆ old: 0.02 ± 0.05 mm), NTG resulted in significant increases in vessel diameter (∆ young: 0.32 ± 0.13 mm, ∆ old: 0.37 ± 0.13 mm) with no differences between age or sex. Similarly, in the MCA, when compared to control (∆ young: 0.07 ± 0.11 mm, ∆ old: 0.02 ± 0.20 mm), NTG resulted in significant increases in vessel diameter (∆ young: 1.01 ± 0.30 mm, ∆ old: 1.00 ± 0.38 mm) with no differences between age or sex. Interestingly, there was no evidence of a relationship between the peripheral and cerebral vascular responses to NTG in terms of either age, sex, or all subjects as a whole. CONCLUSION: Thus, the known age-related vascular dysfunction, in the peripheral and cerebral circulations, does not appear to be the consequence of dysfunctional VSMCs, and there is no evidence of a link between VSMC function in these two vascular beds.

Fisetin Supplementation Improves Age‐Related Vascular Endothelial Function by Suppressing Cellular Senescence and Mitochondrial Oxidative Stress

Age‐related vascular endothelial dysfunction is mediated by excess reactive oxygen species (ROS)—mitochondria (mito) being a key source—which can reduce nitric oxide (NO) bioavailability. Cellular senescence, a fundamental mechanism of aging, may exacerbate mito ROS and be a potential therapeutic target to combat age‐related vascular dysfunction.PurposeTo determine if treatment with the natural flavonoid fisetin improves endothelial function with aging by suppressing cellular senescence, scavenging excess whole‐cell and mito ROS, and increasing NO bioavailability.Methods & ResultsOld (27 mo) male C57BL/6 mice were treated with vehicle (V; 10% EtOH, 30% PEG400 and 60% Phosal 50 PG; n = 9) or fisetin (Fis; 50 mg/kg/day in V; n = 10) by oral gavage following a 1 wk on – 2 wk off – 1 wk on dosing paradigm.Endothelial functionEndothelial function was assessed by ex vivo carotid artery endothelium‐dependent dilation (EDD) and endothelium‐independent dilation (EID) to increasing doses of acetylcholine and sodium nitroprusside, respectively. EDD was greater in Fis vs V treated mice (Peak EDD [%]: 97 ± 1 vs 84 ± 3, P < .05) and no differences were observed among groups in EID (P = .54).Cellular senescenceFis‐treated mice had lower vascular abundance of p16, an established marker of cellular senescence (Fis, .12 ± .01 vs V, .19 ± .02 chemiluminescence units [CU], P < .05). Next, we administered Fis or V to old (27 mo) p16‐3MR mice (a model that allows for clearance of senescent cells with ganciclovir [GCV]; n = 6/group). Fis‐treated p16‐3MR mice had greater EDD (Peak EDD [%]: Fis, 93 ± 2 vs V, 74 ± 5, P < .05). Ex vivo carotid artery incubations with GCV eliminated group differences in EDD, suggesting that Fis reduced senescent cells to improve EDD.Whole‐cell ROSElectron paramagnetic resonance (EPR) spectroscopy was used to assess whole‐cell vascular (aortic) ROS. Fis‐treated mice had lower whole‐cell ROS (Fis, 4703 ± 455 vs V, 8173 ± 1243 amplitude units [AU], P < .05). Incubation with the ROS scavenger TEMPOL eliminated group differences in EDD, implying that Fis ameliorated ROS‐related suppression of EDD.Mito ROSEPR was used to assess vascular mito ROS. Fis‐treated mice had lower mito ROS (Fis, 2527 ±440 vs V, 6603 ± 1956 AU, P < .05). Further, Fis‐treated mice had lower abundance of vascular p‐p66SHC, a recognized marker of mito oxidative stress (Fis, .029 ± .003 vs V, .049 ± .008 CU, P < .05) and greater abundance of Mn superoxide dismutase, a mito antioxidant enzyme (Fis, .41 ± .1 vs V, .20 ± .02 CU, P < .05). Incubation with the mito‐specific antioxidant, MitoQ, eliminated group differences in EDD, implying that Fis ameliorated mito ROS‐related suppression of EDD.NOAddition of the NO‐synthase inhibitor, L‐NAME, abolished group differences in EDD suggesting Fis improved age‐related EDD impairments by increasing NO bioavailability.ConclusionFisetin supplementation may be a novel strategy to target excess cellular senescence and thereby reduce mito ROS to improve NO‐mediated endothelial function with aging.

Waste Clearance in the Brain and Neuroinflammation: A Novel Perspective on Biomarker and Drug Target Discovery in Alzheimer’s Disease

Alzheimer’s disease (AD) is a multifactorial disease with a heterogeneous etiology. The pathology of Alzheimer’s disease is characterized by amyloid-beta and hyperphosphorylated tau, which are necessary for disease progression. Many clinical trials on disease-modifying drugs for AD have failed to indicate their clinical benefits. Recent advances in fundamental research have indicated that neuroinflammation plays an important pathological role in AD. Damage- and pathogen-associated molecular patterns in the brain induce neuroinflammation and inflammasome activation, causing caspase-1-dependent glial and neuronal cell death. These waste products in the brain are eliminated by the glymphatic system via perivascular spaces, the blood-brain barrier, and the blood–cerebrospinal fluid barrier. Age-related vascular dysfunction is associated with an impairment of clearance and barrier functions, leading to neuroinflammation. The proteins involved in waste clearance in the brain and peripheral circulation may be potential biomarkers and drug targets in the early stages of cognitive impairment. This short review focuses on waste clearance dysfunction in AD pathobiology and discusses the improvement of waste clearance as an early intervention in prodromal AD and preclinical stages of dementia.

The Effects of Acute and Chronic Selective Phosphodiesterase 1 Inhibition on Smooth Muscle Cell-Associated Aging Features.

Age-related cardiovascular diseases (CVDs) remain among the leading global causes of death, and vascular smooth muscle cell (VSMC) remodeling plays an essential role in its pathology. Reduced NO-cGMP pathway signaling is a major feature and pathogenic mechanism underlying vasodilator dysfunction. Recently, we identified phosphodiesterase (PDE) 1, an enzyme that hydrolyzes and inactivates the cyclic nucleotides cAMP and cGMP, and thereby provides a potential treatment target for restoring age-related vascular dysfunction due to aging of VSMC. Based on this hypothesis, we here tested the effects of PDE1 inhibition in a model of SMC-specific accelerated aging mice. SMC-KO and their WT littermates received either vehicle or the PDE1 inhibitor lenrispodun for 8 weeks. Vascular function was measured both in vivo (Laser Doppler technique) and ex vivo (organ bath). Moreover, we deployed UV irradiation in cell culture experiments to model accelerated aging in an in vitro situation. SMC-KO mice display a pronounced loss of vasodilator function in the isolated aorta, the cutaneous microvasculature, and mesenteric arteries. Ex vivo, in isolated vascular tissue, we found that PDE1 inhibition with lenrispodun improves vasodilation, while no improvement was observed in isolated aorta taken from mice after chronic treatment in vivo. However, during lenrispodun treatment in vivo, an enhanced microvascular response in association with upregulated cGMP levels was seen. Further, chronic lenrispodun treatment decreased TNF-α and IL-10 plasma levels while the elevated level of IL-6 in SMC-KO mice remained unchanged after treatment. PDE1 and senescence markers, p16 and p21, were increased in both SMC-KO aorta and cultured human VSMC in which DNA was damaged by ultraviolet irradiation. This increase was lowered by chronic lenrispodun. In contrast, lenrispodun increased the level of PDE1A in both situations. In conclusion, we demonstrated that PDE1 inhibition may be therapeutically useful in reversing aspects of age-related VSMC dysfunction by potentiating NO-cGMP signaling, preserving microvascular function, and decreasing senescence. Yet, after chronic treatment, the effects of PDE1 inhibition might be counteracted by the interplay between differential PDE1A and C expression. These results warrant further pharmacodynamic profiling of PDE enzyme regulation during chronic PDE1 inhibitor treatment.

Abstract 13273: Late Senolytic Therapy Improves Cerebrovascular Endothelial Function but Not Cognition in Middle-Aged Severely Atherosclerotic Mice

Introduction: Age-related and atherosclerosis-related vascular dysfunction contributes to neuro-vascular uncoupling, which induces neuronal damage and leads to precocious cognitive loss. This dysfunction could be partially explained by the accumulation of senescent cells (SnC) that remain metabolically active and release many pro-inflammatory factors. Hypothesis: Targeted elimination of SnC with a senolytic restores cerebrovascular function, maintains neuro-vascular coupling and slows cognitive decline. Methods and Results: Nine-month old male atherosclerotic mice (ATX, LDLR -/- ;hApoB +/+ ) were treated (n=13), or not (n=11), for 3 months with navitoclax (50mg/kg), a Bcl2 inhibitor that stimulates global elimination of SnC overexpressing this antiapoptotic pathway. Twelve-month old vehicle-treated ATX mice developed atheroma plaques up to the upper neck portion of the carotid arteries, but not in the cerebral circulation. Three months treatment with navitoclax reduced aortic plaques size (-32%, p<0.001) from 40±2% in vehicle-treated mice to 27±2% in navitoclax mice. At 12 months, endothelial dysfunction was severe in vehicle-treated ATX mice, as evidenced by a strong reduction in flow-mediated dilation (FMD) of pressurized posterior cerebral arteries ex vivo ; navitoclax improved endothelium-dependent dilatation by 50% (for example, FMD at 20 dyn/cm 2 : 58±10%) compared to vehicle-treated mice (FMD: 29±7%; p<0.05). Two cognitive tests were performed: the Y-maze and the novel object recognition tests, to assess exploratory behavior and executive function, respectively. A gradual decline of both exploratory and executive memory was observed from 3 to 6, 9 and 12 months in vehicle-treated ATX mice; navitoclax, however, did not prevent this cognitive decline. Conclusion: Global elimination of SnC slowed atherosclerotic plaque development and restored cerebral endothelial function, demonstrating that senescence contributes to vascular dysfunction. The absence of cognitive improvement with navitoclax suggests an irreversible state of neuronal damage that could not be affected by the late senolytic treatment. To demonstrate the role of SnC in cognitive impairment, an earlier senolytic therapy will be needed.

Abstract 10955: The Effects of Acute and Chronic Selective Phosphodiesterase 1 Inhibition in Associated Smooth Muscle Cell-Specific Aging Features

Introduction: Aging-related vascular smooth muscle cell (VSMC) remodeling plays an essential role in cardiovascular diseases. Reduced NO-cGMP pathway signalling is a major feature and pathogenic mechanism underlying VSMC dysfunction. Recently, we found that phosphodiesterase (PDE) 1, an enzyme that hydrolyzes and inactivates the cyclic nucleotides cAMP and cGMP, is a potential treatment target for restoring age-related deficits in VSMC signalling. We here tested the effects of PDE1 inhibition in SMC-specific Ercc1 DNA repair mutant mice, a model of isolated, accelerated SMC aging. Design and Method: SM22α Cre+ Ercc1 fl/- (SMC-KO) and SM22α Cre+ Ercc1 fl/+ littermate (SMC-LM) mice receive either vehicle or the PDE1 inhibitor lenrispodun (dose 40mg/kg/day) in drinking water for 8 weeks. Reactive hyperemia was measured in-vivo at the age of 14 and 25 weeks. Mice were sacrificed at 26 weeks. The endothelium-dependent (ED) and -independent (EI) relaxation of the thoracic aorta was assessed by wire myography. Moreover, we investigated the effect of PDE1 inhibition on the expression of selected PDEs involved in the vasculature by qPCR. Results: Reactive hyperemia was dramatically reduced in vehicle-treated SMC-KO vs. SMC-LM at the age of 25 weeks (P=0.0002) but was preserved in lenrispodun-treated SMC-KO mice. In vehicle-treated SMC-KO mice, the ED and EI relaxations were greatly diminished compared to SMC-LM (ED: 23.92±4.34 vs. 68.04±4.86, P<0.0001; EI: 57.93±5.31 vs. 87.43±4.01, P=0.0004). Acute administration of lenrispodun in the organ bath improved both ED and EI responses (ED: 38.61±4.04, P=0.001; EI 69.99±4.29 P=0.01) while the relaxations remained unchanged after chronic lenrispodun treatment. In addition, lenrispodun upregulated PDE1a mRNA expression in SMC-KO, while no changes were observed in other PDEs. Conclusions: PDE1 inhibition may therapeutically be useful in restoring age-related vascular dysfunction by preserving microvascular function and potentiating NO-cGMP signaling. Yet, its chronic effects might be counteracted by the upregulation of PDE1a in vasculature. These results warrant further pharmacodynamic profiling of PDE enzyme regulation during chronic PDE1 inhibitor treatment.

Th17/IL-17 induces endothelial cell senescence via activation of NF-κB/p53/Rb signaling pathway

Cellular senescence is a key mechanism of age-related vascular endothelial dysfunction. Interleukin-17A (IL-17A) is an inflammatory cytokine produced by Th17 cells (a subgroup of helper T cells), which is a key factor in the development of atherosclerosis. However, the effect of IL-17A on the senescence of vascular endothelial cells is still unclear. In this study, we aimed to explore the role of IL-17A on endothelial cell senescence and its signaling pathways associated with senescence. The proportion of Th17 cells in the spleen and the expression levels of IL-17A, IL-6, and vascular cell adhesion molecule-1 (VCAM-1) in mice of different ages were increased with aging. In vitro experiments showed that proliferation was inhibited, senescent β-galactosidase and senescence-associated proteins (p16, p19, p21, and p53) of mouse aortic endothelial cells (MAECs) were increased with IL-17A treatment. Blocking the NF-κB pathway with ammonium pyrrolidinedithiocarbamate (PDTC) successfully inhibited IL-17A-induced expression of senescence-associated proteins. In conclusion, our data reveal a previously unsuspected link between IL-17A and endothelial cell senescence, which was mediated by the NF-κB /p53/Rb pathway.Cellular senescence is a key mechanism of age-related vascular endothelial dysfunction. The authors explored the role of IL-17A on endothelial cell senescence and its associated signaling pathways. Their data reveals a previously unsuspected link between IL-17A and endothelial cell senescence, mediated by the NF-κB /p53/Rb signaling pathway.

Transglutaminase 2 Inhibitor LDN 27219 Age-Dependently Lowers Blood Pressure and Improves Endothelium-Dependent Vasodilation in Resistance Arteries.

Transglutaminase 2 (TG2) is an enzyme which in the open conformation exerts transamidase activity, leading to protein cross-linking and fibrosis. In the closed conformation, TG2 participates in transmembrane signaling as a G protein. The unspecific transglutaminase inhibitor cystamine causes vasorelaxation in rat resistance arteries. However, the role of TG2 conformation in vascular function is unknown. We investigated the vascular effects of selective TG2 inhibitors by myography in isolated rat mesenteric and human subcutaneous resistance arteries, patch-clamp studies on vascular smooth muscle cells, and blood pressure measurements in rats and mice. LDN 27219 promoted the closed TG2 conformation and inhibited transamidase activity in mesenteric arteries. In contrast to TG2 inhibitors promoting the open conformation (Z-DON, VA5), LDN 27219 concentration-dependently relaxed rat and resistance human arteries by a mechanism dependent on nitric oxide, large-conductance calcium-activated and voltage-gated potassium channels 7, lowering blood pressure. LDN 27219 also potentiated acetylcholine-induced relaxation by opening potassium channels in the smooth muscle; these effects were abolished by membrane-permeable TG2 inhibitors promoting the open conformation. In isolated arteries from 35- to 40-week-old rats, transamidase activity was increased, and LDN 27219 improved acetylcholine-induced relaxation more than in younger rats. Infusion of LDN 27219 decreased blood pressure more effectively in 35- to 40-week than 12- to 14-week-old anesthetized rats. In summary, pharmacological modulation of TG2 to the closed conformation age-dependently lowers blood pressure and, by opening potassium channels, potentiates endothelium-dependent vasorelaxation. Our findings suggest that promoting the closed conformation of TG2 is a potential strategy to treat age-related vascular dysfunction and lowers blood pressure.

Medin aggregation causes cerebrovascular dysfunction in aging wild-type mice

Medin is the most common amyloid known in humans, as it can be found in blood vessels of the upper body in virtually everybody over 50 years of age. However, it remains unknown whether deposition of Medin plays a causal role in age-related vascular dysfunction. We now report that aggregates of Medin also develop in the aorta and brain vasculature of wild-type mice in an age-dependent manner. Strikingly, genetic deficiency of the Medin precursor protein, MFG-E8, eliminates not only vascular aggregates but also prevents age-associated decline of cerebrovascular function in mice. Given the prevalence of Medin aggregates in the general population and its role in vascular dysfunction with aging, targeting Medin may become a novel approach to sustain healthy aging.

Trimethylamine-N-Oxide Promotes Age-Related Vascular Oxidative Stress and Endothelial Dysfunction in Mice and Healthy Humans.

Age-related vascular endothelial dysfunction is a major antecedent to cardiovascular diseases. We investigated whether increased circulating levels of the gut microbiome-generated metabolite trimethylamine-N-oxide induces endothelial dysfunction with aging. In healthy humans, plasma trimethylamine-N-oxide was higher in middle-aged/older (64±7 years) versus young (22±2 years) adults (6.5±0.7 versus 1.6±0.2 µmol/L) and inversely related to brachial artery flow-mediated dilation (r2=0.29, P<0.00001). In young mice, 6 months of dietary supplementation with trimethylamine-N-oxide induced an aging-like impairment in carotid artery endothelium-dependent dilation to acetylcholine versus control feeding (peak dilation: 79±3% versus 95±3%, P<0.01). This impairment was accompanied by increased vascular nitrotyrosine, a marker of oxidative stress, and reversed by the superoxide dismutase mimetic 4-hydroxy-2,2,6,6-tetramethylpiperidin-1-oxyl. Trimethylamine-N-oxide supplementation also reduced activation of endothelial nitric oxide synthase and impaired nitric oxide-mediated dilation, as assessed with the nitric oxide synthase inhibitor L-NAME (NG-nitro-L-arginine methyl ester). Acute incubation of carotid arteries with trimethylamine-N-oxide recapitulated these events. Next, treatment with 3,3-dimethyl-1-butanol for 8 to 10 weeks to suppress trimethylamine-N-oxide selectively improved endothelium-dependent dilation in old mice to young levels (peak: 90±2%) by normalizing vascular superoxide production, restoring nitric oxide-mediated dilation, and ameliorating superoxide-related suppression of endothelium-dependent dilation. Lastly, among healthy middle-aged/older adults, higher plasma trimethylamine-N-oxide was associated with greater nitrotyrosine abundance in biopsied endothelial cells, and infusion of the antioxidant ascorbic acid restored flow-mediated dilation to young levels, indicating tonic oxidative stress-related suppression of endothelial function with higher circulating trimethylamine-N-oxide. Using multiple experimental approaches in mice and humans, we demonstrate a clear role of trimethylamine-N-oxide in promoting age-related endothelial dysfunction via oxidative stress, which may have implications for prevention of cardiovascular diseases.

Initiation of the Gut Microbiome Targeted Compound 3,3‐Dimethyl‐1‐butanol at Mid‐life Prevents Age‐related Vascular Dysfunction

Aging induces vascular dysfunction (arterial stiffening and endothelial dysfunction) mediated by superoxide‐driven oxidative stress and reduced nitric oxide (NO) bioavailability, which increases risk of cardiovascular (CV) diseases. A potential intervention target is the gut microbiome‐derived metabolite trimethylamine N‐oxide (TMAO), which increases in circulation with advancing age, is associated with higher CV risk, and can be inhibited by the food‐derived compound 3,3‐dimethyl‐1‐butanol (DMB).PurposeTo determine if DMB initiated at mid‐life (i.e., prior to the onset of dysfunction) prevents the development of age‐related vascular dysfunction.MethodsMiddle‐aged (18 mo) male C57BL/6 mice received drinking water with (n=24) or without (control: n=27) 1% DMB until sacrifice at 21, 24, or 27 mo of age (n=7–13/age/treatment). Arterial stiffness was assessed serially in vivo by aortic pulse wave velocity (aPWV). Endothelial function (ex vivo carotid artery endothelium‐dependent dilation [EDD] to acetylcholine), plasma TMAO (LC‐MS) and aortic superoxide (electron paramagnetic resonance spectroscopy) were assessed post‐sacrifice. To determine the prevention of age‐related vascular dysfunction with DMB, functional measures were made in a reference group of young (5 mo) male C57BL/6 mice (YC, n=12).ResultsPlasma TMAO increased with age vs YC (3.1±0.5 μM), but tended to be lower in DMB (5.6±0.5 μM) vs control (6.9±0.8 μM) mice at 24 and 27 mo (p=0.15). In control mice, aPWV was similar to YC (356±14 cm/s) at 18/21 mo (359±12/368±11 cm/s, both p&gt;0.54), but increased with age (24 mo: 386±12 cm/s, p=0.06 vs 18 mo; 27 mo: 432±10 cm/s, p&lt;0.001 vs 18 mo); this increase was attenuated by ~50% in DMB mice (aPWV at 24 mo: 359±9 cm/s; 27 mo: 388±9 cm/s, both p=0.01 vs control). Age‐related declines in peak EDD were apparent in control mice at 24 mo (79±3 vs YC 93±2%, p&lt;0.001) and 27 mo (76±3%, p&lt;0.0001 vs YC) and were fully prevented by DMB (24 mo: 91±3%; 27 mo: 93±1%; both p&lt;0.05 vs control). The effects of DMB at 24 and 27 mo (data combined across these ages) were mediated by reduced aortic superoxide production (control 4.7±0.5 vs DMB 3.1±0.4 AU, p=0.01), which reduced tonic superoxide‐associated suppression of EDD (normalization of EDD with superoxide dismutase mimetic TEMPOL) and restored NO‐mediated dilation (the difference in peak EDD in the absence vs presence of the NO synthase inhibitor L‐NAME: control 45±6 vs DMB 69±4%, p&lt;0.01). There were no differences in smooth muscle sensitivity to NO (peak dilation to sodium nitroprusside; 96±1 vs 98±1%, p=0.12), indicating improvements in EDD were endothelium‐specific.ConclusionSupplementation with DMB from mid‐life through old age in mice prevented the development of arterial stiffening and endothelial dysfunction. DMB may be a promising therapeutic strategy for preventing age‐related vascular dysfunction and thereby reducing CV risk with aging in humans.Support or Funding InformationR01 HL1348870‐02S1, R21 AG060884, F32 HL140875

Vascular age in patients with arterial hypertension

Ageing is considered to be the major and non-modifiable risk factor for the development of hypertension and cardiovascular diseases. During ageing, the vascular system undergoes structural and functional alterations, including endothelial dysfunction, thickening of the vascular wall, reduced distensibility and increased arterial stiffness. Vascular rigidity results from fibrosis and remodelling of the extracellular matrix, processes that are associated with ageing and are amplified in hypertension. These events may be induced by vasoactive agents, such as angiotensin II, endothelin-1, and aldosterone, which are increased in the vasculature during aging and hypertension. Complex interaction between the process of ageing and prohypertensive factors results in accelerated vascular remodelling and fibrosis, as well as increased arterial stiffness. Hypertension accelerates and augments age-related vascular remodelling and dysfunction, and ageing may impact on the severity of vascular damage in hypertension, thus strongly suggesting close interactions between biological ageing and blood pressure elevation. Molecular and cellular mechanisms underlying vascular alterations in ageing and hypertension are common and include aberrant signal transduction, oxidative stress and activation of pro-inflammatory and pro-fibrotic transcription factors. Strategies to suppress age-associated vascular changes can ameliorate vascular damage associated with hypertension. This article looks into vascular alterations occurring during ageing and hypertension, focussing particularly on arterial stiffness and vascular remodelling, also emphasizing the importance of diagnostic methods.