Sex Hormones and Atherosclerosis Prevention in Perimenopausal Women

Endothelial dysfunction, featuring reduced vascular endothelial-dependent dilation (EDD), is
a significant predictor of cardiovascular (CV) events. Reduced EDD is characterized by the
loss of nitric oxide (NO) production. EDD declines with aging in women at a slower rate
compared to men but rapidly declines after menopause and quickly catches up to men,
suggesting a beneficial effect of estrogen (E2). Our research has investigated the mechanisms
underlying endothelial protection of E2 including how hormonal fluctuations during the
peri-to early postmenopausal years contribute to endothelial dysfunction, and how these
hormonal changes might interact with aging. Using a well described experimental model
(systemic infusion of ascorbic acid; AA) to acutely reduce reactive oxygen species (ROS), our
preliminary findings implicate oxidative stress as an underlying mechanism explaining the
impaired EDD during the menopausal transition. Specifically we found improved EDD in response
to AA in late peri- and postmenopausal women but not in pre- or early perimenopausal women.
These data indicate that E2 may protect the vasculature from free radical injury during pre-
and early perimenopausal years. In support of this concept, we demonstrated that AA can
reverse the decrease in EDD that occurs in the setting of short-term E2 deficiency (3-days of
gonadotropin releasing hormone antagonist; GnRHant) in pre-and early perimenopausal women.

Tetrahydrobiopterin (BH4) is a critical co-factor in the production of NO by endothelial
nitric oxide synthase (eNOS), and in the face of deficient levels, uncoupling of eNOS results
in the production of ROS instead of NO. Whether the increased ROS and impaired EDD that
occurs during the peri- and early postmenopausal periods is associated with reduced vascular
BH4 is at present unknown. However, our finding that AA could not improve the reduced EDD
found in early perimenopausal women or restore EDD in peri-and postmenopausal women to
premenopausal levels, indicates that other sources of ROS or causes of low NO are involved in
the impaired EDD across the stages of the menopause transition and aging in women. In vitro
studies demonstrate that the co-administration of BH4 and AA completely prevents the eNOS
uncoupling and decreased NO production in endothelial cells treated with a peroxynitrite
(ONOO-) donor, compared to administering either one alone. The global aim of this renewal is
to follow-up on our previous findings and to determine the role of BH4 as a mediator of
endothelial dysfunction [across the stages of the menopausal transition and aging in women.
This global aim will be addressed by 3 specific aims (SA). Using a cross-sectional design,
SA1 will measure EDD in healthy pre-, peri- and postmenopausal women: 1) under basal
conditions; and 2) following an acute increase in BH4. SA2 will expand on the cross-sectional
comparisons via short-term (10 days) ovarian suppression (GnRhant) in pre-and early
perimenopausal women to isolate the age-independent effects of E2. SA3 will examine whether
co-administration of BH4 and AA fully restore youthful EDD in peri-and postmenopausal women.

SA1: To determine the possible mechanistic involvement of BH4 deficiency in the decline in
endothelial function during the peri-and early postmenopausal period and its potential
relation to oxidative stress. This will be assessed by comparing brachial artery EDD and
plasma and endothelial cell markers of oxidative stress and NO (see methods for details) in
healthy pre-, peri-and postmenopausal women: 1) under basal conditions; and 2) in response to
acute oral BH4 (vs PL) supplementation.

Hypothesis1 (H1): a) EDD will increase in response to BH4 in early peri, late peri and
postmenopausal women but not premenopausal women. b) The increase in EDD in response to BH4
will be associated with a decrease in plasma markers of oxidative stress. c) The reduced EDD
in peri- and postmenopausal women will be associated with lower endothelial cell markers of
NO and higher oxidative Stress.

SA2: To determine whether the decrease in EDD in response to acute ovarian suppression in
pre- and early perimenopausal women is related to reduced BH4 bioavailability and elevated
markers of oxidative stress. This will be assessed by comparing EDD and plasma markers of
oxidative stress after acute oral supplementation of BH4 (or PL) following either acute
ovarian suppression with (GnRHant+E2) or without (GnRHant+PL) E2 add-back. Additionally,
endothelial cell protein expression of markers of BH4 biosynthesis and oxidative stress will
be measured before and after the suppression/add-back paradigm.

H2: a) The expected decrease in EDD and increase in plasma oxidative stress markers following
acute ovarian suppression will be mitigated in the groups treated with oral BH4
(GnRHant+PL+BH4); there will be little or no effect of the addition of BH4 to the E2 add-back
group. b) Ovarian suppression alone will reduce endothelial protein markers of BH4
biosynthesis and will increase markers of oxidative stress. These changes will not be
observed following ovarian suppression with E2 add-back and oral BH4. These sets of
experiments will provide evidence that the oxidative stress-related decrease in EDD in
response to E2 suppression is in part, related to decreased BH4 bioavailability.

SA3: To determine whether the co-administration of BH4 with AA will completely restore EDD in
peri-and postmenopausal women, and in early perimenopausal women following ovarian
suppression.

H3: The co-administration of BH4 with AA will restore EDD to premenopausal levels a) in
peri-and postmenopausal women, and b) in early perimenopausal following acute ovarian
suppression.

Inclusion Criteria:

- age criteria established for pre-, peri- and postmenopausal women, and if
postmenopausal at least 1 year beyond menopause

- resting blood pressure <140/90 mmHg 81; 2) plasma glucose concentrations <110 mg/dl
under fasting conditions

- sedentary or recreationally active (<3 days of vigorous aerobic exercise)

- no use of OCs, HT, or other medications that might influence cardiovascular function

- nonsmokers

- no use of vitamin supplements, NSAIDS or willing to stop use for duration of the study

- not taking any other medications that would interact with cetrotide, E2 patch, or
Kuvan® to confound interpretation of results

Exclusion Criteria:

- history of or active estrogen-dependent neoplasms, acute liver or gallbladder disease,
vaginal bleeding, venous thromboembolism, hypertriglyceridemia, and CVD

- known allergy to transdermal patch, GnRHant (i.e., hypersensitivity to cetrorelix,
extrinsic peptide hormones, mannitol, GnRH, benzyl alcohol - the vehicle for injection
of cetrorelix), or KUVAN

- history of stomach ulcer or bleeding

- other contraindications to HRT, GnRHant, and KUVAN (i.e., taking Levodopa, medications
that can inhibit folate metabolism including methotrexate)

- pregnant or currently breastfeeding

- Other conditions for which individuals will be excluded from the study include:
diabetes, active infection, history of seizures or disease that affects the nervous
system, sepsis or an abnormal resting ECG