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Omer Iqbal, George Fisher, Samir Vira, Daniel Kahn, Daneyal Syed, Jawed Fareed, Charles Bouchard; Increased expression of secreted frizzled-related protein-1 (SFRP-1) and microtubule-associated protein light chain 3 (LC3) in keratoconus. Invest. Ophthalmol. Vis. Sci. 2013;54(15):5301.
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To study the expression of secreted frizzled-related protein-1 (SFRP-1) and Light Chain 3 (LC3), an autophagy marker, in keratoconus and to determine the proteomic profile of normal and keratoconic epithelial and stromal layers by ProteinChip® using SELDI-TOF-MS
Under an IRB approved protocol, surgically discarded and de-identified normal donor (n=10) and keratoconus corneas (n=10) were obtained. A segment of the cornea was fixed in formalin for immunohistochemical staining. From the remaining cornea the endothelium-Descemets membrane was removed. The endothelium free cornea was soaked in prewarmed 20mM EDTA solution for 30 minutes and forceps were used to strip the epithelial and the stromal layers. The samples were homogenized and the protein concentrations adjusted to 1mg/ml. The homogenates were analyzed using SELDI-TOF-MS. The formalin fixed paraffin-embedded corneal samples were cut into sections and mounted on slides. SFRP-1 antibody and LC3 antibody were used to perform immunohistochemical staining.
The proteomic profile showed absence of peaks in the 20-150 kDa range in the keratoconic epithelium. However, there were distinct peaks in keratoconus at 10.8, 12.7, 13.1, 14.6, and 15.8 kDa which were absent in normal samples. High expressivity of SFRP-1 and LC3 was observed in the keratoconus corneas. There also appeared to be a correlation between the expression of SFRP1 and LC3 in keratoconus tissues. Low expressivity of SFRP1 resulted in low expressivity of LC3 while medium-high expressivity of SFRP1 resulted in medium to high expressivity of LC3.
Increased expression of SFRP-1 and LC3 was observed in Keratoconus cornea. Keratocyte autophagy associated with Keratoconus may play a role in the pathogenesis of Keratoconus.
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