Faculty Bibliography

Aquaporin-4 (AQP4) is a basolateral water channel in collecting duct principal cells and assembles into orthogonal array particles (OAPs), the size of which appears to depend on relative expression levels of AQP4 splice variants. Because the higher-order organization of AQP4 was perturbed by vasopressin in Brattleboro rats and phosphorylation sites have been identified on AQP4, we investigated whether vasopressin and forskolin (Fk) affect AQP4 assembly and/or expression in LLC-PK(1) cells stably transfected with the AQP4 splice variant M23, which is responsible for formation of OAPs, and/or the splice variant M1, which does not form OAPs. Our data show that [lys(8)]-vasopressin (LVP) and Fk treatment led to differential increases in expression levels of M23-AQP4 and M1-AQP4 that varied as a function of incubation time. At early time points (day 1) expression of M1 was significantly stimulated (4.5-fold), over that of M23 (1.6-fold), but after 3 days the expression of M23 became predominant (4.1-fold) over that of M1 (1.9-fold). This pattern of stimulation was dependent on an intact AQP4 residue serine 111 and required protein synthesis. In cells expressing both M1 and M23 (M1/M23 approximately 1), with small sized OAPs at the membrane, the LVP/Fk-induced stimulation of M23 was modified and mimicked that of M1 when expressed alone, suggesting a dominant role for M1. In Brattleboro kidney inner medulla, an 8-day chronic exposure to the vasopressin agonist (dDAVP) led to reduction in M1 and a significant increase in M23 immunoblot staining (M1/M23 = 2/3 --> 1/4). These results indicate that AQP4 organization and expression are regulated by vasopressin in vivo and in vitro and demonstrate that the dominant role for M1 is restricted to a one-to-one interaction between AQP4 splice variants that regulates the membrane expression of OAPs.

Glaucomatous neurodegeneration has been associated with the activation of multiple pathogenic mechanisms that can result in RGC death and axonal degeneration. Growing evidence obtained from clinical and experimental studies over the last decade also strongly suggests the involvement of the immune system in the neurodegenerative process of glaucoma. The roles of the immune system in glaucoma have been described as either neuroprotective or neurodestructive. It has been proposed that a critical balance between beneficial protective immunity and harmful sequelae of autoimmune neurodegenerative injury determines the ultimate fate of RGCs in response to various stressors in patients with glaucoma. Here, we review the key role for immunoregulation in cell fate decisions regarding RGC survival in response to glaucomatous tissue stress. Furthermore, we review the mechanisms by which autoimmunity to specific antigens such as heat shock proteins may result in RGC demise in some patients with glaucoma. In these patients, we hypothesized that one form of glaucoma may be an autoimmune optic neuropathy in which an individual's immune system facilitates a somatic or axonal degeneration of RGCs by the very system which normally serves to protect it against stress.

Glaucomatous optic neuropathy causes blindness through the degeneration of retinal ganglion cells (RGCs) and their axons, which comprise the optic nerve. Glaucoma traditionally is associated with elevated intraocular pressure, but often occurs or may progress with intraocular pressure in the normal range. Like other diseases of the CNS, a subset of glaucoma has been proposed to involve an autoimmune component to help explain the loss of RGCs in the absence of elevated intraocular pressure. One hypothesis involves heat shock proteins (HSPs), because increased serum levels of HSP autoantibodies are prominent in some glaucoma patients with normal pressures. In the first direct support of this hypothesis, we found that HSP27 and HSP60 immunization in the Lewis rat induced RGC degeneration and axon loss 1-4 months later in vivo in a pattern with similarities to human glaucoma, including topographic specificity of cell loss. Infiltration of increased numbers of T-cells in the retina occurred much earlier, 14-21 d after HSP immunization, and appeared to be transient. In vitro studies found that T-cells activated by HSP immunization induced RGC apoptosis via the release of the inflammatory cytokine FasL, whereas HSP immunization induced activation of microglia cells and upregulation of the FasL receptor in RGCs. In summary, our results suggest that RGC degeneration in glaucoma for selected individuals likely involves failed immunoregulation of the T-cell-RGC axis and is thus a disturbance of both proapoptotic and protective pathways.

PURPOSE: These studies examined corneal healing rates, Type-IV collagen and zonula occludens membrane-associated protein (ZO-1) expression, as well as aqueous PGE(2) and IL-1 beta concentrations in pigmented rabbits treated with either moxifloxacin 0.5%, gatifloxacin 0.3% or BSS following anterior keratectomy. METHODS: Anterior keratectomy surgery was followed by topical administration with commercial ophthalmic formulations of either moxifloxacin or gatifloxacin or BSS (TID for 96 h). Images of the fluorescein-stained healing corneas were analyzed for wound area. At 48 or 96 h following surgery, aqueous humor samples were collected and analyzed for the inflammatory mediators PGE(2) and IL-1 beta using an ELISA. The corneas were subsequently evaluated using both scanning and transmission electron microscopy. In a second parallel study, corneas were evaluated at both 48 and 96 h for Type-IV collagen and ZO-1 expression using immunohistochemistry. RESULTS: Fluorescein-stained corneal images at 96 h postsurgery demonstrated that 90% +/- 8% re-epithelialization for moxifloxacin, 81% +/- 14% for gatifloxacin, and 88 +/- 6% for BSS((R)) (P > 0.05). PGE(2 )levels in the aqueous humor of fluoroquinolone treated eyes were reduced at 48 h compared to BSS treated eyes. IL-1 beta was undetectable in all samples. No differences in Type-IV collagen or ZO-1 expression were observed between any treatment groups. There were no differences between groups in histological appearance or in ultrastructural healing processes. CONCLUSIONS: These studies demonstrated that the commercial ophthalmic formulations of moxifloxacin and gatifloxacin were similar to each other in their effects on the levels of aqueous humor PGE(2) and rates of corneal wound re-epithelialization.

The aquaporin (AQP) transmembrane proteins facilitate the movement of water across the plasma membrane. In the lens, AQP0 is expressed in fiber cells and AQP1 in the epithelium. Recently, two individuals were identified with congenital polymorphic autosomal dominant cataract, due to a single nucleotide base deletion mutation in the lens AQP0. The deletion modified the reading frame resulting in the addition of a premature stop codon. In the present study, we examined the water permeability properties, trafficking and dominant negative effects as well as cytotoxicity due to the mutant AQP0 (Delta213-AQP0) protein. The membrane water permeability (P(w)) of Delta213-AQP0 expressing oocytes (14+/-1 microm/s) was significantly lower than those expressing WT-AQP0 (25+/-3 microm/s). P(w) of water injected control oocytes was 13+/-2 microm/s. Co-expression of WT-AQP0 with Delta213-AQP0 significantly lowered the P(w) (18+/-3 microm/s) compared to WT-AQP0. With or without the EGFP tag, WT-AQP0 protein localized in the plasma membranes of oocytes and cultured cells whereas Delta213-AQP0 was retained in the ER. Forster Resonance Energy Transfer (FRET) showed that WT-AQP0 partly localized with the co-expressed Delta213-AQP0. Co-localization studies suggest that the mutant AQP0 gained its dominant function by trapping the WT-AQP0 in the ER through hetero-oligomerization. Incubating the cells with chemical chaperones, namely, TMAO and DMSO, did not correct the folding/trafficking defects. Cell death in the Delta213-AQP0 expressing cells was due to necrosis caused by the accumulation of Delta213-AQP0 protein in the ER in cytotoxic proportions. The data show that replacement of the distal end of the 6th TM domain and the C-terminal domain of AQP0 due to the deletion mutation resulted in the impairment of cell membrane P(w), localization of the mutant protein in the ER without trafficking to the plasma membrane, and cytotoxicity due to the accumulation of the mutant protein. Cataracts in patients with this mutation might have resulted from the above mentioned consequences.

The successful development of a therapeutic agent targeting treatment of dry eye syndrome necessitates the demonstration of drug efficacy for both sign and symptom endpoints. As numerous therapeutic strategies incorporate a secretagogue function into their overall mechanism of action, the quantitative assessment of tear production serves as a logical endpoint to anchor "sign" efficacy. Although several methods including the Schirmer, the phenol red thread and tear clearance tests exist, their utility in clinical evaluations of novel therapeutics is unclear. The purpose of this review is to summarize findings and conclusions describing the performance of each of these tests so as to gain insight into which, if any, is most applicable for use in discovering new dry eye therapeutics.

PURPOSE: These studies examined corneal reepithelialization rates and type IV collagen expression in rabbits treated with either moxifloxacin HCl ophthalmic solution 0.5% as base or gatifloxacin 0.3% ophthalmic solution following anterior keratectomy. METHODS: Animals (n = 6 per group) underwent surgery to create an 8-mm anterior keratectomy in the right eye. Rabbits were subsequently dosed with 1 drop, 3 times per day for 4 days with either moxifloxacin, gatifloxacin, or a commercially available irrigating solution. Fluorescein images were collected daily for the duration of the study. Approximately 96 h following surgery, the eyes were processed and evaluated for the presence of type IV collagen using immunohistochemical techniques. In two similar parallel studies, epithelial tissues were collected after the 48-h slit-lamp examination for a quantitative comparison of type IV collagen using either Western blot or quantitative polymerase chain reaction (Q-RT-PCR) techniques. RESULTS: Analysis of fluorescein images demonstrated that there were no significant differences in reepithelialization rates between the groups at any time point. At 96 h, 87%+/- 8% reepithelialization for moxifloxacin-treated eyes was observed compared with 77%+/- 10% for gatifloxacin-treated eyes and 85%+/-14% for BSS-treated eyes. The wound healing rates for the parallel studies demonstrated similar levels of reepithelialization for all groups. No discernable differences in type IV collagen expression were observed between treatment groups in the animals. The Q-RT-PCR analysis yielded no significant quantifiable difference in type IV collagen expression between any of the treatment groups. Expression values for alpha1 type IV collagen relative to the 18 S ribosomal RNA control were 0.0306+/-0.005 for BSS, 0.0251+/-0.002 for moxifloxacin, and 0.0254+/-0.006 for gatifloxacin. CONCLUSIONS: These studies indicate that there are no significant differences in corneal reepithelialization rates and type IV collagen expression between moxifloxacin ophthalmic solution 0.5%, gatifloxacin ophthalmic solution 0.3%, and the commercially available irrigating solution in this anterior keratectomy model.