George Trichonas

Receptor interacting protein kinases mediate retinal detachment-induced photoreceptor necrosis and compensate for inhibition of apoptosis

Apoptosis has been shown to be a significant form of cell loss in many diseases. Detachment of photoreceptors from the retinal pigment epithelium, as seen in various retinal disorders, causes photoreceptor loss and subsequent vision decline. Although caspase-dependent apoptotic pathways are activated after retinal detachment, caspase inhibition by the pan-caspase inhibitor Z-VAD fails to prevent photoreceptor death; thus, we investigated other pathways leading to cell loss. Here, we show that receptor interacting protein (RIP) kinase-mediated necrosis is a significant mode of photoreceptor cell loss in an experimental model of retinal detachment and when caspases are inhibited, RIP-mediated necrosis becomes the predominant form of death. RIP3 expression, a key activator of RIP1 kinase, increased more than 10-fold after retinal detachment. Morphological assessment of detached retinas treated with Z-VAD showed decreased apoptosis but significantly increased necrotic photoreceptor death. RIP1 kinase inhibitor necrostatin-1 or Rip3 deficiency substantially prevented those necrotic changes and reduced oxidative stress and mitochondrial release of apoptosis-inducing factor. Thus, RIP kinase-mediated programmed necrosis is a redundant mechanism of photoreceptor death in addition to apoptosis, and simultaneous inhibition of RIP kinases and caspases is essential for effective neuroprotection and may be a novel therapeutic strategy for treatment of retinal disorders.

In Vivo Evaluation of Laser-Induced Choroidal Neovascularization Using Spectral-Domain Optical Coherence Tomography

PURPOSE To describe the in vivo evolution of laser-induced choroidal neovascularization (CNV) in mice using spectral domain optical coherence tomography (SD-OCT). METHODS Laser photocoagulation was applied to the mouse fundus using a 532-nm diode laser (100, 150, and 200 mW; 100-μm diameter, 0.1-second duration). SD-OCT examination was performed immediately after laser application and at days 3, 5, 7, 14, 21, and 28 after laser. Fluorescein angiography (FA) was performed at day 5, 7, 14, and 28. Acquired SD-OCT images were analyzed to describe morphologic features, measure CNV size and retinal thickness, and assess the frequency of lesions resulting in fluid accumulation. Finally, SD-OCT images were compared to fluorescein angiograms and histologic sections with immunostaining at similar time points. RESULTS SD-OCT allowed visualization of the initial laser damage and the subsequent stages of the injury response. CNV formation reached its maximum size at day 5. By day 7, significant size reduction was observed (P < 0.001), continuing through days 14 and 28. Exudation signs, such as fluid accumulation and increase in retinal thickness, followed the same time course, with a peak at day 5 and a decrease by day 7. Delivery of higher laser energy levels to the RPE/choroid complex resulted in a significant percentage of lesions demonstrating excessive chorioretinal damage without CNV formation. CONCLUSIONS SD-OCT is a fast and reliable tool for the in vivo evaluation of laser-induced CNV, allowing quantification of lesion size and exudation parameters. Moreover, it provides morphologic information that correlates with histologic findings.

AMP-activated Protein Kinase Suppresses Matrix Metalloproteinase-9 Expression in Mouse Embryonic Fibroblasts

Matrix metalloproteinase-9 (MMP-9) plays a critical role in tissue remodeling under both physiological and pathological conditions. Although MMP-9 expression is low in most cells and is tightly controlled, the mechanism of its regulation is poorly understood. We utilized mouse embryonic fibroblasts (MEFs) that were nullizygous for the catalytic α subunit of AMP-activated protein kinase (AMPK), which is a key regulator of energy homeostasis, to identify AMPK as a suppressor of MMP-9 expression. Total AMPKα deletion significantly elevated MMP-9 expression compared with wild-type (WT) MEFs, whereas single knock-out of the isoforms AMPKα1 and AMPKα2 caused minimal change in the level of MMP-9 expression. The suppressive role of AMPK on MMP-9 expression was mediated through both its activity and presence. The AMPK activators 5-amino-4-imidazole carboxamide riboside and A769662 suppressed MMP-9 expression in WT MEFs, and AMPK inhibition by the overexpression of dominant negative (DN) AMPKα elevated MMP-9 expression. However, in AMPKα−/− MEFs transduced with DN AMPKα, MMP-9 expression was suppressed. AMPKα−/− MEFs showed increased phosphorylation of IκBα, expression of IκBα mRNA, nuclear localization of nuclear factor-κB (NF-κB), and DNA-binding activity of NF-κB compared with WT. Consistently, selective NF-κB inhibitors BMS345541 and SM7368 decreased MMP-9 expression in AMPKα−/− MEFs. Overall, our results suggest that both AMPKα isoforms suppress MMP-9 expression and that both the activity and presence of AMPKα contribute to its function as a regulator of MMP-9 expression by inhibiting the NF-κB pathway.

A Novel Nonradioactive Method to Evaluate Vascular Barrier Breakdown and Leakage

PURPOSE To identify a novel, sensitive, nonradioactive leakage assay that can be used in the assessment of retinal vascular permeability in rats and mice. METHODS Breakdown of the vascular barrier was induced by vascular endothelial growth factor (VEGF), lipopolysaccharide (LPS), or diabetes. Biotinylated bovine serum albumin (bBSA) was administered as a tracer. After perfusion with lactated Ringers solution, extravasated bBSA was detected with immunoprecipitation and Western blot analysis or sandwich ELISA. The results were then normalized against the final bBSA plasma concentration, the circulation time, and the protein concentration of the tissue. RESULTS Six hours after VEGF injection, BRB breakdown was quantified in the injected eye and was 2.5-fold higher than in the contralateral phosphate-buffered saline (PBS)-injected eye (n = 6 rats, P < 0.01). Intravitreal LPS injection induced severe inflammation in the directly injected eye and moderate inflammation in the contralateral untreated eye. Leakage was six- and threefold higher, respectively, compared with that in the untreated control animals (n = 5 rats, P < 0.01). Nine-month diabetic rats had a threefold increase in vascular leakage compared with age-matched control animals (n = 6 retinas, P < 0.05). Twenty-four hours after intraperitoneal administration of LPS in mice, the animals showed increased vascular leakage in all tissue organs examined (retina, 1.7-fold; brain, 1.5-fold; and kidney, 1.3-fold). CONCLUSIONS bBSA can serve as an effective alternative to the current methods used for quantitating vascular leakage and especially the blood-retinal barrier breakdown. It is reasonably easy to perform, low in cost, and adaptable to experiments in mice.

Development of an efficiently cleaved, bioactive, highly pure FLAG-tagged recombinant human Mullerian Inhibiting Substance.

Mullerian Inhibiting Substance (MIS), a member of the TGF-beta family, causes regression of the Mullerian duct in male embryos, after binding to Mullerian Inhibiting Substance Receptor II (MISRII). It has also been extensively demonstrated that it can inhibit proliferation of various cancer cell lines such as ovarian, prostate, and breast cancer in vitro and in vivo. Hence, the availability of a recombinant, epitope tagged, bioactive MIS is important for the selection of patients for treatment and for probing novel molecular targets for MIS in various tissues. To this end, we have expressed a recombinant, internally FLAG-tagged form of hMIS with the tag (DYKDDDDK) immediately after the cleavage site (427-428) of MIS at the C-terminus with a modified dibasic cleavage motif sequence. We show that this construct results in a highly pure, endogenously processed (cleaved) FLAG MIS, that causes complete regression of the Mullerian Duct in an organ culture assay. In addition, purified FLAG MIS was able to bind and affinity purify both transfected and endogenous MIS type II receptor. The availability of this fully functional, epitope tagged form of MIS should facilitate scale-up for preclinical and clinical use and should also be used for the study of MIS binding proteins and for tracking in pharmacokinetic studies.

Membrane-bound and soluble Fas ligands have opposite functions in photoreceptor cell death following separation from the retinal pigment epithelium.

Fas ligand (FasL) triggers apoptosis of Fas-positive cells, and previous reports described FasL-induced cell death of Fas-positive photoreceptors following a retinal detachment. However, as FasL exists in membrane-bound (mFasL) and soluble (sFasL) forms, and is expressed on resident microglia and infiltrating monocyte/macrophages, the current study examined the relative contribution of mFasL and sFasL to photoreceptor cell death after induction of experimental retinal detachment in wild-type, knockout (FasL−/−), and mFasL-only knock-in (ΔCS) mice. Retinal detachment in FasL−/− mice resulted in a significant reduction of photoreceptor cell death. In contrast, ΔCS mice displayed significantly more apoptotic photoreceptor cell death. Photoreceptor loss in ΔCS mice was inhibited by a subretinal injection of recombinant sFasL. Thus, Fas/FasL-triggered cell death accounts for a significant amount of photoreceptor cell loss following the retinal detachment. The function of FasL was dependent upon the form of FasL expressed: mFasL triggered photoreceptor cell death, whereas sFasL protected the retina, indicating that enzyme-mediated cleavage of FasL determines, in part, the extent of vision loss following the retinal detachment. Moreover, it also indicates that treatment with sFasL could significantly reduce photoreceptor cell loss in patients with retinal detachment.

Abstract 4363: Identification of ABCB5 multidrug transporter in retinoblastoma

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Purpose: Retinoblastoma (Rb) is the most common intraocular tumor in children. Chemotherapy has improved the outcome for both unilateral and bilateral early stage disease, yet late stage bilateral Rb remains difficult to treat, and metastasis are often fatal. Treatment with carboplatin, vincristine and etoposide is only effective in 40% of bilateral Rb patients and results in a high incidence of secondary malignancies. ATP-binding cassette (ABC) transporters act as efflux pumps of different substrates, including drugs. ABCB5, a member of the ABC-B subfamily, is expressed on both normal tissues (CNS, mammary gland, testis, retina) and in skin melanoma, where it identifies a subpopulation of cancer stem cells with enhanced tumorigenicity. Unexpectedly, the retina displays the highest level of ABCG5 in normal tissues, suggesting it might be expressed in Rb tumors. Here we identify for the first time in Rb, an ABCB5+ subpopulation of cells that we predict will possess drug resistance and/or enhanced tumorigenicity. Methods: Rb143, Rb116, Rb125 and Rb107 cell lines were developed in our laboratory from primary explants recovered from patients with large tumors. Sequencing of the Rb gene (27 exons) was used to validate all Rb cell lines. Flow cytometry was used to identify ABCB5+ cells using a monoclonal anti-ABCB5 antibody or isotype control. Calcein AM and/or Aqua fluorescent dye was used to determine viability. ABCB5 pump function was determined by using Calcein AM, a pump substrate that becomes fluorescent within viable cells. Optical coherence tomography (OCT) was used to quantitate the size of intraocular tumors, following orthotopic injection into the sub-retinal space of NOD-scid IL2rg−/− mice. Results: All Rb cell lines possessed a subpopulation of ABCB5+ cells with a frequency ranging from 3-12%. To determine if the ABCB5 pump was functional, Rb cells were cultured for 30 mins with Calcein AM (ABCB5 pump substrate). ABCB5 positive, but not negative, Rb cells excluded the Calcein AM. The ABCB5 pump mediated Calcein exclusion, which was terminated by the addition of an ABCB5 specific blocking antibody. Rb143 cells (originally 3% ABCB5+ cells) were separated by cell sorting into (i) ABCB5+ cells (90% pure), and (ii) ABCB5- cells (100% pure). To test the growth potential and drug resistance of these two subpopulations in vivo, we developed a murine transient retinal detachment model. Injection of 50μl of PBS into the sub-retinal space induces a retinal detachment, but as the PBS is subsequently absorbed over the next 24 hrs the retina reattaches. Up to 1×106 cells were orthotopically injected beneath the detached retina, resulting in tumor growth in < 1 wk. This model will allow us to determine the drug resistance and growth potential of ABCB5+ and ABCB5- Rb cells. Conclusion: Retinoblastoma possesses a small sub-population of ABCB5+ tumor cells, which displays an active pump with the potential of drug resistance and preferential tumor growth. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4363.