Date of Graduation

5-2015

Document Type

Dissertation (PhD)

Program Affiliation

Immunology

Degree Name

Doctor of Philosophy (PhD)

Advisor/Committee Chair

Eva. M. Sevick-Muraca, Ph.D.

Committee Member

R. Eric Davis, M.D.

Committee Member

Jeffrey E. Gershenwald, M.D.

Committee Member

Philip D. King, Ph.D.

Committee Member

Bradley W. McIntyre, Ph.D.

Abstract

The lymphatic system is essential for the transport of excess fluid, protein, and foreign materials from interstitial tissues to lymph nodes; for immune surveillance, and to maintain fluid homeostasis. Dysregulated lymphatics can be attributed to pathological conditions including tumor metastasis, inflammation, chronic wounds, obesity, blood vascular disorders, and lymphedema. Of these, lymphedema is the most extreme of lymphatic disorders and is represented by a spectrum of symptoms ranging from mild, subtle presentation to severe, disfiguring, overt presentation. Lymphedema is more manageable in the early stages of disease but severely reduces quality of life with progression. Due to lack of molecular knowledge and inadequate imaging techniques to safely, rapidly and non-invasively visualize the lymphatics, lymphedema remains under diagnosed and progresses to the irreversible stage if not diagnosed early.

Candidate gene studies have identified a myriad of genes responsible for lymphedema, however, majority of patients do not harbor mutations in these putative genes, indicating many more unknown genes contribute to the pathology of this disease. In an effort to identify new polymorphisms that possibly effect lymphatic dysfunction, we combined investigational, non-invasive near-infrared fluorescence lymphatic imaging (NIRFLI) and next generation sequencing (NGS), to phenotype and genotype human subjects with familial lymphedema. We discovered that mutations in src homology 2-domain containing 5’-inositol phosphatase-2 (SHIP2), encoded by INPPL1, are associated with lymphatic abnormalities. SHIP2 is a phosphatidyIinositol (3,4,5) triphosphate (PIP3) 5’-phosphatase that negatively controls PIP3 levels thereby inhibiting the PI3K/AKT signaling, a pathway implicated in various lymphatic disorders. Our studies confirm this inhibitory role of SHIP2 against PI3K/AKT in lymphatic endothelial cells, and identify SHIP2 as a potential regulator of MAPK/ERK signaling, another pathway also recently identified as important in lymphatic malformations. Pharmacological inhibition of SHIP2 impedes lymphatic contractility and impairs the normal wound healing processes of lymphangiogenesis and angiogenesis in mice. These studies suggest that SHIP2 could have a previously unidentified effector role in lymphatic dysfunction.

In elucidating the roles of SHIP2 in lymphatic dysfunction, the work presented herein expands our understanding of molecular basis of lymphatic failure which could have clinical implications affecting populations with lymphatic disorders, including the ever increasing population of cancer survivors who experience the chronic, disfiguring and incurable lymphedema.

Keywords

Lymphatic System, Lymphedema, SHIP2, INPPL1, Near Infrared Fluorescence Imaging, Next Generation Sequencing, PI3K/AKT, MAPK/ERK, AS1949490, Cancer Survivorship, Lymphangiogenesis, Angiogenesis, Endothelial Cell Biology