Molecular insights into ECM and collagen assembly disruptions in keratoconus

Abstract

Purpose : The underlying mechanisms of Keratoconus (KC) progression is poorly understood. Collagen fibril assembly at points of nucleation on the cell surface is a highly regulated, complex multistep process requiring cell matrix interaction, removal of the terminal non-collagenous globular domains and stacking of fibrils. We used a monolayer cell culture system to investigate if specific steps in this process are compromised in KC stromal cells.

Methods : Human unaffected donor (DN) and keratoconus patient (KC) stromal keratocytes (n=7) were maintained for 14 days in serum free medium to induce a more keratocyte-like phenotype. Cell layer associated collagen (indication of matrix assembly) was assayed using 1) picro-sirius red staining, 2) hydroxyproline content, 3) Western blotting and 4) MALDI-TOF mass spectrometry. Collagen production, modification and breakdown genes were assayed by qRTPCR. Gelatin zymography was used to quantify MMP activity in the cell culture media. Two-tailed Student’s t-test was used for statistical analysis.

Results : KC cultures harbor less cell layer associated collagen matrix in our in vitro KC model (Hydroxyproline p=0.0449, Sirius red p=0.0195). This difference is not attributed to cell number (p=0.93), density (p=0.32), or activation state (THY-1, α-SMA). Transcriptional levels of COL1A1 are markedly increased in KC cultures. Yet, Type I collagen within the media of KC samples was increased (p=0.01). Collagen processing proteins were examined, HSP47 was unchanged as quantified by western blot, and qPCR. The N-terminal collagen processing enzyme ADAMTS2 followed expression increases of COL1A1 (p=0.0074 and p= 0.0009 respectively), The C-terminal enzyme BMP-1 was found to be unchanged. Type V Collagen, was not found to be changed by qPCR, MS, Immunohistochemical localization or western blotting. MMP2 activity was shown to be increased by 49% in KC media relative to DN cultures (p=0.04). Concordantly collagen lost to the media was higher in KC cultures.

Conclusions : Our results indicate that collagen assembly in KC stromal cell cultures is fundamentally perturbed. We hypothesize that this dysregulation is due to a combination of reduced cell-matrix interactions, insufficiency of BMP1 and the increase of MMPs, and may be related to changes in TGFβ signal as we reported earlier. As such it provides a novel model for investigation of disease progression and trial possible intervention strategies.

This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.