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Endocrinology, Metabolism and Clinical Nutrition

Endothelin Converting Enzyme

Our bone linkage mapping experiments identified a pleiotropic QTL to a small region of chromosome 4. The QTL affected 10 traits related to bone size, shape, and biomechanical performance, but none at the tissue level. In contrast, the QTLs on other chromosomes affect both whole bone and tissue-level properties. This led us to infer that  this QTL acts by modulating radial bone growth. (chromosome 4 linkage image)

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Micro-CT scans of femora from HcB-8 and HcB-23 male mice. Note the difference in size and shape. Scale bar = 1 mm.

Ece1, the gene encoding endothelin converting enzyme 1, is a positional candidate for this QTL. This gene is essential for neural crest cell migration, induction of the Purkinje system, proper development of the cardiac outflow tract during development, and formation of the mandible. ECE1 protein catalyzes the conversion of biologically inert big endothelin 1 to active endothelin 1(ET1). ET1 is a small peptide hormone that mediates blood pressure in a paracrine fashion.

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Skeletal preparations of wild type (left) and Ece1 knockout (right) embryos at 17.5 days of gestation. Bone stains red and cartilage stains blue. Note the severe hypoplasia of the knockout mandible.

Hypothesizing that if Ece1 is the gene underlying the chromosome 4 QTL, we reasoned that this might result in pleiotropy extending to vascular phenotypes. We undertook initial studies in collaboration with Naomi Chesler to explore this possibility. We found that HcB-23 mice have larger, more easily stretched arteries than HcB-8 mice, paralleling the differences observed between bones in these strains.

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Blood pressure in HcB-8 and HcB-23 mice. Measurements made weekly by tail cuff pulse-volume recording.

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Ex vivo carotid artery mechanics. X axis shows intraluminal pressure and Y axis shows relative arterial diameter.

In addition, we have observed that HcB-8 mice have larger hearts, smaller litters, and lower expression of Ece1 and its downstream target, Nos3, in multiple tissues.

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Immunohistochemistry of HcB-8 (A and C) and HcB-23 (B and D) hearts. A and B are stained for ECE1 and C and D are stained for NOS3. Original magnification = 50x.

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Immunohistochemistry of placentas from HcB-8 (left) and HcB-23 (right) mice. Top row is stained for ECE1 and bottom is stained for NOS3. Original magnification = 50x.

We have developed a conditional knockout allele of Ece1. Global Ece1 knockout mice have multiple abnormal phenotypes and are not viable. We are therefore using the conditional knockout to study the bone, vascular, and reproductive phenotypes that differ between HcB-8 and HcB-23 mice. These studies are being done in collaboration with Julian Lombard, Naomi Chesler, Ron Magness, and Rashmi Sood. Work on this project is critically dependent on the Cardiovascular Center. (CKO images)