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PURPOSE Thrombospondin-1 (THBS1) has been suggested as a corneal wound-healing modulator. Therefore, we compromised the integrity of the cornea to elucidate the role of THBS1. METHODS Full-thickness penetrating corneal incisions (1.5 mm) were created in wild type (WT, 129S2/SvPas) and THBS1-deficient mice (Thbs1⁻/⁻), 129S2/SvPas-Thbs1(tm1Hyn)/Thbs1(tm1Hyn)), and allowed to heal up to 1 month, while being monitored by slit-lamp and intravital corneal examinations. Corneas also were examined by transmission electron microscopy and indirect immunofluorescence. To determine how THBS1 was involved in the healing process, we examined THBS1 and α-smooth muscle actin (SMA), a marker of myofibroblasts and myoepithelial cells. RESULTS In WT mice by 1 month, corneas appeared transparent with a thin scar, and endothelium and Descemet's membrane (DM) were restored. In contrast, Thbs1⁻/⁻ corneas exhibited chronic edema and persistent opacity after wounding. The DM and endothelium were not restored, and wound contraction was impaired. The THBS1 was localized in epithelial cells at early stages of the healing process, and in the stroma and endothelial cells during later stages. The SMA-positive epithelial cells and myofibroblasts were observed within the healing area at day 4, peaked at day 14, and disappeared at day 30. The SMA-positive cells were reduced greatly in Thbs1⁻/⁻ mice. CONCLUSIONS In the current study, we demonstrated that corneal restoration is strikingly compromised by a penetrating incision in Thbs1⁻/⁻ mice. The wound results in persistent edema and wound gaping. This appears to be the result of the lack of endothelial migration and DM restoration. In addition, myofibroblast formation is compromised, resulting in the lack of wound contraction.

Integrins are a family of receptors that mediate cell-cell and cell-matrix adhesion,1–3 as well as contributing to a wide variety of cellular responses, including survival, proliferation, and migration.3 These heterodimeric transmembrane glycoproteins have both alpha (α) and beta (β) subunits. The combination of these 2 subunits results in at least 22 different cell surface receptors with wide ligand binding specificities. Integrin αvβ6 is expressed at very low levels in epithelial cells of healthy adult mammals; however, its expression is considerably upregulated in response to injury or inflammation.4,5 Absence of αvβ6 is linked to initiation and progression of periodontal disease,6 regulation of lung and skin inflammation,7 delay in epidermal wound healing,8 and suppression of pulmonary fibrosis.9 In the cornea, expression of αvβ6 is restricted to the epithelium. Under pathologic conditions, such as in patients with bullous keratopathy,10 and during wound healing, αvβ6 is upregulated, suggesting a role in corneal epithelial wound repair11 and keratinocyte migration.12 αvβ6 is a receptor for many of the components of the basement membrane zone (BMZ) including fibronectin, vitronectin, tenascin, and E-cadherins.13,14 It is also the receptor for the Arg-Gly-Asp (RGD) attachment site of both latency-associated peptide (LAP)-TGF-β1 and LAP-TGF-β3.9,15,16 Attachment of αvβ6 to latent TGF-β has been shown in several investigations to be involved in the activation of TGF-β (for review see references 17–20). Indeed, αvβ6-null mice recapitulate much of the phenotype of TGF-β1-null mice.21 Because αvβ6 is known to be expressed in remodeling rat basal corneal epithelium and human bullous keratopathy,10,11 and is an activator of latent TGF-β,9,15,16 we hypothesized that αvβ6 plays an important role in corneal repair when the BMZ is compromised by surgery. We addressed this hypothesis by using adult β6 integrin-deficient mice (β6−/−) and comparing them with wild type (WT) mice.