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Richard M Tempero, Alicia L Connor, Philip M Kelley; Cellular mechanisms of corneal lymphangiogenesis revealed by live imaging. Invest. Ophthalmol. Vis. Sci. 2014;55(13):5047.
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© ARVO (1962-2015); The Authors (2016-present)
The lymphatic vasculature regulates normal physiology and plays an important role in corneal inflammatory disease. Here we investigate the cellular mechanisms and proliferative capacities of newly synthesized lymphatic vessels using inductive genetic lineage tracing studies and quantitative analysis.
Transgenic mice were developed that carry an inducible CRE recombinase-estrogen receptor construct targeted to lymphatic endothelial cells (LECs) and a td tomato reporter gene. We used this inductive transgenic strategy in adult mice to label a sample population of LECs. We followed the fate of their progeny at single cell resolution in real time using intravital microscopy during suture induced corneal lymphangiogenesis. We performed morphologic and quantitative analysis to describe mechanisms of growth.
Using high dose inductive conditions we visualized and quantified a dynamic process of lymphatic vessel growth and contraction, and diverse proliferative capacities in td tomato+ LECs during lymphangiogenesis. The majority of the new growth was generated by sprouting from the preexisting limbal lymphatic vessel. Quantitative analysis of this data showed that the proliferation rate of unbranched lymphatic vessels approximated a linear mathematical function, about 1 cell/day, and that the proliferation rate of branched lymphatic vessels was more complex approximating a geometric function. We used low dose inductive conditions to label a trace frequency of LECs, a strategy that allowed us to visualize clonal expanding td tomato+ lineages within newly synthesized lymphatic vessels. These results suggest that multiple LECs contribute to sprout formation and that branch elongation results from LEC proliferation in a spatially restricted linear, rather than a circumferential, dimension.
The results are consistent with a progenitor model of corneal lymphangiogenesis in which many preexisting LECs support new lymphatic vessel growth in response to inflammatory disease conditions.
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