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Yonathan Garfias, Alfredo Dominguez, Juan Pablo Reyes-Grajeda, Victor Manuel Bautista; Proteomic analysis of the effects of Amniotic Membrane on Human Lymbal Myofibroblasts. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):1862.
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© ARVO (1962-2015); The Authors (2016-present)
Corneal damage observed in herpes simplex keratitis (HSK) is mainly caused by exacerbated immune response to viral components. One of the main lines of defense are the pattern recognition receptors (PRRs) such as TLR3, MDA5 and RIG-1which recognize viral dsRNA and after activation, they are able to promote the production of pro-inflammatory cytokines via NF-κB nuclear translocation. We have previously reported that amniotic membrane (AM) decreases the production of proinflammatory cytokines and PRRs on poly I:C stimulated human limbal myofibroblast (HLM) through the inhibition of NF-κB nuclear translocation. These results explain in part the anti-inflammatory microenvironment that induces the AM transplantation in HSK. The objective of this study was to anlayze the protein changes favored by amniotic membrane on HLM.
HLM were obtained from cadaveric sclero-corneal rims. Frozen AM was enzimatically de-epithelized (dAM). All the assays were carried out using 106 HLM incubated or not with dAM. After dAM exposition, the HLM were recovered to obtain total protein extract and 2D electrophoresis assays were then performed. Subsequently, differential protein sequencing was done by means of MALDI-TOF and Mascot software.
Thirteen proteins were differentially found in HLM with dAM in comparison to HLM seeded on plastic wells. dAM favored de novo expression of three proteins, identified as serum albumin, with three different isoelectric points. In contrast, 6 out of the thirteen differentially expressed proteins were downregulated by dAM: actin 4.7 fold decrease, heat shock cognate 5.4 fold decrease, α-enolase 4.8 fold decrease, glucose-regulated protein 3.8 fold decrease, collagen α-1 4.5 fold decrease, calreticulin 2.9 fold decrease, and the expression of 3 proteins were completely inhibited: 2 variants of cytoplasmic actin and glyceraldehyde-3-phosphate dehydrogenase.
Taken together these results indicate that the AM exerted overlapping functions on HLM given by the protein pattern identified by proteomics. More experiments are needed to determine the nature and functional activity of such molecules.<br /> <br /> Financial Support: This project was supported by CONACYT: SALUD 160286; CIENCIA BASICA 167438; DGAPA-PAPIIT IA203514; CVU: 406706
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