As illustrated by the color photographs taken 3 weeks after transplantation (
Fig. 1A) and immunofluorescent staining of corneal flat mounts (
Fig. 1B), transplant rejection was associated with extensive development of blood (CD31
high/LYVE-1
low) and lymphatic (CD31
low/LYVE-1
high) neovessels, mostly localized around the graft area. The extent of lymphatic vessel formation as well as expression levels of VEGF receptors and ligands were quantified by qRT-PCR in rejected and accepted corneas. As shown in
Figure 1C, qRT-PCR results demonstrated a marked increase in RNA expression of LYVE-1 in rejected compared with accepted corneas (6.90- vs. 1.97-fold increase compared with nontransplanted corneas,
P = 0.006). Expression of VEGF-A (1.84- vs. 0.95-fold increase compared with normal corneas,
P = 0.020), VEGF-C (4.81- vs. 2.34-fold increase compared with normal corneas,
P = 0.002), VEGFR-1 (3.28- vs. 0.63-fold increase compared with normal corneas,
P = 0.0001), and VEGFR-3 (4.33- vs. 1.77-fold increase compared with normal corneas,
P < 0.0001) were also markedly higher in rejected corneas compared with accepted corneas (compared with normal corneas). Despite the marked increase in the expression of VEGFR-2 in transplanted corneas, there was no significant difference in VEGFR-2 expression between accepted and rejected corneas.