Abstract
Purpose :
Chronic inflammation plays a major role in the pathogenesis of Keratoconus (KC), a progressive, bilaterally asymmetric, corneal ectatic disease. Analysis of patient corneas and tears demonstrate that KC pathology is driven by elevated MMP9 and inflammatory cytokines alongside reduced Lysyl Oxidase (LOX) and collagen (COL) levels. Thus, we tested if targeted inhibition of MMP9 can reduce inflammatory signaling and rescue repression of ECM production and regulation.
Methods :
Chronic inflammation was modelled in human corneal epithelial and corneal fibroblast cells (HCE and HCF) with 2 ng/ml rhTNFα for 7 days. Drug treatments were performed for 5-7 days with Cyclosporine A (CsA; immunomodulator with MMP9 inhibitory activity), BAPN (LOX activity inhibitor) or Batimastat (Bst; MMP family specific inhibitor). Treated cells were assessed for changes in expression levels of LOX, MMP9, COLs as well as activation of p65, p38 and ERK1/2 pathway by qPCR and/or immunoblotting. In addition, primary HCF (pHCF) isolated from KC and healthy corneas were treated with either TGFβ1, CsA, BAPN or Bst for 7 days and analyzed for its COL gel contraction ability.
Results :
Inflammatory stimuli resulted in phosphorylation of p65, p38 and ERK1/2, along with an elevation of MMP9 and reduction of LOX, COL IV and VI levels in both HCE and HCF cells. Bst treatment reduced the expression of MMP-9, -1 and -3, but not MMP2, as well as phosphorylation of p38 and ERK1/2. Furthermore, Bst was observed to the reduce mRNA and protein expression of MMP9 while elevating LOX. On the other hand, though CsA reduced TNFα-induced MMP9 levels, it did not induce elevation of LOX or COLs. BAPN did not have any effect on MMP9, LOX or COL levels. Compared to controls, pHCF from KC patients exhibited significantly reduced ability to contract a COL gel, and the gel contraction ability of KC pHCF was improved by Bst and TGFβ1 treatments, but not with BAPN.
Conclusions :
We demonstrate that targeting MMP9 and associated signaling can rescue inflammatory stimuli-induced reduction in LOX and COL expression. Bst induced elevation of LOX with concurrent inhibition of p38 and ERK1/2 suggests that the mechanism of LOX rescue may be transcription factor dependent. Further, improving or maintaining LOX levels resulted in greater collagen stiffening by the corneal fibroblasts. Thus, Bst may be a novel therapeutic modality for treating KC.
This is a 2020 ARVO Annual Meeting abstract.