L-carnitine suppresses transient receptor potential vanilloid type 1 activity and myofibroblast transdifferentiation in human corneal keratocytes.

Corneal stromal wound healing is a well-balanced process promoted by overlapping phases including keratocyte proliferation, inflammatory-related events, and tissue remodeling. L-carnitine as a natural antioxidant has shown potential to reduce stromal fibrosis, yet the underlying pathway is still unknown. Since transient receptor potential vanilloid 1 (TRPV1) is a potential drug target for improving the outcome of inflammatory/fibrogenic wound healing, we investigated if L-carnitine can mediate inhibition of the fibrotic response through suppression of TRPV1 activation in human corneal keratocytes (HCK). We determined TRPV1-induced intracellular calcium transients using fluorescence calcium imaging, channel currents by planar patch-clamping, and cell migration by scratch assay for wound healing. The potential L-carnitine effect on TRPV1-induced myofibroblast transdifferentiation was evaluated by immunocytochemical detection of alpha smooth muscle actin. RT-PCR analysis confirmed TRPV1 mRNA expression in HCK. L-carnitine (1 mmol/l) inhibited either capsaicin (CAP) (10 µmol/l), hypertonic stress (450 mOsmol/l), or thermal increase (>43 °C) induced Ca2+ transients and corresponding increases in TRPV1-induced inward and outward whole-cell currents. This was accompanied by suppression of injury-induced increases in myofibroblast transdifferentiation and cell migration. In conclusion, L-carnitine contributes to inhibit stromal scarring through suppressing an injury-induced intrinsic TRPV1 activity that is linked with induction of myofibroblast transdifferentiation in HCK cells.

Vernal keratoconjunctivitis: a severe allergic eye disease with remodeling changes.

Vernal keratoconjunctivitis (VKC) is an unusually severe sight-threatening allergic eye disease, occurring mainly in children. Conventional therapy for allergic conjunctivitis is generally not adequate for VKC. Pediatricians and allergists are often not familiar with the severe clinical symptoms and signs of VKC. As untreated VKC can lead to permanent visual loss, pediatric allergists should be aware of the management and therapeutic options for this disease to allow patients to enter clinical remission with the least side effects and sequelae. Children with VKC present with severe ocular symptoms, that is, severe eye itching and irritation, constant tearing, red eye, eye discharge, and photophobia. On examination, giant papillae are frequently observed on the upper tarsal conjunctiva (cobblestoning appearance), with some developing gelatinous infiltrations around the limbus surrounding the cornea (Horner-Trantas dot). Conjunctival injections are mostly severe with thick mucus ropy discharge. Eosinophils are the predominant cells found in the tears and eye discharge. Common therapies include topical antihistamines and dual-acting agents, such as lodoxamide and olopatadine. These are infrequently sufficient and topical corticosteroids are often required for the treatment of flare ups. Ocular surface remodeling leads to severe suffering and complications, such as corneal ulcers/scars. Other complications include side effects from chronic topical steroids use, such as increased intraocular pressure, glaucoma, cataract and infections. Alternative therapies for VKC include immunomodulators, such as cyclosporine A and tacrolimus. Surgery is reserved for those with complications and should be handled by ophthalmologists with special expertise. Newer research on the pathogenesis of VKC is reviewed in this article. Vernal keratoconjunctivitis is a very important allergic eye disease in children. Complications and remodeling changes are unique and can lead to blindness. Understanding of pathogenesis of VKC may lead to better therapy for these unfortunate patients.

Franceschetti hereditary recurrent corneal erosion.

PURPOSE: To describe new affected individuals of Franceschetti's original pedigree of hereditary recurrent erosion and to classify a unique entity called Franceschetti corneal dystrophy. DESIGN: Observational case series. METHODS: Slit-lamp examination of 10 affected individuals was conducted. Biomicroscopic examinations were supplemented by peripheral corneal biopsy in 1 affected patient with corneal haze. Tissue was processed for light and electron microscopy and immunohistochemistry was performed. DNA analysis was carried out in 12 affected and 3 nonaffected family members. RESULTS: All affected individuals suffered from severe ocular pain in the first decade of life, attributable to recurrent corneal erosions. Six adult patients developed bilateral diffuse subepithelial opacifications in the central and paracentral cornea. The remaining 4 affected individuals had clear corneas in the pain-free stage of the disorder. Histologic and immunohistochemical examination of the peripheral cornea in a single patient showed a subepithelial, avascular pannus. There was negative staining with Congo red. DNA analysis excluded mutations in the transforming growth factor beta-induced (TGFBI) gene and in the tumor-associated calcium signal transducer 2 (TACSTD2) gene. CONCLUSION: We have extended the pedigree of Franceschetti corneal dystrophy and elaborated its natural history on the basis of clinical examinations. A distinctive feature is the appearance of subepithelial opacities in adult life, accompanied by a decreased frequency of recurrent erosion attacks. Its clinical features appear to distinguish it from most other forms of dominantly inherited recurrent corneal erosion reported in the literature.

EGF suppresses hydrogen peroxide induced Ca2+ influx by inhibiting L-type channel activity in cultured human corneal endothelial cells.

Endogenous generated hydrogen peroxide during eye bank storage limits viability. We determined in cultured human corneal endothelial cells (HCEC) whether: (1) this oxidant induces elevations in intracellular calcium concentration [Ca2+]i; (2) epidermal growth factor (EGF) medium supplementation has a protective effect against peroxide mediated rises in [Ca2+]i. Whereas pathophysiological concentrations of H2O2 (10 mM) induced irreversible large increases in [Ca2+]i, lower concentrations (up to 1 mM) had smaller effects, which were further reduced by exposure to either 5 microM nifedipine or EGF (10 ng ml(-1)). EGF had a larger protective effect against H2O2-induced rises in [Ca2+]i than nifedipine. In addition, icilin, the agonist for the temperature sensitive transient receptor potential protein, TRPM8, had complex dose-dependent effects (i.e. 10 and 50 microM) on [Ca2+]i. At 10 microM, it reversibly elevated [Ca2+]i whereas at 50 microM an opposite effect occurred suggesting complex effects of temperature on endothelial viability. Taken together, H2O2 induces rises in [Ca2+]i that occur through increases in Ca2+ permeation along plasma membrane pathways that include L-type Ca2+ channels as well as other EGF-sensitive pathways. As EGF overcomes H2O2-induced rises in [Ca2+]i, its presence during eye bank storage could improve the outcome of corneal transplant surgery.

Increased endothelial survival of organ-cultured corneas stored in FGF-2-supplemented serum-free medium.

PURPOSE: To investigate the effect of FGF-2 on corneal endothelial cell survival in porcine and human corneas during corneal storage in a serum-free medium. METHODS: Porcine and paired human corneas were stored at 32 degrees C for 9 and 22 days, respectively. One cornea of each pair was stored in a serum-free culture medium, and the mate was preserved in the same medium supplemented with 10 ng/mL FGF-2. Quantitative analysis of corneal damage after storage was determined by the Janus green photometry technique. 5-Bromo-2-deoxyuridine (BrdU) labeling of the endothelium determined the effect of FGF-2 on endothelial proliferation during storage. Additional cell culture studies were performed to elucidate the role of FGF-2 on the incidence of endothelial apoptosis after serum deprivation. RESULTS: When FGF-2 was added to the serum-free medium, the damage rates of porcine endothelia were reduced from 15.1% +/- 8.7% (control) to 6.4% +/- 2.0% after 9 days and from 25.3% +/- 10.2% to 13.6% +/- 4.2% after 22 days of storage. In the human corneas stored during 22 days in FGF-2-supplemented medium, the amount of endothelial damage was 11.8% +/- 3.2%, which was significantly less damage than in the control fellow corneas stored in unsupplemented serum-free medium (19.3% +/- 6.3%; P < 0.01). DNA synthesis was not enhanced in corneas stored in serum-free medium, serum-free medium+FGF-2, or medium containing 10% FCS. Only a few (3.8%) TUNEL-positive endothelial cells were detected in cultures maintained in FGF-2-supplemented serum-free medium compared with a high number (48%) of apoptotic cells in control cultures. CONCLUSIONS: FGF-2 efficiently reduces human corneal endothelial damage that occurs during organ culture storage in a serum-free medium. This effect is truly protective, because no proliferative activity and a decreased rate of apoptosis were determined. FGF-2 emerges as an important component of a future serum-free corneal organ-culture medium established to replace fetal calf serum (FCS) as a potential source of recipient infection.