The expression of MMP13 is regulated by certain cytokines, such as interleukin (IL)-1, IL-6, tumor necrosis factor alpha, transforming growth factor beta, basic fibroblast growth factor, and VEGFa.
12,39–42 The most important family of cytokines implicated in the neovascularization processes is the VEGF family.
43 Vascular endothelial growth factor A and VEGFc are members of the VEGF family and have been demonstrated to promote CNV. Vascular endothelial growth factor A, by binding VEGFr2, and VEGFc, by binding VEGFr2 and VEGFr3, stimulate new blood vessel formation.
1,2,43 Previous studies have indicated that VEGFa is correlated with MMP13 expression.
4,41,44,45 However, the correlation between VEGFc and MMP13 expression has not yet been reported. Because VEGFa, VEGFc, VEGFr2, and VEGFr3 have all been showed to be expressed in the cornea, in addition to VECs,
1,2,46,47 we investigated whether their expression was related to MMP13 expression in alkali-burned rat corneas via qRT-PCR and WB. Interestingly, we found that the mRNA and protein expression of VEGFa and VEGFr2 was not significantly different between normal corneas and alkali-burned corneas, although their expression was clear (
Figs. 5A,
5A[a–c]). However, the mRNA and protein expression of VEGFc and VEGFr3 showed a gradual increase from day 1 to 7 after the alkali burns, similar to the expression of MMP13 mRNA and protein (
Figs. 5A,
5A[d–f] versus
Figs. 1A,
1A[a–c]). These data indicated that MMP13 expression was related to the expression of VEGFc and VEGFr3, but not VEGFa and VEGFr2, in alkali-burned corneas. Because keratocytes were the major cells expressing MMP13 in alkali-burned corneas, to confirm that VEGFc andVEGFr3 could be expressed by keratocytes, in addition to VECs, we performed the sequential detection of IHC for CD146 and ISH for VEGFc and VEGFr3 in normal corneas and alkali-burned corneas at day 3. The results showed that VEGFc (
Figs. 5B[d], 5B[j], purple) and VEGFr3 (
Figs. 5B[e], 5B[k], purple) mRNAs were indeed predominantly expressed in spindle-shaped fibroblast-like keratocytes (some indicated by brown arrows). There was some expression in CD146+ VECs (
Figs. 5B[g], 5B[j], 5B[h], 5B[k]; some indicated by red arrows) of neovessels and other cells in alkali-burned corneas. They were also expressed in the epithelium (
Figs. 5B[d], 5B[e], 5B[j], 5B[k]; some indicated by black arrows) and endothelium (
Figs. 5B[d], 5B[e], 5B[j], 5B[k]; some indicated by blue arrows) in normal and alkali-burned corneas, similar to MMP13 mRNA expression (
Figs. 5B[f], 5B[l], purple). The correlation between the expression of VEGFc/VEGFr3 and MMP13 in alkali-burned corneas strongly indicated that the MMP13 expression of keratocytes could be regulated by VEGFc via VEGFr3 but not VEGFa via VEGFr2. Our results also indicated that the MMP13 expression of keratocytes could be regulated by VEGFc from them keratocytes themselves (autocrine) or other cells in the corneas (paracrine).