Lymph vs. blood angiogenesis: functional differences in*
The physiology of lymphatic regeneration is much less understood than that of angiogenesis, in spite of the importance of lymphangiogenesis in important pathophysiological processes such as tumor metastasis. Indeed, it is generally assumed that many aspects of lymphangiogenesis parallel those in angiohgenesis. This bioengineering study will address the differences between these two processes, using in vitro models as well as a novel animal model of lymphangiogenesis in regenerating skin, developed in our laboratory. Our preliminary observations of lymphatic development using this model indicate that: 1) fluid channel formation occurs prior to lymphatic endothelial cell (LEC) organization, 2) LECs migrate as single cells within the interstitial space before organizing into vessels, 3) protease activity, fluid channeling, VEGF-C protein expression, and LEC migration all occur predominantly from the upstream end, consistent with the direction of lymph flow, and 4) reduction of interstitial flow interrupted lymphatic capillary organization, despite identical epithelial cell migration and angiogenesis. These observations obtained in this model suggest clear and fundamental differences between lymphangiogenesis and blood angiogenesis, where MMP activity at the surface of the sprouting vessel allow capillary progression and where BECs are always connected to their neighboring cells and exposed to a lumen, even while proliferating and advancing. They also represent the first evidence that lymphatic development is influenced by interstitial flow. Based upon preliminary data presented, our working hypothesis is as follows: Interstitial fluid flow is an initiating event in lymphangiogenesis by causing MMP upregulation and transport ("streaming") in the direction of flow, leading to fluid channel formation and directed LEC migration along those channels. The LECs then organize to remodel the crude fluid channels into a functionally optimal network of lymphatic capillaries. This investigation will explore this hypothesis and connect the function of the lymphatics to the process of their formation in several regards, correlating biochemical and biophysical/transport mediators in lymphangiogenesis, with attention to fluid channeling, remodeling of the extracellular matrix (ECM), LEC migration, VEGF-C expression, and lymphatic capillary network formation. The role of protease streaming in channel formation will be clarified, as will the mitogenic vs. morphogenetic function of VEGF-C. In vitro, we will correlate LEC responses to fluid and growth factors with LEC behavior observed in vivo during lymphangiogenesis, and we will compare those results to BEC responses in vitro.