Stroke is a common and multifactorial disease influenced by genetic and environmental risk factors. It is a highly heterogeneous entity consisting of two main types, ischemic (80%) and hemorrhagic (20%) stroke. The most common form of hemorrhagic stroke is intracerebral hemorrhage (ICH). Ischemic stroke mainly results from thrombotic or embolic events, while ICH is caused by the rupture of an artery in the brain. The mean age of first-ever stroke is 75 years (73 vs. 78 years, for men and women, respectively) and the age-specific stroke incidence is higher for men as compared to women, suggesting that hormonal factors confer protection. A large body of experimental and observational studies shows that estrogens exert beneficial effects in the cardiovascular system. However, large, recent, clinical randomized trials have failed to demonstrate a lower risk of stroke with hormone replacement therapy (HRT) in elderly postmenopausal women. It is possible that HRT may only protect a subgroup of women. Here, genetic predisposition might be involved. Stroke incidence is 50% higher in northern compared to southern Sweden, suggesting a genetic predisposition in this population. This relatively homogeneous population displays founder effects, making it well suited for genetic studies. Since 1985, the MONICA and VIP projects have conducted large-scale cardiovascular health surveys in this population. Information about conventional stroke risk determinants and also DNA have been collected, and two prospective, nested case-referent cohorts (113 cases and 226 controls; 275 cases and 549 controls) have been sampled. To investigate whether genes of the estrogen signaling system may be important in stroke development, we performed genetic association studies, including specific functional single nucleotide polymorphisms in the genes for estrogen receptor alpha (ERα, ESR1), and its target genes osteoprotegerin (OPG, TNFRS11B) and interleukin-6 (IL-6, IL6). We found a significant association between the common c.454-397T/T genotype in ESR1 and ICH, remaining after adjustments for conventional stroke risk factors. The c.454-397T/T genotype also associated with increased systolic (SBP) and diastolic blood pressure (DBP). The combination of c.454- 397T/T and either hypertension, increased SBP, or increased DBP boosted this association substantially and significant synergistic effects on ICH risk between this genotype and increased blood pressure were demonstrated. In a second study, we found a similar association between the common OPG-1181C/C genotype and ICH. Cognitive impairments, including spatial memory and learning deficiencies, are common after stroke. Estrogens improve cognitive functions, including memory and learning processes, in postmenopausal women and ovariectomized rodents. Post-ischemic housing of rats in an enriched environment (EE) improves recovery of spatial memory and learning impairments. Both estrogen and EE induce neuroplasticity in the hippocampus. We hypothesized that 17β- estradiol combined with EE would accelerate recovery after experimental focal brain ischemia in ovariectomized rats and that such improvements could be related to expression of nerve growth factor-induced gene A (NGFI-A) in the hippocampus. Five to six weeks after middle cerebral artery occlusion, 17β-estradiol–treated rats housed in an EE showed significant improvements in cognitive function (i.e., shorter latency and path in the Morris water maze task) and significantly higher NGFI-A mRNA expression in bilateral cornu ammonis 1 (CA1) and ipsilateral dentate gyrus (DG) compared to placebo-treated animals in EE. In conclusion, we present evidence for the association between polymorphic variants in the ESR1 and TNFRS11B genes and ICH and show that 17β-estradiol in combination with EE accelerates cognitive functions in a rat stroke model, putatively through upregulation of NGFI-A in hippocampal subregions. These findings may contribute to an increased understanding of the underlying genetic etiology of ICH and may be informative for the primary prevention of this disease. They also provide hope for 17β-estradiol combined with early environmental enrichment as a novel therapeutic option following ischemic stroke.

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La quiescine/sulfhydryl oxydase QSOX1 catalyse la formation de ponts disulfures. In vivo, ses substrats et ses rôles cellulaires restent à déterminer. Les stress oxydants sont notamment impliqués dans les maladies neurodégénératives. L'hormone estradiol-17b (E2) possède des effets neuroprotecteurs. Nos objectifs ont été d'étudier la régulation de l'expression de QSOX1 par E2 et son implication dans les stress oxydants et la neuroprotection par E2.<br />Nous avons tenté d'établir un modèle de protection par E2 des cellules PC12/ERa soumises à un stress oxydant induit par H2O2 ou le complexe Fe(III)-HQ. Malgré différentes conditions testées, aucune protection par E2 n'a pu être obtenue.<br />Nous avons ensuite étudié la régulation de l'expression de QSOX1 dans le cerveau de Rates ovariectomisées traitées ou non par E2. Dans trois aires cérébrales exprimant fortement ERa et ERb, le niveau de messagers QSOX1 diminue en présence de E2.<br />Enfin, nous avons étudié l'implication de QSOX1 dans les stress oxydants. Dans les cellules PC12 soumises au stress oxydant, l'expression des messagers et de la protéine QSOX1 augmente. Suite au stress oxydant, la viabilité des cellules MCF-7 surexprimant QSOX1 diminue moins fortement que celle des cellules contrôles. La diminution de l'apoptose est associée à une moindre dépolarisation des mitochondries dans ces cellules.<br />Nos travaux ont ainsi permis de confirmer l'estrogéno-dépendance de QSOX1 in vivo et de montrer pour la première fois le rôle de QSOX1 dans la protection des cellules contre l'apoptose induite par les stress oxydants. Ces résultats ouvrent de nouvelles perspectives et renforcent l'intérêt de l'étude de QSOX1 dans la neuroprotection par E2.

Sex steroids are inevitable in women. However, long-term exposure to sex steroids increases the risk of breast cancer. A complete understanding of sex steroid control of the breast and how it relates to breast cancer risk is still lacking. Angiogenesis and proteolytic enzyme activity are crucial for the process by which tumors evolve into a vascularized, invasive phenotype. Matrix metalloproteinases are potent matrixdegrading enzymes that affect several steps in tumor progression including angiogenesis. In the female reproductive organs, sex steroids regulate angiogenesis and MMP activity, yet little is known how sex steroids affect these crucial events in normal and malignant breast tissue. This thesis elucidates a link between sex steroids, MMP activity, and angiogenesis. It is shown that estradiol down-regulates while tamoxifen up-regulates the protein expression and activity of MMP-2 and MMP-9 in human breast cancer cells in vitro and in human breast cancer xenografts in vivo. The results further suggest that a biological consequence of this regulation may be modulation of tumor angiogenesis. The net effect of adding tamoxifen to estradiol treatment was an increase in extracellular levels of the endogenous angiogenesis inhibitor endostatin and decreased levels of the tumor promoter TGF-β1 compared to estradiol treatment only. This was accompanied by reduced vasculature and decreased tumor growth. Similarly, a regulatory effect of estradiol and tamoxifen on endostatin generation was observed in normal human breast tissue by whole-tissue culture and microdialysis in human breast tissue in situ. In conclusion, the results presented in this thesis suggest previously unknown mechanisms of action of estradiol and tamoxifen in breast cancer and in normal human breast tissue, and novel means by which estradiol may tip the scale to favor angiogenesis. This knowledge may be important for the understanding of sex steroid dependent breast carcinogenesis and in the future development of tissue-specific preventive as well as therapeutic strategies against breast cancer.

Ovarian cancer is thought to be derived from the ovarian surface epithelium (OSE), but it is often diagnosed during the late stages and therefore the events that contribute to the initiation and progression of ovarian cancer are poorly defined. Epidemiological studies have indicated an association between the female reproductive hormones and ovarian cancer etiology, but the direct effects of 17β-estradiol (E2), progesterone (P4), luteinizing hormone (LH) and follicle stimulating hormone (FSH) on disease pathophysiology are not well understood. A novel transgenic mouse model of ovarian cancer was generated that utilized the Cre/loxP system to inducibly express the oncogene SV40 large and small T-Antigen in the OSE. The tgCAG-LS-TAg mice developed poorly differentiated ovarian tumours with metastasis and ascites throughout the peritoneal space. Although P4 had no effect; E2 significantly accelerated disease progression in tgCAG-LS-TAg mice. The early onset of ovarian cancer was likely mediated by E2’s ability to increase the areas of putative preneoplastic lesions in the OSE. E2 also significantly decreased survival time in ovarian cancer cell xenografts. Microarray analysis of the tumours revealed that E2 mainly affects genes involved in angiogenesis and cellular differentiation, proliferation, and migration. These results suggest that E2 acts on the tumour microenvironment in addition to its direct effects on OSE and ovarian cancer cells. In order to examine the role of the gonadotropins in ovarian cancer progression, the tgCAG-LS-TAg mice were treated with 4-vinylcyclohexene-diepoxide (VCD) to induce menopause. Menopause slowed the progression of ovarian cancer due to a change in the histological subtype from poorly differentiated tumours to Sertoli tumours. Using a transgenic mouse model, it was shown that E2 accelerated ovarian cancer progression, while P4 had little effect on the disease. Menopause (elevated levels of LH and FSH) altered the histological subtype of the ovarian tumours in the tgCAG-LS-TAg mouse model. These results emphasize the importance of generating animal models to accurately recapitulate human disease and utilizing these models to develop novel prevention and treatment strategies for women with ovarian cancer.

Women are twice as likely as men to suffer from depression and anxiety disorders and have an increased risk of onset during periods associated with hormonal changes, such as the postpartum period and the menopausal transition. Furthermore, some women seem more sensitive to normal hormone fluctuations across the menstrual cycle, since approximately 3-5% suffers from premenstrual dysphoric disorder (PMDD). Why these disorders are so common in women has not been established but there is a probable involvement of the ovarian hormones. The aim of this thesis was to investigate the effect of the ovarian hormones on the female brain during different reproductive states using psychological tests known to affect brain activity in different ways. Paper one examined the effect of the ovarian hormones on prepulse inhibition (PPI) on the acoustic startle response (ASR) and comprised cycling women and postmenopausal women. The cycling women had lower levels of PPI compared to postmenopausal women and postmenopausal women with moderate estradiol levels had lower PPI compared to postmenopausal women with low estradiol levels. Paper two examined the effect of anticipation and affective modulation on the ASR in women with PMDD and healthy controls. Women with PMDD have an increased modulation during anticipation of affective pictures compared to healthy controls during the luteal phase of the menstrual cycle. Paper three examined brain activity during response inhibition among women with PMDD and healthy controls by the use of a Go/NoGo task and fMRI. Women with PMDD displayed a decreased activity in the left insula during follicular phase and an increased activity during the luteal phase compared to controls. Paper four comprised women in the postpartum period and non-pregnant controls to examine brain activity during response inhibition. While this study revealed decreased activity at 4 weeks postpartum compared to 48 hours postpartum we cannot ascertain the role of the ovarian steroids, since none of the significant brain areas correlated with ovarian steroid or neurosteroid serum concentrations. The results of this thesis demonstrate that the ovarian hormones, or at least various hormonal states, have a probable impact on how the female brain works.

Estrogen stimulation of the medial amygdala (MEA) of the brain promotes male rat mating behavior. However, selective stimulation of either of the estrogen receptor subtypes found in the MEA (ERα or ERβ) does not support mating behavior. We tested the hypothesis that dual stimulation of ERα and ERβ is required to activate estrogen-dependant neural circuits in the MEA responsible for mating by local treatment of MEA with a combination of selective estrogenic agonists: propyl pyrazole triol (PPT, an ERα agonist ) and diarylpropionitrile (DPN, an ERβ agonist) administered to castrated, DHT maintained male rats. Estradiol (E2) or cholesterol (Chol) MEA implants served as positive and negative controls respectively. The animals receiving a mixture of PPT and DPN into the MEA displayed higher levels of mating behavior than the Chol treated animals but lower levels of mating behavior than the E2 treated animals.

published_or_final_version / Anatomy / Master / Master of Philosophy

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