American Journal of Hypertension Advance Access published March 13, 2014
Original Article
Seven-Day Remote Ischemic Preconditioning Improves Local and Systemic Endothelial Function and Microcirculation in Healthy Humans Helen Jones,1 Nicola Hopkins,1 Tom G. Bailey,1 Daniel J. Green,1,2 N. Timothy Cable,1 and Dick H.J. Thijssen1,3
METHODS Thirteen healthy, young, normotensive male individuals (aged 22 ± 2 years) were assigned to 7-day daily exposure of the arm to IPC (4 × 5 minutes). Assessment of brachial artery endothelial function (using flow-mediated dilation (FMD)) and forearm microcirculation (cutaneous vascular conductance (CVC) at baseline and during local heating) was performed before and after 7 days to examine the local (i.e., intervention arm) and remote (i.e., control arm) effect of IPC. We repeated the assessment tests 8 days after the intervention (Post+8). RESULTS FMD increased after repeated IPC (P = 0.03) and remained significantly elevated at Post+8 in the intervention (5.0 ± 2.2%, 6.1 ± 2.2%,
and 6.6 ± 2.3%) and contralateral arms (5.4 ± 2.2%, 6.0 ± 2.2%, and 7.5 ± 2.2%). Forearm CVC also increased following repeated IPC (P = 0.006) and remained elevated at Post+8 in both arms (intervention: 0.12 ± 0.03, 0.14 ± 0.04, 0.16 ± 0.04 mV/mm Hg; contralateral: 0.14 ± 0.04, 0.015 ± 0.04, 0.17 ± 0.07). No interaction between IPC arm and time was evident for FMD and CVC (both P > 0.05). IPC intervention did not alter CVC responses to local heating (P > 0.05).
Conclusions Daily exposure to IPC for 7 days leads to local and remote improvements in brachial artery FMD and resting skin microcirculation that remain after cessation of the intervention and beyond the late phase of protection. These findings may have clinical relevance for micro- and macrovascular improvements. Keywords: blood pressure; cardiovascular risk; endothelial function; hypertension; microcirculation; remote ischemic preconditioning; vascular adaptation. doi:10.1093/ajh/hpu004
Despite optimal (pharmacological) therapy and risk factor management, cardio- and cerebrovascular diseases remain the leading causes of mortality and morbidity in the Western world.1 Consequently, innovative treatment strategies for protecting against cardiovascular disease are required to improve clinical outcomes. A cycle of repeated bouts of ischemia followed by reperfusion over a short period of time, commonly known as ischemic preconditioning (IPC), has been shown to delay cardiac cell injury2 and protect against myocardial and vascular damage.3 In addition to the local protective effects of IPC,4 protection against tissue damage has also been found in distant vascular areas not directly exposed to the repeated ischemic stimuli.5,6 This well-known phenomenon is commonly referred to as remote
IPC.6–8 These effects suggest that repeated IPC may represent a potent, systemic stimulus for vascular adaptations. Previous studies have demonstrated that acute exposure to IPC leads to increased blood flow in conduit and resistance vessel beds in distant areas (such as the contralateral limb)9 or organs (such as the heart)10,11 and also to enhanced cutaneous tissue oxygen saturation and arterial capillary blood flow.12 Single IPC may acutely change endothelial function,13 although results are conflicting.4,8 Given these acute effects of IPC, repeated exposure to IPC may induce beneficial sustained vascular adaptations. Little is known about the potential of repeated IPC to alter conduit and skin circulatory beds and whether such adaptations also occur in remote vascular beds. Because the IPC stimulus is associated with
Correspondence: Dick H.J. Thijssen ([email protected]).
1Research Institute for Sport and Exercise Science, Liverpool John
Initially submitted October 31, 2013; date of first revision November 20, 2013; accepted for publication January 2, 2014.
Moores University, Liverpool, UK; 2School of Sport Science, Exercise and Health, The University of Western Australia, Crawley, Western Australia, Australia; 3Department of Physiology, RRadboud University Medical Center, Nijmegen, The Netherlands.
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American Journal of Hypertension 1
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Background Ischemic preconditioning (IPC) protects tissue against ischemiainduced injury inside and outside ischemic areas. The purpose was to examine the hypothesis that daily IPC leads to improvement in endothelial function and skin microcirculation not only in the arm exposed to IPC but also in the contralateral arm.
Jones et al.
METHODS Participants
Thirteen healthy males (Table 1) volunteered to participate in this uncontrolled, 7-day IPC intervention study. Participants were recreationally active, were measured by a self-report questionnaire, and typically engaged in low- (e.g., walking) and moderate-intensity (e.g., running and stationary cycling) aerobic activities (2–3 days/week). We excluded smokers and participants with (a history of) cardiovascular disease including hypertension, diabetes, or hypercholesterolemia and participants who were on any medication. All participants provided written informed consent before participation. The study was approved by the institutional ethics committee and adhered to the Declaration of Helsinki (2000). Experimental design
Participants in the IPC intervention group (i.e., 7 days of daily IPC exposure) reported 3 times to our laboratory for assessment of bilateral brachial artery endothelial function Table 1. Descriptive characteristics of the male participants in the intervention (n = 13) Characteristics
Age, y Height, cm
Intervention group
21.5 ± 2.2 178.4 ± 4.8
Weight, kg
75.0 ± 4.6
BMI, kg/m2
22.8 ± 1.0
Systolic blood pressure, mm Hg
138 ± 6
Diastolic blood pressure, mm Hg
73 ± 6
Mean arterial blood pressure, mm Hg
95 ± 5
Pulse pressure, mm Hg
66 ± 7
Data are mean ± SD. Abbreviation: BMI, body mass index.
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(using flow-mediated dilation (FMD)) and bilateral forearm cutaneous vascular conductance (CVC) at rest and during a local heating stimulus (Figure 1).16 Assessment of bilateral brachial artery FMD and bilateral CVC responses was performed before (Pre) and after (Post) 7 days of daily IPC training and also 8 days after the cessation of the intervention (Post+8 days). The IPC intervention consisted of daily exposure to 4 bouts of 5-minute cuff occlusion in a single arm, with the contralateral arm serving as a within-subject control arm. We randomized and counterbalanced the arm that received the IPC intervention between participants (dominant vs. nondominant). Repeated measures were performed at the same time of day to control for diurnal variation in endothelial function.17 Experimental measures
Brachial artery endothelial function. Simultaneous bilateral brachial artery endothelium–dependent function was measured using the FMD technique.18 For this purpose, participants were instructed to abstain from strenuous exercise, caffeine, and alcohol ingestion for 24 hours before reporting to the laboratory. Participants were also asked to fast for 6 hours before each visit. Measurements were performed in the supine position in a quiet, darkened, temperature-controlled room. Measurement began after resting for 20 minutes, followed by assessment of heart rate and blood pressure using an automated sphygmomanometer (GE Pro 300V2; Dinamap, Tampa, FL). To examine brachial artery FMD, both arms were extended and positioned at an angle of approximately 80° from the torso. A rapid inflation and deflation pneumatic cuff (D.E. Hokanson, Bellevue, WA) was positioned on each forearm, immediately distal to the olecranon process to provide a stimulus to forearm ischemia. A 10-MHz multifrequency linear array probe, attached to a high-resolution ultrasound machine (T3000; Terason, Burlington, MA), was then used to image the brachial artery in the distal third of the upper arm. When an optimal image was obtained, the probe was held stable and the ultrasound parameters were set to optimize the longitudinal, B-mode image of the lumen–arterial wall interface. Settings were identical between all FMD assessments. Continuous Doppler velocity assessments were also obtained using the ultrasound machine and were collected using the lowest possible isonation angle (always 200 mm Hg) for 5 minutes. Diameter and flow recordings resumed 30 seconds before cuff deflation and continued for 3 minutes thereafter, in accordance with recent technical specifications.19,20 Brachial artery diameter and blood flow analysis. Analysis of brachial artery diameter was performed using customdesigned edge-detection and wall-tracking software, which is largely independent of investigator bias. Recent articles contain detailed descriptions of our analysis approach.19,20 From synchronized diameter and velocity data, blood flow (the product of lumen cross-sectional area and Doppler velocity) and shear rate (an estimate of shear stress without viscosity) were calculated at 30 Hz. Within-day reproducibility of the FMD possesses a coefficient of variation of 6.7%–10.5%.21
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elevation of circulating growth factors and endothelial progenitor cells,5,14 we hypothesize that repeated IPC will lead to systemic changes in both conduit artery endothelial function and skin microcirculation. Therefore, we examined whether 7 days of daily exposure to unilateral IPC leads to bilateral adaptation of the brachial artery endothelial function and forearm skin microcirculation in healthy, young men. Previous studies have also revealed the presence of a late phase of protection for IPC.8 This late phase begins 12–24 hours after IPC and lasts 3–4 days.15 To determine whether repeated IPC improves endothelial function and cutaneous microcirculation beyond the late phase of protection (of the last IPC session), we assessed endothelial function and forearm microcirculation 8 days after cessation of the IPC intervention. We hypothesize that repeated IPC results in adaptations, resulting in improved endothelial function and elevated microcirculation that are still present 8 days after cessation of the intervention.
Effect of 7-Day IPC on Endothelial Function
5-min occlusion
5-min reperfusion
Post -Brachial FMD -Baseline CVC -CVC responses to local heat
Post+8 -Brachial FMD -Baseline CVC -CVC responses to local heat
Figure 1. Protocol of the study. Every IPC session consisted of 4 periods of 5-minute arterial occlusion (through inflation of a blood pressure cuff around the upper arm to 220 mm Hg) and 5-minute reperfusion. Bilateral assessment of brachial artery FMD and forearm CVC (baseline and in response to heat) is performed not only before the IPC intervention (day 1; Pre) but also immediately after (day 8; Post) and 8 days after the intervention (day 15; Post+8). Abbreviations: CVC, cutaneous vascular conductance; FMD, flow-mediated dilation; IPC, ischemic preconditioning.
Microvascular function measurements. Assessments were performed in a quiet, temperature-controlled room (23 ± 1 °C), with the participants positioned comfortably on a bed. Both arms were positioned approximately 5 cm above heart level, and the laser Doppler probes were positioned on the volar side of the forearm, approximately 5 cm below the elbow crest. The measurement sites for each of the laser Doppler probes were recorded, and efforts were made to use the same places during the 3 consecutive measurements. After an acclimatization period of 30 minutes, microvascular function was examined by recording of local skin blood flow. To obtain an index of skin blood flow, cutaneous red blood cell flux (in millivolts) was measured simultaneously in both forearms using a laser Doppler flowmetry system (Model 413, Periflux 5001 System; Perimed, Stockholm, Sweden). Skin temperature was controlled at the 2 measuring sites with local heating units (Perimed 455; Perimed). To verify whether blood pressure was stable throughout the experimental protocol, blood pressure was measured at 5-minute intervals by an automated sphygmomanometer. After instrumentation, red blood cell flux of both sites was monitored to examine baseline skin blood flow. The temperature of the local heating units at both sites was kept constant at 33 °C during the baseline period. After baseline recording for 10 minutes, local heating protocol was performed simultaneously at both sides. Temperature of the local heating units was increased at a rate of 0.5 °C every 5 seconds to a temperature of 42 °C.16 The protocol was finished after red
blood cell flux in both sites had reached a stable plateau (approximately 30–40 minutes). Skin blood flow response to this protocol is nitric oxide dependent.16 The laser Doppler probe signals were continuously monitored by an online software chart recorder (PSW; Perimed). CVC was calculated as laser Doppler flow (in millivolts) divided by mean arterial pressure (mm Hg) to account for any differences in blood pressure between the groups. Analysis was performed as described elsewhere.22 IPC intervention. The 7-day IPC training protocol consisted of daily exposure to 4 repetitions of inflating an upper arm blood pressure cuff using a rapid cuff inflator (EC-20; D.E. Hokanson) to 220 mm Hg for 5 minutes, followed by deflation of 5 minutes (Figure 1). All sessions were supervised by a researcher to ensure full compliance to the intervention. Statistics
Statistical analyses were performed using SPSS 17.0 (SPSS, Chicago, IL) software. Our primary outcome was endothelial function as measured using the brachial artery FMD. All FMD data were analyzed and presented as covariate controlled for baseline artery diameter measured before the introduction of hyperemia in each test; this approach is more accurate for scaling changes in artery diameter than simple percentage change.23 These data were analyzed using a 2-factor general linear model with repeated measures of IPC (2 levels: IPC arm and contralateral arm) and time (3 levels: American Journal of Hypertension 3
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Pre -Brachial FMD -Baseline CVC -CVC-responses to local heat
Jones et al.
Pre, Post, and Post+8 days). Our primary outcome variable for microvascular endothelial function was CVC. Similarly, a 2-factor general linear model was used with the same repeated measures factors to analyze the summary data (resting baseline, first peak, nadir, and plateau) during the heating protocol. Significant main effects or interactions were followed up using the least significant difference method for pairwise multiple comparisons.24 For all statistical tests, a 2-tailed comparison was used. All data are reported as mean (±SD) unless stated otherwise, and statistical significance was assumed at P 0.05; Table 2). Forearm microvascular function
Local heating. The local heating protocol induced the typical pattern of an initial peak, followed by a nadir and a subsequent plateau in skin flux, which was present during all assessments for all participants in both arms and during all time points. No significant main effects of time, IPC, or interactions between these factors were evident for flux, mean arterial pressure, or CVC at the first peak, the nadir, or the plateau points during heating (P > 0.05) (Table 3). Discussion
Our study provides a number of observations. First, 7-day daily exposure to unilateral IPC of the arm results in significant improvements in conduit artery endothelial function not only in the arm exposed to repeated IPC, but also in
Table 2. Brachial artery FMD before (Pre) and immediately after (Post) the IPC intervention and 8 days after (Post+8) cessation of the intervention in healthy volunteers (n = 13, intervention and contralateral arms) Intervention arm Intervention group
Diameter, mm FMD, % Shear AUC, 103 Time to peak
Pre
3.8 ± 0.6
Post
3.9 ± 0.4
Contralateral arm Post+8
3.9 ± 0.3
Pre
Post
3.9 ± 0.5
3.8 ± 0.4
Two-way ANOVA P values Post+8
3.7 ± 0.4
IPC
Time
IPC × time
0.88
0.42
0.38
5.0 ± 2.2
6.1 ± 2.2
5.4 ± 2.2
6.0 ± 2.2
0.41
0.03
0.71
17.9 ± 12.9
13.6 ± 6.2
16.4 ± 8.1
12.8 ± 5.9
13.1 ± 8.3
16.6 ± 8.6
0.32
0.56
0.47
56 ± 31
47 ± 21
58 ± 35
52 ± 25
49 ± 23
50 ± 18
0.65
0.48
0.37
6.6 ± 2.3*
7.5 ± 2.2*
Data are mean ± SD. Bold numbers represent significant main effects (P < 0.05). Abbreviations: ANOVA, analysis of variance; AUC, area under the curve; FMD, flow-mediated dilation; IPC, ischemic preconditioning. *Post hoc significantly different from Pre (P 0.05).
Jones et al.
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“true” adaptation. To control for this effect, we repeated postintervention measurements (Post+8 days). We found that the increase in brachial artery and skin microcirculation endothelial function remained significantly elevated in both arms 8 days after cessation of the IPC intervention. This finding infers that repeated IPC induces sustained local and remote functional adaptation of the conduit artery endothelium. The late phase of protection is thought to be induced by de novo synthesis of cardioprotective proteins, such as upregulation of inducible nitric oxide synthase.33 These changes may contribute to a higher NO production and improved conduit artery FMD, i.e., a response that is mediated, at least partly, through NO.34 An interesting observation in our study is the significant decline in blood pressure after IPC. A recent study also reported a blood pressure–lowering effect of IPC in normotensive participants after 3 days of daily IPC.35 Interestingly, many of the suggested mechanisms of (remote) IPC, such as hormonal5 and neuronal effects by the autonomic nervous system,8 also contribute to the regulation of the systemic blood pressure. Previous data report an inverse relation between brachial artery FMD and blood pressure.36 Accordingly, the enhanced (systemic) endothelial function in our study may contribute to the lower blood pressure in our participants. Alternatively, methodological aspects may explain the decline in the blood pressure. First, we have adopted a single measure of blood pressure rather than repeated measurements. Secondly, anxiety before the preintervention may have affected measurements and overestimated preintervention values. Limitations. We did not examine endothelium-independent vasodilation. However, we26 and others14 have demonstrated that endothelium-independent function typically does not change over time in healthy participants. Thus, it is unlikely that the improvement in endothelial function in our study is explained by changes in endothelium-independent dilation. Second, we were not able to examine the peak CVC, which some authors use to normalize skin CVC responses. However, peak CVC responses typically do not change across time. Finally, we did not include a control group within the study to control for potential bias in our study. Clinical relevance. Endothelial dysfunction is an important event in the atherosclerotic cascade and predicts cardiovascular endpoints.37,38 Our data demonstrate that IPC, which represents a simple, virtually cost-free, and noninvasive intervention, improves endothelial function in the intervention and contralateral arms in healthy individuals. Although speculative, these local and systemic effects of IPC on endothelial function may have clinical relevance, especially in individuals with impaired micro- or macrovessel functions (e.g., type 2 diabetes), where vascular function is a primary target to improve cardiovascular risk and associated complications. Whether repeated IPC improves micro- and macrovessel functions in individuals with endothelial dysfunction, or in those with conditions that require cardiovascular risk management, is currently unknown, and this should be the subject of future research. Furthermore, such studies should also examine how long adaptations in endothelial function and skin microcirculation remain preserved.
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the contralateral arm. Second, we observed a higher forearm CVC in both forearms. These findings imply that 7-day IPC results in both local and remote improvements in conduit artery endothelial function and forearm CVC. Third, we found that these bilateral improvements in conduit artery endothelial function and forearm CVC persist up to 8 days after intervention. These observations suggest that IPC is an intervention capable of improving endothelial function and skin microcirculation both locally and systemically and that these beneficial effects remain present for at least 7 days after the cessation of the IPC stimulus. IPC is typically used to prevent endothelial dysfunction induced by ischemia-reperfusion injury.4,7,8,25 A single bout of IPC may also acutely change endothelial function,13 although results are conflicting.4,8 Furthermore, preliminary evidence suggests that repeated IPC improves resistance artery endothelial–dependent vasodilation.14 We extend this knowledge with the observation that 1 week of daily IPC leads to a significant improvement in conduit artery endothelial function. The potent vascular effects of daily IPC are further emphasized by the increased forearm CVC. Although these observations provide evidence for vascular adaptations after IPC throughout the vascular tree, we add the novel observation that these vascular adaptations are also present in remote areas. Indeed, a simultaneous increase in endothelial function and CVC was found in the contralateral arm, i.e., the arm not directly exposed to IPC. Thus, our data support the use of IPC to induce local and systemic improvements in conduit artery endothelial function and skin microcirculation in humans. Our study was not designed to examine mechanisms that relate to the local and remote effects of 7 days of IPC. Therefore, we can only speculate about the potential underlying mechanisms. Shear stress represents a major physiological stimulus responsible for improvement in conduit artery function26 and skin microcirculation.27 Although episodic increases in shear stress may contribute (partly) to the adaptations found in the intervention arm, it is unlikely that shear represents the principle mechanism responsible for the remote effects of IPC because elevations in shear in remote areas are only modest. Systemic stimuli or circulating markers activated by IPC more likely explain the remote improvement in conduit artery endothelial function. For example, IPC leads to an increase in vascular endothelial growth factor and endothelial progenitor cells,14 which may improve endothelial function in remote areas.28 Indeed, tissue ischemia29 and shear,30 both prominent during IPC, are key stimuli for endothelial progenitor cell release from bone marrow into the circulation. An alternative explanation relates to reduced levels of oxygen free radicals and inflammation because IPC improves antioxidative defence mechanisms (e.g., increased superoxide dismutase activity) and/ or lowers the generation of oxidative stress.4,31,32 Although future studies should further examine the exact mechanisms by which IPC improves the vasculature, it is likely that systemic pathways contribute to the effects. Because the late phase of protection coincides with our postintervention measurements, the increased FMD and CVC after the 7-day IPC intervention may relate to the late phase of protection of the last IPC bout rather than a
Effect of 7-Day IPC on Endothelial Function
In conclusion, 7 days of daily exposure to IPC leads to local (intervention arm) and systemic (contralateral arm) improvements in conduit artery endothelial function and elevation of resting skin microcirculation, which are present beyond the late phase of protection of IPC. These findings may have clinical relevance because improving endothelial function independently prevents cardiovascular morbidity and mortality.38 Furthermore, repeated IPC increased resting microcirculation by approximately 30%, a clinically relevant improvement, especially because the increase was similar in a remote vascular bed not exposed to IPC. Although future studies should further examine the impact of repeated IPC in clinical groups with endothelial dysfunction, our findings highlight the potential for IPC as a simple, safe, and feasible therapeutic tool.
D.H.J.T. is recipient of the E. Dekker stipend (Netherlands Heart Foundation, 2009T064). D.J.G. is funded by the Australian Research Council (DP 130103793). H.J. and N.H. contributed equally to this work. DISCLOSURE
The authors declared no conflict of interest.
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ACKNOWLEDGMENTS
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8 American Journal of Hypertension
Original Article
Seven-Day Remote Ischemic Preconditioning Improves Local and Systemic Endothelial Function and Microcirculation in Healthy Humans Helen Jones,1 Nicola Hopkins,1 Tom G. Bailey,1 Daniel J. Green,1,2 N. Timothy Cable,1 and Dick H.J. Thijssen1,3
METHODS Thirteen healthy, young, normotensive male individuals (aged 22 ± 2 years) were assigned to 7-day daily exposure of the arm to IPC (4 × 5 minutes). Assessment of brachial artery endothelial function (using flow-mediated dilation (FMD)) and forearm microcirculation (cutaneous vascular conductance (CVC) at baseline and during local heating) was performed before and after 7 days to examine the local (i.e., intervention arm) and remote (i.e., control arm) effect of IPC. We repeated the assessment tests 8 days after the intervention (Post+8). RESULTS FMD increased after repeated IPC (P = 0.03) and remained significantly elevated at Post+8 in the intervention (5.0 ± 2.2%, 6.1 ± 2.2%,
and 6.6 ± 2.3%) and contralateral arms (5.4 ± 2.2%, 6.0 ± 2.2%, and 7.5 ± 2.2%). Forearm CVC also increased following repeated IPC (P = 0.006) and remained elevated at Post+8 in both arms (intervention: 0.12 ± 0.03, 0.14 ± 0.04, 0.16 ± 0.04 mV/mm Hg; contralateral: 0.14 ± 0.04, 0.015 ± 0.04, 0.17 ± 0.07). No interaction between IPC arm and time was evident for FMD and CVC (both P > 0.05). IPC intervention did not alter CVC responses to local heating (P > 0.05).
Conclusions Daily exposure to IPC for 7 days leads to local and remote improvements in brachial artery FMD and resting skin microcirculation that remain after cessation of the intervention and beyond the late phase of protection. These findings may have clinical relevance for micro- and macrovascular improvements. Keywords: blood pressure; cardiovascular risk; endothelial function; hypertension; microcirculation; remote ischemic preconditioning; vascular adaptation. doi:10.1093/ajh/hpu004
Despite optimal (pharmacological) therapy and risk factor management, cardio- and cerebrovascular diseases remain the leading causes of mortality and morbidity in the Western world.1 Consequently, innovative treatment strategies for protecting against cardiovascular disease are required to improve clinical outcomes. A cycle of repeated bouts of ischemia followed by reperfusion over a short period of time, commonly known as ischemic preconditioning (IPC), has been shown to delay cardiac cell injury2 and protect against myocardial and vascular damage.3 In addition to the local protective effects of IPC,4 protection against tissue damage has also been found in distant vascular areas not directly exposed to the repeated ischemic stimuli.5,6 This well-known phenomenon is commonly referred to as remote
IPC.6–8 These effects suggest that repeated IPC may represent a potent, systemic stimulus for vascular adaptations. Previous studies have demonstrated that acute exposure to IPC leads to increased blood flow in conduit and resistance vessel beds in distant areas (such as the contralateral limb)9 or organs (such as the heart)10,11 and also to enhanced cutaneous tissue oxygen saturation and arterial capillary blood flow.12 Single IPC may acutely change endothelial function,13 although results are conflicting.4,8 Given these acute effects of IPC, repeated exposure to IPC may induce beneficial sustained vascular adaptations. Little is known about the potential of repeated IPC to alter conduit and skin circulatory beds and whether such adaptations also occur in remote vascular beds. Because the IPC stimulus is associated with
Correspondence: Dick H.J. Thijssen ([email protected]).
1Research Institute for Sport and Exercise Science, Liverpool John
Initially submitted October 31, 2013; date of first revision November 20, 2013; accepted for publication January 2, 2014.
Moores University, Liverpool, UK; 2School of Sport Science, Exercise and Health, The University of Western Australia, Crawley, Western Australia, Australia; 3Department of Physiology, RRadboud University Medical Center, Nijmegen, The Netherlands.
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American Journal of Hypertension 1
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Background Ischemic preconditioning (IPC) protects tissue against ischemiainduced injury inside and outside ischemic areas. The purpose was to examine the hypothesis that daily IPC leads to improvement in endothelial function and skin microcirculation not only in the arm exposed to IPC but also in the contralateral arm.
Jones et al.
METHODS Participants
Thirteen healthy males (Table 1) volunteered to participate in this uncontrolled, 7-day IPC intervention study. Participants were recreationally active, were measured by a self-report questionnaire, and typically engaged in low- (e.g., walking) and moderate-intensity (e.g., running and stationary cycling) aerobic activities (2–3 days/week). We excluded smokers and participants with (a history of) cardiovascular disease including hypertension, diabetes, or hypercholesterolemia and participants who were on any medication. All participants provided written informed consent before participation. The study was approved by the institutional ethics committee and adhered to the Declaration of Helsinki (2000). Experimental design
Participants in the IPC intervention group (i.e., 7 days of daily IPC exposure) reported 3 times to our laboratory for assessment of bilateral brachial artery endothelial function Table 1. Descriptive characteristics of the male participants in the intervention (n = 13) Characteristics
Age, y Height, cm
Intervention group
21.5 ± 2.2 178.4 ± 4.8
Weight, kg
75.0 ± 4.6
BMI, kg/m2
22.8 ± 1.0
Systolic blood pressure, mm Hg
138 ± 6
Diastolic blood pressure, mm Hg
73 ± 6
Mean arterial blood pressure, mm Hg
95 ± 5
Pulse pressure, mm Hg
66 ± 7
Data are mean ± SD. Abbreviation: BMI, body mass index.
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(using flow-mediated dilation (FMD)) and bilateral forearm cutaneous vascular conductance (CVC) at rest and during a local heating stimulus (Figure 1).16 Assessment of bilateral brachial artery FMD and bilateral CVC responses was performed before (Pre) and after (Post) 7 days of daily IPC training and also 8 days after the cessation of the intervention (Post+8 days). The IPC intervention consisted of daily exposure to 4 bouts of 5-minute cuff occlusion in a single arm, with the contralateral arm serving as a within-subject control arm. We randomized and counterbalanced the arm that received the IPC intervention between participants (dominant vs. nondominant). Repeated measures were performed at the same time of day to control for diurnal variation in endothelial function.17 Experimental measures
Brachial artery endothelial function. Simultaneous bilateral brachial artery endothelium–dependent function was measured using the FMD technique.18 For this purpose, participants were instructed to abstain from strenuous exercise, caffeine, and alcohol ingestion for 24 hours before reporting to the laboratory. Participants were also asked to fast for 6 hours before each visit. Measurements were performed in the supine position in a quiet, darkened, temperature-controlled room. Measurement began after resting for 20 minutes, followed by assessment of heart rate and blood pressure using an automated sphygmomanometer (GE Pro 300V2; Dinamap, Tampa, FL). To examine brachial artery FMD, both arms were extended and positioned at an angle of approximately 80° from the torso. A rapid inflation and deflation pneumatic cuff (D.E. Hokanson, Bellevue, WA) was positioned on each forearm, immediately distal to the olecranon process to provide a stimulus to forearm ischemia. A 10-MHz multifrequency linear array probe, attached to a high-resolution ultrasound machine (T3000; Terason, Burlington, MA), was then used to image the brachial artery in the distal third of the upper arm. When an optimal image was obtained, the probe was held stable and the ultrasound parameters were set to optimize the longitudinal, B-mode image of the lumen–arterial wall interface. Settings were identical between all FMD assessments. Continuous Doppler velocity assessments were also obtained using the ultrasound machine and were collected using the lowest possible isonation angle (always 200 mm Hg) for 5 minutes. Diameter and flow recordings resumed 30 seconds before cuff deflation and continued for 3 minutes thereafter, in accordance with recent technical specifications.19,20 Brachial artery diameter and blood flow analysis. Analysis of brachial artery diameter was performed using customdesigned edge-detection and wall-tracking software, which is largely independent of investigator bias. Recent articles contain detailed descriptions of our analysis approach.19,20 From synchronized diameter and velocity data, blood flow (the product of lumen cross-sectional area and Doppler velocity) and shear rate (an estimate of shear stress without viscosity) were calculated at 30 Hz. Within-day reproducibility of the FMD possesses a coefficient of variation of 6.7%–10.5%.21
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elevation of circulating growth factors and endothelial progenitor cells,5,14 we hypothesize that repeated IPC will lead to systemic changes in both conduit artery endothelial function and skin microcirculation. Therefore, we examined whether 7 days of daily exposure to unilateral IPC leads to bilateral adaptation of the brachial artery endothelial function and forearm skin microcirculation in healthy, young men. Previous studies have also revealed the presence of a late phase of protection for IPC.8 This late phase begins 12–24 hours after IPC and lasts 3–4 days.15 To determine whether repeated IPC improves endothelial function and cutaneous microcirculation beyond the late phase of protection (of the last IPC session), we assessed endothelial function and forearm microcirculation 8 days after cessation of the IPC intervention. We hypothesize that repeated IPC results in adaptations, resulting in improved endothelial function and elevated microcirculation that are still present 8 days after cessation of the intervention.
Effect of 7-Day IPC on Endothelial Function
5-min occlusion
5-min reperfusion
Post -Brachial FMD -Baseline CVC -CVC responses to local heat
Post+8 -Brachial FMD -Baseline CVC -CVC responses to local heat
Figure 1. Protocol of the study. Every IPC session consisted of 4 periods of 5-minute arterial occlusion (through inflation of a blood pressure cuff around the upper arm to 220 mm Hg) and 5-minute reperfusion. Bilateral assessment of brachial artery FMD and forearm CVC (baseline and in response to heat) is performed not only before the IPC intervention (day 1; Pre) but also immediately after (day 8; Post) and 8 days after the intervention (day 15; Post+8). Abbreviations: CVC, cutaneous vascular conductance; FMD, flow-mediated dilation; IPC, ischemic preconditioning.
Microvascular function measurements. Assessments were performed in a quiet, temperature-controlled room (23 ± 1 °C), with the participants positioned comfortably on a bed. Both arms were positioned approximately 5 cm above heart level, and the laser Doppler probes were positioned on the volar side of the forearm, approximately 5 cm below the elbow crest. The measurement sites for each of the laser Doppler probes were recorded, and efforts were made to use the same places during the 3 consecutive measurements. After an acclimatization period of 30 minutes, microvascular function was examined by recording of local skin blood flow. To obtain an index of skin blood flow, cutaneous red blood cell flux (in millivolts) was measured simultaneously in both forearms using a laser Doppler flowmetry system (Model 413, Periflux 5001 System; Perimed, Stockholm, Sweden). Skin temperature was controlled at the 2 measuring sites with local heating units (Perimed 455; Perimed). To verify whether blood pressure was stable throughout the experimental protocol, blood pressure was measured at 5-minute intervals by an automated sphygmomanometer. After instrumentation, red blood cell flux of both sites was monitored to examine baseline skin blood flow. The temperature of the local heating units at both sites was kept constant at 33 °C during the baseline period. After baseline recording for 10 minutes, local heating protocol was performed simultaneously at both sides. Temperature of the local heating units was increased at a rate of 0.5 °C every 5 seconds to a temperature of 42 °C.16 The protocol was finished after red
blood cell flux in both sites had reached a stable plateau (approximately 30–40 minutes). Skin blood flow response to this protocol is nitric oxide dependent.16 The laser Doppler probe signals were continuously monitored by an online software chart recorder (PSW; Perimed). CVC was calculated as laser Doppler flow (in millivolts) divided by mean arterial pressure (mm Hg) to account for any differences in blood pressure between the groups. Analysis was performed as described elsewhere.22 IPC intervention. The 7-day IPC training protocol consisted of daily exposure to 4 repetitions of inflating an upper arm blood pressure cuff using a rapid cuff inflator (EC-20; D.E. Hokanson) to 220 mm Hg for 5 minutes, followed by deflation of 5 minutes (Figure 1). All sessions were supervised by a researcher to ensure full compliance to the intervention. Statistics
Statistical analyses were performed using SPSS 17.0 (SPSS, Chicago, IL) software. Our primary outcome was endothelial function as measured using the brachial artery FMD. All FMD data were analyzed and presented as covariate controlled for baseline artery diameter measured before the introduction of hyperemia in each test; this approach is more accurate for scaling changes in artery diameter than simple percentage change.23 These data were analyzed using a 2-factor general linear model with repeated measures of IPC (2 levels: IPC arm and contralateral arm) and time (3 levels: American Journal of Hypertension 3
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Pre -Brachial FMD -Baseline CVC -CVC-responses to local heat
Jones et al.
Pre, Post, and Post+8 days). Our primary outcome variable for microvascular endothelial function was CVC. Similarly, a 2-factor general linear model was used with the same repeated measures factors to analyze the summary data (resting baseline, first peak, nadir, and plateau) during the heating protocol. Significant main effects or interactions were followed up using the least significant difference method for pairwise multiple comparisons.24 For all statistical tests, a 2-tailed comparison was used. All data are reported as mean (±SD) unless stated otherwise, and statistical significance was assumed at P 0.05; Table 2). Forearm microvascular function
Local heating. The local heating protocol induced the typical pattern of an initial peak, followed by a nadir and a subsequent plateau in skin flux, which was present during all assessments for all participants in both arms and during all time points. No significant main effects of time, IPC, or interactions between these factors were evident for flux, mean arterial pressure, or CVC at the first peak, the nadir, or the plateau points during heating (P > 0.05) (Table 3). Discussion
Our study provides a number of observations. First, 7-day daily exposure to unilateral IPC of the arm results in significant improvements in conduit artery endothelial function not only in the arm exposed to repeated IPC, but also in
Table 2. Brachial artery FMD before (Pre) and immediately after (Post) the IPC intervention and 8 days after (Post+8) cessation of the intervention in healthy volunteers (n = 13, intervention and contralateral arms) Intervention arm Intervention group
Diameter, mm FMD, % Shear AUC, 103 Time to peak
Pre
3.8 ± 0.6
Post
3.9 ± 0.4
Contralateral arm Post+8
3.9 ± 0.3
Pre
Post
3.9 ± 0.5
3.8 ± 0.4
Two-way ANOVA P values Post+8
3.7 ± 0.4
IPC
Time
IPC × time
0.88
0.42
0.38
5.0 ± 2.2
6.1 ± 2.2
5.4 ± 2.2
6.0 ± 2.2
0.41
0.03
0.71
17.9 ± 12.9
13.6 ± 6.2
16.4 ± 8.1
12.8 ± 5.9
13.1 ± 8.3
16.6 ± 8.6
0.32
0.56
0.47
56 ± 31
47 ± 21
58 ± 35
52 ± 25
49 ± 23
50 ± 18
0.65
0.48
0.37
6.6 ± 2.3*
7.5 ± 2.2*
Data are mean ± SD. Bold numbers represent significant main effects (P < 0.05). Abbreviations: ANOVA, analysis of variance; AUC, area under the curve; FMD, flow-mediated dilation; IPC, ischemic preconditioning. *Post hoc significantly different from Pre (P 0.05).
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“true” adaptation. To control for this effect, we repeated postintervention measurements (Post+8 days). We found that the increase in brachial artery and skin microcirculation endothelial function remained significantly elevated in both arms 8 days after cessation of the IPC intervention. This finding infers that repeated IPC induces sustained local and remote functional adaptation of the conduit artery endothelium. The late phase of protection is thought to be induced by de novo synthesis of cardioprotective proteins, such as upregulation of inducible nitric oxide synthase.33 These changes may contribute to a higher NO production and improved conduit artery FMD, i.e., a response that is mediated, at least partly, through NO.34 An interesting observation in our study is the significant decline in blood pressure after IPC. A recent study also reported a blood pressure–lowering effect of IPC in normotensive participants after 3 days of daily IPC.35 Interestingly, many of the suggested mechanisms of (remote) IPC, such as hormonal5 and neuronal effects by the autonomic nervous system,8 also contribute to the regulation of the systemic blood pressure. Previous data report an inverse relation between brachial artery FMD and blood pressure.36 Accordingly, the enhanced (systemic) endothelial function in our study may contribute to the lower blood pressure in our participants. Alternatively, methodological aspects may explain the decline in the blood pressure. First, we have adopted a single measure of blood pressure rather than repeated measurements. Secondly, anxiety before the preintervention may have affected measurements and overestimated preintervention values. Limitations. We did not examine endothelium-independent vasodilation. However, we26 and others14 have demonstrated that endothelium-independent function typically does not change over time in healthy participants. Thus, it is unlikely that the improvement in endothelial function in our study is explained by changes in endothelium-independent dilation. Second, we were not able to examine the peak CVC, which some authors use to normalize skin CVC responses. However, peak CVC responses typically do not change across time. Finally, we did not include a control group within the study to control for potential bias in our study. Clinical relevance. Endothelial dysfunction is an important event in the atherosclerotic cascade and predicts cardiovascular endpoints.37,38 Our data demonstrate that IPC, which represents a simple, virtually cost-free, and noninvasive intervention, improves endothelial function in the intervention and contralateral arms in healthy individuals. Although speculative, these local and systemic effects of IPC on endothelial function may have clinical relevance, especially in individuals with impaired micro- or macrovessel functions (e.g., type 2 diabetes), where vascular function is a primary target to improve cardiovascular risk and associated complications. Whether repeated IPC improves micro- and macrovessel functions in individuals with endothelial dysfunction, or in those with conditions that require cardiovascular risk management, is currently unknown, and this should be the subject of future research. Furthermore, such studies should also examine how long adaptations in endothelial function and skin microcirculation remain preserved.
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the contralateral arm. Second, we observed a higher forearm CVC in both forearms. These findings imply that 7-day IPC results in both local and remote improvements in conduit artery endothelial function and forearm CVC. Third, we found that these bilateral improvements in conduit artery endothelial function and forearm CVC persist up to 8 days after intervention. These observations suggest that IPC is an intervention capable of improving endothelial function and skin microcirculation both locally and systemically and that these beneficial effects remain present for at least 7 days after the cessation of the IPC stimulus. IPC is typically used to prevent endothelial dysfunction induced by ischemia-reperfusion injury.4,7,8,25 A single bout of IPC may also acutely change endothelial function,13 although results are conflicting.4,8 Furthermore, preliminary evidence suggests that repeated IPC improves resistance artery endothelial–dependent vasodilation.14 We extend this knowledge with the observation that 1 week of daily IPC leads to a significant improvement in conduit artery endothelial function. The potent vascular effects of daily IPC are further emphasized by the increased forearm CVC. Although these observations provide evidence for vascular adaptations after IPC throughout the vascular tree, we add the novel observation that these vascular adaptations are also present in remote areas. Indeed, a simultaneous increase in endothelial function and CVC was found in the contralateral arm, i.e., the arm not directly exposed to IPC. Thus, our data support the use of IPC to induce local and systemic improvements in conduit artery endothelial function and skin microcirculation in humans. Our study was not designed to examine mechanisms that relate to the local and remote effects of 7 days of IPC. Therefore, we can only speculate about the potential underlying mechanisms. Shear stress represents a major physiological stimulus responsible for improvement in conduit artery function26 and skin microcirculation.27 Although episodic increases in shear stress may contribute (partly) to the adaptations found in the intervention arm, it is unlikely that shear represents the principle mechanism responsible for the remote effects of IPC because elevations in shear in remote areas are only modest. Systemic stimuli or circulating markers activated by IPC more likely explain the remote improvement in conduit artery endothelial function. For example, IPC leads to an increase in vascular endothelial growth factor and endothelial progenitor cells,14 which may improve endothelial function in remote areas.28 Indeed, tissue ischemia29 and shear,30 both prominent during IPC, are key stimuli for endothelial progenitor cell release from bone marrow into the circulation. An alternative explanation relates to reduced levels of oxygen free radicals and inflammation because IPC improves antioxidative defence mechanisms (e.g., increased superoxide dismutase activity) and/ or lowers the generation of oxidative stress.4,31,32 Although future studies should further examine the exact mechanisms by which IPC improves the vasculature, it is likely that systemic pathways contribute to the effects. Because the late phase of protection coincides with our postintervention measurements, the increased FMD and CVC after the 7-day IPC intervention may relate to the late phase of protection of the last IPC bout rather than a
Effect of 7-Day IPC on Endothelial Function
In conclusion, 7 days of daily exposure to IPC leads to local (intervention arm) and systemic (contralateral arm) improvements in conduit artery endothelial function and elevation of resting skin microcirculation, which are present beyond the late phase of protection of IPC. These findings may have clinical relevance because improving endothelial function independently prevents cardiovascular morbidity and mortality.38 Furthermore, repeated IPC increased resting microcirculation by approximately 30%, a clinically relevant improvement, especially because the increase was similar in a remote vascular bed not exposed to IPC. Although future studies should further examine the impact of repeated IPC in clinical groups with endothelial dysfunction, our findings highlight the potential for IPC as a simple, safe, and feasible therapeutic tool.
D.H.J.T. is recipient of the E. Dekker stipend (Netherlands Heart Foundation, 2009T064). D.J.G. is funded by the Australian Research Council (DP 130103793). H.J. and N.H. contributed equally to this work. DISCLOSURE
The authors declared no conflict of interest.
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ACKNOWLEDGMENTS
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