Showing papers by "Bärbel Rohrer published in 2017"

Targeting the tight junction protein, zonula occludens-1, with the connexin43 mimetic peptide, αCT1, reduces VEGF-dependent RPE pathophysiology

Abstract: A critical target tissue in age-related macular degeneration (AMD) is the retinal pigment epithelium (RPE), which forms the outer blood-retina barrier (BRB). RPE-barrier dysfunction might result from attenuation/disruption of intercellular tight junctions. Zonula occludens-1 (ZO-1) is a major structural protein of intercellular junctions. A connexin43-based peptide mimetic, αCT1, was developed to competitively block interactions at the PDZ2 domain of ZO-1, thereby inhibiting ligands that selectively bind to this domain. We hypothesized that targeting ZO-1 signaling using αCT1 would maintain BRB integrity and reduce RPE pathophysiology by stabilizing gap- and/or tight-junctions. RPE-cell barrier dysfunction was generated in mice using laser photocoagulation triggering choroidal neovascularization (CNV) or bright light exposure leading to morphological damage. αCT1 was delivered via eye drops. αCT1 treatment reduced CNV development and fluid leakage as determined by optical coherence tomography, and damage was correlated with disruption in cellular integrity of surrounding RPE cells. Light damage significantly disrupted RPE cell morphology as determined by ZO-1 and occludin staining and tiling pattern analysis, which was prevented by αCT1 pre-treatment. In vitro experiments using RPE and MDCK monolayers indicated that αCT1 stabilizes tight junctions, independent of its effects on Cx43. Taken together, stabilization of intercellular junctions by αCT1 was effective in ameliorating RPE dysfunction in models of AMD-like pathology.

Anaphylatoxins Activate Ca2+, Akt/PI3-Kinase, and FOXO1/FoxP3 in the Retinal Pigment Epithelium.

Abstract: Purpose: The retinal pigment epithelium (RPE) is a main target for complement activation in age-related macular degeneration. The anaphylatoxins C3a and C5a, have been thought to mostly play a role as chemoattractants for macrophages and immune cells; here we explore whether they trigger RPE alterations. Specifically, we investigated the RPE as a potential immunoregulatory gate, allowing for active changes in the RPE microenvironment in response to complement. Design: in vitro and in vivo analysis of signaling pathways. Methods: Individual activities of and interaction between the two anaphylatoxin-receptors were tested in cultured RPE cells by fluorescence microscopy, western-blot immunohistochemistry. Main outcome measures: Intracellular free calcium, protein phosphorylation, immunostaining of tissues/cells, multiplex secretion assay. Results: Similar to immune cells, anaphylatoxin exposure resulted in increases in free cytosolic Ca2+, PI3-kinase/Akt activation, FoxP3 and FOXO1 phosphorylation and cytokine/chemokine secretion. Differential responses were elicited depending on whether C3a and C5a were co-administered or applied consecutively; and response amplitudes in co-administration experiments ranged from additive, to driven by C5a (C3a+C5a=C5a) or being smaller than those elicited by C3a alone (C3a+C5a

The Retinol Binding Protein Receptor 2 (Rbpr2) is required for Photoreceptor Outer Segment Morphogenesis and Visual Function in Zebrafish.

Abstract: Vitamin A (all-trans retinol) plays critical roles in mammalian development and vision. Since vitamin A is food-derived, tissue-specific uptake and storage mechanism are needed. In the eye, uptake of RBP4-retinol is mediated by the receptor Stra6, whereas the receptor mediating RBP4 binding and retinol transport into the liver has just recently been discovered. Here we examined the role of zebrafish retinol binding protein receptor 2 (Rbpr2) for RBP4-retinol uptake in developing embryos, using eye development and vision as sensitive readouts. In cultured cells, Rbpr2 localized to membranes and promoted RBP4-retinol uptake. In larvae, Rbpr2 expression was detected in developing intestinal enterocytes and liver hepatocytes. Two rbpr2 mutant zebrafish lines, each resulting in Rbpr2 deficiency, exhibit a small eye defect, and systemic malformations including hydrocephaly and cardiac edema, phenotypes associated with vitamin A deficiency. In the retina, Rbpr2 loss resulted in shorter photoreceptor outer segments, mislocalization and decrease in visual pigments, decreased expression of retinoic acid-responsive genes and photoreceptor cell loss, overall leading to a reduction of visual function. Together, these results demonstrate that Rbpr2-mediated RBP4-retinol uptake in developing liver and intestine is necessary to provide sufficient substrate for ocular retinoid production required for photoreceptor cell maintenance and visual function.

Bystander effects elicited by single-cell photo-oxidative blue-light stimulation in retinal pigment epithelium cell networks.

Abstract: 'Bystander effect' refers to the induction of biological effects in cells not directly targeted. The retinal pigment epithelium consists of hexagonal cells, forming a monolayer interconnected by gap junctions (GJs). Oxidative stress initiated in an individual cell by photostimulation (488 nm) triggered changes in reactive oxygen species (ROS), Ca2+ and mitochondrial membrane potential (ψm). The Ca2+ signal was transmitted to neighboring cells slowly and non-uniformly; the ROS signal spread fast and radially. Increased Ca2+ levels were associated with a loss in ψm. GJ blockers prevented the spreading of the Ca2+, but not the ROS-related signal. The GJ-mediated Ca2+ wave was associated with cell death by 24 h, requiring endoplasmic reticulum-mitochondria Ca2+ transfer. Ensuing cell death was correlated with baseline Ca2+ levels, and baseline Ca2+ levels were correlated with pigmentation. Hence, local oxidative stress in a donor cell can trigger changes in certain connected recipient cells, a signal that required GJ communication and an ROS-Ca2+ dual-hit. Finally, damage apparently occurred in susceptible cells, which correlated with baseline Ca2+ levels.

New Insights on Complement Inhibitor CD59 in Mouse Laser-Induced Choroidal Neovascularization: Mislocalization After Injury and Targeted Delivery for Protein Replacement

Abstract: Purpose: The membrane attack complex (MAC) in choriocapillaris (CC) and retinal pigment epithelium (RPE) increase with age and disease (age-related macular degeneration). MAC assembly can be inhibited by CD59, a membrane-bound regulator. Here we further investigated the role of CD59 in murine choroidal neovascularization (CNV), a model involving both CC and RPE, and tested whether CR2-CD59, a soluble targeted form of CD59, provides protection. Methods: Laser-induced CNV was generated in wild type and CD59a-deficient mice (CD59−/−). CNV size was measured by optical coherence tomography, and CR2-CD59 was injected intraperitoneally. Endogenous CD59 localization and MAC deposition were identified by immunohistochemistry and quantified by confocal microscopy. Cell-type-specific responses to MAC were examined in retinal pigment epithelial cells (ARPE-19) and microvascular endothelial cells (HMEC-1). Results: CD59 levels were severely reduced and protein was mislocalized in the RPE surrounding the lesio...

Alginate Microcapsule Technology and Impacts on Cell Therapy Development

Abstract: 1. Department of Cell Biology and Anatomy, School of Medicine, University of South Carolina, Columbia, USA. 2. Department of Ophthalmology, Medical University of South Carolina, Charleston, South Carolina, USA. 3. Ralph H. Johnson VA Medical Center, Charleston, South Carolina, USA. 4. Biomedical Engineering Program, College of Engineering and Computing, University of South Carolina, Columbia, USA.