Pituitary Derived-Intermedin is an Estrogen-Modulated Factor for Reducing Blood Pressure
Pituitary Derived-Intermedin is an Estrogen-Modulated Factor for Reducing Blood Pressure
Calcitonin, calcitonin gene-related peptides (CGRPs), adrenomedullin, and amylin belong to a
 unique group of peptide hormones important for the regulation of calcium balance,
 neurotransmission, cardiovascular homeostasis, and glucose metabolism. We, the investigators
 at Chang Gung Memorial Hospital, recently identified intermedin as a novel peptide hormone
 belonging to this unique peptide ligand family. Adrenomedullin is a 52-amino-acid peptide and
 is one of the most potent vasodilators. Plasma adrenomedullin is elevated in a variety of
 pathological conditions such as hypertension, renal failure, heart failure, and septic shock.
 CGRPα and CGRPβ are 37-amino acid neuropeptides; primarily release from sensory nerves; and
 play important roles in regulating peripheral vascular tone and controlling blood flow in
 various organs. Human mature intermedin encodes 40-amino-acid and is expressed mainly in the
 adrenocorticotrophs of the anterior and intermediate pituitary lobe. Intermedin signals
 through calcitonin receptor-like receptor (CRLR)/receptor activity-modifying protein (RAMP)
 receptor complexes, and CRLR/RAMP signaling has been proven to be critical for vascular tone
 regulation. Based on this finding, we had documented that intraperitoneal administration of
 intermedin dose-dependently suppressed blood pressure in normal Sprague-Dawley rats. In
 addition, our preliminary in vitro and in vivo studies demonstrated that ovariectomy lead to
 a tenfold reduction of intermedin transcript expression in the pituitary in rats whereas
 subsequent estrogen treatment increased pituitary intermedin expression to a level similar to
 that of intact rats. Taken together, we propose that pituitary-derived intermedin is
 regulated by estrogen and exhibits potent hypotensive effects. To address this hypothesis, in
 Specific Aim 1, we will investigate the regulation of secretion and expression of intermedin
 in vitro and in vivo using cultured pituitary cells and oophorectomized rats. In Specific Aim
 2, we will study the molecular mechanism by which estrogen stimulates intermedin gene
 expression. In Specific Aim 3, to demonstrate that the estrogen-dependent regulation of
 intermedin represents a critical link in the regulation of blood pressure in women, we will
 study the relationship between blood pressure and blood intermedin levels using a
 cross-sectional study. Our studies will provide a better understanding of the mechanisms by
 which sex hormones modulate blood pressure and open a new window for postmenopausal
 hypertension treatment.
Specific Aim:

 Validation that intermedin represents an estrogen-regulated vasoactive hormone from the
 pituitary

 Hypothesis:

 Intermedin is a mediator of the vaso-regulatory effects of estrogens.

 Rationale:

 In the Preliminary Study, we showed that intermedin is a pituitary-derived hypotensive factor
 signaling through CRLR/RAMP receptor complexes in vessel endothelium. Based on studies of
 related hormones, adrenomedullin and CGRP, intermedin likely reduces blood pressure by
 exerting a vasodilator effect on vascular beds. Earlier studies have shown that the
 neuropeptide αCGRP is predominately produced in the sensory neuron, and serotonin can
 selectively reduce CGRP secretion by activating the endogenous serotonin (type 1 5-HT1)
 receptor through the calcium signal pathway. Deletion of the CGRP gene in mutant mice
 (αCGRP-/-) leads to the elevation of mean arterial pressure, heart rate, peripheral vascular
 resistance and sympathetic nervous activity as compared to wild-type mice. The other related
 peptide, adrenomedullin (ADM), initially identified from pheochromocytoma, has been shown to
 be expressed in various tissues including adrenal medulla, cardiovascular tissues, lung,
 kidney and placenta. Plasma ADM is elevated in various conditions including cardiovascular
 and renal disorders and septic shock, and it has been hypothesized that ADM may be involved
 in the control of circulation and body fluid dynamics in various organs. Studies of mice
 deficient for ADM showed that ADM is indispensable for cardiovascular development. The
 homozygous knockout ( Adm-/-) mice exhibited an embryonic lethal phenotype.

 Earlier studies of the ADM gene promoter have revealed that, in addition to two transcription
 factors, nuclear factor for interleukin-6 expression (NF-IL6) and activator protein 2 (AP-2),
 the ADM gene expression is regulated by the hypoxia inducible factor-1 (HIF-1). Under hypoxia
 conditions, HIF-1 binds to the hypoxia response elements (HRE) in the ADM gene promoter to
 promote ADM gene expression. Although outside the focus of the present study, we have also
 detected putative HRE sequences in the intermedin gene promoter and demonstrated that hypoxia
 conditions stimulate intermedin gene promoter activity, suggesting that HIF-1 could play a
 role in the regulation of intermedin gene expression. Unlike intermedin, the expression of
 CGRP and ADM is not actively stimulated by estrogens, suggesting that these two related
 peptides are not directly associated with the cardiovascular effects of estrogens. In
 females, 17β-estradiol produced by ovaries is the chief circulating estrogen which increases
 vasodilatation and inhibits the response of blood vessels to injury. Studies by Mercuro et
 al. have shown that surgical-induced menopause causes an increase in peripheral vascular
 resistance and blood pressure. Likewise, a cross sectional study showed that there is a four-
 fold increase in the incidence of hypertension in postmenopausal women as compared to
 premenopausal individuals (40% in post menopausal women vs 10% in premenopausal women). The
 rise in systolic blood pressure per decade was around 5mmHg greater in perimenopausal and
 postmenopausal women as compared with premenopausal women . In addition to demographic
 studies, studies have shown that estrogen could dilate coronary and brachial arteries in
 postmenopausal women. A part of vasodilatation and hypotensive activities of 17β-estradiol
 could be explained by its ability to modulate the expression of genes known to influence
 vascular tone and structure, such as ACE, rennin, and angiotension type I (AT1) receptors.
 However, none of these genes have been shown to be specific direct targets for estrogen
 receptors and are responsible for the vasoprotective effects of estrogens. Based on our
 findings that intermedin is a hypotensive factor and contains putative ERE sequence in the
 promoter region, we propose that intermedin is a pituitary-derived blood pressure modulating
 factor directly regulated by ovarian estrogens. Based on this understanding, we further
 hypothesize that the down regulation of intermedin in aging individuals with estrogen
 deficiency could be responsible for postmenopausal hypertension. Because there is no
 established animal model suitable for the study of naturally occurring menopause, we will
 test our hypothesis by studying the relationship between plasma intermedin concentration and
 blood pressure among women in difference reproductive stages.

 Case Selection, Measurement and Analysis of Biophysical and Biochemical Characteristics

 Premenopausal and Postmenopausal Group:

 Eligible women who visit OB/GYN physicians will be invited for an interview. They will be
 questioned about general characteristics, lifestyle habits (smoking, alcohol consumption,
 physical activity), and medical history. During the interview, blood pressure will be
 measured three times on the right arm, while the participant is seated. A woman is considered
 as post menopausal if her menses have naturally ceased for 12 months (or more), or if she has
 bilateral oophorectomy or hysterectomy without bilateral oophorectomy and aged more than 55
 years. Office BP will be measured using a mercury sphygmomanometer according to the
 recommendations of the American Society of Hypertension (average the three BP readings after
 5 min of quiet sitting position). Biophysical and biochemical characteristics will be
 measured including the last menses period, age, BMI, serum glucose (AC and PC), serum and
 urine creatinine, plasma total cholesterol, HDL, LDL, triglycerides, FSH, LH, 17β-estradiol,
 progesterone, TSH, intermedin, and prolactin. Blood specimens from individual subjects will
 be analyzed in a single assay to minimize variations. Odds ratios (ORs) of elevated blood
 pressure and the corresponding 95% confidence intervals (CIs) will be computed after
 allowance for age. Furthermore, in order to take into account potential reciprocal
 confounding effects of variables considered, factors significantly related with elevated
 blood pressure risk in the age adjusted analysis will be subsequently included in multiple
 logistic regression models fitted by the method of maximum likelihood. Women asking for
 annually regular pap smear examinations without any medical disease will be eligible for this
 study group.

 The reproductive stages will be assigned as:

 -  Premenopausal: no reported change in menstrual cycle pattern;

 -  Early peri-menopausal: reported change in menstrual cycle frequency in preceding year
 with a bleed in the preceding 3 months;

 -  Late peri-menopausal: no menses in the preceding 3-11 months;

 -  Postmenopausal: no menses in the preceding 12 months.

 Surgically-Induced Menopausal Group:

 This protocol will include fertile women who suffer from ovarian pathology without other
 medical disease and are scheduled to undergo bilateral oophorectomy associated with or
 without hysterectomy. Patients who suffer from uterine pathology and undergo hysterectomy
 only will serve as controls. Blood pressure and all biochemical characteristics will be
 measured one week before surgery and 1, 3, 6 and 12 months after operation. All individual
 measurements will be blinded as to the hysterectomized/oophorectomized status of the
 patients. The entire data group will be reported as mean ± SE. Differences in vascular
 parameters between oophorectomized women and controls and between values recorded prior to
 and following surgery will be assessed using paired and unpaired Student's t-tests.

 ART Group:

 Women in ART treatment due to male or tubal factor will be included. The day 3 hormone level
 (E2, progesterone, FSH, LH, TSH, prolactin, inhibin A, inhibin B and activin) and blood
 pressure will be measured one month before ART treatment. After gonadotropin administration,
 all the hormone and blood pressure changes will be measured and recorded at day 3, day 7 and
 the day before hCG injection as well as the day of oocyte retrieval. All biophysical and
 biochemical characteristics will continue to follow up on the 15th day after embryo transfer
 and to the end of the first trimester in the pregnancy group.
Inclusion Criteria:

 -  Premenopausal women: no reported change in menstrual cycle pattern

 -  Early peri-menopausal women: reported change in menstrual cycle frequency in preceding
 year with a bleed in the preceding 3 months

 -  Late peri-menopausal women: no menses in the preceding 3-11 months

 -  Postmenopausal women: no menses in the preceding 12 months

 -  Patients who received oophorectomy

 -  Patients who received hysterectomy for benign uterus tumor

 -  Patients underwent assisted reproductive technology (ART).

 Exclusion Criteria:

 -  Patients underwent chemotherapy or radiotherapy in past two years