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To test the hypothesis that immune activation occurs in glaucoma by comparing concentrations of multiple cytokines in aqueous humor (AH) from patients with primary open angle glaucoma (POAG) and from cataract patients without glaucoma as controls.Cytokine concentrations in AH obtained during surgery were measured using microparticle-based immunoassays. Localized expression of IL-8 protein was investigated by immunohistochemistry of human eyes.Eight cytokines (IL-1β, IL-2, IL-4, IL-5, IL-10, IL-12, IFN-γ, and TNF-α) were below the limits of detection, and two cytokines (IL-18 and IL-15) were detected at low levels or in only a few patients. Although IL-6 was detected in 26 of 30 control patients (median, 2.7 pg/mL) and in 23 of 29 POAG patients (median, 1.6 pg/mL), the difference was not statistically significant. IL-8 was detected in 28 of 30 control patients (median, 1.8 pg/mL) and in all 29 POAG patients (median, 4.9 pg/mL). The higher IL-8 concentration in the AH of POAG patients was statistically significant (P0.001). In pairs of eyes from patients with asymmetric glaucomatous optic nerve damage, IL-8 concentration was higher in the AH of the more severely affected eye (P0.05). Patients with severe visual field defects had higher IL-8 concentrations in the AH than did patients with mild visual field defects. IL-8 protein expression was found in human retina and optic nerve.Concentration of the inflammatory cytokine IL-8 is significantly elevated in the AH of POAG patients, supporting the hypothesis that immune activation occurs in glaucoma.

The third Annual ARVO/Pfizer Ophthalmic Research Institute Conference was held Friday and Saturday, May 4 and 5, 2007 at the Fort Lauderdale Grande Hotel and Yacht Club, Fort Lauderdale, Florida. The conference, funded by the ARVO Foundation for Eye Research through a grant from Pfizer Ophthalmics, provided an opportunity to gather experts from within and outside ophthalmology to develop strategies to address drug delivery to posterior intraocular tissues—a topic of great interest, as the major route of drug delivery is via intravitreous injection. A working group of 33 participants, focused interdisciplinary contributors, 19 observers from ARVO/Pfizer, and clinical and basic ophthalmic researchers convened to identify (1) unmet patient needs regarding current drug delivery, novel treatment of retinal diseases, the potential for novel drug design opportunities, drug delivery methods for targeted localized and sustained release, and delivery of macromolecules (siRNA, DNA); and (2) to evaluate the usefulness of nanoparticles, microbeads, and implants for drug delivery, as well as physical means of drug delivery (ionophoresis, electroporation, and microneedles). Session I: Unmet Needs and New Drug Opportunities in Treating Disorders of the Posterior Segment Session II: Animal Models of Posterior Ocular Diseases Session III: New Drug Design and Delivery Systems: What Do Experts See Beyond the Horizon? Session IV: Ocular Drug Delivery Using Nanoparticles, Microbeads, and Microneedles Session V: Transscleral, Intravitreous, and Suprachoroidal Drug Delivery Session VI: Ocular Tissue Dissection, Modeling, and Ocular Tissue Assays Session VII: Iontophoresis, Electroporation, Electrophoresis, and Photo-acoustic Delivery Each session began with a 10-minute introduction followed by a 30-minute lecture by a distinguished expert. Allan S. Hoffman, ScD, Professor of Bioengineering at the University of Washington, Seattle, presented “Design of Polymer Carriers for Intracellular Delivery of Biomolecular Drugs” and Mansoor Amiji, RPh, PhD, Professor and Associate Chairman of Pharmaceutical Science at Northeastern University (Boston, MA), presented “Nanotechnology for Advanced Drug Delivery.” During the remainder of each session, participants, and attendees discussed pertinent questions, voiced opinions, and identified unanswered questions. These discussions confirmed current and future needs for ocular drug delivery to the posterior segment.

PURPOSE. To evaluate the associations between plasma-soluble Fas ligand (sFasL) and age-related macular degeneration (AMD). METHODS. Plasma samples were obtained from 230 individuals (age range, 45– 85), with or without AMD. The concentrations of sFasL were determined by an enzyme-linked immunosorbent assay (ELISA). The measured sFasL levels were transformed into cubic roots and were fitted into linear regression models against AMD status, with adjustment for age and sex. RESULTS. Plasma sFasL increased with age and AMD. There was a linear correlation between age and the cubic roots of sFasL. The plasma sFasL concentrations in non-AMD subjects ranged from 0 to 1.63 ng/mL (median, 0.69 ng/mL), whereas in patients with AMD, sFasL ranged from 0 to 2.43 ng/mL (median, 0.18 ng/mL). Between the ages of 61 and 84, the subjects with AMD had significantly higher sFasL than did the non-AMD subjects. There was a sexual dimorphism of the plasma sFasL levels. In non-AMD subjects, sFasL was lower in the females. In patients with AMD, sFasL was higher in the females. CONCLUSIONS. An elevation of plasma sFasL with aging may play a role in the development of AMD and is a potential peripheral marker for monitoring disease progression. (Invest Ophthalmol Vis Sci. 2008;49:1345–1349) DOI:10.1167/iovs.07-0308

Purpose To characterize the involvement of Zip2, a zinc transporter protein, in the antioxidant functions of cultured human retinal pigment epithelial (RPE) cells. Methods The expression of zinc transporter proteins was determined by RT-PCR. Intracellular zinc concentration was assessed by staining with a zinc-sensitive dye followed by flow cytometry. Stable overexpression of the transporter protein Zip2 was achieved by transducing ARPE-19 cells with a retroviral vector containing the open reading frame of the human Zip2 gene. Activity of nuclear factor erythroid 2-related factor 2 (Nrf2) was measured using a dual luciferase assay after transient transfection of reporter plasmids containing the antioxidant response element (ARE). Glutamate-cysteine ligase (GCL) expression was measured by quantitative real-time RT-PCR. Results Cultured RPE cells could transport zinc with Zip2 as an influx transporter expressed in ARPE-19 cells and human RPE cells isolated from postmortem donor eyes. The mRNA level of Zip2 was influenced by intracellular and extracellular zinc concentrations. Overexpression of Zip2 resulted in increased Nrf2 activity, higher GCL expression, and increased glutathione synthesis. Conclusions RPE cells can actively uptake zinc through the transporter Zip2, and the increased intracellular zinc upregulates the Nrf2-dependent antioxidant function.

The human retinal pigment epithelial (RPE) cells have low turnover rate and often survive for an individual’s lifespan.1 However, when there is loss of RPE cells via apoptosis, which may occur in chronic eye diseases such as age-related macular degeneration (AMD), retinal function can be compromised. Patchy loss of RPE is one of the classic features of AMD2 and it has been shown that this process involves apoptosis.3 In eyes with wet AMD, apoptotic cells are present in choroidal neovascular membranes.4 The molecular mechanisms controlling mitochondria- and death receptor-mediated apoptosis have been studied extensively5-7 and it has been clearly demonstrated that the Bcl2 family of proteins are central regulators of apoptosis.8-10 Results from recent studies indicate that an immunophilin protein, FKBP8, interacts with Bcl2/ Bcl-XL and regulates the downstream signaling events.11-14 FK506-binding proteins (FKBPs) belong to a major family of immunophilins that bind to immunosuppressive drugs, FK506 and rapamycin, and convey their effects on immune responses and apoptosis.15 At least 15 FKBPs have been identified in humans.16,17 Despite the distinct subcellular localization, all FKBP proteins have a conserved motif, the FK506-binding domain, that mediates peptidyl-prolyl cis-trans isomerase (PPIase) activity and FK506 binding.16,17 FKBPs often function as chaperones or co-chaperones by directly interacting with other proteins. For instance, the prototypical FKBP family member FKBP12 binds to ryanodine receptor (RyR) and inositol 1,4,5-trisphosphate (IP3) receptor, both of which are major intracellular calcium channels.18,19 Accordingly, it plays a key regulatory role in intracellular Ca2+ signaling, and most FKBP12-knockout mice die during embryonic development because of severe cardiomyopathy.20 FKBP8 (also known as FKBP38) was originally isolated from a T-cell cDNA library by a degenerative PCR approach.21 It is expressed in a variety of tissues with the highest abundance in the brain.21 The gene is located on chromosome 19, area p12, and consists of 9 exons.22 Compared with other FKBP family proteins, FKBP8 displays unique features in FK506 binding ability and PPIase activity. Instead of being constitutively active or inactive, its PPIase activity requires prior binding of calcium and calmodulin (CaM) via a CaM binding domain.13 FKBP8 binds to FK506 only in the presence of Ca/CaM.13,23 FKBP8-knockout mice died at embryonic day (E)13.5 and showed defects in eye development.24 Another unique function of FKBP8 is that it interacts with Bcl2 and Bcl-XL.11-13 Although controversial, such interaction has been demonstrated in different types of cells and is believed to be important for the localization and protein stability of Bcl2/Bcl-XL.11-14 The high abundance of FKBP8 in the retina and the requirement of FKBP8 for normal eye development24 suggest that the protein performs important functions in this structure. In the present study, we showed that the FKBP8 gene is expressed in cultured human RPE cells, along with a splice variant lacking the FK506-binding domain. Overexpression of both the full-length and the short form of FKBP8 sensitized RPE cells to thapsigargin-induced apoptosis, but did not affect the transcriptional function of the nuclear factor of activated T cells (NFAT). These data indicate that, by interacting with Bcl2 family proteins, FKBP8 may be a functional component of the apoptotic signaling network in the RPE cells and that such an effect could be independent of FK506.

PURPOSE. To test whether variation in extracellular cysteine (Cys) redox potential (Eh) over the physiologic range occurring in human plasma affects oxidant-induced apoptosis in cultured human retinal pigment epithelial (hRPE) cells. METHODS. The hRPE cells were incubated in culture medium with Eh established over the range of 16 mV (most oxidized) to 158 mV (most reduced) by adding different concentrations of Cys and cystine (CySS) with constant total Cys equivalents. Apoptosis was induced with tert-butylhydroperoxide (tBH). RESULTS. The hRPE cells were sensitized to tBH-induced apoptosis in the more oxidized extracellular conditions (Eh 55 mV) compared with the reduced conditions (Eh 89 mV). Loss of mitochondrial membrane potential (m), release of cytochrome c, and activation of caspase 3 after tBH treatments all increased under the more oxidized conditions. However, the extracellular redox state did not affect expression of Fas or FasL in hRPE cells. CONCLUSIONS. The hRPE cells that are exposed to a more oxidized extracellular redox environment have increased susceptibility to oxidant-induced apoptosis through the intrinsic mitochondrial pathway, which could contribute to an age-related decline in cell populations in the retina and thereby provide a potential mechanism for the degenerative changes that are associated with age-related macular degeneration (ARMD). (Invest Ophthalmol Vis Sci. 2005;46:1054 –1061) DOI:10.1167/ iovs.04-0949