( PDF ) Avaliação da função endotelial e associação com androgênios endógenos em pacientes pós-menopáusicas

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UNIVERSIDADE FEDERAL DO RIO GRANDE DO SUL

FACULDADE DE MEDICINA

PROGRAMA DE PÓS-GRADUAÇÃO EM CIÊNCIAS MÉDICAS: ENDOCRINOLOGIA

AVALIAÇÃO DA FUNÇÃO ENDOTELIAL

E ASSOCIAÇÃO COM ANDROGÊNIOS ENDÓGENOS EM PACIENTES

PÓS-MENOPÁUSICAS

Maria Augusta Maturana

Porto Alegre

2006

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UNIVERSIDADE FEDERAL DO RIO GRANDE DO SUL

FACULDADE DE MEDICINA

PROGRAMA DE PÓS-GRADUAÇÃO EM CIÊNCIAS MÉDICAS: ENDOCRINOLOGIA

Maria Augusta Maturana

AVALIAÇÃO DA FUNÇÃO ENDOTELIAL

E ASSOCIAÇÃO COM ANDROGÊNIOS ENDÓGENOS EM PACIENTES

PÓS-MENOPÁUSICAS

Tese apresentada como requisito parcial

para a obtenção do título de Doutor em

Ciências Médicas: Endocrinologia

Orientadora: Profª. Drª. Poli Mara Spritzer

Co-orientadora: Profª. Drª. Maria Claudia Irigoyen

Porto Alegre

2006

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Este estudo foi realizado na Unidade de Endocrinologia Ginecológica, Serviço

de Endocrinologia do Hospital de Clínicas de Porto Alegre, com o apoio financeiro das

seguintes instituições:

FAPERGS

CNPq

FIPE/HCPA

PRONEX 26/98

À Vitória, minha amada filha.

AGRADECIMENTOS

Para a realização deste trabalho, recebi o incentivo e a colaboração de muitas

pessoas e instituições.

Em especial agradeço:

À Profª Drª Poli Mara Spritzer pela competência e rigor científico na valiosa

orientação deste trabalho. Pela confiança e oportunidade!

À Profª Drª Maria Claudia Irigoyen pela orientação e viabilização de detalhes

importantes na execução deste projeto.

Às pacientes, muitas destas, corajosas mulheres, que enfrentaram pela

primeira vez a vinda à capital para participar deste estudo, o meu sincero

reconhecimento.

Aos meus pais, Leodoro e Selmira, pelos seus ensinamentos e sobretudo, por

seus exemplos de vida. Minhas irmãs, cunhados e sobrinhos: pela amizade e alegria

do nosso convívio.

Ao Dr. Marcelo Rubira pelo empenho e competência na realização dos exames

de dorsal hand vein.

Ao acadêmico Vitor Breda, pela ajuda no atendimento das pacientes e coleta

de dados.

Ao Serviço de Cardiologia do HCPA pelo suporte e espaço físico para a

realização dos exames de dorsal hand vein , em especial a Enfª Eneida Rabelo. E aos

colegas Antônio Marcos V da Silva e Luis Signori pelos vários auxílios na realização

destes exames.

Ao Dr Francisco Lhullier pelas dosagens da endotelina.

Aos colegas e amigos da Unidade de Endocrinologia Ginecológica do HCPA.

Em especial agradeço as palavras sempre amigas do César, Gislaine, Marcelo e

Fabiane.

Aos colegas do Laboratório de Endocrinologia Molecular e Neuroendocrinologia

do Departamento de Fisiologia da UFRGS. E À inesquecível Iracema Vera Soares,

pela cuidado e competência na coleta das amostras.

À gentil Miriam Santa Helena pela colaboração.

Ao Artur e á Vitória: pelo amor, paciência e perseverança! Por serem pessoas

maravilhosamente brilhantes e especiais, e deixarem minha vida muito feliz!

Science without religion is lame, religion without science is blind.

Albert Einstein

APRESENTAÇÂO

A disfunção endotelial está implicada na gênese da aterosclerose e

provavelmente no aumento da incidência de doença cardiovascular em mulheres na

pós- menopausa. Na literatura, ainda há controvérsia a respeito da influência dos

androgênios endógenos, como um dos fatores envolvidos num perfil de risco

cardiovascular nesta população. Nesta tese, estudamos as associações entre

androgênios endógenos, variáveis antropométricas, proteina C-reativa, endotelina-1 e

função endotelial numa amostra de pacientes pós-menopáusicas. A apresentação

desta tese está organizada em três capítulos, conforme o formato sugerido pelo

Programa de Pós-Graduação em Ciências Médicas: Endocrinologia, da Universidade

Federal do Rio Grande do Sul.

O primeiro capítulo é constituído por um trabalho de revisão (artigo 1)

:Menopause, Estrogens and Endothelial Dysfunction: Current Concepts. Este artigo

enfoca aspectos pertinentes à menopausa e hipoestrogenismo, endotélio e suas

funções na homeostase cardiovascular, e modificações na função endotelial na pós-

menopausa. Aborda ainda outros temas, como as ações vasculares e extravasculares

do estrogênio endógeno, e implicações do uso da terapia hormonal na pós-

menopausa. Este manuscrito, já está aceito para publicação no periódico Clinics.

Nos próximos dois capítulos foram inseridos os artigos 2 e 3, referentes a

dados originais que constituem a parte experimental e discussão desta tese

desenvolvida na Unidade de Endocrinologia Ginecológica do Serviço de

Endocrinologia do HCPA.

No artigo 2 “Relationship between endogenous testosterone, endothelin-1 and

C-reactive protein in postmenopausal women” foram incluídas 53 mulheres na pós-

menopausa, com idade superior a 40 anos, excluídas diabéticas, com disfunção

tireoidiana ou hepática. Neste trabalho, o objetivo foi testar a influência dos níveis de

testosterona endógena em marcadores inflamatórios, de função endotelial, e no perfil

metabólico e antropométrico.

O artigo 3 “Free androgen index and endothelial function in postmenopausal

women”, analisa 26 pacientes pós-menopáusicas, não tabagistas ou hipertensas, com

idade inferior a 65 anos, que foram selecionadas entre as pacientes do estudo

anterior, com o objetivo de determinar uma associação entre o índice de androgênios

livres e marcadores vasomotores endoteliais Os exames de avaliação do endotélio

venoso, que fizeram parte deste estudo, foram realizados pelo Dr Marcelo Rubira, no

Serviço de Cardiologia do Hospital de Clínicas de Porto Alegre.

Estes dois manuscritos serão enviados para publicação em periódicos

indexados internacionais.

SUMÁRIO

Artigo 1. Menopause, Estrogens and Endothelial Dysfunction: Current

Concepts.........................................................................................................................9

Artigo 2. Relationship between endogenous testosterone, endothelin-1 and C-

reactive protein in postmenopausal women .................................................................38

Artigo 3. Free androgen index and endothelial function in postmenopausal

women...........................................................................................................................57

ANEXOS .......................................................................................................................80

ARTIGO 1

Menopause, Estrogens and Endothelial Dysfunction: Current Concepts

Maturana MA, Irigoyen MC, Spritzer PM

Clinics.2007;62(1):77-86

Menopause, Estrogens and Endothelial Dysfunction: Current Concepts

Maturana MA

, Irigoyen MC

2,3

, Spritzer PM

1,2

Gynecological Endocrinology Unit, Division of Endocrinology, Hospital de Clínicas de

Porto Alegre, Porto Alegre, Brazil;

Department of Physiology, Universidade Federal do

Rio Grande do Sul , Porto Alegre, Brazil; ;

INCOR, São Paulo, Brazil.

This study was supported by grants from Conselho Nacional de Desenvolvimento

Científico e Tecnológico (CNPq), FAPERGS (Fundação de Apoio à Pesquisa do Rio

Grande do Sul) and PRONEX 26/98 (Programa de Apoio aos Núcleos de Excelência

em Pesquisa).

Running title: Menopause, estrogens and endothelial dysfunction

Corresponding author: Poli Mara Spritzer, M.D., Ph.D.

Department of Physiology, Universidade Federal do Rio Grande do Sul,

Rua Sarmento Leite, 500

90050-170 Porto Alegre, RS, Brazil

Tel: +55-51-3316-3671 / Fax: +55-51-3316-3656.

E-mail: [email protected]

Abstract

Menopause is defined as the permanent cessation of menses. Cardiovascular disease

is the leading cause of death among postmenopausal women in developed countries.

The disparity between the incidence of cardiovascular disease among women in pre-

and post-menopause has been ascribed to the actions of endogenous estrogen on the

cardiovascular system and, particularly, on the vascular endothelium. The endothelium

plays an important role in cardiovascular homeostasis, either in the vascular tonus and

regulation or in coagulation and inflammatory response. Endothelial dysfunction is

implicated in the genesis of atherosclerosis and other chronic disorders, such as

diabetes mellitus and hypertension. The pharmacological use of estrogen exerts

influence on the circulating levels of vascular tonus, inflammatory, pro-thrombotic and

fibrinolytic markers, but the impact of these changes on the atherosclerotic disease is

still uncertain.

Keywords: menopause, endothelial function, cardiovascular disease, estrogen,

hormone therapy

Resumo

A menopausa é definida como a cessação permanente das menstruações. A doença

cardiovascular é a principal causa de mortalidade em mulheres na pós- menopausa,

em países desenvolvidos. A disparidade entre a incidência de doença cardiovascular

entre mulheres na pré e pós-menopausa tem sido atribuída a ações do estrogênio

endógeno sobre o sistema cardiovascular e, em especial, sobre a função do endotélio

vascular. O endotélio tem importante papel na homeostase cardiovascular, seja no

controle do tônus e permeabilidade vascular, ou da coagulação e resposta

inflamatória. A disfunção endotelial está implicada na gênese da aterosclerose e de

outras doenças crônicas, como diabete melito e hipertensão arterial. O uso

farmacológico de estrogênio exerce influência sobre concentrações circulantes de

marcadores do tônus vascular, inflamatórios, pró-trombóticos e fibrinolíticos, porém o

impacto destas alterações sobre a doença aterosclerótica ainda não está determinado.

Palavras chave: menopausa, função endotelial, doença cardiovascular, estrogênio,

terapia hormonal

Menopause is defined by the World Health Organization as the permanent

cessation of menses as a result of the loss of ovarian follicular function or of surgical

removal of ovaries. ¹ The mean age for occurrence of natural menopause is around 50

years.

1,2

The management of the menopausal patient has been a matter of great

concern in the last decades, both in terms of epidemiology and public health and in

medical scientific research. This is largely due to the improved life expectancy, which

allows to predict that the female population can live a third of their lifetime after

menopause. Demographic studies indicate that in 1990 about 467 million women were

at age 50 or over. For year 2030, the estimate is that this group exceeds 1200 million

women.

Hormonal changes accompanying menopause, particularly the decreased

levels of estrogen hormones, have a great physiological impact. Estrogen deficiency

has been associated with vasomotor symptoms, urogenital atrophy, and cognitive

impairment, as well as increased risk of chronic degenerative diseases such as

osteoporosis and Alzheimer's disease.

Menopause and Cardiovascular Disease

CVD remains as the leading cause of death in the twenty first century.

Despite the advances in this area, CVD is still the main cause of death among women

in developed countries. In the United States, over half a million women die of CVD

every year, exceeding the number of deaths among men and the sum total of the next

leading causes of death among women. Coronary arterial disease (CAD) is responsible

for most of the deaths by CVD among women .

Moreover, women have a less

favorable prognosis than men in the presence of coronary event: 40% of the total

coronary events in women are deadly, and 67% of sudden deaths occur in women

without history of coronary disease.

The prevalence of CVD in pre-menopausal women is smaller than in post-

menopausal ones, when there is an exponential increase, with the female risk equaling

the male one at the age of 70 years.

This lag concerning the age period at which the

frequency of cardiovascular events increases among women as compared to men has

been ascribed to the actions of endogenous estrogen on the cardiovascular system,

through mechanisms as yet not completely clarified. On the other hand, the influence of

hormone therapy (HT) during the menopause on the cardiovascular comorbidities is

not well-established. Observational studies showed a reduction of up to 50% in the risk

of CVD in postmenopausal women using HT.

7,8,9

Nevertheless, the results of two large randomized prospective studies refute

the cardioprotective effects of hormone therapy evidenced in observational studies.

The Heart and Estrogen/Progestin Replacement Study (HERS) was a randomized

clinical trial designed to test the effectiveness of HT in the secondary prevention of

coronary heart disease. The results indicate an increase in the coronary events within

the first year of follow-up after acute myocardial infarction (AMI) among patients treated

with a combination of conjugated estrogens/ medroxy progesterone acetate at fixed

doses and mean age of

66.7 years.

Recently, the Women’s Health Initiative (WHI), a

prospective study of primary prevention of CVD, was interrupted early because a

higher incidence of cardiovascular events was demonstrated among the women

randomized for use of HT, than in the group receiving placebo. In this study, although

HT was associated with reduced risk of colorectal cancer and bone fracture, it was

associated as well with increased risk of breast cancer.

Finally, in 2004 the results of

the therapeutic arm of the WHI evaluating the isolated use of conjugated estrogens

versus placebo among 10,739 hysterectomized post-menopausal women were

published. There was increased risk of CVA (rr=1.39 CI: 110-177) but decreased risk of

hip fracture and null effect on the incidence of CVD, as well as potential reduction in

the incidence of breast cancer. Overall, the risk-benefit index was neuter.

Many are the well-known risks for CVD, such as systemic hypertension,

smoking, obesity, sedentary life style, dyslipedemia, stress, family history of CVD,

diabetes mellitus, and insulin resistance.

More recently, endothelial vascular

dysfunction has been on the agenda for its association with CVD, as detailed below.

The Endothelium

The endothelium is a layer of cells lining all the vessels of the organism and

placing itself between the blood and the vascular smooth muscle layer. The endothelial

cells form the more exposed surface of the inner vascular layer, presenting selective

permeability, of non-thrombogenic character, with exuberant metabolic activity, having

also the ability to produce several vasoactive substances.

The main actions exerted by the endothelium can be described as:

• Selective permeability: The endothelium forms a highly selective

permeability barrier, regulating the flow of nutrient substances, many biologically active

molecules, and blood cells.

• Maintenance of a balance between thrombosis and fibrinolysis: The

endothelium normally provides a non-thrombogenic surface due to its ability to form

prostaglandin derivatives, especially prostacyclin, which is a potent vasodilator and an

effective inhibitor of platelet aggregation.

The endothelial cells also secrete agents

that are effective for the lysis of coagula, including the plasminogen, but also

synthesize pro-coagulant agents such as Von Willebrand factor (vWF). Also, they

produce thrombogenic substances such as coagulation factors, adhesion molecules,

plasminogen activator inhibitor-1 (PAI-1), and tromboxan A2.

• Inhibition of cell proliferation of vascular smooth muscles, preventing the

migration of smooth muscle cells through direct mechanisms, such as synthesis of

nitric oxide, which inhibits cell proliferation, and/or indirect ones (platelet action).

17,18

• Active participation in immune response through the release of factors

leading to active contraction, control of plasma extravasation, as well as increase in the

expression of integrins, molecules of adhesion and secretion of cytokines, allowing the

migration of monocytes, lymphocytes and neutrophils and greater local phagocytic

activity.

• Modulation of vascular tonus through the production of numerous

vasoactive substances (Table 1). The regulation of the vasomotor tonus is determined

by a balance of dilation and constriction. Nitric oxide (NO) is the main mediator of

vasomotor tonus in physiological situations. Some stimuli such as the dragging force

produced by the pulsatile blood flow, the pressure of blood against the vascular wall,

and the shear stress contribute to the basal generation of NO.

19,20,21

Endothelial Dysfunction

The term endothelial dysfunction is more frequently used to mean reduction in

endothelium-dependent vasodilatation, associated with diminished bioactivity of local

vasodilating factors, especially NO, but it probably includes such other normal functions

of the endothelium as interaction with leukocytes, platelets and regulatory substances.

Currently, it is a consensus that endothelial dysfunction is the initial event in

atherosclerosis development.

Several conditions such as aging, menopause,

dyslipedemia, high blood pressure and diabetes mellitus are associated with

endothelial dysfunction.

23,24,25,26,27

Techniques for Evaluation of Endothelial Function

There are many techniques to investigate the endothelium, from those that

focus on cell and molecular aspects, through methods involving tissue cultures and

molecular biology tools, to the clinical trials applied to human beings, using invasive

and non-invasive procedures to evaluate endothelium-dependent vasodilatation, or the

determination of plasmatic substances that indicate endothelial activation and damage

(Tables 2 and 3).

Intracoronary studies evaluating endothelium-dependent vasodilatation after

infusion of acetylcholine are considered gold standard techniques in assessing the

endothelial function, but their invasive character precludes their use in large scale.

Thus, taking into account that endothelial dysfunction is present at early stages of

atherosclerosis, and that it involves several arteries, non-invasive tests in peripheral

circulation have been increasingly used.

Three techniques are particularly useful to

estimate the endothelial dysfunction in peripheral circulation: ultrasonography of

brachial artery, impedance pletismography, and dorsal hand vein compliance. Each of

these techniques evaluates the endothelial functistage4(seful tf)]TJ0 -2.306 TD0.0009 96c0.027 Tw sizefusiberiphern[(bra )5.5(ariqu)5.ml hanlerosisbackstage4( )5.-mate

between FMD and a variety of cardiovascular risks, such as smoking, hypertension,

age and hypercholesterolemia.

Other studies have demonstrated a positive

correlation between FMD and central hemodynamic parameters, extension and

prognosis of coronary disease.

29,30

This technique has been used in several studies, but there are limitations

related to environmental and individual factors, such as prandial state and arterial size.

Also, problems related to reproducibility and intraobserver variation are matters of

debate in literature. Rossi et al, 2005, demonstrated variation coefficients of 3.3% and

12.4%, respectively, for basal size and after stimulation, of the brachial artery.

There is as yet no consensus in literature about the parameters of normality for

FMD. Ryliskyte et al (2004) analyzed 115 individuals with low cardiovascular risk and

found that the only independent predictors of FMD were age and vessel size

. These

authors thus suggest that in analyzing results, one must take into account normality

ranges according to age and the caliber of the tested vessel.

Impedance Pletismography and Dorsal Hand Vein Compliance

Compliance and vein occlusion pletismography uses a sensor of stretching to

quantify variations in volume in the forearm or lower limb, taking into account the fact

that variations in volume in these parts are dependent on the local blood flow variation.

Forearm blood flow (FBF) measurements can be done following ischemic (reactive

hyperemia) or pharmacological stimulation, the latter requiring catheterization and drug

infusion into brachial artery (invasive). Flow values can be calculated by manual or

semi-manual analysis using specific software.

This technique has been validated in several studies as a tool to evaluate the

endothelial function: diminished FBF is associated with the increased thickness of the

tunica media of the artery, presence of DAC, and risk factors associated with it.

16,34,35

FBF can also be used to monitor the changes to the endothelial function after

interventions on cardiovascular risk factors.

Environmental and physiological variables can influence the magnitude of FBF

responses, such as room temperature, age, race, hormonal state, phase of menstrual

cycle, anxiety, and prandial state. FBF values variation in a single individual can be

about 12.9%, reflecting the influence of these variables.

37,38

To minimize variability the

tests must be done in a silent room at stable temperature, and the patient must be

fasting.

The dorsal hand vein compliance technique uses measurements of venous

diameter variations, obtained through a linear transducer. Venodilatation curves are

obtained after infusion of acetylcholine or bradykinin (endothelium-dependent

vasodilatation) and nitrate (endothelium-independent vasodilatation) as compared to

basal curves

. As regards safety, risks and accuracy, it is comparable to impedance

pletismography, although it has the advantage of using vasoactive substances at lower

doses, thus avoiding potential systemic confounders

. Again, the results are

influenced by environmental and physiological factors. Greater reproducibility and

smaller intra-patient variability of the test are related to pre-constriction of the tested

vessel, ideally around 80% of vein constriction in basal state (ED

Estrogen, menopause and endothelial function

There is evidence of an association between endothelial dysfunction and

reduced endogenous production of estrogens after natural or surgical menopause or

premature ovarian failure (POF) in women with or without CAD.

27,40,41,42

The actions of endogenous estrogens on the cardiovascular system can be

mediated directly on the vessels or indirectly through the modulation of cardiovascular

risk factors, as well as on the lipid profile (reduction of total cholesterol and LDL,

increase in HDL), as already described more than 20 years ago.

More recently,

studies have demonstrated as well an antioxidant effect by estrogen, reducing LDL

oxidation in vivo and in vitro.

The direct effects of estrogen on the vascular system and which modulate the

vascular tonus comprise 1) acute vasodilatation, increasing the synthesis and

bioactivity of nitric oxide;

44,45

2) long term

modulation of vascular tonus, regulating the

production of prostaglandins and expression of eNOS and the endothelin

gene

; 3)

inhibition of endothelin-induced

vasoconstriction

; and 4) inhibition of sympathetic

activity

In addition to these actions on the vascular tonus, estrogen exerts an

antiproliferative action on the vascular smooth layer.

Also, it appears to have a major

role in vascular remodeling, inhibiting the proliferation of the inner layer after injury

and increasing the expression of contractile proteins in the myocardium.

In other tissues, such as the liver, estrogen can mediate both beneficial

(expression of genes of apoproteins that improve the lipid profile) and adverse effects

(increase in the expression of pro-coagulant factors and decrease of fibrinolytic

factors).

From the clinical point of view, our group has recently reported, in post-

menopausal women who were not on hormone therapy, a positive association between

nitric oxide and free estradiol level, confirming the influence of this steroid on the

endothelial function.

On the other hand, androgen and sex hormone binding globulin (SHBG) levels

have been associated with risk of CVD in pre- and postmenopausal women.

53,54

The

increase in circulating androgens appears to be associated with insulin resistance and

a predictor of diabetes mellitus.

55,56,57

In a previous study, we demonstrated a positive

association, independent of obesity, between testosterone levels and hyperinsulinemia

in post-menopausal women with no clinical evidence of CVD.

Hormone Therapy and Endothelial Function

Disturbances in the endothelial function have an important role in the

physiopathology of atherosclerosis, and several lines of evidence suggest that

interventions in the endothelial function could modify the progress rates of

atherosclerotic disease and the risk of cardiovascular events. A number of studies in

the literature have tested the impact of the use of HT on the endothelial function using

inflammatory, fibrinolytic/thrombogenic markers and functional methods.

40,41,59,60,61

Results from the main randomized studies, HERS 2 and WHI, indicate

increased risk of venous thromboembolism with the use of HT.

8,62

It is important to

highlight that these studies were criticized in at least two respects: patient selection,

which included women in average 10 years older than the age at which HT is usually

recommended (WHI), and the use in both studies of a regimen of HT with drugs,

administration route and fixed doses.

The administration route of HT appears to affect the fibrinolytic activity and the

coagulation markers. The oral route for estrogen therapy is associated with changes in

the levels of coagulation and fibrinolysis markers, especially at the early period of use.

63,64,65

Studies using the estrogen therapy by transdermal route, however, did not

confirm these findings.

66,67

Moreover, Scarabin et al (2003), in a multicenter case-

control study, evaluated 155 post-menopausal women hospitalized because of venous

thromboembolism (VTE) and demonstrated increased risk for VTE with oral, but not

with transdermal, therapy.

Our group showed, in postmenopausal patients, that

reduction of antithrombin III, usually seen with oral estrogen therapy, did not occur with

the use of estradiol-17β in the form of’ percutaneous gel both alone

or in association

with micronized progesterone

(Figure 1). Plasma rennin activity was also similar

before and during this non-oral hormone therapy (Figure 1). These findings have also

shown that progesterone does not appreciably attenuate estradiol-induced beneficial

effects.

In addition, we have recently shown that, in a sample of hypertensive post-

menopausal women, the association of non-oral estradiol-17b and low doses of vaginal

micronized progesterone for one year were both effective on climacteric symptoms and

safe on maintaining blood pressure control and preventing endometrial hyperplasia

70,71

Moreover, in those patients, serial echocardiograph scans showed no change in

left ventricle mass, but a significant reduction in the thickness of the left ventricle

posterior wall was observed

(Table 4).

On the other hand, a number of studies have been calling attention to the effect

of HT in endothelial inflammatory markers, such as decreased expression of adhesion

molecules like ICAM-1, VICAM-1 and E-selectin.

72,73,74

Studies using the oral route have evidenced an increase in the circulating levels

of C-reactive protein (CRP), a well-known inflammatory marker.

73,74,75

However, the

clinical significance of these results has not been totally clarified. Other trials with non-

oral routes reported, on the contrary, stability in CRP levels during HT.

73,76

Acute non-oral use of estradiol may increase endothelium-dependent

vasodilatation.

77,78

Saitta et al. 2001 compared the effects of using 17-β estradiol+

norethisterone, raloxifen or placebo for 6 months in postmenopausal patients

Treated women, in both groups, showed significant increase in endothelium-dependent

vasodilation measured by ultrasonography of brachial artery and in the plasmatic

nitrite/nitrate levels, not observed in placebo users. A significant increase in the NO

metabolites levels has also been shown by Balci et al (2005) with the use of

transdermal 17β estradiol, 100 µg/week for 3 months.

Although there is some evidence towards a favorable action of HT on the

vasomotor endothelial function, the long-term benefits on the natural history of

atherosclerotic disease are yet unpredictable. Ceballos et al, 2000, showed a

significant increase in endothelium-dependent vasodilation in menopausal patients

treated with a combination of transdermal 17β estradiol and micronized progesterone,

but this benefit was lost after 6 months of drug discontinuation.

Few studies in literature have boarded the effect of progestogens on

cardiovascular risk and despite well-recognized benefits of estrogens, controversy

surrounds the risks and negative aspects of combined estrogen and progestogen use

in HT.

The vascular actions of progestins and progesterone are mediated by

progesterone receptors, expressed in endothelial cells and the vascular smooth muscle

as well as through down-regulation of the estradiol receptor.

Concerning

progesterone, evidences suggest that the natural molecule facilitates the

inhibitory

effects of estrogen on vascular smooth muscle proliferation

and may induce

endothelium-dependent vascular relaxation.

45,77

In addition, natural progesterone used

in HT appears to preserve the beneficial actions of estrogen.

8,70,71

In turn, progestins present different pharmacological profiles according to their

molecular structure, dosage and presence of comorbidities. As with estrogens, the

various progestins used in HT may differ significantly as to how closely they mimic their

natural counterparts. For instance, progestin molecules with androgenic properties may

antagonize estrogen-dependent beneficial effects on lipids

and a new molecule with

antimineralocorticoid activity may reduce blood pressure in postmenopausal women

with hypertension.

Progestins added to estrogen therapy seem to increase inflammatory markers.

In addition, medroxyprogesterone acetate associated to conjugated equine

estrogens produce no effects

or inhibit endothelium-dependent vasodilatation

stimulated by estrogens.

These observations have been considered to explain, at

least in part, the adverse results observed on the large prospective, randomized,

placebo-controlled trials of combined HT: WHI and HERS studies. However, the extent

to which findings of these studies of medroxyprogesterone acetate and conjugated

equine estrogens apply to other HT formulations is unclear at present.

Endothelial Dysfunction and CVD Predictors in Menopause

Data from prospective trials have been confirming the hypothesis that

endothelial dysfunction precedes the emergence of chronic disorders. The

MONICA/KORA study (Monitoring of Trends and Determinants in Cardiovascular

Disease/Cooperative Research in the Region of Augsburg), which involved more than

2,000 patients, found an association between increased levels of E-selectin and I-CAM

and increased risk of Diabetes Mellitus Type 2 (DM-2).

Rossi et al (2005), in a follow-

up study of 840 postmenopausal women, showed an adjusted relative risk for DM-2 of

5.87 (confidence interval of 95%:4.34-8.10) in patients with the lower tertile of FMD

(≤4.3).

In addition to effects on endothelial markers, changes in other factors of

cardiovascular risk have been associated with the menopausal transition, such as the

lipid profile

, weight, and body fat distribution.

89,90

The association between

prevalence of cardiovascular risk during peri-menopause and post-menopause and

intimal-medial thickness (IMT) of the carotid was monitored in 314 women by Matthews

et al (2001).

In pre-menopausal patients, arterial and pulse pressure, LDL, HDL,

triglycerides, and BMI values were predictors of TMT and the presence of

atherosclerotic plaque, after 5 years of menses cessation. In post-menopausal

patients, only the increase in pulse pressure was a predictor of IMT.

These findings

support the notion that women at higher risk of CVD can be identified during pre-

menopause, and it is in this period that strategies must be implemented to prevent the

development of atherosclerosis in the postmenopausal years.

Endothelium dysfunction can be curtailed by non-pharmacological measures,

such as physical activity

and weight loss

, or pharmacological ones, such as statins

and angiotensin converting enzyme (ACE).

94.95

The indication of HT use must be

individualized, taking into account the presence of climacteric symptoms and their

impact on inflammatory and coagulation markers, since long-term benefits on

atherosclerotic vascular disease have not yet been determined.

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Legend of Figure 1

Renin activity and antithrombin III in 20 postmenopausal women with mild to moderate

hypertension, before and during 1 year of non-oral natural estradiol and low dose of

micronized progesterone.

Table 1. Endothelial-derived vasoactive factors

Vasodilator Factors Vasoconstrictor Factors

Nitric oxide (NO) Endothelin (ET)

Endothelium-derived hyperpolarizing

factor (EDHF)

Prostanoids (PGH

, TXA

, O

)

Prostacyclin (PGI

) Angiotensin (AII)

Acetylcholine

Bradykinin

Table 2. Markers of Endothelial function

24,91

Endothelial Function Marker

Coagulation

Fibrinogen

vWF

TXA

Fibrinolysis

t-PA

PAI-1

Inflammation

CRP

E-selectin

fibrinogen

ICAMs and VICAMs

IL-6

Vascular Tonus

Plasma Markers

ET-1

vWF: von Willebrand factor; TXA

2,:

tromboxan; A

tPA: tissue plasminogen activator;

PAI-1: plasminogen activation inhibitor-1; CRP: C-reactive protein; ICAMs: intercellular

adhesion molecules; VICAMs: vascular cell adhesion molecules; IL-6: interleukin 6; ET-

1: endothelin-1; NO: Nitric Oxide

Table 3. Functional Tests for endothelial-dependent vasodilation evaluation

24, 90

Functional Tests

Invasive

Non Invasive

Coronary angiography

Pletismography

Positron emission tomography

FMD

Brachial artery ultra-son

Pletismography and dorsal hand

vein compliance method

FMD: flow-mediated vasodilation

Table 4. Echocardiographic variables in 20 hypertensive post-menopausal women

Before HT 6m 12m P

Left ventricle posterior wall (mm)

9.1 ±0.4 8.3 ± 0.3 8.0 ± 0.2

0.042

Ejection fraction (%)

71.3 ± 1.3 67.9 ± 1.2 69.4 ± 1.5

Interventricular septum (mm)

9.2 ± 0.4 8.9 ± 0.4 8.2 ±0.2

Blood pressure control was achieved by administration of amlodipine at individually

adjusted doses. Hormone therapy was introduced in a cyclic regimen (21 of 28 days)

with percutaneous estradiol (1.5 mg/day) and vaginal micronized progesterone (100

mg/day).

100

0612

Mean + SD

100

0612

months

Median

mg/ml/h

Plama renin activity

Antithrombin

III

months

Figure 1

ARTIGO 2

Relationship between endogenous testosterone, endothelin-1 and C-reactive

protein in postmenopausal women

Maria Augusta Maturana, MD;

1,2

Vitor Breda;

Francisco Lhullier, PhD;

1,2

Poli Mara Spritzer, MD, PhD

Relationship between endogenous testosterone, endothelin-1 and C-

reactive protein in postmenopausal women

Maria Augusta Maturana, MD;

1,2

Vitor Breda;

Francisco Lhullier, PhD;

1,2

Poli Mara Spritzer, MD, PhD

Gynecological Endocrinology Unit, Division of Endocrinology, Hospital de Clínicas de

Porto Alegre, Porto Alegre, Brazil;

Department of Physiology, Universidade Federal do

Rio Grande do Sul, Porto Alegre, Brazil;

School of Pharmacy, Pontifícia Universidade

Católica do Rio Grande do Sul, Porto Alegre, Brazil.

Short title: ET-1, CRP and testosterone

Key words: menopause, androgens, endothelin-1, C - reactive protein, cardiovascular risk

factors, endothelial dysfunction

Corresponding author: Poli Mara Spritzer, MD, PhD

Department of Physiology, Universidade Federal do Rio Grande do Sul

Rua Sarmento Leite, 500

90050-170 Porto Alegre, RS, Brazil

Tel: +55-51-3316-3671 / Fax: +55-51-3316-3656

E-mail: [email protected]

ABSTRACT

Objective: To verify whether endogenous testosterone levels are correlated with

endothelial dysfunction markers and metabolic profile in 53 postmenopausal women.

Methods: Total testosterone (TT), endothelin-1 (ET-1), C-reactive protein (CRP) and

metabolic and haemostatic variables were determined.

Results: Mean age was 55 (±5) years. Median time since menopause was 5.5 (3-8)

years. Body mass index and waist circumference were significantly higher in women

with TT ≥ 0.49 ng/mL (group mean) than in women with TT ≤ 0.49 ng/mL (p < 0.005).

Median CRP levels were greater in women with higher TT [1.170 (0.175-2.360) versus

0.175 (0.175-0.610 mg/L, p= 0.039]. Median ET-1 levels were also higher in women

with TT ≥ 0.49 ng/mL [0.84 (0.81-0.97) versus 0.81 (0.74-0.84) pg/mL, p=0.012]. TT

was associated with CRP (r=0.466, p=0.008) and ET-1 (r=0.365, p=0.024). These

correlations were independent of time since menopause and central obesity.

Conclusions: A positive association was observed between testosterone and

endothelial dysfunction markers. Endogenous testosterone in recently postmenopausal

women, even within normal limits, may be part of a proatherogenic profile. Longitudinal

studies are needed to determine if androgenicity represents a risk factor for

cardiovascular disease and to establish the clinical relevance of its association with ET-

1 and CRP in this population.

INTRODUCTION

Although much progress has been made in the prevention and treatment of

cardiovascular disease (CVD), it is still the leading cause of death among

postmenopausal women in developed countries.

Changes in endothelial function play

an important role in the pathophysiology of atherosclerosis,

and there is evidence

suggesting that interventions to improve endothelial function may impact the

progression and the risk of cardiovascular events.

2-5

Circulating inflammatory markers are regarded as manifestations of endothelial

dysfunction and have also been linked to CVD. C-reactive protein (CRP), a reliable and

easily measured marker of inflammation, has been described as a predictor of

cardiovascular events in postmenopausal women.

6-8

Another inflammation marker,

endothelin 1 (ET-1), a peptide isolated from endothelial cells, presents a powerful

vasoconstrictor action. Increased levels of ET-1 have been observed in states of insulin

resistance and in early endothelial dysfunction.

Also, a positive association between

ET-1 levels and androgenicity has been described in women with polycystic ovary

syndrome (PCOS), suggesting a correlation with the early-onset endothelial

dysfunction found in these patients.

In turn, endogenous androgens are thought to be potential mediators of CV risk

in women at midlife, in addition to having been associated with CRP [15-16]. Therefore,

the aim of the present study was to investigate the relationship between testosterone,

CRP and ET-1 levels and the metabolic profile in a group of postmenopausal women.

SUBJECTS AND METHODS

Subjects

The study was carried out with women consulting for climacteric symptoms at

the Gynecological Endocrinology Unit at Hospital de Clínicas de Porto Alegre, Brazil.

Fifty-three postmenopausal women fulfilling all the inclusion criteria were consecutively

enrolled in the study. Inclusion criteria were as follows: 1) menopause, defined as last

menstrual period at least 1 year before the beginning of the study plus follicle

stimulating hormone (FSH) levels higher than 35 IU/L; 2) more than 40 years of age; 3)

no use of any medication known to interfere with hormonal, glucose or lipoprotein

levels in the past 3 months and 4) no use of steroidal or nonsteroidal anti-inflammatory

drugs in the last 15 days. Diabetic patients or patients with thyroid, hepatic or renal

dysfunction were excluded. Five patients were smokers. The study protocol was

approved by the local Ethics Committee, and written, informed consent was obtained

from every subject.

Study protocol

Anthropometric measurements included body weight, height, waist

circumference (waist measured at the midpoint between the lower rib margin and the

iliac crest), hip circumference (recorded at the level of the greater trochanter), waist-to-

hip ratio (WHR), and body mass index (BMI, current measured weight in kg divided by

height in m

). Blood pressure was measured in the supine position after a 10-minute

rest. The same calibrated mercury manometer attached to a 12.5 x 23 cm inflatable

cuff was used in all patients by the same operator, who adopted the 5th Korotkoff

sound to determine diastolic pressure. Hypertension was defined as systolic blood

pressure ≥ 140 mmHg, diastolic blood pressure ≥ 90 mmHg, or current use of

antihypertensive drugs.

After the patients were submitted to a 3-day 300 g carbohydrate diet, two blood

samples were drawn from an antecubital vein for determination of plasma glucose and

insulin: one after overnight fasting, and another 2 hours after the ingestion of 75 g of

glucose. FSH, luteinizing hormone (LH), estradiol, total testosterone (TT), sex hormone

binding globulin (SHBG), dehydroepiandrosterone sulphate

(

DHEAS), fibrinogen, ET-1,

CRP, total and HDL-cholesterol and triglycerides were also determined using the

fasting blood sample. All samples were obtained between 8 and 10 a.m. Free

androgen index (FAI) and homeostasis model assessment (HOMA) index were

calculated as previously reported.

Assays

Total cholesterol, HDL-cholesterol, triglycerides, and glucose were determined

by colorimetric-enzymatic methods using the Bayer 1650 Advia System. LDL

cholesterol was determined indirectly using the formula LDL = total cholesterol - HDL -

triglycerides / 5. Serum LH and FSH were measured by electrochemiluminescence

immunoassay (ECLIA), with intra and interassay coefficients of variation (CV) of 1.8%

and 4.8%, respectively, for LH, and 1.8% and 3.3% for FSH. The sensitivity of the

assays was 0.12 IU/L for LH and 0.05 IU/L for FSH. TT levels were measured with the

RIA method (ICM, Costa Mesa, CA) with an assay sensitivity < 0.2 ng/mL and intra and

interassay CV of 10% and 11.3%, respectively. Free testosterone index was estimated

by dividing TT (nmol/L) by SHBG (nmol/L) x 100. Estradiol was measured by ECLIA

(Roche Diagnostics, Mannheim, Germany), with an assay sensitivity of 5.0 pg/mL and

intra and interassay CV of 5.7% and 6.4%. SHBG was measured by

chemoluminescence enzyme immunoassay (DPC, Los Angeles, CA), with an assay

sensitivity of 0.2 nmol/L and intra and interassay CV of 6.1% and 8.0%, respectively.

Serum insulin levels were measured using ECLIA (Roche Diagnostics, Mannheim,

Germany), with sensitivity of 0.200 µIU/ml and intra and interassay CV of 2.0% and

4.3%, respectively. SDHEA was measured by ECLIA (Roche Diagnostics, Mannheim,

Germany), with sensitivity of 0.10 µg/dL and intra and interassay CV of 2.8% and 6.5%,

respectively. Fibrinogen was measured by the coagulometric method (Diagnostica

Stago, Asnières, France), with sensitivity of 4 s and intra and interassay CV of 3.3%

and 10.0%, respectively. CRP was assayed using stored specimens, with a validated

high-sensitivity nephelometric method (Dade Behring Marburg, Marburg, Germany).

Sensitivity was 0.175 mg/L and intra and interassay CV were 4.4% and 5.7%,

respectively. For data analysis, individual results below the limit of sensitivity were

considered as equal to 0.175 mg/L. ET-1 was assayed using a luminoimmunoassay

(R&D Systems) in stored EDTA plasma samples, with sensitivity of 0.5 pg/mL in our

laboratory, and intra and interassay CV of 4.6% and 6.5%, respectively.

Statistical analysis

Results are expressed as means ± SD or median and interquartile range.

Comparisons between the two group means were analyzed by Student's t-test;

comparisons between median values were analyzed with Mann-Whitney's U test.

Spearman’s rank or Pearson’s correlation coefficient were calculated between

variables using a two-tailed significance test for variables with a Gaussian or non-

Gaussian distribution, respectively. Partial correlations of TT with ET-1 and CRP were

calculated (adjusted for waist circumference and time since menopause).

Comparisons between ratios were carried out using the chi-square test. All

analyses were performed using the Statistical Package for the Social Sciences (SPSS,

Chicago, IL, USA). Data were considered to be significant at p<0.05. Calculation of the

sample size was based on the higher ET-1 levels observed in the pilot study in women

with circulating TT ≥ 0.49 pg/mL. The sample had a power of 90%, with a significance

level of 0.05.

RESULTS

The mean age of participants was of 55 (± 5) years, the age at menopause was

48 (± 3) years, and the median time since menopause was 5.5 (3-8) years. Table 1

presents the anthropometric, metabolic and hormonal profile of participants, showing

that total and LDL cholesterol levels were slightly higher than reference levels.

Metabolic syndrome as defined by NCEP-ATP III criteria

was diagnosed in eight

patients (14.3%). Twelve (21%) had hypertension and 17 (34%) presented impaired

glucose tolerance.

Table 2 presents metabolic and haemostatic variables and markers of

inflammation and endothelial function stratified by testosterone levels in the two

groups. The distribution of metabolic and haemostatic variables and markers of

inflammation and endothelial function was analyzed considering TT levels in relation to

0.49 ng/mL, which was the mean value for this group (TT < 0.49 ng/mL or ≥ 0.49

ng/mL) (Table 2). No differences were found in terms of age and time since

DISCUSSION

In the present study, a significant association between endogenous

testosterone and ET-1 was found in postmenopausal women. To our knowledge, this is

the first time this association is described in recently postmenopausal women

presenting hypertension without clinical evidence of cardiovascular disease. The pro-

inflammatory marker CRP was also found to be associated with testosterone levels in

this population.

Evidence suggests that sex hormones may modulate plasma ET-1 levels.

Webb et al.

found that 17β- estradiol decreased ET-1 levels in the coronary

circulation of postmenopausal women. More recently, Silvestri et al.

also showed a

reduction of ET-1 levels in postmenopausal women under oral hormone therapy. In

turn, high levels of ET-1 were described by Polderman et al.

in a study with female-to-

male transsexuals treated with testosterone.

Orio et al.

have recently described early impairment of endothelial function

pramenwed a significantin(crea)5.3se( ine)5.4(arotid( i tia-mediaP wlal )-5.5(thcknra)5.3ss,l )-5.5an

Fibrinogen has been suggested as an independent risk factor for cardiovascular

disease. Framingham Study data indicate that increases in fibrinogen impose an

independent increment on cardiovascular risk in both sexes.

Additionally, androgens

have been associated with some haemostatic factors,

but there is controversy

concerning the relationship between androgens and fibrinogen.

21,22

In our study,

fibrinogen levels were similar in the two groups stratified by testosterone levels. Taken

together, these data support the notion that fibrinogen is more related to BMI

and

lipoprotein levels than to menopausal status,

at least when postmenopausal women

without clinical cardiovascular disease are considered.

In the present study, TT was independently associated with CRP. CRP is

considered an independent predictor of CVD in both males and postmenopausal

women.

8,25

Our data confirm the results of a recently published cross-sectional study in

which CRP was negatively associated with SHBG and positively associated with

bioavailable testosterone after adjustment for age, BMI, physical activity, alcohol

consumption and tobacco use.

Folsom et al.,

analyzing a sub-sample of the ARIC

study (n=57), showed that, after adjustment for age, race, and case-control status,

mean CRP was 2-fold greater in the highest vs. lowest quartiles of estrone and

androstenedione, and CRP was 2-fold lower across quartiles of SHBG. However,

because of the sample size, not all these associations reached statistical significance.

Conversely, a few studies disconfirm the association between androgenicity

and CRP in specific postmenopausal sub-populations such as older patients referred to

coronary angiography, stratified by the presence or absence of coronary artery

disease.

Joffe et al.

found CRP to be negatively and independently correlated with

SHBG and testosterone in menopausal women who subsequently developed clinical

CVD, but the negative correlation between CRP and testosterone was not present in

those who remained CVD-free. However, the patients in that study were older than our

patients and around 20% were smokers. In addition, a positive association between

CRP and free androgen index was found when the entire group of women not using

hormonal therapy was analyzed. Therefore, it is possible that the association between

testosterone and CRP is not linear across the range of CRP values, appearing only

with lower CRP levels, as suggested by Crandall et al.

While time since menopause was not controlled in the present study, the

patients included were in the early years of menopause, with no clinical evidence of

disease but light hypertension in 22% of the sample. Along with the low levels of CRP,

this profile may also partially explain the low prevalence of metabolic disturbances such

as insulin resistance and dyslipidemia. In a previous study we found an association

between insulin resistance and androgenicity.

However, in that study patients were

older and more obese than those in the present study.

In conclusion, the present results suggest that testosterone levels in recently

postmenopausal women, even within normal limits, may indicate a proatherogenic

profile. Longitudinal studies are needed to determine if androgenicity represents a risk

factor for cardiovascular disease and to establish the clinical relevance of the

association between testosterone, ET-1 and CRP in postmenopausal women.

Acknowledgements

This study was supported by grants from Conselho Nacional de Desenvolvimento

Científico e Tecnológico (CNPq), FAPERGS (Fundação de Apoio à Pesquisa do Rio

Grande do Sul), FIPE-HCPA and PRONEX 26/98 (Programa de Apoio aos Núcleos de

Excelência em Pesquisa).

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Table 1. Baseline characteristics of postmenopausal study participants (n=53)

*Median (and interquartile range); BMI: body mass index.

Mean SD

Age (yr) 55 5

Age at menopause (yr) 48 3

Time since menopause (yr) 5.5 (3-8)*

BMI (kg/m

)274

Waist circumference (cm) 88 10

Estradiol (pg/mL) 13 7

Testosterone (pg/mL) 0.49 0.23

FAI

* 2.9 (2.2-4.6)

Fasting glucose (mg/dL) 93 10

Total cholesterol (mg/dL) 220 37

HDL-C (mg/dL) 57 10

LDL-C (mg/dL) 141 31

Triglycerides (mg/dL) 113 47

Fasting insulin (µIU/mL)

7.6 3

FSH ( mIU/mL) 85 33

Table 2. Distribution of metabolic, haemostatic, inflammatory and vasomotor markers

by total testosterone levels

TT levels (n) < 0.49 (n=32)

≥ 0.49 (n=21)

Age (yr)

55 ± 5 54 ± 5

0.846

Time since menopause* 6 (3-8) 5 (3-9) 0.908

Testosterone (pg/mL)

0.34 ± 0.08 0.69 ± 0.18

<0.001

FAI* 2.5 (1,5-3.2) 5.3 (3.39-6.8) <0.001

HOMA

1.7 ± 0.8 2.7 ± 1.0

0.499

SHBG (nmol/L)* 53 (38-76) 44 (31-73) 0.273

BMI (kg/m

)

25 ±3 27 ± 2

0.005

Waist circumference (cm)

84 ± 7.9 92 ± 7

0.002

Hypertension (%)

†

5 (15) 7 (34) 0.136

Fibrinogen (mg/dl)

323± 122 350 ± 134

0.469

CRP (mg/L)* 0.175 (0.175-0.610) 1.170 (0.175-2.360) 0.039

Endothelin-1 (pg/mL)* 0.81 (0.74-0.84) 0.84 (0.81-0.97) 0.023

TT: total testosterone; BMI: body mass index; CRP: C-reactive protein

Student T test or

Mann-Whitney U test (median IQR:25-75%);

Chi-Square test

Table 3. Cross-sectional association of total testosterone with ET-1 and CRP levels,

adjusted by waist circumference and time since menopause

Testosterone (pg/mL) P

ET-1(pg/mL) 0.365 0.024

CRP (us)(mg/L) 0.466 0.008

ARTIGO 3

Free androgen index and endothelial function in postmenopausal women

Maturana MA

, Rubira MC, Irigoyen MC

2,3

,Spritzer PM

1,2

Free androgen index and endothelial function in postmenopausal women

Maturana MA

, Rubira MC, Irigoyen MC

2,3

,Spritzer PM

1,2

Gynecological Endocrinology Unit, Division of Endocrinology, Hospital de Clínicas de

Porto Alegre, Porto Alegre, Brazil;

Department of Physiology, Universidade Federal

do Rio Grande do Sul , Porto Alegre, Brazil;

INCOR, São Paulo, Brazil.

Running title: FAI and endothelial function in menopause

ABSTRACT

Objective: To assess the influence of free androgen index on endothelial

function in postmenopausal women.

Design: Cross-sectional study. 26 postmenopausal women were submitted

to dorsal hand vein compliance technique. Acetylcholine (Ach) and sodium

nitroprusside (NPS) dose-response curves were constructed to test the endothelium-

dependent and –independent relaxation, respectively. Patients were stratified in 2

groups according to free androgen index (FAI).

Results: Mean age was 54 years (±4) and median time of menopause was

6 years (interquartile range: 3-9). Waist-to-hip ratio was significantly higher in the group

with FAI > 2.5. Maximum vasodilatation (VD) with Ach and with NPS was similar across

the groups stratified by FAI. The median dose of Ach for maximum vasodilatation was

higher in the FAI group > 2.5 (36 (0.36-360) ng/mim) than in the group of FAI ≤ 2.5

(720 (360- 3600) ng/mim) p=0.005. Positive correlations were observed between Ach

doses for maximum VD and FAI (r=0.473, p=0.015), waist (r=0.510, p= 0,011), and

waist-to-hip ratio (r=0.479, p= 0.021). Sex hormone binding globulin (SHBG) was

negatively correlated with Ach doses (rs= - 0.400 p=0.043).

Conclusion: The results from this study suggest that FAI, even if still in the

normal limits, is related to early changes on endothelial function in recently

postmenopausal, healthy women. Longitudinal studies are needed to evaluate the

clinical relevance of these findings.

Key words: menopause, androgens, vasodilation, vascular endothelium, dorsal hand vein

INTRODUCTION

Cardiovascular disease (CVD) is the leading cause of death among men and

postmenopausal women in underdeveloped (Mansur et al., 2006) and developed

countries (Stramba-Badiale et al., 2006; AHA, 2004). Women develop heart disease

later in life than men and this gender disparity in cardiovascular disease has been

interpreted primarily as reflecting estrogen-mediated protection against atherogenesis

(Mendelsohn, 2005).

Recent studies show that androgens can influence cardiovascular risk and

vascular function differently according to the gender disparity. In men, testosterone is

inversely correlated with cardiovascular risk factors, such as arterial wall thickness

(Muller et al., 2004), blood pressure (Svartberg et al., 2004), obesity and metabolic

syndrome (Chen et al., 2006), and presence and extension of coronary disease

(Dobrzycki et al., 2003). In women, androgen levels are positively correlated with risk

factors of CVD in menacme (Coviello et al., 2006; Diamanti-Kandarakis et al., 2006;

Sowers et al., 2005), and in postmenopausal women (Golden et al., 2004; Maturana &

Spritzer, 2002; Phillips et al., 1997)

Endothelial dysfunction is one of the early signs of cardiovascular damage

(Mombouli et al., 1999). In women with hiperandrogenic syndromes, markers of

endothelial dysfunction are present, even in the absence of other cardiovascular risk

factors (Orio et al, 2004). In postmenopausal women, androgens and SHBG are

related to inflammatory endothelial markers (Sutton-Tyrrel et al., 2005; Crandall et al.,

2006). Therefore, the aim of the present study was to assess the influence of free

androgen index on endothelial function in apparently healthy postmenopausal women.

SUBJECTS and METHODS

Subjects

The study was carried out whith women consulting for climateric symptoms at

Gynecological Endocrinology Unit at the Hospital de Clínicas de Porto Alegre, Brazil.

Twenty-six postmenopausal women were enrolled in the study. Inclusion criteria were

as follows: 1) menopause, defined as last menstrual period at least 1 year before the

beginning of the study plus FSH levels higher than 35 IU/L; 2) Age between 40 and 65

years; 3) no use of any medication known to interfere with hormonal, glucose or

lipoprotein levels in the past 3 months and 4) no use of steroidal or nonsteroidal anti-

inflammatory drugs in the last 15 days. Smokers or patients with high blood pressure,

diabetes or thyroid, hepatic or renal dysfunction were excluded. The study protocol was

approved by the local Ethical Committee, and written, informed consent was obtained

from every subject.

Study protocol

Anthropometric measurements included body weight, height, waist

circumference, hip circumference (recorded at the level of the greater trochanter),

waist-to-hip ratio (WHR), and BMI (current measured weight in kg divided by height in

). Blood pressure was measured after a 10-minute rest, with the woman in the sit

position. The same calibrated mercury manometer attached to a 12.5 x 23 cm inflatable

cuff was used in all patients by the same operator, who adopted the 5th Korotkoff

sound to determine diastolic pressure. Hypertension was defined as systolic blood

pressure ≥ 140 mmHg, diastolic blood pressure ≥ 90 mmHg, or current use of

antihypertensive drugs (The JNC 7 Report, 2003).

After the patients were submitted to a 3-day 300 g carbohydrate diet, two blood

samples were drawn from an antecubital vein for determination of serum glucose and

insulin: one after overnight fasting, and then again 2 hours after the ingestion of 75 g of

glucose. All samples were obtained between 8 and 10 a.m. The free androgen index

(FAI) and the HOMA index were calculated as described elsewhere (Frajndlich &

Spritzer, 2005). Patients were stratified by free androgen index (FAI) using as the cutoff

the median FAI obtained in the whole sample of participants.

Assays

Total cholesterol, HDL-cholesterol, triglycerides, and glucose were determined

by colorimetric-enzymatic methods using the Bayer 1650 Advia System. LDL

cholesterol was determined indirectly using the formula LDL = total cholesterol - HDL -

triglycerides / 5. Serum LH and FSH were measured by Electrochemoluminescence

Immunoassay (ECLIA), with intra and inter-assay coefficients of variation (CV) of 1.8%

and 4.8%, respectively for LH, and 1.8% and 3.3% for FSH. The sensitivity of the

assays was 0.12 IU/L for LH and 0.05 IU/L for FSH. TT levels were measured with the

RIA method (ICM, Costa Mesa, CA) with an assay sensitivity < 0.2 ng/mL and intra and

inter-assay CV of 10% and 11.3%, respectively. Free testosterone index was estimated

by dividing TT (nmol/L) by SHBG (nmol/L) x 100. SHBG was measured by

chemoluminescence enzyme immunoassay (DPC, Los Angeles, CA), with an assay

sensitivity of 0.2 nmol/L and intra and inter-assay CV of 6.1% and 8.0%, respectively.

Serum insulin levels were measured using ECLIA (Roche Diagnostics, Mannheim,

Germany), with sensitivity of 0.200 µIU/ml and intra and inter-assay CV of 2.0% and

4.3%, respectively.

Dorsal hand vein technique

The dorsal hand vein technique, as used in our laboratory, has been

described elsewhere (Sabha et al., 1990). Briefly, a 23 G butterfly needle was inserted

into a suitable vein on the back of the hand, with the arm positioned at an upward angle

of 30° to allow the complete emptying of the veins. A tripod, holding a linear variable

differential transformer (LVDT, Schaevitz Engineering Pennsauken, New Jersey, USA)

was mounted on the back of the hand with its central aperture, containing a movable

metal core at a distance of 10 mm downstream from the tip of the needle. The signal

output of the LVDT, which is linearly proportional to the vertical movement of the core,

gave a measurement of the diameter of vein. Readings were taken under a congestive

pressure of 40 mm Hg by inflating a blood pressure cuff placed on the upper portion of

the arm under study. Results were presented as normalized dose-response curves in

which the diameter of the vein during saline infusion is defined as 100% dilation. The

vein was pre-constricted to 70% of baseline size by infusing increasing doses of

phenylephrine (Winthrop Lab, New York,USA;doses: 12-3166 ng/min). This dose rate

of phenylephrine was defined as the ED 70 dose and this degree of constriction was

defined as 0% dilation for the purposes of subsequent calculations. The vasodilation

effects expressed in this study were calculated as a percentage in the range between

0% and 100% dilation. Drugs were infused using a Harvard infusion pump (Harvard

Apparatus, South Natick, MA, USA) at a flow rate of 0.3 mL/min. Blood pressure and

heart rate were monitored in the opposite arm with a Dynamap Bllod Pressure Monitor

(Critikon, Tampa, FL, USA). After pre-constriction of the vein by phenylephrine,

acetylcoline (Ach: 0.36- 3.600 ng/min) (Divisão de Farmácia da Universidade de São

Paulo, Brazil) and/or sodium nitroprusside (SNP) (Biolab Sanus Ltda, Brazil) were

administered with 5 ng/mim infusion rates, to obtain endothelium dependent or

independent mediated responses, respectively. Infusions at each rate lasted for 5 min

with the sphygnomanometer cuff inflated to 45 mmHg for the last 2 min of the infusion.

Statistical analysis

Results are expressed as means ± SD or median and interquartile range.

Comparisons between the two group means were analyzed by Student's t-test;

comparisons between median values were analyzed with Mann-Whitney's U test.

Spearman’s rank or Pearson’s correlation coefficients were calculated between

variables using a two-tailed significance test for variables with a Gaussian or non-

Gaussian distribution, respectively. Analyses were performed using the Statistical

Package for Social Sciences (SPSS, Chicago, IL, USA). Data were considered to be

significant at p<0.05.

RESULTS

The mean age of participants was 54 ± 4 years, the age of menopause was 48

± 3 years, and median time after menopause was 6 (3-9) years. Mean systolic pressure

was 123 ±8 mmHg and mean diastolic pressure was 80 ± 6 mmHg. The mean fasting

glucose was 94± 10 mg/dl and HDL-C was 57 ± 10 mg/dl. Mean testosterone levels

were 0.41 ±0.21 ng/mL and median SHBG levels were 54 (34-78) nmol/L.

Table 1 presents antrophometric, metabolic and vasodilatation variables related

to endothelial function in the two groups stratified by FAI median. While waist and

metabolic variables were similar in the two groups, WHR was higher in the group with

FAI > 2.5. Ach induced maximum flow-dilatation did not differ between the groups, but

women with FAI > 2.5 needed median doses of acetylcholine that were 20 times

greater than those used by the group with FAI < 2.5 to obtain a similar venodilation in

response to Ach infusion.

A positive correlation was found between Ach doses for maximum VD and FAI

(rs=0.473 p=0.015) (Figure 1).

DISCUSSION

The results of the present study show an association between androgenicity and

a state of decreased endothelial sensitivity to acetylcholine in post-menopausal women

without clinical evidence of associated diseases and low cardiovascular risk. Women

with FAI > 2.5 needed median doses of acetylcholine that were 20 times greater than

those used by the group with FAI < 2.5 to obtain a similar venodilation in response to

Ach infusion. To our knowledge, this is the first time this association is reported in

postmenopausal women.

Literature data have shown that natural menopause is positively associated

with changes on endothelial function (Taddei et al., 1996; Sanada et al., 2003; Lima et

al., 2005), although the presence and number of risk factors seem to determine the

degree of endothelial dysfunction (Ishibashi et al., 2006).

Although the present study was performed in a carefully selected healthy

postmenopausal women group, the analysis of the impact of the free androgen index

on venodilation by the dorsal hand vein technique does not exclude the presence of

other factors that potentially alter vascular reactivity (Grundy et al., 1998; Zhang et al.,

2006;Creatsas et al, 2005; Knopp, 2002).

Evidences for a role of testosterone in the development of cardiovascular

disease have increased in the last decades (Liu et al., 2003; Thompson & Khalil, 2003).

Recent studies show that testosterone levels in men are inversely related to

cardiovascular risk factors such as arterial wall thickness (Muller et al., 2004), blood

pressure (Svartberg et al., 2004), obesity and metabolic syndrome (Chen et al., 2006)

and presence and extension of coronary disease (Dobrzycki et al., 2003). In

postmenopausal women, endogenous androgen levels have been associated with

insulin resistance, diabetes, central obesity and hypertension (Maturana & Spritzer,

2002; Ding et al. 2006; Phillips et al.1997), as well as with cardiovascular outcomes

(Rexrode et al., 2003).

In men it is proposed that the positive influence of testosterone upon vascular

reactivity may be observed only when vascular reactivity is sufficiently impaired, as in

the case in atherosclerotic vessels (Webb et al., 1999). In turn, there are few studies

evaluating testosterone levels and vascular reactivity in women. Subcutaneous

physiological testosterone therapy has been shown to increase brachial artery

vasodilation in women already receiving long-term oestrogen replacement therapy

(Worboys et al, 2001). Similar responses have been described in experimental studies

(Honda et al., 1999; Jones et al., 2004). In our population, although women were

separated in two groups with high and low FAI levels, both values were still in normal

limits. It is possible that if the spectrum of FAI levels included subjects with higher

levels of FAI we could detect some differences in maximum venodilation responses.

Endothelial cells release substances acting directly on vascular smooth muscle

(VSM) cells, causing either relaxation or contraction. Several endothelium-derived

substances causing smooth muscle relaxation have been isolated: nitric oxide (NO),

prostacyclin (PGI

), epoxyeicosatrienoic acids and endothelium-derived

hyperpolarizing factor (EDHF). Endothelium-derived vasoconstricting factors are also

secreted, such as prostaglandins, reactive oxygen radicals and endothelin-1 (Luz et al.,

2003; Maturana et al., 2006 submitted).

In the present study, the response to Ach was used to estimate endothelial

functionality, whereas the response to SNP tests the integrity of smooth muscle

function. Ach interacts with M

muscarinic receptor on the endothelial surface, which

initiates a sequence of intracellular events leading to NO synthesis, although

prostacyclin and hiperpolarizing factor release may also be induced. NO diffuses

across the endothelial cell and basement membrane, binds to guanylate cyclase,

leading to an increase in intracellular cyclic guanosine monophosphate and, ultimately,

smoth muscle relaxation and vasodilation. By contrast, SNP decomposes to release

NO, which interacts with the vascular smooth muscle guanyl cyclase to produce

vasodilation in an endothelium-independent way.

Since testosterone modulates the expression of muscarinic acetylcholine

receptor (mAChR) subtypes in different tissues, including smooth muscle cells

(Maróstica et al., 2005; Bush & Borda, 2003), we can speculate that such mechanism

could be involved in the present observation that women with FAI > 2.5 needed

median doses of acetylcholine significantly greater than those used by the group with

FAI < 2.5 to obtain a similar venodilation in response to Ach infusion: women with

higher bioactive androgen concentrations could have different expression of vascular

muscarinic receptor which differently modulates venodilation response.

Vascular tone is defined as the degree of constriction of a

blood vessel relative

to its maximal diameter in the dilated

state. Under basal conditions, most resistance

and capacitance

vessels exhibit some degree of smooth muscle contraction that

determines the diameter or tone of the vessel. Vascular tone

is influenced by both the

endothelium and VSM. Gender differences in vascular tone have been described in a

multitude of vascular beds in both human and experimental animals, suggesting that

sex hormones can be involved (Orshall et al., 2004)

Effects of testosterone are found to be both endothelium dependent and

independent (Khalil, 2005; Jones et al., 2004; Honda et al., 1999; Webb 1999). In

addition, androgen receptors are expressed in endothelium and VSM of vascular tissue

(Khalil, 2005)

Androgens have been related to cardiovascular risk factors and endothelial

dysfunction in women in reproductive years (Meyer et al, 2005; Coviello et al, 2006;

Orio et al, 2004, Nácul et al, 2006) and in post-menopause (Liu et al., 2003;

Reckelhoff & Forteppiani, 2004). In women with polycystic ovary syndrome (PCOS)

impaired endothelial function and decreased endothelium-dependent vasodilation have

been shown (Tarkun et al., 2004). Recent studies have also shown that the endothelial

dysfunction coexist and is influenced by low-grade chronic inflammation in PCOS

(Diamanti-Kandarakis et al., 2006; Brinkwhorth et al., 2006; Carmina et al, 2006; Blake

& Ridker, 2002) as well as in post-menopausal women (Joffe et al., 2006; Crandall et

al, 2006). We have recently shown that androgenicity is related to higher reactive C

protein and endothelin-1 in postmenopausal women (Maturana et al, submitted 2006)

In conclusion, data of the present study indicate that endogenous androgens

may be associated with a reduced vasodilation response to acetylcholine, an early

marker of endothelial dysfunction in apparently healthy postmenopausal women. This

suggests that androgenicity may be part of a risk profile related to endothelial

dysfunction and preclinical cardiovascular disease, observed in the post-menopause.

Acknowledgements

This study was supported by grants from Conselho Nacional de Desenvolvimento

Científico e Tecnológico (CNPq), FAPERGS (Fundação de Apoio à Pesquisa do Rio

Grande do Sul), FIPE-HCPA and PRONEX 26/98 (Programa de Apoio aos Núcleos de

Excelência em Pesquisa).

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Table 1. Distribution of anthropometric, metabolic and vasomotion markers in full

sample and by FAI median

FAI Median

Full sample 1

≤ 2.5 (n=12)

> 2.5 (n=14)

Waist (cm) 86 (8) 82 (8) 89 (8) 0.073

RCQ

0.84 (0.07) 0.81 (0.06) 0.86 (0.07) 0.049

Testosterone

0.41 (±0.21) 0.29 (±0.06) 0.51 (±0.26)

0.009

FAI 2.5 (1.1-2.1) 1.5 (1.1-2.1) 4.7 (2.9-5.7) < 0.001

Total Cholesterol (mg/dL) 221 (42) 217 (40) 224 (44) 0.647

Triglycerides (mg/dL 107 (50) 101 (48) 113 (53) 0.537

HOMA

1.6 (1-2.5) 1.4(1-2.2) 2.2 (1-3.4) 0.301

MaxVD (%) 80 (49) 73 (58) 89 (41) 0.217

Ach Max VD (ng/min)

360 (2.8-990) 36 (0.36-360) 720 (360-3600)

0.005

Max. VD with SNP (%) 146 (55) 144 (58) 145 (57) 0.938

Values are expressed as mean and SD (Student T-test) or median and interquartile

range (Mann-Whitney U test)

MaxVD: maximum venodilation with acetylcholine; Ach

Max VD: acetylcholine doses for maximum VD; VC: venoconstriction after

phenylephrine; Max VD with SNP: maximum venodilation with sodium nitroprusside

Figure 1. Correlation between Ach dose for maximum VD and FAI

FAI

1086420

Ach dose (ng/mim)

4000

3000

2000

1000

-1000

rs=0.473 p=0.015

ANEXOS

HOSPITAL DE CLÍNICAS DE PORT O ALEGRE

UNIDADE DE ENDOCRINOLOGIA GINEC OLÓGICA

DATA: ________________________

I. IDENTIFICAÇÃO:

Registro :_______________

Nome:__________ _____________________________________

DN: ___ /___/____ Idade: ________

Cor: B( ) P( ) PD( )

Endereço: rua_________________________ Nº _______ apto:______________________

Bairro:_________ __________CEP___________Cidade:____________________________

Telefone:______________--

2 ANTECEDENTES

GINECO - OBSTÉTRICOS:

Menarca:

DUM:____/____/____ Idade de menopausa _________ Tempo de

amenorréia:__________

Tipo menstrual no último ano antes de cessar menstruação:

( ) regular: ____l____ dias ( ) irregular ( ) não lembra

Paridade:G___P___A___ lnfertilidade: ( ) sim ( ) não

Método (s) anticoncepcional(is): ( ) ACO Tempo de uso:_____

( ) DIU ( ) Outro ou nenhum

Terapia hormonal prévia: ( ) NÃO

( ) SIM Tipo: _________________ Tempo de uso:______

Tipo: _________________ Tempo de uso: _____

Ano que usou:____________________

Tempo decorrido desde o último tratamento: __________________________________

Sintomas: ( ) Fogachos ( ) Insônia ( ) Dispareunia ( ) IU repetição ( )

Outro___________

3. HISTÓRIA PREGRESSA:

. DCV: ( ) Sim ( ) Não

( ) HAS ( ) Cerebrovascular ( ) outra Qual: _______________

2. Outra:

( ) Diabete melito ( ) tireoideopatia

( ) câncer ( ) Outro

Especificar:_________________________

4. MEDICAÇÕES EM USO:

____________________________________________________________________________

5. Tabagismo: ( ) Não

( ) Sim cig/dia:_______________ tempo de uso: _____________________

( ) História de uso tempo de uso: _____________________

tempo desde que cessou uso: _________

. Álcoolismo: ( ) sim ( )não

História familiar (doença cardiovascular, DM, tireoideopatia, CA ginecológico -

mama, útero, colo, etc):

B. EXAME FÍSICO:

1. CV:_______ FC: __________

Circunferência do braço :___________

PA (braço direito, sentada, após 5 min de repouso): 1ª:: ___________

2ª (corrigida): ___________________

Tireóide:__________________________________________________________________

Mamas: Palpação:__________________________________________________________

Galactorréia: ( ) sim ( ) não

2. MEDIDAS ANTROPOMÉTRICAS:

peso:____________alt:____________ IMC:____________

cint:__________quadril:______ RCQ:______

C. AVALIAÇÃO LABORATORIAL:

glicemia jejum____________glicemia após 75 g de glicose: __________________________

Insulina em jejum:___________Insulina após 75 g de glicose: ________________________

colesterol total:____________HDL:______ LDL:______

triglicerídeos:______________

T4: _____________ TSH: ___________ SDHEA: _____________________

LH:______________ FSH:_________ E2:_________________________

TT: ______________ IAL:_________ SHBG: ____________

Óxido Nítrico___________

Fibrinogênio: ________________ Fator VII:_____________ von

Willebrand:_____________

FV _______________ PAI-1:_________________

AntitrombinaIII:___________________Proteína C:____________ Proteína

S:____________

FUNÇÃO ENDOTELIAL, PARÂMETROS A NTROPOMÉTRICOS, METABÓLI COS E

HORMONAIS EM PA CIENTE S PÓS-MENOP AUSI CAS

Unidade de Endocrinologia Ginecológica/ Serviço de Endocrinologia do HCPA

TERMO DE CONSENTIMENTO

Este estudo tem como objetivo estudar a associação entre alterações no endotélio

(camada interna dos vasos sangüíneos) e idade, tempo decorrido desde a menopausa, níveis

de glicose, lipídeos, hormônios e substâncias que participam na coagulação sangüinea, fatores

de grande importância no aparecimento da doença cardiovascular.

Durante a consulta, será obtido seu histórico médico e verificados sua pressão arterial,

peso, altura e as medidas da cintura e quadril, incluídos no exame físico e serão solicitados

exames. Serão coletadas amostras de sangue que fazem parte da rotina de avaliação para a

mulher na pós-menopausa. Uma fração da amostra de sangue será congelada e utilizada

posteriormente para dosagens que não são realizadas rotineiramente no laboratório do

Hospital de Clínicas.

Faremos também ecografia transvaginal e mamografia bilateral, que fazem parte da

rotina de avaliação em pacientes na pós-menopausa.

Para avaliação da função do endotélio, será realizado através de um exame chamado

pletismografia. Para isto, será puncionada uma veia em cima da sua mão direita, onde será

colocado um soro e depois injetadas algumas medicações. Estas medicações serão muito

diluídas. Assim, a dose que será feita é muito pequena e o efeito causado por elas será única e

exclusivamente no local da punção, não tendo nenhum efeito no seu organismo, ou seja, você

não corre nenhum risco ao recebê-las nestas doses. As medicações irão fazer com que a sua

veia fique mais dilatada (aberta, com uso de acetilcolina ou bradicinina) ou mais estreita

(fechada, com o uso de fenilefrina). Em cima da sua mão será colocado um aparelho que vai

registrar o movimento desta veia quando injetadas estas medicações. Este exame irá durar em

média 3 horas, e a senhora ficará confortavelmente deitado durante todo o procedimento. Os

riscos a que a senhora ficará exposta serão mínimos, incluindo dor tipo “picada de mosquito”

na hora da punção e algum hematoma (mancha roxa) que poderá surgir após a retirada da

agulha da sua veia.

Os resultados dos exames ficarão a sua disposição. Caso queira retirar-se do estudo,

estará livre para fazê-lo em qualquer momento que desejar, sem que isso venha a implicar na

interrupção de seu tratamento. Os dados coletados, além de serem utilizados para indicação

de um melhor tratamento para cada paciente, serão usados para fins de pesquisa e futuras

publicações, reservando-se os preceitos da ética. Se necessário, a senhora poderá entrar em

contato com a Drª Maria Augusta Maturana ou com a Drª Poli Mara Spritzer, coordenadora

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Eu, ......................................................................................concordo voluntariamente em

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estando ciente que serão utilizados com finalidade de pesquisa científica e respeitando os

preceitos da ética.

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