Suppression of In Vivo Neovascularization by the Loss of TRPV1 in Mouse Cornea

To investigate the effects of loss of transient receptor potential vanilloid receptor 1 (TRPV1) on the development of neovascularization in corneal stroma in mice. Blocking TRPV1 receptor did not affect VEGF-dependent neovascularization in cell culture. Lacking TRPV1 inhibited neovascularization in corneal stroma following cauterization. Immunohistochemistry showed that immunoreactivity for active form of TGF 1 and VEGF was detected in subepithelial stroma at the site of cauterization in both genotypes of mice, but the immunoreactivity seemed less marked in mice lacking TRPV1. mRNA expression of VEGF and TGF 1 in a mouse cornea was suppressed by the loss of TRPV1. TRPV1 gene ablation did not affect invasion of neutrophils and macrophage in a cauterized mouse cornea. Blocking TRPV1 signal does not affect angiogenic effects by HUVECs in vitro. TRPV1 signal is, however, involved in expression of angiogenic growth factors in a cauterized mouse cornea and is required for neovascularization in the corneal stroma in vivo

Loss of TRPV4 Function Suppresses Inflammatory Fibrosis Induced by Alkali-Burning Mouse Corneas

<div><p>In humans suffering from pulmonary disease and a mouse model, transient receptor potential vanilloid 4 (TRPV4) channel activation contributes to fibrosis. As a corneal alkali burn induces the same response, we determined if such an effect is also attributable to TRPV4 activation in mice. Accordingly, we determined if the alkali burn wound healing responses in wild-type (WT) mice are different than those in their TRPV4-null (KO) counterpart. Stromal opacification due to fibrosis in KO (n = 128) mice was markedly reduced after 20 days relative to that in WT (n = 157) mice. Immunohistochemistry revealed that increases in polymorphonuclear leukocytes and macrophage infiltration declined in KO mice. Semi-quantitative real time RT-PCR of ocular KO fibroblast cultures identified increases in proinflammatory and monocyte chemoattractant protein-1 chemoattractant gene expression after injury. Biomarker gene expression of fibrosis, collagen1a1 and α-smooth muscle actin were attenuated along with macrophage release of interleukin-6 whereas transforming growth factor β, release was unchanged. Tail vein reciprocal bone marrow transplantation between WT and KO chimera mouse models mice showed that reduced scarring and inflammation in KO mice are due to loss of TRPV4 expression on both corneal resident immune cells, fibroblasts and infiltrating polymorphonuclear leukocytes and macrophages. Intraperitoneal TRPV4 receptor antagonist injection of HC-067047 (10 mg/kg, daily) into WT mice reproduced the KO-phenotype. Taken together, alkali-induced TRPV4 activation contributes to inducing fibrosis and inflammation since corneal transparency recovery was markedly improved in KO mice.</p></div

Identical histology of the wild type (WT) and TRPV4 knockout (KO) mouse corneas.

<p>There are no discernible differences in the corneal histology of WT <b>(A)</b> and <b>(B)</b> KO based on a comparison of hematoxylin-eosin (HE) staining patterns. Scale bar = 100 μm.</p

TRPV4 antagonist (HC-067047) injection attenuates corneal inflammatory fibrosis induced by alkali burn in wild-type (WT) mice.

<p><b>A.</b> 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>B.</b> Eyeball diameter during wound healing after alkali burn shows untreated globes are smaller at 20 days than antagonist treated globes. <b>C and D.</b> 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.</p

Effects of loss of TRPV4 function on TGFβ1 and IL-6 expression in ocular fibroblasts.

<p><b>Comparison of effects of TGFβ1 on VEGF and αSMA expression in WT and TRPV4 KO ocular fibroblasts. A:</b> 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>B:</b> Loss of TRPV4 gene function reduces IL-6 gene expression. <b>C.</b> 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. <b>D:</b> 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 *<i>P</i> < 0.05, Scale bar = 100 μm.</p