Genipin increases extracellular matrix synthesis preventing corneal perforation.

PURPOSE: Corneal melting and perforation are feared sight-threatening complications of infections, autoimmune disease, and severe burns. Assess the use of genipin in treating stromal melt. METHODS: A model for corneal wound healing was created through epithelial debridement and mechanical burring to injure the corneal stromal matrix in adult mice. Murine corneas were then treated with varying concentrations of genipin, a natural occurring crosslinking agent, to investigate the effects that matrix crosslinking using genipin has in wound healing and scar formation. Genipin was used in patients with active corneal melting. RESULTS: Corneas treated with higher concentrations of genipin were found to develop denser stromal scarring in a mouse model. In human corneas, genipin promoted stromal synthesis and prevention of continuous melt. Genipin mechanisms of action create a favorable environment for upregulation of matrix synthesis and corneal scarring. CONCLUSION: Our data suggest that genipin increases matrix synthesis and inhibits the activation of latent transforming growth factor-ß. These findings are translated to patients with severe corneal melting.

Collagen XII regulates stromal wound closure.

The role of collagen XII in regulating injury repair and reestablishment of corneal function is unknown. This manuscript aims to investigate the role(s) of collagen XII in the repair of incisional and debridement injuries in an adult mouse model. Two different types of injury in wild type and Col12a1-/- corneas were created to investigate the effects of collagen XII -in wound repair and scar formation-by using clinical photographs, immunohistology, second harmonic generation imaging and electron microscopy. Results showed that collagen XII is a regulator of wound closure after incisional injuries. Absence of collagen XII retarded wound closure and the wound healing process. These findings show that collagen XII regulates fibrillogenesis, CD68 cell lineage infiltration, and myofibroblast survival following injury. In vitro studies suggest that collagen XII regulates deposition of an early and provisional matrix by interacting with two proteins regulating early matrix deposition: fibronectin and LTBP1(latent transforming growth factor ß binding protein 1). In conclusion, collagen XII regulates tissue repair in corneal incisional wounds. Understanding the function of collagen XII during wound healing has significant translational value.

Downregulation of collagen XI during late postnatal corneal development is followed by upregulation after injury.

Collagen XI plays a role in nucleating collagen fibrils and in controlling fibril diameter. The aim of this research was to elucidate the role that collagen XI plays in corneal fibrillogenesis during development and following injury. The temporal and spatial expression of collagen XI was evaluated in C57BL/6 wild-type mice. For wound-healing studies in adult mice, stromal injuries were created using techniques that avoid caustic chemicals. The temporal expression and spatial localization of collagen XI was studied following injury in a Col11a1 inducible knockout mouse model. We found that collagen XI expression occurs during early maturation and is upregulated after stromal injury in areas of regeneration and remodeling. Abnormal fibrillogenesis with new fibrils of heterogeneous size and shape occurs after injury in a decreased collagen XI matrix. In conclusion, collagen XI is expressed in the stroma during development and following injury in adults, and is a regulator of collagen fibrillogenesis in regenerating corneal tissue.

Collagen XII Regulates Corneal Stromal Structure by Modulating Transforming Growth Factor-ß Activity.

Collagen XII is a regulator of corneal stroma structure and function. The current study examined the role of collagen XII in regulating corneal stromal transforming growth factor (TGF)-ß activation and latency. Specifically, with the use of conventional collagen XII null mouse model, the role of collagen XII in the regulation of TGF-ß latency and activity in vivo was investigated. Functional quantification of latent TGF-ß in stromal matrix was performed by using transformed mink lung reporter cells that produce luciferase as a function of active TGF-ß. Col12a1 knockdown with shRNA was used to test the role of collagen XII in TGF-ß activation. Col12a1-/- hypertrophic stromata were observed with keratocyte hyperplasia. Increased collagen fibril forward signal was found by second harmonic generation microscopy in the absence of collagen XII. Collagen XII regulated mRNA synthesis of Serpine1, Col1a1, and Col5a1 and deposition of collagens in the extracellular matrix. A functional plasminogen activator inhibitor luciferase assay showed that collagen XII is necessary for latent TGF-ß storage in the extracellular matrix and that collagen XII down-regulates active TGF-ß. Collagen XII dictates stromal structure and function by regulating TGF-ß activity. A hypertrophic phenotype in Col12a1-/- corneal tissue can be explained by abnormal up-regulation of TGF-ß activation and decreased latent storage.

Creation and grading of experimental corneal scars in mice models.

PURPOSE: To develop a stromal wound healing model and a reliable scar classification score system that correlates photographic evaluation with changes in the structure and organization of the extracellular matrix. MATERIALS AND METHODS: We tested three stromal injury techniques in adult C57BL/6 mice. Technique 1, a lineal partial thickness keratotomy in the horizontal axis. Technique 2, corneal epithelial and stromal debridement using a diamond burr in the horizontal axis, and technique 3, a combination of techniques 1 and 2. To assess intra-observer and inter-observer agreement between two examiners evaluating formed stromal scars, stereo microscopic photographs of anterior segment were scored by two masked examiners at around 1-month. Depending on the severity of opacification and the area of involvement, scars were classified on a scale from 0 to 3 based on a modified Fantes haze scale. Extracellular matrix composition as well as matrix organization, macrophage infiltration and neovascularization were evaluated with immunofluorescence and second harmonic generation (SHG) microscopy. RESULTS: Technique 1 created mild scars, with a score of 0.5 ± 0.43, while techniques 2 (score 2.1 ± 0.45) and 3 (score 2 ± 0.66), created dense scars with a higher score. A significant difference in scar severity score was noted between the 3 techniques (one way ANOVA, p < 0.0001). Masked graders demonstrated excellent agreement (intraclass correlation = 0.927 [95% confidence interval: 0.87-0.96]). The severity of scars noted at stereo microscopy correlated with the severity of changes in extracellular matrix in the stroma as demonstrated by the expression of collagens I, IV and fibronectin and evaluation of matrix hierarchical organization. In contrast to mild scarring, moderate and severe scars had increased expression of CD31 and CD68, markers of vascular endothelial cells and macrophages, respectively. CONCLUSION: Mouse models of stromal scarring using simple surgical techniques are described. Corneal scars can be consistently classified by two observers. Grading of scar severity positively correlates with changes in extracellular matrix composition, disorganization and cell infiltration.