Expression of neural receptors in mouse meibomian gland.

PURPOSE: To investigate gene and protein expression profiles of neural receptors found in the mouse meibomian gland. RNA and protein levels were determined for neuropeptide Y (NPY) receptor, vasoactive intestinal peptide (VIP) receptor, substance P (SP) receptor, and muscarinic cholinergic receptor (mAChR) subtypes M1-M5 in the mouse meibomian gland. METHODS: Frozen sections of Balb/c mouse eyelids were subjected to laser capture microdissection to isolate pure samples of meibomian gland ductal and acinar cells. Real-time polymerase chain reaction, immunolabeling, and Western blot analysis for SP receptor, VIP receptor, NPY receptor, and mAChR subtypes M1-M5 were performed on meibomian gland ductal and acinar cells. RESULTS: Expression of NPY1 receptor, VIP receptor 1, SP receptor, and all 5 mAChR subtypes was found in all meibomian gland ductal and acinar cells analyzed by real-time polymerase chain reaction. Immunolabeling and Western blot analysis confirmed the presence of NPY1 receptor, VIP receptor 1, SP receptor, and all 5 mAChR subtypes in the meibomian gland. The levels were variable with the duct showing greater levels of NPY1 receptor, SP receptor, and mAChRs 1, 2, 4, and 5 than with the gland. CONCLUSIONS: VIP receptor 1, SP receptor, NPY1 receptor, and mAChR subtypes may be involved in the regulation of meibomian gland secretion. Laser capture microdissection in conjunction with gene expression analysis provides an excellent approach for studying meibomian gland cells about which relatively little is known at the molecular level.

Distinct gene subsets in pterygia formation and recurrence: dissecting complex biological phenomenon using genome wide expression data.

BACKGROUND: Pterygium is a common ocular surface disease characterized by fibrovascular invasion of the cornea and is sight-threatening due to astigmatism, tear film disturbance, or occlusion of the visual axis. However, the mechanisms for formation and post-surgical recurrence of pterygium are not understood, and a valid animal model does not exist. Here, we investigated the possible mechanisms of pterygium pathogenesis and recurrence. METHODS: First we performed a genome wide expression analysis (human Affymetrix Genechip, >22000 genes) with principal component analysis and clustering techniques, and validated expression of key molecules with PCR. The controls for this study were the un-involved conjunctival tissue of the same eye obtained during the surgical resection of the lesions. Interesting molecules were further investigated with immunohistochemistry, Western blots, and comparison with tear proteins from pterygium patients. RESULTS: Principal component analysis in pterygium indicated a signature of matrix-related structural proteins, including fibronectin-1 (both splice-forms), collagen-1A2, keratin-12 and small proline rich protein-1. Immunofluorescence showed strong expression of keratin-6A in all layers, especially the superficial layers, of pterygium epithelium, but absent in the control, with up-regulation and nuclear accumulation of the cell adhesion molecule CD24 in the pterygium epithelium. Western blot shows increased protein expression of beta-microseminoprotein, a protein up-regulated in human cutaneous squamous cell carcinoma. Gene products of 22 up-regulated genes in pterygium have also been found by us in human tears using nano-electrospray-liquid chromatography/mass spectrometry after pterygium surgery. Recurrent disease was associated with up-regulation of sialophorin, a negative regulator of cell adhesion, and never in mitosis a-5, known to be involved in cell motility. CONCLUSION: Aberrant wound healing is therefore a key process in this disease, and strategies in wound remodeling may be appropriate in halting pterygium or its recurrence. For patients demonstrating a profile of 'recurrence', it may be necessary to manage as a poorer prognostic case and perhaps, more adjunctive treatment after resection of the primary lesion.

Phospholipase D in the human ocular surface and in pterygium.

PURPOSE: Pterygium is a fibro-vascular disease of unknown etiology characterized by proliferation and advancement of tissue onto the cornea. Phospholipase Ds (PLDs) are members of an important class of enzymes involved in inflammation and differentiation. In cultured corneal epithelial cells, these enzymes play a role in wound healing, and in other contexts, they suppress apoptosis and increase cell motility. We aimed to study the presence of PLD subtypes in native ocular surface tissue and pterygium. METHODS: This study involved paired control or uninvolved conjunctival and pterygium tissues from 6 patients. Reverse transcription semiquantitative and quantitative polymerase chain reactions were performed to assess transcript levels for PLD1-5 in normal conjunctiva and pterygium tissue. Immunofluorescent staining by using antibodies against PLD1/2 was used to study the expression and tissue distribution. Western blots were performed for protein detection and to confirm the specificity of the antibodies used. RESULTS: PLD1, 2, 3, and 4 transcripts were detected in normal conjunctiva tissue, and types 2, 3, and 4 were upregulated in pterygium. Immunofluorescent staining showed the presence of phospholipase-D1/2 in normal cornea, conjunctival, and pterygial epithelia. In normal cornea and conjunctival epithelia, the expression was mainly localized to the nuclei of the basal and suprabasal epithelial cells, whereas in pterygium, this expression was limited to the cytoplasm and peri-plasma membrane regions. Western blot confirmed the presence of PLD1/2 in proteins extracted from pterygium and conjunctiva tissue. CONCLUSIONS: PLD subtypes are present in human ocular surface epithelium. PLD may be involved in pterygium pathogenesis.