Compositional differences in ECM deposited by Acomys cahirinus and Mus musculus fibroblasts in vitro
Description
The Spiny Mouse (Acomys cahirinus) has the most extensive regenerative capabilities of any known mammal and can regenerate with little to no fibrosis. Key differences between the wounds of Acomys and a scarring counterpart (Mus musculus) include a reduction in fibroblast activation and a less-fibrous ECM composition. We hypothesize that these differences are due in part to reduced stiffness-mediated fibroblast-to-myofibroblast transition (FMT) in Acomys fibroblasts. We sought to investigate the impact of FMT and species differences on ECM composition by fabricating cell-derived matrices (CDMs) from Acomys and Mus fibroblasts. CDMs were generated by culturing Acomys (N=3) or Mus (N=3) fibroblasts on collagen-functionalized PDMS or glass for 16-21 days in the presence of a macromolecular crowder to promote ECM production. To assess the impact of stiffness on ECM composition, CDMs were generated on both “soft” PDMS (Sylgard 527, ~5kPa) and “stiff” PDMS (Sylgard 182, ~1MPa). The CDMs were decellularized, homogenized, and sent to the University of Florida Mass Spectrometry Research and Education Center for label-free quantitative proteomics. The comparisons of interest were species-specific (e.g. Acomys CDMs generated on glass vs. Mus CDMs generated on glass) or stiffness-specific (e.g. Acomys CDMs generated on soft PDMS vs. Acomys CDMs generated on stiff PDMS). The most notable differences were observed when comparing between species. Specifically, there were 186 proteins unique to Acomys CDMs and 169 proteins unique to Mus CDMs on soft PDMS. On stiff PDMS, there were 241 proteins unique to Acomys and 159 unique to Mus. On glass, there were 184 proteins unique to Acomys and 181 proteins unique to Mus. When we filtered for secreted proteins, proteins in Acomys CDMs were less associated with fibrosis. Fibrosis-associated ECM proteins including fibrillin 1, ADAMTS1, SPARC, and galectin 1 were significantly reduced or absent in Acomys CDMs compared to Mus CDMs. In addition, proteins that have been connected to fibrosis resolution, including Col12a1 and clusterin, were upregulated in Acomys CDMs.
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Sylgard 527 and Sylgard 182 PDMS were generated following the manufacturer’s protocol. To prepare the PDMS and glass substrates for cell culture, the PDMS surfaces were charged by plasma treatment then treated with sterile 1% APTMS in ethanol for 10 minutes. Unreacted APTMS was removed by rinsing, then the surfaces were treated with sterile 1% glutaraldehyde in PBS for 30 minutes. Residual glutaraldehyde was removed by rinsing. Finally, the surfaces were treated with 10ug/mL rat tail collagen I for 1 hour at room temperature and sterilized under a bactericidal UV lamp for 30 minutes. Primary dermal fibroblasts from 3 Acomys and 3 Mus pups were seeded at a density of 10,000 cells/cm^3 and cultured for 16-21 days in the presence of 25 mg/mL Ficoll 400 which served as a macromolecular crowder to promote ECM deposition. Following culture, CDMs were decellularized in warm 0.05% Triton X-100 and 20 mM NH4OH for 5 minutes followed by 10 U/mL DNase I for 30 minutes at 37C. CDMs were solubilized using RIPA buffer for 30 minutes on ice, then homogenized with a tissue homogenizer. The RIPA insoluble fraction (remaining pellet following centrifugation) was further solubilized in a membrane solubilization buffer (40 mM Tris-Cl (pH 8.0), 7 M urea, 2 M thiourea, 0.25% w/v ASB-14, and 0.25% NP-40 for 15 minutes). Prior to analysis, both protein fractions were precipitated in ice-cold acetone and resuspended in 2M urea. Nano-LC/MS/MS was performed on a Thermo Scientific Q Exactive HF Obitrap mass spectrometer equipped with an EASY Spray nanospray source (Thermo Scientific) operated in positive ion mode. The LC system was an UltiMateTM 3000 RSLCnano system from Thermo Scientific. All MS/MS spectra were analyzed using the Chimerys node (Thermo Fisher Scientific; Proteome Discoverer 3.0.1.27). Chimerys was set up to search Mus musculus (sp_incl_isoforms TaxID=10090_and_subtaxonomies) (v2023-06-28), Acomys cahirinus (v2024-01-31 with 31141 sequences and 20612094 residues) and Universal Protein Contaminants fasta assuming the digestion enzyme trypsin. A fasta file containing all known protein coding sequences in Acomys was generated using a recently published Acomys genome by Nguyen et al.: https://doi.org/10.1093/g3journal/jkad177. Precursor ion intensity label free quantitation was done using Proteome Discoverer. The two groups in each comparison were compared using a “non-nested” study factor. Normalization was derived by using all peptides. Protein abundances were calculated by summed abundances, meaning the protein abundances are calculated by summing sample abundances of the connected peptide groups. Fisher’s exact test (pairwise ratio-based) was used to calculate p-values with low intensity resampling value imputation included. Adjusted p-values were calculated using Benjamini-Hochberg. Fold-change comparisons and identification of unique protein compositions were performed with Scaffold DDA (version 6.3.0) from Proteome Software.