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Corneal keratocytes, fibroblasts, and myofibroblasts exhibit distinct transcriptional profiles in vitro

ABSTRACT: After stromal injury to the cornea, the release of growth factors and pro-inflammatory cytokines promotes the activation of quiescent keratocytes into a migratory fibroblast and/or fibrotic myofibroblast phenotype. Persistence of the myofibroblast phenotype can lead to corneal fibrosis and scarring, which are leading causes of blindness worldwide. This study aims to establish comprehensive transcriptional profiles for cultured corneal keratocytes, fibroblasts, and myofibroblasts to gain insights into the mechanisms through which these phenotypic changes occur. Primary rabbit corneal keratocytes were cultured in either defined serum-free media (SF), fetal bovine serum (FBS) containing media, or in the presence of TGF-β1 to induce keratocyte, fibroblast, or myofibroblast phenotypes, respectively. Bulk RNA sequencing followed by bioinformatic analyses was performed to identify significant differentially expressed genes (DEGs) and enriched biological pathways for each phenotype. Genes commonly associated with keratocytes, fibroblasts, or myofibroblasts showed high relative expression in SF, FBS, or TGF-β1 culture conditions, respectively. Differential expression and functional analyses revealed novel DEGs for each cell type, as well as enriched pathways indicative of differences in proliferation, apoptosis, extracellular matrix (ECM) synthesis, cell-ECM interactions, cytokine signaling, and cell mechanics. Overall, these data demonstrate distinct transcriptional differences among cultured corneal keratocytes, fibroblasts, and myofibroblasts. We have identified genes and signaling pathways that may play important roles in keratocyte differentiation, including many related to mechanotransduction and ECM biology. Our findings have revealed novel molecular markers for each cell type, as well as possible targets for modulating cell behavior and promoting physiological corneal wound healing.

ORGANISM(S): Oryctolagus cuniculus

PROVIDER: GSE287915 | GEO | 2025/02/21

REPOSITORIES: GEO

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			Action	DRS
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	GSE287915_gene_count.txt.gz	Txt
	GSE287915_gene_fpkm.txt.gz	Txt

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Project description:Over the past decade, our laboratory developed a cat model of corneal wound healing after photorefractive keratectomy (PRK). Recently, we used this model in a combination of in vivo and in vitro approaches to show that myofibroblasts directly inhibit nerve regeneration in the wounded stroma, sub-basal plexus and epithelium. In vitro, two distinct phases to this inhibition have been observed: (1) initial slowing of neurite elongation via a releasable factor, and (2) neurite contact inhibition by myofibroblasts. For phase 1, transforming growth factor beta 1 (TGF-1) was shown to be the soluble, anti-neuritogenic factor released by myofibroblasts, and the specific signaling cascade it triggered to slow neurite elongation was identified. Ultimately, preventing myofibroblast differentiation suppressed both phase 1 and phase 2 inhibition, and resulted in faster nerve regeneration. However, in most cases of accidental injury, patients present after myofibroblast differentiation/fibrosis have occurred. The issue then is no longer preventing fibrosis, but rather overcoming its effects. We recently discovered that the synthetic PPAR ligand and Thiazolidinedione (TZD) drug, Troglitazone, stimulates myofibroblast de-differentiation in vitro and in vivo through a PPAR independent pathway. We now show that this TDZ’s ability to promote myofibroblast de-differentiation can be mimicked by UK-5099, a compound that spares PPAR, instead inhibiting a recently identified alternative target of TZDs: the mitochondrial pyruvate carrier (MPC). The recent literature and our preliminary results thus form a strong premise for refocusing our efforts on mitochondria, and critically testing the hypothesis that inhibiting the MPC facilitates myofibroblast de-differentiation, which together with metabolic remodeling in regenerating nerves can treat both fibrosis and overcome the blocked reinnervation inherent in its context. Work is ongoing to understand the metabolic changes that underlie the ability of TDZs to facilitate wound healing though mito-centric regulation of both myofibroblasts and corneal nerves. Here, we present the results of an RNA sequencing experiment designed to examine changes to the global transcriptome that occur as a result of TGF-β1-induced activation of corneal fibroblasts, and of TDZ- or UK5099-stimulated myofibroblast de-differentiation.

Project description:Pericryptal myofibroblasts in the colon and rectum play an important role in regulating the normal colorectal stem cell niche and facilitating tumour progression. Myofibroblasts have previously mostly been distinguished from normal fibroblasts only by the expression of Î± smooth muscle actin (Î±SMA). We now identify AOC3, a surface monoamine oxidase, as a new marker of myofibroblasts by showing that it is the target protein of the myofibroblast reacting monoclonal antibody (mAb), PR2D3. The normal and tumour tissue distribution and the cell line reactivity of AOC3 match that expected for myofibroblasts. We have shown that the surface expression of AOC3 is sensitive to digestion by trypsin and collagenase and that anti-AOC3 antibodies can be used for FACS sorting of myofibroblasts obtained by non-enzymatic procedures. Whole genome microarray mRNA expression profiles of myofibroblasts and skin fibroblasts revealed four additional genes that are significantly expressed differentially between these two cell types; NKX2-3 and LRRC17 are expressed in myofibroblasts and SHOX2 and TBX5 in skin fibroblasts. Transforming Growth Factor Î² (TGFÎ²) substantially down-regulated AOC3 expression in myofibroblasts but not in skin fibroblasts, in which it dramatically increased the expression of Î±SMA. A knockdown of NKX2-3 in myofibroblasts caused a decrease of myofibroblast-related gene expression and an increased expression of the fibroblast associated gene, SHOX2, suggesting that NKX2-3 is a key mediator for maintaining myofibroblast characteristics. Our results show that colorectal myofibroblasts, as defined by the expression of AOC3, NKX2-3 and other markers, are a distinctly different cell type from TGFÎ² activated fibroblasts. colorectal myofibroblast specific markers and expression profiles were sought by comparing four primary myofibroblast cultures to a panel of four dermal and foreskin fibroblast cell lines Four primary myofibroblast cultures established from adult human colon compared to four skin fibroblast cell lines to identify intestinal myofibroblast specific markers

Project description:Background: The differentiation of pericytes into myofibroblasts causes microvascular degeneration, extracellular matrix (ECM) accumulation, and tissue stiffening, characteristics of fibrotic diseases. It is unclear how pericyte-myofibroblast differentiation is regulated in the microvascular environment. Our previous study established a novel two-dimensional platform for coculturing microvascular endothelial cells (ECs) and pericytes derived from the same tissue. This study investigated how ECM stiffness regulated microvascular ECs, pericytes, and their interactions. Methods: Primary microvessels were cultured in the TGM2D medium. Stiff ECM was prepared by incubating ECM solution in regular culture dishes for one hour followed by PBS wash. Soft ECM with Young’s modulus of approximately 6 kPa was used unless otherwise noted. Bone grafts were prepared from the rat skull. Immunostaining, RNA sequencing, qRT-PCR, western blotting, and knockdown experiments were performed on the cells. Results: Primary microvascular pericytes differentiated into myofibroblasts (NG2+αSMA+) on stiff ECM, even with the TGFβ signaling inhibitor A83-01. Soft ECM and A83-01 cooperatively maintained microvascular stability while inhibiting pericyte-myofibroblast differentiation (NG2+αSMA-/low). We thus defined two pericyte subpopulations: primary (NG2+αSMA-/low) and activated (NG2+αSMA+) pericytes. Soft ECM promoted microvascular regeneration and inhibited fibrosis in bone graft transplantation in vivo. As Integrins are the major mechanosensor, we performed qRT-PCR screening of Integrin family members selected from RNA sequencing data. We found that Integrin β1 (Itgb1) was the major subunit downregulated by soft ECM and A83-01 treatment. Knocking down Itgb1 suppressed myofibroblast differentiation on stiff ECM. Interestingly, ITGB1 phosphorylation (Y783) was mainly located on microvascular ECs on stiff ECM, which promoted EC secretion of paracrine factors, including CTGF, to induce pericyte-myofibroblast differentiation. CTGF knockdown or monoclonal antibody treatment partially reduced myofibroblast differentiation, implying the participation of multiple pathways in fibrosis formation. Conclusions: Microvascular ECs mediate ECM stiffness-induced pericyte-myofibroblast differentiation through paracrine signaling.

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Corneal keratocytes, fibroblasts, and myofibroblasts exhibit distinct transcriptional profiles in vitro

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