The keratocyte: corneal stromal cell with variable repair phenotypes

Abstract

Keratocytes, also known as fibroblasts, are mesencyhmal-derived cells of the corneal stroma. These cells are normally quiescent, but they can readily respond and transition into repair phenotypes following injury. Cytokines and other growth factors that provide autocrine signals for stimulating wound responses in resident cells are typically presented by platelets at the site of an injury. However, due to the avascular nature of the cornea many of the environmental cues are derived from the overlying epithelium. Corneal epithelial-keratocyte cell interactions have thus been extensively studied in numerous in vivo corneal wound healing settings, as well as in in vitro culture models. Exposure to the different epithelial-derived factors, as well as the integrity of the epithelial substratum, are factors known to impact the keratocyte response and determine whether corneal repair will be regenerative or fibrotic in nature. Finally, the recent identification of bone-marrow derived stem cells in the corneal stroma suggests a further complexity in the regulation of the keratocyte phenotype following injury.

Fig. 1

Morphology of the cornea. Hematoxylin and eosin stained cross-sections of the adult mouse cornea showing the three layers of the cornea, the stratified corneal epithelium (CE), the collagenous stroma (S) and the endothelium (E). The keratocytes (arrowheads) are situated between the collagen lamellae in the stroma.

Fig. 2

Cellular interactions during corneal repair. (A) Upon corneal epithelial injury, IL-1α is released from the injured epithelium into the stroma. IL-1α induces some of the underlying stromal keratocytes to undergo cell death, while others are induced to proliferate, secrete MMPs, and transition from a quiescent to an activated phenotype. Due to the absence of a basement membrane, corneal epithelial cells also secrete TGFβ2 into the underlying stroma inducing a subpopulation of keratocytes to undergo transformation into myofibroblasts that secrete ECM. (B) The return of the basement membrane inhibits the release of TGFβ2 into the stroma and the myofibroblast phenotype is no longer observed. The activated keratocytes continue to secrete autocrine IL-1α and remodel the ECM. Abbreviations: MMPs, matrix metalloproteinase; TGFβ2, transforming growth factor beta; ECM, extracellular matrix; IL-1α, interleukin 1 alpha.

Fig. 3

Markers of fibroblast activation are evident in the Le-AP-2α corneal stroma. (A) The proliferating status of the corneal stromal cells was determined by immunostaining for proliferating cell nuclear antigen (PCNA). Little to no proliferative response was observed in the stroma of the wild-type cornea. In contrast, stromal fibroblasts (arrowhead) in the Le-AP-2α mutant cornea exhibited immunoreactivity for PCNA, particularly in regions where the basement membrane was absent. (B) The activated response was further examined by immunofluorescent localization of α-smooth muscle actin (α-SMA) using a CY3-conjugated secondary antibody (red). Cell nuclei were stained with DAPI (blue) to delineate the location of cells. The wild-type corneas did not exhibit any α-SMA protein expression, whereas some fibroblasts beneath the epithelium in the Le-AP-2α mutant corneas showed imunoreactivity to α-SMA. (C) Transforming growth factor-β2 (TGF-β2) was immunolocalized using a FITC-conjugated secondary antibody (green). In wild-type mice TGF-β2 staining was absent in the corneal stroma and only evident in the corneal epithelium. In the Le-AP-2α mutant cornea, both the corneal epithelium and stroma (arrowhead) stained positively for TGF-β2 (green). (D) Immunostaining for phospho-Smad2 (p-Smad2), a downstream effector of TGF-β2 signaling, showed that in wild-type mice, p-Smad2 was localized only in the epithelium, whereas in the Le-AP-2α mutant cornea both the stroma (arrowhead) and epithelium showed immunoreactivity to p-Smad2. Abbreviations: CE, corneal epithelium; S, stroma.