Keratocyte-Derived Myofibroblasts: Functional Differences With Their Fibroblast Precursors

Abstract

Purpose: In this study, we aim to elucidate functional differences between fibroblasts and myofibroblasts derived from a keratocyte lineage to better understand corneal scarring.

Methods: Corneal fibroblasts, derived from a novel triple transgenic conditional KeraRT/tetO-Cre/mTmG mouse strain that allows isolation and tracking of keratocyte lineage, were expanded, and transformed by exposure to transforming growth factor (TGF)-β1 to myofibroblasts. The composition and organization of a fibroblast-built matrix, deposited by fibroblasts in vitro, was analyzed and compared to the composition of an in vitro matrix built by myofibroblasts. Second harmonic generation microscopy (SHG) was used to study collagen organization in deposited matrix. Different extracellular matrix proteins, expressed by fibroblasts or myofibroblasts, were analyzed and quantified. Functional assays compared latent (TGF-β) activation, in vitro wound healing, chemotaxis, and proliferation between fibroblasts and myofibroblasts.

Results: We found significant differences in cell morphology between fibroblasts and myofibroblasts. Fibroblasts expressed and deposited significantly higher quantities of fibril forming corneal collagens I and V. In contrast, myofibroblasts expressed and deposited higher quantities of fibronectin and other non-collagenous matrix components. A significant difference in the activation of latent TGF-β activation exists between fibroblasts and myofibroblasts when measured with a functional luciferase assay. Fibroblasts and myofibroblasts differ in their morphology, extracellular matrix synthesis, and deposition, activation of latent TGF-β, and chemotaxis.

Conclusions: The differences in the expression and deposition of extracellular matrix components by fibroblasts and myofibroblasts are likely related to critical roles they play during different stages of corneal wound healing.

Figure 1.

An inducible triple transgenic conditional KeraRT/tetO-Cre/mTmG mouse allows induction of eGFP expression in vivo by keratocytes after doxycycline-containing diet. (A) Most stromal cells (uninjured cornea) express eGFP (green) after 1 week of oral doxycycline supplementation. The eGFP+ and orange cells are believed to be keratocytes or derived from keratocytes. Some sparse cells in the stroma do not express keratocan and maintain tomato expression, marked by asterisks. (B) Phase contrast (left) and fluorescence (right) microscopy images of expanded corneal fibroblasts showing a mostly homogeneous population of cells, except for interspersed clusters of tdTomato-expressing cells (red). (C) Using flow cytometry, a pure sample of eGFP+ cells can be obtained from cultured fibroblasts. This purified sample consists only of cells derived from a keratocyte lineage. Epi, epithelium; Str, stroma; Endo, endothelium; FACS, fluorescence-activated cell sorting.

Figure 2.

Corneal scars are composed of disorganized extracellular matrix and non-keratocyte cellular components. SHG microscopy was used for high-magnification imaging of collagen organization in mature corneal scars and uninjured corneas. (A) One of the most important properties of a normal cornea is transparency. (B) In an uninjured cornea, keratocytes (eGFP+) are well organized and located in a parallel orientation between the corneal lamellae. Keratocytes make up most cells in the normal uninjured stroma. (C) Higher-magnification image of collagen fibrils in a normal cornea. (D) High-magnification image of a normal mouse cornea including nuclear counterstain. Keratocytes (eGFP+) is predominant population. (E) Cornea with central scar (dashed outline), imaged 9 weeks after full-thickness keratotomy. (F) Within the area of the scar (dashed lines), there is lack of eGFP expression in response to doxycycline induction meaning keratocytes do not populate scars. Asterisks mark limit between keratocytes (eGFP+) and fibroblasts (Tdtomato+) populations. Distinct lamellae are largely absent and collagen matrix is very disorganized in comparison to normal cornea in scars. (G, H) High-magnification images of corneal scar, again showing disorganization of the collagen matrix and the absence of eGFP+ keratocytes. Epi, epithelium; Endo, endothelium.

Figure 3.

Morphological and matrix expression differences between keratocyte derived fibroblasts and myofibroblasts in vitro. (A) Corneal fibroblasts were cultured in DMEM (top), 5% FBS in DMEM (middle), and 5% FBS in DMEM supplemented with 10 ng/mL TGF β1 (bottom). Images shown were obtained by phase contrast (left column) and fluorescence (right column) microscopy. Changes in cellular morphology are evident with different culture conditions, reflecting cellular activity and differentiation. (B) Quantitative RT-PCR shows differences in gene expression between keratocyte-derived fibroblasts and myofibroblasts. Specifically, myofibroblasts showed significantly greater expression of Acta2 and Serpine1 and less expression of Ltbp, Col1a1, and Col5a1 when compared with fibroblasts. No significant difference in Col3a1 expression was found. F, fibroblast; M, myofibroblast; NS, not significant. *:P < 0.05; **: P < 0.01; ***: P < 0.005; ****: P < 0.001. Scale bars = 100 µm.

Figure 4.

Protein analysis confirms higher deposition of most collagens in the “fibroblast matrix” compared to a “myofibroblast matrix”. (A) Heat map of proteomic analysis data comparing the quantities of extracellular matrix proteins deposited by fibroblasts and myofibroblasts. Fibroblasts had higher quantities of most collagens, whereas other non-collagen components were upregulated by myofibroblasts. (B) Western blot confirms that fibroblast matrix contains significantly higher quantities of collagen I and collagen V α1 chains. As expected, myofibroblasts contain significantly higher quantities of αSMA, an important marker of the myofibroblast phenotype. Results were normalized using β-actin. F, fibroblast; M, myofibroblast. *: P < 0.05; **: P < 0.01. (C) Visualization of in vitro fibroblast and myofibroblast extracellular matrix deposition using SHG microscopy. Note how collagen structures are deposited in a more disorganized pattern by myofibroblasts compared to fibroblasts. Nuclei are highlighted in red due to staining with propidium iodide.

Figure 5.

Differences in FN-EDA and αSMA expression between fibroblasts and myofibroblasts. (A) Fluorescence microscopy images of fibroblasts (left) and myofibroblasts (right) immunostained for fibronectin extra domain A (FN-EDA). Nuclei are counterstained with DAPI. (B) Fluorescence microscopy images of fibroblasts (left) and myofibroblasts (right) stained for alpha smooth muscle actin (αSMA). Nuclei are counterstained with DAPI. The inset image shows a higher-magnification view of αSMA-stained myofibroblasts. Green fluorescence in the bottom row of images is due to expression of eGFP by keratocyte-derived cells isolated from I-KeramTmG mice demonstrating these cells are derived from a keratocyte lineage.

Figure 6.

Myofibroblasts are more efficient in activating latent TGF- β from their matrix compared to fibroblasts after thrombin stimulation. This finding confirms that one of the main functions of myofibroblasts is cell contraction during wound closure. (A) Luciferase assay was used to quantify and compare the activation of latent TGF-β in fibroblasts and myofibroblasts. Higher luminescence (expressed as RLU) values indicate greater quantities of active TGF-β in the conditioned medium of cultured cells. Ft-, fibroblasts without thrombin; Ft+, fibroblasts with thrombin added; Mt-, myofibroblasts without thrombin; Mt+, myofibroblasts with thrombin added; D, DMEM only; SB, SB431542 (ALK inhibitor, negative control); T, TGF-β, positive control. (B) Fluorescence microscopy images of keratocyte-derived fibroblasts (top row) and myofibroblasts (bottom row) stained for LTBP1 (purple) and counterstained with DAPI (blue). These images show the deposition of LTBP1 in pericellular “strands” of extracellular matrix components by activated fibroblasts and myofibroblasts.

Figure 7.

Results of wound healing, transwell migration, and MTT cell proliferation assays. (A) Fluorescence microscopy images of fibroblast (left) and myofibroblast (right) monolayers taken immediately, 24 hours, and 48 hours after creation of scratch wounds. Wound edges were outlined automatically in yellow by ImageJ software, which was used to measure wound area. The graphs represent data and comparisons for wound closure (top) and wound closure rate (top) at each time point. Wound closure is calculated as a percentage of the initial wound area, and wound closure rate is calculated as wound closure over time. (B) Results of transwell migration assay for fibroblasts and myofibroblasts. Images were taken of migrated fibroblasts (left) and myofibroblasts (right) on the undersides of transwell insert membranes after a 24-hour incubation period. The bars in the bar graph represent the average number of migrated cells per field for both cell types. (C) Bar graph representing MTT assay absorbance data for fibroblasts and myofibroblasts. Higher absorbance values indicate greater cell metabolic activity. F, fibroblasts; M, myofibroblasts; ns, no significant difference *: P < 0.05; ****: P < 0.001.

Figure 8.

Illustration summarizing some of the main findings of this paper. Compared to fibroblasts, myofibroblasts are characterized by a higher propensity for TGFβ activation, deposition of disordered extracellular matrix (characterized in the figure by randomly oriented and shaped extracellular matrix strands, rather than uniform asterisk-like structures), and contraction of the cells and extracellular matrix. These key differences highlight the unique role of each cell type in physiologic and pathophysiologic processes of wound healing and scar formation.