What have we learned from the DNA & Tissue Bank? A lot!!!

If you have participated in the DNA & Tissue Bank, thank you!!  You have given a tremendous resource to researchers who are using your DNA, tissue samples, and clinical information to make amazing advances in research.  Below are some examples of projects being explored by researchers using our resources:

Example #1

Investigation of CCM3 function – Description provided by Thomas Force, MD, Professor of Cardiology at Thomas Jefferson University, Jefferson Medical College, Philadelphia, PA.


We have been studying the molecular mechanism by which cavernous malformations are generated.  We have a lot of evidence in cells as to how a mutation in PDCD10 leads to the CCM3 malformations.  However, this evidence is only in cultured cells and we need to determine if this could be the mechanism in patients.  We can only accomplish this by directly studying tissue obtained from patients with malformations.


As far as the proposal’s significance, by identifying the mechanism by which PDCD10 mutations lead to malformations, and the specific proteins involved in the process, potential therapeutics could be generated to prevent the development and/or progression of CCMs.

The results of Dr. Force’s research project were published in the Journal of Cell Science in March of 2010.

Example #2

IMMUNE RESPONSE IN HUMAN CEREBRAL CAVERNOUS MALFORMATIONS – Description provided by Issam Awad, MD, Professor of Neurosurgery at the University of Chicago, Chicago IL.


This pilot proposal will characterize cellular and humoral components of the immune response in CCMs.  The hypothesis to be tested is that CCM lesions are associated with immunoglobulin producing B cells and plasma cells.  The Specific Aims propose to characterize the predominant immunoglobulin isotype and distribution of inflammatory cells in CCM lesions, including lesions with and without recent aggressive clinical behavior.


Cerebral cavernous malformations (CCMs) affect more than 1 million Americans, predisposing them to a lifetime risk of hemorrhagic stroke and epilepsy.  Immunoglobulin and other related genes are markedly unregulated within human CCM lesions.  Preliminary work suggests the infiltration of B-lymphocytes and plasma calls within the lesions.  A potential role of the immune response in this disease has not been postulated previously, but would be compelling, given the unique antigenic milieu of CCM lesions with sequestered thrombi and leaky blood-brain barrier, and the numerous examples of immune modulation of angiogenesis in other disease states.  It could explain, in part, why some CCM lesions remain biologically dormant, while others proliferate with serious clinical consequences.  Immune response could play a role in or represent a potential marker of cerebral cavernous malformation lesion proliferation and hemorrhage.  The proposed pilot studies will generated preliminary data for future research aimed at comparing the immune response in quiescent versus clinically aggressive CCM lesions and would stimulated rupture research to indentify autoimmune or extrinsic antigenic triggers involved in CCM disease.

This line of research, how inflammation is involved in CCM disease, has been a focus of Dr. Awad’s laboratory for the past few years.  Bleeding from lesions certainly alters the brain environment immediately surrounding the cavernous angioma.  These researchers have shown that there is a significant and specific influx of immune responding cells (those that produce anti-bodies) to the site of lesions.   While the precise role of these immune cells remains unknown, one might speculate that localized inflammation may be a contributing factor to lesion development.  Therapeutically, this line of research is exciting because if inflammation is shown to cause lesion development, treatments could be developed to target and control localized inflammation to inhibit the growth of new lesions.  More to come…but this is an exciting step in the right direction.

Example #3

INVESTIGATION OF THE TWO-HIT MECHANISM FOR CCM DISEASE PATHOGENESIS – Description provided by Douglas Marchuk, PhD, Professor of Molecular Genetics and Microbiology, Duke University, Durham, NC.


Cerebral cavernous malformations (CCMs) can develop sporadically, but the risk to develop CCMs can also be inherited by mutation in one of thee recently identified genes.  The presence of single lesions in sporadic cases and multiple lesions in inherited cases has led us to hypothesize that CCM lesion formation follows the Knudson two-hit mechanism.  The two-hit mechanism occurs when a person has one inherited mutation in a CCM gene and then acquires another mutation in specific cells.  We believe that acquiring the second mutation is necessary for lesion formation.  We can test our hypothesis by analyzing human CCM lesions tissue for the presence of two mutations.


We propose that CCM lesions formation follows the Knudson two-hit mechanism, in which a person has one inherited mutation in a CCM gene and then acquired another mutation in specific cells.  The two-hit hypothesis for CCM lesions formation is consistent with the repeated observational difference between sporadic and familial CCM; multiple lesions develop in patients with familial CCM, but single lesions develop almost exclusively in sporadic cases.  The two-hit model states that both copies of a gene must be mutated to yield lesion formation.

We propose to test the two-hit hypothesis using CCM lesion tissue samples.  The two-hit hypothesis predicts that each class of CCM lesions (those with inherited mutations in CCM1, CCM2, or CCM3) will show somatic mutation within the lesion.  Additionally, the two-hit hypothesis predicts that sporadic CCM lesions will routinely harbor two distinct somatic mutations within one of the CCM genes.  A key prediction of the two-hit hypothesis regarding the molecular genetic origins of multiple lesions in familial cases is that multiple lesions arise due to independent somatic mutational events.  Through successful completion of these experiments, we hope to more fully understand the underlying fundamental biology of CCM.  Understanding how CCMs develop may eventually aid in future therapies for the condition.

This is the research project that I (Amy Akers) worked on before joining Angioma Alliance, while I was conducting research at Duke.  This work was published in the journal of Human Molecular Genetics in 2009.  Results of this project indicated that CCM lesion genesis follows the hypothesized two-hit model.  Individuals with familial cavernous angioma inherit one mutation in one of the CCM genes from a parent.  This mutation is present throughout the entire body, but because we all have two copies of each of our genes, this single mutation was not sufficient to cause disease onset.  (Not all genetic diseases behave this way; sometimes a mutation of a single copy of a gene is sufficient for disease onset.)  However for Cavernous Angioma, when a second mutation is acquired within the blood vessels of the brain resulting in complete loss of function for CCM1, CCM2, or CCM3, this complete loss of function for one of these necessary genes is what causes lesions to develop.

Example #4

CCM PROTEIN FUNCTION AND ENDOTHELIAL CELL DYNAMICS – Description provided by Gary Johnson, PhD, Professor of Pharmacology at the University of North Carolina at Chapel Hill.


Endothelial cells, the cells that make up blood vessels, maintain vascular integrity through a complex balance of signals, ultimately resulting in appropriate shaping of the cytoskeleton- the network of fibers that gives a cell shape, adhesion, and motility.  Recent research has shown that levels of RhoA, a protein responsible for cytoskeleton dynamics, is greatly increased in endothelial cells missing any of the three CCM proteins in vitro.  Additionally, endothelial cells lacking the CCM proteins show a number of defects in vitro, including lack of tube formation, low levels of migration, and lowered invasion- defects that are consistent with problems rearranging the cytoskeleton.  Using tissue from patient samples, we hope to validate our in vitro findings in humans, and furthermore to explore whether other proteins critical to the cytoskeleton are at appropriate levels in the context of CCM genetic mutation.  This work will provide insight to the molecular mechanisms by which loss of CCM protein leads to the CCM clinical condition.


Clinically, CCMs are characterized by clusters of leaky, dilated blood vessels – in other words, a lack of vascular integrity.  Normally, endothelial cells maintain vascular integrity through a complex balance of signals, ultimately resulting in appropriate shaping of the cytoskeleton – the network of fibers that gives a cell shape, adhesions, and motility.  At a cellular level, many proteins are involved in maintaining the cytoskeleton, but some of the most important are the Rho GTPases.  In recent in vitro studies, the level of one of the members of this family, RhoA, was found to be strikingly elevated in cells lacking any one of the CCM proteins, with resulting defects in endothelial cells, including their ability to migrate, form tubes, and invade though extracellular matrix.  This discovery points to a possible mechanism for development of CCM – increased levels of RhoA cause cytoskeleton dysregulation and consequently endothelial cells are not able to do their job.  Importantly, pharmacological blockade with a Rho kinase inhibitor drug was able to rescue cell function in several in vitro tests.  This suggests that Rho kinase may be a therapeutic target in CCM disease.  The next step is to test this hypothesis in CCM patient samples.  We propose to immunostain for protein levels of RhoA and other important Rho GTPases in human samples.  This research would both serve to validate previous in vitro findings and help us better understand the molecular consequences of CCM mutation.  Better knowledge of the effects of CCM proteins on signaling pathways allows us to test new potentially therapeutic pharmacological inhibitors.

When you think of blood vessels, you likely picture a static structure that simply contains our blood.  But in reality, endothelial cells – those that make up our blood vessels – are active cells.  Take wound healing for example…if you cut yourself, your vessels are damaged, you bleed, and the vessel needs to be mended.  Through a highly coordinated (and very complex!) cascade of genetics and signaling molecules, cells are instructed to change their shape, so they are able to move to a new site where they will grow and divide to mend the broken vessel.  Then, once the fix is completed, another set of signals must tell the cells to go back to their normal resting state.  This project is designed to investigate a similar type of cellular behavior and the signaling molecules (RhoA) that control the endothelial cells.  RhoA, and related molecules are of particular interest to Cavernous Angioma researchers of late because these are the targets for statin drugs that are currently used to treat cholesterol and may become a treatment option for Cavernous Angioma.  More basic research is required to fully understand how statins may affect individual with CA, and this project can provide important insight.  This is the newest project initiated with your samples from the DNA & Tissue bank – stay tuned for results in the coming months…

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