Fig 1.
Expression of transient receptor potential vanilloid 4 (TRPV4) in corneas of wild type mice.
A: TRPV4 expression is more preponderant in the basal epithelial cell layer (arrowheads) in the intact mouse cornea (arrowheads) with some indication of expression in the suprabasal layers. However, there is no indication of its presence in the top tear-side layer. B: Stromal fibroblasts undergo TRPV4 upregulation during healing at day 10 post-alkali burn in (asterisk). Scale bar = 100 μm.
Fig 2.
Identical histology of the wild type (WT) and TRPV4 knockout (KO) mouse corneas.
There are no discernible differences in the corneal histology of WT (A) and (B) KO based on a comparison of hematoxylin-eosin (HE) staining patterns. Scale bar = 100 μm.
Fig 3.
Comparison of WT and TRPV4 knockout alkali-burned corneal wound healing profiles.
A: The corneas of wild type (WT) and TRPV4 KO mice at 0, 5, 10 or 20 days, respectively. At each time point after alkli burn, the degree of opacification in the burned cornea was more prominent in WT mice than TRPV4 KO mice. B: Eyeball diameter resulting from wound healing shows that WT globes are smaller at 20 days than the TRPV4 KO globes. C and D: Histology of burned corneas stained with hematoxylin and eosin (HE) and immunohistochemical findings at day 10 (C) and 20 (D). HE staining shows the burned cornea exhibits more severely disorganized and thicker stroma in WT corneas as compared with TRPV4 KO tissues at both day 10 (C) and 20 (D). Immunohistochemistry suggests that the density of the myeloperoxidase (MPO)-labeled polymorphonuclear neutrophils at day 10 and F4/80-labeled cells (macrophages) at 20 days is greater in a WT cornea as compared with a TRPV4 KO cornea. Healing burned corneas contain many more α-smooth muscle actin (αSMA) positive myofibroblasts in WT mice at day 10 (C) and 20 (D). Scale bar = 100 μm. E: Real-time RT-PCR, showed that mRNA level of MPO was less in the healing stroma of a TRPV4 KO mouse at day 5. F: Expression of F4/80 increased at 20 days in alkali-burned WT corneas. G: mRNA expression of αSMA was significantly suppressed by the loss of TRPV4 in the in vivo healing alkali-burned corneas on days 5 and 10, but not at day 20. Data expressed as mean ± standard error of the mean (SEM) from five specimens in each condition. *P < 0.05, Scale bar = 100 μm.
Fig 4.
Expression pattern of wound healing-related genes in an alkali-burned cornea.
A: Expression of monocyte-chemoattractant protein-1 (MCP-1) was transiently larger at 10 days in an alkali-burned wild-type cornea (WT) than in its TRPV4 KO counterpart but then fell to a level indistinguishable from the TRPV4 KO at 20 days. B: Lacking TRPV4 abolishes interleukin-6 (IL-6) mRNA expression compared to the WT at 5 and 20 days. It is noteworthy that in KO cells IL-6 gene expression was undetectable at 5 and 20 days. C: Difference in VEGF gene expression between WT and KO cell vascular endothelial growth factor (VEGF) mRNA reaches significance at 20 days due to loss of TRPV4 gene function. D: Expression level of transforming growth factor β1 (TGFβ1) was not affected by the loss of TRPV4 function throughout the healing interval. E: Expression of collagen Ia1 mRNA increases after 20 days in the healing alkali-burned WT corneas. Data represent mean ± SEM from five specimens in each condition. *P < 0.05, scale bar = 100 μm
Fig 5.
Effects of loss of TRPV4 function on TGFβ1 and IL-6 expression in ocular fibroblasts.
Comparison of effects of TGFβ1 on VEGF and αSMA expression in WT and TRPV4 KO ocular fibroblasts. A: Loss of TRPV4 gene function does not reduce TGFβ 1 since its levels were the same irrespective of the presence or absence of TRPV4 mRNA expression. B: Loss of TRPV4 gene function reduces IL-6 gene expression. C. Exogenous TGFβ1 increases VEGF mRNA expression levels that are dependent on TRPV4 gene expression. In loss of TRPV4 function fibroblasts, TGFβ1 fails to increase VEGF gene expression. D: Exogenous TGFβ1 induces increases in αSMA expression irrespective of the presence or absence of TRPV4 gene expression. TGFβ1 induced rises were larger in WT ocular fibroblasts than in their TRPV4 KO counterpart. mean ± SEM *P < 0.05, Scale bar = 100 μm.
Fig 6.
Dependence of fibrogenic gene expression on gain of TRPV4 function in peritoneal macrophages.
A: Real-time RT-PCR showed that mRNA of MCP-1 was markedly suppressed by TRPV4 gene ablation. B. Difference in IL-6 gene expression between that in WT fibroblasts and TRPV4 KO macrophage was insignificant C. Difference in TGFβ1 gene expression between that in WT macrophage and TRPV4 KO macrophage was insignificant. D: Difference in VEGF gene expression between that in WT macrophage and TRPV4 KO macrophage were insignificant.
Fig 7.
Time dependent corneal alkali burn-induced wound healing responses in mouse chimeras receiving bone marrow transplantation (BMT).
A: The corneal response to an alkali exposure in chimera mice (WT-to-WT, KO-to-WT and WT-to-KO group) 10 and 20 days after alkali burn. Inflammation (distribution of MPO-labeled PMNs and F4/80-positive macrophages) and myofibroblast distribution (a tissue fibrosis marker) in corneal stroma of chimeric mice of the WT-to-KO and KO-to-WT groups seemed less severe as compared with those in the WT-to-WT group mice. B and C: HE stained histology shows that increases in cell density in the swollen stroma of a WT-to-WT cornea as compared with a KO-to-WT and a KO-to-WT tissue after 10 and 20 days. Immunohistochemistry shows that the cornea of a WT-to-KO and KO-to-WT mouse has less stromal αSMA staining as well as lower levels of MPO and F4/80 immunoreactivity as compared with the mice undergoing WT-to-WT transplantation 10 and 20 days post-alkali burn. Scale bar is 100 μm.
Fig 8.
TRPV4 antagonist (HC-067047) injection attenuates corneal inflammatory fibrosis induced by alkali burn in wild-type (WT) mice.
A. The corneas of WT mice treated with or without HC-030031 at 5, 10 or 20 days. Corneal transparency restoration is markedly improved in the mice treated with a TRPV4 antagonist HC-067047 at each time point. B. Eyeball diameter during wound healing after alkali burn shows untreated globes are smaller at 20 days than antagonist treated globes. C and D. Histology of burned corneas stained with HE and immunohistochemical stained at day 10 (C) and 20 (D). The stromal organization is more disorganized in untreated mice cornea than in antagonist treated mice. The antagonist treated mice cornea has lower levels of infiltration of MPO-labeled neutrophils and F4/80-positive macrophages as well as less marked αSMA staining at each time point. Scale bar is 100 μm.