Key pointsr 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.r We investigated the effects of 8 weeks of repeated hot water immersion ('heat therapy') on various biomarkers of cardiovascular health in young, sedentary humans.r 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.r 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. AbstractThe 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.

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 <0.00001). In young mice, 6 months of dietary supplementation with trimethylamine-N-oxide induced an aging-like impairment in carotid artery endothelium-dependent dilation to acetylcholine versus control feeding (peak dilation: 79±3% versus 95±3%, P <0.01). This impairment was accompanied by increased vascular nitrotyrosine, a marker of oxidative stress, and reversed by the superoxide dismutase mimetic 4-hydroxy-2,2,6,6-tetramethylpiperidin-1-oxyl. Trimethylamine-N-oxide supplementation also reduced activation of endothelial nitric oxide synthase and impaired nitric oxide-mediated dilation, as assessed with the nitric oxide synthase inhibitor L-NAME (N 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.

Key pointsr Age-related arterial dysfunction, characterized by oxidative stress-and inflammation-mediated endothelial dysfunction and arterial stiffening, is the primary risk factor for cardiovascular diseases.Vienna E. Brunt received her PhD in Human Physiology from the University of Oregon in 2016. She is currently a postdoctoral fellow in Dr D. R. Seals' Integrative Physiology of Aging Laboratory at the University of Colorado Boulder. The studies described in the present study represent work carried out as part of an NIH T32 fellowship through the Division of Cardiology at the University of Colorado Denver. Her long-term research goals are to investigate the efficacy of novel interventions for preserving vascular function with ageing, thereby preventing and/or delaying the progression of cardiovascular diseases. Rachel A. Gioscia-Ryan completed her PhD in the Integrative Physiology of Aging Laboratory at the University of Colorado Boulder in 2016 and is currently in medical school at the University of Michigan. She is pursuing a career as a clinician-scientist conducting integrative physiological studies with the aim of improving human health and patient care. * These authors contributed equally to this work.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 ...

Key points• The increased blood flow associated with local heating of the skin is ∼60% dependent on nitric oxide. The remaining ∼40% is unknown.• Endothelial-derived hyperpolarizing factors (EDHFs), a class of vasodilators, are known to contribute to increases in blood flow in other vascular beds.• In the present study, we showed the drug tetraethylammonium (which blocks the channels involved in EDHF-mediated vasodilatation), when given in combination with nitric oxide synthase inhibition, blocked the majority of hyperaemia to local heat, indicating that EDHFs are responsible for the majority of the remaining ∼40% of hyperaemia.• We also showed that about half of the EDHF-component is attributed to a specific type of EDHF, epoxyeicosatrienoic acid (EET), as evidenced using the cytochrome P450 inhibitor sulfaphenazole.• These findings help further our understanding of the mechanisms behind cutaneous thermal hyperaemia.Abstract 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

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) N(ω)-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.

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.

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ArticleLJMU has developed LJMU Research Online for users to access the research output of the University more effectively. Copyright © and Moral Rights for the papers on this site are retained by the individual authors and/or other copyright owners. Users may download and/or print one copy of any article(s) in LJMU Research Online to facilitate their private study or for non-commercial research. You may not engage in further distribution of the material or use it for any profit-making activities or any commercial gain.The version presented here may differ from the published version or from the version of the record. Please see the repository URL above for details on accessing the published version and note that access may require a subscription. Email: minson@uoregon.edu Brunt et al. 2016 Hot water immersion and forearm ischemia-reperfusion 2 ABSTRACT 1 Ischemia-reperfusion (I/R) injury is a primary cause of poor outcomes following ischemic 2 cardiovascular events. We tested whether acute hot water immersion protects against forearm following hot water immersion (7.0±0.7 to 7.7±1.0%; p>0.99). I/R also impaired RH (peak other investigators (22, 60), have shown HSP levels to peak in the range of 1-3 h post-heat stress. LJMU Research Online Acute hot water immersion is protective against impaired vascular function following forearm ischemia-reperfusion in young healthy humans 62As this is the first study to investigate whether heat stress is protective against I/R in humans, we 63 chose to study a non-patient population, since certain disease states or elevated risk may alter 64 vascular function responses to interventions and I/R (53). 65We hypothesized that hot water immersion (plus a 60-min recovery period) would Hot water immersion and forearm ischemia-reperfusion 5 METHODS 70 Ethical Approval 71This study was approved by the Institutional Review Board at the University of Oregon. 72Prior to participation, all subjects provided oral and written informed consent as set forth by the for >24 hours, alcohol and caffeine for >12 hours, and heavy exercise for >24 hours prior to each 83 session. Subjects were instructed to eat a light meal no less than 4 hours prior to each session. 84Female subjects were required to demonstrate a negative pregnancy test prior to each study 85 session, measured using urine hCG. 93For each session, subjects arrived at the laboratory and height and weight were recorded. 94Subjects were then instructed to lay supine and were instrumented with a 3-lead Hot water immersion and forearm ischemia-reperfusion 7 duration of the time-control intervention, but could wear additional clothing or were provided 114 with blankets if desired in order to maintain thermal comfort. 115The hot water immersion intervention consisted of 60 minutes immersion in 40.5°C water 116 followed by 40 minutes of seated recovery. Prior to immersion, euhydration was ensured by a 117 first morning urine specific gravity of 1.02, subjects drank 5 mL . kg -1 prior to entering the hot tub. 118Nude body weight was meas...