Fig 2: VEGF-R2/R3 decoy receptors ablate NGF pellet-mediated corneal lymphangiogenesis.Micropellets loaded with PBS or NGF were positioned in a corneal micropocket on experimental day zero. Fc control or a solution of VEGF-R2/R3 decoy receptors was administered subconjunctivally on days zero, two, and four. Corneas were harvested on day seven, immunostained for Lyve-1 and β-III tubulin, and analyzed by whole mount microscopy. A. 100x epifluorescence images of β-III tubulin+ corneal nerves (top panel) and Lyve-1+ lymphatic vessels (bottom panel). Scale bars = 100 µm. B. Quantification of lymphatic vessel density (left panel) and length of outgrowth from limbus (right panel).

Fig 3: NGF mRNA and protein expression during inflammation and wound recovery. A. NGF expression (mRNA) in corneas was evaluated by qRT-PCR in healthy unmanipulated cornea and during initial inflammation, wound recovery, and days one, three, and seven of recurrent inflammation. B. NGF protein expression in corneal lysates was evaluated by ELISA.

Fig 4: Effects of NGF administration during wound recovery on lymphatic vessel regression, nerve density at corneal wounds, and corneal sensitivity.After removing sutures to stimulate wound recovery, mouse β-NGF or PBS control was injected subconjunctivally every other day for two weeks. Corneas were harvested, immunostained for Lyve-1 and β-III tubulin, and analyzed by whole mount microscopy. A. Epifluorescence images of Lyve-1+ lymphatic vessels (left panel, scale bar = 100 µm, 100x), bright field images showing remaining wound beds at sites of suture placement (center panel, scale bar = 200 µm, 32x), epifluorescence images of β-III tubulin+ nerves (right panel, scale bar = 200 µm, 32x). Expanded detail shows confocal analysis of β-III tubulin+ nerves at wound beds (offset panel far right, scale bar = 100 µm, 200x). B. Analysis of effects of exogenous NGF administration on corneal lymphatic vessel density during wound recovery from images like those in (A). C. Quantification of average remaining wound size following administration of NGF or PBS. D. Nerve density at remaining wound beds quantified from confocal immunofluorescence images. E. Quantification of lymphatic vessel fragments discontinuous with limbal lymphatic vessel. F. Quantification of nerve clusters in wound-recovered cornea following administration of NGF or PBS. G. Measurements of corneal sensitivity throughout wound recovery period with administration of NGF or PBS. H. 200x confocal immunofluorescence micrograph showing lymphatic vessel fragments (arrowheads). Scale bar = 100 µm.

Fig 5: In vitro experiments examining the effects of NGF on LECs. A. Adult human dermal lymphatic endothelial cells (LECs) were seeded in the upper inserts of Boyden chamber migration plates. NGF was added to the lower wells at the indicated concentrations. Migratory cells were quantified. B. Matrigel was placed in tissue culture plate wells. LECs were incubated with the indicated cytokines and then added to wells. Tubes were quantified at four hours. C. RNA was extracted from LECs or positive control adult human skeletal muscle and cDNA was synthesized. Gene expression levels of NGF receptor TrkA were quantified by qRT-PCR. D. Gene expression levels of p75NTR in human skeletal muscle and LECs. E. RIPA lysates were made from LECs treated with the indicated cytokines for 1, 5, 15, or 30 minutes. Western blot analysis was performed for the indicated proteins using β-actin as a loading control.