Calcitonin gene-related peptide protects from soluble fms-like tyrosine kinase-1-induced vascular dysfunction in a preeclampsia mouse model.

Introduction: Preeclampsia (PE) is a hypertensive disorder during pregnancy associated with elevated levels of soluble FMS-like tyrosine kinase (sFLT-1) and increased vascular sensitivity to angiotensin II (ATII). Calcitonin gene-related peptide (CALCA) is a potent vasodilator that inhibits the ATII-induced increase in blood pressure and protects against ATII-induced increases in oxidative stress through a mitochondrial-dependent pathway in male mice. In rodent pregnancy, CALCA facilitates pregnancy-induced vascular adaptation. Most of the vascular effects of CALCA are mediated by vascular smooth muscle cells (VSMCs). We recently reported that CALCA treatment inhibits sFLT-1-induced decreases in cAMP synthesis in omental artery smooth muscle cells (OASMCs) isolated from pregnant women and has relaxant effects in omental arteries (OAs) isolated from pregnant women with preeclamptic (PE) pregnancies. The current study was designed to assess the effects of sFLT-1 on mitochondrial bioenergetics in OASMCs isolated from pregnant women in the presence or absence of CALCA and assess the development of vascular dysfunction in sFLT-1 using a mouse model of PE pregnancy. Methods: OASMCs were isolated from pregnant women to assess the effects of sFLT-1 on mitochondrial bioenergetics and oxidative stress using the Seahorse assay and quantitative PCR. Pregnant mice overexpressing sFLT-1 via adenoviral delivery were used to assess the effects of CALCA infusion on the sFLT-1-induced increase in blood pressure, ATII hypersensitivity, fetal growth restriction, and the elevated albumin-creatinine ratio. Systemic blood pressure was recorded in conscious, freely moving mice using implantable radio telemetry devices. Results: CALCA inhibited the following sFLT-1-induced effects: 1) increased oxidative stress and the decreased oxygen consumption rate (OCR) in response to maximal respiration and ATP synthesis; 2) increases in the expression of mitochondrial enzyme complexes in OASMCs; 3) increased mitochondrial fragmentation in OASMCs; 4) decreased expression of mitophagy-associated PINK1 and DRAM1 mRNA expression in OASMCs; and 5) increased blood pressure, ATII hypersensitivity, fetal growth restriction, and the albumin-creatinine ratio in sFLT-1-overexpressing pregnant mice. Conclusion: CALCA inhibits sFLT-1-induced alterations in mitochondrial bioenergetics in vascular smooth muscle cells and development of maternal vascular dysfunction in a mouse model of PE.

Study the prevalence of rifampicin resistance among pulmonary tuberculosis patients by genexpert assay from a tertiary care hospital of North India.

Background: Rifampicin (RIF) resistance (RR) tuberculosis (TB) has posed a great challenge to TB control programs globally. Evidence of RIF-RR can help as a surrogate marker to find out multidrug-resistance cases. The study aimed to determine the prevalence of RIF-RR in pulmonary TB (PTB) patients over the 4 years at Dr. RPGMC, Tanda, from the year 2018 to 2021. Methods: This was a retrospective study conducted at Dr. RPGMC, Tanda at Kangra, where we checked (from January 2018 to December 2021) clinically suspected PTB patients, whose samples were sent to the laboratory for GeneXpert assay to identify Mycobacterium TB/RIF (MTB/RIF) testing. Results: Of the total 11,774 clinically suspected PTB specimens were collected, and identified by GeneXpert MTB/RIF assay, in which 2358 samples were MTB positive and 9416 were MTB negative. Among 2358 MTB-positive samples, 2240 (95%) samples were RIF sensitive, in which 1553 (65.9%) were males and 687 (29.1%) were females, 76 (3.2%) samples were RIF-RR, in which 51 (2.2%) were males and 25 (1%) were females, and 42 (1.8%) samples were RIF indeterminate, in which 25 (1%) were males and 17 (0.8%) were females. Conclusion: The rate of RIF-RR was found 3.2% of total samples which was more in males. The overall positivity rate was 20%, and the rate of positivity decreased from 32% to 14% over the 4 years in sputum samples. Hence, the GeneXpert assay was found to be very important tool to detect RIF-RR among suspected PTB patients.

Role of adrenomedullin2/ intermedin in pregnancy induced vascular and metabolic adaptation in mice.

Introduction: Adrenomedullin2 (AM2) shares its receptor with Calcitonin gene related peptide and adrenomedullin with overlapping but distinct biological functions. Goal of this study was to assess the specific role of Adrenomedullin2 (AM2) in pregnancy induced vascular and metabolic adaptation using AM2 knockout mice (AM2 -/-). Method : The AM2 -/- mice were successfully generated using Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Nuclease Cas nine system. Phenotype of pregnant AM2 -/- mice was assessed with respect to its fertility, blood pressure regulation, vascular health and metabolic adaptations and compared to the wild type littermates (AM2 +/+). Results : Current data shows that AM2 -/- females are fertile with no significant difference in number of pups/litter compared to the AM2 +/+. However, ablation of AM2 decreases the gestational length and the total number of pups born dead or that die after birth is greater in AM2 -/- mice compared to AM2 +/+ mice (p < 0.05). Further AM2 -/- mice exhibit elevated blood pressure and elevated vascular sensitivity for the contractile responses to angiotensin two and higher serum sFLT-1 trigylcerides levels compared to AM2 +/+(p < 0.05). In addition, AM2 -/- mice develop glucose intolerance with elevated serum levels of Insulin during pregnancy compared to the AM2 +/+mice. Discussion: Current data suggests a physiological role for AM2 in pregnancy induced vascular and metabolic adaptations in mice.

Calcitonin Gene Related Peptide, Adrenomedullin, and Adrenomedullin 2 Function in Uterine Artery During Human Pregnancy.

RATIONALE: Calcitonin gene-related peptide (CGRP) and its family members adrenomedullin (ADM) and adrenomedullin 2 (ADM2; also known as intermedin) support vascular adaptions in rat pregnancy. OBJECTIVE: This study aimed to assess the relaxation response of uterine artery (UA) for CGRP, ADM, and ADM2 in nonpregnant and pregnant women and identify the involved mechanisms. FINDINGS: (1) Segments of UA from nonpregnant women that were precontracted with U46619 (1µM) in vitro are insensitive to the hypotensive effects of CGRP, ADM, and ADM2; (2) CGRP, ADM, and ADM2 (0.1-100nM) dose dependently relax UA segments from pregnant women with efficacy for CGRP > ADM = ADM2; (3) the relaxation responses to CGRP, ADM, and ADM2 are differentially affected by the inhibitors of nitric oxide (NO) synthase (L-NAME), adenylyl cyclase (SQ22536), apamin, and charybdotoxin; (4) UA smooth muscle cells (UASMC) express mRNA for calcitonin receptor-like receptor (CRLR) and receptor activity modifying protein (RAMP)1 and RAMP2 but not RAMP3; (5) receptor heterodimer comprising CRLR/RAMP1 and CRLR/RAMP2 but not CRLR/RAMP3 is present in UA; (6) soluble fms-like tyrosine kinase (sFLT-1) and TNF-α treatment decrease the expression of RAMP1 mRNA (P < 0.05) in UASMC; and (7) sFLT-1 treatment impairs the association of CRLR with all 3 peptides while TNF-α inhibits the interaction of CGRP but not ADM or ADM2 with CRLR in UASMC (P < 0.05). CONCLUSIONS: Relaxation sensitivity of UA for CGRP, ADM, and ADM2 is increased during pregnancy via peptide-specific involvement of NO system and endothelium-derived hyperpolarizing factors; vascular disruptors such as sFLT-1 and TNFα adversely impact their receptor system in UASMC.

Soluble fms-like tyrosine kinase-1 and angiotensin2 target calcitonin gene-related peptide family peptides in maternal vascular smooth muscle cells in pregnancy†.

Calcitonin gene-related peptide (CALCB), adrenomedullin (ADM), and adrenomedullin2 (ADM2) are hypotensive peptides that belong to CALCB family of peptides. Goal of this study was to identify the effect of fms-like tyrosine kinase (sFLT-1) and angiotensin2 (Ang2) on the function of these peptides in OA smooth muscle cells (OASMC) and assess the sensitivity of OA for these peptides in preeclampsia (PE) and normotensive pregnancy. METHODS: Peptide function was assessed by Cyclic adenosine monophosphate (cAMP) assays and wire myograph; mRNA expression by Polymerase chain reaction (PCR) and protein-protein interaction by proximity ligation assay and co-immunoprecipitation. FINDINGS: All three peptides increased cAMP synthesis in the order of efficacy CALCB > ADM = ADM2 and vascular endothelial growth factor (VEGF) mRNA in OASMC (P < 0.05); sFLT-1 mediated decrease in cAMP synthesis (P < 0.05) is differentially rescued by all three CALCB family peptides in OASMC (P < 0.005); sFLT-1 decreased receptor activity-modifying protein (RAMP)1 and RAMP2 mRNA expression (P < 0.05); Ang2 decreased the expression of calcitonin-receptor-like receptor and RAMP1 mRNA and desensitized CALCB and ADM2 receptors in OASMC (P < 0.05); sFLT-1 increased RAMP1and Ang2 type 1 receptor (AT1R) interaction in OASMC which is inhibited in presence of all three peptides; and all three peptides relax OA in PE with enhanced ADM2 response (P < 0.05). CONCLUSION: sFLT-1 and Ang2 impair OASMC mediated functional responses of CALCB family peptides which can be inhibited by respective peptide treatment. The sensitivity of OA for CALCB, ADM, and ADM2-mediated relaxation is retained in PE.

Circulating Adrenomedullin Is Elevated in Gestational Diabetes and Its Role in Impaired Insulin Production by ß-Cells.

Context: Defective pancreatic ß-cell adaptation in pregnancy plays an important role in the pathophysiology of gestational diabetes mellitus (GDM), but the molecular basis remains unclear. Objectives of this study were to determine if circulating levels of adrenomedullin (ADM) in women with GDM are elevated and to assess the effects of ADM on insulin synthesis and secretion by human pancreatic ß-cells. Design: A stable gene product of ADM precursor, midregional pro-adrenomedullin (MR-proADM), was measured in plasma of pregnant women with normal glucose tolerance (NGT, n = 10) or GDM (n = 11). The ß-Lox5 cell line, derived from human pancreatic ß-cells, was transduced with homeodomain transcription factor pancreatic-duodenal homeobox (PDX) factor 1 (PDX1) encoding lentiviral vector and treated with different doses of ADM. mRNA for insulin, ADM, and its receptor components in ß-Lox5 cells and insulin in media were measured. Results: Plasma MR-proADM levels were significantly higher in GDM compared with patients with NGT. Pancreatic ß-Lox5 cells express mRNA for insulin, ADM, and its receptor components. PDX1 transduction and cell-cell contact synergistically promote ß-Lox5 cells insulin mRNA and secretion. Furthermore, ADM dose-dependently inhibited mRNA and secretion of insulin in ß-Lox5 cell aggregates. These inhibitory effects were blocked by ADM antagonist ADM22-52, cAMP-dependent protein kinase A inhibitor KT5720, and Erk inhibitor PD98059, but not by PI-3K the inhibitor wortmannin. Conclusions: Circulating ADM concentrations were elevated in pregnant women with GDM. ADM suppresses insulin synthesis and secretion by pancreatic ß-cells in vitro. Thus, increased circulating ADM may contribute to the defective adaptation of ß-cells in diabetic pregnancies, and blockade of ADM actions with its antagonists may improve ß-cell functions.

Adipose Tissue Inflammation and Adrenomedullin Overexpression Contribute to Lipid Dysregulation in Diabetic Pregnancies.

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.

Calcitonin Gene-Related Peptide Rescues Proximity Associations of Its Receptor Components, Calcitonin Receptor-Like Receptor and Receptor Activity-Modifying Protein 1, in Rat Uterine Artery Smooth Muscle Cells Exposed to Tumor Necrosis Factor Alpha.

Calcitonin gene-related peptide (CALCB), adrenomedullin (ADM), and ADM2/intermedin play critical roles in vascular adaptation during pregnancy through calcitonin receptor-like receptor (CALCRL) and receptor activity-modifying proteins (RAMPs). This study was designed to assess the predominant RAMP that associates with CALCRL to form a functional receptor in the rat uterine artery smooth muscle (RUASM). We also determined if these receptor component associations are decreased by tumor necrosis factor (TNF) alpha and if CALCB, ADM, or ADM2 can rescue CALCRL/RAMP associations. Using proximity ligation assay in RUASM cells, this study shows that CALCRL predominantly associates with RAMP1 forming a CALCB receptor, and minimally with RAMP2 and RAMP3 that confer specificity for ADM and ADM2. However, knockdown of RAMP1 mRNA increases the interaction between CALCRL and RAMP3 without affecting the association of CALCRL and RAMP2. Furthermore, CALCB, ADM, and ADM2 have no effects on the associations of CALCRL with any of the RAMPs in RUASM cells. Interestingly, CALCB reverses the TNFalpha-induced decreases in CALCRL/RAMP1 associations. Furthermore, CALCB increases ERK1/2 phosphorylation in a time-dependent manner in RUASM, and the protective effect of CALCB on TNFalpha-induced inhibition of CALCRL/RAMP1 associations was significantly blocked in presence of ERK inhibitor (PD98059). In conclusion, this study demonstrates that CALCRL predominantly associates with RAMP1 forming a CALCB-specific receptor complex in RUASM cells, which is dissociated by TNFalpha. Rescue of TNFalpha-induced dissociation of CALCRL/RAMP1 complex by CALCB in RUASM cells suggests a potential use of CALCB in developing therapeutic strategies for pregnancy-related complications that are vulnerable to abnormal levels of TNFalpha, such as fetal growth restriction and preeclampsia.

Adrenomedullin2 (ADM2)/Intermedin (IMD): A Potential Role in the Pathophysiology of Preeclampsia.

CONTEXT: It is not known whether decreases in trophoblast invasion promoting the peptide, adrenomedullin2 (ADM2) system is associated with preeclampsia (PreE). OBJECTIVE: The objective of the study was to assess the changes in ADM2 levels in plasma, placenta, and amniotic fluid (AF) and its receptor components in placenta from PreE pregnancy compared with the age-matched normal and study the effect of ADM2 on the synthesis of nitric oxide (NO), endothelial nitric oxide synthase (eNOS), and matrix-metalloproteinase (MMP)-2 and MMP-9 in trophoblast cells. RESULTS: PreE is associated with a decreased expression of ADM2 in plasma and placenta (P < .05); ADM2 interacts with a seven-transmembrane G protein-coupled receptor, calcitonin receptor-like receptor (CRLR) in HTR-8/SVneo cells; placental expression of ADM2/CRLR complex is lower in PreE; mRNA for CRLR and receptor activity-modifying protein-3 are lower, whereas receptor activity-modifying protein-2 is higher in the PreE placenta (P < .05); ADM2 levels in the second trimester are lower in the AF from pregnant women who develop PreE later in gestation (P < .05); ADM2 is localized to the epithelium of the amnion and the ectoderm and mesoderm of the chorion in term fetal membranes; ADM2 increases NO production, eNOS, and MMP2/9-immunoreactivity, whereas ADM2 knockdown inhibits the expression of eNOS and MMP2/9 mRNA and S-nitrosylation in HTR-8/SVneo cells; and ADM2-induced increases in MMP2/9 activity is inhibited by L-nitro-arginine methyl ester in HTR-8SV/neo cells. CONCLUSION: Decreases in the ADM2 system in PreE at term, in AF from pregnant women during the second trimester who develop PreE later in gestation, and ADM2-induced increases in the NO and MMP-2/9 levels in trophoblast cells suggest a potential role for ADM2 via the NO-MMP system in the pathophysiology of PreE.

Impaired Vasodilatory Responses of Omental Arteries to CGRP Family Peptides in Pregnancies Complicated by Fetal Growth Restriction.

RATIONALE: Calcitonin gene-related peptide (CGRP), adrenomedullin (ADM), and adrenomedullin2 (ADM2)/intermedin are potent vasorelaxant peptides considered to play a role in the adaptive mechanisms in rat pregnancy through increased vasodilation in mesenteric and uterine artery. OBJECTIVE: This study was designed to demonstrate the response of omental arteries (OA) to vasoactive peptides CGRP, ADM, and ADM2 in pregnancy complications such as fetal growth restriction (FGR), and assess the changes in the expression of their receptor components in segments of OA from FGR pregnancy compared to the control. FINDINGS: The findings for this study are: 1) relaxation responses of OA were higher for bradykinin (78.55 ± 3.91 vs 52.67 ± 2.19; P < .05) in pregnancy with FGR compared to the normal, 2) relaxation response of OA segments to CGRP was similar with no change in the expression of G-protein couple receptor-calcitonin receptor-like receptor complex in normal healthy pregnancy and pregnancy complicated by FGR, 3) maximal relaxation response of OA were significantly (P < .05) lower for both ADM (18.2 ± 6.7 vs 38 ± 2.5) and ADM2 (26.9 ± 6.7 vs 48 ± 2.6) along with decreases in their respective ligand-receptor complex in FGR compared to the normal pregnancies, 4) expression of calcitonin receptor-like receptor mRNA was higher but its immunoreactivity was lower in OA from FGR pregnancy compared to the normal, and 5) mRNA and protein levels of RAMP1, RAMP2, and RAMP3 were lower in OA isolated from FGR pregnancies compared to the normal. CONCLUSION: The current study demonstrates that FGR is associated with an increase in the sensitivity of OA to bradykinin and decreased sensitivity for ADM and ADM2 ligand-receptor system with no change in the response for CGRP compared to the normal healthy pregnancy, and suggests a potential role for ADM and ADM2 in the pathophysiology of maternal vasculature in FGR pregnancy.

Pregnancy Increases Relaxation in Human Omental Arteries to the CGRP Family of Peptides.

Calcitonin gene-related peptide (CALCB) and its family members adrenomedullin (ADM) and intermedin (ADM2) play important roles in maintaining vascular adaptations during pregnancy in animal models. The present study was designed to evaluate the responses of omental arteries to CALCB, ADM, and ADM2 in pregnant and nonpregnant women, and to determine the mechanisms involved. By using resistance omental arteries collected from nonpregnant women (n = 15) during laparotomy and from term pregnant women (n = 15) at cesarean delivery, this study shows that the receptor components--calcitonin receptor-like receptor (CALCRL) and receptor activity-modifying proteins (RAMPs) 1, 2 and 3--are localized to endothelial and smooth muscle cells in omental arteries, with increased expressions of both mRNA and protein in pregnant compared with nonpregnant women. The myography study demonstrated that CALCB, ADM, and ADM2 (0.1-100 nM) dose dependently relax U46619 (1 muM) precontracted omental artery segments, and the maximum possible effects to CALCB and ADM2, but not to ADM, are significantly enhanced in pregnant compared with nonpregnant women. Further, the vasodilatory responses to CALCB, ADM, and ADM2 are reduced by inhibitors of nitric oxide (NO) synthase (L-NAME), adenylyl cyclase (SQ22536), voltage-activated potassium channels (4-aminopyrodin and tetrabutylammonium), Ca(2+)-activated potassium channel (charybdotoxin), and cyclooxygenase (indomethacin). In conclusion, the CALCB family of peptides, CALCB and ADM2, increase human omental artery relaxation during pregnancy through diverse mechanisms, including NO, endothelium-derived hyperpolarizing factors (EDHFs) and prostaglandins, and thus could contribute to the vascular adaptations during pregnancy in the human.

Adrenomedullin 2 (ADM2) Regulates Mucin 1 at the Maternal-Fetal Interface in Human Pregnancy.

Association of an altered expression of placental mucin 1 (MUC1) with first-trimester spontaneous abortion and its regulation in placenta by an invasion-promoting peptide, adrenomedullin 2 (ADM2), is not known. The objective of this study was to assess 1) the association of MUC1 mRNA expression in the placental villi and decidua with first-trimester spontaneous abortion, 2) the effects of ADM2 on the expression of MUC1 in trophoblast cells in the presence or absence of hypoxia, 3) the effects of ADM2 on expression of MUC1 in decidual stromal cells (DSCs), and 4) if ADM2 regulates the expression of MUC1 and MMP2 protein in trophoblastic spheroids. Data demonstrate that 1) expression of MUC1 mRNA in villous tissue is higher in spontaneous abortion compared to age-matched electively terminated pregnancies (P > 0.05), 2) ADM2 decreases the expression of MUC1 mRNA and protein in trophoblast cells and spheroids with concomitant increases in MMP2 immunoreactivity in the spheroids, 3) ADM2 decreases hypoxia-induced increases in MUC1 immunoreactivity in trophoblast cells, 4) decidual MUC1 mRNA expression is lower in spontaneous compared to elective abortions (P < 0.05), and 5) DSCs express MUC1 mRNA and protein and ADM2 decreases the expression of MUC1 mRNA and protein in DSCs. Taken together, this study demonstrates that first-trimester spontaneous abortion is associated with increases in MUC1 expression in villi and decreases in the decidual tissues, and suggests that ADM2 may contribute to the physiology of embryo implantation and placental growth via increasing MMP2 and decreasing MUC1 expression to facilitate trophoblast invasion.

Involvement of Receptor Activity-Modifying Protein 3 (RAMP3) in the Vascular Actions of Adrenomedullin in Rat Mesenteric Artery Smooth Muscle Cells.

CALCB, ADM, and ADM2 are potent vasodilators that share a seven-transmembrane GPCR, calcitonin receptor-like receptor (CALCRL), whose ligand specificity is dictated by the presence of one of the three receptor activity-modifying proteins (RAMPs). We assessed the relative pharmacologic potency of these peptides in mesenteric artery smooth muscle cells (VSMCs) and the specific RAMP that mediates the effect of ADM in VSMCs. VSMCs, with or without RAMP knockdown, were treated with CALCB, ADM, or ADM2 in the presence or absence of their antagonists, CALCB8-37, ADM22-52, and ADM217-47, respectively, to assess the relative effect of peptides on cAMP production and their pharmacologic potency. Proximity ligation assay was used to assess the specific RAMP that associates with CALCRL to mediate the actions of ADM in VSMCs. All three peptides induced cAMP generation in VSMCs and the order of their potency is CALCB > ADM > ADM2. Effects of CALCB were blocked by CALCB8-37, ADM effects were blocked by CALCB8-37 and ADM217-47 but not ADM22-52, and ADM2 effects were blocked by all three antagonists. Knockdown of RAMP2 was ineffective, whereas knockdown of RAMP3 inhibited ADM-induced cAMP production in VSMCs, suggesting involvement of RAMP3 with CALCRL to mediate ADM effects. Absence of both RAMP2 and RAMP3 further increased CALCB-induced cAMP synthesis compared to control (P < 0.05). ADM increased CALCRL and RAMP3 association and RAMP3 knockdown inhibited the interaction of ADM with CALCRL.

Adrenomedullin2 (ADM2)/intermedin (IMD) in rat ovary: changes in estrous cycle and pregnancy and its role in ovulation and steroidogenesis.

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.

Calcitonin gene related family peptides: importance in normal placental and fetal development.

Synchronized molecular and cellular events occur between the uterus and the implanting embryo to facilitate successful pregnancy outcome. Nevertheless, the molecular signaling network that coordinates strategies for successful decidualization, placentation and fetal growth are not well understood. The discovery of calcitonin/calcitonin gene-related peptides (CT/CGRP) highlighted new signaling mediators in various physiological processes, including reproduction. It is known that CGRP family peptides including CGRP, adrenomedulin and intermedin play regulatory functions during implantation, trophoblast proliferation and invasion, and fetal organogenesis. In addition, all the CGRP family peptides and their receptor components are found to be expressed in decidual, placental and fetal tissues. Additionally, plasma levels of peptides of the CGRP family were found to fluctuate during normal gestation and to induce placental cellular differentiation, proliferation, and critical hormone signaling. Moreover, aberrant signaling of these CGRP family peptides during gestation has been associated with pregnancy disorders. It indicates the existence of a possible regulatory role for these molecules during decidualization and placentation processes, which are known to be particularly vulnerable. In this review, the influence of the CGRP family peptides in these critical processes is explored and discussed.

Calcitonin gene-related family peptides in vascular adaptations, uteroplacental circulation, and fetal growth.

Maternal vascular adaptations, implantation of embryo, and placental growth and development are crucial for overall well-being of the fetus and are controlled by a range of signals, including growth factors and steroid hormones. The calcitonin (CT)/calcitonin gene-related peptide (CGRP) family peptides have been the focus of emerging studies, and these peptides appear to mediate some of the critical functions during pregnancy. Three peptides of the CT/CGRP family, CGRP, adrenomedullin, and intermedin, working through their overlapping receptor components, exert significant positive effects on vascular adaptations during pregnancy, uteroplacental vascular functions, and fetal growth. Many of the effects of these peptides are regulated by sex steroid hormones. Use of peptide antagonist in animals, together with genetic animal models, strongly implicates the importance of these 3 peptides in human pregnancy and related complications. However, insights into the underlying mechanisms of their actions on fetal-placental growth are limited by the lack of specificity of currently available antagonists. Future studies with specific knockdown of receptor components and/or peptides should be helpful for better understanding of these mechanisms and for the development of target-specific therapies to prevent pregnancy complications.

Intermedin/adrenomedullin 2 is associated with implantation and placentation via trophoblast invasion in human pregnancy.

RATIONALE: Intermedin (IMD) is a novel peptide expressed in trophoblast cells in human placenta and enhances the invasion, migration, and human leukocyte antigen class I, G (HLA-G) expression in first-trimester HTR-8SV/neo cells. We recently reported that infusion of IMD antagonist in pregnant rats is detrimental to pregnancy outcome, resulting in impaired fetoplacental growth and deformed placental vasculature. OBJECTIVE: This study was undertaken to assess expression of IMD and its involvement in human implantation and early placentation and assess whether its expression is altered in spontaneous abortion. FINDINGS AND CONCLUSIONS: We demonstrate for the first time that IMD is present in day 5 embryonic secretome; villous and decidual expression of IMD is higher at 6-8 weeks after a decline as gestation advances toward the second trimester; first-trimester spontaneous abortion is associated with a lower expression of IMD in serum, villi, and decidua; IMD stimulates the invasive capacity of first-trimester primary Extravillous cytotrophoblast cells; and IMD decreases elevated levels of tumor suppressor Kangia-1 in decidual explants from first-trimester spontaneous abortion. In conclusion, this study is the first to demonstrate a potential involvement of IMD in human embryo implantation and placental development via regulation of trophoblast invasion at the maternal-fetal interface and suggests a physiological role for this novel peptide in establishment of human pregnancy.

Adrenomedullin 2/intermedin regulates HLA-G in human trophoblasts.

Adrenomedullin 2 (ADM2), also referred to as intermedin (IMD), is expressed in trophoblast cells in human placenta and enhances the invasion and migration of first-trimester HTR-8SV/neo cells. Further infusion of ADM2 antagonist in pregnant rat causes fetoplacental growth restriction, suggesting a role for ADM2 in maintaining a successful pregnancy. This study was undertaken to assess whether ADM2 protein is present in decidual tissue and colocalized with HLA-G-positive cytotrophoblast cells and natural killer cells; to assess whether ADM2 regulates expression of HLA-G in trophoblast cells; and to identify whether mitogen-activated protein kinase (MAPK) signaling pathway is involved in ADM2-induced trophoblast cell invasion and migration. Using immunohistochemical methods and RT-PCR, this study shows that ADM2 protein is colocalized with HLA-G-expressing cytotrophoblast cells as well as with NCAM1 (CD56) immunoreactivity in human first-trimester decidual tissue, and that ADM2 mRNA is expressed in peripheral blood natural killer cells. Further, ADM2 dose dependently increases the expression of HLA-G antigen in HTR-8SV/neo cells as well as in term placental villi explants, suggesting involvement of ADM2 in the regulation of HLA-G in trophoblast cells. In addition, interference with the activity of RAF and MAPK3/1 by their inhibitors, manumycin and U0126, respectively, reduces ADM2-induced HTR-8SV/neo cell invasion and migration. In summary, this study suggests a potential involvement for ADM2 in regulating HLA-G antigen at the maternal-fetal interface in human pregnancy and facilitating trophoblast invasion and migration via MAPK3/1 phosphorylation.

Potential role of intermedin/adrenomedullin 2 in early embryonic development in rats.

Adrenomedullin2 (ADM2), also referred to as Intermedin (IMD) is expressed in trophoblast cells in human placenta and enhances the invasion and migration of first trimester HTR-8/SV-neo cells. Recently we demonstrated that infusion of IMD antagonist in pregnant rats causes feto-placental growth restriction suggesting a role for IMD in maintaining a successful pregnancy. Therefore, this study was undertaken to assess if IMD has a functional role in embryo implantation in a rat model. We show that IMD mRNA is expressed in rat implantation sites and its expression is significantly higher on day 15 in placenta compared to days 18-22. Infusion of IMD antagonist IMD17₋47 from day 3 of pregnancy causes a significant decrease in the weights of day 9 implantation sites as well as serum levels of 17ß-estradiol, progesterone, nitric oxide and serum MMP2 and MMP9 gelatinase activity. Further, expression of MMP2, MMP9, VEGF and PLGF protein levels are significantly downregulated in the implantation sites of IMD antagonist treated rats. This study suggests a potential involvement of IMD in regulating the factors that are critical for implantation and growth of the embryo and thus in establishment of normal rat pregnancy.

Adrenomedullin relaxes rat uterine artery: mechanisms and influence of pregnancy and estradiol.

Uterine arteries play a major role in regulating uteroplacental blood flow. Failure to maintain blood flow to the uteroplacental compartment during pregnancy often results in intrauterine growth retardation. Immunohistochemical staining of adrenomedullin (AM), an endogenous vasoactive peptide, in uterine artery was intense in pregnant compared to nonpregnant rats, but it is not known whether AM directly relaxes uterine artery or not. In this study, we elucidated the mechanisms of uterine artery relaxation by AM and its regulation by pregnancy and female sex steroids. AM was able to relax uterine artery, and this relaxation was influenced positively by pregnancy and estradiol as evidenced by the increased pD(2) and E(max) values of AM. Both pregnancy and estradiol treatment to ovariectomized rats amplified RAMP(3) expression in uterine arteries while progesterone had no effect. AM-induced uterine artery relaxation is predominantly endothelium-dependent. The AM receptor antagonist CGRP(8-37) is more potent than AM(22-52) in inhibiting the AM relaxation, indicating the involvement of AM(2) receptor subtype. Moreover, AM uses the classical nitric oxide-cyclic guanosine monophosphate pathway along with K(Ca) channels to mediate the vasodilatory effect in uterine artery. In conclusion, sensitivity of uterine artery to AM-induced relaxation is increased with pregnancy or estradiol treatment by increasing RAMP(3) expression, suggesting an important role for AM in regulating the uterine hemodynamics, probably maintaining uterine blood flow during pregnancy and in pre- and postmenopausal cardiovascular adaptation differences.