At the groundbreaking National Institutes of Health (NIH) HHT meeting in June 2006, it was our pleasure to award two new HHT research grants. There had been an extensive peer review of ten proposals. The two winning projects were:
Mechanism of Disease $50,000 Research Grant Award
TGF-B Activates eNOS and Regulates Vasomotor Function by an endoglin-and Alk-1' Dependent Mechanism
Dr. Michelle Letarte
Toronto Hospital for Sick Children, Canada
According to Dr. Letarte, “The genes mutated in HHT are endoglin and ALK1. The products of these genes, the endoglin and ALK1 proteins, are receptors for a potent factor called TGF-ß. We now have recent evidence that this TGF-ß factor may control the dilatation of blood vessels. We hypothesize that this normal process of regulation of vessel dilatation by TGF-ß is defective in HHT and can eventually lead to the clinical manifestations of disease. We will use the mouse models of HHT1 and HHT2 and test blood vessels isolated from these mice to see how they respond to TGF-ß compared to those of normal mice. We will then identify the molecules and pathways responsible for the effects of TGF-ß in cells from mice and patients with HHT. Our experiments should provide a better understanding of the mechanisms responsible for the early events in the disease and pave the way to novel therapeutic interventions.
Our specific objectives are:
- To measure the effects of TGF-b1 on eNOS-dependent vasodilation in small resistance arteries isolated from Eng+/- (HHT1 model) and control mice, using a perfusion myograph.
- To test whether TGF-b1 regulation of eNOS-dependent vasodilation is similarly altered in HHT2. Vasodilation will be measured on small resistance arteries from alk1+/- and control mice.
- To characterize the mechanism of TGF-b1-induced eNOS activation in murine endothelial cells (normal and deficient endoglin) and in human umbilical vein endothelial cells (HUVEC) from newborns with a molecular diagnostic of HHT1 or HHT2. We will measure the effects of TGF-b1 on eNOS enzymatic activity, eNOS-Hsp90 association, and eNOS phosphorylation using specific anti-phospho-eNOS antibodies. We will test for the association of ALK1 with eNOS and/or Hsp90 and see if its overexpression in endothelial cells amplifies the activation of eNOS by TGF-b1.
Dr. Letarte states, “We are very grateful to the HHT International Foundation for awarding us this grant and thank all of you for your kind donations. Our daily efforts are devoted to understanding the underlying mechanisms of HHT through basic research. We could not do it without your generous financial and moral support.”
Treatment of Disease $50,000 Research Grant Award
Differentiation of Eng-/- Embryonic Stem Cells in Embryoid Bodies, a Paradigm for Vascuaogenesis/Angiogenesis
Defects in HHT and a Potential Drug Screen
Professor Christine Mummery
Utrecht, The Netherlands
This study will examine the effects of Thalidomide on blood vessel structure. According to Professor Mummery, “By studying the effects of thalidomide on vessel stabilization, we propose (1) to validate the Eng-/vasculogenesis/angiogenesis assay as a useful method in screening potential HHT drugs, particularly those directed towards improving vessel robustness and (2) to determine the mechanisms underlying vessel stabilization. We will attempt to generate a human model of HHT development in vitro.”
(1) Mechanism underlying vessel stabilization. By using FACS sorting, ECs and SMCs will be isolated from 18 day Eng-/- EBs treated or otherwise with thalidomide. Transcriptional profiles will be compared both by microarray analysis and by real time RT-PCR to identify signaling pathways that may underlie the effects observed.
(2) Validation of the EB vasculogenesis/angiogenesis assay for screening HHT drugs. To date our experiments have been performed using only one of each wt, Eng+/- and Eng-/- ES cell lines. To validate our model, multiple new mES cell lines lacking or heterozygous for endoglin will be generated from blastocysts isolated from Eng+/- crosses and EB differentiation in presence of thalidomide will be repeated.
(3) Since we have expertise in the differentiation of multiple human ESC (HESC) lines into mesoderm derivatives, including vascular cells, in EBs we will test the effects of thalidomide in an analogous model to validate and extend our findings on vessel stabilization to human. Time permitting and the context of other studies to optimize siRNA-mediated knockdown in HESCs, we will attempt to generate a human model of HHT development in vitro.
OUTCOMES published in Nature Medicine