The gut microbiome as a modulator of arterial function and age-related arterial dysfunction.

The arterial system is integral to the proper function of all other organs and tissues. Arterial function is impaired with aging, and arterial dysfunction contributes to the development of numerous age-related diseases, including cardiovascular diseases. The gut microbiome has emerged as an important regulator of both normal host physiological function and impairments in function with aging. The purpose of this review is to summarize more recently published literature demonstrating the role of the gut microbiome in supporting normal arterial development and function and in modulating arterial dysfunction with aging in the absence of overt disease. The gut microbiome can be altered due to a variety of exposures, including physiological aging processes. We explore mechanisms by which the gut microbiome may contribute to age-related arterial dysfunction, with a focus on changes in various gut microbiome-related compounds in circulation. In addition, we discuss how modulating circulating levels of these compounds may be a viable therapeutic approach for improving artery function with aging. Finally, we identify and discuss various experimental considerations and research gaps/areas of future research.

Role of the circulating milieu in age-related arterial dysfunction: a novel ex vivo approach.

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

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.

Apigenin restores endothelial function by ameliorating oxidative stress, reverses aortic stiffening, and mitigates vascular inflammation with aging.

We assessed the efficacy of oral supplementation with the flavanoid apigenin on arterial function during aging and identified critical mechanisms of action. Young (6 mo) and old (27 mo) C57BL/6N mice (model of arterial aging) consumed drinking water containing vehicle (0.2% carboxymethylcellulose; 10 young and 7 old) or apigenin (0.5 mg/mL in vehicle; 10 young and 9 old) for 6 wk. In vehicle-treated animals, isolated carotid artery endothelium-dependent dilation (EDD), bioassay of endothelial function, was impaired in old versus young (70% ± 9% vs. 92% ± 1%, P < 0.0001) due to reduced nitric oxide (NO) bioavailability. Old mice had greater arterial reactive oxygen species (ROS) production and oxidative stress (higher nitrotyrosine) associated with greater nicotinamide adenine dinucleotide phosphate oxidase (oxidant enzyme) and lower superoxide dismutase 1 and 2 (antioxidant enzymes); ex vivo administration of Tempol (antioxidant) restored EDD to young levels, indicating ROS-mediated suppression of EDD. Old animals also had greater aortic stiffness as indicated by higher aortic pulse wave velocity (PWV, 434 ± 9 vs. 346 ± 5 cm/s, P < 0.0001) due to greater intrinsic aortic wall stiffness associated with lower elastin levels and higher collagen, advanced glycation end products (AGEs), and proinflammatory cytokine abundance. In old mice, apigenin restored EDD (96% ± 2%) by increasing NO bioavailability, normalized arterial ROS, oxidative stress, and antioxidant expression, and abolished ROS inhibition of EDD. Moreover, apigenin prevented foam cell formation in vitro (initiating step in atherosclerosis) and mitigated age-associated aortic stiffening (PWV 373 ± 5 cm/s) by normalizing aortic intrinsic wall stiffness, collagen, elastin, AGEs, and inflammation. Thus, apigenin is a promising therapeutic for arterial aging.NEW & NOTEWORTHY Our study provides novel evidence that oral apigenin supplementation can reverse two clinically important indicators of arterial dysfunction with age, namely, vascular endothelial dysfunction and large elastic artery stiffening, and prevents foam cell formation in an established cell culture model of early atherosclerosis. Importantly, our results provide extensive insight into the biological mechanisms of apigenin action, including increased nitric oxide bioavailability, normalization of age-related increases in arterial ROS production and oxidative stress, reversal of age-associated aortic intrinsic mechanical wall stiffening and adverse remodeling of the extracellular matrix, and suppression of vascular inflammation. Given that apigenin is commercially available as a dietary supplement in humans, these preclinical findings provide the experimental basis for future translational studies assessing the potential of apigenin to treat arterial dysfunction and reduce cardiovascular disease risk with aging.

Brachial and carotid hemodynamic response to hot water immersion in men and women.

This study sought to compare the brachial and carotid hemodynamic response to hot water immersion (HWI) between healthy young men and women. Ten women (W) and 11 men (M) (24 ± 4 yr) completed a 60-min HWI session immersed to the level of the sternum in 40°C water. Brachial and carotid artery hemodynamics (Doppler ultrasound) were measured at baseline (seated rest) and every 15 min throughout HWI. Within the brachial artery, total shear rate was elevated to a greater extent in women [+479 (+364, +594) s-1] than in men [+292 (+222, +361) s-1] during HWI (P = 0.005). As shear rate is inversely proportional to blood vessel diameter and directly proportional to blood flow velocity, the sex difference in brachial shear response to HWI was the result of a smaller brachial diameter among women at baseline (P < 0.0001) and throughout HWI (main effect of sex, P < 0.0001) and a greater increase in brachial velocity seen in women [+48 (+36, +61) cm/s] compared with men [+35 (+27, +43) cm/s] with HWI (P = 0.047) which allowed for a similar increase in brachial blood flow between sexes [M: +369 (+287, +451) mL/min, W: +364 (+243, +486) mL/min, P = 0.943]. In contrast, no differences were seen between sexes in carotid total shear rate, flow, velocity, or diameter at baseline or throughout HWI. These data indicate the presence of an artery-specific sex difference in the hemodynamic response to a single bout of HWI.

Suppression of the gut microbiome ameliorates age-related arterial dysfunction and oxidative stress in mice.

KEY POINTS: Age-related arterial dysfunction, characterized by oxidative stress- and inflammation-mediated endothelial dysfunction and arterial stiffening, is the primary risk factor for cardiovascular diseases. To investigate whether age-related changes in the gut microbiome may mediate arterial dysfunction, we suppressed gut microbiota in young and old mice with a cocktail of broad-spectrum, poorly-absorbed antibiotics in drinking water for 3-4 weeks. In old mice, antibiotic treatment reversed endothelial dysfunction and arterial stiffening and attenuated vascular oxidative stress and inflammation. To provide insight into age-related changes in gut microbiota that may underlie these observations, we show that ageing altered the abundance of microbial taxa associated with gut dysbiosis and increased plasma levels of the adverse gut-derived metabolite trimethylamine N-oxide. The results of the present study provide the first proof-of-concept evidence that the gut microbiome is an important mediator of age-related arterial dysfunction and therefore may be a promising therapeutic target for preserving arterial function with ageing, thereby reducing the risk of cardiovascular diseases. ABSTRACT: Oxidative stress-mediated arterial dysfunction (e.g. endothelial dysfunction and large elastic artery stiffening) is the primary mechanism driving age-related cardiovascular diseases. Accumulating evidence suggests the gut microbiome modulates host physiology because dysregulation ('gut dysbiosis') has systemic consequences, including promotion of oxidative stress. The present study aimed to determine whether the gut microbiome modulates arterial function with ageing. We measured arterial function in young and older mice after 3-4 weeks of treatment with broad-spectrum, poorly-absorbed antibiotics to suppress the gut microbiome. To identify potential mechanistic links between the gut microbiome and age-related arterial dysfunction, we sequenced microbiota from young and older mice and measured plasma levels of the adverse gut-derived metabolite trimethylamine N-oxide (TMAO). In old mice, antibiotics reversed endothelial dysfunction [area-under-the-curve carotid artery dilatation to acetylcholine in young: 345 ± 16 AU vs. old control (OC): 220 ± 34 AU, P < 0.01; vs. old antibiotic-treated (OA): 334 ± 15 AU; P < 0.01 vs. OC] and arterial stiffening (aortic pulse wave velocity in young: 3.62 ± 0.15 m  s-1  vs. OC: 4.43 ± 0.38 m  s-1 ; vs. OA: 3.52 ± 0.35 m  s-1 ; P = 0.03). These improvements were accompanied by lower oxidative stress and greater antioxidant enzyme expression. Ageing altered the abundance of gut microbial taxa associated with gut dysbiosis. Lastly, plasma TMAO was higher with ageing (young: 2.6 ± 0.4 µmol  L-1   vs. OC: 7.2 ± 2.0 µmol  L-1 ; P < 0.0001) and suppressed by antibiotic treatment (OA: 1.2 ± 0.2 µmol  L-1 ; P < 0.0001 vs. OC). The results of the present study provide the first evidence for the gut microbiome being an important mediator of age-related arterial dysfunction and oxidative stress and suggest that therapeutic strategies targeting gut microbiome health may hold promise for preserving arterial function and reducing cardiovascular risk with ageing in humans.

Heat therapy reduces sympathetic activity and improves cardiovascular risk profile in women who are obese with polycystic ovary syndrome.

Polycystic ovary syndrome (PCOS) affects up to 15% of women and is associated with increased risk of obesity and cardiovascular disease. Repeated passive heat exposure [termed "heat therapy" (HT)] is a lifestyle intervention with the potential to reduce cardiovascular risk in obesity and PCOS. Women with obesity (n = 18) with PCOS [age 27 ± 4 yr, body mass index (BMI) 41.3 ± 4.7 kg/m2] were matched for age and BMI, then assigned to HT (n = 9) or time control (CON; n = 9). HT subjects underwent 30 one-hour hot tub sessions over 8-10 wk, whereas CON subjects did not undergo HT. Muscle sympathetic nerve activity (MSNA), blood pressure, cholesterol, C-reactive protein, and markers of vascular function were assessed at the start (Pre) and end (Post) of 8-10 wk. These measures included carotid and femoral artery wall thickness and flow-mediated dilation (FMD), measured both before and after 20 min of ischemia-20 min of reperfusion (I/R) stress. HT subjects exhibited reduced MSNA burst frequency (Pre: 20 ± 8 bursts/min, Post: 13 ± 5 bursts/min, P = 0.012), systolic (Pre: 124 ± 5 mmHg, Post: 114 ± 6 mmHg; P < 0.001) and diastolic blood pressure (Pre: 77 ± 6 mmHg, Post: 68 ± 3 mmHg; P < 0.001), C-reactive protein (Pre: 19.4 ± 13.7 nmol/L, Post: 15.2 ± 12.3 nmol/L; P = 0.018), total cholesterol (Pre: 5.4 ± 1.1 mmol/L, Post: 5.0 ± 0.8 mmol/L; P = 0.028), carotid wall thickness (Pre: 0.054 ± 0.005 cm, Post: 0.044 ± 0.005 cm; P = 0.010), and femoral wall thickness (Pre: 0.056 ± 0.009 cm, Post: 0.042 ± 0.005 cm; P = 0.003). FMD significantly improved in HT subjects over time following I/R (Pre: 5.6 ± 2.5%, Post: 9.5 ± 1.7%; P < 0.001). No parameters changed over time in CON, and BMI did not change in either group. These findings indicate that HT reduces sympathetic nerve activity, provides protection from I/R stress, and substantially improves cardiovascular risk profiles in women who are obese with PCOS.

Passive heat therapy protects against endothelial cell hypoxia-reoxygenation via effects of elevations in temperature and circulating factors.

KEY POINTS: Accumulating evidence indicates that passive heat therapy (chronic use of hot tubs or saunas) has widespread physiological benefits, including enhanced resistance against novel stressors ('stress resistance'). Using a cell culture model to isolate the key stimuli that are likely to underlie physiological adaptation with heat therapy, we showed that both mild elevations in temperature (to 39°C) and exposure to serum from human subjects who have undergone 8 weeks of heat therapy (i.e. altered circulating factors) independently prevented oxidative and inflammatory stress associated with hypoxia-reoxygenation in cultured endothelial cells. Our results elucidate some of the mechanisms (i.e. direct effects of temperature vs. circulating factors) by which heat therapy seems to improve resistance against oxidative and inflammatory stress. Heat therapy may be a promising intervention for reducing cellular damage following ischaemic events, which has broad implications for patients with cardiovascular diseases and conditions characterized by 'chronic' ischaemia (e.g. peripheral artery disease, metabolic diseases, obesity). ABSTRACT: Repeated exposure to passive heat stress ('heat therapy') has widespread physiological benefits, including cellular protection against novel stressors. Increased heat shock protein (HSP) expression and upregulation of circulating factors may impart this protection. We tested the isolated abilities of mild heat pretreatment and serum from human subjects (n = 10) who had undergone 8 weeks of heat therapy to protect against cellular stress following hypoxia-reoxygenation (H/R), a model of ischaemic cardiovascular events. Cultured human umbilical vein endothelial cells were incubated for 24 h at 37°C (control), 39°C (heat pretreatment) or 37°C with 10% serum collected before and after 8 weeks of passive heat therapy (four to five times per week to increase rectal temperature to ≥ 38.5°C for 60 min). Cells were then collected before and after incubation at 1% O2 for 16 h (hypoxia; 37°C), followed by 20% O2 for 4 h (reoxygenation; 37°C) and assessed for markers of cell stress. In control cells, H/R increased nuclear NF-κB p65 protein (i.e. activation) by 106 ± 38%, increased IL-6 release by 37 ± 8% and increased superoxide production by 272 ± 45%. Both heat pretreatment and exposure to heat therapy serum prevented H/R-induced NF-κB activation and attenuated superoxide production; by contrast, only exposure to serum attenuated IL-6 release. H/R also decreased cytoplasmic haemeoxygenase-1 (HO-1) protein (known to suppress NF-κB), in control cells (-25 ± 8%), whereas HO-1 protein increased following H/R in cells pretreated with heat or serum-exposed, providing a possible mechanism of protection against H/R. These data indicate heat therapy is capable of imparting resistance against inflammatory and oxidative stress via direct heat and humoral factors.

Keynote lecture: strategies for optimal cardiovascular aging.

This review summarizes the opening keynote presentation overview of the American Physiological Society Conference on Cardiovascular Aging: New Frontiers and Old Friends held in Westminster, CO, in August 2017. Age is the primary risk factor for cardiovascular diseases (CVDs). Without effective intervention, future increases in the number of older adults will translate to a greater prevalence of CVDs and related disorders. Advancing age increases the risk of CVDs partly via direct effects on the heart and through increases in blood pressure; however, much of the risk is mediated by vascular dysfunction, including large elastic artery stiffening and both macro- and microvascular endothelial dysfunction. Although excessive superoxide-related oxidative stress and chronic low-grade inflammation are the major processes driving cardiovascular aging, the upstream mechanisms involved represent new frontiers of investigation and potential therapeutic targets. Lifestyle practices, including aerobic exercise, energy intake (caloric) restriction, and healthy diet composition, are the most evidence-based strategies (old friends) for optimal cardiovascular aging, but adherence is poor in some groups. Healthy lifestyle "mimicking" approaches, including novel forms of physical training, intermittent fasting paradigms, exercise/healthy diet-inspired nutraceuticals (functional foods and natural supplements), as well as controlled environmental stress exposure (e.g., heat therapy), may hold promise but are unproven. Mitigating the adverse effects of aging on cardiovascular function and health is a high biomedical priority.

Passive heat therapy improves endothelial function, arterial stiffness and blood pressure in sedentary humans.

KEY POINTS: A recent 30 year prospective study showed that lifelong sauna use reduces cardiovascular-related and all-cause mortality; however, the specific cardiovascular adaptations that cause this chronic protection are currently unknown. We investigated the effects of 8 weeks of repeated hot water immersion ('heat therapy') on various biomarkers of cardiovascular health in young, sedentary humans. We showed that, relative to a sham group which participated in thermoneutral water immersion, heat therapy increased flow-mediated dilatation, reduced arterial stiffness, reduced mean arterial and diastolic blood pressure, and reduced carotid intima media thickness, with changes all on par or greater than what is typically observed in sedentary subjects with exercise training. Our results show for the first time that heat therapy has widespread and robust effects on vascular function, and as such, could be a viable treatment option for improving cardiovascular health in a variety of patient populations, particularly those with limited exercise tolerance and/or capabilities. ABSTRACT: The majority of cardiovascular diseases are characterized by disorders of the arteries, predominantly caused by endothelial dysfunction and arterial stiffening. Intermittent hot water immersion ('heat therapy') results in elevations in core temperature and changes in cardiovascular haemodynamics, such as cardiac output and vascular shear stress, that are similar to exercise, and thus may provide an alternative means of improving health which could be utilized by patients with low exercise tolerance and/or capabilities. We sought to comprehensively assess the effects of 8 weeks of heat therapy on biomarkers of vascular function in young, sedentary subjects. Twenty young, sedentary subjects were assigned to participate in 8 weeks (4-5 times per week) of heat therapy (n = 10; immersion in a 40.5°C bath sufficient to maintain rectal temperature ≥ 38.5°C for 60 min per session) or thermoneutral water immersion (n = 10; sham). Eight weeks of heat therapy increased flow-mediated dilatation from 5.6 ± 0.3 to 10.9 ± 1.0% (P < 0.01) and superficial femoral dynamic arterial compliance from 0.06 ± 0.01 to 0.09 ±0.01 mm(2)  mmHg(-1) (P = 0.03), and reduced (i.e. improved) aortic pulse wave velocity from 7.1 ± 0.3 to 6.1 ± 0.3 m s(-1) (P = 0.03), carotid intima media thickness from 0.43 ± 0.01 to 0.37 ± 0.01 mm (P < 0.001), and mean arterial blood pressure from 83 ± 1 to 78 ± 2 mmHg (P = 0.02). No changes were observed in the sham group or for carotid arterial compliance, superficial femoral intima media thickness or endothelium-independent dilatation. Heat therapy improved endothelium-dependent dilatation, arterial stiffness, intima media thickness and blood pressure, indicating improved cardiovascular health. These data suggest heat therapy may provide a simple and effective tool for improving cardiovascular health in various populations.

Cutaneous blood flow during intradermal NO administration in young and older adults: roles for calcium-activated potassium channels and cyclooxygenase?

Nitric oxide (NO) increases cutaneous blood flow; however, the underpinning mechanism(s) remains to be elucidated. We hypothesized that the cutaneous blood flow response during intradermal administration of sodium nitroprusside (SNP, a NO donor) is regulated by calcium-activated potassium (KCa) channels and cyclooxygenase (COX) in young adults. We also hypothesized that these contributions are diminished in older adults given that aging can downregulate KCa channels and reduce COX-derived vasodilator prostanoids. In 10 young (23 ± 5 yr) and 10 older (54 ± 4 yr) adults, cutaneous vascular conductance (CVC) was measured at four forearm skin sites infused with 1) Ringer (Control), 2) 50 mM tetraethylammonium (TEA), a nonspecific KCa channel blocker, 3) 10 mM ketorolac, a nonspecific COX inhibitor, or 4) 50 mM TEA + 10 mM ketorolac via intradermal microdialysis. All skin sites were coinfused with incremental doses of SNP (0.005, 0.05, 0.5, 5, and 50 mM each for 25 min). During SNP administration, CVC was similar at the ketorolac site (0.005-50 mM, all P > 0.05) relative to Control, but lower at the TEA and TEA + ketorolac sites (0.005-0.05 mM, all P < 0.05) in young adults. In older adults, ketorolac increased CVC relative to Control during 0.005-0.05 mM SNP administration (all P < 0.05), but this increase was not observed when TEA was coadministered (all P > 0.05). Furthermore, TEA alone did not modulate CVC during any concentration of SNP administration in older adults (all P > 0.05). We show that during low-dose NO administration (e.g., 0.005-0.05 mM), KCa channels contribute to cutaneous blood flow regulation in young adults; however, in older adults, COX inhibition increases cutaneous blood flow through a KCa channel-dependent mechanism.

Acute hot water immersion is protective against impaired vascular function following forearm ischemia-reperfusion in young healthy humans.

Ischemia-reperfusion (I/R) injury is a primary cause of poor outcomes following ischemic cardiovascular events. We tested whether acute hot water immersion protects against forearm vascular I/R. Ten (5 male, 5 female) young (23 ± 2 yr), healthy subjects participated in two trials in random order 7-21 days apart, involving: 1) 60 min of seated rest (control), or 2) 60 min of immersion in 40.5°C water (peak rectal temperature: 38.9 ± 0.2°C). I/R was achieved 70 min following each intervention by inflating an upper arm cuff to 250 mmHg for 20 min followed by 20 min of reperfusion. Brachial artery flow-mediated dilation (FMD) and forearm postocclusive reactive hyperemia (RH) were measured as markers of macrovascular and microvascular function at three time points: 1) preintervention, 2) 60 min postintervention, and 3) post-I/R. Neither time control nor hot water immersion alone affected FMD (both, P > 0.99). I/R reduced FMD from 7.4 ± 0.7 to 5.4 ± 0.6% (P = 0.03), and this reduction was prevented following hot water immersion (7.0 ± 0.7 to 7.7 ± 1.0%; P > 0.99). I/R also impaired RH (peak vascular conductance: 2.6 ± 0.5 to 2.0 ± 0.4 ml·min-1·mmHg-1, P = 0.003), resulting in a reduced shear stimulus (SRAUC·10-3: 22.5 ± 2.4 to 16.9 ± 2.4, P = 0.04). The post-I/R reduction in peak RH was prevented by hot water immersion (2.5 ± 0.4 to 2.3 ± 0.4 ml·min-1·mmHg-1; P = 0.33). We observed a decline in brachial artery dilator function post-I/R, which may be (partly) related to damage incurred downstream in the microvasculature, as indicated by impaired RH and shear stimulus. Hot water immersion was protective against reductions in FMD and RH post-I/R, suggesting heat stress induces vascular changes consistent with reducing I/R injury following ischemic events.

Tempol improves cutaneous thermal hyperemia through increasing nitric oxide bioavailability in young smokers.

We recently found that young cigarette smokers display cutaneous vascular dysfunction relative to nonsmokers, which is partially due to reduced nitric oxide (NO) synthase (NOS)-dependent vasodilation. In this study, we tested the hypothesis that reducing oxidative stress improves NO bioavailability, enhancing cutaneous vascular function in young smokers. Ten healthy young male smokers, who had smoked for 6.3 ± 0.7 yr with an average daily consumption of 9.1 ± 0.7 cigarettes, were tested. Cutaneous vascular conductance (CVC) during local heating to 42°C at a rate of 0.1°C/s was evaluated as laser-Doppler flux divided by mean arterial blood pressure and normalized to maximal CVC, induced by local heating to 44°C plus sodium nitroprusside administration. We evaluated plateau CVC during local heating, which is known to be highly dependent on NO, at four intradermal microdialysis sites with 1) Ringer solution (control); 2) 10 µM 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl (tempol), a superoxide dismutase mimetic; 3) 10 mM N(ω)-nitro-l-arginine (l-NNA), a nonspecific NOS inhibitor; and 4) a combination of 10 µM tempol and 10 mM l-NNA. Tempol increased plateau CVC compared with the Ringer solution site (90.0 ± 2.3 vs. 77.6 ± 3.9%maximum, P = 0.028). Plateau CVC at the combination site (56.8 ± 4.5%maximum) was lower than the Ringer solution site (P < 0.001) and was not different from the l-NNA site (55.1 ± 4.6%maximum, P = 0.978), indicating the tempol effect was exclusively NO dependent. These data suggest that in young smokers, reducing oxidative stress improves cutaneous thermal hyperemia to local heating by enhancing NO production.

Intermittent supplementation with fisetin improves arterial function in old mice by decreasing cellular senescence.

Cellular senescence and the senescence-associated secretory phenotype (SASP) contribute to age-related arterial dysfunction, in part, by promoting oxidative stress and inflammation, which reduce the bioavailability of the vasodilatory molecule nitric oxide (NO). In the present study, we assessed the efficacy of fisetin, a natural compound, as a senolytic to reduce vascular cell senescence and SASP factors and improve arterial function in old mice. We found that fisetin decreased cellular senescence in human endothelial cell culture. In old mice, vascular cell senescence and SASP-related inflammation were lower 1 week after the final dose of oral intermittent (1 week on-2 weeks off-1 weeks on dosing) fisetin supplementation. Old fisetin-supplemented mice had higher endothelial function. Leveraging old p16-3MR mice, a transgenic model allowing genetic clearance of p16INK4A -positive senescent cells, we found that ex vivo removal of senescent cells from arteries isolated from vehicle- but not fisetin-treated mice increased endothelium-dependent dilation, demonstrating that fisetin improved endothelial function through senolysis. Enhanced endothelial function with fisetin was mediated by increased NO bioavailability and reduced cellular- and mitochondrial-related oxidative stress. Arterial stiffness was lower in fisetin-treated mice. Ex vivo genetic senolysis in aorta rings from p16-3MR mice did not further reduce mechanical wall stiffness in fisetin-treated mice, demonstrating lower arterial stiffness after fisetin was due to senolysis. Lower arterial stiffness with fisetin was accompanied by favorable arterial wall remodeling. The findings from this study identify fisetin as promising therapy for clinical translation to target excess cell senescence to treat age-related arterial dysfunction.

Impaired acetylcholine-induced cutaneous vasodilation in young smokers: roles of nitric oxide and prostanoids.

Cigarette smoking attenuates acetylcholine (ACh)-induced cutaneous vasodilation in humans, but the underlying mechanisms are unknown. We tested the hypothesis that smokers have impaired nitric oxide (NO)- and cyclooxygenase (COX)-dependent cutaneous vasodilation to ACh infusion. Twelve young smokers, who have smoked more than 5.2 ± 0.7 yr with an average daily consumption of 11.4 ± 1.2 cigarettes, and 12 nonsmokers were tested. Age, body mass index, and resting mean arterial pressure were similar between the groups. Cutaneous vascular conductance (CVC) was evaluated as laser-Doppler flux divided by mean arterial pressure, normalized to maximal CVC (local heating to 43.0°C plus sodium nitroprusside administration). We evaluated the increase in CVC from baseline to peak (CVCΔpeak) and area under the curve of CVC (CVCAUC) during a bolus infusion (1 min) of 137.5 µM ACh at four intradermal microdialysis sites: 1) Ringer (control), 2) 10 mM N(G)-nitro-l-arginine methyl ester (l-NAME; NO synthase inhibitor), 3) 10 mM ketorolac (COX inhibitor), and 4) combination of l-NAME + ketorolac. CVCΔpeak and CVCAUC at the Ringer site in nonsmokers were greater than in smokers (CVCΔpeak, 42.9 ± 5.1 vs. 22.3 ± 3.5%max, P < 0.05; and CVCAUC, 8,085 ± 1,055 vs. 3,145 ± 539%max·s, P < 0.05). In nonsmokers, CVCΔpeak and CVCAUC at the l-NAME site were lower than the Ringer site (CVCΔpeak, 29.5 ± 6.2%max, P < 0.05; and CVCAUC, 5,377 ± 1,109%max·s, P < 0.05), but in smokers, there were no differences between the Ringer and l-NAME sites (CVCΔpeak, 16.8 ± 4.3%max, P = 0.11; and CVCAUC, 2,679 ± 785%max·s, P = 0.30). CVCΔpeak and CVCAUC were reduced with ketorolac in nonsmokers (CVCΔpeak, 13.3 ± 3.6%max, P < 0.05; and CVCAUC, 1,967 ± 527%max·s, P < 0.05) and smokers (CVCΔpeak, 7.8 ± 1.8%max, P < 0.05; and CVCAUC, 1,246 ± 305%max·s, P < 0.05) and at the combination site in nonsmokers (CVCΔpeak, 15.9 ± 3.1%max, P < 0.05; and CVCAUC, 2,660 ± 512%max·s, P < 0.05) and smokers (CVCΔpeak, 11.5 ± 2.6%max, P < 0.05; and CVCAUC, 1,693 ± 409%max·s, P < 0.05), but the magnitudes were greater in nonsmokers (P < 0.05). These results suggest that impaired ACh-induced skin vasodilation in young smokers is related to diminished NO- and COX-dependent vasodilation.

Short-term administration of progesterone and estradiol independently alter carotid-vasomotor, but not carotid-cardiac, baroreflex function in young women.

The individual effects of estrogen and progesterone on baroreflex function remain poorly understood. We sought to determine how estradiol (E2) and progesterone (P4) independently alter the carotid-cardiac and carotid-vasomotor baroreflexes in young women by using a hormone suppression and exogenous add-back design. Thirty-two young women were divided into two groups and studied under three conditions: 1) after 4 days of endogenous hormone suppression with a gonadotropin releasing hormone antagonist (control condition), 2) after continued suppression and 3 to 4 days of supplementation with either 200 mg/day oral progesterone (N = 16) or 0.1 to 0.2 mg/day transdermal 17ß-estradiol (N = 16), and 3) after continued suppression and 3 to 4 days of supplementation with both hormones. Changes in heart rate (HR), mean arterial pressure (MAP), and femoral vascular conductance (FVC) were measured in response to 5 s of +50 mmHg external neck pressure to unload the carotid baroreceptors. Significant hormone effects on the change in HR, MAP, and FVC from baseline at the onset of neck pressure were determined using mixed model covariate analyses accounting for P4 and E2 plasma concentrations. Neither P4 (P = 0.95) nor E2 (P = 0.95) affected the HR response to neck pressure. Higher P4 concentrations were associated with an attenuated fall in FVC (P = 0.01), whereas higher E2 concentrations were associated with an augmented fall in FVC (P = 0.02). Higher E2 was also associated with an augmented rise in MAP (P = 0.01). We conclude that progesterone blunts whereas estradiol enhances carotid-vasomotor baroreflex sensitivity, perhaps explaining why no differences in sympathetic baroreflex sensitivity are commonly reported between low and high combined hormone phases of the menstrual cycle.

Circulating interleukin-37 declines with aging in healthy humans: relations to healthspan indicators and IL37 gene SNPs.

Aging is characterized by declines in physiological function that increase risk of age-associated diseases and limit healthspan, mediated in part by chronic low-grade inflammation. Interleukin (IL)-37 suppresses inflammation in pathophysiological states but has not been studied in the context of aging in otherwise healthy humans. Thus, we investigated associations between IL-37 and markers of healthspan in 271 young (18-39 years; n = 41), middle-aged (40-64 years; n = 162), and older (65 + years; n = 68) adults free of overt clinical disease. After conducting a thorough validation of AdipoGen's IL-37 ELISA, we found that plasma IL-37 is lower in older adults (young: 339 ± 240, middle-aged: 345 ± 234; older: 258 ± 175 pg/mL; P = 0.048), despite elevations in pro-inflammatory markers. As such, the ratios of circulating IL-37 to pro-inflammatory markers were considerably lower in older adults (e.g., IL-37 to C-reactive protein: young, 888 ± 918 vs. older, 337 ± 293; P = 0.02), indicating impaired IL-37 responsiveness to a pro-inflammatory state with aging and consistent with the notion of immunosenescence. These ratios were related to multiple indicators of healthspan, including positively to cardiorespiratory fitness (P < 0.01) and negatively to markers of adiposity, blood pressure, and blood glucose (all P < 0.05). Lastly, we correlated single-nucleotide polymorphisms (SNPs) in the IL37 and ILR8 (the co-receptor for IL-37) genes and found that variants in IL37 SNPs tended to be associated with blood pressure and adiposity (P = 0.08-0.09) but did not explain inter-individual variability in circulating IL-37 concentrations across age (P ≥ 0.23). Overall, our findings provide novel insights into a possible role of IL-37 in biological aging in humans.