Impaired Endothelial Function in Patients with Postural Tachycardia Syndrome

Abstract

The purpose of this study is to evaluate endothelial function in postural tachycardia syndrome (PoTS), a poorly understood chronic condition characterized by a state of consistent orthostatic tachycardia (delta heart rate ≥30 beats per minute) upon standing without orthostatic hypotension. Nineteen PoTS patients and nine healthy controls were studied after 3 days of a fixed, caffeine-free, normal sodium (150 milliequivalents/day) diet. All participants underwent autonomic function testing including sinus arrhythmia, Valsalva maneuver, hyperventilation, cold pressor, handgrip, and a standing test with catecholamine measurements, followed by endothelial function testing. We analyzed three measures of endothelial function: percent brachial flow mediated dilation (FMD), Digital Pulsatile Arterial Tonometry (EndoPAT), and post-ischemic percent leg blood flow (%LBF). FMD was significantly lower in PoTS patients (6.23 ± 3.54 % for PoTS) than in healthy controls (10.6 ± 4.37 % for Controls vs, p=0.014). PoTS and Controls had similar EndoPAT (1.93 ± 0.40 Arbitrary Units for Controls vs 2.13 ± 0.63 Arbitrary Units for PoTS). PoTS had similar, but suggestive %LBF to Controls (313 ± 158 % for PoTS vs 468 ± 236 % for Controls, p=0.098). Patients with PoTS have significantly reduced FMD compared to healthy controls, suggesting that PoTS is characterized by endothelial dysfunction in conduit arteries.

INTRODUCTION

Postural Tachycardia Syndrome (PoTS) is a disorder characterized by a chronic state of consistent orthostatic tachycardia (delta HR ≥30bpm) associated with symptoms upon standing in the absence of orthostatic hypotension ¹. This syndrome disproportionately affects young women, with 80-85% of cases occurring in women, typically of childbearing age ². Estimates of the prevalence of this disorder vary from 500,000 ³ to 3,000,000 ² individuals affected in the United States. Typical symptoms of PoTS include tachycardia, exercise intolerance, lightheadedness, extreme fatigue, headache and mental clouding ¹. Even the performance of daily activities like bathing can be impaired for PoTS patients. As of now, there is no medication approved by the FDA for PoTS and all current treatments are considered “off label” ¹.

The pathophysiology of PoTS is complex and not yet fully understood. Among other things, increased sympathetic activity has been shown to be present in some cases ⁴. We and others have shown that increased sympathetic activity can have a negative impact on endothelial function ^5,6. We propose that this same process may be at work in PoTS patients, and may result in diminished endothelial function in PoTS.

Endothelial dysfunction is described as the inability of arteries to appropriately dilate in response to an endothelial stimulus ⁷. Nitric Oxide (NO) is arguably the strongest local vasodilator and is an important component of endothelial function⁸. In the past, we have shown basal nitric oxide function in PoTS to be similar to that of controls ⁹, and that inhibition of tonic NO release by L-NMMA under autonomic blockade produced similar responses in PoTS and controls, indicating that there is no impairment of NO bioavailability in PoTS ⁹.

In addition to tonic NO, the endothelial system can also respond to stimulated NO in the presence of shear stress. Because tonic NO function is normal in PoTS, our goal in this study is to measure endothelial function following shear stress, an increase in blood flow that increases pressure in a vessel. In healthy individuals, shear stress triggers the stimulated release of NO to facilitate vasodilation and restore normal levels of pressure on vessel walls. We hypothesize that patients with PoTS will have decreased endothelial function (and therefore decreased stimulated NO release) compared with healthy control subjects. To test this hypothesis, we measured endothelial function in different vascular beds (conduit arteries, resistance vessels and microcirculation) and different anatomic sites (upper and lower limbs) in healthy females and a cohort of PoTS patients following ischemia-induced shear stress.

METHODS

The data that support the findings of this study are available from the corresponding author upon reasonable request. This study was approved by the Vanderbilt University Institutional Review Board. Written informed consent was obtained from all participants prior to enrollment. This study was registered at ClinicalTrials.gov (ClinicalTrials.gov identifier: NCT01308099).

Subjects.

Nineteen PoTS patients (1 male and 18 female) aged 18-53 years (pre-menopausal for females) and nine healthy female volunteers with similar age and body weight were enrolled in this study. PoTS patients seen at Vanderbilt Autonomic Dysfunction Center in the last 6 years before the start of the study were invited to participate in this study. Healthy controls were enrolled after PoTS patient enrollment was completed. These were selected based on similar age and weight to the PoTS group. The diagnosis of PoTS was made according to current guidelines ¹⁰. Participants were excluded if they were smokers, had significant cardiovascular, pulmonary, hepatic, or hematological disease by history or screening results, or were pregnant. All subjects were provided with a caffeine free, normal sodium (150 mEq/day) diet for for ≥3 days before testing. The inpatient diet for PoTS patients was additionally low monoamine, methylxanthine free and had fixed potassium (70 mEq). The healthy volunteers were studied in an outpatient setting and abstained from methylxanthine containing products. Subjects were screened in a quiet, temperature-controlled room (22-23°C) after an 8-hour fasting period. Medications affecting blood pressure, blood volume, the immune system, and autonomic function were withheld for ≥5 half-lives.

Procedures.

General Description

Studies of enrolled participants were performed in the morning at least 2 hours after a light breakfast. Subjects were in the supine position for 30 minutes before starting data collection.

The severity of autonomic impairment was assessed by standardized autonomic testing as previously described ^11,12. This included a standing stress test, respiratory sinus arrhythmia, and Valsalva maneuver. Sinus arrythmia was measured during controlled breathing (at 6 breaths/min) and was obtained by dividing the longest to the shortest RR intervals. Brachial blood pressure and heart rate were determined at intervals, using an automated cuff-oscillometric sphygmomanometer (VitalGuard 450C, Ivy Biomedical, Branford, CT), and continuously with the finger clamp method (Nexfin, BMEYE, Amsterdam, the Netherlands) and ECG (VitalGuard450C). Cardiovascular signals were digitized using a Windaq system (DA-220; DATAQ Instruments). During the standing test, blood samples were obtained for norepinephrine while patients were supine and upright, as described previously ¹³. Plasma norepinephrine was measured by high-performance liquid chromatography with electrochemical detection ¹⁴.

Assessment of Endothelial Function.

On a different day, subjects were again brought to the Vanderbilt Autonomic Dysfunction Center to evaluate endothelial function non-invasively in the brachial artery using flow mediated dilation (FMD), in resistance vessels of the legs using venous occlusion plethysmography to measure leg blood flow (LBF), and in the microcirculation using digital pulsatile amplitude tonometry (EndoPAT) following reactive hyperemia.

Flow Mediated Dilation (FMD).

Baseline and peak brachial arterial diameters (BAD) were measured before and following ischemic reactive hyperemia. The absolute change (peak-baseline) and percent change [(peak-baseline)/baseline] x 100% (FMD) were determined as described previously ¹⁵. Time to peak dilation (seconds) was also recorded. Briefly, an ultrasound probe was placed over the brachial artery 3-5 cm above the elbow and baseline diameter was obtained for 60 seconds (iU22; Phillips, Bothell, WA). After this, a brachial blood pressure cuff was inflated distal to the probe for 5 min to induce forearm ischemia. Following release of the cuff, arterial diameter was continuously measured for 180 seconds to capture the peak dilatory response to ischemia. Images were analyzed by an investigator blinded to the study using an automated brachial artery edge and wall detection software (Brachial analyzer 5.0; Medical Imaging Applications LLC, Iowa City, IA), as previously described ¹⁵. In order to maximize the resolution of the brachial artery diameter, we sacrificed flow measurements in the brachial artery.

Leg Blood Flow (LBF).

The percent change in muscle blood flow following ischemia is a measurement of endothelial function in resistance vessels ¹⁶. The forearm has commonly been used as the measurement site ^16,17. However, prior work by Stewart and colleagues found that some PoTS patients have reduced blood flow to the legs ¹⁸. To assess whether this reduced blood flow would be seen in our population or manifest as decreased endothelial function in the resistance vessels of the legs, we adapted our forearm blood flow procedures to the leg ^5,19,20. Briefly, mercury insilastic strain gauges were wrapped around the widest circumference of the calf and inflatable cuffs were placed above and below the strain gauge to prevent venous outflow from the region of interest and to limit arterial flow to the distal limb. The distal cuff (around the ankle) was inflated to 200 mmHg and the proximal cuff (two centimeters above the flexion of the knee) was inflated to ischemic levels for 5 minutes. This was then followed by intermittent rapid inflation (50 mm Hg) and deflation in a 16-second cycle, for the last two minutes of the measurement. Rapid cuff inflation was achieved using a commercially available air source (Hokanson EC4, DE; Hokanson Inc, Indianapolis; IN). This method allows measurement of baseline and peak post-ischemic blood flow (ml/100ml/min), and percent change in blood flow [(peak-baseline)/baseline] × 100% per minute (%LBF).

Digital Pulsatile Arterial Tonometry.

Peripheral microvascular endothelial function responses to ischemia in the hand was assessed using peripheral artery tonometry (PAT) with an Endo-PAT device (Endo-PAT 2000, Itamar-Medical, Caesarea; Israel) ^21,22. In this method, a probe was placed on a finger of each hand to measure pulse volume by PAT, and ischemia was applied to one of the upper arms. The Reactive Hyperemia Index (RHI, a ratio of pulse volume before and after ischemia in the affected limb normalized by measurements in the contralateral arm) indicates peripheral microvascular endothelial function ^16,23. The device also calculated the augmentation index (AI, a ratio of the second to first measured systolic pulse pressures) as an indicator of arterial stiffness ¹⁶.

Missing Data

All 28 patients and controls completed the autonomic evaluation and standing stress test. LBF was measured in 8 controls and 12 PoTS patients. RHI and AI were missing for one PoTS patient.

Statistical Analysis

We hypothesized that PoTS patients would have a blunted response in FMD (primary outcome) as compared to controls. FMD (%) is defined as the percentage change in brachial artery [(Peak-Baseline/Baseline)x100]. Secondary outcomes included %LBF and RHI. The difference in FMD between PoTS and controls was assumed to be clinically significant at 5%. Data are presented as mean ± SD, unless otherwise stated. Comparisons between groups were obtained by performing Mann Whitney U tests. Within groups, differences were compared with Wilcoxon signed-rank test. Analyses were performed using SPSS version 26.0 (IBM Corp) and Stata version 13 (StataCorp LLC).

RESULTS

Subjects characteristics (Tables 1 and 2)

Table 1.

Patient Characteristics

Characteristic	Controls (n=9)	POTS (n=19)	p
Age, years	29.67 ± 5.00	31.16 ± 10.18	0.962
Gender, proportion of female	1.00	0.95
Height, cm	161.41 ± 8.40	166.64 ± 7.56	0.110
Weight, kg	58.57 ± 9.36	63.44 ± 14.10	0.699
Body Mass Index (BMI), kg/m2	22.39 ± 2.45	22.72 ± 4.05	0.962
Triglycerides, mg/dL	78.00 ± 2.83	139.67 ± 72.95	1.000
HDL Cholesterol, mg/dL	53.00 ± 18.38	47.33 ± 10.02	0.400
LDL Cholesterol, mg/dL	86.50 ± 26.16	97.33 ± 13.32	1.000
Glucose, mg/dL	72.95 ± 27.93	90.50 ± 13.03	0.227
Insulin Resistance (HOMA2 IR)	1.75 ± 0.53	2.51 ± 1.71	0.852
Insulin, mg/dL	11.55 ± 0.59	18.61 ± 7.60	0.812
HR supine, bpm	65.33 ± 14.09	71.89 ± 11.15	0.142
SBP supine, mmHg	103.11 ± 7.69	105.21 ± 10.48	0.847
DBP supine, mmHg	62.56 ± 1.96	62.68 ± 5.59	0.847
Mean Arterial Pressure (MAP), mmHg	76.07 ± 6.02	76.06 ± 5.41	0.902
Cardiac Output (CO), L/min	4.52 ± 0.63	5.66 ± 0.72	0.019*
Stroke Volume (SV), mL/beat	66.00 ± 12.94	78.70 ± 15.42	0.165

Data are presented as mean ± standard deviation (SD). HDL represents high-density lipoprotein and LDL represents low-density lipoprotein. SBP represents systolic blood pressure, DBP represents diastolic blood pressure, and HR represents heart rate.

^*represents significantly different between groups as determined by non parametric Mann Whitney U Test p<0.05.

¹represents significantly different from supine to standing as determined by Wilcoxon Signed Rank Test p <0.05.

Table 2.

Autonomic Function and Orthostatic Stress Testing

	Controls (n=9)	POTS (n=19)	p
Autonomic function tests
Sinus arrhythmia ratio, A.U.	1.43 ± 0.20	1.32 ± 0.13	0.194
Valsalva HR ratio, A.U.	1.72 ± 0.37	1.80 ± 0.40	0.527
Valsalva phase II, early ΔSBP, mmHg	−12.89 ± 16.07	−28.58 ± 18.321	0.037*
Valsalva phase II, late ΔSBP, mmHg	−1.44 ± 18.03	−14.05 ± 16.261	0.142
Valsalva phase IV ΔSBP, mmHg	19.44 ± 16.261	29.16 ± 18.811	0.308
Hyperventilation ΔSBP, mmHg	−6.78 ± 8.66	−12.26 ± 15.841	0.383
Handgrip ΔSBP, mmHg	3.00 ± 38.58	17.68 ± 11.60	0.468
Cold pressor ΔSBP, mmHg	13.00 ± 9.41	−17.11 ± 10.81	<0.001*
Standing Stress Test
Supine SBP, mmHg	103.11 ± 7.69	105.21 ± 10.48	0.847
Maximum Standing SBP	113.11 ± 13.041	111.68 ± 18.63	0.923
Δ SBP standing, mmHg	10 ± 9.53	6.47 ± 15.39	0.923
Supine DBP, mmHg	62.56 ± 5.88	62.68 ± 5.59	0.847
Maximum Standing DBP	76.56 ± 6.751	70.95 ± 13.581	0.308
Δ DBP standing, mmHg	14.00 ± 4.50	8.26 ± 11.92	0.357
Supine HR, bpm	65.33 ± 14.09	71.89 ± 11.15	0.142
Maximum Standing HR, bpm	100.56 ± 11.831	122.89 ± 20.831	0.005*
Δ HR standing, bpm	35.22 ± 17.55	51.00 ± 16.83	0.054
Supine Norepinephrine, pg/mL	130.56 ± 43.17	193.22 ± 123.20	0.212
Maximum Standing Norepinephrine, pg/mL	495.11 ± 120.211	777.94 ± 460.971	0.176
ΔNorepinephrine, pg/mL	364.56 ± 111.71	553.95 ± 384.00	0.332

Data are presented as mean ± standard deviation (SD). SBP represents systolic blood pressure, DBP represents diastolic blood pressure, and HR represents heart rate.

^*represents significantly different between groups as determined by non parametric Mann Whitney U Test p<0.05.

¹represents significantly different from supine to standing as determined by Wilcoxon Signed Rank Test p <0.05.

We enrolled 19 PoTS patients (95% female) and 9 healthy female controls, 18-53 years old (pre-menopausal for females). Demographic data and supine parameters of all participants are shown in Table 1. There were no significant differences in age (31.2 ± 10.2 years for PoTS vs 29.7 ± 5.0 years for Controls, p=0.962) or body mass index (22.7 ± 4.1 kg/m² for PoTS vs 22.4 ± 2.5 kg/m² for Controls, p=0.962; Table 1). The PoTS subjects were otherwise metabolically healthy and did not have increased body mass or lipid disorders.

Autonomic Testing

Results from autonomic testing are described in Table 2. BP responses to early phase II of the Valsalva were significantly greater in PoTS (−28.6 ± 18.3 mmHg for PoTS vs −12.9 ± 16.1 mmHg for Controls, p=0.037). Additionally, BP responses to the cold pressor test at the 1 minute interval were also significantly higher for PoTS (−17.1 ± 10.8 mmHg for PoTS vs 13.0 ± 9.4 mmHg for Controls, p<0.001). These are both signs of autonomic dysfunction in PoTS potentially resulting from prolonged sympathetic activation. There were no differences between groups in sinus arrhythmia ratio (1.32 ± 0.13 for PoTS vs 1.43 ± 0.20 for Controls, p=0.194), BP responses to phase IV of the Valsalva (29.2 ± 18.8 mmHg for PoTS vs 19.4 ± 16.3 mmHg for Controls, p=0.308), hyperventilation (−12.3 ± 15.8 mmHg for PoTS vs −6.8 ± 8.7 mmHg for Controls, p=0.383), and handgrip tests (17.7 ± 11.6 mmHg for PoTS vs 3.00 ± 38.6 mmHg for Controls, p=0.468).

Consistent with diagnostic criteria, PoTS had greater maximum upright heart rates (123 ± 21 for PoTS vs 101 ± 12 bpm for Controls, p=0.005) and change in heart rate upon standing (51 ± 17 bpm for PoTS vs 35 ± 18 bpm for Controls; p=0.054) compared to Controls (fig 1). Supine and upright norepinephrine levels were similar between groups, with both groups experiencing a significant increase in norepinephrine levels upon standing (193 ± 123 pg/mL supine vs 778 ± 461 pg/mL standing for PoTS; p<0.001; 131 ± 43 pg/mL supine vs 495 ± 120 pg/mL standing for Controls, p=0.008, fig. 1).

Fig. 1.

Panel A shows Norepinephrine (NE) values for PoTS (circles) and controls (HC, squares) during supine (open) and standing (Up, closed). Panel B shows heart rate (HR) values for PoTS (circles) and controls (HC, squares) during supine (open) and standing (closed). *, p value for the difference between supine and standing; ǂ, p value for the difference at standing between PoTS and controls.

Assessment of Endothelial Function (Figure 2, Table 3)

Fig. 2.

Panel A shows differences in percent flow mediated dilation (FMD) between PoTS (open circles) and controls (HC, closed squares). Panel B shows differences in percent leg blood flow (%LBF) between PoTS (open circles) and controls (HC, closed squares). Panel C shows differences in reactive hyperemic index (RHI) between PoTS (open circles) and controls (HC, closed squares). Dotted line at 1.67 indicates the cut-off value for endothelial dysfunction. *, p value for the difference between PoTS and controls.

Table 3.

Assesment of Endothelial Function

	Controls (n=9)	POTS (n=19)	p
FMD
Baseline Diameter, mm	3.01 ± 0.25	3.18 ± 0.43	0.595
Peak Diameter, mm	3.33 ± 0.31	3.38 ± 0.43	0.809
FMD, %	10.62 ± 4.37	6.23 ± 3.54	0.014*
Time to Peak, seconds	42.71 ± 9.85	54.44 ± 13.13	0.033*
LBF
Baseline, mL/100mL/min	2.44 ± 0.88	2.57 ± 1.06	1.000
Post Ischemia, mL/100mL/min	13.92 ± 6.86	10.07 ± 4.78	0.181
%LBF, %	468.02 ± 236.11	312.83 ± 157.97	0.098
Time to Return to Baseline, min	3.92 ± 0.59	3.79 ± 0.86	0.792
EndoPAT
RHI, A.U.	1.93 ± 0.40	2.13 ± 0.63	0.495
AI, A.U.	0.37 ± 13.49	−1.68 ± 15.38	0.561

Data are presented as mean ± standard deviation (SD). SBP represents systolic blood pressure, DBP represents diastolic blood pressure, and HR represents heart rate. FMD represents Percent Flow Mediated Dilation, %LBF represents Percent Leg Blood Flow, RHI represents Reactive Hyperemia Index, and AI represents Augmentation Index.

^*represents significantly different between groups as determined by non parametric Mann Whitney U Test p<0.05.

¹represents significantly different from supine to standing as determined by Wilcoxon Signed Rank Test p <0.05.

Conduit artery vessel function.

Endothelial function as measured by FMD was significantly lower in PoTS than in healthy controls (6.23 ± 3.54 % for PoTS vs 10.6 ± 4.4 % for Controls, p=0.014). PoTS also experienced a longer time to peak than healthy controls (54.4 ± 13.1 seconds for PoTS vs 42.7 ± 9.9 seconds for Controls, p=0.033). Baseline (3.18 ± 0.43 mm for PoTS vs 3.01 ± 0.25 mm for Controls, p=0.595) and peak diameters (3.38 ± 0.43 mm for PoTS vs 3.33 ± 0.31 mm for Controls, p=0.809) were similar between groups.

Resistance artery vessel function.

PoTS tended to have lower %LBF than healthy controls (313 ± 158 % for PoTS vs 468 ± 236 % for Controls, p=0.098), but this difference did not reach statistical significance. Baseline (2.57 ± 1.07 for PoTS vs 2.44 ± 0.88 for Controls, p=1.000) and post-ischemic flows (10.1 ± 4.8 for PoTS vs 13.9 ± 6.9 for Controls, p=0.181) were not different between groups.

Microvascular function.

Peripheral microvascular endothelial function responses to ischemia in the hand were similar between PoTS and Controls, with a RHI of 2.13 ± 0.63 A.U. for PoTS vs 1.93 ± 0.40 A.U. for Controls (p=0.495) and AI of −1.68 ± 15.38 for PoTS vs 0.37 ± 13.49 for Controls (p=0.561).

DISCUSSION

The main finding of the present study is that under carefully controlled conditions, PoTS patients have decreased percent flow mediated dilation in the brachial artery, suggesting impaired conduit artery endothelial function. Functional, healthy blood vessels respond to shear stress by dilating to maintain constant blood pressure in the presence of increased blood volume ²⁴. A decreased dilatory response to post-ischemic shear stress is indicative of reduced endothelial function (and may imply a decrease in NO bioavailability). Based on our previous findings, and the fact that increased sympathetic activity has been linked both to PoTS and to decreased endothelial function, we hypothesized that the PoTS group would have decreased post-ischemic vasodilation as compared to the healthy group.

The primary outcome of this study, FMD, supports our hypothesis that patients with PoTS have endothelial dysfunction compared to healthy controls. FMD of young healthy subjects has been seen to range from 0.20% to 19.2%, according to a literature review of 219 studies ²⁵. It is clear that there is wide variation across studies and overlap between populations ²⁵. However, we set reference values such as location of occlusion cuff and time frame for peak response measurement to minimize individual variation. Using our rigorous technique, we found an impaired conduit artery endothelial response in PoTS, i.e. lower FMD, compared to healthy controls despite unavoidable variability inherent in the FMD method. This confirms our hypothesis that PoTS patients have a decreased dilatory response to shear stress, thus indicating the presence of impaired endothelial dysfunction and stimulated NO release.

In the resistance vessels of the legs, we found that PoTS subjects tended to have a decreased (313 ± 158%) dilatory response to hyperemia as compared to healthy subjects (468 ± 236%) although this difference was not statistically significant (p=0.098) (Table 3). It is possible that these data didn’t reach significance because of individual variation and relatively small sample size. It is also possible that we did not enroll “low flow” PoTS patients, who would be expected to experience lower LBF than controls. Stewart et al. have described sub-populations of PoTS patients with low leg blood flow (“low flow”) and high leg blood flow (“high flow”) ²⁶. They postulated that the low-flow patients might have a hyperadrenergic state, while the high flow patients might have a partial autonomic neuropathy involving their lower limbs. Interestingly, the Ross group showed a differential response to midodrine between these 2 PoTS patient groups²⁷. It is possible that our findings would have been different if we had enrolled a greater number of “low-flow” PoTS patients.

We did not find differences in either EndoPAT or AI between groups. An EndoPAT under 1.67 is typically accepted as a sign of endothelial dysfunction ²⁸. However, neither the control nor the PoTS group reached this threshold (Table 3). EndoPAT is a technique used to assess peripheral microvascular endothelial function, but only 61% of what is measured by EndoPAT is explained by NO ²³. It is possible that this measure of endothelial function represents aspects of EndoPAT not dependent on NO. This index also measures microvascular endothelial function, which might have different mechanisms than the conduit artery endothelial function measured by FMD, and which are perhaps not impaired in PoTS. Furthermore, PoTS is not a homogeneous disorder—it manifests differently in different patients—and does not exist in isolation. Our results may be impacted by this variation and potential concomitant diseases we did not screen for.

In our cohort of PoTS patients, we found evidence of increased sympathetic activity indicated by a greater maximum standing heart rate during orthostatic stress testing (123 ± 21 bpm for PoTS vs 101 ± 12 bpm for Controls, p=0.005, Table 2). However, our PoTS patients did not have hypertension or even increased blood pressure as compared to age- and gender-matched controls, which ruled out endothelial damage as the cause of conduit artery endothelial dysfunction. Additionally, we were unable to determine whether the level of endothelial dysfunction and severity of sympathetic activation are correlated because we did not measure muscle sympathetic nervous activity (MSNA) in this population and there are not great indirect measures of sympathetic activity. We can only speculate as to this association. We failed to observe significant differences in resistance vessels in the legs or in the microcirculation. This could be important in the pathophysiology of PoTS, but the results need to be validated in a study designed to test differences in different vascular beds, which was not the objective of the present study. It is also possible that we did not enroll “low flow” PoTS patients, who would be expected to experience lower LBF than controls. Beyond the evidence of increased sympathetic activity that we found, there are three potential mechanisms that may contribute to the observed conduit artery endothelial dysfunction.

First, this decreased dilatory response could be the result of Angiotensin II levels. Angiotensin II levels were shown to be increased in low-flow PoTS patients ²⁹ despite the known renin-aldosterone paradox ³⁰. These levels may not be unique to low-flow PoTS patients, but there would need to be another study which measured these levels in a heterogeneous PoTS population to know for sure. Angiotensin II is also known to induce oxidative stress through direct stimulation of endothelial NADPH oxidases and indirect mitochondrial ROS production, among other mechanisms ³¹. The increased oxidative stress and ROS production lead to the the reduced availability of tetrahydrobiopterin (−BH4), a cofactor for nitric oxide synthesis by nitric oxide synthase. This leads to the production of superoxide radical instead of nitric oxide. Superoxide in turn causes further ROS production as well as reacts with the available nitric oxide to form peroxynitrite (another oxidizing agent). The excess oxidants lead to tyrosine nitration causing mitochondrial dysfunction, endothelial cell dysfunction, and apoptosis, which contribute to the abnormal flow mediated dilation ^32-34. The reduced dilation could also be related to a reduced myogenic flow-mediated arteriolar dilation response that is mediated in part by calcium-dependent smooth muscle contraction where nitric oxide plays a role ^35,36.

Another aspect to be considered in the context of our results is sex and gender. Sex and gender differences are of critical importance in many diseases ³⁷. Sex and gender appear to play a role in PoTS considering the high incidence in young females compared to males. Potentially significant in the pathophysiology of PoTS, sex differences have been reported in the renin-angiotensin system (RAS) and in the regulation of blood pressure ³⁷. Although the precise mechanisms which cause these differences are not completely understood, it is believed that sex hormones play a role, with estrogens possibly increasing the synthesis of angiotensinogen but decreasing the synthesis of renin and angiotensin-converting enzyme (ACE). Further, it has been reported that healthy premenopausal females have a greater production of whole body NO compared with age-matched men potentially because renal NO syntheses are enhanced by estrogens ³⁷. These observations in healthy females contradict our observations of females with PoTS, suggesting estrogen dysfunction (decreased bioavailability or faulty receptors) as a potential mechanism of PoTS causing the renin-aldosterone paradox and reduced NO bioavailability. Future studies should take into account levels of estrogen and testosterone as well as menstrual phase of females with PoTS.

Another possible explanation for the symptoms of PoTS is an increased level of asymmetric dimethylarginine (ADMA) in PoTS patients. ADMA is an endogenous competitive inhibitor of eNOS. Levels of plasma ADMA are inversely related to endothelium-dependent vasodilation. ADMA is eliminated via excretion through the kidneys, which reiterates the possible implication of the kidneys in PoTS introduced by Raj et al. in response to the renin-aldosterone paradox³⁰. Oxidative stress also increases the plasma ADMA levels, which ultimately decreases the release of NO by inhibiting NOS.

In conclusion, our data suggest that PoTS patients have endothelial dysfunction of the brachial artery. We did not however, find any significant differences in microvascular endothelial function or in endothelial function in the leg. This could have implications for the pathophysiology of PoTS. If our data are confirmed that PoTS patients have conduit vessel endothelial dysfunction, then this would become a future therapeutic target for PoTS, and a metric to be tracked in future PoTS investigations. Targeting endothelial dysfunction in PoTS will not be easy. Many of the traditional markers of endothelial function are not often present in PoTS (e.g. obesity). However, many PoTS patients do have high levels of sympathetic tone. It may be important to determine whether treatments that lower sympathetic tone (either resting or evoked) lead to improvements in endothelial function.

LIMITATIONS

We did not collect more information regarding body composition in both groups beyond height, weight, and BMI. Therefore, physical activity and fitness of individual subjects may be a confounding variable.

Cardiac output was reduced in patients, which may have affected brachial artery flow. However, to maximize the resolution of brachial artery diameter measurements, we sacrificed flow measurements. Future studies should investigate the impact of shear stress on brachial artery flow.

Since non-endothelium mediated vasodilation was not assessed, we can’t exclude that the differences in FMD observed between groups were endothelium independent.

In future studies, we will assess in more detail the microvascular circulation, as postischemic increases in flow are not solely mediated by endothelial NO. We will investigate the response of EndoPat to sympathetically mediated vasoconstriction in peripheral vessels.

PERSPECTIVES

We found that PoTS patients, many of whom have increased sympathetic activity, also showed impaired endothelial function in the brachial artery, but not in the microcirculation of the hand or the legs. The importance of this finding for the pathophysiology of PoTS needs to be further explored with studies looking at the different physiological properties of each vascular bed in this population.

NOVELTY AND SIGNIFICANCE.

What is new?

• Our original contribution is evidence for conduit vessel endothelial dysfunction in PoTS.

• This is significant because it provides new insight into the pathophysiology of PoTS and gives researchers a new target for potential therapies to treat this enigmatic and debilitating disease.

• This study also provides new guidance for further research on endothelial function in PoTS in different vascular beds.

What is relevant?

• Although good long-term data are lacking, the PoTS population may be at risk for developing hypertension given that a fair number of patients have increased sympathetic activity, have a high sodium diet, and are typically sedentary.

• These pathophysiological mechanisms, and therefore PoTS, are important to the readers of Hypertension.

Summary

• Our data suggest that PoTS patients have endothelial dysfunction of the brachial artery. We did not find any significant differences in endothelial function of arteries in the leg or in microvascular endothelial function.