The corneal wound healing response, including the development of stromal opacity in some eyes, is a process that often leads to scarring that occurs after injury, surgery or infection to the cornea. Immediately after epithelial and stromal injury, a complex sequence of processes contributes to wound repair and regeneration of normal corneal structure and function. In some corneas, however, often depending on the type and extent of injury, the response may also lead to the development of mature vimentin+ α-smooth muscle actin+ desmin+ myofibroblasts. Myofibroblasts are specialized fibroblastic cells generated in the cornea from keratocyte-derived or bone marrow-derived precursor cells. The disorganized extracellular matrix components secreted by myofibroblasts, in addition to decreased expression of corneal crystallins in these cells, are central biological processes that result in corneal stromal fibrosis associated with opacity or “haze”. Several factors are associated with myofibroblast generation and haze development after PRK surgery in rabbits, a reproducible model of scarring, including the amount of tissue ablated, which may relate to the extent of keratocyte apoptosis in the early response to injury, irregularity of stromal surface after surgery, and changes on corneal stromal proteoglycans, but normal regeneration of the epithelial basement membrane (EBM) appears to be a critical factor determining whether a cornea heals with relative transparency or vision-limiting stromal opacity. Structural and functional abnormalities of the regenerated EBM facilitate prolonged entry of epithelium-derived growth factors such as transforming growth factor β (TGF-β) and platelet-derived growth factor (PDGF) into the stroma that both drive development of mature myofibroblasts from precursor cells and lead to persistence of the cells in the anterior stroma. A major discovery that has contributed to our understanding of haze development is that keratocytes and corneal fibroblasts, but not myofibroblasts, produce large amounts of critical EBM components, such as nidogen-1, nidogen-2 and perlecan, that are essential for complete regeneration of a normal EBM once laminin secreted by epithelial cells self-polymerizes into a nascent EBM. Mature myofibroblasts that become established in the anterior stroma are a barrier to keratocyte contributions to the nascent EBM. These myofibroblasts, and the opacity they produce, often persist for months or years after the injury. Transparency is subsequently restored when the EBM is completely regenerated, myofibroblasts are deprived of TGFβ and undergo apoptosis, and the keratocytes re-occupy the anterior stroma and reabsorb disordered extracellular matrix. The aim of this review is to highlight factors involved in the generation of stromal haze and its subsequent removal.

Thrombospondin-4 (TSP-4) attracted renewed attention recently as a result of assignment of new functions to this matricellular protein in cardiovascular, muscular, and nervous systems. We have previously reported that TSP-4 promotes local vascular inflammation in a mouse atherosclerosis model. A common variant of TSP-4, P387-TSP-4, was associated with increased cardiovascular disease risk in human population studies. In a mouse atherosclerosis model, TSP-4 had profound effect on accumulation of macrophages in lesions, which prompted us to examine its effects on macrophages in more detail. We examined the effects of A387-TSP-4 and P387-TSP-4 on mouse macrophages in cell culture and in vivo in the model of LPS-induced peritonitis. In tissues and in cell culture, TSP-4 expression was associated with inflammation: TSP-4 expression was upregulated in peritoneal tissues in LPS-induced peritonitis, and pro-inflammatory signals, INFγ, GM-CSF, and LPS, induced TSP-4 expression in macrophages in vivo and in cell culture. Deficiency in TSP-4 in macrophages from Thbs4 −/− mice reduced the expression of pro-inflammatory macrophage markers, suggesting that TSP-4 facilitates macrophage differentiation into a pro-inflammatory phenotype. Expression of TSP-4, especially more active P387-TSP-4, was associated with higher cellular apoptosis. Cultured macrophages displayed increased adhesion to TSP-4 and reduced migration in presence of TSP-4, and these responses were further increased with P387 variant. We concluded that TSP-4 expression in macrophages increases their accumulation in tissues during the acute inflammatory process and supports macrophage differentiation into a pro-inflammatory phenotype. In a model of acute inflammation, TSP-4 supports pro-inflammatory macrophage apoptosis, a response that is closely related to their pro-inflammatory activity and release of pro-inflammatory signals. P387-TSP-4 was found to be the more active form of TSP-4 in all examined functions.

The epithelial basement membrane (BM) is a specialized extracellular matrix that has been shown to have a critical role in corneal development, wound healing, and disease. Although the epithelial BM contributes to corneal homeostasis, relatively little is know about non-epithelial production of its components that may be important in defective regeneration of the epithelial basement membrane associated with opacity after photorefractive keratectomy. The purpose of the current study was to investigate stromal production of corneal epithelial BM proteins in wounded human corneas using immunohistochemistry. A total of five unwounded control eyes and five 30-minute epithelial-wounded corneas were obtained from fresh corneoscleral buttons removed from human eyes enucleated due to choroidal melanoma with normal anterior segments. In the wounded corneas, an eight mm patch of central corneal epithelium and epithelial BM was removed with a Beaver blade when the patient was under general anesthesia. Immunohistochemical analyses were performed to detect perlecan and nidogen-2 proteins–important components of the epithelial BM lamina lucida and lamina densa zones. Perlecan and nidogen-2 proteins were detected in the BM itself and at low levels in keratocytes in all unwounded corneas. After epithelial injury, both perlecan and nidogen-2 was expressed at high levels in stromal keratocytes, including superficial keratocytes in the early phases of apoptosis. Thus, after epithelial and epithelial BM injury, stromal keratocytes contribute important perlecan and nidogen-2 components to the regenerating epithelial BM.