Congenital diaphragmatic hernia (CDH) is a birth defect that occurs when the diaphragm does not fully form, allowing organs to enter the chest cavity and interfere with lung growth. CDH strikes 1 in every 2500 births independently of race, religion, socioeconomic background, and prenatal care, and is a significant cause of pediatric mortality. Its etiology is heterogeneous and poorly understood. We recently found that altering heparan sulfate in endothelial cells in the mouse results in a CDH-like phenotype. Mutations altering a protein called Slit3 also cause a diaphragmatic hernia resembling septum transversum CDH. Slit proteins are expressed during development and act as signals to adjacent tissues by binding to receptor tyrosine kinases, called Robo. Robo1 and Robo4 are expressed on endothelial cells and the binding of Slits to these receptors may play a role in endothelial cell growth and angiogenesis. Slit proteins bind to heparan sulfate facilitating the interaction between Slit and Robo and subsequent signaling. Thus, our central hypothesis is that a deficiency of endothelial heparan sulfate may decrease the binding affinity of Slit3 to Robo receptors, resulting in impaired signaling and altered angiogenesis. A decrease in vascularization of the diaphragm in turn results in loss of integrity and eventual hernia of the central tendon creating a condition resembling CDH. This study explores the novel idea of endothelial heparan sulfate facilitating Slit/Robo signaling in angiogenesis, and may uncover a new etiology for CDH. To address this hypothesis, we have the following specific aims:
Aim 1. Examine early histological signs of aberrant diaphragmatic development in mice containing an endothelial-specific knockout in heparan sulfate biosynthesis. In this aim, we will examine the histology of the diaphragm before and after overt herniation. These studies will involve examination of the architecture of the diaphragm and the formation of muscle, tendon, nerve, and blood vessels in the diaphragm during fetal development.
Aim 2. Examine Slit3 interaction with heparan sulfate and its role in signaling through Robo receptors expressed on endothelial cells. Recombinant Slit3 protein has been produced in cultured cells and shown to bind heparin-Sepharose. The binding of recombinant Slit3 protein to wildtype and heparan sulfate-deficient endothelial cells will be tested. Slit protein initiated signal transduction will be measured as well.
Aim 3. Combine Slit3 or Robo mutations with endothelial alteration of heparan sulfate and examine the time of onset, penetrance and severity of herniation. Heparan sulfate-deficient mice will be crossed with Slit3+/- and Robo+/- mice to test if the combination of these mutations potentiates the CDH phenotype (onset, penetrance, and severity). ? ? ?
| Zhang, Bing; Xiao, Wenyuan; Qiu, Hong et al. (2014) Heparan sulfate deficiency disrupts developmental angiogenesis and causes congenital diaphragmatic hernia. J Clin Invest 124:209-21 |
| Zhang, Bing; Dietrich, Ursula M; Geng, Jian-Guo et al. (2009) Repulsive axon guidance molecule Slit3 is a novel angiogenic factor. Blood 114:4300-9 |
| Fuster, Mark M; Wang, Lianchun; Castagnola, Janice et al. (2007) Genetic alteration of endothelial heparan sulfate selectively inhibits tumor angiogenesis. J Cell Biol 177:539-49 |
