Purchase this article with an account.
Paramananda Saikia, Madison Juszczak, Luciana L Dibbin, Rodrigo Carlos Oliveira, Carla S Medeiros, Belinda Willard, Jang Geeng-Fu, Jack S Crabb, John W Crabb, Steven E Wilson; Proteome of keratocyte-derived versus bone marrow-derived myofibroblasts. Are they different?. Invest. Ophthalmol. Vis. Sci. 2019;60(9):5254.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
The aim of the current study is to determine whether keratocyte-derived myofibroblasts are different from bone marrow-derived myofibroblasts at the protein expression level.
Primary keratocyte-derived corneal fibroblasts were isolated from four New Zeland white rabbit eye purchased from Peel-Freeze, USA. Tibia and femur of rabbits were removed and bone marrow (BM) cells were harvested by standard methods. Corneal fibroblasts and BM cells were treated with 20 ng/ml TGF-β for 14 days and greater than 99.9% of cells derived from both precursor sources were alpha-smooth muscle actin (α-SMA) + using immunocytochemistry. Proteins were identified by LC MS/MS and quantified with iTRAQ tags. Ingenuity Pathway Analysis (IPA) were used to identify biologically relevant networks and several major pathways or networks. Proteomics results were corroborated by Western blot analysis.
Proteins with 2 or more peptides per protein (n=2700) were quantified by LC MS/MS ITRAQ technology. The relative amounts of proteins in myofibroblasts derived from corneas was compared to those derived from bone marrow. The data suggest 7-12% difference in the proteome of myofibroblasts from cornea compared to bone marrow. Western blot analysis used to independently evaluate the abundance of proteins corroborated the ITRAQ quantitation by demonstrating increased collagen VII and decreased Collagen III and Collagen XI in myofibroblast derived from keratocytes compared to myofibroblasts derived from bone marrow cells. Ingenuity Pathway Analysis of significantly elevated and significantly decreased proteins in the myofibroblast implicated tissue development, organ morphology and connective tissue development pathways as the highest scoring networks.
Myofibroblasts that differentiated from keratocytes or bone marrow cells both express the myofibroblast marker α-SMA. Quantitative proteomic analyses corroborated by Western blot analyses support 7-12% proteomic difference between myofibroblasts derived from keratocytes versus bone marrow. The result suggest that the proteome of these two different myofibroblasts are very similar but could have somewhat differing roles in the stromal fibrosis response to corneal injury. Further studies will be needed to confirm the differential roles of the myofibroblast derived from keratocytes versus bone marrow.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.
This PDF is available to Subscribers Only