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Adrenomedullin (ADM) is an evolutionarily conserved multifunctional peptide hormone that regulates implantation, embryo spacing, and placentation in humans and rodents. However, the potential roles of ADM in implantation and placentation in pigs, as a litter-bearing species, are not known. This study determined abundances of ADM in uterine luminal fluid, and the patterns of expression of ADM and its receptor components (CALCRL, RAMP2, RAMP3, and ACKR3) in uteri from cyclic and pregnant gilts, as well as conceptuses (embryonic/fetus and its extra-embryonic membranes) during the peri-implantation period of pregnancy. Total recoverable ADM was greater in the uterine fluid of pregnant compared with cyclic gilts between Days 10 and 16 post-estrus and was from uterine luminal epithelial (LE) and conceptus trophectoderm (Tr) cells. Uterine expression of CALCRL, RAMP2, and ACKR3 were affected by day (P < 0.05), pregnant status (P < 0.01) and/or day x status (P < 0.05). Within porcine conceptuses, the expression of CALCRL, RAMP2, and ACKR3 increased between Days 10 and 16 of pregnancy. Using an established porcine trophectoderm (pTr1) cell line, it was determined that 10-7 M ADM stimulated proliferation of pTr1 cells (P < 0.05) at 48 h, and increased phosphorylated mechanistic target of rapamycin (p-MTOR) and 4E binding protein 1 (p-4EBP1) by 6.1- and 4.9-fold (P < 0.0001), respectively. These novel results indicate a significant role for ADM in uterine receptivity for implantation and conceptus growth and development in pigs. They also provide a framework for future studies of ADM signaling to affect proliferation and migration of Tr cells, spacing of blastocysts, implantation, and placentation in pigs., (© The Author(s) 2021. Published by Oxford University Press on behalf of Society for the Study of Reproduction. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Preeclampsia (PE) is a placental disorder caused by endothelial dysfunction via trophoblast inadequate invasion activity. Adrenomedullin (ADM) and ADM2 are multifunctional peptides that can support vascular activity and placental growth. However, correlation between ADMs and trophoblast functions is currently unclear. The objective of this study was to analyze changes in expression of ADMs in placenta and HTR-8/SVneo trophoblast cells under hypoxia and their effects on invasion activity of trophoblast cells and expression of HLA-G. In placental tissues of PE, expression levels of ADM and HLA-G were significantly increased (P < 0.05) whereas expression of ADM2 was decreased compared to that in normal term placenta. Under hypoxia, expression levels of ADM, ADM2, and HLA-G and invasion ability of trophoblast cells were increased in hypoxia-inducible factor-1 (HIF-1α)- dependent manner (P < 0.05). Treatment with ADMs agonists reduced HIF-1α activity whereas enhanced invasion ability under hypoxia. However, they were not changed after cotreatment of ADMs and HIF-1α inhibitor, YC-1, although expression levels of invasion-related genes MMP2, MMP9, and Rac1 were altered (P < 0.05). ADMs also increased HLA-G expression under normoxia whereasADM2 or cotreatment of ADMs under hypoxia attenuated HLA-G expression (P < 0.05). Our findings demonstrate that altered expression of ADMs plays a critical role in placental physiology, especially in trophoblast invasion and immune-modulation under hypoxia., (© The Author(s) 2020. Published by Oxford University Press on behalf of Society for the Study of Reproduction.)

In glioblastomas, apoptosis inhibitor proteins (IAPs) are involved in apoptotic and nonapoptotic processes. We previously showed that IAP inhibition induced a loss of stemness and glioblastoma stem cells differentiation by activating nuclear factor-κB under normoxic conditions. Hypoxia has been shown to modulate drug efficacy. Here, we investigated how IAPs participate in glioblastoma stem-like cell maintenance and fate under hypoxia. We showed that in a hypoxic environment, IAPs inhibition by GDC-0152, a small-molecule IAPs inhibitor, triggered stem-like cell apoptosis and decreased proliferation in four human glioblastoma cell lines. We set up a three-dimensional glioblastoma spheroid model in which time-of-flight secondary ion mass spectrometry analyses revealed a decrease in oxygen levels between the periphery and core. We observed low proliferative and apoptotic cells located close to the hypoxic core of the spheres and glial fibrillary acidic protein + cells at their periphery. These oxygen-dependent GDC-0152 antitumoral effects have been confirmed on human glioblastoma explants. Notably, serine-threonine kinase activation analysis revealed that under hypoxic conditions, IAP inhibition activated ataxia telangiectasia and Rad3-related protein signaling. Our findings provide new insights into the dual mechanism of action of IAP inhibitors that depends on oxygen level and are relevant to their therapeutic application in tumors. Stem Cells 2019;37:731-742., (©AlphaMed Press 2019.)

Context: Impaired maternal lipid metabolism in gestational diabetes mellitus (GDM) has detrimental effects on maternal health and fetal growth. We previously reported the excessive expression of adrenomedullin (ADM) and its receptors in GDM adipose tissues compared with normal glucose-tolerant pregnancies. In the present study, we determined the mechanisms underlying enhanced expression of ADM and its receptors., Design: Omental adipose tissue (OAT) samples were collected from women during cesarian section of term pregnancy with nonoverweight (NOW; n = 9), overweight (OW; n = 8), obese (OBS; n = 10), and GDM (n = 10) status., Results: The expression of ADM and its receptors was greater in OATs from GDM than from women who were NOW, OW, and OBS. The expression of adipokines, leptin, and resistin were significantly increased, but adiponectin was decreased in OATs from patients with GDM compared with those without GDM. Macrophage infiltration and TNF-α expression were greater in OAT from pregnant women with GDM than in pregnant women without GDM. Furthermore, TNF-α dose dependently increased mRNA for ADM and its receptor components calcitonin receptor-like receptor and receptor activity-modifying proteins 2 and 3 in OAT explants from women who were NOW. Human adipocytes treated with ADM significantly increased glycerol release in culture medium, and the increases of glycerol in culture medium of OAT from women with GDM were attenuated by ADM antagonists, ADM22-52., Conclusions: Increased macrophage infiltration and TNF-α expression in adipose tissue from GDM, but not from OBS, tissues stimulate ADM and its receptor overexpression, leading to enhanced lipolysis and hyperlipidemia. This might contribute to fetal macrosomia and adiposity in diabetic pregnancies.

Neointimal hyperplasia is the primary lesion underlying atherosclerosis and restenosis after coronary intervention. We previously described the essential angiogenic function of the adrenomedullin (AM)-receptor activity-modifying protein (RAMP) 2 system. In the present study, we assessed the vasoprotective actions of the endogenous AM-RAMP2 system using a wire-induced vascular injury model. We found that neointima formation and vascular smooth muscle cell proliferation were enhanced in RAMP2+/- male mice. The injured vessels from RAMP2+/- mice showed greater macrophage infiltration, inflammatory cytokine expression, and oxidative stress than vessels from wild-type mice and less re-endothelialization. After endothelial cell-specific RAMP2 deletion in drug-inducible endothelial cell-specific RAMP2-/- (DI-E-RAMP2-/-) male mice, we observed markedly greater neointima formation than in control mice. In addition, neointima formation after vessel injury was enhanced in mice receiving bone marrow transplants from RAMP2+/- or DI-E-RAMP2-/- mice, indicating that bone marrow-derived cells contributed to the enhanced neointima formation. Finally, we found that the AM-RAMP2 system augmented proliferation and migration of endothelial progenitor cells. These results demonstrate that the AM-RAMP2 system exerts crucial vasoprotective effects after vascular injury and could be a therapeutic target for the treatment of vascular diseases., (Copyright © 2017 Endocrine Society.)

The practical application of engineered nanomaterials or nanoparticles like polyamidoamine (PAMAM) dendrimers has been promoted in medical devices or industrial uses. The safety of PAMAM dendrimers needs to be assessed when used as a drug carrier to treat brain disease. However, the effects of PAMAM on the human nervous system remain unknown. In this study, human neural progenitor cells cultured as a 3D neurosphere model were used to study the effects of PAMAM dendrimers on the nervous system. Neurospheres were exposed to different G4-PAMAM dendrimers for 72 h at concentrations of 0.3, 1, 3, and 10 μg/ml. The biodistribution was investigated using fluorescence-labeled PAMAM dendrimers, and gene expression was evaluated using microarray analysis followed by pathway and network analysis. Results showed that PAMAM dendrimer nanoparticles can penetrate into neurospheres via superficial cells on them. PAMAM-NH2 but not PAMAM-SC can inhibit neurosphere growth. A reduced number of MAP2-positive cells in flare regions were inhibited after 10 days of differentiation, indicating an inhibitory effect of PAMAM-NH2 on cell proliferation and neuronal migration. A microarray assay showed 32 dendrimer toxicity-related genes, with network analysis showing 3 independent networks of the selected gene targets. Inducible immediate early gene early growth response gene 1 (Egr1), insulin-like growth factor-binding protein 3 (IGFBP3), tissue factor pathway inhibitor (TFPI2), and adrenomedullin (ADM) were the key genes in each network, and the expression of these genes was significantly down regulated. These findings suggest that exposure of neurospheres to PAMAM-NH2 dendrimers affects cell proliferation and migration through pathways regulated by Egr1, IGFBP3, TFPI2, and ADM., (© The Author 2016. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

Adrenomedullin2 (ADM2) is reported to facilitate embryo implantation and placental development. Therefore, the current study was undertaken to identify if ADM2 has a functional role in ovary to facilitate its reproductive actions. This study shows that the expression of ADM2 is differentially regulated in rat estrous cycle and that ADM2 increases the synthesis and secretion of 17beta-estradiol accompanied with an increase in the expression of steroidogenic factor 1 (Sf1), estrogen receptor Esr1, and enzymes involved in steroidogenesis in equine chorionic gonadotropin (eCG)-treated rat ovaries. In addition, inhibition of endogenous ADM2 function in eCG-treated immature rats caused impaired ovulation. Furthermore, the mRNA expression of Adm2 and receptor activity modifying protein 3 is higher in the ovary on Day 18 compared to nonpregnant and pregnant rats on Day 22. ADM2-like immunoreactivity is localized in granulosa cells, blood vessels, oocytes, cumulous oophorus, and corpus luteum of pregnant ovaries, suggesting a potential role for ADM2 in the ovary. This is supported by the presence of ADM2-like immunoreactivity in the corpus luteum during pregnancy and a decline in aromatase immunoreactivity in corpus luteum on Day 9 of gestation in rats infused with ADM2 antagonist during implantation and decidualization phase. Taken together, this study suggests a potential involvement of ADM2 in the rat ovary in regulating synthesis of estradiol to support ovulation and facilitate efficient implantation and placental development for a successful pregnancy., (© 2015 by the Society for the Study of Reproduction, Inc.)

Adrenomedullin (ADM) may regulate seminal vesicle fluid secretion, and this may affect sperm quality. In this study, we investigated the effect of ADM on chloride secretion in the mouse seminal vesicle. The presence of ADM in mouse seminal vesicle was confirmed using immunostaining, and the molecular species was determined using gel filtration chromatography coupled with enzyme-linked assay for ADM. The effects of ADM on chloride secretion were studied by short-circuit current technique in a whole-mount preparation of mouse seminal vesicle in an Ussing chamber. The effects of specific ADM and calcitonin gene-related peptide (CGRP) receptor antagonists were investigated. Whether the ADM effect depended on the cAMP- and/or calcium-activated chloride channel was also studied using specific chloride channel blockers. The results showed that ADM was present in seminal vesicle epithelial cells. The major molecular species was precursor in the mouse seminal vesicle. ADM increased short-circuit current through the calcium-activated chloride channel in mouse seminal vesicle, and CGRP receptor was involved. We conclude that ADM may regulate chloride and fluid secretion from the seminal vesicle, which may affect the composition of the seminal plasma bathing the sperm and, hence, fertility., (© 2014 by the Society for the Study of Reproduction, Inc.)

Context: The risk of developing cancer is regulated by genetic variants, including polymorphisms. Characterizing such variants may help in developing protocols for personalized medicine., Objective: Adrenomedullin is a regulatory peptide involved in cancer promotion and progression. Carriers of a single nucleotide polymorphism (SNP) in the proximity of the adrenomedullin gene have lower levels of circulating peptide. The aim of the present work was to investigate whether carriers of this SNP (rs4910118) are protected against cancer., Design: This was a retrospective study. DNA samples were obtained from the Carlos III DNA National Bank (University of Salamanca, Salamanca, Spain)., Setting: Samples represent a variety of donors and patients from Spain., Patients or Other Participants: DNA from patients with breast cancer (n = 238), patients with lung cancer (n = 348), patients with cardiac insufficiency (n = 474), and healthy donors of advanced age (n = 500) was used., Interventions: All samples were genotyped using double-mismatch PCR, and confirmation was achieved by direct sequencing., Main Outcome Measures: The minor allele frequency was calculated in all groups. The Pearson χ(2) was used to compare SNP frequencies., Results: Of 1560 samples, 14 had the minor allele, with a minor allele frequency in healthy donors of 0.90%. Patients with cancer had a statistically significantly lower frequency than healthy donors (odds ratio = 0.216, 95% confidence interval = 0.048-0.967, P = .028)., Conclusions: Carriers of the minor allele have a 4.6-fold lower risk of developing cancer than homozygotes for the major allele. Knowledge of the rs4910118 genotype may be useful for stratifying patients in clinical trials and for designing prevention strategies.

Context: Tubal ectopic pregnancy (tEP) is currently the leading cause of pregnancy-related deaths during the first trimester. Our current knowledge on the molecular pathogenesis is limited., Objective: The objective of the study was to find out the possible role of adrenomedullin (ADM) in the pathogenesis of tEP., Design: This was an experimental in vitro study on oviductal tissue., Setting: The study was conducted at a university teaching hospital., Patients and Interventions: Patients included those having oviducts removed surgically during salpingectomy for tEP or hysterectomy for benign gynecological conditions. Oviductal tissues were incubated in hormonal condition mimicking early pregnancy before used for in vitro experiments., Main Outcome Measures: Plasma ADM concentration, oviductal expression of ADM and its receptors, ciliary beat frequency, smooth muscle contraction were measured., Results: The ciliary beat frequency and frequency of muscle contraction were lower in the oviducts from patients with tEP than those from simulated normal pregnancy. The plasma and oviductal tissue ADM levels were also lower. The decreases in ciliary beat and frequency of contraction were restored to normal after ADM treatment., Conclusions: The results suggest that the lower ADM level in the oviducts of tEP may lead to the decrease in ciliary beating and muscle contraction, with the result that the embryo is retained and implanted in the oviduct. Our findings explain for the first time the etiology of tubal pregnancy on the basis of an impairment of the transport of the fertilized ovum resulting from an ADM deficiency and raise the possibility of using the plasma ADM level as a predictor for tubal ectopic pregnancy.

Objective: Adrenomedullin (ADM) plays an important role in inflammation and is a marker of future cardiovascular events. We studied common single nucleotide polymorphisms (SNPs) in the gene encoding ADM and their relationship with the plasma levels of ADM and other inflammatory markers., Design and Methods: Plasma ADM, interleukin 6 (IL6), fibrinogen, and C-reactive protein (CRP) were measured in 476 subjects from the population-based Hong Kong Cardiovascular Risk Factor Prevalence Study-2. Four tag SNPs in ADM were genotyped., Results: Plasma ADM level increased with decreasing plasma IL6 level (β=-0.116, P=0.014). Plasma ADM level was not related to plasma levels of CRP and fibrinogen, and other clinical characteristics, except age (P=0.049). The four SNPs, rs3814700, rs11042725, rs34354539, and rs4910118, had minor allele frequencies of 31.1, 28.7, 33.8, and 23.4% respectively. Carriers of the minor allele of rs4910118 had a mean plasma ADM level that was 10.5% (95% confidential interval: 2.5-17.8%) lower than the non-carriers (β=-0.115, P=0.011). Haplotype analysis revealed a similar significant association with plasma ADM (P=0.040). In multivariate analysis, the presence of the minor allele of rs4910118, but not plasma IL6, was independently associated with plasma ADM (P=0.010)., Conclusion: Plasma ADM correlates with plasma IL6 level, consistent with its role in inflammation. It is related to an SNP common in Chinese, independent of other covariates. ADM genotype should be included in future studies of cardiovascular risk prediction.

After menstruation, the endometrium has a remarkable capacity for repair, but the factors involved remain undefined. We hypothesize adrenomedullin (AM) plays a role in this process. Premenstrually progesterone levels decline, stimulating prostaglandin (PG) synthesis, vasoconstriction, and hypoxia. This study aimed to determine 1) AM expression throughout the menstrual (M) cycle and 2) its regulation by PG and hypoxia. Human endometrial biopsies (n = 51) were collected with ethical approval and consent. AM mRNA expression was examined by quantitative RT-PCR and was found to be selectively elevated in endometrium from the menstrual (M) phase (P < 0.001). AM immunohistochemical staining was maximal in M and proliferative (P) endometrium. Culture of secretory, but not P, explants with 100 nm PGF(2α) or hypoxia (0.5% O2) increased AM mRNA (P < 0.05). P explants were induced to increase AM expression using in vitro progesterone withdrawal but required the presence of hypoxia (P < 0.05). Short hairpin sequences against hypoxia-inducible factor-1α (HIF-1α) inhibited AM hypoxic up-regulation but did not alter PGF(2α)-induced expression. The AM receptor was immunolocalized to endothelial cells in both lymphatic and blood vessels. Conditioned medium from PGF(2α)-treated cells increased endothelial cell proliferation and branching (P < 0.05). This was abolished by AM receptor antagonists. In conclusion, AM is elevated at the time of endometrial repair and induces both angiogenesis and lymphangiogenesis by stimulating endothelial cell proliferation and tube formation. In the human endometrium, AM expression is up-regulated by two mechanisms: a HIF-1α-mediated hypoxic induction and a HIF-1α-independent PGF(2α) pathway. These physiological mechanisms may provide novel therapeutic targets for disorders such as heavy menstrual bleeding.

Adrenomedullin (AM) is a regulatory peptide, coded by the adm gene, which is involved in numerous physiological processes, including pain sensitivity. Previous studies have shown that intrathecal injection of AM induced hyperalgesia in the rat. Here, we explore pain sensitivity in a mouse conditional knockout for adm in neurons of the central nervous system, including the spinal cord and dorsal root ganglia. Double immunofluorescence in wild-type (WT) animals shows that AM immunoreactivity is found in calcitonin gene-related peptide-positive neurons of the dorsal root ganglia but not in neurons that bind isolectin B4. Mice lacking adm had modified expression of canonical sensorial neuropeptides, having significantly more calcitonin gene-related peptide and less substance P and enkephalin than their WT littermates. Furthermore, the spinal cord of adm knockout mice expressed higher levels of the AM receptor components, suggesting a compensation attempt to deal with the lack of afferent AM signaling. Behavioral nociceptive tests also found differences between genotypes. In the tail-flick test, which measures mostly spinal reflexes, the adm-null animals had a longer latency than their WT counterparts. On the other hand, in the hotplate test, which requires encephalic processing, mice lacking adm had shorter latencies than normal littermates. These results suggest that AM acts as a nociceptive modulator in spinal reflexes, whereas it may have an analgesic function at higher cognitive levels. This study confirms the important role of AM in pain sensitivity processing but unveils a more complex scenario than previously surmised.

Context: Adrenomedullin (ADM) has been found expressed in the mouse oviduct and might play a role in reproduction., Objective: The objective of the study was to demonstrate the expression of ADM in the human oviduct, investigate its regulation by steroidal hormones and spermatozoa contact, and study its effect on ciliary beat frequency (CBF) in the human oviduct. DESIGN, SETTING, PATIENTS, AND INTERVENTIONS: Oviducts from women undergoing hysterectomy for benign diseases in a university hospital were collected. The oviducts were treated with estradiol and/or progesterone to simulate different phases of the ovarian cycle. ADM expression was studied at the peptide and mRNA levels by immunohistochemistry and RT-PCR, respectively. CBF was measured after treatment with graded concentrations of ADM and its antagonists. Cells from OE-E6/E7, an immortalized oviductal cell line, as well as oviductal tissue were cocultured with and without direct contact with capacitated human spermatozoa to compare oviductal cell ADM expression levels. CBF was also analyzed in oviductal tissue after spermatozoa-oviduct coincubation., Results: ADM expression was the highest in the isthmic region and in a hormonal environment simulating the early luteal phase. CBF was increased by ADM in a dose-dependent manner, which was blocked by ADM and calcitonin-gene-related peptide receptor antagonists. Direct contact with spermatozoa in coculture resulted in higher ADM expression in OE-E6/E7 cell line and oviductal tissue and higher CBF in oviductal epithelium., Conclusions: ADM expression in the human oviduct is hormone dependent and is up-regulated by sperm contact. ADM stimulates ciliary motility of the human oviduct.

Estradiol (E(2)) rapidly and strongly induces vascular endothelial growth factor (VEGF) transcription in uterine endometrial epithelial cells in vivo. We have shown that this is mediated by both the estrogen receptor-alpha and hypoxia-inducible factor (HIF)-1alpha. By contrast, E(2) induces little or no VEGF expression in cultured breast or endometrial cancer cells, which lack HIF-1alpha due to the abnormally high concentration of oxygen ( approximately 20%) to which they are exposed. To test the hypothesis that restoring HIF-1alpha in cultured cells would restore the ability of E(2) to induce VEGF expression, we treated human endometrial cancer cells (ECC-1) with cobalt chloride (CoCl(2);100 microm), which prevents oxygen-induced HIF-1alpha degradation. HIF-1alpha was absent in untreated ECC-1 cells but detectable by 4 h after treatment with CoCl(2) alone, as was a significant increase in VEGF mRNA. E(2) plus CoCl(2) induced detectable HIF-1alpha expression at 2 h and an even higher level than that induced by CoCl(2) alone at 4 h; this HIF-1alpha was localized in the nuclei. This was accompanied by increasing VEGF expression, with the increase at 4 h severalfold higher than that induced by CoCl(2) alone and was concurrent with recruitment of both HIF-1alpha and estrogen receptor-alpha to the VEGF promoter. These results confirm that HIF-1alpha plays an essential role in E(2)-induced expression of VEGF. Through the induction of increased microvascular permeability and the consequent exudation of plasma growth factors, VEGF in turn may play an essential role in cancer cell proliferation in vivo.

Adrenomedullin (AM) is a multifunctional peptide vasodilator that signals through a G-protein-coupled receptor when the receptor, called calcitonin receptor-like receptor (CL), is associated with a receptor activity-modifying protein 2 (RAMP2). We demonstrated previously that haploinsufficieny for each of these genes led to reduced maternal fertility, and that even a modest genetic reduction of AM peptide caused maternal defects in implantation, placentation, and fetal growth. Here, we further demonstrate that Adm(+/-) female mice displayed reduced pregnancy success rates that were not caused by defects in folliculogenesis, ovulation, or fertilization. The poor fertility of Adm(+/-) female mice could not be rescued by transfer of wild-type blastocysts, which suggested an underlying defect in uterine receptivity. In fact, we found that Adm, Calcrl, and Ramp2 gene expressions are tightly and spatiotemporally regulated in the luminal epithelial cells of the uterus during the estrus cycle and the peri-implantation period. RAMP3, which also generates an AM receptor when associated with CL, had a diametrically opposite expression pattern than that of Adm, Calcrl, and Ramp2 and was most robustly induced in the stroma of the uterus. Finally, we discovered that Adm(+/-) female mice have a substantially reduced number of pinopodes on the uterine luminal epithelial surface, which is indicative and possibly causative of the poor uterine receptivity. Taken together, our studies identify a new class of pharmacologically tractable proteins that are involved in establishing uterine receptivity through the regulation of pinopode formation.

The present study demonstrates the expression of adrenomedullin (ADM) in the reproductive system of the female rat and its effect on the secretion of estradiol and progesterone. Ovarian ADM and Adm mRNA levels were decreased at estrus, whereas oviductal Adm mRNA levels were low at proestrus. Both tissues were shown to coexpress mRNAs encoding the calcitonin receptor-like receptor and receptor activity-modifying protein 1 (Ramp1), Ramp2, and Ramp3. Gel filtration chromatography of ovarian extracts showed two peaks, with the predominant one eluting at the position of authentic rat ADM (1-50) at estrus and at the position of ADM precursor at diestrus. Positive ADM immunostaining was localized in the granulosa and theca cells of the follicle and corpora lutea of the ovary. Adrenomedullin inhibited FSH-induced estradiol secretion in 2-day-old follicles and also suppressed eCG-stimulated progesterone release in corpora lutea. The inhibitory effect of ADM on the follicles and the corpora lutea was abolished by calcitonin gene-related peptide (8-37) and ADM (22-52), respectively. The presence of ADM and the gene expression of ADM and its receptor components in the female reproductive system suggest a paracrine effect of ADM on ovarian steroidogenesis.

Adrenomedullin (AM) and proadrenomedullin N-terminal 20 peptide (PAMP) are secretory hormones, but it is not unusual to find them in intracellular compartments. Using yeast-2 hybrid technology, we found interactions between AM and several microtubule-associated proteins (MAPs), and between PAMP and tubulin. Expression of fluorescent-tagged AM and PAMP as well as immunofluorescence for the native peptides showed a complete decoration of the microtubules and colocalization with other MAPs. PAMP, but not AM, bound to tubulin in vitro and destabilized tubulin polymerization. Down-regulation of the gene coding for both AM and PAMP through small interfering RNA technology resulted in morphological changes, microtubule stabilization, increase in posttranslational modifications of tubulin such as acetylation and detyrosination, reduction in cell motility, and partial arrest at the G2 phase of the cell cycle, when compared with cells transfected with the same vector carrying a scrambled sequence. These results show that PAMP is a novel MAP, whereas AM may be exerting more subtle effects in regulating cytoskeleton function.

The present study demonstrates the expression of adrenomedullin (ADM) in the rat Sertoli cells and its effect on inhibin production. The regulation of ADM by FSH and its interaction with endothelin 1 (EDN1) in the rat Sertoli cells have also been established. Primary culture of Sertoli cells secreted 414+/-27 pg immunoreactive ADM per 10(6) cells in 24 h and expressed Adm mRNA. In addition, the Sertoli cell was shown to co-express mRNAs encoding for the calcitonin receptor-like receptor (CALCRL) and receptor activity-modifying proteins (RAMPs) 1-3. These may account for the specific binding of ADM to the Sertoli cells. Administration of ADM to Sertoli cells resulted in an enhancement of basal and FSH-stimulated inhibin B production. On the other hand, the production of ADM and the mRNA levels of Calcrl and Ramp2 in the Sertoli cells were suppressed by FSH. The results suggest that ADM, via its control in the secretion of inhibin B, may play a role in regulating spermatogenesis as well as the hypothalamus-pituitary-gonad feedback system. In addition, like in the Leydig cell, ADM and EDN1 were found to regulate the production of each other in opposite directions in the Sertoli cells, suggesting the presence of yet another local regulatory mechanism in the rat testis that may be important in modulating testicular functions regulated by gonadotropins.

Based on the finding of gene expression of adrenomedullin (Adm) and its receptor components in the rat testis, a paracrine effect of ADM on testicular steroidogenesis has been suggested by our group. The present study demonstrates the gene expression of Adm and the effect of ADM on testosterone production in the Leydig cell. The regulation of ADM by hCG and its interaction with endothelin 1 (EDN1) in the rat Leydig cells are also observed. Primary culture of Leydig cells produced Adm mRNA and secreted 275+/-19 pg immunoreactive ADM per 10(6) cells in 24 h. In addition, the Leydig cell was shown to coexpress mRNAs encoding for the calcitonin receptor-like receptor (CALCRL) and receptor activity-modifying protein (RAMP1, RAMP2, and RAMP3). These may account for the specific binding of ADM to the Leydig cells. Administration of ADM to Leydig cells resulted in an inhibition of hCG- and EDN1-stimulated testosterone production. Correlated with this, ADM reduced EDN1 production, whereas its production was increased by EDN1. Furthermore, the production of ADM and the mRNA levels of Calcrl and Ramp2 were suppressed by hCG. Our results suggest that ADM has an autocrine effect on Leydig cell steroidogenesis, possibly by interacting with EDN1 and under the control of gonadotropin. We propose that there is an ADM/EDN1 local regulatory mechanism that may be important in modulating the control of testicular functions by gonadotropins.

Adrenomedullin (ADM) has been shown to be present in the human and rat male reproductive systems. This study demonstrates the expression of ADM in the rat testis and its effect on the secretion of testosterone. Whole testicular extracts had 5.43 +/- 0.42 fmol of immunoreactive ADM per milligram of protein and 84 +/- 8 fg of ADM mRNA per picogram of Actb (beta-actin) mRNA. Immunocytochemical studies showed positive ADM immunostaining in the Leydig cells and in the Sertoli cells. Gel filtration chromatography of testicular extracts showed two peaks, with the predominant one eluting at the position of the ADM precursor. Furthermore, the testis was shown to coexpress mRNAs encoding the calcitonin receptor-like receptor and receptor activity modifying protein 1 (Ramp1), Ramp2, and Ramp3. These account for the specific binding of ADM to the testis, which was partially inhibited by human ADM (22-52) and by human calcitonin gene-related peptide (8-37), the ADM and calcitonin gene-related peptide receptor antagonists, respectively. Administration of ADM to testicular blocks in vitro resulted in a dose-dependent inhibition of hCG-stimulated release of testosterone, which was abolished by the administration of ADM (22-52). Our results suggest a paracrine effect of ADM on testicular steroidogenesis.

Recent progress has been made in the identification of the osteoblastic cellular niche for hematopoietic stem cells (HSCs) within the bone marrow (BM). Attempts to identify the soluble factors that regulate HSC self-renewal have been less successful. We have demonstrated that primary human brain endothelial cells (HUBECs) support the ex vivo amplification of primitive human BM and cord blood cells capable of repopulating non-obese diabetic/severe combined immunodeficient repopulating (SCID) mice (SCID repopulating cells [SRCs]). In this study, we sought to characterize the soluble hematopoietic activity produced by HUBECs and to identify the growth factors secreted by HUBECs that contribute to this HSC-supportive effect. Extended noncontact HUBEC cultures supported an eight-fold increase in SRCs when combined with thrombopoietin, stem cell factor, and Flt-3 ligand compared with input CD34(+) cells or cytokines alone. Gene expression analysis of HUBEC biological replicates identified 65 differentially expressed, nonredundant transcripts without annotated hematopoietic activity. Gene ontology studies of the HUBEC transcriptome revealed a high concentration of genes encoding extracellular proteins with cell-cell signaling function. Functional analyses demonstrated that adrenomedullin, a vasodilatory hormone, synergized with stem cell factor and Flt-3 ligand to induce the proliferation of primitive human CD34(+)CD38(-)lin(-) cells and promoted the expansion of CD34(+) progenitors in culture. These data demonstrate the potential of primary HUBECs as a reservoir for the discovery of novel secreted proteins that regulate human hematopoiesis.