Quantitative proteomic comparison of myofibroblasts derived from bone marrow and cornea

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.

Proteomics of Primary Uveal Melanoma: Insights into Metastasis and Protein Biomarkers

“…The protein pellets were resuspended in 100 mM TEAB buffer containing 0.5 mM CaCl 2 and were digested overnight at 37 °C with trypsin (initially with 2% trypsin ( w / w ), followed in 2 h with another 2% ( w / w ), and the next day with another 1% ( w / w ) for 2h). Following proteolysis, soluble peptides were quantified by AccQ-Tag amino acid analysis [ 67 , 68 ]. Equal amounts of each of the 13 choroid specimens from the UM eyes were pooled to form a single reference control sample for the proteomic analysis of the 100 pUM.…”

“…iTRAQ labeling with an 8-plex iTRAQ kit was performed as previously described [ 33 , 68 , 69 , 70 , 71 ]. The choroid specimens from the UM eyes were first analyzed by LC MS/MS relative to the choroid from disease-free eyes.…”

Molecular Changes Underlying Hypertrophic Scarring Following Burns Involve Specific Deregulations at All Wound Healing Stages (Inflammation, Proliferation and Maturation)

“…Many of them differentiate into α-SMA-expressing myofibroblasts ( Figure 5 ) [ 133 ]. In addition, myofibroblasts may also be derived from other sources, for example, epithelial cells through the EMT, epidermal stem cells [ 134 ], bone marrow-derived mesenchymal stem cells [ 135 ], and from endothelial cells through the process called endothelial-mesenchymal transition [ 136 ].…”

Interleukin-1 and Transforming Growth Factor Beta: Commonly Opposing, but Sometimes Supporting, Master Regulators of the Corneal Wound Healing Response to Injury

“… 125 , 126 Recent studies, however, have shown that corneal fibroblast-derived and fibrocyte-derived myofibroblasts are not equivalent cells, but likely cooperate in the generation of tissue fibrosis. 127 …”

“…125,126 Recent studies, however, have shown that corneal fibroblast-derived and fibrocyte-derived myofibroblasts are not equivalent cells, but likely cooperate in the generation of tissue fibrosis. 127 Myofibroblasts are rarely seen in unwounded corneas. 1,9 Interestingly, if TGFβ is overexpressed at high levels in the lens in vivo, myofibroblasts and fibrosis are generated in the overlying cornea.…”