Genetically Engineered Animal Models in Study of Human Disease
The overall scientific goal of our laboratory is to develop and use genetically engineered animal models to better understand and treat human disease. We are currently investigating the physiological role of a newly identified peptide vasodilator called adrenomedullin (Adm) and its associated receptor and signaling proteins. Adm has been implicated in a wide variety of normal physiological
processes, including maintenance of basal vascular tone, regulation of salt and water appetite, cellular proliferation and anti-microbial defense. Most noteworthy is the finding that plasma levels of Adm are elevated in patients with many types of diseases, including essential hypertension and sepsis, suggesting that elevations in Adm are compensatory to other primary cardiovascular stresses. Recently, it has become clear that Adm is also important in pregnancy (another form of cardiovascular stress) since circulating levels of Adm are five fold higher during normal pregnancy and fall back to non-pregnant values within 48 hours after delivery. Furthermore,
the pattern of Adm expression suggests an important role for this peptide in the establishment of maternal/fetal circulation and the maintenance of pregnancy. The vasodilatory properties of Adm coupled with its expression in fetoplacental tissues have led to the suggestion that decreased Adm levels may contribute to the development of preeclampsia, a common and severe pregnancy-induced hypertensive condition. Thus, Adm is quickly becoming recognized as a broadly expressed peptide hormone that impacts on many systems under normal and pathological conditions.

To elucidate the physiological functions of Adm as they relate to blood pressure regulation and reproduction we have used gene targeting methods to generate a series of mouse lines that have 0 through 4 copies of the Adm gene and therefore have decreased and increased levels of Adm peptide. We
find that although changes in Adm gene expression do not alter basal BP, there is an effect on the ability to compensate for pharmacologically-induced changes in BP. The Adm null animals are not viable and die in utero from extreme hydrops fetalis (see figure) and unusual cardiovascular defects. These findings consequently demonstrate an essential role for Adm in embryonic development and suggest that absence of Adm may be a cause of nonimmune hydrops fetalis in humans.

Clinical studies have suggested that decreased levels of Adm from fetoplacental tissues may be a cause of preeclampsia, a systemic maternal disease that originates from shallow trophoblast cell invasion and placentation. Preliminary studies using the Adm+/- female mice have revealed several pathological features associated with preeclampsia in humans. For example, Adm+/- females show reduced fertility, intrauterine growth retardation, twinning and aberrant trophoblast cell migration- an essential component of placentogenesis. These findings suggest that a reduction in Adm, either systemically or in the placenta, may affect normal placental development and eventually compromise the ability to compensate for physiologically-induced changes in BP, such as those that occur during pregnancy.

Current and Future Projects

1. Define the role of Adm in pregnancy and placental development. In doing so, develop new methods for high-throughput screening of placental defects by gene expression analysis. We are currently performing breeding schemes and embryo transfer experiments to determine if the placental defects described above are due to decreased Adm levels from the maternal or fetal compartment. However, it is likely that both sources of Adm contribute to establishing the appropriate local concentration of Adm required for effective maternal/fetal circulation. Therefore, in addition to using classical qualitative methods for characterizing placental defects (histology and immunohistochemistry) we plan to develop a quantitative approach that uses real-time RT-PCR to examine numerous genetic markers of placental function. Assessing placental development and function through a panel of quantitative gene expression analysis is a novel approach
that will greatly advance the current repertoire of tools for phenotyping placental defects. In the future, we plan to use
this approach to perform high-throughput mutagenesis screening for genetic mouse models with defects in placental development and function that may be related to
preeclampsia in humans.

2. Use gene targeting techniques to modulate endogenous gene expression in a manner that more closely recapitulates quantitative physiological changes in gene expression. One of our major goals will be to design and implement generally applicable principles of gene regulation in order to improve targeted transgenics and eventually precisely regulate endogenous gene expression through homologous recombination. In collaboration with other laboratories at UNC-CH, we have developed a "gene titration" vector that can be used to alter the mRNA stability (and consequently, protein levels) of any given gene through homologous recombination (see figure). For example, replacement of the 3’UTR of the Adm gene with that of bovine growth hormone (a long-lived mRNA) or c-fos (a short-lived mRNA) will result in mouse lines with markedly varied levels of Adm expression. The first model, with increased Adm expression, is in many respects superior to conventional transgenic approaches because the endogenous promoter remains functionally intact. The second model, with decreased (but not absent) Adm expression, has the added potential of being generated in a tissue-specific manner by breeding to transgenic mice expressing cre recombinase. With these models we expect to exacerbate the Adm+/- reproductive defects and further explore the protective cardiovascular functions of the Adm peptide. Of greater significance, however, will be the application of these principles to endogenously targeted genes, resulting in new and improved methods for generating physiologically relevant, genetic mouse models of human disease.

3. Explore the mechanisms of Adm G-protein coupled receptor (GPCR) signaling. Recent studies have revealed that Adm mediates its physiological effects through a novel paradigm in signal transduction. In 1998, a new class of proteins, called receptor activity modifying proteins (RAMPs), were identified and shown to impose specificity to a common calcitonin-receptor-like receptor (CRLR). As depicted in the cartoon, association of CRLR with RAMP1 causes the receptor to function as a CGRP receptor, whereas association with RAMP2/3 makes it an Adm receptor. Moreover, the cell-specific expression of the RAMPs determines a cell's fate to respond to either Adm or CGRP. Using conventional targeted disruption and the quantitative gene targeting approaches described above, we will examine the physiological role of CRLR and its associated RAMPs in relation to cardiovascular and reproductive physiology. Questions we plan to address are: 1. Do the RAMPs govern the specificity of other GPCRs? 2. Do RAMP2 and RAMP3 have distinct or overlapping biological functions? 3. Do quantitative changes in the levels of RAMP expression alter a cell’s affinity for Adm or CGRP signaling? and 4. Do the RAMPs associate or function with other G-proteins (studied by biochemical and in vitro reconstitution approaches)?