A nano-phytochemical ophthalmic solution for marked improvement of corneal wound healing in healthy or diabetic mice

Aim: To formulate a novel nano-phytochemical ophthalmic solution to promote corneal wound healing. Methods: Dipotassium glycyrrhizinate (DG) and palmatine (PAL) were used to formulate this formulation marked as DG-PAL, and its efficacy and mechanisms for promoting corneal wound healing were evaluated in mice. Results: DG-PAL was easily fabricated with excellent physical profiles. In in vivo efficiency evaluations, DG-PAL demonstrated an excellent promoting effect on corneal epithelial/nerve wound healing in both healthy and diabetic mice. These effects were involved in the DG-PAL-induced decreased expression levels of HMGB1 and its signaling-related factors in the corneas and trigeminal neurons of the healthy or diabetic mice. Conclusion: DG-PAL possibly represents a promising ophthalmic solution for promoting corneal wound healing.

β-blocker eye drops affect ocular surface through β2 adrenoceptor of corneal limbal stem cells

Background Topical application of β-blocker eye drops induces damage to the ocular surface in clinical. However, the mechanism involved remains incompletely understood. The purpose of this study was to investigate the influence and mechanism of β-blocker eye drops on corneal epithelial wound healing. Methods Corneal epithelial wound healing models were constructed by epithelial scraping including in the limbal region and unceasingly received eye drops containing 5 mg/mL β-blocker levobunolol, β1-adrenoceptor (β1AR)-specific antagonist atenolol or β2-adrenoceptor (β2AR)-specific antagonist ICI 118, 551. For the migration assay, the murine corneal epithelial stem/progenitor cells (TKE2) were wounded and subsequently incubated with levobunolol, atenolol, or ICI 118, 551. The proliferation and colony formation abilities of TKE2 cells treated with levobunolol, atenolol, or ICI 118, 551 were investigated by CCK-8 kit and crystal violet staining. The differentiation marker Cytokeratin 3 (CK3), the stem cell markers-Cytokeratin 14 (CK14) and Cytokeratin 19 (CK19), and corneal epithelium regeneration-related signaling including in Ki67 and the phosphorylated epithelial growth factor receptor (pEGFR) and phosphorylated extracellular signal-regulated kinase 1/2 (pERK1/2) were assessed by immunofluorescence staining. Results Levobunolol and ICI 118, 551 impaired corneal wound healing, decreased the expressions of CK3, CK14, and CK19 after limbal region scraping in vivo and reduced the migration and proliferation of TKE2 in vitro, whereas atenolol had no significant effect. Moreover, levobunolol and ICI 118, 551 inhibited corneal wound healing by mediating the expression of Ki67, and the phosphorylation of EGFR and ERK1/2 in the limbal and regenerated corneal epithelium. Conclusion β-blocker eye drops impaired corneal wound healing by inhibiting the β2AR of limbal stem cells, which decreased corneal epithelial regeneration-related signaling. Therefore, a selective β1AR antagonist might be a good choice for glaucoma treatment to avoid ocular surface damage.

Contribution of the Transcription Factors Sp1/Sp3 and AP-1 to Clusterin Gene Expression during Corneal Wound Healing of Tissue-Engineered Human Corneas

In order to reduce the need for donor corneas, understanding of corneal wound healing and development of an entirely tissue-engineered human cornea (hTECs) is of prime importance. In this study, we exploited the hTEC to determine how deep wound healing affects the transcriptional pattern of corneal epithelial cells through microarray analyses. We demonstrated that the gene encoding clusterin (CLU) has its expression dramatically repressed during closure of hTEC wounds. Western blot analyses confirmed a strong reduction in the expression of the clusterin isoforms after corneal damage and suggest that repression of CLU gene expression might be a prerequisite to hTEC wound closure. Transfection with segments from the human CLU gene promoter revealed the presence of three regulatory regions: a basal promoter and two more distal negative regulatory regions. The basal promoter bears DNA binding sites for very potent transcription factors (TFs): Activator Protein-1 (AP-1) and Specificity protein-1 and 3 (Sp1/Sp3). By exploiting electrophoretic mobility shift assays (EMSA), we demonstrated that AP-1 and Sp1/Sp3 have their DNA binding site overlapping with one another in the basal promoter of the CLU gene in hCECs. Interestingly, expression of both these TFs is reduced (at the protein level) during hTEC wound healing, thereby contributing to the extinction of CLU gene expression during that process. The results of this study contribute to a better understanding of the molecular mechanisms accounting for the repression of CLU gene expression during corneal wound healing.

Extracellular Vesicles in the Cornea: Insights from Other Tissues

Extracellular vesicles (EVs) are phospholipid bilayer-bound particles secreted by cells that have been found to be important in mediating cell-cell communication, signal transduction, and extracellular matrix remodeling. Their role in both physiological and pathological processes has been established in different tissues throughout the human body. The human cornea functions as a transparent and refractive barrier that protects the intraocular elements from the external environment. Injury, infection, or disease may cause the loss of corneal clarity by altering extracellular matrix organization within the stroma that may lead to detrimental effects on visual acuity. Over the years, numerous studies have identified many of the growth factors (e.g., transforming growth factor-β1, thrombospondin-1, and platelet-derived growth factor) important in corneal wound healing and scarring. However, the functional role of bound factors encapsulated in EVs in the context of corneal biology is less defined. In this review, we describe the discovery and characterization of EVs in the cornea. We focus on EV-matrix interactions, potential functions during corneal wound healing, and the bioactivity of mesenchymal stem cell-derived EVs. We also discuss the development of EVs as stable, drug-loaded therapeutics for ocular applications.