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

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

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

Adrenomedullin Signaling Pathway Polymorphisms and Adverse Pregnancy Outcomes

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

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

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

Loss of <i>Ramp2</i> alters mRNA expression of RAMP2-associating GPCRs, including <i>Pthr1</i>.

<p>(A) mRNA expression of RAMP2-interacting GPCRs from e13.5 wild type and <i>Ramp2</i>^<i>-/-</i> placentas (n≥3 placentas per genotype). (B) PTH1R protein expression by immunoblot of e13.5 tissue lysates from wild type and <i>Ramp2</i>^<i>-/-</i> placentas. (C and D), Immunofluorescence for PTH1R in the labyrinth of wild type and <i>Ramp2</i>^<i>-/-</i> placentas. Scale bars, 100 μm. <i>Pth1r</i>, parathyroid hormone receptor 1; <i>Casr</i>, calcium-sensing receptor; <i>Ctr</i>, calcitonin receptor; <i>Calcrl</i>, calcitonin receptor-like receptor; <i>Gcgr</i>, glucagon receptor; <i>Vpac1r</i>, vasoactive intestinal peptide receptor 1; <i>Crhr1</i>, corticotrophin releasing hormone receptor 1. *p<0.01.</p

Loss of <i>Ramp2</i> leads to a proliferation defect in the labyrinth layer.

<p>(A and B) Immunofluorescence for BrdU in e13.5 placentas. Scale bars, 500<b>μ</b>m. (C and D) Immunofluorescence for Ki67 in the labyrinth of e13.5 placentas. Scale bars, 500 μm. (E) Ratio of <i>Bax</i> to <i>Bcl2</i> mRNA expression normalized to <i>Gapdh</i> as measured by qRT-PCR (n≥5 placentas per genotype). lb, labyrinth; jz, junctional zone; dec, decidua.</p

<i>Ramp2</i> is essential for proper spiral artery remodeling.

<p>(A and B), Immunofluorescence for smooth muscle actin in e13.5 littermate placentas (n≥6 placentas per genotype). Arrows point to retained smooth muscle actin expression in spiral arteries of <i>Ramp2</i>^<i>-/-</i> placentas. Scale bars, 100 μm. (C and D), Immunofluorescence for DBA lectin, a marker of uNK cells, in e13.5 littermate placentas. Scale bars, 100<b>μ</b>m. (E) Quantification of uNK cells from e13.5 placentas (n≥3 placentas per genotype). ns, not significant.</p