Inhibition of a sympathetic stimulus (i.e., sympatholysis) during forearm exercise is reduced with age in women. This age-related alteration has not been characterized in the lower extremity vasculature of women, and the potential for blunting of the conduit artery dilatory response to a sudden increase in shear stress [flow-mediated dilation (FMD)] has not been examined in older adults of either sex. In the present study, we assessed popliteal artery diameter and velocity (Doppler ultrasound) in 16 young (23 ± 1 yr) and 14 older (69 ± 1 yr) women after 5 min of distal calf occlusion (FMD), 3 min of hand immersion in ice water [cold pressor test (CPT)], and 5 min of distal calf occlusion combined with hand immersion in ice water (FMD+CPT). Peak popliteal conductance after 5-min ischemia was not significantly different in young vs. older women. During the combined stimulus (FMD+CPT), the magnitude of vasoconstriction in the calf (reduction in peak popliteal artery conductance) was similar (5–8%), despite reduced resting adrenergic sensitivity to CPT [young (Y): −27.3 ± 3.8%; older (O): −15.8 ± 2.2%; P < 0.05] and blunted muscle sympathetic nerve activity responses to CPT (Y: 12.7 ± 3.6 bursts/min; O: 7.8 ± 2.5 bursts/min; P < 0.05) in older women. Popliteal FMD, normalized to the shear stimulus, was attenuated by 60–70% in older women. Peak popliteal diameter, measured during the combined stimulus (FMD+CPT), was blunted in young but not in older women (Y FMD: 5.5 ± 0.1 mm; Y FMD+CPT: 5.4 ± 0.1 mm; P = 0.03; O FMD: 5.8 ± 0.2 mm; O FMD+CPT: 5.8 ± 0.2 mm). These results confirm previous findings of diminished reactivity in the conduit arteries of older humans and provide the first evidence of reduced sympatholysis in the leg resistance vasculature of older women.
- vascular reactivity
- endothelial function
dynamic large muscle mass exercise evokes two competing influences: vasodilation in the working muscle vasculature, the purpose of which is to meet metabolic demand, and sympathetic vasoconstriction in blood vessels of both inactive and active circulations to maintain systemic blood pressure (41, 43, 46). A characteristic of dynamic exercise, therefore, is that vascular conductance in the exercising muscle will be determined in part by the balance between vasoconstriction and vasodilation, such that metabolic vasodilators inhibit the vascular effects of augmented sympathetic outflow (commonly referred to as functional sympatholysis; Refs. 18, 39, 43).
There is accumulating evidence to suggest that sympatholysis is reduced with age during two-leg cycling in men (22), handgrip exercise in men (12), and handgrip exercise in women (17); this could be one mechanism underlying age-related alterations in muscle blood flow and vascular control during exercise (36). An alternative approach to studying the balance between vasodilation and augmented sympathetic tone that has been used in young adults involves the application of an acute sympathetic stimulus during brachial artery flow-mediated dilation (FMD) (14, 19, 25, 48, 49). Doppler ultrasound-derived measurements of FMD, the dilatory response to local ischemia, offer the advantage of investigating vasodilation through both the resistance arteriole response to occlusion (the immediate hyperemic response on release of occlusion, as quantified by changes in flow and vascular conductance) as well as the conduit artery response to increased fluid shear stress (the percent dilation of the conduit, normalized to the shear stimulus). In addition, a carefully timed sympathetic stimulus can be applied such that the peak constrictor stimulus coincides reproducibly with peak dilatory responses. Finally, FMD can be studied in the lower-extremity vasculature, a vascular bed that is more relevant to the control of blood flow and systemic pressure during large muscle mass exercise, without being confounded by age-related alterations in exercise tolerance, baroreceptor responsiveness, or central limitations (e.g., cardiac output, systemic pressure) to leg vasodilation.
Accordingly, we sought to determine the effect of an acute elevation in sympathetic tone (cold pressor test, CPT) on both the ischemia-induced dilation of resistance vessels and the conduit artery response in the popliteal artery of young vs. older women, as the effect of age on lower-limb sympatholysis has not been characterized in women. In light of the above-mentioned studies (14, 17), we hypothesized that both resistance vessel dilation and popliteal artery FMD would be blunted to a greater extent in older compared with young women, suggestive of reduced sympatholysis with age.
Sixteen young (20–30 yr) and fourteen older (62–74 yr) women completed the study. All subjects were nonobese (body mass index ≤ 30 kg/m2) nonsmokers and had clinically normal blood chemistry (i.e., hemoglobin concentrations ranged from 11.6 to 14.8 g/dl, total cholesterol ≤240 mg/dl, LDL cholesterol ≤150 mg/dl) and resting supine ankle-brachial index (ABI) ratings (ABI between 0.90 and 1.30; VP2000, Colin Medical). All subjects were normotensive (resting blood pressure ≤140/90 mmHg) and were neither extremely sedentary nor extremely fit [i.e., had cycle ergomenter peak oxygen uptake (V̇o2 peak) values between 20% and 80% of age-predicted norms; Ref. 2]. Subjects were free of overt chronic diseases as evaluated by medical history questionnaire, a physical examination, and resting ECG. Percent body fat, estimated by dual-energy X-ray absorptiometry (model QDR 4500W, Hologic, Waltham, MA), did not differ between groups. Additionally, no subjects were taking medications having significant hemodynamic effects, including oral contraceptives (young) or hormone replacement therapy (older) for at least the last 12 mo. Younger subjects were studied in days 1–7 of their menstrual cycle to minimize the influence of female hormones. On study day, subjects were asked to refrain from caffeine, aspirin, ibuprofen, or herbal supplements for at least 12 h before testing. All subjects gave their written, informed consent to participate. This study was approved by the Office for Research Protections and the Institutional Review Board at Pennsylvania State University. Subject characteristics are presented in Table 1.
Subjects also completed a physical activity questionnaire to assess routine physical activity [Yale Physical Activity Questionnaire (13) for older and Baecke Questionnaire of Habitual Physical Activity (4) for young]. None of the subjects had participated in moderate- to high-intensity aerobic exercise >3 days/wk or regular lower-body resistance training >2 days/wk during the past 12 mo. To objectively quantify aerobic fitness status, all of the subjects performed a continuous incremental leg cycle ergometer test (Lode) to maximal exertion to determine V̇o2 peak, as described in detail previously (35).
All subjects participated in parts 1–4 of the experiment (see Fig. 1) on a single study visit in which Doppler ultrasound was used to assess popliteal diameters and velocities during FMD, CPT, and CPT superimposed on FMD (FMD+CPT). The room was thermoneutral, with temperature consistently maintained at 23.5 ± 0.1°C. Blood pressure was measured at heart level in the dominant arm with an automatic blood pressure monitor (Omron HEM-705CP; Vernon Hills, IL), and heart rate was continuously monitored with a three-lead electrocardiogram. Each part of the experiment was separated by a 15-min rest period. A subset of young and older subjects allowed us to obtain direct recordings of muscle sympathetic nerve activity (MSNA) on a separate day (see part 5 below).
Part 1 (FMD alone).
Popliteal FMD was measured with the subject lying prone with a small pillow under the nondominant ankle. A rapid inflation/deflation pneumatic cuff (D. E. Hokanson; Bellevue, WA) was placed around the calf, 2–3 in. distal to the popliteal fossa. The artery was imaged immediately proximal to the bifurcation (usually at or slightly above the popliteal fossa) with a 6- to 11-MHz multifrequency linear array probe attached to an Acuson Aspen duplex ultrasound imaging system (Siemens, New York, NY); once a satisfactory image using optimal B-mode imaging was obtained, the placement of the probe was marked on the leg to ensure that the site of measurement did not change during the trial. Ultrasound parameters were not changed during the study. Doppler velocity was also measured with the Aspen, using a 60° angle of insonation held constant throughout the study. After the subject had rested in the prone position for 10 min, resting popliteal artery diameter and velocity were measured for 1 min before inflation of the pneumatic cuff. The cuff was then inflated to ∼300 mmHg for 5 min; diameter and velocity recordings resumed 1 min before cuff deflation and continued for 3 min after inflation.
Part 2 (cold pressor stimulation alone).
Baseline Doppler measurements (diameter and velocity) were taken for 1 min. The subject was then instructed to immerse her dominant hand (up to the wrist) in a 0–1°C mixture of ice and water for 3 min while maintaining a steady, relaxed breathing pattern, consistent with previous studies using this sympathetic stimulus (44, 54). Diameters and velocities were measured throughout the ice water immersion, and measurements continued for 3 min of recovery after the subject removed her hand from the ice-water mixture.
Part 3 (FMD + cold pressor stimulation).
Baseline Doppler measurements (diameter and velocity) were taken for 1 min. The FMD protocol was repeated in an identical fashion except that CPT (as described above) was superimposed on the protocol such that ice water immersion started at minute 3.5 of the 5-min occlusion and continued for 3 min until 1.5 min after cuff release. This timing ensured that a sufficient sympathetic stimulus would occur both during resistance arteriole dilation (i.e., occlusion) as well as during the ensuing conduit artery dilation so that both components of FMD could be examined with respect to CPT (44, 54). Recovery data were collected for 3 min after the end of CPT.
Part 4 (sublingual nitroglycerin).
Endothelium-independent dilation of the leg was assessed through administration of 0.4 mg sublingual nitroglycerin (NTG), a NO donor eliciting maximal dilation representative of smooth muscle function. After 1 min of baseline diameter measurements, diameters were measured continuously for 10 min after administration of NTG.
Part 5 (MSNA responses to cold pressor stimulation).
On a separate visit, a subset of subjects (7 young and 7 older) participated in part 5. To assess neural sympathetic outflow, peroneal nerve MSNA was measured continuously before, during, and after CPT. CPT was repeated as described above, with the exception that resting measurements were obtained for 3 rather than 1 min to quantify MSNA. Heart rate, blood pressure, and MSNA measurements were taken for 3 min before (rest) and 3 min after (recovery) the 3-min 0–1°C ice water immersion of the dominant hand (CPT).
Measurements and Calculations
Measurement of popliteal artery diameter and velocity.
For FMD measurements, popliteal artery diameters were measured for 1 min at rest, during the last minute of occlusion, and as the highest diameter observed during 3 min of postinflation imaging. Consistent with the literature, the peak diameter was observed between 50 and 75 s in most subjects (5, 8). Diameter measurements were sampled at end diastole (ECG gating was used to select images that were trigged by the R wave of the cardiac cycle) with Brachial Imager software (Medical Imaging Applications; Iowa City, IA). Posttest analysis of diameters was performed with edge-detection software (Brachial Analyzer Software, Medical Imaging Applications); briefly, the technician (always the same and blind to any subject information) selected a region of interest along the arterial wall, and the edge of the wall was detected by pixel density and represented by a line of best fit. Each sequence of images was reviewed by the technician and adjusted to ensure that diameter measurements were always calculated from the intima-lumen interface at both the distal and proximal vessel wall. Resting and occlusion diameters were calculated as a 10-s average of images taken over the 1-min baseline and last minute of occlusion, respectively. The peak diameter was calculated by identifying the postocclusion image with the largest diameter and averaging that image with the five preceding and five following images. Blood flow velocity was measured as the average velocity during 10 s of rest, the average velocity during 10 s of the last minute of occlusion, and the average (peak) velocity measured over the first 10 s after cuff release. Velocity measurements continued until 45 s after cuff release, at which time two-dimensional (2D) imaging was optimized for diameter measurements. Again, imaging software (Brachial Analyzer Software, Medical Imaging Applications) was used to automatically trace the velocity-time integral of time-averaged, angle-corrected maximum velocities (highest velocity across the cardiac cycle). For NTG measurements, the resting diameter was calculated as a 10-s average of images taken over the 1-min baseline and the peak diameter was calculated as the post-NTG image with the largest diameter, again averaging that image with the five preceding and five following images.
Assessment of dilation.
FMD was calculated as the absolute and percent change in diameter from either rest or the last minute of occlusion to peak. We calculated postocclusion area under the curve (AUC) shear rate (SR), using lower-resolution diameter imaging simultaneously with velocity measurements for 45 s after cuff release (until it was necessary to switch into high-resolution 2D mode to image peak diameters), as recent evidence suggests that AUC measurements may more closely approximate the shear stimulus than peak measurements (37, 38). FMD was then normalized to the 45-s AUC SR (4*velocity/diameter). NTG dilation was calculated as the percent change in dilation relative to baseline.
Calculation of popliteal blood flow and vascular conductance.
Resting, occlusion, and peak popliteal blood flow (PBF) were derived from the formula: where PBF is in milliliters per minute, blood velocity is in meters per second, brachial diameter is in millimeters, and 60 is used to convert from milliliters per second to milliliters per minute. Resting, occlusion, and peak popliteal vascular conductance (PVC) were calculated as PBF/mean arterial pressure (MAP).
Assessment of blunted vasodilatory responses during cold pressor stimulation.
To examine the influence of CPT on peak reactive hyperemia, we calculated the percent reduction in hyperemic PVC during sympathetic stimulation as: where the difference between PVC measured after 5-min calf occlusion and PVC measured when CPT was superimposed on occlusion is divided by PVC measured after 5-min calf occlusion. Additionally, we compared this value with the PVC percent reduction relative to rest observed during CPT alone (PVCrest − PVCCPT/PVCrest * 100) to account for any age-related differences in baseline adrenergic sensitivity. This approach has been used previously to compare vasoconstrictor responsiveness in young vs. older subjects (12).
To examine the influence of CPT on FMD, we examined all components of the dilatory response—absolute diameter changes, percent diameter changes relative to rest or occlusion, 45-s AUC SR, and percent diameter changes normalized to the AUC SR—between FMD and FMD+CPT. This approach has been used previously to assess the influence of a sympathetic stimulus on FMD (14, 25).
Muscle sympathetic nerve activity.
Multiunit postganglionic recordings of muscle sympathetic nerve activity (MSNA) were made with sterile 200-μm-diameter Tungsten microelectrodes of 2- to 3-MΩ impedance inserted percutaneously into muscle nerve fascicles of the peroneal nerve of the dominant leg, as described previously (34). Briefly, a recording electrode was placed in the peroneal nerve at the fibular head or the popliteal fossa, and a reference electrode was placed subcutaneously 2–3 cm from the recording electrode, with the subject in the supine position. The nerve signal was amplified (40,000–70,000), band-pass filtered (0.7 kHz high pass; 2–3 kHz low pass), and then full-wave rectified and smoothed with a resistance-capacitance circuit (time constant 0.1 s) to produce a recording of “integrated” MSNA. The recording electrode was adjusted until clear sympathetic bursts were recorded and the signal-to-noise ratio of the band-pass filtered neurogram (peak-to-peak burst amplitude relative to baseline noise) exceeded 2. The microneurographer evaluated the quality of MSNA recordings based on accepted criteria, including 1) pulse synchrony, 2) facilitation during the hypotensive phase of the Valsalva maneuver and suppression during the hypertensive overshoot after release, 3) increases in response to breath holding, and 4) insensitivity to emotional stimuli (loud noises or stressful mental arithmetic) and stroking the skin. ECG (78534A; Hewlett Packard), beat-to-beat blood pressure (radial tonometry of the nondominant hand; Colin, Medical Instruments), and neural recordings were stored on a chart recorder (MT95K2; Astromed) and VCR tape (Vetter) for later analysis. The amplitude of each mass sympathetic discharge was quantified by digitization of the resistance-capacitance-filtered neurogram (SigmaScan, version 2.1, Jandel Scientific). Sympathetic activity was expressed as the frequency of discharges (bursts/min).
Assessment of sympathetic stimulus.
The quotient of the percent change in peak popliteal conductance from FMD to FMD+CPT conditions and the absolute change in MSNA were calculated to determine the relationship between reductions in peak vascular conductance and the stimulus for vasoconstriction.
We determined between-condition Doppler ultrasound measurement reproducibility as well as between-condition and between-visit CPT reproducibility (estimated by peak MAP) according to the following formula (1, 10): Baseline popliteal measurement reproducibility (n = 30) was 0.17% for resting diameters and 5.18% for resting SR. FMD popliteal measurement reproducibility (n = 10; subjects studied on a different visit) was 9.54% for peak diameters, 16.05% for peak SR, and 19.7% for FMD. Peak MAP reproducibility was 0.48% between conditions for study visit 1 and 0.86% between study visit 1 and study visit 2.
Statistical analyses were performed with Minitab (Minitab, State College, PA) and SPSS (SPSS 13.0, Chicago, IL) software. All data are reported as means ± SE, and significance was set at P < 0.05. A Student's t-test for independent groups and Tukey post hoc analysis were used to compare baseline differences between young and older groups. Repeated-measures ANOVA models were used to assess the effects of within-group differences (i.e., values at different time points or conditions) and between-group differences (i.e., influence of age) on different response variables such as PBF, PVC, MAP, MSNA, and normalized FMD.
Influence of Age on Peak Reactive Hyperemia and Vascular Conductance
After cuff release, peak blood flow [young (Y): 850 ± 85 ml/min; older (O): 848 ± 69 ml/min] and peak vascular conductance (Y: 10.0 ± 0.8 ml·min−1·mmHg−1; O: 9.5 ± 0.8 ml·min−1·mmHg−1) were similar between young and older women (P > 0.40 for both comparisons).
Effects of Cold Pressor Stimulation of Resistance Vessels of Young vs. Older Women
Young subjects demonstrated a trend toward a greater percent reduction in resting blood flow (Y: −7.0 ± 3.7%; O: −3.3 ± 4.5%; P = 0.09) and a significantly greater percent reduction in PVC during CPT (P = 0.03) (Fig. 2). After superimposition of CPT on FMD, peak blood flow was significantly augmented relative to FMD alone (Y: P < 0.01; O: P = 0.04) in both subject groups (Y: 1,016 ± 80 ml/min; O: 1,039 ± 109 ml/min); however, there was no difference in peak blood flow between subject groups (P = 0.86). PVC was still similar in both age groups (Y: 9.2 ± 0.6 ml·min−1·mmHg−1; O: 9.0 ± 0.9 ml·min−1·mmHg−1; P = 0.58) and was reduced similarly (P = 0.79) respective to FMD alone. In a subset of subjects matched for baseline adrenergic responsiveness to CPT, older subjects demonstrated a significantly greater (P = 0.04) reduction in peak popliteal conductance during FMD+CPT.
Influence of Age on FMD Responses
Absolute diameter responses are shown in Fig. 3. Because of the significant dilation observed during occlusion in older women we estimated FMD as both the percent dilation (%D) from rest to peak as well as occlusion to peak conditions (%D from rest: Y, 8.4 ± 0.9%; O, 3.2 ± 0.4%; %D from occlusion: Y, 8.1 ± 0.9%; O, 2.3 ± 0.5%; both P < 0.01). The 45-s AUC SR was attenuated (P < 0.01) in older subjects (Y AUC: 15,930 ± 877 s−1; O AUC: 13,351 ± 1,088 s−1). Normalization of resting or occlusion FMD to the 45-s AUC SR, which is believed to most closely represent the shear stimulus (38), still yielded an age-associated decrease in the dilatory response to 5-min distal occlusion (Fig. 4).
Effects of Cold Pressor Stimulation on Conduit Artery Responses of Young vs. Older Women
There were no changes in popliteal diameter during or after CPT alone in either young or older subjects. When CPT was superimposed on FMD, the dilatory response measured relative to baseline was not different from the FMD condition in young (P = 0.31) and older (P = 0.20) subjects (Y: 7.5 ± 1.0%; O: 2.0 ± 0.6%). However, the dilatory response measured relative to occlusion was blunted in young (P = 0.05) but not older (P = 0.36) subjects (Y: 6.3 ± 0.8%; O: 1.6 ± 0.8%), as young subjects displayed a significant popliteal dilation during occlusion and a reduced peak diameter (Fig. 3). AUC SR was unchanged (P = 0.37 and 0.38, respectively) between conditions in young and older women (Y FMD+CPT: 15,070 ± 867 s−1; O FMD+CPT: 12,123 ± 800 s−1); however, in the young subjects peak SR was significantly higher and the decay curve was significantly steeper when CPT was superimposed on FMD (Fig. 5), leading to a similar estimate of AUC SR despite different response curve characteristics. These SR changes were not observed in older subjects. Finally, the resting dilatory response normalized to the AUC SR was not significantly reduced in either subject population (P = 0.60 and 0.26 for young and older, respectively), whereas the occlusion dilatory response normalized to the AUC SR was significantly reduced in young but not older subjects (Fig. 6). In a subset of subjects (n = 7/group) matched for resting FMD (%D/s−1: Y, 0.00035 ± 0.00003; O, 0.00033 ± 0.00004), peak diameter was still unchanged in older women (FMD peak diameter: 5.96 ± 0.21 mm; FMD+CPT peak diameter: 5.90 ± 0.23 mm; P = 0.47) such that normalized FMD estimated relative to occlusion was attenuated in young but not older women (Y FMD: 0.00065 ± 0.0002 %D/s−1, Y FMD+CPT: 0.00050 ± 0.0001 %D/s−1; O FMD: 0.00010 ± 0.0001 %D/s−1; O FMD+CPT: 0.00010 ± 0.0001 %D/s−1).
Influence of Age on Sublingual NTG Responses
Endothelium-independent dilation was reduced in older subjects (Fig. 7) such that normalization of FMD (calculated relative to rest or occlusion diameter) to NTG-induced dilation abolished age-group differences [resting FMD%/NTG%: Y, 1.1 ± 0.2%, O 1.0 ± 0.3% (P = 0.79); occlusion FMD%/NTG%: Y, 1.1 ± 0.2%, O 0.7 ± 0.3% (P = 0.30)].
Influence of Age on Blood Pressure and MSNA (n = 7/group) Responses to Cold Pressor Stimulation
While MAP was significantly higher in older subjects at most time points before, during, or after CPT, the change from rest to peak measured during CPT was similar in young vs. older subjects (Y ΔMAP: 22.5 ± 2.3 mmHg; O ΔMAP: 24.9 ± 2.6 mmHg; P = 0.50). In the subset of subjects in whom MSNA was measured, young and older subjects exhibited similar peak MSNA during CPT (Y: 26.6 ± 5.7 bursts/min; O: 36.3 ± 2.3 bursts/min; P > 0.60), but young subjects demonstrated a greater change in MSNA from rest to peak measured during CPT (Y ΔMSNA: 12.7 ± 3.6 bursts/min; O ΔMSNA: 7.8 ± 2.5 bursts/min; P < 0.05) as a result of lower resting MSNA (Y: 13.8 ± 2.9 bursts/min; O: 28.5 ± 2.9 bursts/min; P < 0.01). In this subset of women, the reduction in peak popliteal conductance following CPT (Y: −5.9 ± 5.2%; O: −8.4 ± 13.6%) divided by the absolute change in MSNA was greater in older women (Y: −0.7 ± 0.8 %·burst−1·min−1; O: −5.6 ± 4.8 %·burst−1·min−1), although this age difference was not statistically significant (P = 0.21) because of the limited sample size.
Recent evidence suggests that the blunting of sympathetic vasoconstriction in resistance vessels (sympatholysis) may be reduced in exercising limbs of older adults. This age-related alteration has not been characterized in the lower-extremity vasculature of women, and the potential for blunting of the conduit artery dilatory response to a sudden increase in shear stress (i.e., FMD) has not been examined in older adults of either sex. The present study utilized a standardized hyperemic stimulus (5 min of distal calf ischemia) and application of a robust sympathetic vasoconstrictor stimulus (3 min of cold pressor stimulation spanning the last 1.5 min of ischemia and the first 1.5 min after cuff release) in healthy young vs. older women to test the hypothesis that aging reduces lower-limb sympatholysis in women. Popliteal artery FMD responses to sublingual NTG were also studied to determine whether there may be a smooth muscle contribution to any age-dependent dilatory responses. The primary new findings were that 1) lower-limb resistance vessel sympatholysis appears to be less effective in healthy older vs. younger women when the vasculature is vasodilated in response to 5 min of ischemia and 2) FMD of the popliteal artery is altered during acute sympathetic stimulation in young, but not older, women.
Is the Vasodilatory Response to 5 min of Ischemia Preserved in the Lower Legs of Older Women?
After 5 min of distal cuff occlusion, there was no age difference in reactive hyperemia (expressed as either peak popliteal artery blood flow or conductance). These findings differ from previous findings of blunted calf hyperemia with age following exhaustive ischemic exercise (27) and 10 min of passive proximal cuff occlusion (40) in women. These disparities are likely attributable to 1) the different dilator stimulus used in the present study, as both ischemic calf exercise and 10 min of proximal occlusion evoke significantly greater and more sustained hyperemia than 5 min of distal occlusion (27), and 2) differences in blood flow measurement techniques between the present study (Doppler ultrasound) and the aforementioned studies (plethysmography).
Is There Evidence of Reduced Sympatholysis in Lower-Leg Resistance Vessels of Older Women?
In agreement with the well-documented age-related decrease in resting adrenergic responsiveness (12, 45), CPT reduced resting popliteal conductance in older women by ∼15% compared with the ∼28% reduction observed in young women. After superimposition of CPT on FMD, the percent reduction in peak popliteal conductance relative to FMD alone was similar in young vs. older women (∼5–8%) (Fig. 2), which is notable given that the increase in MSNA (i.e., efferent sympathetic stimulus) was blunted in older women. Furthermore, in a subgroup comparison of nine young and nine older women who had similar reductions (20 ± 3%) in popliteal conductance to CPT at rest, older women had approximately twice the reduction in peak popliteal conductance compared with young women when CPT was superimposed on FMD (∼12% vs. 6%) (Fig. 2). In addition, superimposing CPT on FMD resulted in a significant augmentation of the peak SR in young (Fig. 5) but not older women. Since CPT raises systemic pressure, and calculations of SR do not account for perfusion pressure, a plausible explanation for this finding is that CPT augmented peak SR in young women because of an intact sympatholysis combined with increased perfusion pressure. By contrast, the increased perfusion pressure in older women did not translate into an increase in peak SR, even though increases in MAP during CPT were identical in both subject groups. Taken collectively, these results provide evidence to suggest that sympatholysis is blunted with age in the lower-extremity resistance vasculature in women.
The metabolic milieu [i.e., release of vasoactive substances such as NO, prostaglandins (PG), endothelium-derived hyperpolarizing factor (EDHF), and endothelin (ET)] of the calf resistance vasculature immediately before and after cuff release is not known, especially since the release of dilators and constrictors is transient and may change with age, sex, oxidative stress, and metabolite bioavailability (20, 24, 51, 52). Certainly, one attractive hypothesis is that release of NO, which may underlie peak reactive hyperemia (15, 23), attenuates adrenergic vasoconstriction (7, 9) such that there is an age-related decrease in the NO-mediated modulation of CPT in the calf resistance vasculature. However, other interactions in the resistance vasculature among dilators, constrictors, and/or ion channels (18) may also underlie metabolic inhibition of an acute sympathetic stimulus, rendering specific conclusions difficult without further research.
Effect of Age on Popliteal FMD
Given the significant popliteal dilation observed in older women during cuff occlusion in this study (Fig. 3) as well as our previous study (32), attributable perhaps to enhanced myogenic responsiveness with age (26), we estimated peak dilation relative to occlusion as well as baseline. As stated previously, our rationale for this approach is that inasmuch as FMD represents the dilatory response to the increase in fluid shear stress after occlusion, the expression of dilation should only represent changes in vessel diameter occurring in direct relation to the shear stimulus rather than those evoked through occlusion, such as changes in transmural pressure (32). Relative to occlusion diameter, older women exhibited an ∼70% lower popliteal FMD than their younger counterparts. Calculating FMD relative to resting diameter resulted in a 60% reduction in conduit dilation in older vs. young women. Interestingly, the shear stimulus, estimated as the 45-s post-cuff release AUC to reflect current thought concerning the true shear stimulus for FMD (38), was significantly lower in older women because of the similar blood velocity responses to ischemia yet larger overall diameters. Taking into account the reduced shear stimulus by normalizing the FMD response to these estimations of the shear stimulus (Fig. 4) did not influence the magnitude of the age-associated reduction in FMD, in agreement with our recently published findings (32). Cumulatively, these data suggest that conduit artery dilation is influenced by age in women similar to what has been observed in the forearm (6, 21), resulting in a net dilatory deficit.
The blunted conduit artery dilation in older women could represent increased popliteal stiffening (11, 53), an altered balance of dilator (reduced) and constrictor (increased) release (28, 47, 52), or diminished smooth muscle responsiveness (3, 30, 42). The latter is supported by the reduced popliteal dilation we observed in older women (Fig. 4) in response to NTG administration, as NTG is a NO donor that evokes endothelium-independent dilation, assessing smooth muscle function. However, while the similar percent reduction in FMD and NTG responses makes it tempting to attribute blunted FMD in older women entirely to smooth muscle dysfunction, we would caution that this line of reasoning may be an oversimplification as the pathways underlying FMD in the leg, with age, and in women have not been elucidated. For example, it is possible that FMD is not NO dependent in the lower-extremity vasculature of women and thus blunted FMD encompasses alterations in dilator pathways (e.g., EDHF and PG) that are not assessed by or comparable to the smooth muscle response to a NO donor.
Effects of Acute Sympathetic Stimulation on Popliteal FMD.
In young women, superimposing CPT on FMD resulted in 1) a significant dilation during occlusion and reduction in peak diameter (Fig. 3), 2) an alteration in the postocclusion SR profile (Fig. 5) and 3) an ∼20% decrease in normalized popliteal FMD, when data were calculated relative to occlusion (Fig. 6). When FMD was calculated relative to rest, normalized FMD was not significantly influenced by adrenergic stimulation in young women. By contrast, popliteal dilation estimated relative to rest or occlusion was not significantly blunted in older women after superimposition of CPT on FMD (Fig. 6). These findings underscore the importance of analyzing FMD with respect to both resting and occlusion diameters since changes in conduit artery diameter during occlusion occur through different mechanisms as those evoked by reactive hyperemia and may affect estimation and assessment of endothelium-dependent dilation. Additionally, the application of CPT on FMD in the popliteal artery in young women did not yield results similar to what has been published in the brachial artery in young men [i.e., in young men, CPT blunts FMD with no change in shear rate or occlusion diameter (14)], highlighting the importance of recognizing limb and sex differences in vascular regulation.
The differential effect of CPT on diameter measured during occlusion is puzzling, given that there were no changes in popliteal diameter during CPT alone in young women that would yield insight into a possible mechanism underlying the dilation, such as greater β-adrenergic stimulation (50). An alternative possibility is that interactions between myogenic tone and norepinephrine-induced vasoconstriction, similar to what has been observed in studies of isolated rat arterioles (16, 29), lead to facilitation of the myogenic response to occlusion in young women that cannot occur in older women because of their already augmented myogenic response to occlusion and/or reduced adrenergic sensitivity. However, human data on this topic are lacking.
Contrary to our hypothesis, peak diameters were reduced in young but not older women. To investigate the possibility that this finding might be an artifact of a baseline effect in older women (i.e., the already reduced FMD observed in older women under normal conditions may be prohibitive of further reductions in the dilatory response when CPT is superimposed on FMD), we examined diameter changes in seven young and seven older women matched for baseline FMD. The observed findings (augmented dilation during occlusion, reduced peak popliteal diameter) persisted in young, but not older women, suggesting that a baseline effect cannot completely explain our results. Alternative explanations are that the dilatory mechanisms underlying popliteal FMD differ in young vs. older women such that CPT only influences the endothelium-dependent dilators utilized by young women (55), vasodilation of the popliteal artery unmasks a reduction in conduit adrenergic sensitivity in older women that was not detectable at rest given the absence of conduit artery responses to CPT alone in either subject group, or the altered SR profile in young women provided a reduced dilatory stimulus even though total AUC was similar between conditions. Finally, given that there is evidence suggesting that the popliteal artery stiffens significantly with age in women (11, 53), it is possible that our findings represent a general, age-related reduction in popliteal vascular reactivity, as indicated by the blunted responses both to occlusion and to superimposition of CPT on FMD.
During the primary study visit (parts 1–4), the order of experiments was not randomized and did not change between subjects since we did not want the systemic release of epinephrine induced by CPT to affect our baseline measurements (48). However, we do not believe that this study design limited interpretation of our findings, as popliteal diameters and velocities always returned to baseline before the next protocol was started and pilot testing demonstrated similar influence of CPT and/or FMD on the popliteal artery irrespective of time course.
We chose to administer CPT to acutely raise sympathetic tone based on previous results suggesting that the absolute increase in MSNA as well as norepinephrine and epinephrine release to CPT are not significantly altered by age in women (22, 31, 33). Interestingly, we observed that our subset of older women displayed blunted changes in MSNA during CPT, resulting from higher resting MSNA accompanied by a similar peak response to CPT. Thus it is possible that the present findings were influenced by the nature of the sympathetic stimulus. However, given the list of unknowns concerning age and adrenergic stimulation—the efficacy of the efferent signal that stimulates norepinephrine release, neuronal reuptake and spillover, transmission of norepinephrine into the intimal-medial layer, adrenergic receptor density and distribution, and downstream signaling efficacy—we cannot distinguish between findings directly applicable to age and those attributable to an age-stimulus interaction.
Finally, our Doppler ultrasound machine samples the peak, or center, blood velocity envelope rather than an intensity-weighted mean blood velocity, as described previously (32). Estimating PBF, conductance, and SR with peak blood velocity could introduce error into our measurements should velocity parabolas be different between the two subject groups. To this end, Silber et al. have collected MRI-based femoral artery data on 63 young and older healthy subjects (men and women) suggesting that velocity profiles are similar (H. A. Silber, personal communication). Thus we do not believe that sampling peak blood velocity introduces age-related bias into our measurements.
This study provides evidence that reactivity of the lower-leg conduit vasculature as well as inhibition of sympathetic vasconstriction in the leg resistance vasculature are diminished with age in women. In particular, the latter finding suggests that control of leg vascular conductance during conditions in which release of dilator and constrictor substances are acutely augmented is altered with age such that metabolic inhibition of sympathetic neural outflow is less effective in older compared with young women.
This research was supported by an ACSM Foundation Research Grant (FRG) from the American College of Sports Medicine Foundation, National Institute on Aging (NIA) Grant R01-AG-018246 (D. N. Proctor), NIA Interdisciplinary Training in Gerontology Grant T32-AG-00048 (B. A. Parker), National Aeronautics and Space Administration Grant NNJ04HF45G (J. A. Pawelczyk), and Division of Research Resources Grant M01-RR-10732 (GCRC).
We acknowledge the assistance of Aaron Mishkin with data collection, Drs. Sheila West and Penny Kris-Etherton for use of the Doppler ultrasound machine, as well as the University Park General Clinical Research Center (GCRC) clinical staff.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
- Copyright © 2007 by the American Physiological Society