Estrogen-based drug therapy in cardiovascular diseases has been difficult because it has not been possible to separate the wanted vasculoprotective effect from the unwanted effects of the hormone to the reproductive system. Here, we demonstrate that, after endothelial denudation of rat carotid artery, the mRNA of the classical estrogen receptor (ER␣) is constitutively expressed at a low level whereas the expression of the novel ER␤ mRNA increases >40-fold. Under in situ hybridization and immunohistochemistry, ER␤ mRNA and protein colocalize with the smooth muscle cells in the media and neointima. Treatment of ovariectomized female rats with the isof lavone phytoestrogen genistein, which shows 20-fold higher binding affinity to ER␤ than to ER␣, or with 17␤-estradiol, which does not differentiate between the two receptors, provides similar dosedependent vasculoprotective effect in rat carotid injury model. In addition in concentrations <10 M, both ligands are equally inhibitory to the replication and migration of smooth muscle cells in vitro. However, only treatment with 17␤-estradiol, but not with genistein, is accompanied with a dose-dependent uterotrophic effect. The results suggest that preferential targeting to ER␤ will provide vasculoprotective estrogen analogs devoid of effects to the reproductive system. Vascular intimal dysplasia and remodeling are characteristic features of injury after percutaneous transluminal coronary angioplasty (1) and in chronic allograft rejection (2, 3). The initial response to injury is inflammatory and involves the attraction of lymphocytes, macrophages, and thrombocytes to the site of injury and the secretion of cytokines, eicosanoids, and growth factors (4). Under the influence of growth factors and cytokines, smooth muscle cells (SMCs) proliferate and migrate into the intima and contribute to intimal hyperplasia and vascular stenosis.Estrogen has several protective effects on the vascular wall. Some of these are rapid, presumably direct membrane effects whereas others require transcriptional activation of genes (5, 6). The inhibitory effect of estrogen on the replication, migration, and extracellular matrix deposition by vascular SMC, the key events in vascular fibroproliferative dysplasias, is presumably a genomic effect mediated through a variety of mechanisms, including regulation of several growth factors and͞or their receptors and possibly by a direct antiproliferative effect of estrogen on SMC (5, 7).The development of vasculoprotective drug therapies based on the protective effect of estrogen has been difficult, as it has not been possible to differentiate the desired vasculoprotective effect of estrogen from its effects on the reproductive system. In this communication, we demonstrate that this is possible by preferential targeting to the novel estrogen receptor ␤ (ER␤). MATERIALS AND METHODSCarotid Denudation Injury. Carotid denudations were made to Wistar rats purchased from the Laboratory Animal Center (University of Helsinki). The details of the operation...

Our previous studies have shown that transgenic male mice expressing human P450 aromatase (AROM+) are infertile. In the present study, we followed the testis phenotype up to 15 months of age in these mice. The testes of the old AROM+ mice showed Leydig cell hypertrophy and hyperplasia, as indicated by the staining for steroidogenic enzymes and androgen and estrogen receptors. However, the Leydig cell adenomas did not show signs of malignization. In contrast, we observed a marked increase in the number of activated macrophages in the testicular interstitium of the aging AROM+ mice. The macrophages were further shown to express high levels of CD68 (a monocyte/macrophage marker) and secrete TNFalpha, indicating strong activation, presumably by estrogen exposure. The increased activity of the macrophages was associated with Leydig cell depletion (analyzed at the age of 9 and 15 months) and an increased number of mast cells and fibrosis in the testicular interstitium. Interestingly, similar findings have been made in testes of infertile men. Hence, the aging AROM+ males present with a phenocopy of inflammation-associated infertility in men, providing a model for further studies on the putative link among estrogens, orchitis, and infertility.

The data indicate that the increase in testicular DCN found in male infertility is a consequence of actions of MC-derived tryptase. We propose that the increases in DCN may consequently imbalance the paracrine signaling pathways in human testis.

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Disturbed action of sex steroid hormones, i.e. androgens and estrogens, is involved in the pathogenesis of various severe diseases in humans. Interestingly, recent studies have provided data further supporting the hypothesis that the circulating hormone concentrations do not explain all physiological and pathological processes observed in hormone-dependent tissues, while the intratissue sex steroid concentrations are determined by the expression of steroid metabolising enzymes in the neighbouring cells (paracrine action) and/or by target cells themselves (intracrine action). This local sex steroid production is also a valuable treatment option for developing novel therapies against hormonal diseases. Hydroxysteroid (17b) dehydrogenases (HSD17Bs) compose a family of 14 enzymes that catalyse the conversion between the low-active 17-keto steroids and the highly active 17b-hydroxy steroids. The enzymes frequently expressed in sex steroid target tissues are, thus, potential drug targets in order to lower the local sex steroid concentrations. The present review summarises the recent data obtained for the role of HSD17B1, HSD17B2, HSD17B7 and HSD17B12 enzymes in various metabolic pathways and their physiological and pathophysiological roles as revealed by the recently generated genetically modified mouse models. Our data, together with that provided by others, show that, in addition to having a role in sex steroid metabolism, several of these HSD17B enzymes possess key roles in other metabolic processes: for example, HD17B7 is essential for cholesterol biosynthesis and HSD17B12 is involved in elongation of fatty acids. Additional studies in vitro and in vivo are to be carried out in order to fully define the metabolic role of the HSD17B enzymes and to evaluate their value as drug targets.

Deteriorated male reproductive health has been connected to overexposure to estrogens or to imbalanced androgen-estrogen ratio. Transgenic male mice expressing human aromatase (AROM ϩ mice) serve as an apt model for the study of the consequences of an altered androgenestrogen ratio. Our previous studies with AROM ϩ mice showed that low androgen levels together with high estrogen levels result in cryptorchidism and infertility. In the present study, the AROM ϩ mice were shown to have severe abnormalities in the structure and function of Leydig cells before the appearance of spermatogenic failure. Decreased expression of adult-type Leydig cell markers (Ptgds, Vcam1, Insl3, Klk21, Star, Cyp17a1, and Hsd17b3) indicated an immature developmental stage of the Leydig cells, which appears to be the first estrogen-dependent alteration. Genes involved in steroidogenesis (Star, Cyp17a1, and Hsd17b3) were suppressed despite normal LH levels. The low expression level of kallikreins 21, 24, and 27 potentially further inhibited Leydig cell function via remodeling extracellular matrix composition. In connection with disrupted steroidogenesis, Leydig cells showed enlarged mitochondria, a reduced amount of smooth endoplasmic reticulum, and an accumulation of cholesterol and precursors for cholesterol synthesis. The results of studies with AROM ϩ mice crossed with estrogen receptor ␣ or ␤ (ER␣ and ER␤, respectively) knockout mice lead to the conclusion that the structural and functional disorders caused by estrogen exposure were mediated via ER␣, whereas ER␤ was not involved. (Endocrinology 150: [2865][2866][2867][2868][2869][2870][2871][2872] 2009)

Strong gain-of-function mutations have not been identified in humans in the FSH receptor (FSHR), whereas such mutations are common among many other G protein-coupled receptors. In order to predict consequences of such mutations on humans, we first identified constitutively activated mutants of the mouse (m) Fshr and then expressed them under the human anti-Müllerian hormone promoter in transgenic mice or created knock-in mutation into the mouse genome. We show here that mutations of Asp580 in the mFSHR significantly increase the basal receptor activity. D580H and D580Y mutations of mFSHR bind FSH, but the activity of the former is neither ligand-dependent nor promiscuous towards LH/human choriogonadotropin stimulation. Transgenic expression of mFshr(D580H) in granulosa cells leads to abnormal ovarian structure and function in the form of hemorrhagic cysts, accelerated loss of small follicles, augmented granulosa cell proliferation, increased estradiol biosynthesis, and occasional luteinized unruptured follicles or teratomas. The most affected mFshr(D580H) females are infertile with disturbed estrous cycle and decreased gonadotropin and increased prolactin levels. Increased estradiol and prolactin apparently underlie the enhanced development of the mammary glands, adenomatous pituitary growth, and lipofuscin accumulation in the adrenal gland. The influence of the mFSHR(D580Y) mutation is milder, mainly causing hemorrhagic cysts in transgenic mFSHR(D580Y) and mFSHR(D580Y) -knock-in mice. The results demonstrate that gain-of-function mutations of the FSHR in mice bring about distinct and clear changes in ovarian function, informative in the search of similar mutations in humans.

As tools for quantitative label-free mass spectrometry (MS) rapidly develop, a consensus about the best practices is not apparent. In the work described here we compared popular statistical methods for detecting differential protein expression from quantitative MS data using both controlled experiments with known quantitative differences for specific proteins used as standards as well as "real" experiments where differences in protein abundance are not known a priori. Our results suggest that data-driven reproducibility-optimization can consistently produce reliable differential expression rankings for label-free proteome tools and are straightforward in their application.