Adrenomedullin signaling pathway polymorphisms and adverse pregnancy outcomes.

OBJECTIVE: Reduced maternal plasma levels of the peptide vasodilator adrenomedullin have been associated with adverse pregnancy outcomes. We measured the extent to which genetic polymorphisms in the adrenomedullin signaling pathway are associated with birth weight, glycemic regulation, and preeclampsia risk. STUDY DESIGN: We genotyped 1,353 women in the Pregnancy, Infection, and Nutrition Postpartum Study for 37 ancestry-informative markers and for single-nucleotide polymorphisms in adrenomedullin (ADM), complement factor H variant (CFH), and calcitonin receptor-like receptor (CALCRL). We used linear and logistic regression to model the association between genotype and birth weight, glucose loading test (GLT) results, preeclampsia, and gestational diabetes (GDM). All models were adjusted for pregravid body mass index, maternal age, and probability of Yoruban ancestry. p values of < 0.05 were considered statistically significant. RESULTS: Among Caucasian women, ADM rs57153895, a proxy for rs11042725, was associated with reduced birth weight z-score. Among African-American women, ADM rs57153895 was associated with increased birth weight z-score. Two CALCRL variants were associated with GDM risk. CFH rs1061170 was associated with higher GLT results and increased preeclampsia risk. CONCLUSION: Consistent with studies of plasma adrenomedullin and adverse pregnancy outcomes, we found associations between variants in the adrenomedullin signaling pathway and birth weight, glycemic regulation, and preeclampsia.

Loss of receptor activity-modifying protein 3 exacerbates cardiac hypertrophy and transition to heart failure in a sex-dependent manner.

Sex differences exist in the hypertrophic response, cardiac remodeling, and transition to heart failure of hypertensive patients, and while some of these differences are likely influenced by estrogen, the genetic pathways downstream of estrogen that impact on cardioprotection have yet to be fully elucidated. We have previously shown that the cardioprotective effects of adrenomedullin (AM), an emerging clinical biomarker for cardiovascular disease severity, vary with sex in mouse models. AM signaling during cardiovascular stress is strongly modulated by receptor activity-modifying protein 3 (RAMP3) via its interaction with the G protein-coupled receptor calcitonin receptor-like receptor (CLR). Like AM, RAMP3 expression is potently regulated by estrogen, and so we sought to determine the consequences of genetic Ramp3 loss on cardiac adaptation to chronic hypertension, with a particular focus on characterizing potential sex differences. We generated and bred RAMP3(-/-) mice to RenTgMK mice that consistently display severe angiotensin II-mediated CV disease and compared CV disease progression in RenTgMK to that of RenTgMK:RAMP3(-/-) offspring. As expected, RAMP3 gene expression was higher in cardiovascular tissues of RenTgMK mice and more strongly up-regulated in female RenTgMK mice relative to wildtype controls. RAMP3 loss did not affect the development of hypertension or the presence and severity of perivascular and interstitial fibrosis in the left ventricle (LV). However, echocardiography revealed that while RenTgMK mice developed concentric cardiac hypertrophy with sustained systolic function, male RenTgMK:RAMP3(-/-) mice showed evidence of LV chamber dilatation and depressed systolic function, suggestive of cardiac decompensation. Consistent with these measures of heart failure, male RenTgMK:RAMP3(-/-) mice had increased cardiac apoptosis and elevated activation of Akt. These phenotypes were not present in female RenTgMK:RAMP3(-/-) mice. Collectively, these data demonstrate a sex-dependant, cardioprotective role of RAMP3 in the setting of chronic hypertension.

Loss of receptor activity-modifying protein 2 in mice causes placental dysfunction and alters PTH1R regulation.

Receptor activity-modifying protein 2 (Ramp2) is a single-pass transmembrane protein that heterodimerizes with several family B G-protein coupled receptors to alter their function. Ramp2 has been primarily characterized in association with calcitonin receptor-like receptor (Calcrl, CLR), forming the canonical receptor complex for the endocrine peptide adrenomedullin (Adm, AM). However, we previously demonstrated that Ramp2+/- female mice display a constellation of endocrine-related phenotypes that are distinct from those of Adm+/- and Calcrl+/- mice, implying that RAMP2 has physiological functions beyond its canonical complex. Here, we localize Ramp2 expression in the mouse placenta, finding that Ramp2 is robustly expressed in the fetal labyrinth layer, and then characterize the effects of loss of Ramp2 on placental development. Consistent with the expression pattern of Ramp2 in the placenta, Ramp2-/- placentas have a thinner labyrinth layer with significantly fewer trophoblast cells secondary to a reduction in trophoblast proliferation. We also find that absence of Ramp2 leads to failed spiral artery remodeling unaccompanied by changes in the uterine natural killer cell population. Furthermore, we assess changes in gene expression of other RAMP2-associated G-protein coupled receptors (GPCRs), concluding that Ramp2 loss decreases parathyroid hormone 1 receptor (Pthr1) expression and causes a blunted response to systemic parathyroid hormone (PTH) administration in mice. Ultimately, these studies provide in vivo evidence of a role for RAMP2 in placental development distinct from the RAMP2-CLR/AM signaling paradigm and identify additional pathways underlying the endocrine and fertility defects of the previously characterized Ramp2 heterozygous adult females.

G-protein-coupled receptor 30 interacts with receptor activity-modifying protein 3 and confers sex-dependent cardioprotection.

Receptor activity-modifying protein 3 (RAMP3) is a single-pass transmembrane protein known to interact with and affect the trafficking of several G-protein-coupled receptors (GPCRs). We sought to determine whether RAMP3 interacts with GPR30, also known as G-protein-coupled estrogen receptor 1. GPR30 is a GPCR that binds estradiol and has important roles in cardiovascular and endocrine physiology. Using bioluminescence resonance energy transfer titration studies, co-immunoprecipitation, and confocal microscopy, we show that GPR30 and RAMP3 interact. Furthermore, the presence of GPR30 leads to increased expression of RAMP3 at the plasma membrane in HEK293 cells. In vivo, there are marked sex differences in the subcellular localization of GPR30 in cardiac cells, and the hearts of Ramp3(-/-) mice also show signs of GPR30 mislocalization. To determine whether this interaction might play a role in cardiovascular disease, we treated Ramp3(+)(/)(+) and Ramp3(-/-) mice on a heart disease-prone genetic background with G-1, a specific agonist for GPR30. Importantly, this in vivo activation of GPR30 resulted in a significant reduction in cardiac hypertrophy and perivascular fibrosis that is both RAMP3 and sex dependent. Our results demonstrate that GPR30-RAMP3 interaction has functional consequences on the localization of these proteins both in vitro and in vivo and that RAMP3 is required for GPR30-mediated cardioprotection.

Fetal-derived adrenomedullin mediates the innate immune milieu of the placenta.

The remodeling of maternal uterine spiral arteries (SAs) is an essential process for ensuring low-resistance, high-capacitance blood flow to the growing fetus. Failure of SAs to remodel is causally associated with preeclampsia, a common and life-threatening complication of pregnancy that is harmful to both mother and fetus. Here, using both loss-of-function and gain-of-function genetic mouse models, we show that expression of the pregnancy-related peptide adrenomedullin (AM) by fetal trophoblast cells is necessary and sufficient to promote appropriate recruitment and activation of maternal uterine NK (uNK) cells to the placenta and ultimately facilitate remodeling of maternal SAs. Placentas that lacked either AM or its receptor exhibited reduced fetal vessel branching in the labyrinth, failed SA remodeling and reendothelialization, and markedly reduced numbers of maternal uNK cells. In contrast, overexpression of AM caused a reversal of these phenotypes with a concomitant increase in uNK cell content in vivo. Moreover, AM dose-dependently stimulated the secretion of numerous chemokines, cytokines, and MMPs from uNK cells, which in turn induced VSMC apoptosis. These data identify an essential function for fetal-derived factors in the maternal vascular adaptation to pregnancy and underscore the importance of exploring AM as a biomarker and therapeutic agent for preeclampsia.

Adrenomedullin and pregnancy: perspectives from animal models to humans.

A healthy pregnancy requires strict coordination of genetic, physiologic and environmental factors. The relatively common incidence of infertility and pregnancy complications has resulted in increased interest in understanding the mechanisms that underlie normal versus abnormal pregnancy. The peptide hormone adrenomedullin (AM) has recently been the focus of some exciting breakthroughs in the pregnancy field. Supported by mechanistic studies in genetic animal models, there continues to be a growing body of evidence demonstrating the importance of AM protein levels in a variety of human pregnancy complications. With more extensive mechanistic studies and improved consistency in clinical measurements of AM, there is great potential for the development of AM as a clinically-relevant biomarker in pregnancy and pregnancy complications.

Adrenomedullin Function in Vascular Endothelial Cells: Insights from Genetic Mouse Models.

Adrenomedullin is a highly conserved peptide implicated in a variety of physiological processes ranging from pregnancy and embryonic development to tumor progression. This review highlights past and present studies that have contributed to our current appreciation of the important roles adrenomedullin plays in both normal and disease conditions. We provide a particular emphasis on the functions of adrenomedullin in vascular endothelial cells and how experimental approaches in genetic mouse models have helped to drive the field forward.

The ADAM10 prodomain is a specific inhibitor of ADAM10 proteolytic activity and inhibits cellular shedding events.

ADAM10 is a disintegrin metalloproteinase that processes amyloid precursor protein and ErbB ligands and is involved in the shedding of many type I and type II single membrane-spanning proteins. Like tumor necrosis factor-alpha-converting enzyme (TACE or ADAM17), ADAM10 is expressed as a zymogen, and removal of the prodomain results in its activation. Here we report that the recombinant mouse ADAM10 prodomain, purified from Escherichia coli, is a potent competitive inhibitor of the human ADAM10 catalytic/disintegrin domain, with a K(i) of 48 nM. Moreover, the mouse ADAM10 prodomain is a selective inhibitor as it only weakly inhibits other ADAM family proteinases in the micromolar range and does not inhibit members of the matrix metalloproteinase family under similar conditions. Mouse prodomains of TACE and ADAM8 do not inhibit their respective enzymes, indicating that ADAM10 inhibition by its prodomain is unique. In cell-based assays we show that the ADAM10 prodomain inhibits betacellulin shedding, demonstrating that it could be of potential use as a therapeutic agent to treat cancer.