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Corneal sections were stained with antibodies against α-SMA (red) to label myofibroblasts, antibodies against fibronectin (green), and 4′6′-diamidino-2-phenylondole dihydrochloride (DAPI) (blue) was used to label cell nuclei. (A–C) Photomicrographs of ex vivo cat corneal sections of the graft-host interface (arrows) on post-operative days 0 (A), 4 (B), and 9 (C)(scale bar for A – C = 0.2 mm). (D and E) The graft-host interface on days 0 (D) and 9 (E) (scale bar for D & E = 0.2 mm). (F) The central stroma of an unoperated cat cornea demonstrated an absence of α-SMA and mild diffuse fibronectin staining. (G and H) Incisional paracenteses wounds on day 0 (G) and day 9 (H) (scale bar for F – H = 0.4 mm). (I) An incisional paracentesis wound on day 9 at high magnification (scale bar for I = 0.2 mm). Note the lack of α-SMA staining at the graft-host interface (C and E), but positive α-SMA staining at the incisional wound (H and I) on day 9. On day 0, 4 hours after DSAEK, fibronectin staining was present extracellularly along the interface and also appeared to co-localize with DAPI in the cells of the adjacent host stroma (A and D). On day 9 post-DSAEK, there was faint fibronectin staining near the host stromal cells, but the interface fibronectin staining is much fainter and more consistent with the unoperated control.
Source publication
To evaluate myofibroblast differentiation as an etiology of haze at the graft-host interface in a cat model of Descemet's Stripping Automated Endothelial Keratoplasty (DSAEK).
DSAEK was performed on 10 eyes of 5 adult domestic short-hair cats. In vivo corneal imaging with slit lamp, confocal, and optical coherence tomography (OCT) were performed tw...
Citations
... However, they did not investigate the change of a-SMA expression before 24 h. The previous study also indicated that the protein expression of fibroblasts, like SMA and fibronectin, would change from 4 to 12 h of various stimuli (Weis et al., 2013;Gao et al., 2014). Differential time of protein expression in fibroblasts might be related to many factors, such as cell type and strength of the stimulus. ...
Scleral extracellular matrix (ECM) remodeling is thought to play a critical role in the pathogenesis of glaucoma. Mechanical strain induced by elevated intraocular pressure can promote myofibroblast differentiation of fibroblasts and result in scleral ECM remodeling; however, the underlying mechanism remains poorly understood. Yes-associated protein (YAP) is a mechanosensory protein and the key downstream transcriptional effector of the Hippo signaling pathway. Here, we investigated the role of YAP in mechanical strain-induced myofibroblast transformation during glaucoma scleral ECM remodeling. Integrative bioinformatics analyses were performed to identify the key pathways for the ECM remodeling of the sclera in glaucoma. Sprague–Dawley rats were used to establish a chronic ocular hypertension model, and the expression of collagen type I (COL1) and YAP in the sclera was analyzed by immunohistochemical analysis and Western blotting. Furthermore, human scleral fibroblasts (HSFs) were cultured and subjected to mechanical strain. In groups with or without the YAP siRNA or YAP inhibitor, cell proliferation, migration capacity, and the expression levels of YAP, COL1, and α-smooth muscle actin (α-SMA) were evaluated by Cell Counting Kit-8 assay, scratch assay, and Western blotting. The interactions between YAP and Smad3 were demonstrated by coimmunoprecipitation, and the expression levels of COL1 and α-SMA were evaluated in groups treated with or without the Smad3 inhibitor. We first revealed that the Hippo signaling pathway may be involved in mechanical strain-induced scleral ECM remodeling through bioinformatics analysis. Furthermore, the in vivo study showed upregulated YAP, COL1, and α-SMA expression in the hypertensive sclera of rats. In vitro, mechanical strain increased YAP and COL1 expression in HSFs and promoted myofibroblast differentiation. After YAP knockdown or inhibition with verteporfin, mechanical strain-induced fibrotic changes in HSFs were markedly suppressed. Additionally, YAP showed a protein interaction with Smad3, and the upregulation of a-SMA and COL1 in response to mechanical strain was also significantly downregulated following the inhibition of Smad3. In conclusion, mechanical strain activated scleral myofibroblast differentiation via YAP. The YAP pathway may play an important role in regulating scleral myofibroblast differentiation and ECM remodeling of the sclera in glaucoma.
... Myofibroblasts fill the wound in an interwoven and interconnected network pattern, and express filaments of α-smooth muscle actin (α-SMA) to promote wound contraction (6). Myofibroblasts are less translucent than static keratocytes and they further decrease stromal transparency by altering the organization and composition of the extracellular matrix (ECM) (7). Vimentin is one of the many intermediate filaments of mesenchymal cells such as fibroblasts and endothelial cells (8). ...
... It is generally accepted that the interaction between epithelial and mesenchymal cells is crucial in abnormal wound repair (7). In our model, HMS evidently breaks the normal interaction of epithelium and matrix. ...
The aim of the study was to investigate wound healing and scar formation in rabbit corneal lamellar wounds repaired with simple interrupted sutures (SIS) or horizontal mattress sutures (HMS). Two parallel ‘I’-shaped lamellar cornea wounds were created in one eye of 40 white New Zealand rabbits, while 5 uninjured rabbits were sacrificed to serve as normal controls. One side of the wounds, in the test rabbits, was closed with SIS, while the other side was treated with HMS. Ten days later, the stitches were removed under anesthesia. The animals were sacrificed on days 14 and 21, and months 3 and 6 after the suturing surgery, and corneal samples were subjected to histological and immunofluorescent studies: α-smooth muscle actin (α-SMA) and vimentin were used to detect myofibroblasts and fibroblasts, respectively, and collagen type I and Ⅲ was used to detect extracellular matrix (ECM) deposition. Relevant mRNA levels were assessed by quantitative polymerase chain reaction (qPCR) to elucidate the differences in wound healing and formation of fibrosis. Macroscopic and hematoxylin and eosin staining observations showed that the two sides of the wounds developed the most prominent fibrotic tissue on day 21. The immunofluorescence and qPCR results showed that HMS wounds produced increased α-SMA, vimentin and collagen type Ⅲ compared to the SIS wounds on day 14 or 21. The collagen type I expression showed no distinctive difference in SIS and HMS wounds. In conclusion, corneal lamellar wounds treated with SIS developed less fibrotic-related proteins and related mRNA in the early stages of wound healing than wounds treated with HMS. Although differences were not distinct after 3 months, the results of the present study suggest a benefit in choosing SIS over HMS, as at least the initial fibrotic process seems more benign with SIS. Corneal wounds should be carefully sutured, ensuring the tissue is well aligned.
Purpose:
This study aims to evaluate visual outcome after Descemet's membrane stripping automated endothelial keratoplasty (DSAEK) and relate to interface and corneal higher-order aberrations (HOAs).
Materials and methods:
We enrolled 16 eyes of 16 patients (eight males and eight females) in this interventional case series and followed DSAEK operation for about two to 20 months. OCULUS Pentacam, as well as other ophthalmic evaluations in follow-up visits, examined interface reflectivity and HOAs. Statistical relations were analyzed.
Results:
There was statistically significant correlation between interface reflectivity and best corrected visual acuity (BCVA) (r = 0.56, P = 0.021). Pachymetry (central corneal thickness) and BCVA had a moderate correlation (r = 0.6, P = 0.013). There was no statistically significant correlation between pachymetry and follow-up time (r = -0.36, P = 0.16). Negative correlation between follow-up and interface reflectivity was also not statistically significant (r = -0.24, P = 0.35). Coma had a significant correlation with BCVA in cornea and cornea front maps (r = 0.74, P = 0.009 and r = 0.71, P = 0.013, respectively).
Conclusion:
Significant correlation between interface reflectivity and BSCVA was found, and anterior corneal HOAs are significantly higher than posterior HOAs.
Purpose:
The purpose of this review was to provide detailed insights into the pathophysiology of myofibroblast-mediated fibrosis (scarring or late haze) after corneal injury, surgery, or infection.
Method:
Literature review.
Results:
The epithelium and epithelial basement membrane (EBM) and/or endothelium and Descemet's basement membrane (BM) are commonly disrupted after corneal injuries, surgeries, and infections. Regeneration of these critical regulatory structures relies on the coordinated production of BM components, including laminins, nidogens, perlecan, and collagen type IV by epithelial, endothelial, and keratocyte cells. Whether a cornea, or an area in the cornea, heals with transparency or fibrosis may be determined by whether there is injury to one or both corneal basement membranes (EBM and/or Descemet's BM) and delayed or defective regeneration or replacement of the BM. These opaque myofibroblasts, and the disordered extracellular matrix these cells produce, persist in the stroma until the EBM and/or Descemet's BM is regenerated or replaced.
Conclusions:
Corneal stromal fibrosis (also termed "stromal scarring" or "late haze") occurs as a consequence of BM injury and defective regeneration in both the anterior (EBM) and posterior (Descemet's BM) cornea. The resolution of fibrosis and return of stromal transparency depends on reestablished BM structure and function. It is hypothesized that defective regeneration of the EBM or Descemet's BM allows key profibrotic growth factors, including transforming growth factor beta-1 (TGF-β1) and TGF-β2, to penetrate the stroma at sustained levels necessary to drive the development and maintenance of mature opacity-producing myofibroblasts from myofibroblast precursors cells, and studies suggest that perlecan and collagen type IV are the critical components in EBM and Descemet's BM that bind TGF-β1, TGF-β2, platelet-derived growth factor, and possibly other growth factors, and regulate their bioavailability and function during homeostasis and corneal wound healing.
