Abstract
Purpose :
Corneal chemical burn is characterized by the presence of activated keratocytes (KC) which produce a variety of cytokine and growth factors resulting in the accumulation of extracellular matrix (ECM), and consequently leading to corneal fibrosis. KCs are derived from the corneal stroma which rapidly transit into activated myofibroblasts (MFBs) following stimulation. We hypothesized that selective blocking of activated KC would ameliorate corneal fibrosis and associated complications. Recently, we discovered that utilizing an engineered recombinant TRAIL (TRAILPEG) selectively induces apoptosis in activated MFBs, but not in normal cells in experimental models of liver and pancreatic fibrosis. To this end, we applied our hypothesis in experimental models of corneal fibrosis.
Methods :
Primary human and rat KCs were activated with TGF-β and treated with TRAILPEG. Apoptosis assay, Western blot (WB), and gene expression by qRT-PCR (Acta2, Col1a2, Dr4, Dr5, and Tgf-β) were performed to analyze the efficacy of TRAILPEG. Immunofluorescence staining and flow cytometry were used to determine whether DRs will be colocalized with α-SMA in activated KCs. The corneal alkali-burn injury was induced by application of NaOH onto the cornea of Lewis rats (n=4). Briefly, the filter paper was soaked in 1 N NaOH and then placed on the central cornea for 60 sec. 5 days later, the rats were assessed by the pathophysiological score, WB, and immunofluorescence to verify the TRAIL signaling on the sections by DRs/α-SMA dual-staining. Student’s t-test was used for statistical analysis.
Results :
We investigated the effects of TRAILPEG in TGF-β activated KCs and the role of TRAIL signaling in DR-mediated apoptosis. Activated KCs highly express fibrogenic markers including α-SMA and collagen with a membrane expression of TRAIL receptors. Selectively TRAIL-induced apoptosis was observed by WST-1 (60%) and caspase activity assay, whereas changes were almost negligible in normal KCs after treating with TRAILPEG. To validate the clinical feasibility of the TRAILPEG in vivo, a moderate chemical burn injury was induced by NaOH. We found highly upregulated levels of α-SMA and colocalization of DR5+ cells in the α-SMA+ area when directly compared to the control cornea.
Conclusions :
This study indicated that targeting a TRAIL signaling to eliminate activated KCs, originators for corneal fibrosis, can possibly provide an effective therapy for corneal chemical burn patients treatment.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.