Corneal myofibroblast generation from bone marrow-derived cells

Abstract

The purpose of this study was to determine whether bone marrow-derived cells can differentiate into myofibroblasts, as defined by alpha smooth muscle actin (SMA) expression, that arise in the corneal stroma after irregular phototherapeutic keratectomy and whose presence within the cornea is associated with corneal stromal haze. C57BL/6J-GFP chimeric mice were generated through bone marrow transplantation from donor mice that expressed enhanced green fluorescent protein (GFP) in a high proportion of their bone marrow-derived cells. Twenty-four GFP chimeric mice underwent haze-generating corneal epithelial scrape followed by irregular phototherapeutic keratectomy (PTK) with an excimer laser in one eye. Mice were euthanized at 2 weeks or 4 weeks after PTK and the treated and control contralateral eyes were removed and cryo-preserved for sectioning for immunocytochemistry. Double immunocytochemistry for GFP and myofibroblast marker alpha smooth muscle actin (SMA) were performed and the number of SMA+GFP+, SMA+GFP−, SMA −GFP+ and SMA−GFP− cells, as well as the number of DAPI+ cell nuclei, per 400X field of stroma was determined in the central, mid-peripheral and peri-limbal cornea. In this mouse model, there were no SMA+ cells and only a few GFP+ cells detected in unwounded control corneas. No SMA+ cells were detected in the stroma at two weeks after irregular PTK, even though there were numerous GFP+ cells present. At 4 weeks after irregular PTK, all corneas developed mild to moderately severe corneal haze. In each of the three regions of the corneas examined, there were on average more than 9X more SMA+GFP+ than SMA+GFP− myofibroblasts. This difference was significant (p <0.01). There were significantly more (p <0.01) SMA−GFP+ cells, which likely include inflammatory cells, than SMA+GFP+ or SMA+GFP− cells, although SMA−GFP− cells represent the largest population of cells in the corneas. In this mouse model, the majority of myofibroblasts developed from bone marrow-derived cells. It is possible that all myofibroblasts in these animals developed from bone marrow-derived cells since mouse chimeras produced using this method had only 60% to 95% of bone marrow-derived cells that were GFP+ and it is not possible to achieve 100% chimerization. This model, therefore, cannot exclude the possibility of myofibroblasts also developed from keratocytes and/or corneal fibroblasts.

Substratum Topography Modulates Corneal Fibroblast to Myofibroblast Transformation

“…38 Currently, it is unclear what percentage of myofibroblasts is of bone marrow cell origin during in vivo corneal wounding. In our study, all myofibroblasts were derived from corneal fibroblasts, and it is unknown whether myofibroblasts of differing origins will respond in a similar manner to micro-and nanoscale topographic cues.…”

Mitomycin C: Biological Effects and Use in Refractive Surgery

“…52 This may have important implications for devising new treatments to prevent haze and may also provide a biological explanation for the cellular difference in humans between steroid-responsive haze and steroid-unresponsive haze. Our working hypothesis is that the myofibroblasts in steroid-responsive haze are primarily derived from bone marrow-derived cells and, therefore, retain the capacity to undergo cell death in response to corticosteroids.…”

“…26,50,51,62 Scattering light more intensely than quiescent keratocytes and producing large amounts of disorganized extracellular matrix, myofibroblasts play an important role in corneal haze. 72 Recently, Barbosa et al 52 have shown in a chimeric mouse model that bone marrow-derived cells can differentiate into corneal stromal myofibroblasts associated with haze after phototherapeutic keratectomy (PTK).…”

“…160 An intact basement membrane serves as a barrier to block penetration of cytokines, such as TGF-b and IL-1, into the corneal stroma and, therefore, modulate the overall wound healing response. Myofibroblasts are derived from keratocytes or bone marrow-derived cells 52 under the influence of TGF-b. One study provided evidence that tear fluid TGF-b1 levels and corneal haze are lower in patients who had Epi-LASIK compared with LASEK at 1, 3, and 5 days postoperatively, 161 but this length of follow-up is insufficient to monitor the appearance of myofibroblast-related late haze.…”

The Myofibroblast: TGFβ-1, A Conductor which Plays a Key Role in Fibrosis by Regulating the Balance between PPARγ and the Canonical WNT Pathway

“…Non fibroblastic cell lineages [27][28][29] can differentiate into myofibroblasts through the processes of epithelial-mesenchymal transition [30] and endothelial-mesenchymal transition [31]. MSCs are myofibroblast precursors in several pathological states [32]. An important particular case is the presence of myofibroblasts in normal human placenta, a tissue in which the population of contractile myofibroblasts is dominant in placental stem villi [33].…”