Alteration in Carotid Arterial Stiffness During Passive Leg Raising May Reflect Vascular Endothelial Function

Abstract

Background: Flow-mediated dilation (FMD) is the established parameter of endothelial function but requires skill and specialized equipment. This study aimed to investigate whether changes in carotid artery ultrasound parameters during passive leg raising (PLR) could reflect FMD values.

Methods and Results: Thirty-six adult males underwent standard FMD measurement. After 15 min of rest, a carotid artery ultrasound was performed to measure the maximal common carotid artery (CCA) diameter and stiffness parameter β. The PLR maneuver was then performed, and the change in these parameters (∆CCA_PLR and ∆β_PLR) was calculated. There were 6 participants with decreased FMD value (<4%). While the maximal CCA diameter remained unchanged during PLR (P=0.54), the stiffness parameter β significantly decreased during PLR compared with baseline (P=0.014). Among several carotid artery ultrasound parameters, ∆β_PLR correlated most strongly with FMD (r=−0.70; P<0.001). Receiver operating characteristic analysis showed that ∆β_PLR predicted decreased FMD with an area under the curve of 0.89, sensitivity of 87%, and specificity of 83% at an optimal cut-off of 4.7%.

Conclusions: Change in carotid arterial stiffness parameter β during the PLR maneuver correlated with FMD, suggesting it may serve as an alternative indicator for endothelial function.

Central Figure

Outline of this study.

Atherosclerosis is known to be the basis of various cardiovascular diseases. Deaths related to atherosclerosis are also very common worldwide, and the benefits of preventing the development of atherosclerosis are significant.^1–4 Vascular endothelial dysfunction is the first stage of atherosclerosis, and several conditions and lifestyle habits, such as hypertension, diabetes, smoking, and lack of exercise, are known as factors that promote endothelial dysfunction.⁵ Vascular endothelial dysfunction can recover to an adequate level for a patient’s age by improving lifestyle habits.^3,6,7 Therefore, early detection of vascular endothelial dysfunction and its elimination through lifestyle modification is important because it improves individual life expectancy and quality of life.

Flow-mediated dilation (FMD) is one of the established standard methods for quantifying vascular endothelial function.^5,8–10 In this test, the forearm is tied for 5 min and then released, which transiently increases brachial artery blood flow and simultaneously increases the shear stress on the vascular endothelium. The increase in shear stress induces nitric oxide (NO) release resulting in dilation of the brachial artery diameter. The FMD value is determined as the change in vessel diameter before and after blood flow is interrupted. The disadvantages of this test are that it requires sufficient skill on the part of the sonographer and that special equipment is recommended to obtain a stable examination.^10,11

The passive leg raising (PLR) maneuver shifts blood pooled in the lower limb veins to the upper body and is commonly used in clinical practice, particularly for its role in increasing the cardiac preload. A previous study investigated changes in brachial artery diameter during PLR as an alternative to FMD.¹² The study reported that although changes in the brachial artery during PLR were significantly correlated with FMD values, the dilating effect was approximately half that of the standard FMD procedure.¹²

Carotid artery ultrasound is closer to the surface of the body and allows for easier acquisition of fine ultrasound images of the vessel wall compared with the brachial artery. The intima-media complex thickness (IMT) of the carotid artery is widely used in routine clinical practice as a method of detecting more advanced atherosclerosis.^13,14 In addition, by measuring the maximum and minimum carotid artery diameters and simultaneously measuring blood pressure, it is also possible to measure the stiffness parameter β.^15,16 We hypothesized that changes in the carotid artery parameters such as diameter and stiffness parameter β following PLR might correspond to FMD values. This study aimed to test this hypothesis.

Methods

Study Participants

The participants were healthy male volunteers (n=36; mean age 23.3±6.9 years) who were fully informed regarding their participation in the study and provided written informed consent to participate in the study of their own free will, in accordance with the Helsinki Declaration of 1964 and later versions. The exclusion criterion was set as poor ultrasonography imaging, but no one was excluded. Smoking history was assessed, and participants were divided into 3 categories: never; former; and current smokers. This prospective study was approved by the Research Ethics Committee of Japan Healthcare University (No. R04-41).

Measurement of FMD

Based on technical guidelines, the standard FMD procedure was performed using an Aplio a Verifia ultrasound system equipped with a 18L7 linear probe (Canon Medical Systems, Otawara, Japan).^10,11,17 The probe was fastened with a locking device (MIST-100H, Saraya Co., Ltd, Osaka, Japan). Participants were placed in a supine position and rested for 10 min with their arm in a comfortable position for imaging the brachial artery. The brachial arterial longitudinal images were taken and the baseline diameter at end-systole was measured as an average value of 3 points (Figure 1).¹¹ The forearm was then tied for 5 min using a specific blood pressure monitor (MIST-2000, Saraya Co., Ltd, Osaka, Japan). The cuff occlusion pressure was automatically adjusted to 50 mmHg above systolic blood pressure. After 5 min, the cuff was released and brachial arterial longitudinal images were recorded for 3 min. After the examination, brachial arterial diameter was measured in the same manner as the baseline every 10s, and the FMD value was calculated as the ratio of the difference between the maximal diameter after cuff release and baseline diameter to the baseline diameter. In the present study, FMD examinations were conducted at various times and without any dietary or smoking restrictions.

Figure 1.

Measurement of brachial artery diameter to assess flow-mediated dilation.

Carotid Artery Ultrasound With PLR

After the FMD measurement, the participants were rested for 15 min in a supine position, and a carotid artery ultrasound was then performed with the same machine and probe. The examiner took the image of the right common carotid artery (CCA) and measured the maximal and minimal diameters and IMT in accordance with current guidelines.^13,18 Systolic and diastolic blood pressures were also measured, and the stiffness parameter β was calculated as follows:

  

$$\beta =\ln (\text{Ps/Pd})/[\Delta \text{D/Dd],}$$

where Ps is systolic blood pressure, Pd is diastolic blood pressure, ∆D is the difference between maximal and minimal diameters of the CCA, and Dd is the minimal diameter of the CCA.

After acquiring baseline images, the PLR maneuver was performed. The participants elevated their leg onto a 20 cm footstool, and 1 min later the examiner measured blood pressure and acquired right CCA images. The maximal and minimal CCA diameters during the PLR maneuver were measured and stiffness parameter β was also calculated. The change in maximal CCA diameter and stiffness parameter β during the PLR maneuver (∆CCA_PLR and ∆β_PLR, respectively) were calculated as follows:

  

$$\begin{array}{l}\Delta {\text{CCA}}_{\text{PLR}}=\frac{MaximalCCAdiameterduringPLR-BaselineCCAdiameter}{BaselineCCAdiameter}\\ \times 100(\%)\end{array}$$

  

$$\Delta {\beta }_{\text{PLR}}=\frac{\beta diameterduringPLR-Baseline\beta }{Baseline\beta }\times 100(\%).$$

Statistical Analysis

Statistical analysis was performed using a standard statistical software package (IBM SPSS ver. 27 for Windows; IBM Co., Armonk, NY, USA). All numerical data is presented as mean±standard deviation (SD). Changes in carotid artery ultrasound parameters from baseline to during the PLR maneuver were analyzed using a paired t-test. Differences between the smoking and non-smoking groups were assessed using Welch’s t-test. Relationships between pairs of parameters were evaluated using linear correlation and regression analysis. A receiver operating characteristic (ROC) curve analysis was performed to evaluate its ability to predict the decreased FMD value (<4%). Multivariate regression analysis was performed to find the independent determinants of ∆CCA_PLR and ∆β_PLR. Inter- and intra-observer reproducibility for the maximal CCA diameter, β, and FMD were studied in 15 randomly selected participants. For all statistical tests, a P value of <0.05 was considered statistically significant.

Results

The characteristics and ultrasonographic parameters of the study participants are summarized in Table 1. Among the participants, 8 were current smokers, 2 were former smokers (for 8 and 10 years, respectively), and the remaining 26 had no history of smoking. Three participants had a body mass index >25 kg/m². FMD values and carotid artery ultrasound parameters are also summarized in Table 1. There were 6 participants with a reduced FMD value (<4%).

Table 1.

Participant Characteristics and Ultrasound Parameters

	Mean±SD	Range
Parameter
Age (years)	23.3±6.9	19–46
Height (cm)	172.5±5.2	162–182
Weight (kg)	62.7±11.2	51–103
Body surface area (cm2)	1.74±0.15	1.54–2.19
BMI (kg/m2)	21.0±3.3	17.4–33.2
Body circumference (mm)	75.6±8.9	62.7–102.6
SBP (mmHg)	120±11	97–145
DBP (mmHg)	73±10	51–99
Heart rate (beats/min)	63±11	42–97
SBP during PLR (mmHg)	119±10	105–144
DBP during PLR (mmHg)	71±9	53–94
Heart rate during PLR (beats/min)	63±9	45–84
Ultrasound parameter
Pre BA diameter (mm)	3.6±0.4	2.8–4.7
Post BA diameter (mm)	3.9±0.5	3.0–4.9
FMD (%)	6.2±2.2	1.6–11.4
IMT (mm)	0.48±0.09	0.3–0.7
Maximal CCA diameter at rest (mm)	6.5±0.5	5.7–8.4
Minimal CCA diameter at rest (mm)	5.7±0.5	5.0–7.5
Stiffness parameter β at rest	4.2±1.0	2.2–6.4
Maximal CCA diameter during PLR (mm)	6.5±0.5	5.7–8.6
Minimal CCA diameter during PLR (mm)	5.7±0.5	4.8–7.5
Stiffness parameter β during PLR	3.9±0.9	2.3–7.5
ΔCCAPLR (%)	0.3±2.7	−6.8 to 7.4
ΔβPLR (%)	−5.2±13.6	−31.4 to 25.6

Δβ_PLR, difference between stiffness parameter β at rest and during PLR; ΔCCA_PLR, difference in between maximal CCA diameter at rest and during PLR; BA, brachial artery; BMI, body mass index; CCA, common carotid artery; DBP, diastolic blood pressure; FMD, flow-mediated dilation; IMT, intima-media thickness; PLR, passive leg raising; SBP, systolic blood pressure.

Changes from baseline to during the PLR maneuver were analyzed using a paired t-test. Although systolic blood pressure (P=0.47), the maximal CCA diameter (P=0.54), and the minimal CCA diameter (P=0.06) did not differ between baseline and during PLR, diastolic blood pressure was significantly lower during PLR than baseline (P=0.037). As a result, the stiffness parameter β was significantly lower during PLR compared with baseline (P=0.014).

Correlation coefficients between FMD and blood pressure and carotid artery ultrasound parameters are summarized in Table 2. All of the blood pressure parameters did not significantly correlate with the FMD value. Among the carotid artery ultrasound parameters, ∆β_PLR was most strongly correlated with FMD (Figure 2). Multivariate regression analysis among age, body mass index, systolic blood pressure at rest, and FMD value revealed that FMD was an independent predictor of ∆CCA_PLR and ∆β_PLR (Table 3).

Table 2.

Correlation Between FMD and the Carotid Artery Ultrasound Parameters

Parameter	r	P value
SBP at rest	0.09	0.62
DBP at rest	−0.12	0.51
SBP during PLR	0.04	0.80
DBP during PLR	−0.04	0.81
ΔSBP	−0.06	0.72
ΔDBP	0.15	0.39
Maximal CCA diameter at rest	−0.18	0.30
Minimal CCA diameter at rest	−0.16	0.35
Stiffness parameter β at rest	0.31	0.08
Maximal CCA diameter during PLR	−0.03	0.85
Minimal CCA diameter during PLR	−0.01	0.53
Stiffness parameter β during PLR	−0.15	0.38
ΔCCAPLR	0.41	<0.05
ΔβPLR	−0.70	<0.001

BP, blood pressure. Other abbreviations as in Table 1.

Figure 2.

Correlation between ∆β_PLR and FMD value. ∆β_PLR, difference between stiffness parameter β at rest and during PLR; FMD, flow-mediated dilation; PLR, passive leg raising.

Table 3.

Multivariate Regression Analysis for the Predictors of ΔCCA_PLR and ∆β_PLR

Parameter	ΔCCAPLR			ΔβPLR
	Coefficient (β)	Standard error	P value	Coefficient (β)	Standard error	P value
Intercept	−7.25	4.97	0.16	13.8	20.3	0.50
Age	−0.85	0.07	0.21	0.36	0.27	0.19
BMI	0.27	0.13	<0.05	−0.10	0.54	0.85
SBP at rest	0.003	0.04	0.94	0.02	0.17	0.92
FMD	0.54	0.19	<0.01	−4.40	0.77	<0.001

Corrected R² was 0.20 (P<0.05) for ΔCCA_PLR, and 0.53 (P<0.001) for Δβ_PLR. Abbreviations as in Table 1.

The results of the ROC analysis for detecting reduced FMD using ∆β_PLR are shown in Figure 3. The area under the curve of ∆β_PLR was 0.89, with a sensitivity of 87%, and specificity of 83% at the optimal cut-off value of 4.7%, whereas that of ∆CCA_PLR was 0.79, with a sensitivity of 100%, and specificity of 63% at the optimal cut-off value of 0.2%.

Figure 3.

Receiver operator characteristic (ROC) curve analyses. ROC curves of ∆β_PLR (red line) and ∆CCA_PLR (dashed blue line) have been plotted for the differentiation of participant with reduced FMD value (<4%) from those without. ∆β_PLR, difference between stiffness parameter β at rest and during PLR; ∆CCA_PLR, difference in between maximal CCA diameter at rest and during PLR; CCA, common carotid artery; FMD, flow-mediated dilation; PLR, passive leg raising.

The impact of smoking on FMD and changes in the carotid artery ultrasound parameters during the PLR maneuver were examined. In the smoking group (8 current and 2 former smokers), FMD was significantly lower (4.6±1.6% vs 6.9±2.1%; P<0.01) and ∆β_PLR was significantly greater (4.5±13.3% vs −8.9±12.0%; P<0.05) than in the non-smoking group, whereas ∆CCA_PLR was comparable between the groups (0.06±3.9% vs 0.34±2.2; P=0.83).

The inter- and intra-observer reproducibility are summarized in Table 4. Inter- and intra-observer variability were excellent for maximal CCA diameter and good for β. The ICC for FMD was good for intra-observer comparison and moderate for inter-observer comparison.

Table 4.

Inter- and Intra-Observer Reproducibility

Parameter	Inter-observer			Intra-observer
	ICC	95% CI	P value	ICC	95% CI	P value
Maximal CCA diameter	0.96	0.86–0.99	<0.001	0.95	0.87–0.98	<0.001
β	0.78	0.46–0.92	<0.001	0.82	0.54–0.93	<0.001
FMD (%)	0.64	0.21–0.86	0.004	0.89	0.70–0.96	<0.001

CI, confidence interval. Other abbreviations as in Table 1.

Discussion

In the present study we found that the changes in carotid artery stiffness parameter β during the PLR maneuver (∆β_PLR) were closely correlated with the FMD value, and the effect of smoking on FMD and ∆β_PLR was comparable. These results showed that ∆β_PLR has a potential candidate parameter reflecting vascular endothelial function.

Possible Mechanism of Change in Stiffness Parameter β During PLR Reflects Vascular Endothelial Function

The PLR maneuver has the effect of returning blood pooled in the lower limb and abdominal compartment back to the upper body. Several studies have shown that stroke volume and cardiac output increase by approximately 10% during the PLR maneuver. This maneuver is used in clinical situations, such as for critically ill patients to assess fluid responsiveness, and in cardiovascular disease patients to increase preload of the heart.^19–21 The PLR maneuver can be similar to the FMD procedure in terms of transient increases in blood flow. Kamran et al. reported that the brachial artery diameter significantly increased during the PLR maneuver; however, the degree of dilation was approximately half that observed in the FMD procedure.¹² They also examined the change of heart rate variability analysis before and during PLR and elucidated that any heart rate variability parameters such as SD of the NN intervals and the ratio of low-frequency to high-frequency heart rate variability remained unchanged upon PLR maneuver, and concluded that brachial diameter dilation during the PLR maneuver is induced by an endothelial-dependent process rather than a baroreceptor process. We hypothesized that, similar to the brachial artery, the CCA might be affected by the PLR maneuver because both share the characteristic of being elastic arteries; however, the maximal CCA diameter was comparable between baseline and during the PLR maneuver, while diastolic blood pressure and stiffness parameter β significantly decreased during PLR. These results suggest that, in response to transient increases in carotid artery blood flow, NO was released, which reduced the elasticity of the carotid arteries but may not have had enough effect to increase the maximal CCA diameter. As stiffness parameter β is a sensitive marker that assesses extensibility while considering blood pressure changes, alterations in stiffness parameter β during the PLR maneuver (∆β_PLR) could reflect vascular endothelial function.

In the present study, participants with smoking habits have lower FMD values and higher ∆β_PLR values. Smoking is a strong promoter of the progression of atherosclerosis.^5,22–24 We did not require that participants cease smoking because we were particularly interested in the relationships between FMD values and the changes in carotid artery parameters during the PLR maneuver in this study. Both FMD and ∆β_PLR, which were highly correlated with each other, showed significant differences between the smoking and non-smoking groups. These results may provide a reason to believe that ∆β_PLR could be an alternative indicator for FMD value.

Clinical Implications of This Study

Vascular endothelial dysfunction is an abnormality that occurs in the early stages of atherosclerosis and is the main cause of all atherosclerotic diseases such as ischemic heart disease, which is the leading cause of death worldwide.^1–5 In developed countries with well-developed emergency medical services, even if a patient is saved after myocardial infarction, the patient requires continuous outpatient care. Vascular endothelial function can improve to an age-appropriate level at an early stage through lifestyle modification. Therefore, in the prevention of arteriosclerosis-related diseases, quantitative evaluation of the degree of vascular endothelial dysfunction and correction of the dysfunction are highly important in terms of preventing the development of subsequent arteriosclerotic diseases. The FMD value is a well-established parameter of vascular endothelial function, and many studies have reported its usefulness;^{5,8–10,22–25} however, it has limitations, such as requiring specialized equipment for stable measurements and the need for dietary restrictions. Carotid artery ultrasound is widely used to detect advanced atherosclerosis, such as thickened IMT, stroke-causing plaques, and carotid stenosis. This study shows that by applying a simple PLR maneuver during carotid artery ultrasound examination, ∆β_PLR can be measured as a surrogate marker of endothelial function in addition to conventional parameters. This approach has the potential to improve individuals’ quality of life by identifying patients with worsening ∆β_PLR at an early stage and preventing the progression of atherosclerotic disease through lifestyle modifications.

Study Limitations

This study has several limitations. First, as described above, we did not limit the participants to any dietary or smoking restrictions, and the examination time was at any given point in the day because our primary focus in this study was to explore the parameter of carotid artery ultrasound during the PLR maneuver, which can be used for the surrogate for the FMD value. Further study is necessary to examine whether the daily variation in FMD and ∆β_PLR remains parallel. Second, in the present study, only relatively young male adults were included because FMD values in women are influenced by estrogen.²⁶ Further studies are needed to confirm the generalizability of ∆β_PLR in women, the elderly, and patients with cardiovascular disease. Third, the risk factors for cardiovascular disease were not fully investigated, therefore, we cannot exclude the possibility that the relationship between the 2 is not a direct relationship, but caused by other factors. Fourth, carotid artery blood flow could not be compared between the baseline and during the PLR maneuver because we could not set the pulsed-wave Doppler angle to be the same. Also, it is a limitation that we did not measure the brachial artery diameter during the PLR maneuver. Recently, vascular wall shear stress has been evaluated by using novel ultrasonographic imaging.^27,28 Further studies utilizing such novel methods are needed to establish the usefulness of ∆β_PLR, which could address these limitations.

Conclusions

Change in carotid arterial stiffness parameter β during passive leg raising strongly correlated with FMD value. This parameter may be an alternative indicator for FMD.

Acknowledgments

During the preparation of this work, the authors used several AI-assisted technologies such as DeepL, Grammarly, and Chat-GPT to check English grammar. After using these tools, the authors reviewed and edited the content as needed and take full responsibility for the content of the publication.

Disclosures

None to declare.

IRB Information

This prospective study was approved by the Ethics Committee of the Faculty of Health Sciences at Japan Healthcare University (No. R04-41).

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