Kathleen M. Caron

Adrenomedullin signaling is necessary for murine lymphatic vascular development

The lymphatic vascular system mediates fluid homeostasis, immune defense, and tumor metastasis. Only a handful of genes are known to affect the development of the lymphatic vasculature, and even fewer represent therapeutic targets for lymphatic diseases. Adrenomedullin (AM) is a multifunctional peptide vasodilator that transduces its effects through the calcitonin receptor-like receptor (calcrl) when the receptor is associated with a receptor activity-modifying protein (RAMP2). Here we report on the involvement of these genes in lymphangiogenesis. AM-, calcrl-, or RAMP2-null mice died mid-gestation after development of interstitial lymphedema. This conserved phenotype provided in vivo evidence that these components were required for AM signaling during embryogenesis. A conditional knockout line with loss of calcrl in endothelial cells confirmed an essential role for AM signaling in vascular development. Loss of AM signaling resulted in abnormal jugular lymphatic vessels due to reduction in lymphatic endothelial cell proliferation. Furthermore, AM caused enhanced activation of ERK signaling in human lymphatic versus blood endothelial cells, likely due to induction of CALCRL gene expression by the lymphatic transcriptional regulator Prox1. Collectively, our studies identify a class of genes involved in lymphangiogenesis that represent a pharmacologically tractable system for the treatment of lymphedema or inhibition of tumor metastasis.

Hydrops Fetalis, Cardiovascular Defects, and Embryonic Lethality in Mice Lacking the Calcitonin Receptor-Like Receptor Gene

ABSTRACT Adrenomedullin (AM) is a multifunctional peptide vasodilator that is essential for life. To date, numerous in vitro studies have suggested that AM can mediate its biological effects through at least three different receptors. To determine the in vivo importance of the most likely candidate receptor, calcitonin receptor-like receptor, a gene-targeted knockout model of the gene was generated. Mice heterozygous for the targeted Calcrl allele appear normal, survive to adulthood, and reproduce. However, heterozygote matings fail to produce viable Calcrl −/− pups, demonstrating that Calcrl is essential for survival. Timed matings confirmed that Calcrl −/− embryos die between embryonic day 13.5 (E13.5) and E14.5 of gestation. The Calcrl −/− embryos exhibit extreme hydrops fetalis and cardiovascular defects, including thin vascular smooth muscle walls and small, disorganized hearts remarkably similar to the previously characterized AM −/− phenotype. In vivo assays of cellular proliferation and apoptosis in the hearts and vasculature of Calcrl −/− and AM −/− embryos support the concept that AM signaling is a crucial mediator of cardiovascular development. The Calcrl gene targeted mice provide the first in vivo genetic evidence that CLR functions as an AM receptor during embryonic development.

Important role of endogenous norepinephrine and epinephrine in the development of in vivo pressure-overload cardiac hypertrophy.

OBJECTIVESnWe sought to define the role of norepinephrine and epinephrine in the development of cardiac hypertrophy and to determine whether the absence of circulating catecholamines alters the activation of downstream myocardial signaling pathways.nnnBACKGROUNDnCardiac hypertrophy is associated with elevated plasma catecholamine levels and an increase in cardiac morbidity and mortality. Although considerable evidence suggests that G-protein-coupled receptors are involved in the hypertrophic response, it remains controversial whether catecholamines are required for the development of in vivo cardiac hypertrophy.nnnMETHODSnWe performed transverse aortic constriction (TAC) in dopamine beta-hydroxylase knockout mice (Dbh(-/-), genetically altered mice that are completely devoid of endogenous norepinephrine and epinephrine) and littermate control mice. After induction of cardiac hypertrophy, the mitogen-activated protein kinase (MAPK) signaling pathways were measured in pressure-overloaded/wild-type and Dbh(-/-) hearts.nnnRESULTSnCompared with the control animals, cardiac hypertrophy was significantly blunted in Dbh(-/-) mice, which was not associated with altered cardiac function, as assessed by transthoracic echocardiography in conscious mice. The extracellularly regulated kinase (ERK 1/2), c-jun-NH(2)-terminal kinase (JNK) and p38 MAPK pathways were all activated by two- to threefold after TAC in the control animals. In contrast, induction of the three pathways (ERK 1/2, JNK and p38) was completely abolished in Dbh(-/-) mice.nnnCONCLUSIONSnThese data demonstrate a nearly complete requirement of endogenous norepinephrine and epinephrine for the induction of in vivo pressure-overload cardiac hypertrophy and for the activation of hypertrophic signaling pathways.

Reduced maternal expression of adrenomedullin disrupts fertility, placentation, and fetal growth in mice

Adrenomedullin (AM) is a multifunctional peptide vasodilator that is essential for life. Plasma AM expression dramatically increases during pregnancy, and alterations in its levels are associated with complications of pregnancy including fetal growth restriction (FGR) and preeclampsia. Using AM+/- female mice with genetically reduced AM expression, we demonstrate that fetal growth and placental development are seriously compromised by this modest decrease in expression. AM+/- female mice had reduced fertility characterized by FGR. The incidence of FGR was also influenced by the genotype of the embryo, since AM-/- embryos were more often affected than either AM+/- or AM+/+ embryos. We demonstrate that fetal trophoblast cells and the maternal uterine wall have coordinated and localized increases in AM gene expression at the time of implantation. Placentas from growth-restricted embryos showed defects in trophoblast cell invasion, similar to defects that underlie human preeclampsia and placenta accreta. Our data provide a genetic in vivo model to implicate both maternal and, to a lesser extent, embryonic levels of AM in the processes of implantation, placentation, and subsequent fetal growth. This study provides the first genetic evidence to our knowledge to suggest that a modest reduction in human AM expression during pregnancy may have an unfavorable impact on reproduction.

Immune mechanisms at the maternal-fetal interface: perspectives and challenges

Leaders gathered at the US National Institutes of Health in November 2014 to discuss recent advances and emerging research areas in aspects of maternal-fetal immunity that may affect fetal development and pregnancy success.

Receptor Activity-modifying Proteins 2 and 3 Have Distinct Physiological Functions from Embryogenesis to Old Age

RAMPs (receptor activity modifying proteins) impart remarkable effects on G protein-coupled receptor (GPCR) signaling. First identified through an interaction with the calcitonin receptor-like receptor (CLR), these single transmembrane proteins are now known to modulate the in vitro ligand binding affinity, trafficking, and second messenger pathways of numerous GPCRs. Consequently, the receptor-RAMP interface represents an attractive pharmacological target for the treatment of disease. Although the three known mammalian RAMPs differ in their sequences and tissue expression, results from in vitro biochemical and pharmacological studies suggest that they have overlapping effects on the GPCRs with which they interact. Therefore, to determine whether RAMP2 and RAMP3 have distinct functions in vivo, we generated mice with targeted deletions of either the RAMP2 or RAMP3 gene. Strikingly, we found that, although RAMP2 is required for survival, mice that lack RAMP3 appear normal until old age, at which point they have decreased weight. In addition, mice with reduced expression of RAMP2 (but not RAMP3) display remarkable subfertility. Thus, each gene has functions in vivo that cannot be accomplished by the other. Because RAMP2, RAMP3, and CLR transduce the signaling of the two potent vasodilators adrenomedullin and calcitonin gene-related peptide, we tested the effects of our genetic modifications on blood pressure, and no effects were detected. Nevertheless, our studies reveal that RAMP2 and RAMP3 have distinct physiological functions throughout embryogenesis, adulthood, and old age, and the mice we have generated provide novel genetic tools to further explore the utility of the receptor-RAMP interface as a pharmacological target.

A genetically clamped renin transgene for the induction of hypertension

Experimental analysis of the effects of individual components of complex mammalian systems is frequently impeded by compensatory adjustments that animals make to achieve homeostasis. We here introduce a genetic procedure for eliminating this type of impediment, by using as an example the development and testing of a transgene for “genetically clamping” the expression of renin, the major homeostatically responding component of the renin–angiotensin system, one of the most important regulators of blood pressure. To obtain a renin transgene whose expression is genetically clamped at a constant level, we have used single-copy chosen-site gene targeting to insert into a liver-specific locus a single copy of a modified mouse renin transgene driven by a liver-specific promoter/enhancer. The resulting transgene expresses renin ectopically at a constant high level in the liver and leads to elevated plasma levels of prorenin and active renin. The transgenic mice display high blood pressure, enhanced thirst, high urine output, proteinuria, and kidney damage. Treatment with the angiotensin II type I receptor antagonist, losartan, reduces the hypertension, albuminuria, and kidney damage, but does not affect expression of the transgene. This genetically clamped renin transgene can be used in models in which hypertension and its complications need to be investigated in a high prorenin/renin environment that is not subject to homeostatic compensations by the animal when other factors are changed.

Restoration of Podocyte Structure and Improvement of Chronic Renal Disease in Transgenic Mice Overexpressing Renin

Background Proteinuria is a major marker of the decline of renal function and an important risk factor of coronary heart disease. Elevated proteinuria is associated to the disruption of slit-diaphragm and loss of podocyte foot processes, structural alterations that are considered irreversible. The objective of the present study was to investigate whether proteinuria can be reversed and to identify the structural modifications and the gene/protein regulation associated to this reversal. Methodology/Principal Findings We used a novel transgenic strain of mouse (RenTg) that overexpresses renin at a constant high level. At the age of 12-month, RenTg mice showed established lesions typical of chronic renal disease such as peri-vascular and periglomerular inflammation, glomerular ischemia, glomerulosclerosis, mesangial expansion and tubular dilation. Ultrastructural analysis indicated abnormal heterogeneity of basement membrane thickness and disappearance of podocyte foot processes. These structural alterations were accompanied by decreased expressions of proteins specific of podocyte (nephrin, podocin), or tubular epithelial cell (E-cadherin and megalin) integrity. In addition, since TGFβ is considered the major pro-fibrotic agent in renal disease and since exogenous administration of BMP7 is reported to antagonize the TGFβ-induced phenotype changes in kidney, we have screened the expressions of several genes belonging in the TGFβ/BMP superfamily. We found that the endogenous inhibitors of BMPs such as noggin and Usag-1 were several-fold activated inhibiting the action of BMPs and thus reinforcing the deleterious action of TGFβ.Treatment with an AT1 receptor antagonist, at dose that did not decrease arterial pressure, gradually reduced albuminuria. This decrease was accompanied by re-expression of podocin, nephrin, E-cadherin and megalin, and reappearance of podocyte foot processes. In addition, expressions of noggin and Usag-1 were markedly decreased, permitting thus activation of the beneficial action of BMPs. Conclusions/Significance These findings show that proteinuria and alterations in the expression of proteins involved in the integrity and function of glomerular and renal epithelial phenotype are reversible events when the local action of angiotensin II is blocked, and provide hope that chronic renal disease can be efficiently treated.

RASA3 is a critical inhibitor of RAP1-dependent platelet activation

The small GTPase RAP1 is critical for platelet activation and thrombus formation. RAP1 activity in platelets is controlled by the GEF CalDAG-GEFI and an unknown regulator that operates downstream of the adenosine diphosphate (ADP) receptor, P2Y12, a target of antithrombotic therapy. Here, we provide evidence that the GAP, RASA3, inhibits platelet activation and provides a link between P2Y12 and activation of the RAP1 signaling pathway. In mice, reduced expression of RASA3 led to premature platelet activation and markedly reduced the life span of circulating platelets. The increased platelet turnover and the resulting thrombocytopenia were reversed by concomitant deletion of the gene encoding CalDAG-GEFI. Rasa3 mutant platelets were hyperresponsive to agonist stimulation, both in vitro and in vivo. Moreover, activation of Rasa3 mutant platelets occurred independently of ADP feedback signaling and was insensitive to inhibitors of P2Y12 or PI3 kinase. Together, our results indicate that RASA3 ensures that circulating platelets remain quiescent by restraining CalDAG-GEFI/RAP1 signaling and suggest that P2Y12 signaling is required to inhibit RASA3 and enable sustained RAP1-dependent platelet activation and thrombus formation at sites of vascular injury. These findings provide insight into the antithrombotic effect of P2Y12 inhibitors and may lead to improved diagnosis and treatment of platelet-related disorders.

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.