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Item does not contain fulltext 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

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.

We provide novel evidence that increased circulating concentrations of the gut microbiome-derived metabolite trimethylamine N-oxide (TMAO) contribute to vascular dysfunction associated with consumption of a Western-style diet and that this dysfunction can be prevented by suppressing TMAO with DMB, thereby supporting translation of this compound to humans. Furthermore, to our knowledge, we present the first evidence of the role of TMAO in mediating impairments in endurance exercise tolerance and increased frailty in any context.

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.

Aging is associated with stiffening of the large elastic arteries and consequent increases in systolic blood pressure (SBP), which together increase cardiovascular disease risk; however, the upstream mechanisms are incompletely understood. Using complementary translational approaches in mice and humans, we investigated the role of the gut microbiome-derived metabolite trimethylamine N-oxide (TMAO) in age-related aortic stiffening and increased SBP. Aortic stiffness was measured using carotid-femoral or aortic pulse wave velocity (PWV) in humans and mice, respectively. Study 1: Plasma TMAO concentrations were elevated ( P r 2 =0.15, P r 2 =0.09, P

Cardiovascular diseases (CVD) are the leading cause of death worldwide, and novel therapies are drastically needed to prevent or delay the onset of CVD to reduce the societal and healthcare burdens associated with these chronic diseases. One such therapy is “heat therapy,” or chronic, repeated use of hot baths or saunas. Although using heat exposure to improve health is not a new concept, it has received renewed attention in recent years as a growing number of studies have demonstrated robust and widespread beneficial effects of heat therapy on cardiovascular health. Here, we review the existing literature, with particular focus on the molecular mechanisms that underscore the cardiovascular benefits of this practice.

Aortic stiffening is a major independent risk factor for cardiovascular diseases, kidney dysfunction, and cognitive impairment. Doxorubicin chemotherapy-treated cancer survivors have greater aortic stiffness relative to healthy controls, but the mechanisms by which doxorubicin induces arterial stiffening are unknown. We tested the hypothesis that doxorubicin increases aortic stiffness by increasing intrinsic mechanical wall stiffness due to proinflammatory signaling-induced adverse structural changes, including collagen deposition (fibrosis), elastin fragmentation, and formation of AGEs (advanced glycation end products). In vivo aortic stiffness (assessed via aortic pulse wave velocity), aortic intrinsic wall stiffness (ex vivo assessment of elastic modulus), and potential underlying mechanisms were assessed 4 weeks after administration of doxorubicin (10 mg/kg) or vehicle (saline) in young adult male C57BL6/J mice. Aortic pulse wave velocity increased by ~30% following doxorubicin (pre: 341±18 versus post: 431±28 cm/s, mean±SEM, P =0.001) and aortic elastic modulus was ~100% higher following doxorubicin (5438±445 kPa) versus vehicle (2659±433 kPa; P =0.003). These effects of doxorubicin were associated with an ~3-fold greater formation of AGEs ( P =0.01) and an ~50% reduction in elastin ( P =0.01), whereas collagen deposition was unaffected. Doxorubicin increased aortic proinflammatory cytokines ( P =0.03) without a compensatory increase in the anti-inflammatory cytokine interleukin-10. Direct ex vivo exposure of aorta rings to doxorubicin mimicked the increase in aortic elastic modulus observed in vivo with doxorubicin, whereas TNF-α (tumor necrosis factor-α) inhibition prevented this response. Doxorubicin induces aortic stiffening in vivo due, in part, to an increase in intrinsic wall stiffness associated with elastin degradation and AGEs formation and mediated by TNF-α-dependent vascular inflammation.

BACKGROUND Doxorubicin (DOXO) chemotherapy increases risk for cardiovascular disease in part by inducing endothelial dysfunction in conduit arteries. However, the mechanisms mediating DOXO-associated endothelial dysfunction in (intact) arteries and treatment strategies are not established. OBJECTIVES We tested the hypothesis that DOXO impairs endothelial function in conduit arteries via excessive mitochondrial reactive oxygen species (ROS) and that these effects could be prevented by treatment with a mitochondrial-targeted antioxidant (MitoQ). METHODS Endothelial function (endothelium-dependent dilation [EDD] to acetylcholine) and vascular mitochondrial ROS were assessed 4 weeks following administration (10 mg/kg intraperitoneal injection) of DOXO. A separate cohort of mice received chronic (4 weeks) oral supplementation with MitoQ (drinking water) for 4 weeks following DOXO. RESULTS EDD in isolated pressurized carotid arteries was 55% lower 4 weeks following DOXO (peak EDD, DOXO: 42 ± 7% vs. sham: 94 ± 3%; p = 0.006). Vascular mitochondrial ROS was 52% higher and manganese (mitochondrial) superoxide dismutase was 70% lower after DOXO versus sham (p = 0.0008). Endothelial function was rescued by administration of the mitochondrial-targeted antioxidant, MitoQ, to the perfusate. Exposure to plasma from DOXO-treated mice increased mitochondrial ROS in cultured endothelial cells. Analyses of plasma showed differences in oxidative stress-related metabolites and a marked reduction in vascular endothelial growth factor A in DOXO mice, and restoring vascular endothelial growth factor A to sham levels normalized mitochondrial ROS in endothelial cells incubated with plasma from DOXO mice. Oral MitoQ supplementation following DOXO prevented the reduction in EDD (97 ± 1%; p = 0.002 vs. DOXO alone) by ameliorating mitochondrial ROS suppression of EDD. CONCLUSIONS DOXO-induced endothelial dysfunction in conduit arteries is mediated by excessive mitochondrial ROS and ameliorated by mitochondrial-specific antioxidant treatment. Mitochondrial ROS is a viable therapeutic target for mitigating arterial dysfunction with DOXO. (J Am Coll Cardiol CardioOnc 2020;2:475–88) © 2020 The Authors. Published by Elsevier on behalf of the American College of Cardiology Foundation.

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 ( r 2 =0.29, P P G -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.

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.

The current study was performed to examine if heat treatment (HT) has beneficial effects on the exaggerated exercise pressor reflex in rats with peripheral artery disease (PAD). We further determined if the temperature-sensitive P2X receptor is involved in the effects of HT. The pressor response to static muscle contraction and α,β-methylene ATP (αβ-me ATP, a P2Xagonist) was examined. Western blot analysis was used to determine the protein levels of P2X(3) in the dorsal root ganglion (DRG), and the whole cell patch clamp was used to examine the amplitude of P2X currents in the DRG neurons. The basal muscle temperature (T(m)) was lower in PAD rats than in control rats. T(m) was increased by ~1.5°C and this increase was maintained for 30 min. This HT protocol was performed tweice daily for three continuous days. A greater blood pressure (BP) response to contraction was observed in PAD rats. HT attenuated the amplification of the BP response in PAD rats. HT also attenuated the enhancement of the BP response induced by the arterial injection of αβ-me ATP in PAD rats. In addition, HT attenuated the upregulation of the P2X(3) and increased P2X currents in the DRG neurons of PAD rats. In conclusion, previous heat exposure plays an inhibitory role in modifying the exaggeration of the exercise pressor reflex in PAD and a reduction of the activity of the P2X receptor pathway is probably a part of mechanisms leading to the beneficial effects of HT.

Rationale: Passive heat therapy improves vascular endothelial function, likely via enhanced nitric oxide (NO) bioavailability, although the mechanistic stimuli driving these changes are unknown. Objective: To determine the isolated effects of circulating (serum) factors on endothelial cell function, particularly angiogenesis, and NO bioavailability. Methods and Results: Cultured human umbilical vein endothelial cells (HUVECs) were exposed to serum collected from 20 healthy young (22 ± 1 years) adults before (0 wk), after one session of water immersion (Acute HT), and after 8 wk of either heat therapy (N = 10; 36 sessions of hot water immersion; session 1 peak rectal temperature: 39.0 ± 0.03°C) or sham (N = 10; 36 sessions of thermoneutral water immersion). Serum collected following acute heat exposure and heat therapy improved endothelial cell angiogenesis (Matrigel bioassay total tubule length per frame, 0 wk: 69.3 ± 1.9 mm vs. Acute HT: 72.8 ± 1.4 mm, p = 0.04; vs. 8 wk: 73.0 ± 1.4 mm, p = 0.03), with no effects of sham serum. Enhanced angiogenesis was NO-mediated, as addition of the NO synthase (NOS) inhibitor L-NNA to the culture media abolished differences in tubule formation across conditions (0 wk: 71.3 ± 1.8 mm, Acute HT: 71.6 ± 1.9 mm, 8 wk: 70.5 ± 1.6 mm, p = 0.69). In separate experiments, we found that abundance of endothelial NOS (eNOS) was unaffected by Acute HT serum (p = 0.71), but increased by 8 wk heat therapy serum (1.4 ± 0.1-fold from 0 wk, p < 0.01). Furthermore, increases in eNOS were related to improvements in endothelial tubule formation (r(2) = 0.61, p < 0.01). Conclusions: Passive heat therapy beneficially alters circulating factors that promote NO-mediated angiogenesis in endothelial cells and increase eNOS abundance. These changes may contribute to improvements in vascular function with heat therapy observed in vivo. Abbreviations: Ang-1: angiopoietin-1; ANOVA: analysis of variance; bFGF: basic fibroblast growth factor; CV: cardiovascular; CVD: cardiovascular diseases; eNOS: endothelial nitric oxide synthase; HSPs: heat shock proteins; HT: heat therapy; HUVECs: human umbilical endothelial cells; L-NNA: Nω-nitro-L-arginine; MnSOD: manganese superoxide dismutase; NO: nitric oxide; NOS: nitric oxide synthase; PBMCs: peripheral blood mononuclear cells; RM: repeated measures; sFlt-1: soluble VEGF receptor; SOD: superoxide dismutase; TGF-β: transforming growth factor- β; VEGF: vascular endothelial growth factor

Aging is associated with declines in cognitive performance, which are mediated in part by neuroinflammation, characterized by astrocyte activation and higher levels of pro-inflammatory cytokines; however, the upstream drivers are unknown. We investigated the potential role of the gut microbiome–derived metabolite trimethylamine N-oxide (TMAO) in modulating neuroinflammation and cognitive function with aging. Study 1: In middle-aged and older humans (65 ± 7 years), plasma TMAO levels were inversely related to performance on NIH Toolbox Cognition Battery tests of memory and fluid cognition (both r(2) = 0.07, p

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.

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.

Aging is associated with vascular endothelial dysfunction, reduced exercise tolerance, and impaired whole‐body glucose metabolism. Interleukin‐37 (IL‐37), an anti‐inflammatory cytokine of the interleukin‐1 family, exerts salutary physiological effects in young mice independent of its inflammation‐suppressing properties. Here, we assess the efficacy of IL‐37 treatment for improving physiological function in older age. Old mice (26–28 months) received daily intraperitoneal injections of recombinant human IL‐37 (recIL‐37; 1 µg/200 ml PBS) or vehicle (200 ml PBS) for 10–14 days. Vascular endothelial function (ex vivo carotid artery dilation to increasing doses of acetylcholine, ACh) was enhanced in recIL‐37 vs. vehicle‐treated mice via increased nitric oxide (NO) bioavailability (all p, Physiological function declines with aging, increasing risk of chronic diseases and disability. We show for the first time that treatment with a novel anti‐inflammatory compound interleukin‐37 (IL‐37) improves multiple physiological functions in old mice, and identify potential roles of reduced superoxide production, improved oxidative metabolism and circulating factors in mediating improvements. Our findings support IL‐37 therapy as a novel strategy for improving diverse physiological functions in old age.

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.

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

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

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.

Age-related increases in aortic stiffness, assessed by pulse wave velocity (PWV), predict cardiovascular (CV)-related mortality, but the upstream drivers are incompletely understood. Purpose: To determine if higher circulating levels of the gut microbiome-derived metabolite trimethylamine-N-oxide (TMAO) contribute to age-related aortic stiffening. Methods and Results: Plasma TMAO concentrations were higher in healthy middle-aged-to-older (45–79 y; N = 83) vs. young (18–27 y; N = 14) humans (6.3 ± 0.6 vs. 1.8 ± 0.3 μM; p < 0.01) and positively related to carotid-femoral (c-f) PWV (r2 = 0.15, p < 0.0001). To determine the role of TMAO in established age-related aortic stiffness, we supplemented old mice (27 mo; N = 12–16/group) with 1% 3,3-dimethyl-1-butanol (DMB; suppresses microbiota-dependent TMAO production) in drinking water for 8–10 weeks vs. normal drinking water (control). Relative to young mice (3 mo; N = 23), old mice had higher aortic (a) PWV (412 ± 17 vs. 349 ± 11 cm/s; p < 0.01), but DMB had no effect on aPWV (p = 0.58 vs. control) despite suppressing plasma TMAO (control: 8.7 ± 6.3 vs. DMB: 4.3 ± 1.2 µM, p = 0.07) to young levels (3.8 ± 2.6 µM). Next, to determine if TMAO contributes to the development of aortic stiffening, we initiated DMB at mid-life (18 mo; i.e., before the onset of stiffening; N = 8–21/age/treatment). aPWV was similar between young and 18 month-old mice (363 ± 5 cm/s; p = 0.58), but increased progressively with age in control mice (24 mo: 401 ± 13 cm/s, p = 0.03 vs. young; 27 mo: 442 ± 10 cm/s, p < 0.001 vs. young), whereas age-related increases in PWV were considerably attenuated by DMB (24 mo: 359 ± 9 cm/s; 27 mo: 388 ± 10 cm/s, both p < 0.01 vs. control). Conclusions: Age-related increases in TMAO contribute to the development of aortic stiffness. TMAO-targeted interventions initiated in mid-life may prevent/delay age-related aortic stiffening and reduce CV risk. Funding: HL134887-02S1, AG060884, HL140875, AG000279.

Cutaneous acetylcholine (ACh)-mediated dilation is commonly used to assess microvascular function, but the mechanisms of dilation are poorly understood. Depending on dose and method of administration, nitric oxide (NO) and prostanoids are involved to varying extents and the roles of endothelial-derived hyperpolarizing factors (EDHFs) are unclear. In the present study, five incremental doses of ACh (0.01-100 mM) were delivered either as a 1-min bolus ( protocol 1, n = 12) or as a ≥20-min continuous infusion ( protocol 2, n = 10) via microdialysis fibers infused with 1) lactated Ringer, 2) tetraethylammonium (TEA) [a calcium-activated potassium channel (KCa) and EDHF inhibitor], 3) L-NNA+ketorolac [NO synthase (NOS) and cyclooxygenase (COX) inhibitors], and 4) TEA+L-NNA+Ketorolac. The hyperemic response was characterized as peak and area under the curve (AUC) cutaneous vascular conductance (CVC) for bolus infusions or plateau CVC for continuous infusions, and reported as %maximal CVC. In protocol 1, TEA, alone and combined with NOS+COX inhibition, attenuated peak CVC (100 mM Ringer 59 ± 6% vs. TEA 43 ± 5%, P < 0.05; L-NNA+ketorolac 35 ± 4% vs. TEA+L-NNA+ketorolac 25 ± 4%, P < 0.05) and AUC (Ringer 25,414 ± 3,528 vs. TEA 21,403 ± 3,416%·s, P < 0.05; L-NNA+ketorolac 25,628 ± 3,828%.s vs. TEA+L-NNA+ketorolac 20,772 ± 3,711%·s, P < 0.05), although these effects were only significant at the highest dose of ACh. At lower doses, TEA lengthened the total time of the hyperemic response (10 mM Ringer 609 ± 78 s vs. TEA 860 ± 67 s, P < 0.05). In protocol 2, TEA alone did not affect plateau CVC, but attenuated plateau in combination with NOS+COX inhibition (100 mM 50.4 ± 6.6% vs. 30.9 ± 6.3%, P < 0.05). Therefore, EDHFs contribute to cutaneous ACh-mediated dilation, but their relative contribution is altered by the dose and infusion procedure.

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 Nω-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.

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 17β-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.

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 NG-nitro-l-arginine methyl ester (l-NAME; NO synthase inhibitor), 3) 10 mM ketorolac (COX inhibitor), and 4) combination of l-NAME + ketorolac. CVCΔpeakand CVCAUCat 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, CVCΔpeakand CVCAUCat 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). CVCΔpeakand CVCAUCwere 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.

The aim of the present study was to determine whether 10 days of repeated local heating could induce peripheral adaptations in the cutaneous vasculature and to investigate potential mechanisms of adaptation. We also assessed maximal forearm blood flow to determine whether repeated local heating affects maximal dilator capacity. Before and after 10 days of heat training consisting of 1-h exposures of the forearm to 42°C water or 32°C water (control) in the contralateral arm (randomized and counterbalanced), we assessed hyperemia to rapid local heating of the skin ( n = 14 recreationally active young subjects). In addition, sequential doses of acetylcholine (ACh, 1 and 10 mM) were infused in a subset of subjects ( n = 7) via microdialysis to study potential nonthermal microvascular adaptations following 10 days of repeated forearm heat training. Skin blood flow was assessed using laser-Doppler flowmetry, and cutaneous vascular conductance (CVC) was calculated as laser-Doppler red blood cell flux divided by mean arterial pressure. Maximal cutaneous vasodilation was achieved by heating the arm in a water-spray device for 45 min and assessed using venous occlusion plethysmography. Forearm vascular conductance (FVC) was calculated as forearm blood flow divided by mean arterial pressure. Repeated forearm heating did not increase plateau percent maximal CVC (CVCmax) responses to local heating (89 ± 3 vs. 89 ± 2% CVCmax, P = 0.19), 1 mM ACh (43 ± 9 vs. 53 ± 7% CVCmax, P = 0.76), or 10 mM ACh (61 ± 9 vs. 85 ± 7% CVCmax, P = 0.37, by 2-way repeated-measures ANOVA). There was a main effect of time at 10 mM ACh ( P = 0.03). Maximal FVC remained unchanged (0.12 ± 0.02 vs. 0.14 ± 0.02 FVC, P = 0.30). No differences were observed in the control arm. Ten days of repeated forearm heating in recreationally active young adults did not improve the microvascular responsiveness to ACh or local heating. NEW & NOTEWORTHY We show for the first time that 10 days of repeated forearm heating is not sufficient to improve cutaneous vascular responsiveness in recreationally active young adults. In addition, this is the first study to investigate cutaneous cholinergic sensitivity and forearm blood flow following repeated local heat exposure. Our data add to the limited studies regarding repeated local heating of the cutaneous vasculature.

Passive heat therapy (repeated hot tub or sauna use) reduces cardiovascular risk, but its effects on the mechanisms underlying improvements in microvascular function have yet to be studied. We investigated the effects of heat therapy on microvascular function and whether improvements were related to changes in nitric oxide (NO) bioavailability using cutaneous microdialysis. Eighteen young, sedentary, otherwise healthy subjects participated in 8 wk of heat therapy (hot water immersion to maintain rectal temperature ≥38.5°C for 60 min/session; n = 9) or thermoneutral water immersion (sham, n = 9), and participated in experiments before and after the 8-wk intervention in which forearm cutaneous hyperemia to 39°C local heating was assessed at three microdialysis sites receiving 1) Lactated Ringer's (Control), 2) Nω-nitro-l-arginine (l-NNA; nonspecific NO synthase inhibitor), and 3) 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl (Tempol), a superoxide dismutase mimetic. The arm used for microdialysis experiments remained out of the water at all times. Data are means ± SE cutaneous vascular conductance (CVC = laser Doppler flux/mean arterial pressure), presented as percent maximal CVC (% CVCmax). Heat therapy increased local heating plateau from 42 ± 6 to 53 ± 6% CVCmax ( P < 0.001) and increased NO-dependent dilation (difference in plateau between Control and l-NNA sites) from 26 ± 6 to 38 ± 4% CVCmax ( P < 0.01), while no changes were observed in the sham group. When data were pooled across all subjects at 0 wk, Tempol had no effect on the local heating response ( P = 0.53 vs. Control). There were no changes at the Tempol site across interventions ( P = 0.58). Passive heat therapy improves cutaneous microvascular function by improving NO-dependent dilation, which may have clinical implications.

While it is accepted that NO is responsible for ∼60% of the plateau in cutaneous thermal hyperaemia, a large portion of the response remains unknown. We sought to determine whether the remaining ∼40% could be attributed to EDHF-mediated activation of KCa channels, and whether the epoxyeicosatrienoic acids (EETs), derived via cytochrome P450, were the predominant EDHF active in the response. Four microdialysis fibres were placed in the forearm skin of 20 subjects. In Protocol 1 (n = 10): (1) Control, (2) N(G)-nitro-l-arginine methyl ester (l-NAME), (3) a KCa channel inhibitor, tetraethylammonium (TEA), and (4) TEA + l-NAME. In Protocol 2 (n = 10): (1) Control, (2) l-NAME, (3) a cytochrome P450 inhibitor, sulfaphenazole, and (4) sulfaphenazole + l-NAME. Local heating to 42°C was performed and skin blood flow was measured with laser Doppler flowmetry. Data are presented as the percentage of maximal cutaneous vascular conductance (CVC). All drug sites attenuated plateau CVC from the control site (86 ± 1%) to 79 ± 3% with sulfaphenazole (P = 0.02 from control), 71 ± 3% with TEA (P = 0.01 from control), and further to 38 ± 2% with l-NAME (P < 0.001 from control, P < 0.001 from TEA). Plateau was largely attenuated with sulfaphenazole + l-NAME (24 ± 2%; P = 0.002 from l-NAME), and nearly abolished with l-NAME + TEA (13 ± 2%; P = 0.001 from sulfaphenazole + l-NAME), which was not different from baseline (P = 0.14). Furthermore, the initial peak was just 17 ± 2% with TEA + l-NAME (P < 0.001 from l-NAME). These data suggest EDHFs are responsible for a large portion of initial peak and the remaining 40% of the plateau phase, as administration of TEA in combination with l-NAME abolished the majority of hyperaemia. These data also suggest EETs contribute to about half of the EDHF response.

Advancing age remains the primary risk factor for cardiovascular diseases (CVD). Given the growing number of older individuals in our society, CVD presents a prevalent and burdensome consequence This article is protected by copyright. All rights reserved

Please cite this paper as: Brunt, Miner, Meendering, Kaplan, and Minson (2011). 17β-Estradiol and Progesterone Independently Augment Cutaneous Thermal Hyperemia But Not Reactive Hyperemia. Microcirculation 18(5), 347–355. Abstract Objective: We examined the impact of estradiol and progesterone on skin LH and RH in 25 healthy women. Methods: Subjects were studied three times over 10–12 days. Endogenous sex hormones were suppressed with a GnRHa. Subjects were studied on day 4 of suppression (study day 1), three to four days later following treatment with either 17β-estradiol or progesterone (study day 2), and another three to four days later, following treatment with both estradiol and progesterone (study day 3). Subjects underwent identical LH and RH protocols on all study days. LH is characterized by an initial peak in blood flow, followed by a prolonged plateau. A brief nadir is seen between the phases. Results: Blood flow values are expressed as percent maximum CVC. Estradiol alone increased initial peak CVC from 71 ± 2% to 79 ± 2% (p = 0.001). Progesterone alone increased initial peak CVC from 72 ± 2% to 78 ± 2% (p = 0.046). Neither estradiol nor progesterone increased plateau CVC. No significant changes were seen between study days 2 and 3 for either group. No differences were observed in RH. Conclusions: Both estradiol and progesterone increased initial peak CVC during LH, without altering plateau CVC. There was no additive effect of estradiol and progesterone.

Nitroxide small molecule agents are in development as preventative or therapeutic pharmaceutical drugs for age-related macular degeneration and cardiovascular disease, which are two major diseases of aging. These aging diseases are associated with patient genetics, smoking, diet, oxidative stress and chronic inflammation. Nitroxide drugs preventing aging-, smoking-, high sugar or high fat diet-, or radiation- and other environmental-induced pathophysiological conditions in aging disease are reviewed. Tempol (TP), Tempol Hydroxylamine (TP-H), and TP-H prodrug (OT-551) are evaluated in (1) nonsmokers versus smokers with cutaneous microvascular dysfunction, rapidly reversed by cutaneous TP; (2) elderly cancer patients at risk for radiation-induced skin burns or hair loss, prevented by topical TP; and (3) elderly smoker or nonsmoker Age-related Macular Degeneration (AMD) patients at risk for vision loss, prevented by daily eye drops of OT-551. The human data indicates safety and efficacy for these nitroxide drugs. Both TP and TP-H topically penetrate and function in skin or mucosa, protecting and treating radiation burns and hair loss or smoking-induced cutaneous vascular dysfunction. TP and TP-H do not penetrate the cornea, while OT-551 does effectively penetrate and travels to the back of the eye, preserving visual acuity and reducing low luminance deficit and night vision loss in dry AMD smokers and non-smoker patients. Topical, oral or injectable drug formulations are discussed.

The gut microbiome, an emerging mediator of host physiological function, is adversely altered by aging and many diseases, termed “gut dysbiosis.” One consequence of gut dysbiosis is elevated circulating levels of the gut-derived metabolite trimethylamine N-oxide (TMAO), which has been directly linked to cardiovascular (CV) risk, including the development of atherosclerosis. However, it is unknown whether TMAO mediates arterial dysfunction that precedes the onset of clinical disease, and if so, the underlying mechanisms. Purpose: To determine whether TMAO independently induces large elastic artery stiffening and endothelial dysfunction via increased superoxide-related oxidative stress. Method: Twenty young (6 mo) male C57BL/6 mice were fed a chemically-defined choline (0.08–0.09%) diet supplemented without (Control; N = 9) or with (N = 11) 0.12% TMAO for 6 months. Arterial stiffness was assessed as aortic pulse wave velocity (aPWV). Endothelial function was evaluated ex vivo as carotid artery endothelium-dependent dilation (EDD) to increasing doses of acetylcholine (10−9 to 10−4M) in the absence or presence of the superoxide dismutase mimetic TEMPOL. Results: TMAO increased aPWV (Control: 392 ± 20 vs. TMAO: 483 ± 32 cm/sec, p = 0.04) and impaired EDD (peak dilation, Control: 93.7 ± 3.2 vs. TMAO: 79.9 ± 3.4%, p = 0.01). Suppression of oxidative stress with TEMPOL restored EDD in TMAO-treated animals (peak dilation: 92.1 ± 4.7%, p = 0.46 vs. Control). Conclusions: TMAO independently induces large elastic artery stiffening and endothelial dysfunction in mice. Dysfunction appears to occur through increases in oxidative stress. These data may explain, at least in part, why TMAO increases CV risk and provide a potential target for prevention/treatment of arterial dysfunction. Supported by R01 HL134887 & T32 HL007822.

Local cutaneous heating produces vasodilation that is largely nitric oxide (NO) dependent. We showed that angiotensin II (ANG II) attenuates this by an ANG II receptor, type 1 (AT1R)-dependent mechanism that is reversible with the antioxidant ascorbate, indicating oxidative stress. Reactive oxygen species (ROS) produced by ANG II employ NADPH and xanthine oxidase pathways. To determine whether these mechanisms pertain to skin, we measured cutaneous local heating with 10 μM ANG II, using apocynin to inhibit NADPH oxidase and allopurinol to inhibit xanthine oxidase. We also inhibited superoxide with tempol, and H2O2 with ebselen. We heated the skin of the calf in 8 healthy volunteers (24.5–29.9 yr old) to 42°C and measured local blood flow to assess the percentage of maximum cutaneous vascular conductance. We remeasured while perfusing allopurinol, apocynin, ebselen, and tempol through individual microdialysis catheters. This was then repeated with ANG II combined with antioxidant drugs. tempol and apocynin alone had no effect on the heat response. Allopurinol enhanced the entire response (125% of heat alone), while ebselen suppressed the heat plateau (76% of heat alone). ANG II alone caused significant attenuation of the entire heat response (52%). When added to ANG II, Allopurinol partially reversed the ANG II attenuation. Heat with ebselen and ANG II were similar to heat and ANG II; ebselen only partially reversed the ANG II attenuation. Apocynin and tempol each partially reversed the attenuation caused by ANG II. This suggests that ROS, produced by ANG II via NADPH and xanthine oxidase pathways, modulates the response of skin to the application of heat, and thus contributes to the control of local cutaneous blood flow.

Cutaneous hyperemia in response to rapid skin local heating to 42°C has been used extensively to assess microvascular function. However, the response is dependent on both nitric oxide (NO) and endothelial-derived hyperpolarizing factors (EDHFs), and increases cutaneous vascular conductance (CVC) to ∼90–95% maximum in healthy subjects, preventing the study of potential means to improve cutaneous function. We sought to identify an improved protocol for isolating NO-dependent dilation. We compared nine heating protocols (combinations of three target temperatures: 36°C, 39°C, and 42°C, and three rates of heating: 0.1°C/s, 0.1°C/10 s, 0.1°C/min) in order to select two protocols to study in more depth ( protocol 1; N = 6). Then, CVC was measured at four microdialysis sites receiving: 1) lactated Ringer solution (Control), 2) 50-mM tetraethylammonium (TEA) to inhibit EDHFs, 3) 20-mM nitro-L-arginine methyl ester (L-NAME) to inhibit NO synthase, and 4) TEA+L-NAME, in response to local heating either to 39°C at 0.1°C/s ( protocol 2; N = 10) or 42°C at 0.1°C/min ( protocol 3; N = 8). Rapid heating to 39°C increased CVC to 43.1 ± 5.2%CVCmax (Control), which was attenuated by L-NAME (11.4 ± 2.8%CVCmax; P < 0.001) such that 82.8 ± 4.2% of the plateau was attributable to NO. During gradual heating, 81.5 ± 3.3% of vasodilation was attributable to NO at 40°C, but at 42°C only 32.7 ± 7.8% of vasodilation was attributable to NO. TEA+L-NAME attenuated CVC beyond L-NAME at temperatures >40°C (43.4 ± 4.5%CVCmax at 42°C, P < 0.001 vs. L-NAME), suggesting a role of EDHFs at higher temperatures. Our findings suggest local heating to 39°C offers an improved approach for isolating NO-dependent dilation and/or assessing perturbations that may improve microvascular function.