Identification of novel treatments for a hereditary stroke disorder using quantitative phenotypic fingerprinting
Creators: name: Gibson, Christopher C.
Publisher: name: University of Utah
Year: 2015
Subjects: Drug discovery
Drug repurposing
Endothelium
High-content
Screening
Stroke

description: Destabilization of the endothelial monolayer lining blood vessels has profound consequences on organismal homeostasis. Vascular instability plays a well-known role in the pathophysiology of diseases from sepsis to stroke. A variety of factors are known to influence endothelial function, including the extracellular milieu, biomechanical factors, various molecular pathways, as well as genetic elements. Many factors promoting vascular instability have been described; however, far fewer studies have identified factors promoting stability. No overarching theory of vascular stability has yet been proposed that takes into account extracellular, biomechanical, molecular, and genetic variables. In this dissertation, I detail studies of extracellular matrix cues, molecular pathways, and genetic factors in an attempt to identify commonalities associated with regulation of vascular stability. I first show that the extracellular matrix protein elastin normalizes endothelial cell function. Next, I demonstrate a critical role of the small GTPase ARF6 in mediating cytokine-induced endothelial instability. I also identify a crucial role of ARF6 in mediating transduction of mechanical signals in the endothelium. Finally, I describe a central role of various genes associated with a human disease, Cerebral Cavernous Malformation (CCM), in endothelial stability. During the course of these studies, I developed and utilized a variety of tools not often found in molecular biology labs. I built new apparatuses and wrote software programs to answer the questions I had, rather than relying on what had already been built by others. Perhaps my largest contribution to the Li laboratory has been to propagate the use of these new systems to molecular biologists who may have a better capability to ask important questions, and who now have the ability to answer their own questions more quickly, more efficiently, and without the inherent bias associated with the standard protocols used in the majority of molecular biology labs. Finally, during my last two years in the laboratory, I further refined and integrated the tools I developed with new tools available from the Broad Institute to quantitatively evaluate endothelial stability through measurement of both structural and functional phenotypes. Using quantification of structural and functional phenotypes, I identified multiple drugs that ameliorate in vitro CCM models, and found two drugs that significantly reduce the formation of lesions in murine models of CCM disease. Further, I found that one of these compounds was protective against diverse destabilizing cues in the endothelium. These discoveries are important for the study of CCM disease, for patients with CCM disease, and have implications for many other diseases in the future. Overall, my studies resulted in specific new mechanistic insights into a wide-variety of factors that promote endothelial stability. More important is the development of a multilayered strategy and platform that can be applied to study effects of extracellular, biomechanical, molecular, and genetic cues on the endothelium either alone or in any combination. In the immediate future, this platform will serve as a vehicle for creating an over-arching model of vascular stability. Beyond this application, this same platform can be rapidly scaled to answer broad questions about factors crucial in health and disease across a wide variety of other cell and organ types. I intend to use the approach I developed and describe in my dissertation to try to untangle how all genes, all proteins, all diseases, and all drugs interact - a lofty goal to be sure.