Mechanism of Disease: $50,000 Research Grant
Primary Cultures of Endothelial and Monocytic Cells Derived from HHT Patients: An Open Window to Unravel the Pathogeniticy of HHT
Blood vessels are lined with endothelial cells that express two proteins known as endoglin and ALK-1. Mutations in endoglin or ALK-1 genes interfere with the expression and function of these proteins in endothelial cells, affecting the vessels network and giving rise to the Hereditary Hemorrhagic Telangiectasia (HHT). Thus, isolation of endothelial cells derived from HHT patients to determine endoglin/ALK-1 levels and activity is crucial to understand how the disease develops. So far, most of the studies have been carried out in endothelial cells from umbilical veins of affected newborns. However, since the prevalence of HHT increases with age, probably these newborn endothelial cells are not a good model system. Instead, endothelial cells from adult patients with known HHT symptoms would be a more appropriate model of choice. In this project, we propose to isolate and characterize endothelial cells derived from blood samples of HHT patients. The same samples will be used to isolate the blood cells called monocytes, which have been shown to express endoglin, although at lower levels than endothelial cells. Both cell types will be used to determine whether the levels and function of endoglin and ALK-1 are affected in HHT patients and whether there is a correlation between these properties and the severity of the disease in the individual patient. Also, the impact of endoglin or ALK-1 mutations on endothelial gene expression using microarrays, will be assessed. These studies may help to understand the mechanisms of the disease, as well as to design new diagnostic methods.
Outcomes: According to Dr. Bernabeu, "Blood vessels are lined with endothelial cells that express two proteins known as endoglin and ALK-1. Mutations in endoglin or ALK-1 genes interfere with the expression and function of these proteins in endothelial cells, affecting the vessels network and giving rise to Hereditary Hemorrhagic Telangiectasia (HHT).
We have isolated and characterized for the first time, circulating endothelial cells derived from blood samples of HHT patients. These cells show functional abnormalities that may explain the vascular lesions of those with HHT. To further investigate the altered function of these cells, we have analyzed the changes in their gene expression pattern (a sort of fingerprint of the cell). This study has allowed the identification of several genes specifically modified in endothelial cells from HHT patients. The genes affected are involved in different cellular processes relevant to the generation of the vascular lesion. Overall, these represent pioneer studies on the establishment of a culture method to isolate endothelial cells derived from HHT patients. Further studies on the genetic fingerprint of these cells will help to explain the molecular mechanism of the disease, which in turn may contribute to the design of novel therapeutic approaches to cure the disease.
Once a culture method to isolate endothelial cells derived from HHT patients is established, this method can be reproduced by other laboratories and these cells can be used for further biochemical and functional analyses. In addition, studies on the specific genes whose expression is affected in HHT cells are necessary. It is crucial to understand why these genes are disregulated and how the protein’s function is affected in the HHT cell.
This study constitutes a solid base to apply for a larger grant. In fact, at the end of this year, I will apply for a three- year grant on an HHT related subject to the Spanish Ministry of Education and Science for a total amount of approx. $300,000 USD.”
- Journal Human Mutation
- Journal Cardiovascular Research - 2005
- Journal Cardiovascular Research - 2006
- Journal Molecular Diagnostics and Genetics
Treatment of Disease: $50,000 Research Grant
Identification of Risk Factors for AVM Formation in HHT
Although mutations in one of two genes, endoglin and ACVRL1, cause HHT, questions pertaining to vascular lesion formation in HHT remain largely unanswered. These vascular lesions in HHT develop in discrete locations, and often increase in number and size with age. The focal nature of these lesions and their age-dependent appearance suggests that yet undiscovered “factors” initiate lesion development. The identification of these initiation factors could lead to new approaches and therapies to reduce the incidence of vascular lesion formation.
According to Dr. Marchuk, "My laboratory has recently shown that older mice heterozygous for an engineered acvrl1 mutation show many features of the human HHT phenotype. Using a genetic approach, we will cross the ACVRL1 mutation into various genetic backgrounds that will enable us to test specific hypotheses concerning the nature of the initiation factor(s) for lesion formation. These genetically sensitized backgrounds are mutant mice that exhibit various potential physiological triggers for lesion formation, including (1) local alterations in blood pressure, (2) hypoxia of the surrounding tissue, (3) local inflammation of the endothelium, or (4) a second somatic mutation in the wild-type copy of the gene."
One (or more) of these sensitized genetic backgrounds may show an increase in AVM formation, lending further support for that particular trigger for lesion formation. Some of these physiological triggers can be reduced by currently-available pharmacological treatments, lending some hope of a long-term treatment outcome from this work. In addition, this work may lead to a more highly-penetrant animal model for HHT for further investigation into the mechanisms of HHT pathogenesis.
Outcomes: Despite the discoveries of the HHT genes, many questions remain unanswered concerning the role of these genes in the pathogenesis of HHT. The most critical question is an explanation for the focal nature of the vascular lesions associated with this disorder. Germline mutations only predispose the individual to the development of the discrete focal vascular lesions. The aim of our proposal was to identify the “trigger(s)” for vascular lesion development.
Various hypotheses have been proposed to explain the actual trigger for lesion development. These include:
- Elevated Blood Pressure (cross with Agt tg and eNos null mice)
- Tissue Hypoxia (cross with Csf2 and EpoR null mice)
- Local Inflammation (cross with IL2 and IL10 null mice) d. Loss of Heterozygosity at Acvrl1 (Cross with p53 null and Blm hypomorph mice)
According to Dr. Marchuk, “We did not find that loss of eNos, IL10, or p53 greatly exacerbated the phenotype of HHT mice. But we can say with some confidence that in the existing HHT mouse models, these various triggers do not appear to be the limiting factor in causing the HHT lesions. After performing the initial crosses to create the appropriate mutant mice, we began aging the mice. Here again we chose the 6 month end point to examine the mice. This final cross is ongoing and we have thus far analyzed only 9 of the double mutant mice. However, we are cautiously excited about our initial results with this cross. In about 1/3 of the mutant mice, we have found grossly dilated vessels on the surface of the liver. We have seen this level of dilation occasionally before, but we have never routinely seen this phenotype in HHT mice at such an early age."
Dr. Marchuk's team is deeply grateful for this funding from the HHT Foundation which made their work possible. "We hope sometime in the future to leverage your investment in our work with a funded NIH grant proposal to continue our work on identifying the trigger for vascular lesion formation in HHT.”