Rebecca E. Johansson

Microvascular function in younger adults with obesity and metabolic syndrome: role of oxidative stress

Older adults with cardiovascular disease exhibit microvascular dysfunction and increased levels of reactive oxygen species (ROS). We hypothesized that microvascular impairments begin early in the disease process and can be improved by scavenging ROS. Forearm blood flow (Doppler ultrasound) was measured in 45 young (32 ± 2 yr old) adults (n = 15/group) classified as lean, obese, and metabolic syndrome (MetSyn). Vasodilation in response to endothelial (ACh) and vascular smooth muscle [nitroprusside (NTP) and epoprostenol (Epo)] agonists was tested before and after intra-arterial infusion of ascorbic acid to scavenge ROS. Vasodilation was assessed as a rise in relative vascular conductance (ml·min(-1)·dl(-1)·100 mmHg(-1)). ACh and NTP responses were preserved (P = 0.825 and P = 0.924, respectively), whereas Epo responses were lower in obese and MetSyn adults (P < 0.05) than in lean controls. Scavenging of ROS via infusion of ascorbic acid resulted in an increase in ACh-mediated (P < 0.001) and NTP-mediated (P < 0.001) relative vascular conductance across all groups, suggesting that oxidative stress influences vascular responsiveness in adults with and without overt cardiovascular disease risk. Ascorbic acid had no effect on Epo-mediated vasodilation (P = 0.267). These results suggest that obese and MetSyn adults exhibit preserved endothelium-dependent vasodilation with reduced dependence on prostacyclin and are consistent with an upregulation of compensatory vascular control mechanisms.

β-Adrenergic-mediated vasodilation in young men and women: Cyclooxygenase restrains nitric oxide synthase

We tested the hypothesis that women exhibit greater vasodilator responses to β-adrenoceptor stimulation compared with men. We further hypothesized women exhibit a greater contribution of nitric oxide synthase and cyclooxygenase to β-adrenergic-mediated vasodilation compared with men. Forearm blood flow (Doppler ultrasound) was measured in young men (n = 29, 26 ± 1 yr) and women (n = 33, 25 ± 1 yr) during intra-arterial infusion of isoproterenol (β-adrenergic agonist). In subset of subjects, isoproterenol responses were examined before and after local inhibition of nitric oxide synthase [N(G)-monomethyl-l-arginine (l-NMMA); 6 male/10 female] and/or cyclooxygenase (ketorolac; 5 male/5 female). Vascular conductance (blood flow ÷ mean arterial pressure) was calculated to assess vasodilation. Vascular conductance increased with isoproterenol infusion (P < 0.01), and this effect was not different between men and women (P = 0.41). l-NMMA infusion had no effect on isoproterenol-mediated dilation in men (P > 0.99) or women (P = 0.21). In contrast, ketorolac infusion markedly increased isoproterenol-mediated responses in both men (P < 0.01) and women (P = 0.04) and this rise was lost with subsequent l-NMMA infusion (men, P < 0.01; women, P < 0.05). β-Adrenergic vasodilation is not different between men and women and sex differences in the independent contribution of nitric oxide synthase and cyclooxygenase to β-mediated vasodilation are not present. However, these data are the first to demonstrate β-adrenoceptor activation of cyclooxygenase suppresses nitric oxide synthase signaling in human forearm microcirculation and may have important implications for neurovascular control in both health and disease.

Greater Beta-Adrenergic Receptor Mediated Vasodilation in Women Using Oral Contraceptives

Background: β-adrenergic receptors play an important role in mitigating the pressor effects of sympathetic nervous system activity in young women. Based on recent data showing oral contraceptive use in women abolishes the relationship between muscle sympathetic nervous system activity and blood pressure, we hypothesized forearm blood flow responses to a β-adrenergic receptor agonist would be greater in young women currently using oral contraceptives (OC+, n = 13) when compared to those not using oral contraceptives (OC–, n = 10). Methods: Women (18–35 years) were studied during the early follicular phase of the menstrual cycle (days 1–5) or placebo phase of oral contraceptive use. Forearm blood flow (FBF, Doppler ultrasound) and mean arterial blood pressure (MAP, brachial arterial catheter) were measured at baseline and during graded brachial artery infusion of the β-adrenergic receptor agonist, Isoproterenol (ISO), as well as Acetylcholine (ACH, endothelium-dependent vasodilation) and Nitroprusside (NTP, endothelium-independent vasodilation). Forearm vascular conductance was calculated (FVC = FBF/MAP, ml/min/100 mmHg) and the rise in FVC from baseline during infusion quantified vasodilation (ΔFVC = FVCinfusion − FVCbaseline). Results: ISO increased FVC in both groups (p < 0.01) and ISO-mediated ΔFVC was greater in OC+ compared to OC– (Main effect of group, p = 0.02). Expressing data as FVC and FBF resulted in similar conclusions. FVC responses to both ACH and NTP were also greater in OC+ compared to OC–. Conclusions: These data are the first to demonstrate greater β-adrenergic receptor-mediated vasodilation in the forearm of women currently using oral contraceptives (placebo phase) when compared to those not using oral contraceptives (early follicular phase), and suggest oral contraceptive use influences neurovascular control.

Preserved Microvascular Endothelial Function in Young, Obese Adults with Functional Loss of Nitric Oxide Signaling

Data indicate endothelium-dependent dilation (EDD) may be preserved in the skeletal muscle microcirculation of young, obese adults. Preserved EDD might be mediated by compensatory mechanisms, impeding insight into preclinical vascular dysfunction. We aimed to determine the functional roles of nitric oxide synthase (NOS) and cyclooxygenase (COX) toward EDD in younger obese adults. We first hypothesized EDD would be preserved in young, obese adults. Further, we hypothesized a reduced contribution of NOS in young, obese adults would be replaced by increased COX signaling. Microvascular EDD was assessed with Doppler ultrasound and brachial artery infusion of acetylcholine (ACh) in younger (27 ± 1 year) obese (n = 29) and lean (n = 46) humans. Individual and combined contributions of NOS and COX were examined with intra-arterial infusions of l-NMMA and ketorolac, respectively. Vasodilation was quantified as an increase in forearm vascular conductance (ΔFVC). Arterial endothelial cell biopsies were analyzed for protein expression of endothelial nitric oxide synthase (eNOS). ΔFVC to ACh was similar between groups. After l-NMMA, ΔFVC to ACh was greater in obese adults (p < 0.05). There were no group differences in ΔFVC to ACh with ketorolac. With combined NOS-COX inhibition, ΔFVC was greater in obese adults at the intermediate dose of ACh. Surprisingly, arterial endothelial cell eNOS and phosphorylated eNOS were similar between groups. Younger obese adults exhibit preserved EDD and eNOS expression despite functional dissociation of NOS-mediated vasodilation and similar COX signaling. Compensatory NOS- and COX-independent vasodilatory mechanisms conceal reduced NOS contributions in otherwise healthy obese adults early in life, which may contribute to vascular dysfunction.

Exercise-mediated vasodilation in human obesity and metabolic syndrome: effect of acute ascorbic acid infusion.

We tested the hypothesis that infusion of ascorbic acid (AA), a potent antioxidant, would alter vasodilator responses to exercise in human obesity and metabolic syndrome (MetSyn). Forearm blood flow (FBF, Doppler ultrasound) was measured in lean, obese, and MetSyn adults (n = 39, 32 ± 2 yr). A brachial artery catheter was inserted for blood pressure monitoring and local infusion of AA. FBF was measured during dynamic handgrip exercise (15% maximal effort) with and without AA infusion. To account for group differences in blood pressure and forearm size, and to assess vasodilation, forearm vascular conductance (FVC = FBF/mean arterial blood pressure/lean forearm mass) was calculated. We examined the time to achieve steady-state FVC (mean response time, MRT) and the rise in FVC from rest to steady-state exercise (Δ, exercise - rest) before and during acute AA infusion. The MRT (P = 0.26) and steady-state vasodilator responses to exercise (ΔFVC, P = 0.31) were not different between groups. Intra-arterial infusion of AA resulted in a significant increase in plasma total antioxidant capacity (174 ± 37%). AA infusion did not alter MRT or steady-state FVC in any group (P = 0.90 and P = 0.85, respectively). Interestingly, higher levels of C-reactive protein predicted longer MRT (r = 0.52, P < 0.01) and a greater reduction in MRT with AA infusion (r = -0.43, P = 0.02). We concluded that AA infusion during moderate-intensity, rhythmic forearm exercise does not alter the time course or magnitude of exercise-mediated vasodilation in groups of young lean, obese, or MetSyn adults. However, systemic inflammation may limit the MRT to exercise, which can be improved with AA.

Ageing uncompensated: exercise, nitric oxide and hypoxia.

The vast majority of research comparing skeletal muscle haemodynamics during exercise in young and older adults has been done under normoxic conditions. These studies provided novel insight into regulatory mechanisms of skeletal muscle blood flow, but leave untested circumstances when arterial oxygen saturation may be limited by environmental conditions or medical pathologies. Enhanced vasodilatation in response to hypoxia has been termed compensatory vasodilatation. Compensatory vasodilatation allows for an adequate amount of oxygen to be delivered to the working musculature and is, in part, nitric oxide (NO) dependent (Casey et al. 2010). Ageing is associated with changes in cardiovascular regulation that may alter NO-mediated dilatation (Schrage et al. 2007) and thereby reduce vasodilatory responses to hypoxia during exercise. Any alterations to the regulation of skeletal muscle blood flow may limit functional capacity and impair the ability to perform exercise with ageing. It was previously unknown whether older adults exhibit reduced compensatory vasodilatation or the regulatory mechanisms responsible for any impairment. In a recent article in The Journal of Physiology, Casey et al. (2011) explored the contribution of NO to forearm vasodilatation during exercise in ageing humans under conditions of hypoxia. To test the hypothesis that skeletal muscle compensatory vasodilatation is blunted in ageing humans, Casey et al. (2011) assessed forearm vascular conductance (FVC) in 11 healthy older subjects (55–70 years). FVC was measured under normoxic and hypoxic (80% arterial oxygen saturation) conditions during graded forearm exercise (10% and 20% of maximal effort); data were compared with findings from young adults presented previously (Casey et al. 2010). Results showed hypoxic compensatory vasodilatation (% change in ΔFVC) was maintained in older adults at 10% and absent at 20% effort. These findings suggest attenuated hypoxic vasodilatation in ageing humans presents with increasing exercise intensity. Casey et al. predicted that altered NO signalling in the ageing group contributed to the attenuated compensatory vasodilatation. To test this hypothesis, the group infused l-NMMA at rest to inhibit NO synthesis. In the young adults, l-NMMA reduced compensatory vasodilatation to hypoxic exercise at 10% and 20% effort (Casey et al. 2010); l-NMMA had no effect on compensatory vasodilatation in the older adults. This form of analysis suggests that reduced NO signalling may be responsible for attenuated compensatory vasodilatation with ageing. However, it is important to acknowledge when analysed as a change in FVC (ΔFVC), the older group showed significantly reduced hypoxic FVC with l-NMMA infusion at 10% effort (but not at 20%). Thus, a portion of the increase of FVC at 10% effort in older adults may be due to NO signaling; however, any increase in FVC at 20% cannot be attributed to NO considering l-NMMA did not alter vascular responses. The authors further explored the vasodilatory role of NO in ageing by infusing acetylcholine during l-NMMA or saline administration. l-NMMA reduced endothelial-dependent vasodilator responses to acetylcholine in the young adults compared to the response with saline (Casey et al. 2010). Conversely, l-NMMA did not significantly alter the vasodilator response in the older adults; this again suggests attenuated NO signalling with ageing, although irrespective of hypoxia. In summary, novel findings from the current study demonstrate that hypoxia-induced compensatory vasodilatation is attenuated in ageing subjects during moderate-intensity exercise (20% effort). The authors attributed the decrease in compensatory vasodilatation to diminished NO signalling. This conclusion was reinforced by the lack of significant reduction in ΔFVC with l-NMMA infusion at 20% effort. The authors provide readers with a thorough analysis of data from ageing adults, including absolute FVC measures and both delta (Δ) and per cent (%) changes from normoxic levels. The representation of hypoxia-mediated compensatory vasodilatation (per cent change in ΔFVC from respective normoxic responses) may seem convoluted. However, it is probably the most appropriate means to compare results between groups with varying absolute flows. In this way, variability in resting flow is heeded and results are presented within the context of normoxic exercise vasodilatation. However, considering the hypothesis of the current study, the authors presented few direct statistical comparisons of ageing and young subjects – leaving conclusions based solely upon compensatory (% change in ΔFVC) analysis. Providing additional between-group analyses from available data could have added key information regarding apparent differences in haemodynamic responses to hypoxia. Whereas these findings suggest the importance of NO to hypoxic vasodilatation is attenuated with ageing, the methods used do not allow for understanding of vascular control during hypoxic exercise. When nitric oxide synthase (NOS) inhibition occurs prior to the onset of hypoxia at rest, redundant signals are capable of compensating to produce a normal hyperaemic response (Markwald et al. 2011). However, when combined with exercise, Casey and colleagues suggest NOS inhibition attenuates hypoxic exercise dilatation in young adults (2010) while minimally impacting older adults (2011). Though novel, these findings cannot rule out the possibility of compensatory mechanisms capable of masking the normal contribution of NO to steady-state flow. Thus, current results may underestimate the importance of NO to hypoxic dilatation in either study population. Follow-up studies should consider local inhibition of NO synthesis during hypoxic handgrip exercise, in addition to the exploration of compensatory mechanisms. Consistent with the concept of signal redundancy, the possible contribution of β-adrenergic or prostaglandin-mediated vasodilatation remains untested. In young adults, β-receptor activation is responsible for a substantial portion of hypoxic dilatation during low-intensity forearm exercise (10% effort, Wilkins et al. 2008); a portion of this dilatation occurs through an NO-dependent pathway. As a result, the exact mechanisms behind NO-mediated compensatory vasodilatation at 10% effort may be group-specific if altered β-adrenergic sensitivity or receptor expression occur with ageing. Furthermore, prostaglandins are known to play a role in resting hypoxic vasodilatation in young adults (Markwald et al. 2011) and their contribution to normoxic exercise vasodilatation is reduced with age (Schrage et al. 2007). Future studies are necessary to fill the gap in knowledge and elucidate the individual roles of both β-adrenergic and prostaglandin-mediated mechanisms on hypoxic vasodilatation in ageing adults. Neurovascular control of hypoxia-mediated blood flow is a balance between adrenergic constriction and dilatation. Thus, it is important to acknowledge that ageing subjects presented 2-fold higher venous noradrenaline levels compared to young adults – probably due, in part, to age-related increases in sympathetic nerve activity. Given the observed increase, it is possible that enhanced adrenergic vasoconstriction contributed to blunted compensatory vasodilatation in the ageing subjects. However, the authors show similar heart rate and blood pressure responses to hypoxia between groups (Casey et al. 2011). Whereas these observations are unlikely to confound current conclusions, one should consider the potential clinical implications in disease states presenting increased sympathetic nerve activity (diabetes, heart failure, hypertension). Interestingly, the authors note significant between-group differences in baseline blood pressures. Given that both ageing and hypertension are known to increase sympathetic nerve activity and independently reduce NO-mediated dilatation, the current cohort does not allow for differentiation between these manifestations. Despite attenuated compensatory vasodilatation in ageing adults, results from the current study suggest that oxygen consumption is maintained during forearm exercise. Thus, older adults are able to meet metabolic demand through combined changes in oxygen delivery and extraction. Although vasodilatory responses to environmental stressors and the mechanisms behind vascular control are altered with ageing, reduced haemodynamics do not appear to compromise older adults’ ability to meet oxygen demand. Whether this continues to apply at higher exercise intensities or under more severe hypoxia, and the clinical implications of such changes, have yet to be assessed. In conclusion, Casey et al. (2011) provide novel insight to hypoxia-mediated dilatation during forearm exercise and the effects of ageing on mechanisms of vascular control. This, combined with their previous work, begins to describe complex interactions in vascular control. Future studies might aim to examine the relative importance of alternative vascular control mechanisms and the effect of differences in sympathetic nerve activity between groups. Clinically, this information provides further understanding of the impact of ageing and the role of NO in skeletal muscle blood flow under circumstances when local or systemic arterial oxygen saturation may be limited by exercise, environmental conditions or medical pathologies.

Increased leg blood flow and improved femoral artery shear patterns in metabolic syndrome after a diet and exercise programme

Altered vascular shear profiles may contribute to the development of atherosclerosis. Physical activity promotes anti‐atherogenic shear patterns, resulting in reduced cardiovascular disease risk. Adults with metabolic syndrome (MetSyn) are at increased risk of developing atherosclerosis and cardiovascular disease. Thus, we hypothesized that conduit artery antegrade shear rate (ASR) would increase and retrograde shear rate (RSR) and oscillatory shear indices (OSI) would decrease in MetSyn patients (n = 16, 51 ± 2 years) after participation in a diet and exercise programme (DEP).

ATP‐mediated vasodilatation: all thanks to potassium?

Emerging evidence suggests circulating adenosine triphosphate (ATP) plays an important role in local blood flow control. ATP is primarily thought to bind to P2Y receptors on the endothelium, and activate downstream vasodilator pathways. Specific mechanisms behind ATP-mediated vasodilatation are currently unknown in humans; however, research supports the contribution of nitric oxide (NO), prostaglandins (PGs) and endothelial-derived hyperpolarization (EDH) via one or several potassium (K+) channels. Despite consistent findings in vitro, the contribution of NO and PGs to ATP-mediated vasodilatation in vivo is contradictory. Specifically, Crecelius et al. (2011) demonstrated NO and PGs to contribute to only ∼20–30% of the vasodilatory response to exogenous ATP in healthy humans, suggesting other mechanisms must be largely responsible for the increase in blood flow. Using evidence from in vitro research identifying a role for K+ channel activation in ATP-mediated vasodilation, Crecelius et al. (2012) continued to advance our understanding of local blood flow control in a recent article published in The Journal of Physiology. K+ efflux from small and intermediate-conductance Ca2+-activated K+ channels increases myoendothelial K+ concentration. This increase in K+ concentration results in activation of Na+/K+-ATPase and KIR channels leading to hyperpolarization of vascular smooth muscle cells. In vitro research has shown that application of ATP evokes local and conducted vasodilatation via these pathways. Thus, Crecelius et al. thought outside the NO/PG box by testing the hypotheses that ATP-mediated vasodilatation in the forearm is largely independent of NO and PG synthesis and occurs via Na+/K+ -ATPase and KIR channel activation in humans. To test their hypotheses, the authors assessed forearm vascular conductance (FVC) to intra-arterial infusions using standard venous occlusion plethysmography (VOP) procedures in 33 young, healthy adults. To confirm that ATP-mediated vasodilatation is largely independent of NO and PGs, and to focus on the role of K+ channels, Crecelius et al. used three separate but related protocols. First, the group tested the effect of combined NO synthase (NOS) and cyclooxygenase (COX) inhibition (l-NMMA and ketorolac, respectively) on ATP-mediated vasodilatation (n = 8). Confirming previous findings (Crecelius et al. 2011), combined NOS and COX inhibition significantly reduced FVC at rest, but not during ATP infusion. Thus, ATP vasodilatation appears to be largely independent of NO and PGs. The authors also examined K+-mediated vasodilatation using KCl. As expected, KCl infusion increased FVC; yet surprisingly, the relative response of KCl infusion was greater after NOS–COX inhibition. The second protocol examined the contribution of alternative downstream vascular control mechanisms to ATP-mediated vasodilatation via Na+/K+-ATPase and KIR channel inhibition. ATP (n = 8) or KCl (n = 6) were infused with and without ouabain and BaCl2 (Na+/K+-ATPase and KIR channel inhibitors, respectively). Co-infusion abolished forearm vasodilatation to KCl, and, as the authors hypothesized, oubain and BaCl2 attenuated vasodilatation in response to ATP (56% reduction). To test the independent effect of KIR channels on ATP-mediated smooth muscle relaxation, BaCl2 was administered alone in six additional subjects. The authors observed a significant reduction (51%) in the vasodilatory response to ATP. Taken together, inhibition of KIR had relatively the same effect as combined Na+/K+-ATPase and KIR inhibition, indicating KIR channels are a key player in ATP-mediated vasodilatation in the resting forearm circulation. However, the authors chose to compare double blockade of Na+/K+-ATPase and KIR channels vs. single blockade of KIR channels in separate research cohorts. Future studies might aim to examine the effect of single and double blockade in the same subset of subjects in order to control for inter-subject variability in blood flow responses to different pharmacological interventions. Additionally, figures directly comparing the two trials were not provided. A helpful approach to compare data from multiple protocols might be providing figures comparing percentage inhibition across doses and between various drug conditions. Such data presentation could provide readers with key information regarding the efficacy of inhibition and might allow insight into the relative importance of various K+ channels. In summary, Crecelius and colleagues provided novel and exciting research regarding specific mechanisms of ATP-mediated dilatation. They verified previous results (Crecelius et al. 2011) that blood flow responses to ATP in the forearm are largely independent of NO and PG synthesis. In addition, this research group was the first to identify vascular hyperpolarization in the forearm via KIR channel activation as one of the primary pathways underlying the vasodilator mechanisms of intravascular ATP in humans. Interestingly, blockade of KIR, with or without Na+/K+-ATPase blockade, did not completely abolish ATP-induced vasodilatation. One possible explanation for these findings is that ATP might have the ability to promote dilatation via auxiliary down-stream pathways. For example, ATP has been shown to stimulate epoxyeicosatrienoic acid (EETs) release from erythrocytes (Jiang et al. 2007), BKCa channel hyperpolarization of the smooth muscle, and subsequent vasodilatation independent of KIR channels. Therefore, a protocol utilizing blockade of KIR channels with and without blockade of cytochrome P450 epoxygenase (using miconazole or sulfaphenazole) could be considered in follow-up studies. This protocol would demonstrate the relative contribution of BKCa channel vasodilatation in response to ATP. The results from the current study have potential to provide insight to other applications regarding ATP-mediated vasodilatation such as exercise and disease. One exciting area to consider is the role of ATP dilatation and its associated pathways during steady-state exercise. ATP is thought to be released from red blood cells in response to a decrease in blood oxygen content and mechanical deformation and, thus, can contribute to the blood flow response seen with exercise. Future studies could test whether KIR channels are also a primary player in ATP-mediated dilatation during exercise. This study holds potential to provide clinical insight into patient populations such as those with hypertension. Adults with hypertension have been shown to exhibit endothelial dysfunction due, in part, to impairments in NO bioavailability, but other mechanisms contributing to the endothelial dysfunction have yet to be fully explored. Research in animal models suggests KIR channel function is impaired in the cerebral and mesenteric circulation during hypertension (Haddy et al. 2006). It is interesting to consider the potential for KIR channel impairments in other vascular beds, which could be explored in hypertensive humans using a protocol similar to that of the Crecelius group. If KIR channel function in response to ATP is indeed impaired in hypertensive humans, it might be interesting to consider the idea that expression and/or activity of BKCa channels is enhanced in this population. This could be in order to compensate for a reduction in KATP, Kv and KIR channel function in an attempt to ultimately restrict increases in vascular tone (Sobey, 2001). In conclusion, novel studies by Crecelius et al. uncovered one of the major players (KIR) of ATP-mediated vasodilatation. Their research allows for new avenues to test alternative downstream vasodilatory mechanisms and novel interactions between NO, PGs, EETs and KIR channels in a variety of vascular beds and cardiovascular conditions.