Age-related Endothelial Dysfunction Research Articles

Epigenetic clock in the aorta and age-related endothelial dysfunction in mice.

While epigenetic age (EA) of mouse blood can be determined using DNA methylation analysis at three CpG sites in the Prima1, Hsf4 and Kcns1 genes it is not known whether this approach is useful for predicting vascular biological age. In this study we validated the 3-CpG estimator for age prediction in mouse blood, developed a new predictive model for EA in mouse aorta, and assessed whether epigenetic age acceleration (EAA) measured with blood and aorta samples correlates with age-dependent endothelial dysfunction. Endothelial function was characterized in vivo by MRI in 8-96-week-old C57BL/6 mice. Arterial stiffness was measured by USG-doppler. EA-related changes within 41 CpG sites in Prima1, Kcns1 and Hsf4 loci, were analyzed in the aorta and blood using bisulfite amplicon high-throughput sequencing. Progressive age-dependent endothelial dysfunction and changes in arterial stiffness were observed in 36-96-week-old C57BL/6 mice. Methylation levels of the investigated loci correlated with chronological age in blood and the aorta. The new model for EA estimation in aorta included three cytosines located in the Kcns1 and Hsf4, explained R2 = 87.8% of the variation in age, and predicted age with an mean absolute error of 9.6weeks in the independent test set. EAA in the aorta was associated with endothelial dysfunction in the abdominal aorta and femoral arterywhat was consistent with the EAA direction estimated in blood samples. The rate of vascular biological ageing in mice, reflected by the age-dependent systemic endothelial dysfunction, could be estimated using DNA methylation measurements at three loci in aorta and blood samples.

Endothelin-1 Contributes to Reduced Endothelial Function in Middle-aged/Older Men with Low Testosterone

Male aging is associated with a general decline in serum testosterone (T) concentrations. Low endogenous T is associated with increased cardiovascular disease risk, and we recently demonstrated accelerated age-related endothelial dysfunction, measured by brachial artery flow mediated dilation (FMD), in middle-aged/older (MA/O) men with low T compared with age-matched men with normal T. Endothlin-1 (ET-1) is a potent vasoconstrictor that is modulated by sex hormones and contributes to age-associated endothelial dysfunction. However, whether ET-1 contributes to reduced FMD in MA/O men with low T has yet to be determined. PURPOSE: to determine if ET-1 contributes to reduced FMD in MA/O men with low T. HYPOTHESIS: we hypothesized that MA/O men with low T would have higher ET-1 concentrations compared with young and MA/O men with normal T. Additionally, we hypothesized that there would be an inverse relation between ET-1 and FMD. METHODS: 60 men were categorized as either young (18-40 years, T: 400-1000 ng/dL) or MA/O (50-75 years) with low (<300ng/dL) or normal (400-1000 ng/dL) serum total T. T was determined from a venous blood draw using a one-step competitive enzyme linked immunosorbent assay. Endothelial function was determined using brachial artery FMD and ET-1 concentrations were determined using radioimmunoassay from a venous blood sample. RESULTS: T concentrations were significantly lower in MA/O men with low T (269±48 ng/dL) compared to young (500±58 ng/dL; p<0.001) or MA/O men with normal T (512±115, ng/dL; p<0.001). There was no difference in T between MA/O men with normal T and young men (p=0.995). MA/O men were similar in age (low T=59±8, normal T=60±6 years; p=0.996) and older than young men (29±4 years; p<0.001 compared to MA/O with both low and normal T). Age-associated reductions in FMD (young=7.3±1.3 vs. MA/O normal T=5.7±2.2%; p=0.019), were exaggerated in MA/O men with low T (4.0±1.7%; p=0.010 vs. MA/O normal T, p<0.001 vs. young). ET-1 concentrations were similar between young and MA/O men with normal T (young= 5.6±0.9 vs. MA/O normal T=6.0±1.4 pg/mL; p=0.768), and higher in MA/O men with low T (7.7±2.8 pg/mL; p=0.002 vs. young, p=0.016 vs. MA/O normal T). There was a moderate inverse association between FMD and ET-1 (r=-0.374, p=0.003). CONCLUSIONS: These data suggest that the greater age-associated endothelial dysfunction in MA/O men with low T may be related to elevations in ET-1. Future studies should examine the effects of low T on endothelin receptors ETA and ETB, as well as factors that could explain the increase in ET-1 in MA/O men with low T. Supported by NIH Grants R01AG049762, U54AG062319, and T32AG000279-16A1 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.

PS-B12-12: NOVEL PATHOPHYSIOLOGICAL ROLES OF A-SYNUCLEIN IN AGE-RELATED VASCULAR ENDOTHELIAL DYSFUNCTION AND E NHANCED PERMEABILITY.

Although α-synuclein (SNCA) is a well-known pathological molecule involved in synucleinopathy in neurons, its physiological roles remain largely unknown. We reported that serum SNCA levels have a close inverse correlation with blood pressure and age, which indicates the involvement of SNCA in age-related endothelial dysfunction. Therefore, this study aimed to elucidate the molecular functions of SNCA in the endothelium. We confirmed that SNCA was expressed in and secreted from endothelial cells (ECs). Exogenous treatment with recombinant SNCA (rSNCA) activated the Akt-eNOS axis and increased nitric oxide (NO) production in ECs. Treatment with rSNCA also suppressed TNF-α and palmitic acid induced NF-κB activation, leading to the suppression of VCAM-1 upregulation and restoration of eNOS downregulation in ECs. As for endogenous SNCA expression, replicative senescence resulted in the attenuation of SNCA expression in cultured ECs, similar to the effects of physiological aging on mice aortas. The siRNA-mediated silencing of SNCA consistently resulted in senescent phenotypes, such as eNOS downregulation, increased β; gal activity, decreased Sirt1 expression, and increased p53 expression, in ECs. Mechanistically, loss-of-function of SNCA upregulated the expression of β;-site amyloid precursor protein cleaving enzyme 1 (BACE1), which was reported to exacerbate eNOS inactivation and permeability in ECs. Addition of SNAP, NO donor, rescued the increment of BACE1, which indicated eNOS downregulation by loss-of-function of SNCA caused BECE1 increment in ECs. Ex vivo assessment of endothelial functions using aortic rings revealed impaired endothelium-dependent acetylcholine-induced relaxation in SNCA knockout (KO) mice. Furthermore, SNCA KO mice, especially those on a high fat diet, displayed elevated blood pressure compared with wild-type (WT) mice; this could be eNOS dysfunction dependent because of the lower difference caused by L NAME administration. In addition, increased depositions of immunoglobulin were found in the aortas of SNCA KO compared with those of WT mice, which indicated enhanced endothelial permeability by in vivo loss-of-function of SNCA. Thus, these results indicate that exogenous and endogenous SNCA in ECs might physiologically maintain vascular integrity, and age-related endothelial dysfunction and enhanced permeability might be partially ascribed to loss-of-function of SNCA in ECs.

Advances in the study of the vascular protective effects and molecular mechanisms of hawthorn (Crataegus anamesa Sarg.) extracts in cardiovascular diseases.

Hawthorn belongs to the rose family and is a type of functional food. It contains various chemicals, including flavonoids, terpenoids, and organic acid compounds. This study aimed to review the vascular protective effects and molecular mechanisms of hawthorn and its extracts on cardiovascular diseases (CVDs). Hawthorn has a wide range of biological functions. Evidence suggests that the active components of HE reduce oxidative stress and inflammation, regulate lipid levels to prevent lipid accumulation, and inhibit free cholesterol accumulation in macrophages and foam cell formation. Additionally, hawthorn extract (HE) can protect vascular endothelial function, regulate endothelial dysfunction, and promote vascular endothelial relaxation. It has also been reported that the effective components of hawthorn can prevent age-related endothelial dysfunction, increase cellular calcium levels, cause antiplatelet aggregation, and promote antithrombosis. In clinical trials, HE has been proved to reduce the adverse effects of CVDs on blood lipids, blood pressure, left ventricular ejection fraction, heart rate, and exercise tolerance. Previous studies have pointed to the benefits of hawthorn and its extracts in treating atherosclerosis and other vascular diseases. Therefore, as both medicine and food, hawthorn can be used as a new drug source for treating cardiovascular diseases.

Ageing and endothelium-mediated vascular dysfunction: the role of the NADPH oxidases.

The present study aimed to determine the isoform-specific role of the NADPH oxidases (NOX) in the endothelium-mediated vascular dysfunction associated with ageing. Endothelium-dependent [intraluminal flow- and acetylcholine (ACh)-induced] vasodilatation in human skeletal muscle feed arteries (SMFAs) of young (24±1 years, n=16), middle aged (45±1 years, n=18) and old (76±2 years, n=21) subjects was assessed in vitro with and without the inhibition of NOX1 (ML090), NOX2 (gp91) and NOX4 (plumbagin). To identify the role of nitric oxide (NO) bioavailability in these responses, NO synthase blockade (l-NG -monomethyl arginine citrate) was utilized. SMFA NOX1, NOX2 and NOX4 protein expression was determined by western blotting. Age related endothelium-dependent vasodilatory dysfunction was evident in response to flow (young: 69±3; middle aged: 51±3; old: 27±3%, P<0.05) and ACh (young: 89±2; middle aged: 72±3; old: 45±4%, P<0.05). NOX1 inhibition had no effect on SMFA vasodilatation, whereas NOX2 inhibition restored flow- and ACh-induced vasodilatation in the middle aged and the old SMFAs (middle aged+gp91: 69±3; 86±3, old+gp91: 65±5; 83±2%, P<0.05) and NOX4 inhibition tended to restore these vasodilatory responses in these two groups, but neither achieved statistical significance (P ≈ 0.06). l-NG -monomethyl arginine citrate negated the restorative effects of NOX2 and NOX4 blockade. Only NOX2 and NOX4 protein expression was significantly greater in the two older groups and inversely related to vascular function (r=0.48 to 0.93, P<0.05). NOX2 and, to a lesser extent, NOX4 appear to play an important, probably NO-mediated, role in age-related endothelial dysfunction. KEY POINTS: The present study aimed to determine the isoform-specific role of the NADPH oxidases (NOX) in the endothelium-mediated vascular dysfunction associated with ageing. Age related endothelium-dependent vasodilatory dysfunction was evident in skeletal muscle feed arteries in response to both flow and acetylcholine. NOX2 inhibition (gp91) restored endothelium-dependent vasodilatation in the middle aged and the old skeletal muscle feed arteries, and NOX4 inhibition (plumbagin) tended to restore these vasodilatory responses in these two groups. Nitric oxide synthase inhibition negated the restorative effects of NOX2 and NOX4 blockade. NOX2 and NOX4 protein expression was significantly greater in the two older groups and inversely related to vascular function. NOX2 and, to a lesser extent, NOX4 appear to play an important, probably nitric oxide-mediated, role in age-related endothelial dysfunction and could be important therapeutic targets to maintain vascular health with ageing.

Novel pathophysiological roles of α-synuclein in age-related vascular endothelial dysfunction.

Although α-synuclein (SNCA) is a well-known pathological molecule involved in synucleinopathy in neurons, its physiological roles remain largely unknown. We reported that serum SNCA levels have a close inverse correlation with blood pressure and age, which indicates the involvement of SNCA in age-related endothelial dysfunction. Therefore, this study aimed to elucidate the molecular functions of SNCA in the endothelium. We confirmed that SNCA was expressed in and secreted from endothelial cells (ECs). Exogenous treatment with recombinant SNCA (rSNCA) activated the Akt-eNOS axis and increased nitric oxide production in ECs. Treatment with rSNCA also suppressed TNF-α- and palmitic acid-induced NF-κB activation, leading to the suppression of VCAM-1 upregulation and restoration of eNOS downregulation in ECs. As for endogenous SNCA expression, replicative senescence resulted in the attenuation of SNCA expression in cultured ECs, similar to the effects of physiological aging on mice aortas. The siRNA-mediated silencing of SNCA consistently resulted in senescent phenotypes, such as eNOS downregulation, increased β-gal activity, decreased Sirt1 expression, and increased p53 expression, in ECs. Ex vivo assessment of endothelial functions using aortic rings revealed impaired endothelium-dependent acetylcholine-induced relaxation in SNCA knockout (KO) mice. Furthermore, SNCA KO mice, especially those on a high-fat diet, displayed elevated blood pressure compared with wild-type mice; this could be eNOS dysfunction-dependent because of the lower difference caused by L-NAME administration. These results indicate that exogenous and endogenous SNCA in ECs might physiologically maintain vascular integrity, and age-related endothelial dysfunction might be partially ascribed to loss-of-function of SNCA in ECs.

Targeting SIRT1 Rescues Age- and Obesity-Induced Microvascular Dysfunction in Ex Vivo Human Vessels.

Background:Experimental evidence suggests a key role of SIRT1 (silent information regulator 1) in age- and metabolic-related vascular dysfunction. Whether these effects hold true in the human microvasculature is unknown. We aimed to investigate the SIRT1 role in very early stages of age- and obesity-related microvascular dysfunction in humans.Methods:Ninety-five subjects undergoing elective laparoscopic surgery were recruited and stratified based on their body mass index status (above or below 30 kg/m2) and age (above or below 40 years) in 4 groups: Young Nonobese, Young Obese, Old Nonobese, and Old Obese. We measured small resistance arteries’ endothelial function by pressurized micromyography before and after incubation with a SIRT1 agonist (SRT1720) and a mitochondria reactive oxygen species (mtROS) scavenger (MitoTEMPO). We assessed vascular levels of mtROS and nitric oxide availability by confocal microscopy and vascular gene expression of SIRT1 and mitochondrial proteins by qPCR. Chromatin immunoprecipitation assay was employed to investigate SIRT1-dependent epigenetic regulation of mitochondrial proteins.Results:Compared with Young Nonobese, obese and older patients showed lower vascular expression of SIRT1 and antioxidant proteins (FOXO3 [forkhead box protein O3] and SOD2) and higher expression of pro-oxidant and aging mitochondria proteins p66Shc and Arginase II. Old Obese, Young Obese and Old Nonobese groups endothelial dysfunction was rescued by SRT1720. The restoration was comparable to the one obtained with mitoTEMPO. These effects were explained by SIRT1-dependent chromatin changes leading to reduced p66Shc expression and upregulation of proteins involved in mitochondria respiratory chain.Conclusions:SIRT1 is a novel central modulator of the earliest microvascular damage induced by age and obesity. Through a complex epigenetic control mainly involving p66Shc and Arginase II, it influences mtROS levels, NO availability, and the expression of proteins of the mitochondria respiratory chain. Therapeutic modulation of SIRT1 restores obesity- and age-related endothelial dysfunction. Early targeting of SIRT1 might represent a crucial strategy to prevent age- and obesity-related microvascular dysfunction.

TARGETING SIRT1 RESCUES AGE- AND OBESITY-INDUCED HUMAN MICROVASCULAR DYSFUNCTION

Experimental evidence suggests a relevant role of SIRT1 in age- and metabolic-related vascular dysfunction. Whether these effects hold true in the human microvasculature is unknown. We aimed to investigate the role of SIRT1 in age- and obesity-related microvascular dysfunction in humans.Individuals (n = 95) undergoing laparoscopic surgery were stratified by BMI status (above or below 30 Kg/m2) and age (above or below 40 years) and classified as young control (YC), young obese (YO), old control (OC) and old obese (OO). Endothelial function was assessed in small resistance arteries by pressurized micromyography, before and after incubation with a SIRT1 agonist (SRT1720) and a mitochondria reactive oxygen species (mtROS) scavenger (MitoTEMPO). We assessed vascular levels of mtROS and nitric oxide availability by confocal microscopy and vascular gene expression of SIRT1 and mitochondrial proteins by qPCR. ChIP assay was employed to investigate SIRT1-dependent epigenetic regulation of mitochondrial proteins.Endothelial function showed a graded decrease from YC to OO, with reduced nitric oxide availability and increased mtROS levels. Similarly, SIRT1 and mitochondrial antioxidant proteins (FOXO3 and SOD2) expression levels were reduced while there was a higher expression of pro-oxidant and ageing mitochondria proteins p66Shc and Arginase II. OO, YO and OC endothelial function was rescued by SRT1720 (Figure). The improvement in endothelial function showed a linear relationship with age, BMI and vascular emodelling. The restoration was comparable to the one obtained with mitoTEMPO in the same groups. These effects were explained by SIRT1-dependent chromatin changes leading to reduced p66Shc expression and upregulation of proteins involved in mitochondria respiratory chain: ATP synthase 6, cytochrome B, NADH dehydrogenase 2, NADH dehydrogenase 5.Restoring SIRT1 activity rescues obesity and age-related endothelial dysfunction. Its epigenetic control on mitochondrial pathways influences mtROS levels, NO availability, and the expression of the mitochondria respiratory chain proteins. SIRT1 is a novel central regulator of a homeostatic axis connecting vascular phenotype to systemic metabolism and ageing and to exposure to external adverse stimuli. Early targeting of SIRT1 might represent a crucial strategy to prevent the microvascular dysfunction related to age and obesity.

Changes in Gut Microbiome Composition with Healthy Aging in Humans: Links to Vascular Endothelial Function

Aging is the primary risk factor for cardiovascular diseases, primarily due to development of vascular endothelial dysfunction. The gut microbiome is a strong influencer of host physiology, but few studies have investigated how gut microbiome composition changes with primary (healthy) aging in humans, or how such changes may influence endothelial function.PURPOSETo: 1) determine changes in gut microbiome composition and their relation to endothelial function in healthy late middle‐aged to older (MA/O) vs. young (Y) adults; and 2) investigate potential mechanisms of this link.METHODS &amp; RESULTSN=14/group (MA/O: 60‐79 yrs; Y: 18‐29 yrs). Data are mean ± SE. Gut microbiome composition was assessed via fecal 16S rRNA sequencing. α‐diversity, phylogenetic diversity within each sample, was higher in MA/O vs Y adults (Faith’s PD: 22.2 ± 1.8 vs 15.5 ± 1.3, P = 0.02). β‐diversity, difference in overall composition between samples, was also altered with aging (PERMANOVA: P &lt; 0.05; unweighted UniFrac). Both α‐diversity (R = ‐0.60, P = 0.04) and β‐diversity (R = ‐0.58, P = 0.04) were inversely related to age‐related impairments in endothelial function, measured by brachial artery flow‐mediated dilation (MA/O: 4.5 ± 0.4 vs Y: 7.9 ± 1.7%, P &lt; 0.05).Potential Mechanisms: In preliminary analyses (unpaired t‐tests), changes in gut microbiome composition were accompanied by altered relative abundance of gram‐negative bacteria (e.g., Bacteroides [MA/O: 33 ± 4% vs Y: 20 ± 4%, P = 0.02]) and Enterobacteriaceae [MA/O: 1.5 ± 0.1% vs Y: 0.4 ± 0.1%, P = 0.02]), which contain pro‐inflammatory lipopolysaccharide (LPS) in their cell walls. Translocation of LPS into systemic circulation is facilitated by increased intestinal permeability, which we found was higher with aging, as measured by a lactulose‐mannitol (L:M) test (1‐hour serum L:M ratio, MA/O: 0.012 ± 0.002 vs Y: 0.007 ± 0.001, P = 0.04). As such, plasma LPS‐binding protein, a readily detectable marker of LPS in peripheral blood, was higher in MA/O vs Y adults (31.3 ± 4.1 vs 17.7 ± 2.0 ng/mL, P = 0.01). Once in circulation, LPS is recognized as a pathogen‐associated molecular pattern and can trigger an inflammatory response. Consistent with this, circulating levels of the pro‐inflammatory markers IL‐6 [MA/O: 1.56 ± 0.29 vs Y: 0.70 ± 0.13 pg/ml, P = 0.01] and CRP [MA/O: 1.65 ± 0.27 vs Y: 0.78 ± 0.11 pg/ml, P = 0.02]) were increased with aging. In biopsied venous endothelial cells from a subset of subjects (n = 4‐8/group), abundance of phosphorylated (i.e., activated) NF□B was higher in MA/O vs Y adults (0.40 ± 0.05 vs 0.31 ± 0.04 ng/ml, P = 0.24), with no difference in total NFkB, indicating potentially increased vascular inflammation.CONCLUSIONSOur findings represent initial evidence in humans that the gut microbiome changes with healthy aging and may be an important mediator of age‐related endothelial dysfunction, possibly via increasing circulating LPS and vascular inflammation.

Intake of coated EPA:DHA 6:1 nanoparticles improves age-related endothelial dysfunction by restoring the endothelial formation of NO and improving oxidative stress: Role of the local angiotensin system

Omega-3 polyunsaturated fatty acids including eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) have been shown to reduce cardiovascular risk. This study investigated whether nanoencapsulation of EPA:DHA 6:1 is associated with an improved beneficial effect on age-related endothelial dysfunction. Thoracic aorta and main mesenteric artery from young and middle-aged rats that received daily by gavage 100 mg/kg of either native or coated form for 1-week were investigated. Vascular reactivity and level of oxidative stress and target proteins were assessed using organ chambers, dihydroethidium and immunofluorescence staining, respectively. Compared to the native form, the coated EPA:DHA 6:1 nanoparticles treatment improved to a greater extent NO-mediated relaxation and contractile response, and the level of vascular oxidative stress and expression of ACE/AT1R in the aged artery. The nanoencapsulation of EPA:DHA 6:1 promotes a greater beneficial effect on age-related endothelial dysfunction involving improved NO-mediated relaxations and normalization of the pro-oxidant local angiotensin system.

The role of transient receptor potential ankyrin 1 in age-related endothelial dysfunction.

Oxidative stress plays a key role in age-related vascular disease. The present study aimed to investigate the role of an antioxidant channel, transient receptor potential ankyrin 1 (TRPA1), in age-related endothelial dysfunction. Human umbilical vein endothelial cells (HUVECs) were grown to induce replicative senescence, and 6-month-old young, 12-month-old middle-aged, and 24-month-old aged mice were used. TRPA1 was downregulated in senescent HUVECs, so were endothelial nitric oxide synthase (eNOS), nuclear factor erythroid 2-related factor 2 (Nrf2), and uncoupling protein 2 (UCP2). Activating TRPA1 with cinnamaldehyde prevented downregulation of eNOS, Nrf2, and UCP2, inhibited superoxide production and apoptosis, and preserved nitric oxide bioavailability in senescent HUVECs. TRPA1, phosphorylated eNOS, Nrf2 and UCP2 were significantly downregulated in aged aortas compared with young aortas after a compensatory upregulation in middle-aged aortas. Dietary administration of cinnamaldehyde for 12months prevented mitochondrial dysfunction, improved endothelium-dependent relaxation, and increased expression of eNOS, Nrf2, and UCP2 in aged aortas. Importantly, the effects of cinnamaldehyde can be blocked by a TRPA1 antagonist HC-030031. These findings suggest that TRPA1 may play a critical role in age-related endothelial dysfunction and may become a therapeutic target for the treatment and prevention of age-related vascular disease.

Oxidative Stress and Inflammation Are Associated With Age-Related Endothelial Dysfunction in Men With Low Testosterone.

Vascular aging, including endothelial dysfunction secondary to oxidative stress and inflammation, increases the risk for age-associated cardiovascular disease (CVD). Low testosterone in middle-aged/older men is associated with increased CVD risk. We hypothesized that low testosterone contributes to age-associated endothelial dysfunction, related in part to greater oxidative stress and inflammation. This cross-sectional study included 58 healthy, nonsmoking men categorized as young (N = 20; age 29 ± 4 years; testosterone 500 ± 58 ng/dL), middle-aged/older with higher testosterone (N = 20; age 60 ± 6 years; testosterone 512 ± 115 ng/dL), and middle-aged/older lower testosterone (N = 18; age 59 ± 8 years; testosterone 269 ± 48 ng/dL). Brachial artery flow-mediated dilation (FMDBA) was measured during acute infusion of saline (control) and vitamin C (antioxidant). Markers of oxidative stress (total antioxidant status and oxidized low-density lipoprotein cholesterol), inflammation (interleukin [IL]-6 and C-reactive protein [CRP]), and androgen deficiency symptoms were also examined. During saline, FMDBA was reduced in middle-aged/older compared with young, regardless of testosterone status (P < 0.001). FMDBA was reduced in middle-aged/older lower testosterone (3.7% ± 2.0%) compared with middle-aged/older higher testosterone (5.7% ± 2.2%; P = 0.021), independent of symptoms. Vitamin C increased FMDBA (to 5.3% ± 1.6%; P = 0.022) in middle-aged/older lower testosterone but had no effect in young (P = 0.992) or middle-aged/older higher testosterone (P = 0.250). FMDBA correlated with serum testosterone (r = 0.45; P < 0.001), IL-6 (r = -0.41; P = 0.002), and CRP (r = -0.28; P = 0.041). Healthy middle-aged/older men with low testosterone appear to have greater age-associated endothelial dysfunction, related in part to greater oxidative stress and inflammation. These data suggest that low testosterone concentrations may contribute to accelerated vascular aging in men.

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.

Local endothelial DNA repair deficiency causes aging-resembling endothelial-specific dysfunction.

We previously identified genomic instability as a causative factor for vascular aging. In the present study, we determined which vascular aging outcomes are due to local endothelial DNA damage, which was accomplished by genetic removal of ERCC1 (excision repair cross-complementation group 1) DNA repair in mice (EC-knockout (EC-KO) mice). EC-KO showed a progressive decrease in microvascular dilation of the skin, increased microvascular leakage in the kidney, decreased lung perfusion, and increased aortic stiffness compared with wild-type (WT). EC-KO showed expression of DNA damage and potential senescence marker p21 exclusively in the endothelium, as demonstrated in aorta. Also the kidney showed p21-positive cells. Vasodilator responses measured in organ baths were decreased in aorta, iliac and coronary artery EC-KO compared with WT, of which coronary artery was the earliest to be affected. Nitric oxide-mediated endothelium-dependent vasodilation was abolished in aorta and coronary artery, whereas endothelium-derived hyperpolarization and responses to exogenous nitric oxide (NO) were intact. EC-KO showed increased superoxide production compared with WT, as measured in lung tissue, rich in endothelial cells (ECs). Arterial systolic blood pressure (BP) was increased at 3 months, but normal at 5 months, at which age cardiac output (CO) was decreased. Since no further signs of cardiac dysfunction were detected, this decrease might be an adaptation to prevent an increase in BP. In summary, a selective DNA repair defect in the endothelium produces features of age-related endothelial dysfunction, largely attributed to loss of endothelium-derived NO. Increased superoxide generation might contribute to the observed changes affecting end organ perfusion, as demonstrated in kidney and lung.

Habitual Exercise and ETB Receptor Responses in Postmenopausal Women

Age‐related declines in endothelial function can be reversed with habitual exercise (HEx) in older men by attenuating the vasoconstricting effects of endothelin‐1 (ET‐1), attributable in part by enhanced ETA receptor activity. Our laboratory has recently demonstrated that ET‐1 contributes to age‐related endothelial dysfunction in postmenopausal women (PMW) via the ETB receptor (ETBR). Although there is evidence for sex‐specific differences in ET‐1 receptor activity with aging, the impact of habitual exercise (HEx) on ET‐1 receptor responses in PMW has yet to be established.PURPOSETo test the hypothesis that ETBR responses would be attenuated in HEx PMW.METHODSIn a retrospective analysis, 30 PMW were grouped by self‐reported weekly exercise. We measured vasodilatory responses to local heating of the skin (laser Doppler flowmetry) during microdialysis perfusions of lactated Ringer’s (Control) and ETBR blockade (BQ‐788, 300nM) in 15 low (LEx: 2±2 d/wk, 57±4 yrs) and 15 high (HEx: 6±1 d/wk, 60±5 yrs) frequency exercisers. Cutaneous vascular conductance (CVC) was calculated during the plateau phase of local heating (42°C), and normalized to maximal vasodilation achieved by perfusion of sodium nitroprusside (28mM) and heating to 43°C. We analyzed the impact of weekly exercise frequency on vasodilatory responses to ETBR blockade using a two‐way ANOVA. Data presented are mean ± standard deviation.RESULTSGroups were well‐matched for body mass index (BMI) (LEx: 25±4 vs HEx: 24±2 kg/m2, P=0.31), resting heart rate (LEx: 60±8 vs HEx: 54±9 bpm, P=0.14), and resting mean arterial pressure (LEx: 90±10 vs HEx: 90±8 mmHg, P=0.96). Microvascular endothelial function was similar between LEx (control: 87±9 %CVCmax) and HEx (control: 88±6 %CVCmax, P=0.86). ETBR blockade improved vasodilation in LEx (BQ‐788: 93±7 %CVCmax, P=0.01 vs. control) but not in HEx (BQ‐788: 91±7 %CVCmax, P=0.11). To account for differences in age and time since menopause, we performed a sub‐analysis of age and BMI‐matched early PMW (&lt;5 years since menopause) PMW (LEx, n=4: 53±2 yrs, 24±4 kg/m2; HEx, n=5: 53±3 yrs, 23±2 kg/m2). This sub‐analysis revealed a similar trend of enhanced vasodilation during ETBR blockade in LEx (control: 88±3 vs BQ‐788: 96±4 %CVCmax) but not HEx PMW (control: 87±6 vs BQ‐788: 85±13 %CVCmax; drug*exercise, P=0.07).CONCLUSIONBlocking ETBR improved microvascular endothelial function in LEx, indicating a removal of the vasoconstricting effects of ETBR in LEx but not HEx PMW. Therefore, these data suggest that exercise may attenuate ETBR‐mediated vasoconstriction in PMW. Additional data are needed to determine the impact of exercise on ETA receptor responses in PMW.Support or Funding InformationSupported by NIH R01 HL 146558, P20 GM 113125, AHA 16SDG30700015, and University of Delaware Research Foundation

Oral Intake of EPA:DHA 6:1 by Middle-Aged Rats for One Week Improves Age-Related Endothelial Dysfunction in Both the Femoral Artery and Vein: Role of Cyclooxygenases.

In humans, aging is associated with endothelial dysfunction and an increased risk of venous thromboembolism. Although intake of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) at a ratio of 6:1 by old rats improved the endothelial dysfunction in arteries, the impact on veins remains unclear. Eight-month-old male Wistar rats were either untreated or orally administered corn oil, EPA:DHA 1:1, or EPA:DHA 6:1 (500 mg/kg/d) for seven days. Vascular reactivity was studied by myography. In middle-aged femoral artery rings, acetylcholine caused a partial relaxation at low concentrations and a contractile response at high concentrations, whereas in the old femoral vein only a partial relaxation was observed. The EPA:DHA 6:1 treatment blunted the contractile response to acetylcholine in the middle-aged femoral artery and both EPA:DHA 6:1 and 1:1 increased the relaxation to acetylcholine in the old femoral vein. No such effects were observed with corn oil. Both the non-selective cyclooxygenase inhibitor indomethacin and the COX-1 inhibitor SC-560 increased the relaxation to acetylcholine in the middle-aged femoral artery whereas the COX-2 inhibitor NS-398 increased that in the middle-aged femoral vein. In conclusion, our results indicate that aging is associated with an endothelial dysfunction in the femoral artery and vein, which can be improved by EPA:DHA 6:1 treatment—most likely via a cyclooxygenase-dependent mechanism.

Intake of omega-3 formulation EPA:DHA 6:1 by old rats for 2 weeks improved endothelium-dependent relaxations and normalized the expression level of ACE/AT1R/NADPH oxidase and the formation of ROS in the mesenteric artery

Omega-3 polyunsaturated fatty acids (PUFAs) including eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) have been shown to protect the cardiovascular system, in part, by stimulating the endothelial formation of nitric oxide (NO). EPA:DHA 6:1 has been identified as a potent omega 3 PUFA formulation to induce endothelium-dependent vasorelaxation and activation of endothelial NO synthase (eNOS). This study examined whether intake of EPA:DHA 6:1 (500 mg/kg/day) for 2 weeks improves an established endothelial dysfunction in old rats (20 months old), and, if so, the underlying mechanism was subsequently determined.In the main mesenteric artery rings, an endothelial dysfunction characterized by a blunted NO component, an abolished endothelium-dependent hyperpolarization component, and increased endothelium-dependent contractile responses (EDCFs) are observed in old rats compared to young rats. Age-related endothelial dysfunction was associated with increased vascular formation of reactive oxygen species (ROS) and expression of eNOS, components of the local angiotensin system, senescence markers, and cyclooxygenase-2 (COX-2), and the downregulation of COX-1. The EPA:DHA 6:1 treatment improved the NO-mediated relaxation, reduced the EDCF-dependent contractile response and the vascular formation of ROS, and normalized the expression level of all target proteins in the old arterial wall.Thus, the present findings indicate that a 2-week intake of EPA:DHA 6:1 by old rats restored endothelium-dependent NO-mediated relaxations, most likely, by preventing the upregulation of the local angiotensin system and the subsequent formation of ROS.

Aging Alters Cerebrovascular Endothelial GPCR and K+ Channel Function: Divergent Role of Biological Sex.

Age-related dementia entails impaired blood flow to and throughout the brain due, in part, to reduced endothelial nitric oxide signaling. However, it is unknown whether sex affects cerebrovascular Gq-protein-coupled receptors (GPCRs) and K+ channels underlying endothelium-derived hyperpolarization (EDH) during progressive aging. Thus, we simultaneously evaluated intracellular Ca2+ ([Ca2+]i) and membrane potential (Vm) of intact endothelial tubes freshly isolated from posterior cerebral arteries of young (4-6 mo), middle-aged (12-16 mo), and old (24-28 mo) male and female C57BL/6 mice. Purinergic receptor function (vs. muscarinic) was dominant and enhanced for [Ca2+]i increases in old females versus old males. However, Ca2+-sensitive K+ channel function as defined by NS309-evoked Vm hyperpolarization was mildly impaired in females versus males during old age. This sex-based contrast in declined function of GPCRs and K+ channels to produce EDH may support a greater ability for physiological endothelial GPCR function to maintain optimal cerebral blood flow in females versus males during old age. As reflective of the pattern of cerebral blood flow decline in human subjects, inward-rectifying K+ (KIR) channel function decreased with progressive age regardless of sex. Combined age-related analyses masked male versus female aging and, contrary to expectation, hydrogen peroxide played a minimal role. Altogether, we conclude a sex-based divergence in cerebrovascular endothelial GPCR and K+ channel function while highlighting a previously unidentified form of age-related endothelial dysfunction as reduced KIR channel function.

P2250P66Shc mediates the influence of Sirt-1 on age and obesity related endothelial function

Abstract Background Sirtuins are histone deacetylase downregulated in conditions of excessive calory intake, with important roles in controlling cellular ageing pathways. Among their different isoforms, Sirt-1 is the most studied and experimental models suggest it could contribute to the downregulation of the ageing protein p66Shc in conditions of reduced energy intake, ultimately resulting in a reduced mitochondrial free radical production (mtROS). Purpose We assessed the combined influence of ageing and obesity (a condition of excessive calory intake) on the expression and functional activity of Sirt-1 in humans small resistance arteries, as well as whether the effects of Sirt-1 on endothelial function might depend from the regulation of p66Shc activity and its influence on mtROS. Methods Small resistance arteries were isolated from subcutaneous tissue of obese subjects and healthy controls undergoing bariatric surgery of inguinal hernia repair, respectively. The obese and control groups were further divided into young (&lt;40 years old) and old (&gt;40 years old) subjects. Arteries were mounted on a pressurised myograph and endothelial function was assessed using dose-response curves to acetylcholine before and after incubation with SRT1720 (selective agonist of Sirt-1), L-NAME (eNOS inhibitor), MitoTEMPO (mtROS scavenger) and gp91ds-tat (NADPH oxidase inhibitor), alone or in combination. The expression of Sirt-1 and p66Shc in the vascular wall of obese and control subjects was assessed by qPCR while the ChIP assay was used to assess the binding of Sirt-1 to the promoter region of p66Shc. Results Endothelial function was lower in older than younger and in obese than control groups (p&lt;0.001). Incubation with SRT1720 did not affect endothelial function in young controls, slightly improved endothelial function in young obese (p&lt;0.01) and induced a substantial improvement in endothelial function in both the old control and old obese groups (p&lt;0.001). Pre-incubation with L-NAME suggested that the effects of Sirt-1 were dependent from an improved vascular NO availability. Incubation with gp91ds-tat improved endothelial function in both obese groups and the old control group (p&lt;0.01), but to a lesser extent than SRT1720. Conversely, incubation with MitoTEMPO induced the same improvement of endothelial function than SRT1720 in all study groups and, when vessels were preincubated with MitoTEMPO, the addition of Sirt-1 did not produce further improvement of endothelial function. The expression of Sirt-1 and p66Shc was increased in the vessels from obese subjects compared to the non-obese group (p&lt;0.001), while the ChIP assay revealed a lower binding of Sirt-1 to the promoter region of p66Shc in obese than controls subjects (p&lt;0.001). Conclusions Our results suggest that Sirt-1 has an important role in the obesity and age-related endothelial dysfunction, throughout epigenetic regulation of p66Shc and, thus, controlling the production of mtROS. Acknowledgement/Funding Ministero dell'istruzione, dell'università e della ricerca

Treatment with the poly(ADP-ribose) polymerase inhibitor PJ-34 improves cerebromicrovascular endothelial function, neurovascular coupling responses and cognitive performance in aged mice, supporting the NAD+ depletion hypothesis of neurovascular aging.

Adjustment of cerebral blood flow (CBF) to neuronal activity via neurovascular coupling (NVC) plays an important role in the maintenance of healthy cognitive function. Strong evidence demonstrates that age-related cerebromicrovascular endothelial dysfunction and consequential impairment of NVC responses contribute importantly to cognitive decline. Recent studies demonstrate that NAD+ availability decreases with age in the vasculature and that supplemental NAD+ precursors can ameliorate cerebrovascular dysfunction, rescuing NVC responses and improving cognitive performance in aged mice. The mechanisms underlying the age-related decline in [NAD+] in cells of the neurovascular unit are likely multifaceted and may include increased utilization of NAD+ by activated poly (ADP-ribose) polymerase (PARP-1). The present study was designed to test the hypothesis that inhibition of PARP-1 activity may confer protective effects on neurovascular function in aging, similar to the recently demonstrated protective effects of treatment with the NAD+ precursor nicotinamide mononucleotide (NMN). To test this hypothesis, 24-month-old C57BL/6 mice were treated with PJ-34, a potent PARP inhibitor, for 2weeks. 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. Treatment with PJ-34 improved NVC responses by increasing endothelial NO-mediated vasodilation, which was associated with significantly improved spatial working memory. PJ-34 treatment also improved endothelium-dependent acetylcholine-induced relaxation of aorta rings. Thus, PARP-1 activation, likely by decreasing NAD+ availability, contributes to age-related endothelial dysfunction and neurovascular uncoupling, exacerbating cognitive decline. The cerebromicrovascular protective effects of pharmacological inhibition of PARP-1 highlight the preventive and therapeutic potential of treatments that restore NAD+ homeostasis as effective interventions in patients at risk for vascular cognitive impairment (VCI).