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Abstract
Myofibroblasts are fibroblastic cells that function in wound healing, tissue repair and fibrosis, and arise from bone marrow (BM)-derived fibrocytes and a variety of local progenitor cells. In the cornea, myofibroblasts are derived primarily from stromal keratocytes and from BM-derived fibrocytes after epithelial-stromal and endothelial-stromal injuries. Quantitative proteomic comparison of mature alpha-smooth muscle actin (α-SMA)+ myofibroblasts (verified by immunocytochemistry for vimentin, α-SMA, desmin, and vinculin) generated from rabbit corneal fibroblasts treated with transforming growth factor (TGF) beta-1 or generated directly from cultured BM treated with TGF beta-1 was pursued for insights into possible functional differences. Paired cornea-derived and BM-derived α-SMA+ myofibroblast primary cultures were generated from four New Zealand white rabbits and confirmed to be myofibroblasts by immunocytochemistry. Paired cornea- and BM-derived myofibroblast specimens from each rabbit were analyzed by LC MS/MS iTRAQ technology using an Orbitrap Fusion Lumos Tribrid mass spectrometer, the Mascot search engine, the weighted average quantification method and the UniProt rabbit and human databases. From 2329 proteins quantified with ≥ 2 unique peptides from ≥ 3 rabbits, a total of 673 differentially expressed (DE) proteins were identified. Bioinformatic analysis of DE proteins with Ingenuity Pathway Analysis implicate progenitor-dependent functional differences in myofibroblasts that could impact tissue development. Our results suggest BM-derived myofibroblasts may be more prone to the formation of excessive cellular and extracellular material that are characteristic of fibrosis.
Highlights
Myofibroblasts are fibroblastic cells that function in wound healing, tissue repair and fibrosis, and arise from bone marrow (BM)-derived fibrocytes and a variety of local progenitor cells
Myofibroblast identity and homogeneity were confirmed by immunocytochemistry (Fig. 1) and paired cornea- and BM-derived myofibroblast specimens were analyzed by LC MS/MS Isobaric Tag for Relative and Absolute Quantification (iTRAQ) technology, yielding a total of 2420 proteins quantified, of which 2329 were quantified in ≥ 3 rabbits
The mean relative abundance of proteins quantified in all the myofibroblast samples is presented in Supplemental Table S5, including sample frequency, standard error of the mean, and moderated p values adjusted for multiple testing, A total 673 differentially expressed (DE) proteins were identified, as illustrated by Volcano plot (Fig. 3) and itemized in Supplemental Table S5 with color coding
Summary
Myofibroblasts are fibroblastic cells that function in wound healing, tissue repair and fibrosis, and arise from bone marrow (BM)-derived fibrocytes and a variety of local progenitor cells. Quantitative proteomic comparison of mature alpha-smooth muscle actin (α-SMA)+ myofibroblasts (verified by immunocytochemistry for vimentin, α-SMA, desmin, and vinculin) generated from rabbit corneal fibroblasts treated with transforming growth factor (TGF) beta-1 or generated directly from cultured BM treated with TGF beta-1 was pursued for insights into possible functional differences. This study pursued quantitative proteomic comparison of rabbit myofibroblasts using LC MS/MS Isobaric Tag for Relative and Absolute Quantification (iTRAQ) technology for further insights into whether myofibroblasts that differentiate from corneal keratocytes are functionally different from myofibroblasts that differentiate from BM-derived cells
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