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.

Effects of Combined Inorganic Nitrate and Nitrite Supplementation on Cardiorespiratory Fitness and Skeletal Muscle Oxidative Capacity in Type 2 Diabetes: A Pilot Randomized Controlled Trial.

Nitric oxide (NO) stimulates mitochondrial biogenesis in skeletal muscle. However, NO metabolism is disrupted in individuals with type 2 diabetes mellitus (T2DM) potentially contributing to their decreased cardiorespiratory fitness (i.e., VO2max) and skeletal muscle oxidative capacity. We used a randomized, double-blind, placebo-controlled, 8-week trial with beetroot juice containing nitrate (NO3−) and nitrite (NO2−) (250 mg and 20 mg/day) to test potential benefits on VO2max and skeletal muscle oxidative capacity in T2DM. T2DM (N = 36, Age = 59 ± 9 years; BMI = 31.9 ± 5.0 kg/m2) and age- and BMI-matched non-diabetic controls (N = 15, Age = 60 ± 9 years; BMI = 29.5 ± 4.6 kg/m2) were studied. Mitochondrial respiratory capacity was assessed in muscle biopsies from a subgroup of T2DM and controls (N = 19 and N = 10, respectively). At baseline, T2DM had higher plasma NO3− (100%; p < 0.001) and lower plasma NO2− levels (−46.8%; p < 0.0001) than controls. VO2max was lower in T2DM (−26.4%; p < 0.001), as was maximal carbohydrate- and fatty acid-supported oxygen consumption in permeabilized muscle fibers (−26.1% and −25.5%, respectively; p < 0.05). NO3−/NO2− supplementation increased VO2max (5.3%; p < 0.01). Further, circulating NO2−, but not NO3−, positively correlated with VO2max after supplementation (R2= 0.40; p < 0.05). Within the NO3−/NO2− group, 42% of subjects presented improvements in both carbohydrate- and fatty acid-supported oxygen consumption in skeletal muscle (vs. 0% in placebo; p < 0.05). VO2max improvements in these individuals tended to be larger than in the rest of the NO3−/NO2− group (1.21 ± 0.51 mL/(kg*min) vs. 0.31 ± 0.10 mL/(kg*min); p = 0.09). NO3−/NO2− supplementation increases VO2max in T2DM individuals and improvements in skeletal muscle oxidative capacity appear to occur in those with more pronounced increases in VO2max.

Dietary Inorganic Nitrate/Nitrite Supplementation Reduces Central and Peripheral Blood Pressure in Patients With Type 2 Diabetes Mellitus.

BACKGROUND: Patients with type 2 diabetes mellitus (T2DM) have increased cardiovascular risk due to elevated blood pressure (BP). As low levels of nitric oxide (NO) may contribute to increased BP, we determined if increasing NO bioavailability via eight weeks of supplementation with beetroot juice containing inorganic nitrate/nitrite (4.03 mmol nitrate, 0.29 mmol nitrite) improves peripheral and central BP relative to nitrate/nitrite-depleted beetroot juice. METHODS: Peripheral and central BP were assessed at heart-level in supine subjects using a brachial artery catheter and applanation tonometry, respectively. RESULTS: Nitrate/nitrite supplementation reduced peripheral systolic BP (148 ± 16 to 142 ± 18 mm Hg, P < 0.05) but not placebo (150 ± 19 to 149 ± 17 mm Hg, P = 0.93); however, diastolic BP was unaffected (supplement-by-time P = 0.08). Central systolic BP (131 ± 16 to 127 ± 17 mm Hg) and augmented pressure (13.3 ± 6.6 to 11.6 ± 6.9 mm Hg, both P < 0.05) were reduced after nitrate/nitrite, but not placebo (134 ± 17 to 135 ± 16 mm Hg, P = 0.62; 14.1 ± 6.6 to 15.2 ± 7.4 mm Hg, P = 0.20); central diastolic BP was unchanged by the interventions (supplement-by-time P = 0.16). Inorganic nitrate/nitrite also reduced AIx (24.3 ± 9.9% to 21.0 ± 9.6%) whereas no changes were observed following placebo (24.6 ± 9.3% to 25.6 ± 9.9%, P = 0.46). CONCLUSIONS: Inorganic nitrate/nitrite supplementation improves peripheral and central BP as well as AIx in T2DM. CLINICAL TRIALS REGISTRATION: Trial Number NCT02804932.

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.

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.

Annexin A6 and NPC1 regulate LDL-inducible cell migration and distribution of focal adhesions.

Cholesterol is considered indispensable for cell motility, but how physiological cholesterol pools enable cells to move forward remains to be clarified. The majority of cells obtain cholesterol from the uptake of Low-Density lipoproteins (LDL) and here we demonstrate that LDL stimulates A431 squamous epithelial carcinoma and Chinese hamster ovary (CHO) cell migration and invasion. LDL also potentiated epidermal growth factor (EGF) -stimulated A431 cell migration as well as A431 invasion in 3-dimensional environments, using organotypic assays. Blocking cholesterol export from late endosomes (LE), using Niemann Pick Type C1 (NPC1) mutant cells, pharmacological NPC1 inhibition or overexpression of the annexin A6 (AnxA6) scaffold protein, compromised LDL-inducible migration and invasion. Nevertheless, NPC1 mutant cells established focal adhesions (FA) that contain activated focal adhesion kinase (pY397FAK, pY861FAK), vinculin and paxillin. Compared to controls, NPC1 mutants display increased FA numbers throughout the cell body, but lack LDL-inducible FA formation at cell edges. Strikingly, AnxA6 depletion in NPC1 mutant cells, which restores late endosomal cholesterol export in these cells, increases their cell motility and association of the cholesterol biosensor D4H with active FAK at cell edges, indicating that AnxA6-regulated transport routes contribute to cholesterol delivery to FA structures, thereby improving NPC1 mutant cell migratory behaviour.

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.

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.

A non-genetic, cell cycle-dependent mechanism of platinum resistance in lung adenocarcinoma.

We previously used a pulse-based in vitro assay to unveil targetable signalling pathways associated with innate cisplatin resistance in lung adenocarcinoma (Hastings et al., 2020). Here, we advanced this model system and identified a non-genetic mechanism of resistance that drives recovery and regrowth in a subset of cells. Using RNAseq and a suite of biosensors to track single-cell fates both in vitro and in vivo, we identified that early S phase cells have a greater ability to maintain proliferative capacity, which correlated with reduced DNA damage over multiple generations. In contrast, cells in G1, late S or those treated with PARP/RAD51 inhibitors, maintained higher levels of DNA damage and underwent prolonged S/G2 phase arrest and senescence. Combined with our previous work, these data indicate that there is a non-genetic mechanism of resistance in human lung adenocarcinoma that is dependent on the cell cycle stage at the time of cisplatin exposure.

Glycemic management is inversely related to skeletal muscle microvascular endothelial function in patients with type 2 diabetes.

Microvascular endothelial dysfunction precipitates cardiovascular disease mortality in patients with type 2 diabetes mellitus (T2DM). However, the relationship between glycemic management and microvascular endothelial function of these patients remains ill defined. We investigated the association between skeletal muscle microvascular endothelial function with glycemic management (HbA1c) and responses to an oral glucose challenge (OGTT) in 30 patients with T2DM (59 ± 9 years, 31.2 ± 5.1 kg/m2 , HbA1c = 7.3 ± 1.3%). On study day 1, microvascular endothelial function was quantified as the increase (Δ from rest) in forearm vascular conductance (FVC, ml/min/100 mmHg) during intra-arterial acetylcholine infusion at 4.0 and 8.0 µg/dl forearm volume/min (ACh4 and ACh8, respectively). [Glucose] and [insulin] were measured in a fasted state as well as following a 75 g OGTT on a second day with an additional fasting blood sample collected to measure HbA1c. FVC increased (Δ) 221 ± 118 and 251 ± 144 ml/min/100 mm Hg during ACh4 and ACh8 trials, respectively (p < 0.05 between doses). [Glucose] and [insulin] increased at the 1-h time point, relative to fasting levels, and remained elevated 2 h post-consumption (p < 0.05 for both variables and time points). [Glucose] nor [insulin], fasting or during the OGTT, were associated with ΔFVC during ACh4 or ACh8, respectively (p = 0.11-0.86), although HbA1c was inversely related (r = -0.47 and -0.46, respectively, p < 0.01 for both). Patients whose HbA1c met the ADA's therapeutic target of ≤7.0% had greater ΔFVC to ACh4 (272 ± 147 vs. 182 ± 74 ml/100 mm Hg/min) and ACh8 (324 ± 171 vs. 196 ± 90 ml/100 mm Hg/min, p < 0.05 for both trials) compared to those with >7.0%, respectively. Our data show glycemic management is related to acetylcholine-mediated vasodilation (e.g., microvascular endothelial function) in skeletal muscle of patients with T2DM.

Hypertension preserves the magnitude of microvascular flow-mediated dilation following transient elevation in intraluminal pressure.

OBJECTIVE: The objective of this study was to measure flow-mediated dilation (FMD) prior to and following transient increases in intraluminal pressure (IILP) in resistance arterioles isolated from subjects with and without coronary artery disease (CAD) (CAD and non-CAD) and non-CAD subjects with hypertension. METHODS: Arterioles were isolated from discarded surgical tissues (adipose and atrial) from patients without coronary artery disease (non-CAD; ≤1 risk factor, excluding hypertension), with CAD, and non-CAD patients with hypertension (hypertension as the only risk factor). To simulate transient hypertension, increased IILP was generated (150 mmHg, 30 min) by gravity. Arterioles were constricted with endothelin-1, followed by FMD and endothelial-independent dilation prior to and following exposure to IILP. RESULTS: IILP reduced FMD in non-CAD and CAD arterioles relative to pre-IILP (p <.05 at 100 cmH2 O). In contrast, arterioles from non-CAD hypertensive subjects exhibited no reduction in maximal FMD following IILP (p = .84 at 100 cmH2 O). FMD was reduced by L-NAME prior to IILP in non-CAD hypertensive patients (p < .05 at 100 cmH2 O); however, following IILP, FMD was inhibited by peg-cat (p < .05 at 100 cmH2 O), indicating a switch from NO to H2 O2 as the mechanism of dilation. CONCLUSIONS: Acute exposure (30 min) to IILP (150 mmHg) attenuates the magnitude of FMD in non-CAD and CAD resistance arterioles. The presence of clinically diagnosed hypertension in non-CAD resistance arterioles preserves the magnitude of FMD following IILP as a result of a compensatory switch from NO to H2 O2 as the mechanism of dilation.

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.

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.

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.

Effects of Whey Protein Supplementation on Aortic Stiffness, Cerebral Blood Flow, and Cognitive Function in Community-Dwelling Older Adults: Findings from the ANCHORS A-WHEY Clinical Trial.

ANCHORS A-WHEY was a 12-week randomized controlled trial (RCT) designed to examine the effect of whey protein on large artery stiffness, cerebrovascular responses to cognitive activity and cognitive function in older adults. METHODS: 99 older adults (mean ± SD; age 67 ± 6 years, BMI 27.2 ± 4.7kg/m2, 45% female) were randomly assigned to 50g/daily of whey protein isolate (WPI) or an iso-caloric carbohydrate (CHO) control for 12 weeks (NCT01956994). Aortic stiffness was determined as carotid-femoral pulse wave velocity (cfPWV). Aortic hemodynamic load was assessed as the product of aortic systolic blood pressure and heart rate (Ao SBP × HR). Cerebrovascular response to cognitive activity was assessed as change in middle-cerebral artery (MCA) blood velocity pulsatility index (PI) during a cognitive perturbation (Stroop task). Cognitive function was assessed using a computerized neurocognitive battery. RESULTS: cfPWV increased slightly in CHO and significantly decreased in WPI (p < 0.05). Ao SBP × HR was unaltered in CHO but decreased significantly in WPI (p < 0.05). Although emotion recognition selectively improved with WPI (p < 0.05), WPI had no effect on other domains of cognitive function or MCA PI response to cognitive activity (p > 0.05 for all). CONCLUSIONS: Compared to CHO, WPI supplementation results in favorable reductions in aortic stiffness and aortic hemodynamic load with limited effects on cognitive function and cerebrovascular function in community-dwelling 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.

Age-Associated Differences in Central Artery Responsiveness to Sympathoexcitatory Stimuli.

BACKGROUND: Age-associated arterial stiffening may be the result of greater tonic sympathetic nerve activity. However, age-associated changes in central artery responsiveness to sympathoexcitatory stimuli are understudied. Therefore, we examined changes in central artery stiffness and wave reflection in response to sympathoexcitatory stimuli in young and older adults. METHODS: Fourteen young (25 ± 4 years) and 15 older (68 ± 4 years) subjects completed 3 minutes of the cold pressor test (CPT) and lower-body negative pressure (LBNP) separated by 15 minutes. Carotid-femoral pulse wave velocity (cfPWV), central augmentation pressure (cAP), and augmentation index (AIx) were measured in duplicate during rest and the final minute of each perturbation. RESULTS: Young subjects had lower baseline cfPWV, cAP, and AIx than older subjects (P < 0.05 for all). During the CPT mean arterial pressure (MAP), cfPWV, cAP, and AIx increased in both groups (P < 0.05 for all); however, changes (Δ) in MAP (18 ± 7 vs. 9 ± 5 mm Hg), cfPWV (1.3 ± 0.7 vs. 0.6 ± 0.9 m/s), cAP (4 ± 2 vs. 6 ± 3 mm Hg), and AIx (18 ± 9% vs. 7 ± 4%) were greater in young vs. older subjects, respectively (P < 0.05 for all). With MAP as a covariate, cfPWV, cAP, and AIx responses to the CPT were no longer significantly different between groups. During LBNP, changes in MAP (-1 ± 3 vs. -3 ± 5 mm Hg), cfPWV (0.5 ± 0.3 vs. 0.5 ± 0.7 m/s), cAP (-2 ± 2 vs. -2 ± 3 mm Hg), and AIx (-7 ± 7% vs. -3 ± 6%) were similar between young and older groups, respectively (P > 0.05 for all). CONCLUSIONS: Collectively, our data suggest the sympathetic nervous system may directly modulate central hemodynamics and that age-associated differences in central artery responsiveness to sympathoexcitatory stimuli are largely attributable to differential blood pressure responses.