Lipid phosphate phosphatase 3 maintains NO-mediated flow-mediated dilatation in human adipose resistance arterioles.

Microvascular dysfunction predicts adverse cardiovascular events despite absence of large vessel disease. A shift in the mediator of flow-mediated dilatation (FMD) from nitric oxide (NO) to mitochondrial-derived hydrogen peroxide (H2 O2 ) occurs in arterioles from patients with coronary artery disease (CAD). The underlying mechanisms governing this shift are not completely defined. Lipid phosphate phosphatase 3 (LPP3) is a transmembrane protein that dephosphorylates lysophosphatidic acid, a bioactive lipid, causing a receptor-mediated increase in reactive oxygen species. A single nucleotide loss-of-function polymorphism in the gene coding for LPP3 (rs17114036) is associated with elevated risk for CAD, independent of traditional risk factors. LPP3 is suppressed by miR-92a, which is elevated in the circulation of patients with CAD. Repression of LPP3 increases vascular inflammation and atherosclerosis in animal models. We investigated the role of LPP3 and miR-92a as a mechanism for microvascular dysfunction in CAD. We hypothesized that modulation of LPP3 is critically involved in the disease-associated shift in mediator of FMD. LPP3 protein expression was reduced in left ventricle tissue from CAD relative to non-CAD patients (P = 0.004), with mRNA expression unchanged (P = 0.96). Reducing LPP3 expression (non-CAD) caused a shift from NO to H2 O2 (% maximal dilatation: Control 78.1 ± 11.4% vs. Peg-Cat 30.0 ± 11.2%; P < 0.0001). miR-92a is elevated in CAD arterioles (fold change: 1.9 ± 0.01 P = 0.04), while inhibition of miR-92a restored NO-mediated FMD (CAD), and enhancing miR-92a expression (non-CAD) elicited H2 O2 -mediated dilatation (P < 0.0001). Our data suggests LPP3 is crucial in the disease-associated switch in the mediator of FMD. KEY POINTS: Lipid phosphate phosphatase 3 (LPP3) expression is reduced in heart tissue patients with coronary artery disease (CAD). Loss of LPP3 in CAD is associated with an increase in the LPP3 inhibitor, miR-92a. Inhibition of LPP3 in the microvasculature of healthy patients mimics the CAD flow-mediated dilatation (FMD) phenotype. Inhibition of miR-92a restores nitric oxide-mediated FMD in the microvasculature of CAD patients.

XBP1 maintains beta cell identity, represses beta-to-alpha cell transdifferentiation and protects against diabetic beta cell failure during metabolic stress in mice.

AIMS/HYPOTHESIS: Pancreatic beta cell dedifferentiation, transdifferentiation into other islet cells and apoptosis have been implicated in beta cell failure in type 2 diabetes, although the mechanisms are poorly defined. The endoplasmic reticulum stress response factor X-box binding protein 1 (XBP1) is a major regulator of the unfolded protein response. XBP1 expression is reduced in islets of people with type 2 diabetes, but its role in adult differentiated beta cells is unclear. Here, we assessed the effects of Xbp1 deletion in adult beta cells and tested whether XBP1-mediated unfolded protein response makes a necessary contribution to beta cell compensation in insulin resistance states. METHODS: Mice with inducible beta cell-specific Xbp1 deletion were studied under normal (chow diet) or metabolic stress (high-fat diet or obesity) conditions. Glucose tolerance, insulin secretion, islet gene expression, alpha cell mass, beta cell mass and apoptosis were assessed. Lineage tracing was used to determine beta cell fate. RESULTS: Deletion of Xbp1 in adult mouse beta cells led to beta cell dedifferentiation, beta-to-alpha cell transdifferentiation and increased alpha cell mass. Cell lineage-specific analyses revealed that Xbp1 deletion deactivated beta cell identity genes (insulin, Pdx1, Nkx6.1, Beta2, Foxo1) and derepressed beta cell dedifferentiation (Aldh1a3) and alpha cell (glucagon, Arx, Irx2) genes. Xbp1 deletion in beta cells of obese ob/ob or high-fat diet-fed mice triggered diabetes and worsened glucose intolerance by disrupting insulin secretory capacity. Furthermore, Xbp1 deletion increased beta cell apoptosis under metabolic stress conditions by attenuating the antioxidant response. CONCLUSIONS/INTERPRETATION: These findings indicate that XBP1 maintains beta cell identity, represses beta-to-alpha cell transdifferentiation and is required for beta cell compensation and prevention of diabetes in insulin resistance states.

Critical Interaction Between Telomerase and Autophagy in Mediating Flow-Induced Human Arteriolar Vasodilation.

OBJECTIVE: Coronary artery disease (CAD) is associated with a compensatory switch in mechanism of flow-mediated dilation (FMD) from nitric oxide (NO) to H2O2. The underlying mechanism responsible for the pathological shift is not well understood, and recent reports directly implicate telomerase and indirectly support a role for autophagy. We hypothesize that autophagy is critical for shear stress-induced release of NO and is a crucial component of for the pathway by which telomerase regulates FMD. Approach and Results: Human left ventricular, atrial, and adipose resistance arterioles were collected for videomicroscopy and immunoblotting. FMD and autophagic flux were measured in arterioles treated with autophagy modulators alone, and in tandem with telomerase-activity modulators. LC3B II/I was higher in left ventricular tissue from patients with CAD compared with non-CAD (2.8±0.2 versus 1.0±0.2-fold change; P<0.05), although p62 was similar between groups. Shear stress increased Lysotracker fluorescence in non-CAD arterioles, with no effect in CAD arterioles. Inhibition of autophagy in non-CAD arterioles induced a switch from NO to H2O2, while activation of autophagy restored NO-mediated vasodilation in CAD arterioles. In the presence of an autophagy activator, telomerase inhibitor prevented the expected switch (Control: 82±4%; NG-Nitro-l-arginine methyl ester: 36±5%; polyethylene glycol catalase: 80±3). Telomerase activation was unable to restore NO-mediated FMD in the presence of autophagy inhibition in CAD arterioles (control: 72±7%; NG-Nitro-l-arginine methyl ester: 79±7%; polyethylene glycol catalase: 38±9%). CONCLUSIONS: We provide novel evidence that autophagy is responsible for the pathological switch in dilator mechanism in CAD arterioles, demonstrating that autophagy acts downstream of telomerase as a common denominator in determining the mechanism of FMD.

Sodium nitrate supplementation improves blood pressure reactivity in patients with peripheral artery disease.

BACKGROUND & AIMS: Peripheral artery disease (PAD) is characterized by elevated blood pressure (BP), low nitric oxide availability (NO), and exaggerated pressor responses to sympatho-excitatory stressors. Inorganic nitrate reduces peripheral BP in healthy and chronically diseased populations. The objective of this study was to investigate the effects of eight-weeks of sodium nitrate (NaNO3) supplementation on indices of BP in PAD patients. METHODS: 21 patients with PAD were recruited to participate in this study, undergoing 8-weeks of NaNO3 (n = 13; 73 ± 9 years) or placebo (n = 8; 69 ± 10 years) supplementation. BP responsiveness to a cold pressor test (CPT) were examined prior to and following the supplementation period. The systolic BP response (change from rest) during the first (26 ± 10 vs. 19 ± 11 mmHg) and second minutes (32 ± 10 vs. 26 ± 12 mmHg) of CPT were reduced following NaNO3 (P < 0.05 for both) but not after placebo (first minute: 22 ± 10 vs. 24 ± 10 mmHg, P = 0.30; second minute 26 ± 10 vs 27 ± 10 mmHg, P = 0.72) supplementation. CONCLUSION: Our data suggest that eight-weeks of NaNO3 supplementation reduces BP responsiveness to sympatho-excitatory stimuli. CLINICAL TRIALS REGISTRATION NUMBER: NCT01983826.

Transcriptome profiles of stem-like cells from primary breast cancers allow identification of ITGA7 as a predictive marker of chemotherapy response.

BACKGROUND: Breast cancer stem cells (BCSCs) are drivers of therapy-resistance, therefore are responsible for poor survival. Molecular signatures of BCSCs from primary cancers remain undefined. Here, we identify the consistent transcriptome of primary BCSCs shared across breast cancer subtypes, and we examine the clinical relevance of ITGA7, one of the genes differentially expressed in BCSCs. METHODS: Primary BCSCs were assessed using immunohistochemistry and fluorescently labelled using Aldefluor (n = 17). Transcriptomes of fluorescently sorted BCSCs and matched non-stem cancer cells were determined using RNA-seq (n = 6). ITGA7 expression was examined in breast cancers using immunohistochemistry (n = 305), and its functional role was tested using siRNA in breast cancer cells. RESULTS: Proportions of BCSCs varied from 0 to 9.4%. 38 genes were significantly differentially expressed in BCSCs; genes were enriched for functions in vessel morphogenesis, motility, and metabolism. ITGA7 was found to be significantly downregulated in BCSCs, and low expression significantly correlated with reduced survival in patients treated with chemotherapy, and with chemoresistance in breast cancer cells in vitro. CONCLUSIONS: This study is the first to define the molecular profile of BCSCs from a range of primary breast cancers. ITGA7 acts as a predictive marker for chemotherapy response, in accordance with its downregulation in BCSCs.

Modulation of p66Shc impairs cerebrovascular myogenic tone in low renin but not low nitric oxide models of systemic hypertension.

Cerebral blood flow and perfusion are tightly maintained through autoregulation despite changes in transmural pressure. Oxidative stress impairs cerebral blood flow, precipitating cerebrovascular events. Phosphorylation of the adaptor protein p66Shc increases mitochondrial-derived oxidative stress. The effect of p66Shc gain or loss of function in nonhypertensive rats is unclear. We hypothesized that p66Shc gain of function would impair autoregulation of cerebral microcirculation under physiological and pathological conditions. Three previously established transgenic [salt-sensitive (SS) background] p66Shc rats were used, p66-Del/SS (express p66Shc with a nine-amino acid deletion), p66Shc-knockout (KO)/SS (frameshift premature termination codon), and p66Shc signaling and knock-in substitution of Ser36Ala (p66Shc-S36A)/SS (substitution of Ser36Ala). The p66Shc-Del were also bred on Sprague-Dawley (SD) backgrounds (p66-Del/SD), and a subset was exposed to a hypertensive stimulus [NG-nitro-l-arginine methyl ester (l-NAME)] for 4 wk. Active and passive diameters to increasing transmural pressure were measured and myogenic tone was calculated in all groups (SS and SD). Myogenic responses to increasing pressure were impaired in p66Shc-Del/SS rats relative to wild-type (WT)/SS and knock-in substitution of Ser36Ala (S36A; P < 0.05). p66-Del/SD rats did not demonstrate changes in active/passive diameters or myogenic tone relative to WT/SD but did demonstrate attenuated passive diameter responses to higher transmural pressure relative to p66-Del/SS. Four weeks of a hypertensive stimulus (l-NAME) did not alter active or passive diameter responses to increasing transmural pressure (P = 0.86-0.99), but increased myogenic responses relative to p66-Del/SD (P < 0.05). Collectively, we demonstrate the functional impact of p66Shc within the cerebral circulation and demonstrate that the genetic background of p66Shc rats largely drives changes in cerebrovascular function.NEW & NOTEWORTHY We demonstrate that the modulation of p66Shc signaling impairs cerebral artery myogenic tone in a low renin model of hypertension. This impairment is dependent upon the genetic background, as modulated p66Shc signaling in Sprague-Dawley rats does not impair cerebral artery myogenic tone.

Autophagy, TERT, and mitochondrial dysfunction in hyperoxia.

Ventilation with gases containing enhanced fractions of oxygen is the cornerstone of therapy for patients with hypoxia and acute respiratory distress syndrome. Yet, hyperoxia treatment increases free reactive oxygen species (ROS)-induced lung injury, which is reported to disrupt autophagy/mitophagy. Altered extranuclear activity of the catalytic subunit of telomerase, telomerase reverse transcriptase (TERT), plays a protective role in ROS injury and autophagy in the systemic and coronary endothelium. We investigated interactions between autophagy/mitophagy and TERT that contribute to mitochondrial dysfunction and pulmonary injury in cultured rat lung microvascular endothelial cells (RLMVECs) exposed in vitro, and rat lungs exposed in vivo to hyperoxia for 48 h. Hyperoxia-induced mitochondrial damage in rat lungs [TOMM20, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)], which was paralleled by increased markers of inflammation [myeloperoxidase (MPO), IL-1ß, TLR9], impaired autophagy signaling (Beclin-1, LC3B-II/1, and p62), and decreased the expression of TERT. Mitochondrial-specific autophagy (mitophagy) was not altered, as hyperoxia increased expression of Pink1 but not Parkin. Hyperoxia-induced mitochondrial damage (TOMM20) was more pronounced in rats that lack the catalytic subunit of TERT and resulted in a reduction in cellular proliferation rather than cell death in RLMVECs. Activation of TERT or autophagy individually offset mitochondrial damage (MTT). Combined activation/inhibition failed to alleviate hyperoxic-induced mitochondrial damage in vitro, whereas activation of autophagy in vivo decreased mitochondrial damage (MTT) in both wild type (WT) and rats lacking TERT. Functionally, activation of either TERT or autophagy preserved transendothelial membrane resistance. Altogether, these observations show that activation of autophagy/mitophagy and/or TERT mitigate loss of mitochondrial function and barrier integrity in hyperoxia.NEW & NOTEWORTHY In cultured pulmonary artery endothelial cells and in lungs exposed in vivo to hyperoxia, autophagy is activated, but clearance of autophagosomes is impaired in a manner that suggests cross talk between TERT and autophagy. Stimulation of autophagy prevents hyperoxia-induced decreases in mitochondrial metabolism and sustains monolayer resistance. Hyperoxia increases mitochondrial outer membrane (TOMM20) protein, decreases mitochondrial function, and reduces cellular proliferation without increasing cell death.

Greater α1-adrenergic-mediated vasoconstriction in contracting skeletal muscle of patients with type 2 diabetes.

Patients with type 2 diabetes mellitus (T2DM) exhibit diminished exercise capacity likely attributable to reduced skeletal muscle blood flow (i.e., exercise hyperemia). A potential underlying mechanism of the impaired hyperemic response to exercise could be inadequate blunting of sympathetic-mediated vasoconstriction (i.e., poor functional sympatholysis). Therefore, we studied the hyperemic and vasodilatory responses to handgrip exercise in patients with T2DM as well as vasoconstriction to selective α-agonist infusion. Forearm blood flow (FBF) and vascular conductance (FVC) were examined in patients with T2DM (n = 30) as well as nondiabetic controls (n = 15) with similar age (59 ± 9 vs. 60 ± 9 yr, P = 0.69) and body mass index (31.4 ± 5.2 vs. 29.5 ± 4.6 kg/m2, P = 0.48). Intra-arterial infusion of phenylephrine (α1-agonist) and dexmedetomidine (α2-agonist) were used to induce vasoconstriction: [(FVCwith drug - FVCpredrug)/FVCpredrug × 100%]. Subjects completed rest and dynamic handgrip exercise (20% of maximum) trials per α-agonist. Patients with T2DM had smaller increases (Δ from rest) in FBF (147 ± 71 vs. 199 ± 63 ml/min) and FVC (126 ± 58 vs. 176 ± 50 ml·min-1·100 mmHg-1, P < 0.01 for both) during exercise compared with controls, respectively. During exercise, patients with T2DM had greater α1- (-16.9 ± 5.9 vs. -11.3 ± 3.8%) and α2-mediated vasoconstriction (-23.5 ± 7.1 vs. -19.0 ± 6.5%, P < 0.05 for both) versus controls. The magnitude of sympatholysis (Δ in %vasoconstriction between exercise and rest) for PE was lower (worse) in patients with T2DM versus controls (14.9 ± 12.2 vs. 23.1 ± 8.1%, P < 0.05) whereas groups were similar during DEX trials (24.6 ± 12.3 vs. 27.6 ± 13.4%, P = 0.47). Our data suggest patients with T2DM have attenuated hyperemic and vasodilatory responses to exercise, which could be attributable to greater α1-mediated vasoconstriction in contracting skeletal muscle.NEW & NOTEWORTHY Findings presented in this article are the first to show patients with type 2 diabetes mellitus have blunted hyperemic and vasodilatory responses to dynamic handgrip exercise. Moreover, we illustrate greater α1-adrenergic-mediated vasoconstriction may contribute to our initial observations. Collectively, these data suggest patients with type 2 diabetes may have impaired functional sympatholysis, which can contribute to their reduced exercise capacity.

Vascular autophagy in health and disease.

Homeostasis is maintained within organisms through the physiological recycling process of autophagy, a catabolic process that is intricately involved in the mobilization of nutrients during starvation, recycling of cellular cargo, as well as initiation of cellular death pathways. Specific to the cardiovascular system, autophagy responds to both chemical (e.g. free radicals) and mechanical stressors (e.g. shear stress). It is imperative to note that autophagy is not a static process, and measurement of autophagic flux provides a more comprehensive investigation into the role of autophagy. The overarching themes emerging from decades of autophagy research are that basal levels of autophagic flux are critical, physiological stressors may increase or decrease autophagic flux, and more importantly, aberrant deviations from basal autophagy may elicit detrimental effects. Autophagy has predominantly been examined within cardiac or vascular smooth muscle tissue within the context of disease development and progression. Autophagic flux within the endothelium holds an important role in maintaining vascular function, demonstrated by the necessary role for intact autophagic flux for shear-induced release of nitric oxide however the underlying mechanisms have yet to be elucidated. Within this review, we theorize that autophagy itself does not solely control vascular homeostasis, rather, it works in concert with mitochondria, telomerase, and lipids to maintain physiological function. The primary emphasis of this review is on the role of autophagy within the human vasculature, and the integrative effects with physiological processes and diseases as they relate to the vascular structure and function.

A subset of interleukin-21+ chemokine receptor CCR9+ T helper cells target accessory organs of the digestive system in autoimmunity.

This study describes a CD4+ T helper (Th) cell subset marked by coexpression of the cytokine interleukin 21 (IL-21) and the gut-homing chemokine receptor CCR9. Although CCR9+ Th cells were observed in healthy mice and humans, they were enriched in the inflamed pancreas and salivary glands of NOD mice and in the circulation of Sjögren's syndrome patients. CCR9+ Th cells expressed large amounts of IL-21, inducible T cell costimulator (ICOS), and the transcription factors Bcl6 and Maf, and also supported antibody production from B cells, thereby resembling T follicular B helper (Tfh) cells. However, in contrast to Tfh cells, CCR9+ Th cells displayed limited expression of CXCR5 and the targets of CCR9+ Th cells were CD8+ T cells whose responsiveness to IL-21 was necessary for the development of diabetes. Thus, CCR9+ Th cells are a subset of IL-21-producing T helper cells that influence regional specification of autoimmune diseases that affect accessory organs of the digestive system.

Dietary nitrate does not acutely enhance skeletal muscle blood flow and vasodilation in the lower limbs of older adults during single-limb exercise.

PURPOSE: Blood flow (BF) and vasodilator responses to knee-extension exercise are attenuated in older adults across an exercise transient (onset, kinetics, and steady-state), and reduced nitric oxide bioavailability (NO) has been hypothesized to be a primary mechanism contributing to this attenuation. We tested the hypothesis acute dietary nitrate (NO3-) supplementation (~ 4.03 mmol NO3- and 0.29 mmol NO2-) would improve leg vasodilator responses across an exercise transient during lower limb exercise in older adults. METHODS: Older (n = 10) untrained adults performed single and rhythmic knee-extension contractions at 20% and 40% work-rate maximum (WRmax) prior to and 2-h after consuming a NO3- or placebo beverage in a double-blind, randomized fashion. Femoral artery BF was measured by Doppler ultrasound. Vascular conductance was calculated using BF and mean arterial pressure. RESULTS: Acute ingestion of dietary NO3- enhanced plasma [NO3-] and [NO2-] (P < 0.05). Neither dietary NO3- or placebo enhanced vasodilator responses at the onset of exercise or during steady state at 20% and 40% WRmax (P > 0.05). Leg vasodilator kinetics during rhythmic exercise remained unchanged following NO3- and placebo ingestion (P > 0.05). CONCLUSIONS: The key findings of this study are that despite increasing plasma [NO3-] and [NO2-], acute dietary NO3- intake had no effect on (1) rapid hyperaemic or vasodilator responses at the onset of exercise; (2) hyperaemic and vasodilator responses during steady-state submaximal exercise; or (3) kinetics of vasodilation preceding steady-state responses. Collectively, these findings suggest that low dose dietary NO3- supplementation does not improve hyperaemic and vasodilator responses across an exercise transient in older adults.

Adipocyte-specific deletion of IL-6 does not attenuate obesity-induced weight gain or glucose intolerance in mice.

It has been suggested that interleukin-6 (IL-6) produced by adipocytes in obesity leads to liver insulin resistance, although this hypothesis has never been definitively tested. Accordingly, we did so by generating adipocyte-specific IL-6-deficient (AdipoIL-6-/-) mice and studying them in the context of diet-induced and genetic obesity. Mice carrying two floxed alleles of IL-6 (C57Bl/6J) were crossed with Cre recombinase-overexpressing mice driven by the adiponectin promoter to generate AdipoIL-6-/- mice. AdipoIL-6-/- and floxed littermate controls were fed a standard chow or high-fat diet (HFD) for 16 wk and comprehensively metabolically phenotyped. In addition to a diet-induced obesity model, we also examined the role of adipocyte-derived IL-6 in a genetic model of obesity and insulin resistance by crossing the AdipoIL-6-/- mice with leptin-deficient (ob/ob) mice. As expected, mice on HFD and ob/ob mice displayed marked weight gain and increased fat mass compared with chow-fed and ob/+ (littermate control) animals, respectively. However, deletion of IL-6 from adipocytes in either model had no effect on glucose tolerance or fasting hyperinsulinemia. We concluded that adipocyte-specific IL-6 does not contribute to whole body glucose intolerance in obese mice.

ß-catenin is important for the development of an insulin responsive pool of GLUT4 glucose transporters in 3T3-L1 adipocytes.

GLUT4 is unique among specialized glucose transporters in being exclusively expressed in muscle and adipocytes. In the absence of insulin the distribution of GLUT4 is preferentially intracellular and insulin stimulation results in the movement of GLUT4 containing vesicles to the plasma membrane. This process is responsible for the insulin stimulation of glucose uptake in muscle and fat. While signalling pathways triggering the translocation of GLUT4 are well understood, the mechanisms regulating the intracellular retention of GLUT4 are less well understood. Here we report a role for ß-catenin in this process. In 3T3-L1 adipocytes in which ß-catenin is depleted, the levels of GLUT4 at and near the plasma membrane rise in unstimulated cells while the subsequent increase in GLUT4 at the plasma membrane upon insulin stimulation is reduced. Small molecule approaches to acutely activate or inhibit ß-catenin give results that support the results obtained with siRNA and these changes are accompanied by matching changes in glucose transport into these cells. Together these results indicate that ß-catenin is a previously unrecognized regulator of the mechanisms that control the insulin sensitive pool of GLUT4 transporters inside these adipocyte cells.

Impaired modulation of postjunctional α1 - but not α2 -adrenergic vasoconstriction in contracting forearm muscle of postmenopausal women.

KEY POINTS: Contraction-mediated blunting of postjunctional α-adrenergic vasoconstriction (functional sympatholysis) is attenuated in skeletal muscle of ageing males, brought on by altered postjunctional α1 - and α2 -adrenergic receptor sensitivity. The extent to which postjunctional α-adrenergic vasoconstriction occurs in the forearms at rest and during exercise in postmenopausal women remains unknown. The novel findings indicate that contraction-mediated blunting of α1 - (via intra-arterial infusion of phenylephrine) but not α2 -adrenergic (via intra-arterial infusion of dexmedetomidine) vasoconstriction was attenuated in postmenopausal women compared to young women. Additional important findings revealed that postjunctional α-adrenergic vasoconstrictor responsiveness at rest does not appear to be affected by age in women. Collectively, these results contribute to our understanding of local neurovascular control at rest and during exercise with age in women. ABSTRACT: Contraction-mediated blunting of postjunctional α-adrenergic vasoconstriction (functional sympatholysis) is attenuated in older males; however, direct confirmation of this effect remains unknown in postmenopausal women (PMW). The present study examined whether PMW exhibit augmented postjunctional α-adrenergic receptor vasoconstriction at rest and during forearm exercise compared to young women (YW). Eight YW (24 ± 1 years) and eight PMW (65 ± 1 years) completed a series of randomized experimental trials: (1) at rest, (2) under high flow (adenosine infusion) conditions and (3) during 6 min of forearm exercise at relative (20% of maximum) and absolute (7 kg) intensities. Phenylephrine (α1 -agonist) or dexmedetomidine (α2 -agonist) was administered during the last 3 min of each trial to elicit α-adrenergic vasoconstriction. Forearm vascular conductance (FVC) was calculated from blood flow and blood pressure. Vasoconstrictor responsiveness was identified as the change in FVC (%) during α-adrenergic agonist infusions from baseline (resting trial) or from steady-state conditions (high flow and exercise trials). During resting and high flow trials, the %FVC during α1 - and α2 -agonist stimulation was similar between YW and PMW. During exercise, α1 -mediated vasoconstriction was blunted in YW vs. PMW at relative (-6 ± 2% vs. -15 ± 3%) and absolute (-4 ± 2% vs. -14 ± 5%) workloads, such that blood flow and FVC were lower in PMW (P < 0.05 for all). Conversely, α2 -mediated vasoconstriction was similar between YW and PMW at relative (-22 ± 3% vs. -22 ± 4%; P > 0.05) and absolute (-19 ± 3% vs. -18 ± 4%; P > 0.05) workloads. Collectively, these findings demonstrate that despite similar α-adrenergic vasoconstrictor responsiveness at rest, PMW have a decreased ability to attenuate α1 -adrenergic vasoconstriction in contracting skeletal muscle.

Eight weeks of nitrate supplementation improves blood flow and reduces the exaggerated pressor response during forearm exercise in peripheral artery disease.

Peripheral artery disease (PAD) is characterized by a reduced blood flow (BF) and an elevated blood pressure (pressor) response during lower extremity exercise. Although PAD is evident in the upper extremities, no studies have determined BF and pressor responses during upper extremity exercise in PAD. Emerging evidence suggests that inorganic nitrate supplementation may serve as an alternative dietary strategy to boost nitric oxide bioavailability, improving exercising BF and pressor responses during exercise. The present study investigated 1) BF and pressor responses to forearm exercise in patients with PAD ( n = 21) relative to healthy age-matched control subjects ( n = 16) and 2) whether 8 wk of NaNO3 supplementation influenced BF and pressor responses to forearm exercise in patients with PAD. Patients with moderate to severe PAD were randomly assigned to a NaNO3 (1 g/day, n = 13)-treated group or a placebo (microcrystalline cellulose, n = 8)-treated group. Brachial artery forearm BF (FBF; via Doppler) and blood pressure (via finger plethysmography) were measured during mild-intensity (~3.5-kg) and moderate-intensity (~7-kg) handgrip exercise. The absolute change (from baseline) in FBF was reduced (except in the 3.5-kg condition) and BP responses were increased in patients with PAD compared with healthy control subjects in 3.5- and 7-kg conditions (all P < 0.05). Plasma nitrate and nitrite were elevated, exercising (7-kg) ΔFBF was improved (from 141 ± 17 to 172 ± 20 ml/min), and mean arterial pressure response was reduced (from 13 ± 1 to 9 ± 1 mmHg, P < 0.05) in patients with PAD that received NaNO3 supplementation for 8 wk relative to those that received placebo. These results suggest that the BF limitation and exaggerated pressor response to moderate-intensity forearm exercise in patients with PAD are improved with 8 wk of NaNO3 supplementation. NEW & NOTEWORTHY Peripheral artery disease (PAD) results in an exaggerated pressor response and reduced blood flow during lower limb exercise; however, the effect of PAD in the upper limbs has remained unknown. These results suggest that 8 wk of inorganic nitrate supplementation improves the blood flow limitation and exaggerated pressor response to moderate-intensity forearm exercise in PAD.

Inorganic nitrate supplementation attenuates peripheral chemoreflex sensitivity but does not improve cardiovagal baroreflex sensitivity in older adults.

Aging is associated with increased peripheral chemoreceptor activity, reduced nitric oxide (NO) bioavailability, and attenuation of cardiovagal baroreflex sensitivity (BRS), collectively increasing the risk of cardiovascular disease. Evidence suggests that NO may attenuate peripheral chemoreflex sensitivity and increase BRS. Exogenous inorganic nitrate ([Formula: see text]) increases NO bioavailability via the [Formula: see text]-[Formula: see text]-NO pathway. Our hypothesis was that inorganic [Formula: see text] supplementation would attenuate peripheral chemoreflex sensitivity and enhance spontaneous cardiovagal BRS in older adults. We used a randomized, placebo-controlled crossover design in which 13 older (67 ± 3 yr old) adults ingested beetroot powder containing (BRA) or devoid of (BRP) [Formula: see text] and [Formula: see text] daily over 4 wk. Spontaneous cardiovagal BRS was assessed over 15 min of rest and was quantified using the sequence method. Chemoreflex sensitivity was assessed via ~5 min of hypoxia (10% fraction of inspired O2) and reported as the slope of the relationship between O2 saturation (%[Formula: see text]) and minute ventilation (in l/min) or heart rate (in beats/min). Ventilatory responsiveness to hypoxia was reduced after BRA (from -0.14 ± 0.04 to -0.05 ± 0.02 l·min-1·%[Formula: see text]-1, P = 0.01) versus BRP (from -0.10 ± 0.05 to -0.11 ± 0.05 l·min-1·%[Formula: see text]-1, P = 0.80), with no differences in heart rate responsiveness (BRA: from -0.47 ± 0.06 to -0.33 ± 0.04 beats·min-1·%[Formula: see text]-1, BRP: from -0.48 ± 0.07 to -0.42 ± 0.06 beats·min-1·%[Formula: see text]-1) between conditions (interaction effect, P = 0.41). Spontaneous cardiovagal BRS was unchanged after BRA and BRP (interaction effects, P = 0.69, 0.94, and 0.39 for all, up, and down sequences, respectively), despite a reduction in resting systolic and mean arterial blood pressure in the experimental (BRA) group ( P < 0.01 for both). These findings illustrate that inorganic [Formula: see text] supplementation attenuates peripheral chemoreflex sensitivity without concomitant change in spontaneous cardiovagal BRS in older adults. NEW & NOTEWORTHY Exogenous inorganic nitrate supplementation attenuates ventilatory, but not heart rate, responsiveness to abbreviated hypoxic exposure in older adults. Additionally, inorganic nitrate reduces systolic and mean arterial blood pressure without affecting spontaneous cardiovagal baroreflex sensitivity. These findings suggest that inorganic nitrate may attenuate sympathetically oriented pathologies associated with aging.

Age-associated impairments in contraction-induced rapid-onset vasodilatation within the forearm are independent of mechanical factors.

NEW FINDINGS: What is the central question of this study? We examined whether the mechanical contribution to contraction-induced rapid-onset vasodilatation (ROV) differed with age and whether ROV is associated with peripheral artery stiffness. Furthermore, we examined how manipulation of perfusion pressure modulates ROV in young and older adults. What is the main finding and its importance? The mechanical contribution to ROV is similar in young and older adults. Conversely, peripheral arterial stiffness is not associated with ROV. Enhancing perfusion pressure augments ROV to a similar extent in young and older adults. These results suggest that age-related attenuations in ROV are not attributable to a mechanical component and that ROV responses are independent of peripheral artery stiffness. ABSTRACT: Contraction-induced rapid-onset vasodilatation (ROV) is modulated by perfusion and transmural pressure in young adults; however, this effect remains unknown in older adults. The present study examined the mechanical contribution to ROV in young versus older adults, the influence of perfusion pressure and whether these responses are associated with arterial stiffness. Forearm vascular conductance (in millilitres per minute per 100 mmHg) was measured in 12 healthy young (24 ± 4 years old) and 12 older (67 ± 3 years old) adults during: (i) single dynamic contractions at 20% of maximal voluntary contraction; and (ii) single external mechanical compression of the forearm (200 mmHg) positioned above, at and below heart level. Carotid-radial pulse-wave velocity characterized upper limb arterial stiffness. Total ROV responses to single muscle contractions and single external mechanical compressions were attenuated in older adults at heart level (P < 0.05); however, the relative mechanical contribution to contraction-induced peak (46 ± 14 versus 40 ± 18%; P = 0.21) and total (37 ± 21 versus 32 ± 18%; P = 0.27) responses were not different between young and older adults. Reducing or enhancing perfusion pressure altered ROV responses to a similar extent between young and older adults (P < 0.05). Upper limb arterial stiffness was not associated with peak (r = 0.02; P = 0.93) or total vascular conductance (r = -0.01; P = 0.96) in the group as a whole. Our data suggest that: (i) age-associated attenuations in ROV are not attributable to a mechanical component; (ii) enhancing perfusion pressure augments ROV to a similar extent between young and older adults; and (iii) basal upper limb arterial stiffness is not associated with the vasodilator responses after a single skeletal muscle contraction in young and older adults.

Reduced blood pressure responsiveness to skeletal muscle metaboreflex activation in older adults following inorganic nitrate supplementation.

The vasoactive molecule nitric oxide (NO) contributes to regulation of blood pressure (BP) at rest and during exercise. Age-related exaggerated increased BP responses during exercise have been proposed to be due in part to a decreased NO bioavailability and possibly an enhanced skeletal muscle metaboreflex. In the present study we sought to determine if age-related differences in BP responses to skeletal muscle metaboreflex activation exist. Additionally, since NO bioavailability can be improved with exogenous nitrate (NO3-) via the nitrate-nitrite-NO pathway, we tested the hypothesis that inorganic NO3- supplementation would reduce BP responses to muscle metaboreflex activation in healthy older adults. 13 older adults (67 ±â€¯1 years) participated in a randomized, double-blind, placebo controlled crossover study consisting of four weeks of NO3- supplementation [beetroot powder; 250 mg (∼4.03 mmol) of NO3- and 20 mg (∼0.29 mmol) of NO2-] and four weeks of placebo (beetroot powder devoid of NO3-/NO2-). Skeletal muscle metaboreflex testing consisted of isometric handgrip exercise (IHG) at 30% of maximal voluntary contraction immediately followed by post exercise forearm ischemia (PEI), which was achieved by inflation of a rapid pressure cuff (240 mmHg) around the upper arm. BP responses were analyzed as the change (Δ) from baseline to the end of IHG and PEI. An additional 10 young adults (25 ±â€¯1 years) were recruited to serve as a reference cohort and address if BP responses to skeletal muscle metaboreflex activation were greater with aging. BP responses to IHG were similar between the young and older adults. However, older adults demonstrated a greater increase in systolic BP during PEI (P < 0.05). Plasma NO3- and NO2-were increased following NO3- supplementation in older adults (P < 0.01). ΔSystolic BP (19 ±â€¯2 vs. 13±3 mmHg, P < 0.05), ΔDiastolic BP (7 ±â€¯1 vs. 5±1 mmHg, P < 0.05) and ΔMean arterial pressure (11 ±â€¯1 vs. 8±2 mmHg, P < 0.05) were reduced during PEI following four weeks of NO3-supplementation, whereas placebo had no effect on ΔSystolic BP (16 ±â€¯2 vs. 17±2 mmHg), ΔDiastolic BP (5 ±â€¯1 vs. 7±1 mmHg), and ΔMean arterial pressure (8 ±â€¯1 vs. 10±1 mmHg) during PEI (all P > 0.05). These data suggest that inorganic NO3- supplementation attenuates skeletal muscle metaboreflex mediated increases in BP during exercise in older adults.

Inorganic nitrate supplementation enhances functional capacity and lower-limb microvascular reactivity in patients with peripheral artery disease.

Peripheral artery disease (PAD) is characterized by functional and vascular impairments as well as elevated levels of inflammation which are associated with reduced nitric oxide (NO) bioavailability. Inorganic nitrate supplementation boosts NO bioavailability potentially improving functional and vasodilatory capacities and may reduce inflammation. Twenty-one patients with PAD were randomly assigned to sodium nitrate (NaNO3) or placebo supplementation groups for eight-weeks. Outcome measures included a 6-min walk test (6 MWT), blood flow and vasodilator function in the forearm and calf, as well as plasma inflammatory and adhesion biomarker concentrations. NaNO3 elevated plasma nitrate (32.3 ±â€¯20.0 to 379.8 ±â€¯204.6 µM) and nitrite (192.2 ±â€¯51.8 to 353.1 ±â€¯134.2 nM), improved 6 MWT performance (387 ±â€¯90 to 425 ±â€¯82 m), peak calf blood flow (BFPeak; 11.6 ±â€¯4.9 to 14.1 ±â€¯5.1 mL/dL tissue/min), and peak calf vascular conductance (VCPeak; 11.1 ±â€¯4.3 to 14.2 ±â€¯4.9 mL/dL tissue/min/mmHg) (p < 0.05 for all). Improvements in calf BFPeak (r = 0.70, p < 0.05) and VCPeak (r = 0.61, p < 0.05) correlated with changes in 6 MWT distance. Placebo supplementation did not change plasma nitrate or nitrite, 6 MWT, calf BFPeak, or calf VCPeak. Forearm vascular function nor inflammatory and adhesion biomarker concentrations changed in either group. Eight-weeks of NaNO3 supplementation improves vasodilatory capacity in the lower-limbs of patients with PAD, which correlated with improvement in functional capacity.

An actin filament population defined by the tropomyosin Tpm3.1 regulates glucose uptake.

Actin has an ill-defined role in the trafficking of GLUT4 glucose transporter vesicles to the plasma membrane (PM). We have identified novel actin filaments defined by the tropomyosin Tpm3.1 at glucose uptake sites in white adipose tissue (WAT) and skeletal muscle. In Tpm 3.1-overexpressing mice, insulin-stimulated glucose uptake was increased; while Tpm3.1-null mice they were more sensitive to the impact of high-fat diet on glucose uptake. Inhibition of Tpm3.1 function in 3T3-L1 adipocytes abrogates insulin-stimulated GLUT4 translocation and glucose uptake. In WAT, the amount of filamentous actin is determined by Tpm3.1 levels and is paralleled by changes in exocyst component (sec8) and Myo1c levels. In adipocytes, Tpm3.1 localizes with MyoIIA, but not Myo1c, and it inhibits Myo1c binding to actin. We propose that Tpm3.1 determines the amount of cortical actin that can engage MyoIIA and generate contractile force, and in parallel limits the interaction of Myo1c with actin filaments. The balance between these actin filament populations may determine the efficiency of movement and/or fusion of GLUT4 vesicles with the PM.