Suture placement and alkali burn to the cornea are often used to induce inflammatory corneal neovascularization (CorNV) models in animals. This study compares the changes in genome-wide gene expression under these two CorNV conditions in mice.CorNV were induced in Balb/c mice by three interrupted 10-0 sutures placed at sites about 1 mm from the corneal apex, or by alkali burns that were 2 mm in size in the central area of the cornea. At the points in time when neovascularization progressed most quickly, some eyeballs were subjected to histological staining to examine CorNV and inflammatory cells infiltration, and some corneas were harvested to extract mRNA for microarray assay. After normalization and filtering, the microarray data were subject to statistical analysis using Significance Analysis of Microarray software, and interested genes were annotated using the Database for Annotation, Visualization, and Integrated Discovery (DAVID) program. The expression change of classical proangiogenic molecule like vascular endothelial growth factor (VEGF) and antiangiogenic molecule like pigment epithelium-derived factor (PEDF) was further verified using western blotting.Suture placement induced CorNV in the areas between the suture and limbus, but did not affect the transparency of the yet unvasuclarized areas of the corneas. In contrast, alkali burn caused edema and total loss of transparency of the whole cornea. Histology showed that sutures only caused localized epithelial loss and inflammatory infiltration between the suture and limbus, but chemical burn depleted the whole epithelial layer of the central cornea and caused heavy cellular infiltration of the whole cornea. At day 5 after suture placement, 1,055 differentially expressed probes were identified, out of which 586 probes were upregulated and 469 probes were downregulated. At a comparable time point, namely on day 6 after the alkali burn to the corneas, 472 probes were upregulated and 389 probes were downregulated. Among these differentially expressed probes, a significant portion (530 probes in total, including 286 upregulated and 244 downregulated probes) showed a similar pattern of change in both models. Annotation (using DAVID) of the overlapping differential genes revealed that the significant enrichment gene ontology terms were "chemotaxis" and "immune response" for the upregulated genes, and "oxidation reduction" and "programmed cell death" for the downregulated genes. Some genes or gene families (e.g., S100A family or α-, β-, or γ-crystallin family) that had not been related to corneal pathogenesis or neovascularization were also revealed to be involved in CorNV. VEGF was upregulated and PEDF was stable as shown with western blotting.Sutures and alkali burn to the corneas produced types of damage that affected transparency differentially, but gene profiling revealed similar patterns of changes in gene expression in these two CorNV models. Further studies of the primary genes found to be involved in CorNV will supplement current understanding about the pathogenesis of neovascularization diseases.
Abstract Descemet’s membrane (DM) helps maintain phenotype and function of corneal endothelial cells under physiological conditions, while little is known about the function of DM in corneal endothelial wound healing process. In the current study, we performed in vivo rabbit corneal endothelial cell (CEC) injury via CEC scraping, in which DM remained intact after CECs removal, or via DM stripping, in which DM was removed together with CECs. We found rabbit corneas in the CEC scraping group healed with transparency restoration, while there was posterior fibrosis tissue formation in the corneas after DM stripping on day 14. Following CEC scraping on day 3, cells that had migrated toward the central cornea underwent a transient fibrotic endothelial-mesenchymal transition (EMT) which was reversed back to an endothelial phenotype on day 14. However, in the corneas injured via DM stripping, most of the cells in the posterior fibrosis tissue did not originate from the corneal endothelium, and they maintained fibroblastic phenotype on day 14. We concluded that corneal endothelial wound healing in rabbits has different outcomes depending upon the presence or absence of Descemet’s membrane. Descemet’s membrane supports corneal endothelial cell regeneration in rabbits after endothelial injury.
To solidify the involvement of Saa-related pathway in corneal neovascularization (CorNV). The pathogenesis of inflammatory CorNV is not fully understood yet, and our previous study implicated that serum amyloid A (Saa) 1 (Saa1) and Saa3 were among the genes up-regulated upon CorNV induction in mice.Microarray data obtained during our profiling project on CorNV were analyzed for the genes encoding the four SAA family members (Saa1-4), six reported SAA receptors (formyl peptide receptor 2, Tlr2, Tlr4, Cd36, Scarb1, P2rx7) and seven matrix metallopeptidases (Mmp) 1a, 1b, 2, 3, 9, 10, 13 reportedly to be expressed upon SAA pathway activation. The baseline expression or changes of interested genes were further confirmed in animals with CorNV using molecular or histological methods. CorNV was induced in Balb/c and C57BL/6 mice by placing either three interrupted 10-0 sutures or a 2 mm filter paper soaked with sodium hydroxide in the central area of the cornea. At desired time points, the corneas were harvested for histology examination or for extraction of mRNA and protein. The mRNA levels of Saa1, Saa3, Fpr2, Mmp2 and Mmp3 in corneas were detected using quantitative reverse transcription-PCR, and SAA3 protein in tissues detected using immunohistochemistry or western blotting.Microarray data analysis revealed that Saa1, Saa3, Fpr2, Mmp2, Mmp3 messengers were readily detected in normal corneas and significantly up-regulated upon CorNV induction. The changes of these five genes were confirmed with real-time PCR assay. On the contrary, other SAA members (Saa2, Saa4), other SAA receptors (Tlr2, Tlr4, Cd36, P2rx7, etc), or other Mmps (Mmp1a, Mmp1b, Mmp9, Mmp10, Mmp13) did not show consistent changes. Immunohistochemistry study and western blotting further confirmed the expression of SAA3 products in normal corneas as well as their up-regulation in corneas with CorNV.SAA-FPR2 pathway composing genes were expressed in normal murine corneas and, upon inflammatory stimuli challenge to the corneas, their expressions were up-regulated, suggesting their roles in pathogenesis of CorNV. The potential usefulness of SAA-FPR2 targets in future management of CorNV-related diseases deserves investigation.
How corneal transparency is formed/maintained remains largely unclear. A group of enzymes which are referred to as enzymatic crystallins were proposed to contribute to corneal transparency in various animals. This study investigated whether the three classical lens crystallins, namely α-, β-, and γ-crystallins, exist in mouse and human corneas.Mice, human tissues, and cultured corneal cells were studied. The expression of lens crystallins in corneas or in cultured corneal cells were detected at the mRNA level by quantitative reverse transcription-PCR (QRT-PCR) and at the protein level by immunohistochemistry or western blotting. To check the effect of exogenous factor on expression of lens crystallins, cultured corneal cells were challenged with lipopolysaccharide or hydrogen peroxide and the expression of lens crystallins was monitored.QRT-PCR revealed that the relative expression level of lens crystallins in C57BL/6 corneas were higher than in Balb/c corneas. Immunohistochemistry study showed that expression of αA-crystallin started from the embryonic stage, lasted untill old age, and was largely restricted to the epithelium or endothelium of the corneas. β- and γ-crystallins also were found in murine corneal epithelium. Upon treatment with lipopolysaccharide or hydrogen peroxide of cultured corneal epithelial cells, lens crystallins expression was significantly increased as detected by QRT-PCR or western blot assay. Further, both fetal corneal epithelial cultures and limbal stem cell cultures from adult human tissues were positive for lens crystallin immunofluorescence or immunohistochemistry staining.Lens crystallins are expressed in mammalian corneas and cultured corneal cells. The expression levels depended on the animal strains or cell status. The physiologic and pathological significance of lens crystallins in corneas deserves more investigation.
Abstract Amniotic membranes (AMs) are widely used as a corneal epithelial tissue carrier in reconstruction surgery. However, the engineered tissue transparency is low due to the translucent thick underlying AM stroma. To overcome this drawback, we developed an ultra-thin AM (UAM) by using collagenase IV to strip away from the epithelial denuded AM (DAM) some of the stroma. By thinning the stroma to about 30 μm, its moist and dry forms were rendered acellular, optically clear and its collagen framework became compacted and inerratic. Engineered rabbit corneal epithelial cell (RCEC) sheets generated through expansion of limbal epithelial cells on UAM were more transparent and thicker than those expanded on DAM. Moreover, ΔNp63 and ABCG2 gene expression was greater in tissue engineered cell sheets expanded on UAM than on DAM. Furthermore, 2 weeks after surgery, the cornea grafted with UAM based cell sheets showed higher transparency and more stratified epithelium than the cornea grafted with DAM based cell sheets. Taken together, tissue engineered corneal epithelium generated on UAM has a preferable outcome because the transplanted tissue is more transparent and better resembles the phenotype of the native tissue than that obtained by using DAM for this procedure. UAM preserves compact layer of the amniotic membrane and maybe an ideal substrate for corneal epithelial tissue engineering.
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