Tetrahydrobiopterin improves endothelial function and decreases arterial stiffness in estrogen-deficient postmenopausal women

Abstract

The mechanisms mediating arterial stiffening with aging and menopause are not completely understood. We determined whether administration of tetrahydrobiopterin (BH₄), a critical cofactor for endothelial nitric oxide synthase to produce nitric oxide, would increase vascular endothelial-dependent vasodilatory tone and decrease arterial stiffness in estrogen-deficient postmenopausal women. Additionally, we examined whether the beneficial effects of estrogen on vascular function were possibly related to BH₄. Arterial stiffness (carotid artery compliance) and endothelial-dependent vasodilation [brachial artery flow-mediated dilation (FMD)] were measured in postmenopausal (n = 24; 57 ± 1 yr, mean ± SE) and eumenorrheic premenopausal (n = 9; 33 ± 2 yr) women before and 3 h after the oral administration of BH₄. Subsequently, in postmenopausal women, vascular testing (before and after BH₄) was repeated following randomization to either 2 days of transdermal estradiol or placebo. Baseline carotid artery compliance and brachial artery FMD were lower in postmenopausal than in premenopausal women (P < 0.0001). BH₄ administration increased carotid artery compliance (0.61 ± 0.05 to 0.73 ± 0.04 mm²·mmHg⁻¹·10⁻¹ vs. baseline, P < 0.0001) and brachial artery FMD (P < 0.001) in postmenopausal women but had no effect in premenopausal women (P = 0.62). Carotid artery compliance (0.59 ± 0.05 to 0.78 ± 0.06 mm²·mmHg⁻¹·10⁻¹, P < 0.001) and FMD increased in postmenopausal women in response to estradiol (P = 0.02) but were not further improved with the coadministration of BH₄, possibly because estrogen increased BH₄ bioavailability. Carotid artery compliance and FMD increased with BH₄ in the placebo group (P = 0.02). Although speculative, these results suggest that reduced vascular BH₄ may be an important contributor to arterial stiffening in estrogen-deficient postmenopausal women, related in part to reduced endothelial-dependent vasodilatory tone.

arterial stiffening, a biomarker of vascular aging, is a major risk factor for the development of cardiovascular disease (CVD) (24, 47). In women and men, the stiffness of large elastic arteries increases with advancing age, even in the absence of clinical CVD (31, 35, 43). However, in women, the age-associated increase in arterial stiffness appears to be augmented around the menopause transition presumably because of estrogen deficiency (35, 43, 54). Indeed, estrogen-deficient postmenopausal women show greater arterial stiffening compared with age-matched premenopausal women (54), and the age-associated increase in arterial stiffening is attenuated in postmenopausal women on chronic menopausal hormone therapy (MHT) compared with non-MHT users (26, 31, 35, 42). Thus, determining the mechanisms by which aging and estrogen deficiency contribute to arterial stiffening in women is clinically important.

Tetrahydrobiopterin (BH₄) is an essential cofactor for endothelial nitric oxide synthase (eNOS) to synthesize nitric oxide (NO), a potent endothelial-derived vasodilator and regulator of arterial stiffness (1, 55). Reduced BH₄ bioavailability leads to the uncoupling of eNOS and favors the production of reactive oxygen species (ROS) instead of NO (25). In this regard, we previously demonstrated that oxidative stress contributes to arterial stiffening in estrogen-deficient postmenopausal women (33). BH₄ supplementation improves vascular endothelial-dependent vasodilation in healthy older men (10). However, it is unknown whether BH₄ modulates endothelial vasodilatory function in postmenopausal women or whether BH₄ is mechanistically linked to arterial stiffening.

Accordingly, we tested the hypothesis that reduced BH₄ contributes to the impairment in endothelial vasodilatory function and arterial stiffening in estrogen-deficient postmenopausal women. Additionally, because estrogen is associated with a lower production of ROS and ovariectomized rats have lower aortic content of BH₄ compared with estrogen-replete rats (22, 23, 29), we examined whether the beneficial effects of estrogen on vascular function may be related to BH₄.

METHODS

Study population.

We studied 33 healthy women: 24 postmenopausal (51–65 yr of age) who were estrogen-deficient and 9 premenopausal women (24–40 yr of age) who had regular menstrual cycles (21–35 days). All women were sedentary or recreationally active (not exercising regularly >2 days/wk) and had not used oral contraceptives or MHT for at least 6 mo before study entry. Participants were included if they were nonsmokers, normotensive (resting blood pressure <140/90 mmHg), nondiabetic [fasted plasma glucose concentrations <7.0 mmol/l (126 mg/dl)], had a fasted low-density lipoprotein (LDL)-cholesterol <4.1 mmol/l (160 mg/dl), a body mass index (BMI) <36 kg/m², and were free of overt chronic diseases as assessed by medical history, physical examination, standard blood chemistries, and hematological evaluation. Participants had not taken any cardiovascular or lipid-lowering medications for at least 6 mo or aspirin, nonsteroidal anti-inflammatory medications, or vitamin supplements for at least 4 wk. Postmenopausal women with contraindications to estrogen (e.g., history of active estrogen-dependent neoplasms, thromboembolism) were excluded. All subjects gave their written informed consent to participate. All procedures were reviewed and approved by the University of Colorado Denver Colorado Multiple Institutional Review Board.

Measurements.

Subjects were studied in the supine position following an overnight fast with proper hydration (water drinking only) and abstinence from caffeine. The participants' normal dietary patterns were maintained, including sodium intake for the 2-day period immediately before any measurements. Premenopausal women were tested 7–10 days after onset of menstruation (i.e., midfollicular phase). The study took place at the University of Colorado Denver Colorado Clinical Translational Sciences Institute (CCTSI) Clinical and Translational Research Center (CTRC).

Arterial stiffness.

Carotid artery compliance was determined using high-resolution ultrasound imaging as previously described (50). To determine whether the effects of the interventions were selective for the carotid arteries, arterial compliance of the brachial artery, a medium-sized muscular artery, was also measured. Carotid and brachial images were analyzed for arterial distention using Vascular Analysis Tools version 5.5. All images were coded by number, blinded to group assignment, and analyzed by the same individual.

Brachial artery blood pressure.

Peripheral arterial blood pressure was measured in triplicate in the seated and the supine positions with a semiautomated device (Dinamap; Johnson & Johnson) over the brachial artery, as previously described (50).

Vascular endothelial-dependent vasodilation.

Ultrasound measurements of brachial artery flow-mediated dilation (FMD) were performed according to the method originally described by Celermajer et al. (3) and as previously described (9). Brachial artery FMD was determined using duplex ultrasonography (GE Vivid I) using a multifrequency linear-array transducer as previously described (3, 9). Briefly, a pediatric cuff was placed on the upper forearm, and brachial artery images were acquired ∼3–6 cm above the antecubital fossa. The ultrasound probe was clamped to ensure the location of the same arterial segment with the serial measurements and to avoid any involuntary movement. After obtaining concurrent measures of baseline brachial artery diameter and blood flow velocity, reactive hyperemia was produced by inflating the cuff up to 250 mmHg of pressure for 5 min followed by rapid deflation. After the release of the arterial occlusion, Doppler blood flow velocity was acquired, and B-mode ultrasound brachial artery diameter images were measured continuously for 2 min. Brachial artery diameter and blood flow velocity were analyzed using a commercially available software package and analyzed by the same individual (Vascular Analysis Tools 5.5.1; Medical Imaging Applications, Iowa City, IA). The initial 10 velocity waveform envelopes after cuff deflation were averaged to obtain the time-averaged peak velocity. Brachial artery peak hyperemic shear rate was calculated as the peak mean blood velocity divided by occlusion diameter. All procedures conformed strictly with recently published guidelines for assessing FMD in human subjects (5).

Body composition, metabolic risk factors, sex hormones, and oxidative stress markers.

Total fat mass and fat-free mass were determined using dual energy x-ray absorptiometry (DPX-IQ; Lunar). Minimal waist and hip circumferences were measured according to previously published guidelines, and the waist-to-hip ratio was calculated (27).

Fasting plasma concentrations of insulin and total (Roche Diagnostic Systems, Indianapolis, IN)- and high-density lipoprotein (Diagnostic Chemicals, Oxford, CT) cholesterol were determined using enzymatic/colorimetric methods, and LDL-cholesterol was determined using the Friedewald equation (12). Serum concentration of estradiol was measured using chemiluminescence, and plasma endothelin-1 was measured using an enzyme-linked immunoassay (14). Oxidized LDL, an indirect measure of oxidative stress, was determined with enzyme-linked immunosorbent plate assays (Alpco Diagnostics, Windham, NH). Total antioxidant status (TAS), a measure of the overall antioxidant defenses, was determined on serum samples using the Randox Laboratories (Oceanside, CA) enzymatic kit, and glutathione peroxidase, an antioxidant found in the cytoplasm and mitochondria, was measured using the RANDOX RANSEL glutathione peroxidase assay (Randox Laboratories). All assays were performed by the University of Colorado Denver CCTSI CTRC core laboratory.

Experimental design.

To determine whether BH₄ deficiency contributes to reduced endothelial vasodilatory function and arterial stiffening in estrogen-deficient postmenopausal women, measurements were obtained in premenopausal and postmenopausal women before and 3 h after oral BH₄ [(6R)-5,6,7,8-tetrahydro-l-biopterin dihydrochloride; Schirks Laboratories] dosing (10 mg/kg body wt). Three hours was chosen because that is the time when BH₄ reaches its maximal plasma concentrations (11). To examine whether the improvements in vascular function with estrogen could be related to BH₄, postmenopausal women were randomly assigned to either transdermal placebo or estradiol (0.05 mg/day, Climara; Bayer HealthCare Pharmaceuticals) for 2 days, and then vascular measurements were repeated before and 3 h after BH₄.

Statistical analysis.

Unpaired t-tests were used to assess group differences in subject characteristics, humoral vasoactive factors, oxidative stress markers, and baseline brachial artery EDD and carotid arterial compliance between premenopausal and postmenopausal women. Paired t-tests were used to determine the effects of oral BH₄ and transdermal estradiol or placebo on vasodilatory function. Secondary analyses were performed using two-group t-tests on the changes in carotid artery compliance and brachial artery FMD with BH₄ and transdermal estradiol or placebo. ANCOVA was used to adjust for potential confounders. Exploratory analyses were performed using Pearson product-moment correlations to test for the presence of significant linear bivariate relations between variables of interest. Data analysis was performed with SPSS software, version 18.0.

RESULTS

Subject characteristics.

Postmenopausal women were on average 8.7 ± 1.2 yr postmenopause. BMI, systolic blood pressure, fasted glucose, total and LDL cholesterol were higher, and estradiol was lower in postmenopausal compared with premenopausal women (all P < 0.05). Plasma endothelin-1 tended to be higher in postmenopausal women (P = 0.08). There were no other significant group differences in body composition, fasted insulin, oxidized LDL, or antioxidant concentrations (Table 1).

Table 1.

Subject characteristics of pre- and postmenopausal women

Variable	Premenopausal	Postmenopausal
n	9	24
Age, yr	33 ± 2	57 ± 1*
Body mass, kg	63.1 ± 3.4	67.1 ± 2.1
BMI	22.7 ± 1.3	26.0 ± 0.9*
Body fat, %	31 ± 3	37 ± 2
Waist circumference, cm	77 ± 5	83 ± 3
WHR	0.81 ± 0.03	0.83 ± 0.02
Systolic BP, mmHg	103 ± 5	120 ± 3*
Diastolic BP, mmHg	67 ± 4	73 ± 2
Total cholesterol, mmol/l	4.3 ± 0.3	5.0 ± 0.2*
LDL-cholesterol, mmol/l	2.4 ± 0.2	2.9 ± 0.1*
HDL-cholesterol, mmol/l	1.5 ± 0.1	1.6 ± 0.1
Fasting insulin, pmol/l	52 ± 13	77 ± 8
Fasting glucose, mmol/l	4.5 ± 0.1	4.9 ± 0.1*
Estradiol, pmol/l	286 ± 86	122 ± 53*
Endothelin-1, pg/ml	5.8 ± 0.5	6.6 ± 0.3
Oxidized LDL, U/l	48.1 ± 5.4	53.9 ± 3.1
TAS, mmol/l	1.35 ± 0.05	1.40 ± 0.03
Glutathione peroxidase, U/l	6,173 ± 395	6,906 ± 228

Values are means ± SE; n, no. of subjects. BMI, body mass index; WHR, waist-to-hip ratio; BP, blood pressure; LDL, low-density liproprotein; HDL, high-density lipoprotein; TAS, total antioxidant status.

^*P < 0.05 vs. premenopausal.

BH₄ administration in premenopausal and postmenopausal women.

Baseline carotid artery compliance (i.e., before BH₄ administration) was lower (P < 0.0001) in the postmenopausal women when compared with premenopausal women (Fig. 1). The reduced baseline carotid artery compliance in the postmenopausal women was associated with a lower baseline brachial artery FMD (before BH₄ administration, P < 0.0001 vs. premenopausal women; Fig. 2). Carotid artery compliance was strongly correlated with brachial artery FMD (r=0.66; P < 0.0001) in the pooled women, as well as within the postmenopausal women (r=0.47, P = 0.02). Baseline brachial artery compliance was not different between pre- and postmenopausal women (0.10 ± 0.02 vs. 0.10 ± 0.01 mm²·mmHg⁻¹·10⁻¹, respectively, P = 0.98), nor was it correlated with carotid artery compliance (r=−0.08, P = 0.64) or brachial artery FMD (r=−0.13, P = 0.49).

Fig. 1.

Carotid artery compliance before and after oral tetrahydrobiopterin (BH₄) administration in premenopausal and postmenopausal women. Data are reported as means ± SE. *P < 0.0001 vs. premenopausal women. †P < 0.001 vs. baseline of the same group.

Fig. 2.

Brachial artery flow-mediated dilation (FMD) before and after oral BH₄ administration in premenopausal and postmenopausal women. Data are reported as means ± SE. *P < 0.0001 vs. premenopausal women. †P < 0.001 vs. baseline of the same group.

BH₄ administration increased carotid artery compliance by 24 ± 5% in the postmenopausal women (vs. baseline, P < 0.001) but had no effect in the premenopausal women (P = 0.62; Fig. 1) or on brachial artery compliance of either group (P = 0.52, data not shown). The increase in carotid artery compliance corresponded with a 64 ± 11% increase in brachial artery FMD with BH₄ administration in the postmenopausal women (P < 0.001; Fig. 2) but did not have a significant effect in the premenopausal women (P = 0.10). Moreover, in the postmenopausal women, the changes in carotid artery compliance and in FMD from baseline in response to BH₄ administration were correlated with each other (r=0.54, P = 0.01). There was no change in carotid or brachial artery lumen diameter, brachial artery peak hyperemic shear rates, arterial blood pressure, or heart rate with BH₄ administration (Table 2).

Table 2.

Carotid and brachial artery parameters and hemodynamics in premenopausal and postmenopausal women

Variable	Premenopausal	Postmenopausal
Carotid diameter, mm
Baseline	5.78 ± 0.15	5.95 ± 0.09
After BH4	5.73 ± 0.15	5.93 ± 0.09
Carotid distention, mm
Baseline	0.58 ± 0.03	0.33 ± 0.02*
After BH4	0.59 ± 0.03	0.39 ± 0.02†
Supine systolic BP, mmHg
Baseline	104 ± 5	123 ± 3*
After BH4	101 ± 5	123 ± 3
Diastolic BP, mmHg
Baseline	63 ± 3	71 ± 2*
After BH4	61 ± 3	72 ± 3
Pulse pressure, mmHg
Baseline	40 ± 4	52 ± 2*
After BH4	41 ± 3	51 ± 2
Brachial diameter, mm
Baseline	3.3 ± 0.2	3.8 ± 0.1*
After BH4	3.1 ± 0.2	3.7 ± 0.1
Peak shear rate§, s−1
Baseline	135 ± 11	125 ± 7
After BH4	132 ± 9	114 ± 6

Data are means ± SE. BH₄, tetrahydrobiopterin.

^*P < 0.05 vs. premenopausal.

^†P < 0.05 vs. baseline of the same group;

^§n = 20 postmenopausal and 9 premenopausal women.

Administration of estradiol with and without BH₄ in previously estrogen-deficient postmenopausal women.

There were no differences in characteristics of the postmenopausal women randomized to transdermal estradiol or placebo (Table 3), with the exception that baseline brachial artery FMD was significantly lower in the estradiol group compared with the placebo group (P = 0.01; Fig. 4). Carotid artery compliance and brachial artery FMD increased in response to transdermal estradiol [P < 0.001 (Fig. 3) and P = 0.02 (Fig. 4), respectively] but did not change with placebo (P = 0.20 and 0.54, respectively). Brachial artery compliance remained unchanged in both groups (P = 0.26 and 0.78, respectively, data not shown). The change in carotid artery compliance in response to transdermal estradiol was strongly correlated with the change in FMD (r=0.64, P = 0.03). With the exception of an increase in serum estradiol concentration and decrease in fasted insulin concentration and systolic blood pressure with transdermal estradiol (both P < 0.05; Tables 4 and 5), there were no changes in any other humoral factor or hemodynamics with estradiol treatment. The changes in fasted insulin and systolic blood pressure and the time past menopause were not correlated with the change in carotid artery compliance or brachial artery FMD. Moreover, the change in carotid artery compliance and brachial artery FMD with estradiol was still significant (P = 0.001 and P = 0.04, respectively) after adjustment for these potential confounders. Oral BH₄ had no effect on carotid artery compliance (P = 0.10; Fig. 3) or brachial artery FMD in postmenopausal women after treatment with transdermal estradiol (P = 0.91; Fig. 4). In contrast, oral BH₄ increased carotid compliance (P = 0.006; Fig. 3) and brachial artery FMD (P = 0.02; Fig. 4) in placebo-treated postmenopausal women.

Table 3.

Subject characteristics for the postmenopausal women who participated in the estradiol and placebo intervention study

Variable	Estradiol	Placebo
n	12	10
Age, yr	57 ± 1	58 ± 1
Menopause duration, yr	9.2 ± 1.7	8.4 ± 1.7
Prior HT use, %	50	60
Body mass, kg	69.6 ± 3.1	64.6 ± 3.1
BMI	26.5 ± 1.3	25.5 ± 1.3
Body fat, %	39 ± 2	35 ± 2
Waist circumference, cm	83 ± 6	83 ± 6
WHR	0.85 ± 0.03	0.81 ± 0.03
Systolic BP, mmHg	122 ± 4	118 ± 4
Diastolic BP, mmHg	75 ± 3	71 ± 3
Total cholesterol, mmol/l	4.9 ± 0.2	5.1 ± 0.2
LDL-cholesterol, mmol/l	2.8 ± 0.2	2.9 ± 0.2
HDL-cholesterol, mmol/l	1.6 ± 0.1	1.7 ± 0.1
Fasting insulin, pmol/l	82 ± 12	72 ± 12
Fasting glucose, mmol/l	4.9 ± 0.1	4.9 ± 0.1

Data are means ± SE; n, no. of subjects. HT, hormone therapy.

Fig. 4.

Brachial artery FMD at baseline and following transdermal placebo or estradiol, and after oral BH₄ administration in postmenopausal women. *P < 0.001 vs. placebo women. †P < 0.001 vs. baseline of the same group.

Fig. 3.

Carotid artery compliance at baseline and following transdermal placebo or estradiol (E₂) and after oral BH₄ administration in postmenopausal women. Data are reported as means ± SE. *P < 0.001 vs. placebo women.

Table 4.

Circulating humoral factors before and after the interventions of estradiol or placebo

Variable	Estradiol	Placebo
Estradiol, pmol/l
Baseline	66 ± 8	65 ± 9
Follow up	297 ± 25*	62 ± 28
Glucose, mmol/l
Baseline	5.1 ± 0.1	4.9 ± 0.1
Follow up	5.0 ± 0.1	4.8 ± 0.1
Insulin, pmol/l
Baseline	91 ± 10	74 ± 11
Follow up	79 ± 10*	69 ± 11
Oxidized LDL, U/l
Baseline	53.3 ± 4.7	58.8 ± 4.7
Follow up	56.2 ± 4.1	57.2 ± 4.1
TAS, mmol/l
Baseline	1.44 ± 0.04	1.36 ± 0.04
Follow up	1.40 ± 0.04	1.41 ± 0.04
Endothelin-1, pg/ml
Baseline	6.5 ± 0.4	6.8 ± 0.4
Follow up	6.2 ± 0.3	7.4 ± 0.3
Glutathione peroxidase, U/l
Baseline	7,081 ± 364	6,600 ± 364
Follow up	6,691 ± 353	6,139 ± 353

All data are means ± SE.

^*P < 0.05 vs. baseline of the same group.

Table 5.

Carotid and brachial artery parameters and hemodynamics after estradiol and placebo treatment

Variable	Estrogen	Placebo
Carotid diameter, mm
Baseline	6.03 ± 0.13	5.92 ± 0.14
Follow up	5.96 ± 0.12	5.94 ± 0.13
Follow up + BH4	5.88 ± 0.13	5.88 ± 0.14
Carotid distension, mm
Baseline	0.33 ± 0.02	0.35 ± 0.03
Follow up	0.39 ± 0.02†	0.35 ± 0.0.2
Follow up + BH4	0.38 ± 0.02	0.41 ± 0.02†
Supine systolic BP, mmHg
Baseline	128 ± 5	113 ± 5
Follow up	120 ± 5†	115 ± 5
Follow up + BH4	122 ± 5	114 ± 5
Diastolic BP, mmHg
Baseline	75 ± 2	65 ± 3
Follow up	72 ± 3	68 ± 3
Follow up + BH4	72 ± 2	65 ± 4
Pulse pressure, mmHg
Baseline	53 ± 3	48 ± 4
Follow up	48 ± 3†	48 ± 4
Follow up + BH4	50 ± 3	48 ± 3
Brachial diameter, mm
Baseline	3.9 ± 0.13	3.5 ± 0.2
Follow up	3.8 ± 0.1	3.5 ± 0.2
Follow up + BH4	3.8 ± 0.2	3.5 ± 0.2
Peak shear rate§, s−1
Baseline	113 ± 11	143 ± 12
Follow up	108 ± 12	125 ± 12
Follow up + BH4	109 ± 13	127 ± 14

All data are means ± SE.

^†P < 0.05 vs. baseline of the same group;

^§n = 9 estradiol and 8 placebo.

DISCUSSION

The findings of the present study provide novel insight into the mechanisms contributing to large artery stiffening in estrogen-deficient postmenopausal women. First, administration of BH₄, a critical cofactor for eNOS functioning and NO production, increased carotid artery compliance in estrogen-deficient postmenopausal, but not premenopausal, women. Second, the increase in carotid artery compliance with BH₄ was significantly related to the increase in endothelial-dependent vasodilation with BH₄ administration. Third, while short-term transdermal estradiol increased carotid artery compliance and endothelial-dependent vasodilation in previously estrogen-deficient postmenopausal women, coadministration of BH₄ with estradiol does not augment vascular function further. Collectively, these findings suggest that reduced BH₄ bioavailability contributes to large artery stiffening in estrogen-deficient postmenopausal women in part through reduced endothelial vasodilatory function. Finally, our results also suggest that the beneficial effects of estrogen on arterial stiffening in previously estrogen-deficient postmenopausal women may be mediated by improved endothelial vasodilatory tone, secondary to increasing BH₄ bioavailability.

Arterial stiffening in estrogen-deficient postmenopausal women: BH₄ and reduced endothelial-dependent vasodilatory tone.

In the present study, baseline carotid artery compliance was ∼50% lower in healthy estrogen-deficient postmenopausal women compared with premenopausal women, whereas brachial artery compliance was not different between the groups, consistent with our and other's previous observations on central and peripheral arterial stiffness in older adults (2, 49, 51). The differential effects of arterial stiffening between the two arterial segments can be explained by differences in geometry and mechanical properties of elastic and muscular arteries and their distinct roles in hemodynamic regulation (37, 49). Arterial compliance is primarily determined by the intrinsic viscoelastic properties of the arterial wall (37). Compared with muscular arteries (i.e., brachial), which have a greater ratio of collagen to elastin content and, thus, are more rigid, the central arteries (i.e., aorta and carotid) are more elastic in nature, which allows them to “buffer” the pulsatile systolic output through elastic recoil, thereby sustaining continuous blood flow to the periphery.

The mechanisms that contribute to large elastic arterial stiffening in estrogen-deficient postmenopausal women are not completely understood but likely involve structural changes within the arterial wall, including increased collagen and reduced and fragmented elastin content (21, 36) and increased intimal-medial thickness (IMT) (32, 40). However, increased IMT does not appear to restrict compliance unless it exceeds a threshold of thickness representing atherosclerotic manifestations (40). The arterial wall is also composed of vascular smooth muscle cells that vasodilate and vasoconstrict in response to a number of circulating and local vasoactive mediators, including those released by the vascular endothelium. Indeed, endothelial-derived substances, including those that cause vasodilation (e.g., NO) and vasoconstriction (e.g., endothelin-1) of vascular smooth muscle cells, have been shown to modulate arterial stiffness (46, 55). As such, functional changes in endothelial regulation of vascular smooth muscle cell tone may also be involved. In this regard, aging and estrogen deficiency are associated with a reduced vasodilatory capacity of the vascular endothelium of which the loss of endothelial-derived NO bioavailability is a cardinal feature (4, 13, 48, 52). Consistent with this, we found that brachial artery FMD, a measure of systemic endothelial-dependent vasodilation mediated predominantly by NO (5, 8), was ∼50% lower in estrogen-deficient postmenopausal women compared with premenopausal women. Moreover, brachial artery FMD was strongly correlated with carotid artery compliance both in the pooled women and within the postmenopausal women, supporting the idea that large elastic arterial stiffening in estrogen-deficient postmenopausal women is mediated, in part, by an elevated state of vascular smooth muscle cell vasoconstrictor tone, possibly related to reduced NO bioavailability. In contrast, brachial artery FMD was not correlated with brachial artery compliance. This finding may be related to the fact that muscular arteries have a rich sympathetic innervation, and sympathetic activation and NO competitively impact brachial artery mechanics, possibly as a regulatory control feature to modify flow transit time through the vascular bed and back to the heart (41).

We previously reported that administration of supraphysiological concentrations of the potent antioxidant ascorbic acid increases carotid artery compliance in estrogen-deficient postmenopausal women, suggesting that oxidative stress contributes mechanistically to the large elastic artery stiffening in estrogen-deficient postmenopausal women (33, 34). We speculated that the mechanism by which ascorbic acid improved carotid artery compliance was likely mediated, in part, through protecting NO scavenging by ROS, which would in turn increase NO bioavailability and the tonic state of vascular smooth muscle cell vasorelaxation (19, 38). One of the sources of increased ROS production in the arterial wall has been attributed to “eNOS uncoupling,” a phenomenon that occurs when there are inadequate levels of BH₄, a critical cofactor for eNOS enzymatic function to produce NO from the interaction with l-arginine (1, 53). BH₄ can be limited when there is decreased de novo synthesis from guanosine 5′-triphosphate (GTP) and/or via reduced regeneration of BH₄ by dihydrofolate reductase (7). Additionally, BH₄ can be rapidly oxidized by peroxynitrite and other ROS to its inactive form dihydrobiopterin, creating a vicious cycle of vascular oxidative stress (1, 53). In the present study, we found that acute supplementation of BH₄ increased brachial artery FMD and carotid artery compliance in estrogen-deficient postmenopausal women but not in premenopausal women. Moreover, the improvements in carotid artery compliance and brachial artery FMD in response to BH₄ were strongly correlated with each other, suggesting that reduced BH₄ bioavailability may contribute to large elastic arterial stiffening in estrogen-deficient postmenopausal women through reduced endothelial vasodilatory tone. These results extend our previous findings by providing novel insight into a potential mechanism by which oxidative stress contributes to the large elastic artery stiffening in estrogen-deficient postmenopausal women. Interestingly, the magnitude of the improvement in carotid artery compliance with BH₄ (24%) was similar to what we reported previously with acute ascorbic acid (∼26%) (33). Because ascorbic acid is capable of stabilizing eNOS by recycling BH₃ to BH₄ (20), it is possible that part of the improvement in carotid artery compliance that we previously reported (33) may be through recycling intracellular BH₄ and preventing eNOS uncoupling, in addition to its antioxidant properties.

Vascular effects of estrogen in postmenopausal women: possible role of BH₄.

In the present study, estrogen supplementation, but not placebo, increased carotid artery compliance (reduced arterial stiffness) in postmenopausal women, consistent with previous investigations demonstrating reduced arterial stiffening with chronic MHT (26, 31, 35, 42). The results of the present study provide preliminary insight into the potential mechanisms by which estrogen may modulate arterial stiffening in postmenopausal women. Estrogen preserves vascular endothelial vasodilatory function in part by enhancing NO release and by protecting NO from inactivation by ROS (29). Animal studies demonstrate that estrogen supplementation prevents the decrease in NO release with ovariectomy, in part by restoring BH₄ bioavailability and decreasing the overproduction of superoxide anion (22, 23). Thus, we reasoned that the modulatory influence of estrogen on arterial stiffness may be through replenishing vascular BH₄ and increasing vasodilatory tone. In support of this, in the present study, the increase in carotid artery compliance in response to estrogen was associated with a marked increase in endothelial-dependent vasodilation. Moreover, while oral BH₄ improved brachial artery FMD and carotid artery compliance in women randomized to placebo, there was no further improvement in brachial artery FMD and carotid artery compliance following oral BH₄ in women randomized to estrogen. Thus, these findings suggest that the favorable influence of estrogen on arterial stiffening in postmenopausal women may be related in part to increased vasodilatory tone, possibly mediated by increased vascular BH₄ and NO bioavailability.

Although caution needs to be used in the interpretation of the present study's findings, there is reason to speculate that estrogen increases the bioavailability of BH₄ in postmenopausal women. Both GTP cyclohydrolase I (GTPCH I), the rate-limiting enzyme for de novo BH₄ synthesis, and BH₄ are reduced in mesenteric arteries isolated from aged mice compared with younger mice, and estrogen has been shown to counteract the downregulation of GTPCH I in bovine aortic endothelial cells incubated with high glucose (30, 56). Additionally, because estrogen has direct antioxidant effects in vitro and in vivo, estrogen may maintain and/or replenish BH₄ by preventing its oxidation by ROS (18, 23, 45). In the present study, plasma markers of oxidative stress (oxidized LDL) and antioxidants (TAS, glutathione peroxidase) did not change with estrogen supplementation. However, these indirect systemic plasma biomarkers lack sensitivity and may not accurately reflect the amount of oxidative stress and antioxidants in the vasculature. Additionally, it is possible that estrogen modulated other sources of ROS and/or endogenous antioxidants that we did not measure. Thus, it is plausible that estrogen could replenish BH₄ by increasing de novo BH₄ synthesis via stimulating or increasing GTPCH I, increasing the regeneration of BH₄, and/or decreasing ROS and the subsequent oxidation of BH₄.

Experimental considerations and limitations.

We recognize that mechanisms other than BH₄ could contribute to the decrease in arterial stiffness with estrogen supplementation, including the modulation of vasoconstrictor hormones and local factors. In the present study, plasma endothelin-1 concentrations tended to be higher in postmenopausal compared with premenopausal women but did not change with estrogen supplementation. However, plasma concentrations do not necessarily reflect differences at the local vascular level or vascular responsiveness to these factors. As such, we cannot rule out the possibility that these or other vasoactive factors were modulated with estrogen supplementation. Structural changes within the arterial wall (i.e., elastin-collagen composition) are thought to occur over a period of years, making it unlikely that 2 days of estrogen supplementation decreased arterial stiffness by such mechanism (39). Estrogen may have also decreased arterial stiffness in the postmenopausal women in part via its antioxidant effects that do not modulate BH₄.

The fact that carotid artery compliance and brachial artery FMD were not restored to premenopausal levels with either BH₄ or estrogen indicates that other mechanisms other than BH₄ deficiency contribute to arterial stiffening and reduced endothelial vasodilatory function in postmenopausal women. Possible mechanisms include other vasoconstrictor hormones and local factors discussed above, as well as extracellular matrix-linked structural alterations within the arterial wall that cannot be modified with short-term BH₄ or estrogen. Additionally, it is possible that the small effect of BH₄ on carotid artery compliance, and to a lesser extent brachial artery FMD, was the result of a suboptimal dose of BH₄ and that a higher dose of BH₄ may have resulted in a greater improvement in arterial compliance. The dose of BH₄ used in the present study (10 mg/kg of body wt) has been shown to increase plasma biopterin levels by ∼50-fold (11) and restore brachial artery FMD in sedentary older men (10), as well as in hypercholesterolemic men and women (6). In contrast to the study conducted in older men, brachial artery FMD was not restored with BH₄ administration in our postmenopausal women even though a similar dose of BH₄ was used (10). Thus, it is plausible that a higher dose of BH₄ may be necessary to restore endothelial vasodilatory function in postmenopausal women or that other mechanisms are contributing to the reduced brachial artery FMD in postmenopausal women. It is also possible that there may be a ceiling effect for how much brachial artery FMD can be improved with any intervention in postmenopausal women.

There are several limitations associated with the present study. First, we did not measure plasma or vascular BH₄ levels. Thus, we cannot demonstrate that there were group differences at baseline or that oral BH₄ and estrogen supplementation replenished vascular BH₄ in postmenopausal women. Second, we used brachial artery FMD to mechanistically examine whether increased NO bioavailability mediated the improvements in carotid artery compliance with BH₄ and estrogen instead of directly measuring NO and vasodilatory tone in the carotid artery. Thus, because of the different vascular districts, we cannot infer a causal relationship or rule out other mechanisms possibly involved in the endothelial-dependent vasodilatory improvement with BH₄ or estrogen (e.g., prostacyclin). Additionally, it is possible that the improvement in brachial artery FMD was related to a more compliant brachial artery. However, brachial artery compliance did not change with BH₄ or estrogen administration, making it unlikely that the improvement in FMD with BH₄ and estrogen was related to such mechanism, but rather likely related to increased NO bioavailability. The lack of change in brachial artery compliance with the interventions was not surprising given that baseline brachial artery compliance was not different between the groups. Nonetheless, it is possible that any improvements in brachial compliance could have been offset by competitive neurogenic input (41). Third, we did not measure endothelial-independent vasodilation; thus, it is possible that estrogen and BH₄ could have increased vasodilatory function through actions on vascular smooth muscle cells. However, previous studies have demonstrated no improvement in endothelial-independent vasodilation with BH₄ administration (10, 15–17, 28, 44). Finally, we did not conduct a randomized placebo controlled study with oral BH₄ as previously done (6, 10), and, thus, improvements in vascular function in the estrogen-deficient postmenopausal women may have been because of a “placebo effect.” However, oral BH₄ improved vascular function in the groups of women that we hypothesized would have reduced BH₄ (i.e., estrogen-deficient postmenopausal and not premenopausal women at baseline; and in postmenopausal women following placebo treatment and not estrogen treatment), making it unlikely that our findings are related to a placebo effect.

In conclusion, our findings suggest that reduced vascular BH₄ contributes to arterial stiffening in estrogen-deficient postmenopausal women, related in part to reduced endothelial vasodilatory function. Moreover, BH₄ maintenance may be an underlying mechanism by which estrogen benefits endothelial-dependent vasodilation and arterial stiffness; however, further study is needed in postmenopausal women for a definitive causal link. These findings provide new mechanistic insight into the processes that mediate the biological changes in arterial stiffening in estrogen-deficient postmenopausal women that may help guide future sex-specific therapies for the prevention of CVD.

GRANTS

This study was supported by the National Institutes of Health Awards AG-027678 and Colorado Clinical Translational Sciences Institute (CCTSI) RR-025780, a University of Colorado Denver (UCD) Center for Women's Health Research Junior Faculty Development Award, and a UCD Women's Health Research Pilot Project Grant.

DISCLOSURES

The authors have nothing to disclose.

AUTHOR CONTRIBUTIONS

K. Moreau: conceived and designed research, acquired and managed the data, analyzed and interpreted the data, performed statistical analyses, handled funding and supervision and drafted the manuscript; A. Meditz: provided medical oversight, made critical revision of the manuscript for important intellectual content; K. Deane: provided medical oversight and made critical revision of the manuscript for important intellectual content; and W. Kohrt: data management, interpreted data, and made critical revision of the manuscript for important intellectual content.