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James William Foster, Vishal Shinde, Uri Soiberman, Yassine Jamil Daoud, Fares Alsaleh, Gajanan Sathe, Jun Wan, Jiang Qian, Albert S Jun, Akhilesh Pandey, Shukti Chakravarti; Molecular insights into ECM and collagen assembly disruptions in keratoconus. Invest. Ophthalmol. Vis. Sci. 2017;58(8):3904.
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© ARVO (1962-2015); The Authors (2016-present)
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.
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.
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.
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.
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