A role for topographic cues in the organization of collagenous matrix by corneal fibroblasts and stem cells

Human corneal fibroblasts (HCF) and corneal stromal stem cells (CSSC) each secrete and organize a thick stroma-like extracellular matrix in response to different substrata, but neither cell type organizes matrix on tissue-culture polystyrene. This study compared cell differentiation and extracellular matrix secreted by these two cell types when they were cultured on identical substrata, polycarbonate Transwell filters. After 4 weeks in culture, both cell types upregulated expression of genes marking differentiated keratocytes (KERA, CHST6, AQP1, B3GNT7). Absolute expression levels of these genes and secretion of keratan sulfate proteoglycans were significantly greater in CSSC than HCF. Both cultures produced extensive extracellular matrix of aligned collagen fibrils types I and V, exhibiting cornea-like lamellar structure. Unlike HCF, CSSC produced little matrix in the presence of serum. Construct thickness and collagen organization was enhanced by TGF-β3. Scanning electron microscopic examination of the polycarbonate membrane revealed shallow parallel grooves with spacing of 200-300 nm, similar to the topography of aligned nanofiber substratum which we previously showed to induce matrix organization by CSSC. These results demonstrate that both corneal fibroblasts and stromal stem cells respond to a specific pattern of topographical cues by secreting highly organized extracellular matrix typical of corneal stroma. The data also suggest that the potential for matrix secretion and organization may not be directly related to the expression of molecular markers used to identify differentiated keratocytes. © 2014 Karamichos et al

Eccentric lamellar keratolimbal grafts harvested with a manually guided microkeratome

Background: To perform lamellar keratolimbal allograft transplantation in a one- step procedure with a single graft, we investigated the feasibility of harvesting eccentric lamellar keratolimbal grafts from conventionally processed corneoscleral buttons using a manually guided microkeratome in conjunction with an artificial anterior chamber system. Methods: We used the Moria LSK- One microkeratome and the automated lamellar therapeutic keratoplasty ( ALTK) system ( Antony, France). Ten human donor eyes were used to obtain single- piece lamellar keratolimbal grafts. Specimens were processed for light and electron microscopy. Results: Eccentric keratolimbal grafts could be obtained from all human donor buttons. Grafts include a crescent- shaped limbal and a large corneal portion. No visible damage to the limbal region was discernible. Conclusion: Our data show that the LSK- One microkeratome in conjunction with the ALTK system allows harvesting eccentric keratolimbal grafts from donor corneoscleral buttons. Copyright (c) 2007 S. Karger AG, Basel

Corneal Epithelium Expresses a Variant of P2X7 Receptor in Health and Disease

Improper wound repair of the corneal epithelium can alter refraction of light resulting in impaired vision. We have shown that ATP is released after injury, activates purinergic receptor signaling pathways and plays a major role in wound closure. In many cells or tissues, ATP activates P2X7 receptors leading to cation fluxes and cytotoxicity. The corneal epithelium is an excellent model to study the expression of both the full-length P2X7 form (defined as the canonical receptor) and its truncated forms. When Ca2+ mobilization is induced by BzATP, a P2X7 agonist, it is attenuated in the presence of extracellular Mg2+ or Zn2+, negligible in the absence of extracellular Ca2+, and inhibited by the competitive P2X7 receptor inhibitor, A438079. BzATP enhanced phosphorylation of ERK. Together these responses indicate the presence of a canonical or full-length P2X7 receptor. In addition BzATP enhanced epithelial cell migration, and transfection with siRNA to the P2X7 receptor reduced cell migration. Furthermore, sustained activation did not induce dye uptake indicating the presence of truncated or variant forms that lack the ability to form large pores. Reverse transcription-polymerase chain reaction and Northern blot analysis revealed a P2X7 splice variant. Western blots identified a full-length and truncated form, and the expression pattern changed as cultures progressed from monolayer to stratified. Cross-linking gels demonstrated the presence of homo- and heterotrimers. We examined epithelium from age matched diabetic and non-diabetic corneas patients and detected a 4-fold increase in P2X7 mRNA from diabetic corneal epithelium compared to non-diabetic controls and an increased trend in expression of P2X7variant mRNA. Taken together, these data indicate that corneal epithelial cells express full-length and truncated forms of P2X7, which ultimately allows P2X7 to function as a multifaceted receptor that can mediate cell proliferation and migration or cell death

Nerve growth factor promotes corneal epithelial migration by enhancing expression of matrix metalloprotease-9.

PURPOSE. Nerve growth factor (NGF) is a neuropeptide essential for the development, survival, growth, and differentiation of corneal cells. Its effects are mediated by both TrkA and p75 receptors. Clinically relevant use of NGF was introduced to treat neurotrophic ulcerations in patients. Herein, we examine the mechanisms by which NGF enhances epithelial wound healing both in vivo and in vitro. METHODS. An animal model using adult hens was implemented for the in vivo experiments. Laser ablation keratectomy was performed and animals were observed for up to 7 days. Epithelial healing was measured with fluorescein. In addition, proliferation was measured using BrdU incorporation and both TrkA and matrix metalloprotease-9 (MMP-9) expression were measured by immunohistochemistry (IHC) and Western blot (WB). In vitro experiments were carried out with telomerase-immortalized human corneal epithelial cells (HCLE). The rate of proliferation was measured using a colorimetric assay and BrdU incorporation. Realtime migration was evaluated with an inverted microscope. MMP-9 expression was evaluated by immunocytochemistry (ICC), WB, zymography, and RT-PCR. Finally, beta-4 integrin (b4) expression was assessed by ICC and WB. RESULTS. Faster epithelial healing was observed in NGF-treated corneas compared with controls (P < 0.01). These corneas showed increased proliferation, TrkA upregulation, and enhanced MMP-9 presence (P < 0.01). In vitro, faster spreading and migration were observed in response to NGF (P < 0.01). Enhanced proliferation, as well as enhanced TrkA and MMP-9 expression, and decreased b4 levels were observed after adding NGF (P < 0.01). CONCLUSIONS. NGF plays a major role during the epithelial healing process by promoting migration, a process that is accelerated by cell spreading. This effect is mediated by both the upregulation of MMP-9 and cleavage of b4 integrin

Comparison of expression profiles induced by dust mite in airway epithelia reveals a common pathway

Background: Airway epithelial cells have shown to be active participants in the defense against pathogens by producing signaling and other regulatory molecules in response to the encounter. Methods: In previous manuscripts, we have studied the effect of house dust mite (HDM) extract on both an epithelial cell-line (H292) and primary nasal epithelial cell. When we compare these responses we conclude that the H292 cells more closely resemble nasal epithelium of healthy controls (share 107 probe-sets) than of allergic individuals (share 17 probe-sets). Results: Interestingly, probably because of an absent intraindividual variation between samples, more probe-sets (8280) change expression significantly in H292 than in either healthy (555) or allergic (401) epithelium. Conclusions: A direct comparison of all the responses in these epithelial cells reveals a core-response to HDM of just 29 genes. These genes (CCL20, IL-8, CXCL2, CXCL1, IL-1B, AREG, TNFAIP3, HBEGF, PTGS2, BMP2, LDLR, PLAUR, PLAU, NFKB2, NFKB1, JUN, ATF3, EGR1, NPC1, TICAM1, EPHA2, CTGF, DUSP1, SPRY1, TLR-3, complement factor C3, IVNS1ABP, SerpinB3, and PSAT1) have described links with allergy or inflammation and may even describe the well-established relationship between viral infections and allergic exacerbations or allergy development