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Rabab Sharif, Mariam Khaled, JIn-Xiong She, Yutao Liu, Dimitrios Karamichos; Exosomes and their miRNA/protein contents in Keratoconus. Invest. Ophthalmol. Vis. Sci. 2019;60(9):314. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
Keratoconus (KC) is a corneal thinning disorder and a leading cause of corneal transplantation worldwide. Exosomes are small extracellular vesicles (30-150nm) secreted by various cell types to mediate intracellular communication via their content of proteins, lipids, and nucleic acids. We aim to investigate the role of exosomes secreted by stromal fibroblasts in KC pathogenesis through characterization of these exosomes, their miRNA and protein contents.
Using human corneal fibroblasts (HCFs) and human Keratoconus fibroblasts (HKCs) derived from four healthy donors and four KC patients. We collected serum free media to isolate exosomes using serial ultracentrifugation. Using nanoparticle tracking analysis (NTA) with ZetaView, we compared the size and quantity of isolated exosomes. Different exosomal markers were identified using electron microscope (EM) (CD81) and western blot (CD9 and CD63). We extracted RNA from exosomes, and RNA quality and quantity was determined using the Bioanalyzer system. The expression profiles of 368 miRNAs in the 8 exosome samples were determined by qRT-PCR using Exiqon Human panel I assays. Mass spectrometry was used for exosomal protein profiling.
We successfully characterized exosomes isolated from both HCFs and HKCs using complimentary techniques. NTA and EM showed no significant differences in the size, quantity, or morphology between exosomes secreted by HCFs or HKCs. Exosomal markers, CD81, CD63 and CD9, were expressed in all exosome samples, but with different expression levels. Furthermore, we detected the expression of 80-150 miRNAs (Ct < 36) in all exosome samples. In KC-derived exosome samples, miR-328-3p and miR-299-5p showed unique expression, let-7f-5p and let-7a-5p had a 2-fold increase and let-7c-5p had a 4-fold increase. Protein profiling revealed 13 proteins with differential expression between HCF and HKC samples. Pathway analyses using WebGestalt and Ingenuity Pathway Analysis indicated the potential role of PDGFRβ signaling pathway in KC pathogenesis via exosome secretion.
For the first time, we were able to differentiate between exosomes secreted by corneal fibroblasts derived from patients with or without KC. Our successful characterization of exosomes miRNA and protein content provides us with new and unprecedented pathophysiological understanding of KC.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.
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