Molecular mechanisms regulating lymphangiogenesis

Lymphangiogenesis, described as growth of nascent lymphatic vessels in vivo, and proliferation, migration, tube formation of lymphatic endothelial cells (LECs) in vitro, plays an important physiological role in homeostasis, metabolism and immunity. Lymphatic vessel formation has also been implicated in a number of pathological conditions including cancer metastasis, lymphedema and impaired wound healing. Elucidating molecular mechanisms that regulate LEC motility and its relationship to lymphangiogenesis will play an important part in understanding lymphatic biology and in studying disease associated with altered lymphatic vessel architecture or function. Cancer is the most commonly diagnosed malignancy and the leading cause of mortality around the globe. A key turning point in the course of cancer progression is the development of metastatic potential as most cancer related deaths are not due to the primary tumor, but rather to metastases. A better understanding of molecular mechanisms that underlie the process of cancer metastasis to lymph nodes will most likely result in improved therapeutic options for patients and thus help reduce the burden of the disease. This project focuses on discovering the molecular mechanisms that regulate the spread of cancer to lymph nodes and lymphangiogenesis, and will identify potential new targets for therapy. Lymph node metastasis is the first route of cancer cell dissemination, and may provide a bridgehead that subsequently results in tumor cell seeding into the lymphatic system to distant organs, which in turn leads to one of the major causes of morbidity associated with cancer progression. Currently, there are no effective therapies against these metastases. The delineation of the processes that result in lymphangiogenesis, including the roles of LECs in vitro, and the interaction between cancer cells and lymphatic environment in vivo is anticipated to lead to novel therapeutic strategies. The first aim of this study was to identify signaling pathways regulating lymph node metastasis via vascular endothelial growth factor receptor (VEGFR)-3. This was investigated in vitro using human LECs (hLECs), and in vivo using clinical material. The direct association of VEGFR-3/ phosphatidylinositol 3-kinase (PI3K) was shown for the first time in the metastatic small cell lung carcinoma samples. Second aim of the study was to isolate and characterise murine LECs (mLECs) from dermis and prostate. We have shown that regulation of lymphangiogenesis by VEGF family ligands is conserved in mLECs and hLECs. This finding was an important discovery into how mLECs are regulated and will provide bridgehead into using these mLECs as tools for studying lymphatic system related diseases such as cancer. NADPH oxidases (Nox) family are emerging as novel regulators of tumor angiogenesis and were previously shown to regulate blood vascular endothelial signaling via reactive oxygen species (ROS). Nox2 was described as a major regulator of migration and proliferation of vascular endothelial cells. In the third aim of the study, using the techniques described in Aim 2, mLECs were derived from Nox2-/- mice and wild-type mice in order to define the role of Nox2. Key signaling pathways regulated by Nox2/VEGFR-2 that modulate murine and human LEC migration and tube formation were identified. This work provides an important insight into the role of Nox2 as a potential regulator of lymphangiogenesis. These findings add to the current knowledge of VEGF receptor signaling pathways, and introduce a potentially important new player, Nox2. Hence, targeting VEGF receptor family/Nox2 may provide new emerging therapeutic targets and could have beneficial clinical effects in treatment of lymphatic associated disorders.