Magnetic resonance imaging in an experimental model of human ovarian cancer demonstrating altered microvascular permeability after inhibition of vascular endothelial growth factor.

OBJECTIVE: Magnetic resonance imaging enhanced with macromolecular contrast medium was used to monitor effects of angiogenesis inhibition on tumor microvascular permeability and ascites volume in an athymic rat model of human ovarian cancer. STUDY DESIGN: Groups of 6 athymic rats implanted intraperitoneally with SKOV-3, a human ovarian cancer cell line, were treated through a 14-day course with antibody to vascular endothelial growth factor or with saline solution for control animals. Dynamic magnetic resonance imaging was performed with a 92,000-d contrast agent, albumin-(gadolinium-diethylenetriaminepentaacetic acid)(30). Vascular permeability was estimated from dynamic enhancement data that were analyzed with a unidirectional 2-compartment kinetic model. RESULTS: Animals treated with vascular endothelial growth factor antibody accumulated significantly smaller volumes of peritoneal ascites (P <.05) and showed significantly lower magnetic resonance imaging-assayed tumor microvascular permeabilities (P <.05) than did control animals. CONCLUSION: Magnetic resonance imaging enhanced with a macromolecular contrast agent in an athymic rat model of human ovarian cancer treated with anti-vascular endothelial growth factor antibody can be used to measure a reduction in tumor microvascular permeability, corresponding to a reduction in ascites production.

Corticotropin-releasing hormone stimulates P450 17alpha-hydroxylase/17,20-lyase in human fetal adrenal cells via protein kinase C.

CRH directly stimulates dehydroepiandrosterone sulfate (DHEAS) production in human fetal adrenal cells. In the human fetal and adult pituitary, CRH acts via protein kinase A (PKA). We determined the CRH signal transduction pathway in fetal adrenal cells, i.e. whether CRH modulates human fetal adrenal steroidogenesis via PKA and/or protein kinase C (PKC). In primary cultures, CRH increased inositol trisphosphate. After CRH treatment, inositol tris-, bis-, and monophosphates increased within 1 min, reaching maximal levels at 5 min. In contrast, PGF2alpha, known to act via PKC, induced a sustained response for up to 20 min. The response to CRH was dose dependent, maximal at 1 micromol/L at both 1 and 5 min. CRH increased DHEAS production, with a much lesser effect on cortisol. CRH did not stimulate inositol phospholipid in adult adrenal glands, suggesting that this pathway is unique to the fetal adrenal. CRH increased messenger ribonucleic acid encoding 17alpha-hydroxylase/17,20 lyase (P450c17), but not 3beta-hydroxysteroid dehydrogenase/delta(4-5) isomerase. However, 3betaHSD expression was stimulated by ACTH. PKC, but not PKA, inhibitors blocked CRH-stimulated P450c17 induction, whereas PKA inhibitors blocked ACTH-stimulated cortisol. Thus, CRH is coupled to the phospholipase C-inositol phosphate second messenger system and preferentially induces the expression of P450c17 and DHEAS, suggesting a unique role of CRH regulating human fetal adrenal function via PKC.

Phytoestrogens alter adrenocortical function: genistein and daidzein suppress glucocorticoid and stimulate androgen production by cultured adrenal cortical cells.

Phytoestrogens influence a variety of biological processes. As 17beta-estradiol alters adrenocortical cell function, we examined whether the dietary phytoestrogens, genistein and daidzein, have related effects. In cultured human fetal and postnatal adrenal cortical cells, genistein and daidzein (both 0.4-40 micromol/L) decreased ACTH-stimulated cortisol production to basal levels (ED50, 1-4 micromol/L). In the adult adrenocortical cell line, H295, genistein, daidzein, and 17beta-estradiol (10 micromol/L) decreased cAMP-stimulated cortisol synthesis in a similar fashion. Neither genistein nor daidzein altered basal or ACTH-stimulated dehydroepiandosterone sulfate (DHEA-S) production in fetal adrenocortical cells, whereas in postnatal adrenocortical cells, DHEA and DHEA-S were markedly increased (ED50, 1-4 micromol/L). In H295 cells, basal and cAMP-stimulated DHEA production were similarly increased by the phytoestrogens and 17beta-estradiol. Genistein and daidzein did not affect the expression of steroid-metabolizing enzymes. However, genistein and daidzein specifically inhibited the activity of 21-hydroxylase (P450c21); the activities of other steroidogenic enzymes were not affected. Thus, phytoestrogens may decrease cortisol synthesis by suppressing the activity of P450c21 and, as a consequence, increase DHEA/DHEA-S synthesis by shunting metabolites away from the glucocorticoid synthetic pathway. Therefore, consumption of foods containing phytoestrogens may alter adrenocortical function by decreasing cortisol and increasing androgen production.

Proliferation and apoptosis in the human adrenal cortex during the fetal and perinatal periods: implications for growth and remodeling.

After 10-15 weeks of gestation, the human fetal adrenal cortex undergoes rapid growth due to enlargement of a specialized cortical compartment known as the fetal zone (FZ). Soon after birth, the FZ regresses and the adult zonation pattern develops at least in part from cells derived from the persistent definitive zone (DZ), a thin layer of tightly packed cells surrounding the FZ. We postulated that growth of the fetal adrenal cortex involves zone-specific cellular hyperplasia, whereas the postnatal involution of the FZ is due to apoptosis. Therefore, we investigated the pattern of cellular proliferation and death in the FZ and DZ of the human fetal and postnatal adrenal cortex using immunohistochemical staining for proliferating cell nuclear antigen as a marker of mitosis and in situ detection of DNA fragmentation as a marker of apoptosis. Between 10-14 weeks' gestation, the mitotic indexes (percentage of proliferating cell nuclear antigen-positive cells) in the DZ (26.46 +/- 2.95%) and in the FZ (21.26 +/- 2.57%) were not significantly different. Between 15-20 weeks gestation, the mitotic index increased significantly (P < 0.05) in both zones (FZ, 33.84 +/- 5.21%; DZ, 67.45 +/- 7.58%) relative to levels before 15 weeks. This increase persisted between 21-24 weeks gestation (FZ, 39.5 +/- 4.22%; DZ, 58.63 +/- 6.83%). Interestingly, after 14 weeks, the mitotic index of the DZ was significantly greater (P < 0.05) than that of the FZ. In adrenal specimens obtained from infants born prematurely and treated in utero with glucocorticoid, the mitotic indexes in the FZ and DZ were significantly decreased. At all stages of gestation, no apoptotic nuclei were detected in the DZ. However, scattered apoptotic nuclei were detected in the central portions of the FZ. The number of apoptotic nuclei in the inner FZ increased with advancing gestation and was maximal during the first postnatal month. To identify factors that may regulate apoptosis, primary cultures of midgestation FZ cells were treated with activin A and transforming growth factor-beta (TGFbeta). Activin A and TGFbeta both induced apoptotic cell death, as assessed by internucleosomal DNA cleavage (DNA laddering). Induction of apoptosis by activin A was prevented by concomitant addition of follistatin, an activin-binding protein. Taken together, these data indicate that 1) growth of the human fetal adrenal cortex involves cellular hyperplasia, mainly in the DZ and to a lesser extent in the FZ, which is probably dependent on ACTH; and 2) apoptosis occurs predominantly in the inner cortical compartment and may be responsible for the rapid regression of the FZ after birth, a process that may be regulated by activin A and/or TGFbeta.

Role of vascular endothelial growth factor in ovarian cancer: inhibition of ascites formation by immunoneutralization.

Ovarian cancer is characterized by the rapid growth of solid intraperitoneal tumors and large volumes of ascitic fluid. Vascular endothelial growth factor (VEGF) augments tumor growth by inducing neovascularization and may stimulate ascites formation by increasing vascular permeability. We examined the role of VEGF in ovarian carcinoma using in vivo models in which intraperitoneal or subcutaneous tumors were induced in immunodeficient mice using the human ovarian carcinoma cell line SKOV-3. After tumor engraftment (7 to 10 days), some mice were treated with a function-blocking VEGF antibody (A4.6.1) specific for human VEGF. A4.6.1 significantly (P < 0.05) inhibited subcutaneous SKOV-3 tumor growth compared with controls. However, tumor growth resumed when A4.6.1 treatment was discontinued. In mice bearing intraperitoneal tumors (IP mice), ascites production and intraperitoneal carcinomatosis were detected 3 to 7 weeks after SKOV-3 inoculation. Importantly, A4.6.1 completely inhibited ascites production in IP mice, although it only partially inhibited intraperitoneal tumor growth. Tumor burden was variable in A4.6.1-treated IP mice; some had minimal tumor, whereas in others tumor burden was similar to that of controls. When A4.6.1 treatment was stopped, IP mice rapidly (within 2 weeks) developed ascites and became cachectic. These data suggest that in ovarian cancer, tumor-derived VEGF is obligatory for ascites formation but not for intraperitoneal tumor growth. Neutralization of VEGF activity may have clinical application in inhibiting malignant ascites formation in ovarian cancer.

Corticotropin-releasing hormone directly and preferentially stimulates dehydroepiandrosterone sulfate secretion by human fetal adrenal cortical cells.

Estrogens produced by the placenta play a pivotal role in the endocrine control of pregnancy and induce many of the key changes involved in parturition. The placentae of humans and higher primates use the C19 androgen dehydroepiandrosterone sulfate (DHEA-S) supplied by the fetal adrenals as the principal substrate for estrogen synthesis. Thus, secretion of androgens by the fetal adrenals may be central to the process of primate parturition. The timing of human parturition also is correlated with placental CRH concentrations in the maternal circulation. Because the mechanisms that regulate DHEA-S production by the fetal adrenals are incompletely understood, we examined whether there is a functional relationship between CRH and steroid production by human fetal adrenal cortical cells. Using Northern blot analysis, we detected messenger RNA transcripts (2.7 kb) encoding the type-1 CRH receptor in total RNA extracted from midgestation human fetal adrenals, suggesting that the fetal adrenal cortex may be directly responsive to CRH. To test this, primary cultures of human fetal adrenal cortical cells were exposed to human CRH. Human CRH increased DHEA-S production by cultured human fetal adrenal cortical cells in a dose-dependent fashion, with an ED50 of 10-100 pmol/L. Human CRH was as effective as ACTH at stimulating DHEA-S production; however, it was 70% less potent than ACTH at stimulating cortisol production, indicating that its actions were preferentially directed toward increasing DHEA-S synthesis. Consistent with this thesis, we found that CRH increased abundance of messenger RNA encoding cytochrome P450 cholesterol side-chain cleavage and 17alpha-hydroxylase/17,20 lyase but not 3beta-hydroxysteroid dehydrogenase in adrenal cells. CRH did not alter cell number, indicating that it is not mitogenic for fetal adrenal cortical cells. These data demonstrate a direct functional interaction between CRH and the fetal adrenal. Therefore, placental CRH production, which rises exponentially during human pregnancy, may play a key role in promoting DHEA-S production by the fetal adrenals, which could lead to increasing placental estrogen synthesis and contribute to the process of parturition in humans.

Corticotropin regulates vascular endothelial growth factor expression in human fetal adrenal cortical cells.

The human adrenal cortex has a complex vasculature that is essential for growth, organ maintenance, and access of secreted hormones to the circulation. Growth and function of the adrenal cortex are regulated by corticotropin (ACTH), the actions of which are in part mediated by locally produced growth factors. As cortical growth and vascularization must increase in a coordinated manner, we hypothesized that ACTH also influences adrenal cortical angiogenesis by stimulating the local expression of specific angiogenic factors. Vascular endothelial growth factor (VEGF) is a potent endothelial cell-specific angiogenic peptide, the expression of which has been detected in adrenal cortical cells. Therefore, we examined the localization of VEGF expression in the midgestation (16-20 weeks) human fetal adrenal cortex and determined whether VEGF expression and secretion by isolated human fetal adrenal cortical cells are regulated by ACTH. By immunohistochemical analysis, strong cytoplasmic staining for VEGF was detected in scattered clusters of fetal zone (inner cortical compartment) cells. In contrast, cells in the outer, definitive zone of the cortex stained only weakly for VEGF. The predominant staining for VEGF in the fetal zone correlated with the extensive vasculature of this zone as detected by immunohistochemical staining for von Willebrand factor, which is specific for endothelial cells. In primary cultures of human fetal adrenal cortical cells, ACTH (1 nmol/L) and forskolin (10 micromol/L) increased the abundance of messenger ribonucleic acid transcripts encoding VEGF, as assessed by Northern and slot blot analyses. The stimulatory effect of ACTH and forskolin on VEGF gene expression occurred within 2 h of agonist exposure and persisted for at least 24 h. ACTH and forskolin also increased VEGF protein secretion by fetal adrenal cortical cells, as assessed by enzyme-linked immunosorbent assay for VEGF in fetal adrenal cortical cell-conditioned medium. A significant (P < 0.05) increase in VEGF secretion was detected as early as 8 h after ACTH or forskolin treatment. By 24 h after the addition of ACTH or forskolin, VEGF secreted from isolated human fetal adrenal cells was increased 5- to 6-fold. These data demonstrate that the human fetal adrenal cortex, particularly the cells of the inner fetal zone, express VEGF and that VEGF expression and secretion by these cells are directly regulated by ACTH and the activation of adenylate cyclase. Thus, VEGF may be a local regulator of adrenal cortical angiogenesis and an important mediator of the tropic action of ACTH, ensuring the coordination of ACTH-stimulated cortical growth and vascularization.

The regulation and role of fetal adrenal development in human pregnancy.

The rapid growth of the human fetal adrenal gland, which is primarily a reflection of the growth of the unique fetal zone, is regulated by ACTH acting indirectly to stimulate the expression of locally produced growth factors, of which IGF-II and bFGF appear to play key roles. Through most of gestation, the outer definitive zone appears to function as a reservoir of progenitor cells which move centripetally to populate the rest of the gland. At the end of pregnancy, the fetal zone undergoes senescence through an apoptotic process. Activin and TGF-beta are capable of inducing apoptosis in the fetal zone. Corticotropin-releasing hormone, which is produced by the placenta in markedly increased amounts at the end of gestation, may orchestrate a variety of processes, including direct stimulation of fetal adrenal steroidogenesis, culminating in the initiation of parturition.

Developmental and functional biology of the primate fetal adrenal cortex.

The unique characteristics of the primate (particularly human) fetal adrenal were first realized in the early 1900s when its morphology was examined in detail and compared with that of other species. The unusual architecture of the human fetal adrenal cortex, with its unique and disproportionately enlarged fetal zone, its compact definitive zone, and its dramatic remodeling soon after birth captured the interest of developmental anatomists. Many detailed anatomical studies describing the morphology of the developing human fetal adrenal were reported between 1920 and 1960, and these morphological descriptions have not changed significantly. More recently, it has become clear that fetal adrenal cortical growth involves cellular hypertrophy, hyperplasia, apoptosis, and migration and is best described by the migration theory, i.e. cells proliferate in the periphery, migrate centripetally, differentiate during their migration to form the functional cortical zones, and then likely undergo apoptosis in the center of the cortex. Consistent with this model, cells of intermediate phenotype, arranged in columnar cords typical of migration, have been identified between the definitive and fetal zones. This cortical area has been referred to as the transitional zone and, based on the expression of steroidogenic enzymes, we consider it to be a functionally distinct cortical zone. Elegant experiments during the 1950s and 1960s demonstrated the central role of the primate fetal adrenal cortex in establishing the estrogenic milieu of pregnancy. Those findings were among the first indications of the function and physiological role of the human fetal adrenal cortex and led Diczfalusy and co-workers to propose the concept of the feto-placental unit, in which DHEA-S produced by the fetal adrenal cortex is used by the placenta for estrogen synthesis. Tissue and cell culture techniques, together with improved steroid assays, revealed that the fetal zone is the primary source of DHEA-S, and that its steroidogenic activity is regulated by ACTH. In recent years, function of the human and rhesus monkey fetal adrenal cortical zones has been reexamined by assessing the localization and ontogeny of steroidogenic enzyme expression. The primate fetal adrenal cortex is composed of three functionally distinct zones: 1) the fetal zone, which throughout gestation does not express 3 beta HSD but does express P450scc and P450c17 required for DHEA-S synthesis; 2) the transitional zone, which early in gestation is functionally identical to the fetal zone but late in gestation (after 25-30 weeks) expresses 3 beta HSD, P450scc, and P450c17, and therefore is the likely site of glucocorticoid synthesis, and 3) the definitive zone, which lacks P450c17 throughout gestation but late in gestation (after 22-24 weeks) expresses 3 beta HSD and P450scc, and therefore is the likely site of mineralocorticoid synthesis. Indirect evidence, based on effects of P450c21 deficiency and maternal estriol concentrations, indicate that the fetal adrenal cortex produces cortisol and DHEA-S early in gestation (6-12 weeks). However, controversy exists as to whether cortisol is produced de novo or derived from the metabolism of progesterone, as data regarding the expression of 3 beta HSD in the fetal adrenal cortex early in gestation are conflicting. During the 1960s, Liggins and colleagues demonstrated that in the sheep, cortisol secreted by the fetal adrenal cortex late in gestation regulates maturation of the fetus and initiates the cascade of events leading to parturition. Those pioneering discoveries provided insight into the mechanism underlying the timing of parturition and therefore were of particular interest to obstetricians and perinatologists confronted with the problems of preterm labor. However, although cortisol emanating from the fetal adrenal cortex promotes fetal maturation in primates as it does in sheep, its role in the regulation of primate parturition, unlike that in sheep

Insulin-like growth factors augment steroid production and expression of steroidogenic enzymes in human fetal adrenal cortical cells: implications for adrenal androgen regulation.

The fetal zone is a unique adrenal cortical compartment that exists only during fetal life in humans and higher primates and produces large amounts of the adrenal androgen dehydroepiandrosterone sulfate (DHEA-S). Growth of the fetal zone is primarily regulated by ACTH, the actions of which are mediated in part by locally produced autocrine/paracrine growth factors. We previously demonstrated that one of these growth factors, insulin-like growth factor II (IGF-II), is mitogenic for cultured fetal zone cells and is produced in high abundance by these cells in response to ACTH. In the present study, we determined whether IGF-II also modulates the differentiated function of fetal zone cells. We examined the effects of recombinant human IGF-II and the closely related peptide, IGF-I, on 1) basal and agonist-stimulated [ACTH-(1-24), forskolin, or 8-bromo-cAMP] cortisol and DHEA-S production, 2) basal and ACTH-stimulated steady state abundance of messenger ribonucleic acids (mRNAs) encoding the steroidogenic enzymes cytochrome P450 side-chain cleavage (P450scc) and cytochrome P450 17alpha-hydroxylase/17,20-lyase (P450c17), and 3) basal and ACTH-stimulated steady state abundance of mRNA encoding the ACTH receptor. Basal cortisol (23.93 +/- 1.20 pmol/10(5) cells x 24 h) and DHEA-S (548.87 +/- 43.17 pmol/10(5) cells x 24 h) productions were significantly (P < 0.05) increased by IGF-I (2.3- and 1.8-fold, respectively) and IGF-II (2.8- and 1.8-fold, respectively). As expected, ACTH, forskolin, and cAMP markedly increased the production of cortisol by 26-, 10-, and 13-fold, respectively, and that of DHEA-S by 5.4-, 4.6-, and 5.5-fold, respectively, compared with basal levels. IGF-II (100 ng/mL) significantly (P < 0.001) increased ACTH-, forskolin-, and cAMP-stimulated production of cortisol by 2.4-, 4.3-, and 3.2-fold, respectively, and that of DHEA-S by 1.4, 1.6-, and 1.4-fold, respectively. IGF-I (100 ng/mL) had similar effects as IGF-II and significantly (P < 0.001) increased ACTH-, forskolin-, and cAMP-stimulated production of cortisol by 2.8-, 3.9-, and 3.1-fold, respectively, and that of DHEA-S by 1.3-, 1.6-, and 1.4-fold, respectively. The similar potencies of IGF-I and IGF-II suggest that the actions of these factors were mediated via a common receptor, most likely the type I IGF receptor. The effects of IGF-II on ACTH-stimulated steroid production were dose-dependent (EC50, 0.5-1.0 nmol/L), and IGF-II markedly increased the steroidogenic responsiveness of fetal zone cells to ACTH. With respect to cortisol production, IGF-II shifted the ACTH dose-response curve to the left by 1 log10 order of magnitude. IGF-II also increased ACTH-stimulated abundance of mRNA encoding P450scc (1.9-fold) and P450c17 (2.2-fold). Basal expression of P450scc was not affected by IGF-II. In contrast, basal expression of P450c17 was increased 2.2-fold by IGF-II and IGF-I in a dose-responsive fashion. Neither IGF-I nor IGF-II affected basal or ACTH-stimulated abundance of mRNA encoding the ACTH receptor, suggesting that the increase in ACTH responsiveness was not mediated by an increase in ACTH-binding capacity. Taken together, these data indicate that activation of the type I IGF receptor increases ACTH responsiveness in fetal zone cells by modulating ACTH signal transduction at some point distal to ACTH receptor activation. These data also indicate that locally produced IGF-II modulates fetal adrenal cortical cell function by increasing responsiveness to ACTH and possibly (based on its direct stimulation of P450c17 expression) augmenting the potential for adrenal androgen synthesis. Thus, activation of the type I IGF receptor on adrenal cortical cells may play a pivotal role in adrenal androgen production, both physiologically in utero and at adrenarche, and in pathophysiological conditions ofhyperandrogenemia, such as the polycystic ovary syndrome.

Role of growth factors in the developmental regulation of the human fetal adrenal cortex.

Development of the human fetal adrenals is characterized by rapid growth and high levels of steroidogenic activity during the latter two-thirds of pregnancy. By midgestation, the human fetal adrenals are composed of two distinct cortical zones: the predominant fetal zone, which occupies 80-90% of the cortical volume and produces large amounts of the delta 5-steroid dehydroepiandrosterone sulfate, and the narrow definitive zone, which surrounds the fetal zone. Late in gestation, the peripheral portion of the fetal zone develops into a third, functionally distinct compartment, the transitional zone, which is the likely site of cortisol synthesis. Soon after birth, the adrenal cortex is remodeled and the fetal zone disappears. The adult cortical zones are thought to arise from the definitive zone, which persists postnatally. Development of the human fetal adrenals is regulated primarily by corticortropin (ACTH) secreted from the fetal pituitary. However, as ACTH is not a mitogen per se, its proliferative actions on human fetal adrenal cortical cells are thought to be mediated by autocrine/paracrine growth factors produced by adrenal cortical cells in response to ACTH. In addition, these growth factors appear to modulate the functional response of fetal adrenal cortical cells to ACTH. The roles of several growth factors, including the insulin like growth factors I and II (IGF-I and IGF-II), epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), activin, inhibin, and the transforming growth factors alpha and beta (TGF-alpha and TGF-beta) have been examined. In cultured human fetal adrenal cortical cells, EGF, bFGF, and IGF-I and -II are mitogenic, whereas activin and TGF-beta inhibit proliferation. IGF-II, activin, and TGF-beta also modulate ACTH-stimulated steroidogenesis. Human fetal adrenal cortical cells express IGF-II, bFGF and the activin/inhibin subunits, and the abundance of mRNAs for each of these factors is up-regulated by ACTH, suggesting that these growth factors are autocrine/paracrine mediators of ACTH action. Thus, although human adrenal development is primarily regulated by ACTH, its actions appear to be mediated/modulated by a cohort of locally expressed growth factors, the net effect of which results in the unique growth and steroidogenic activity of the human fetal adrenal cortex.

Functional maturation of the primate fetal adrenal in vivo. II. Ontogeny of corticosteroid synthesis is dependent upon specific zonal expression of 3 beta-hydroxysteroid dehydrogenase/isomerase.

Cortisol, produced by the primate fetal adrenal, regulates the maturation of organ systems necessary for extrauterine life. During most of primate pregnancy, however, the fetal adrenal lacks the enzyme 3 beta-hydroxysteroid dehydrogenase/isomerase (3 beta HSD), which is essential for cortisol synthesis. Therefore, we used immunohistochemistry and in situ hybridization techniques to investigate the developmental expression of 3 beta HSD in the fetal rhesus monkey adrenal from 109 days' gestation until term (165 +/- 5 days) and assessed the role of ACTH in the induction of its expression and localization. We also examined whether ACTH regulates the expression of two other steroidogenic enzymes, cytochrome P450 cholesterol side-chain cleavage (P450scc) and P450 17 alpha-hydroxylase, 17/20-lyase (P450c17), in the fetal rhesus monkey adrenal. To stimulate ACTH secretion from the fetal pituitary in vivo, we administered metyrapone to late gestation fetal rhesus monkeys for 3-7 days. Adrenals were collected from untreated fetuses at 109-125 days (n = 5), 130-148 days (n = 7), 155-172 days (n = 4), and after metyrapone treatment at 135-137 days (n = 4). The cortical width and total amount of 3 beta HSD staining were measured using an image analysis system. 3 beta HSD was localized primarily in the definitive zone cells of the adrenal from fetuses between 109-148 days, whereas at term (155-172 days), 3 beta HSD was localized in both definitive and transitional zone cells. The cortical width and total amount of 3 beta HSD staining in the adrenal increased significantly (P < 0.05) between 148 days (137 +/- 14 microns and 3,689 +/- 522 grains) and 155 days (315 +/- 61 microns and 7,321 +/- 2,008 grains). Interestingly, in metyrapone-treated fetuses at 135-137 days, 3 beta HSD messenger RNA (mRNA) and protein were localized extensively in both the definitive and transitional zones, a pattern seen only in term fetal adrenals in untreated animals. In addition, metyrapone treatment significantly (P < 0.05) increased cortical width (386 +/- 95 microns) and total 3 beta HSD immunostaining (29,063 +/- 13,692 grains) compared with age-matched controls. In contrast to 3 beta HSD, P450scc mRNA was detected in the definitive, transitional, and fetal zones, and its expression was not altered after metyrapone treatment. P450c17 mRNA was detected in the transitional and fetal zones, and the relative abundance was greater in the transitional zone. The relative abundance of P450c17 mRNA was increased in the fetal zone after metyrapone treatment. In summary, at term or after metyrapone treatment, expression of 3 beta HSD is induced in the transitional zone of the fetal rhesus monkey adrenal gland, an indication of functional maturation of the primate adrenal cortex. These data suggest that the ontogenetic increase in fetal pituitary ACTH secretion plays an important role in the induction of 3 beta HSD expression in the transitional zone.

Functional maturation of the primate fetal adrenal in vivo: I. Role of insulin-like growth factors (IGFs), IGF-I receptor, and IGF binding proteins in growth regulation.

The rapid growth of the primate fetal adrenal from midgestation until term is regulated by ACTH secreted by the fetal pituitary. Previous studies suggest that the trophic actions of ACTH are mediated by insulin-like growth factor II (IGF-II) synthesized by fetal adrenal cortical cells. To characterize further the role of IGF-II in the regulation of fetal adrenal growth, we investigated the expression of the messenger RNAs (mRNAs) encoding IGF-I, IGF-II, IGF-I receptor (IGF-IR) and IGF binding protein (IGFBP) 1-6 in the fetal rhesus monkey adrenal in vivo from 109 days of gestation until term (165 +/- 5 days) using in situ hybridization. To assess the role of ACTH in the regulation of expression of the IGF system in vivo, we administered metyrapone (3-7 days) to late gestation fetal rhesus monkeys (n = 4) in utero to increase fetal pituitary ACTH secretion. IGF-II mRNA was abundant in the definitive, transitional and fetal zones of the adrenal cortex from 109 days until term. IGF-IR mRNA was expressed in the definitive, transitional and fetal zones and decreased to nondetectable levels at term. IGFBP-2 and IGFBP-6 mRNAs were expressed in the definitive, transitional, and fetal zones, whereas IGFBP-1, -3, -4, and -5 were not detected in adrenal cells. The effects of increasing ACTH secretion on the growth of the specific zones of the adrenal were determined using morphometric techniques. Metyrapone treatment approximately doubled adrenal weight, which was due to an increase in the area of the definitive, transitional, and fetal zones with decreased cell density of the definitive, transitional, and fetal zones compared with controls and not due to a change in total cell number. Therefore, the increase in adrenal weight after metyrapone treatment was due to hypertrophy of the three cortical zones; there was no effect on adrenal medullary growth. The relative abundance of the mRNAs encoding IGF-II and the IGF-IR was increased after metyrapone treatment, whereas the localization and relative abundance of IGFBP 1-6 mRNAs were not altered by metyrapone treatment. We conclude that the ontogenetic increase in adrenal growth may be regulated, at least in part, by locally synthesized IGF-II, and the cessation of adrenal growth that occurs at term may be mediated by the decrease in the IGF-IR. The adrenal cortical expression of IGFBP-2 and IGFBP-6 suggests that these IGFBPs may modulate the IGF-IGF-IR interaction. Metyrapone treatment, which likely increased fetal pituitary ACTH secretion, causes a coordinated increase in expression of IGF-II and IGF-IR in fetal adrenal cortical cells, which may be an important mechanism of regulation of fetal adrenal cortical growth.

Localization and regulation of corticotropin receptor expression in the midgestation human fetal adrenal cortex: implications for in utero homeostasis.

Developmental changes in the responsiveness of the fetal adrenals to corticotropin (ACTH) play an important role in the regulation of the fetal hypothalamic-pituitary-adrenal axis. Responsiveness of adrenal cortical cells to ACTH is dependent on the extent of ACTH receptor expression. Therefore, we examined the localization and regulation of ACTH receptor expression in the midgestation (16-24 weeks) human fetal adrenal cortex. In situ hybridization analysis was used to localize messenger RNA (mRNA) encoding the ACTH receptor in sections of human fetal adrenal glands. Messenger RNA encoding the ACTH receptor was localized in cells from all cortical zones; abundance was higher in definitive zone than in fetal zone cells and was least abundant in the more central portions of the cortex. Regulation of ACTH receptor expression was studied using Northern blot analysis of total RNA extracted from primary cultures of fetal and definitive zone cells. Two major (1.5 and 3.5 kilobases) and, upon stimulation with ACTH, 3 minor (4.0, 6.0 and 10.0 kb) ACTH receptor mRNA transcripts were detected in RNA from fetal and definitive zone cells. In both cell types, ACTH-(1-24) increased the abundance of mRNA encoding the ACTH receptor 10- to 20-fold compared with untreated cells. The effects of ACTH-(1-24) on ACTH receptor expression in fetal zone cells were time- and dose-dependent. The ED50 for the stimulation of ACTH receptor expression by ACTH-(1-24) was 1-10 pM, and maximal response to 0.1 nm ACTH-(1-24) was detected after 12-16 h. Eight-bromoadenosine cAMP and forskolin also stimulated ACTH receptor expression in fetal zone cells and closely mimicked the effects of ACTH-(1-24). In contrast, stimulation of protein kinase C with 12-O-tetradecanoyl phorbol 13-acetate had no effect on ACTH receptor expression. Changes in ACTH receptor expression in response to ACTH-(1-24), cAMP and forskolin were paralleled by changes in expression of the P450 cholesterol side chain cleavage (P450scc) enzyme. These data demonstrate that expression of the ACTH receptor by the human fetal adrenal cortex is up-regulated by its own ligand and that this effect is mediated by a cAMP-dependent mechanism. In addition, the coordinate stimulation of ACTH receptor and P450scc expression by ACTH indicates that the gene for the ACTH receptor is one of a specific cohort of genes regulated by ACTH that are required to facilitate fetal adrenal cortical response to ACTH. ACTH regulation of its own receptor may represent a mechanism by which fetal adrenal responsiveness to ACTH is maintained and possibly enhanced during fetal development.

Vascular endothelial growth factor localization in human ovary and fallopian tubes: possible role in reproductive function and ovarian cyst formation.

Vascular endothelial growth factor (VEGF) is a potent angiogenic factor that also increases vascular permeability. We hypothesized that VEGF plays a role in the regulation of cyclic ovarian angiogenesis in women, and that its ability to increase vascular permeability may be an important factor in the production of fallopian tube effluent and fluid formation in ovarian cysts. To examine these hypotheses, we assessed VEGF expression in ovaries and fallopian tubes from premenopausal (n = 10) and postmenopausal (n = 4) women. Immunohistochemical analysis for VEGF was performed using a rabbit polyclonal antibody directed against human VEGF. In normal ovaries from premenopausal women, VEGF within healthy follicles was localized to the thecal cell layer, with minimal VEGF peptide detected in the granulosa cell layer. VEGF was not expressed in atretic follicles or a degenerating corpus luteum. However, intense VEGF immunostaining was observed within the highly vascularized corpora lutea in all specimens examined. In normal ovaries from postmenopausal women, VEGF was detected only in epithelial inclusion cysts and a serous cystadenoma. In specimens from both pre- and postmenopausal women, the luminal epithelium of the fallopian tube as well as smooth muscle cells and pericytes lining small and large blood vessels within the tube and hilum of the ovary exhibited specific staining for VEGF. Based on these data, we suggest that during reproductive life, VEGF plays a role in the growth and maintenance of the ovarian follicle and corpus luteum by mediating angiogenesis. In addition, VEGF within the fallopian tube luminal epithelium may increase vascular permeability and modulate tubal luminal secretions. Similarly, VEGF in the epithelial lining of benign ovarian neoplasms may contribute to fluid formation in ovarian cysts.

In the human fetus, vascular endothelial growth factor is expressed in epithelial cells and myocytes, but not vascular endothelium: implications for mode of action.

Vascular endothelial growth factor (VEGF) is potentially an important regulator of angiogenesis, particularly during the extensive tissue growth and remodeling that occur in utero. In the present study, we have investigated the role of VEGF during human fetal development by analyzing the distribution of VEGF messenger RNA as well as the tissue- and cell-specific localization of VEGF peptide in the human midgestation (16-22 weeks) fetus. As a comparison, we conducted parallel studies on several human adult tissues. Messenger RNA encoding VEGF was detected in all fetal tissues studied and was most abundant in human fetal lung, kidney, and spleen; moderately abundant in heart, adrenal, pancreas, intestine, liver, testis, skin, muscle, and brain; and minimally detected in thymus and placenta. VEGF peptide, detected by immunohistochemistry, always was intracytoplasmic and localized principally in epithelial cells and myocytes, including the smooth muscle cells lining blood vessels. VEGF was not detected in vascular endothelial cells. As the cellular localization of VEGF in several human adult tissues was similar to that found in the cognate fetal tissues, VEGF is probably important not only in angiogenesis, but also in the maintenance of existing vessels. As VEGF was localized primarily in epithelial cells and myocytes and not in endothelial cells, these data are consistent with a paracrine mechanism of action whereby VEGF secreted by nonendothelial cells modulates activities in adjacent vascular endothelium.

Activin expression by cultured human retinal pigment epithelial cells.

PURPOSE: To determine whether human retinal pigment epithelial (hRPE) cells produce activin, a growth factor in the transforming growth factor beta family, and to characterize growth regulatory effects of activin on retinal pigment epithelium. METHODS: mRNA expression was examined using polymerase chain reaction with primers specific for the beta A and beta B chains of activin and by slot blot analysis with a probe specific for the beta A chain. Protein localization was determined immunocytochemically using antibodies specific for the beta A chain of activin and intact activin A. The effect of activin A on DNA synthesis was studied by measuring (3H) thymidine incorporation after cells were exposed to recombinant human activin A (rhA). Growth regulatory effects of rhA on hRPE cells were examined with cell growth assays. RESULTS: beta A mRNA was expressed constitutively in 8/8 cells lines tested. beta B mRNA was not expressed in any of the six cell lines tested but was expressed in human ovarian granulosa cell controls. Positive immunostaining was observed for both the beta A chain and intact activin A. (3H) thymidine incorporation was inhibited 44% (P < 0.025), 45% (P < 0.025), and 44% (P < 0.015) when RPE cells were exposed to 100 ng/ml rhA and grown in serum-free medium, medium with 0.5% serum, and 1% serum, respectively. Cell growth was inhibited 33.2% (P = 0.0001) after RPE cells were exposed to 100 ng/ml rhA for 8 days. CONCLUSIONS: These results suggest that activin A can act as an autocrine-paracrine growth regulator in RPE cells and may help control cellular growth in ocular development and proliferative eye disease.

Localization of cytochrome P450 cholesterol side-chain cleavage, cytochrome P450 17 alpha-hydroxylase/17, 20-lyase, and 3 beta-hydroxysteroid dehydrogenase isomerase steroidogenic enzymes in human and rhesus monkey fetal adrenal glands: reappraisal of functional zonation.

We examined the in situ localization of key steroidogenic enzymes in adrenal gland sections from midgestation (17-24 weeks) human fetuses and late gestation (130-142 days; term = 165 days) rhesus monkey fetuses. The rhesus monkey fetal adrenals were used as a model for the late gestation human fetal adrenal. The enzymes examined were cytochrome P450 cholesterol side-chain cleavage (P450scc), cytochrome P450 17 alpha-hydroxylase/17,20-lyase (P450c17), and 3 beta-hydroxysteroid dehydrogenase/isomerase (3 beta HSD). In human fetal adrenals, P450scc and P450c17 proteins and mRNAs were detected only in fetal zone (innermost cortical zone) and transitional zone (between the fetal and definitive zone) cells, not in definitive zone cells. Expression of 3 beta HSD was not detected in any cortical zone cells in midgestation human fetal adrenals. In rhesus monkey fetal adrenals, a similar pattern of P450scc and P450c17 expression was observed in the fetal and transitional zones. In the definitive zone cells of rhesus monkey fetal adrenals, expression of both P450scc and 3 beta HSD was detected. In addition, low levels of 3 beta HSD expression could be detected in some transitional zone cells. P450c17 expression was lacking in definitive zone cells from rhesus monkey fetal adrenals. These data suggest that early in gestation, cortisol is not produced by the human fetal adrenal cortex in vivo (because it does not express 3 beta HSD), whereas androgen production occurs in the transitional and fetal zones (which express P450scc and P450c17). Later in gestation, the definitive zone may produce minearlocorticoids (because it expresses P450scc and 3 beta HSD, but lacks P450c17), and the transitional zone may produce glucocorticoids (it expresses P450scc, P450c17, and 3 beta HSD), whereas the fetal zone continues to produce androgens. Thus, late in gestation the functional zonation of the human fetal adrenal cortex may be similar to that of the adult, with the definitive zone being analogous to the nascent zona glomerulosa, the transitional zone analogous to the zona fasciculata, and the fetal zone analogous to the zona reticularis.

Interaction of insulin-like growth factor-II and estradiol directs steroidogenesis in the human fetal adrenal toward dehydroepiandrosterone sulfate production.

We examined the regulation of steroid production in fetal zone cells from midgestation (16-21 weeks) human fetal adrenal glands to elucidate the mechanism by which these cells secrete large quantities of dehydroepiandrosterone sulfate (DHAS) and little cortisol in response to ACTH. Our underlying hypothesis is that estrogen and insulin-like to ACTH. Our underlying hypothesis is that estrogen and insulin-like growth factor-II (IGF-II) modulate the steroidogenic response of fetal zone cells to ACTH, driving steroid production toward DHAS rather than cortisol. We also hypothesize that the effects of IGF-II and estrogen on steroidogenesis are achieved by modulating the expression of key enzymes in the steroidogenic pathway. Basal cortisol secretion by cultured fetal zone cells was below the limit of assay sensitivity (< 0.54 pmol/10(5) cells.24 h), whereas basal DHAS secretion was 210.8 +/- 41.0 pmol/10(5) cells.24 h (mean +/- SE). ACTH-(1-24) increased the secretion of cortisol to 228.96 +/- 6.75 pmol/10(5) cells.24 h and that of DHAS to 2039.8 +/- 121.7 pmol/10(5) cells.24 h. Neither IGF-II nor estradiol (E2) affected basal (no added ACTH) steroid secretion by fetal zone cells. IGF-II increased ACTH-stimulated cortisol and DHAS secretion by fetal zone cells in a dose-dependent fashion. In contrast, E2 at high concentrations (1-10 mumol/L) decreased ACTH-stimulated cortisol production to basal levels, but increased ACTH-stimulated DHAS production 1.5- to 2-fold. Combinations of IGF-II (100 ng/mL) and E2 (1 mumol/L) increased ACTH-stimulated cortisol and DHAS secretion by 1.5- to 2-fold compared with control values. However, compared with cultures exposed to IGF-II alone, inclusion of E2 decreased ACTH-stimulated cortisol secretion by about 60% and increased ACTH-stimulated DHAS secretion by about 50%. IGF-II increased the abundance of ACTH-stimulated mRNAs encoding cholesterol side-chain cleavage cytochrome P450 (P450scc), 17 alpha hydroxylase/17,20 lyase P450 (P450c17), and 3 beta-hydroxysteroid dehydrogenase (3 beta HSD). In addition, IGF-II increased the abundance of mRNA encoding P450c17 under basal conditions, but did not affect the basal expression of P450scc or 3 beta HSD. E2 had no effect on basal expression of these steroidogenic enzymes, but increased the abundance of ACTH-stimulated mRNA encoding P450scc and P450c17. The abundance of mRNA encoding 3 beta HSD was not affected by E2. The effect of IGF-II and E2 in combination on steroidogenic enzyme mRNA abundance was not different from that of IGF-II alone. These data indicate that IGF-II increases ACTH-stimulated steroid production in fetal zone cells by increasing the expression of key steroidogenic enzymes.(ABSTRACT TRUNCATED AT 400 WORDS)