Galectins and their involvement in ocular disease and development

Galectins are carbohydrate binding proteins with high affinity to ß-galactoside containing glycoconjugates. Understanding of the functions of galectins has grown steadily over the past decade, as a result of substantial advancements in the field of glycobiology. Galectins have been shown to be versatile molecules that participate in a range of important biological systems, including inflammation, neovascularisation and fibrosis. These processes are of particular importance in ocular tissues, where a major theme of recent research has been to divert diseases away from pathways which result in loss of function into pathways of repair and regeneration. This review summarises our current understanding galectins in the context important ocular diseases, followed by an update on current clinical studies and future directions

Stem Cell Therapies for Retinal Repair and Regeneration (Chapter 12)

Neural cell damage is the main feature of retinal degenerative disorders and constitutes the major cause of blindness in patients affected by retinal disease. Present treatments aim to prevent disease progression but do not reverse lost vision, for which stem cell-based therapies are the only hope for restoration or maintenance of visual function in individuals affected by severe disease. This chapter summarizes recent progress in the stem cell field and describes advances made on the clinical application of these cells for treatment of retinal degeneration. In addition, it highlights research being actively pursued to promote endogenous regeneration of the neural retinaas an alternative to transplantation

Insights into the design of spray systems for cell therapies for retinal disease using computational modelling

Chronic eye diseases are the main cause of vision loss among adults. Among these, retinal degenerative diseases affect millions of people globally, causing permanent loss of cells and organ dysfunction. Despite recent progress in developing stem cell therapies for retinal diseases, methods for delivery remain an area of intense research. Aerosol technology is a promising technique with the potential to spray cells evenly and directly across the retinal surface, promoting cell attachment and survival. Here we implement mathematical modelling of the spraying process to develop organ-specific spraying parameters in this therapeutic scenario. Firstly, we characterise the rheological parameters for a typical hydrogel used for spraying cells. These parameters are then integrated into a 3D computational model of an adult human eye under realistic surgical conditions. Simulation results provide quantitative relationships between the volume flow rate of the cell-laden hydrogel, external pressure needed for aerosolization, angle of the spraying, and properties of the cell delivery. An experimental assessment is also carried out to explore the impact of spraying under the regimes identified by the computational model on cell viability. This is the first stage towards using computational models to inform the design of spray systems to deliver cell therapies onto the human retina

In vitro characterization of a spontaneously immortalized human Muller cell line (MIO-M1)

purpose. To characterize a spontaneously immortalized human Müller cell line and to determine whether it retains the characteristics of primary isolated cells without undergoing differentiation in vitro. methods. An immortalized cell line obtained from human retina was investigated for the expression of known markers of Müller cells, including cellular retinaldehyde binding protein (CRALBP), glutamine synthetase, epidermal growth factor receptor (EGF-R), α-smooth muscle actin (α-SMA), and glial fibrillary acidic protein (GFAP). Also examined were the morphologic features of these cells, by scanning and transmission electron microscopy, and their functional characteristics, by electrogenic responses to glutamate. In addition, comparative studies were made of these cells with primary cultures of freshly isolated human Müller cells. results. The cells expressed CRALBP, EGF-R, glutamine synthetase, and α-SMA, as judged by confocal microscopy and Western blot analysis of cell lysates. Western blot analysis did not detect GFAP in cell lysates, but confocal microscopy showed that occasional cells expressed GFAP after detachment from the monolayer. The morphologic features of the cells examined, as judged by scanning and transmission electron microscopy, resemble those of cells derived from primary cell cultures. They possess villous projections on their apical surfaces and contain loose bundles of microtubules aligned parallel to one another and the long axis of the cell process. Characteristically, they contain abundant deposits of glycogen particles that do not differ from those seen in primary isolated cells. Preliminary recordings with intracellular electrodes revealed that these cells have properties similar to those described for mammalian Müller cells and depolarize in response to l-glutamate without significant change in membrane resistance, consistent with the well-established electrogenic uptake of this amino acid. conclusions. A spontaneously immortalized Müller cell line was characterized that retains the characteristics of primary isolated cells in culture. To the authors’ knowledge, it constitutes the first human Müller cell line reported in the literature. It has been named MIO-M1 (Moorfields/Institute of Ophthalmology-Müller 1) after the authors’ institution. Availability of this human cell line will facilitate studies designed to obtain a better understanding of the role of Müller cells in normal and pathologic conditions

Strain Specific Responses in a Microbead Rat Model of Experimental Glaucoma

Purpose: A major challenge in glaucoma research is the lack of reproducible animal models of RGC and optic nerve damage, the characteristic features of this condition. We therefore examined the glaucomatous responses of two different rat strains, the Brown Norway (BN) and Lister Hooded (LH) rats, to high intraocular pressure (IOP) induced by injection of magnetic beads into the anterior chamber. Methods: Magnetic microsphere suspensions (20 µl of 5–20 mg/ml) were injected into the anterior chamber of BN (n = 9) or LH (N = 15) rats. Animals from each strain were divided into three groups, each receiving a different dose of microspheres. IOP was measured over 4 weeks using a rebound tonometer. Retinal ganglion cell (RGC) damage and function were assessed using scotopic electroretinograms (ERGs), retinal flatmounts and optic nerve histology. ANOVA and Student’s t-tests were used to analyse the data. Results: A significant elevation in IOP was observed in BN rats receiving injections of 20 mg (37.18 ± 12.28 mmHg) or 10 mg microspheres/ml (36.95 ± 13.63 mmHg) when compared with controls (19.63 ± 4.29 mmHg) (p < .001) over 2 weeks. This correlated with a significant impairment of RGC function, as determined by scotopic ERG (p < .001), reduction in axon number (p < .05) and lower RGC density (P < .05) in animals receiving 20 mg or 10 mg microspheres/ml as compared with controls. LH rats receiving similar microsphere doses showed reduced scotopic ERG function (p < .001) after 2 weeks. No changes in IOP was seen in this strain, although a reduction in axon density was observed in optic nerve cross-sections (p < .05). Initial changes in IOP and ERG responses observed in BN rats remained unchanged for a duration of 7 weeks. In LH animals, ERG responses were decreased at 1–2 weeks and returned to control levels after 5 weeks. Conclusions: Although this model was easily reproducible in BN rats, the phenotype of injury observed in LH rats was very different from that observed in BN animals. We suggest that differences in the glaucomatous response observed in these two strains may be ascribed to anatomical and physiological differences and merits further investigation

Prospects for the application of Müller glia and their derivatives in retinal regenerative therapies

Neural cell death is the main feature of all retinal degenerative disorders that lead to blindness. Despite therapeutic advances, progression of retinal disease cannot always be prevented, and once neuronal cell damage occurs, visual loss cannot be reversed. Recent research in the stem cell field, and the identification of Müller glia with stem cell characteristics in the human eye, have provided hope for the use of these cells in retinal therapies to restore vision. Müller glial cells, which are the major structural cells of the retina, play a very important role in retinal homeostasis during health and disease. They are responsible for the spontaneous retinal regeneration observed in zebrafish and lower vertebrates during early postnatal life, and despite the presence of Müller glia with stem cell characteristics in the adult mammalian retina, there is no evidence that they promote regeneration in humans. Like many other stem cells and neurons derived from pluripotent stem cells, Müller glia with stem cell potential do not differentiate into retinal neurons or integrate into the retina when transplanted into the vitreous of experimental animals with retinal degeneration. However, despite their lack of integration, grafted Müller glia have been shown to induce partial restoration of visual function in spontaneous or induced experimental models of photoreceptor or retinal ganglion cell damage. This improvement in visual function observed after Müller cell transplantation has been ascribed to the release of neuroprotective factors that promote the repair and survival of damaged neurons. Due to the development and availability of pluripotent stem cell lines for therapeutic uses, derivation of Müller cells from retinal organoids formed by iPSC and ESC has provided more realistic prospects for the application of these cells to retinal therapies. Several opportunities for research in the regenerative field have also been unlocked in recent years due to a better understanding of the genomic and proteomic profiles of the developing and regenerating retina in zebrafish, providing the basis for further studies of the human retina. In addition, the increased interest on the nature and function of cellular organelle release and the characterization of molecular components of exosomes released by Müller glia, may help us to design new approaches that could be applied to the development of more effective treatments for retinal degenerative diseases

Downregulation of the Canonical WNT Signaling Pathway by TGF beta 1 Inhibits Photoreceptor Differentiation of Adult Human Muller Glia with Stem Cell Characteristics

Muller glia are responsible for the retina regeneration observed in zebrafish. Although the human retina harbors Muller glia with stem cell characteristics, there is no evidence that they regenerate the retina after disease or injury. Transforming growth factor-b (TGFb) and Wnt signaling regulate retinal neurogenesis and inflammation, but their roles in the neural differentiation of human Mu¨ller stem cells (hMSC) are not known. We examined hMSC lines in vitro for the expression of various Wnt signaling components and for their modulation by TGFb1, as well as the effect of this cytokine on the photoreceptor differentiation of these cells. Culture of hMSC with a combination of factors that induce photoreceptor differentiation of hMSC (FGF2, taurine, retinoic acid, and insulin-like growth factor type1; FTRI), markedly upregulated the expression of components of the canonical Wnt signaling pathway, including WNT2B, DKK1, and active b-CATENIN. Although FTRI did not modify mRNA expression of WNT5B, a component of the noncanonical/planar cell polarity Wnt pathway, it upregulated its secretion. Furthermore, TGFb1 not only decreased WNT2B expression, but also inhibited FTRI-induced photoreceptor differentiation of hMSC, as determined by expression of the photoreceptor markers NR2E3, RHODOPSIN, and RECOVERIN. Inhibition of TGFb1 signaling by an ALK5 inhibitor prevented TGFb1-induced changes in the expression of the two Wnt ligands examined. More importantly, inhibition of the canonical WNT signaling by XAV-939 prevented FTRI-induced photoreceptor differentiation. These observations suggest that TGFb may play a key role in preventing neural differentiation of hMSC and may constitute a potential target for induction of endogenous regeneration of the human retina

Effects of fluoroquinolones and tetracyclines on mitochondria of human retinal MIO-M1 cells

Our goal was to explore the detrimental impacts of ciprofloxacin (CPFX) and tetracycline (TETRA) on human retinal Müller (MIO-M1) cells in vitro. Cells were exposed to 30, 60 and 120 μg/ml of CPFX and TETRA. The cellular metabolism was measured with the MTT assay. The JC-1 and CM-H2DCFDA assays were used to evaluate the levels of mitochondrial membrane potential (MMP) and ROS (reactive oxygen species), respectively. Mitochondrial DNA (mtDNA) copy number, along with gene expression levels associated with apoptotic (BAX, BCL2-L13, BCL2, CASP-3 and CASP-9), inflammatory (IL-6, IL-1β, TGF-α, TGF-β1 and TGF-β2) and antioxidant pathways (SOD2, SOD3, GPX3 and NOX4) were analyzed via Quantitative Real-Time PCR (qRT-PCR). Bioenergetic profiles were measured using the Seahorse® XF Flux Analyzer. Cells exposed 24 h to 120 μg/ml TETRA demonstrated higher cellular metabolism compared to vehicle-treated cells. At each time points, (i) all TETRA concentrations reduced MMP levels and (ii) ROS levels were reduced by TETRA 120 μg/ml treatment. TETRA caused (i) higher expression of CASP-3, CASP-9, TGF-α, IL-1B, GPX3 and SOD3 but (ii) decreased levels of TGF-B2 and SOD2. ATP production and spare respiratory capacity declined with TETRA treatment. Cellular metabolism was reduced with CPFX 120 μg/ml in all cultures and 60 μg/ml after 72 h. The CPFX 120 μg/ml reduced MMP in all cultures and ROS levels (72 h). CPFX treatment (i) increased expression of CASP-3, CASP-9, and BCL2-L13, (ii) elevated the basal oxygen consumption rate, and (iii) lowered the mtDNA copy numbers and expression levels of TGF-B2, IL-6 and IL-1B compared to vehicle-control cells. We conclude that clinically relevant dosages of bactericidal and bacteriostatic antibiotics can have negative effects on the cellular metabolism and mitochondrial membrane potential of the retinal MIO-M1 cells in vitro. It is noteworthy to mention that apoptotic and inflammatory pathways in exposed cells were affected significantly This is the first study showing the negative impact of fluoroquinolones and tetracyclines on mitochondrial behavior of human retinal MIO-M1 cells

Comparison of proteomic profiles in the zebrafish retina during experimental degeneration and regeneration

Zebrafish spontaneously regenerate the retina after injury. Although the gene expression profile has been extensively studied in this species during regeneration, this does not reflect protein function. To further understand the regenerative process in the zebrafish, we compared the proteomic profile of the retina during injury and upon regeneration. Using two-dimensional difference gel electrophoresis (2D-DIGE) and label-free quantitative proteomics (quadrupole time of flight LC-MS/MS), we analysed the retina of adult longfin wildtype zebrafish at 0, 3 and 18 days after Ouabain injection. Gene ontology analysis indicates reduced metabolic processing, and increase in fibrin clot formation, with significant upregulation of fibrinogen gamma polypeptide, apolipoproteins A-Ib and A-II, galectin-1, and vitellogenin-6 during degeneration when compared to normal retina. In addition, cytoskeleton and membrane transport proteins were considerably altered during regeneration, with the highest fold upregulation observed for tubulin beta 2 A, histone H2B and brain type fatty acid binding protein. Key proteins identified in this study may play an important role in the regeneration of the zebrafish retina and investigations on the potential regulation of these proteins may lead to the design of protocols to promote endogenous regeneration of the mammalian retina following retinal degenerative disease