Carl Sheridan

Matrix Metalloproteinases : A Role in the Contraction of Vitreo-Retinal Scar Tissue.

The most common cause of failure of retinal reattachment surgery is formation of fibrocellular contractile membranes on both surfaces of the neuroretina. This intraocular fibrosis, known as proliferative vitreoretinopathy, results in a blinding tractional retinal detachment because of the contractile nature of the membrane. Contractility is a cell-mediated event that is thought to be dependent on locomotion and adhesion to the extracellular matrix. Interactions between cells and the extracellular matrix can be influenced by matrix metalloproteinases (MMPs) and we investigated the role of MMPs in two in vitro models (two- and three-dimensional) of human retinal pigment epithelial (RPE) cell-mediated contraction. MMP activity was detected using enzyme-linked immunosorbent assays and zymography techniques that revealed MMP-1, -2, -3, and -9 positivity during the collagen matrix contraction assays. RPE-populated collagen matrix contraction (three-dimensional) was inhibited using a cocktail of anti-MMP antibodies and with Galardin (a broad-spectrum MMP inhibitor). Galardin inhibition was dose-dependent, reversible, and dependent on cell number. MMP inhibitors had no effect on contraction when RPEs were seeded on two-dimensional collagen matrices or on cellular adhesion to collagen type I. Our results suggest that MMP activity may be required for three-dimensional but not two-dimensional RPE-collagen matrix contraction.

Pathobiology of epiretinal and subretinal membranes: possible roles for the matricellular proteins thrombospondin 1 and osteonectin (SPARC).

Epiretinal and subretinal membranes are fibrocellular proliferations which form on the surfaces of the neuroretina as a sequel to a variety of ocular diseases. When these proliferations complicate rhegmatogenous retinal detachment (a condition known as proliferative vitreoretinopathy or PVR), the membranes often contain numerous retinal pigment epithelial (RPE) cells and a variety of extracellular proteins. The extracellular proteins include adhesive proteins like collagen, laminin and fibronectin. In addition, several matricellular proteins with potential counter-adhesive functions are present in the membranes. Two such matricellular proteins, thrombospondin 1 and osteonectin (or SPARC: Secreted Protein Acidic and Rich in Cysteine), tend to be co-distributed with the RPE cells in PVR membranes. By virtue of their counter-adhesive properties, thrombospondin 1 and SPARC may reduce RPE cell-matrix adhesion and so permit key RPE cellular activities (for example, migration or shape change) in periretinal membrane development. Furthermore, within a ‘cocktail’ containing other proteins such as the metalloproteinases and growth factors like the scatter factor/hepatocyte growth factor family, matricellular proteins may play a role in the RPE cell dissociation from Bruch’s membrane, which characterises early PVR.

Expression of hypoxia-inducible factor−1α and −2α in human choroidal neovascular membranes

PurposeUp-regulation of pro-angiogenic cytokine expression occurring secondary to hypoxia in physiologic and pathophysiologic conditions is mediated by the family of transcription regulators know as hypoxia inducible factors (HIF). The present study was undertaken to investigate the expression of HIF occurring in human choroidal neovascularization (CNV) and the posterior segment of young and old eyes.MethodsSurgically excised CNV from patients with either age-related macular degeneration (AMD; n = 9), punctuate inner choroidopathy (PIC; n = 3) and young normal eyes were immunohistochemically probed with monoclonal antibodies against HIF−1α and −2α and compared to that for cell markers specific for vascular endothelial cells (CD34), macrophages (CD68), retinal pigment epithelial cells (RPE; panel cytokeratins/CK18) and VEGF. Following secondary antibody amplification, reactions were visualized with fast red chromogen.ResultsCellular immunoreactivity of membranes for HIF−2α was strong in eight out of nine AMD specimens but it was only weakly positive for HIF−1α in five specimens. In contrast, two out of three PIC specimens were weakly positive for HIF−1α but demonstrated no staining for HIF−2α. Immunohistochemical analysis revealed areas within the CNV membranes that were predominantly immunopositive for CD68 and cytokeratin indicating the presence of RPE and/or macrophages and that these cells strongly co-localized with the presence of HIF and VEGF. No immunochemical co-localization was observed with HIF and the endothelial cell marker CD34 in any membranes studied. Normal globes also demonstrated HIF−2 positivity to be predominantly localized to the central RPE rather than peripheral RPE irrespective of age of donor.ConclusionsThe localization of HIF expression supports the concept that hypoxia is a major stimulus for the development of submacular wound healing and within this context CNV is but one component of this process.

Expression of ADAMTS metalloproteinases in the retinal pigment epithelium derived cell line ARPE-19: Transcriptional regulation by TNFα

ADAMTS (A Disintegrin-like And Metalloprotease domain with ThromboSpondin type I motifs) are multidomain proteins with demonstrated metalloproteinase functionality and have potential roles in embryonic development, angiogenesis and cartilage degradation. We present here investigations of ADAMTS expression in an ocular cell type, ARPE-19, with a view to implicating them in retinal matrix turnover. Expression analysis was undertaken using a combination of reverse transcription polymerase chain reaction (RT-PCR) and Northern blotting experiments, which together detected the expression of mRNAs for several ADAMTS proteins, all of which have active site motifs characteristic of matrix metalloproteases (MMPs). These included ADAMTS1, ADAMTS2, ADAMTS3, ADAMTS5, ADAMTS6, ADAMTS7 and ADAMTS9. The expression of mRNA isoforms for ADAMTS7 and ADAMTS9 were also detected. Following stimulation with TNFalpha, ADAMTS1, ADAMTS6 and both ADAMTS9 transcripts expressed in ARPE-19 cells showed a potent upregulation. The expression of ADAMTS genes in ARPE-19 cells and the transcriptional stimulation of some family members by TNFalpha may implicate them in inflammatory eye disease and the compromise of retinal matrix structure, which is evident in age-related macular degeneration (ARMD) and other retinal pathologies.

Non-vascular vitreoretinopathy : The cells and the cellular basis of contraction

Background: We consider epiretinal membranes in terms of the two repair processes of gliosis and fibrosis and look at the cellular basis of contraction.Methods: Pathological material removed at surgery was examined by a range of morphological procedures. Cultures of fibroblasts, retinal pigment epithelium cells and retinal glia were subjected to bioassays which relate to behavioural activities in scar formation.Results and conclusions: Our findings highlight the importance of activities such as migration and adhesion in the formation of epiretinal membranes, and also show that these activities are central to our understanding of contraction.

Progenitors for the Corneal Endothelium and Trabecular Meshwork: A Potential Source for Personalized Stem Cell Therapy in Corneal Endothelial Diseases and Glaucoma

Several adult stem cell types have been found in different parts of the eye, including the corneal epithelium, conjunctiva, and retina. In addition to these, there have been accumulating evidence that some stem-like cells reside in the transition area between the peripheral corneal endothelium (CE) and the anterior nonfiltering portion of the trabecular meshwork (TM), which is known as the Schwalbes Ring region. These stem/progenitor cells may supply new cells for the CE and TM. In fact, the CE and TM share certain similarities in terms of their embryonic origin and proliferative capacity in vivo. In this paper, we discuss the putative stem cell source which has the potential for replacement of lost and nonfunctional cells in CE diseases and glaucoma. The future development of personalized stem cell therapies for the CE and TM may reduce the requirement of corneal grafts and surgical treatments in glaucoma.

Inflammatory Mediators and Angiogenic Factors in Choroidal Neovascularization: Pathogenetic Interactions and Therapeutic Implications

Choroidal neovascularization (CNV) is a common and severe complication in heterogeneous diseases affecting the posterior segment of the eye, the most frequent being represented by age-related macular degeneration. Although the term may suggest just a vascular pathological condition, CNV is more properly definable as an aberrant tissue invasion of endothelial and inflammatory cells, in which both angiogenesis and inflammation are involved. Experimental and clinical evidences show that vascular endothelial growth factor is a key signal in promoting angiogenesis. However, many other molecules, distinctive of the inflammatory response, act as neovascular activators in CNV. These include fibroblast growth factor, transforming growth factor, tumor necrosis factor, interleukins, and complement. This paper reviews the role of inflammatory mediators and angiogenic factors in the development of CNV, proposing pathogenetic assumptions of mutual interaction. As an extension of this concept, new therapeutic approaches geared to have an effect on both the vascular and the extravascular components of CNV are discussed.

Basement membranes and artificial substrates in cell transplantation

change influenced by both genetic predisposition and environmental factors and focused at the level of Bruch’s membrane. A number of age-related changes occur at the level of Bruch’s membrane [26, 32]. Morphological and biochemical studies have demonstrated that the changes that occur to Bruch’s membrane are typified by increased thickness and the progressive accumulation of deposits (such as drusen—see later) within the inner layers of the membrane. Bruch’s membrane is a basement membrane complex located between the retinal pigment epithelium (RPE) and the choroid. It is a pentalaminar structure with a central elastin layer bordered on either side by a collagenous zone. There is the basal lamina of the RPE at the innermost collagenous layer of Bruch’s membrane and a second basal lamina, produced by the endothelial cells of the choriocapillaris, that is juxtaposed to the outer collagenous layer. Although much more remains to be learned, the basic ultrastructural organization and biochemical composition of Bruch’s membrane are similar to basement membrane complexes in other tissues, such as the choroid plexus, kidney glomerulus, and airway alveoli, where an epithelial–endothelial juxtaposition occurs. All ionic exchange and metabolic traffic from the neural retina and RPE to the choroidal capillaries, and vice versa, must traverse Bruch’s membrane, thus leaving the neural retina vulnerable to any disruptions of those processes [3]. It is well documented that Bruch’s membrane undergoes a number of changes throughout life, including increased thickening, protein cross-linking and reduced permeability to nutrients as well as increased amounts of lipid deposition and the accumulation of basal laminar deposits and drusen (for more detailed reviews see [13, 22, 86]. Drusen are insoluble deposits that accumulate at the interface between Bruch’s membrane and the RPE. Clinically, drusen are divided into two main morphologic phenotypes—hard and soft. Hard drusen are hemispherical structures with well-defined edges, whereas soft This article will concentrate largely on the current developments in the area of cell transplantations presented at the 1st Workshop for Cell Transplantation in Age-related Macular Degeneration. In particular, this brief review will address our current understanding of the role of cell–matrix interactions by covering the pathobiology of normal ageing Bruch’s membrane; some of the problems faced at the time of surgery from a basement membrane prospective; the dedifferentiation and differentiation of RPE cells; and how the use of artificial substrates may address several of these issues. We will concentrate on problems related to age-related macular degeneration (AMD), the leading cause of irreversible blindness in Europe, America and other industrialized nations. AMD is likely to be a family of disorders, rather than a single biologic entity, characterized by the progressive loss of sight in the central portion of the visual field [6, 56, 71, 81].

Does the Presence of an Epiretinal Membrane Alter the Cleavage Plane during Internal Limiting Membrane Peeling

PURPOSE To determine whether the presence of a clinically and/or microscopically detectable epiretinal membrane (ERM) alters the cleavage plane during internal limiting membrane (ILM) peeling. DESIGN Retrospective, observational, immunohistochemical study of ILM specimens using archival formalin-fixed, paraffin-embedded tissue. PARTICIPANTS Fifty-one patients who had had ILM excision. METHODS Fifty-one ILM specimens peeled during vitrectomy for various etiologies were examined by light microscopy. The removal of ILM was assisted using Trypan blue (n = 30), indocyanine green (n = 7), or brilliant blue G (n = 14). Monoclonal antibodies to glial fibrillary acidic protein and to neurofilament protein were used to detect glial or neuronal cells respectively on the vitreous or retinal surfaces of the ILM. Specimens were divided into 2 groups: ILM peeled for full-thickness macular hole (MH; n = 31) and ILM peeled after removal of clinically detectable ERM (n = 20). MAIN OUTCOME MEASURES Primary outcome measure was the localization of immunohistochemical markers to neuronal or glial cells on the vitreous or retinal surfaces of ILM. The secondary outcome measure was the correlation of the results of the primary measure with the dyes used to facilitate ILM peeling. RESULTS Glial and/or neuronal cells were detected on the retinal surface of the ILM in 10 of 31 (32%) of the MH ILM specimens and in 13 of 20 (65%) of the ILM peeled after ERM excision; the difference was significant (P = 0.02). There was no association between the presence of neuronal and glial cells with the type of dye used (P = 0.2). Of the 23 ILM specimens with cells attached to the retinal surface, 21 (91%) were associated with clinical and/or histologic evidence of ERM and 2 (9%) were not. The correlation between the presence of cells on the vitreous and the retinal surfaces of ILM was high (P<0.0001). CONCLUSIONS The findings suggest that ERM may be associated with sub-ILM changes that alter the plane of separation during ILM peeling. This study does not confirm any influence of dyes on the cleavage plane during surgery.

SDF1-alpha is associated with VEGFR-2 in human choroidal neovascularisation

Endothelial progenitor cells (EPCs) have been shown to contribute to experimentally induced choroidal neovascularisation (CNV) in animal models. The recruitment pathway for EPCs is dependent on the chemokine stromal cell derived factor 1-alpha (SDF) and its receptor CXCR4 on the progenitor cell. We examined 23 specimens of CNV occurring secondary to a variety of aetiologies (10 secondary to age-related macular degeneration (AMD), 4 inflammatory, 4 idiopathic and 5 melanoma-associated) for the presence and distribution of SDF and CXCR4 in order to determine if this pathway may play a role in neovascularisation. Specimens were examined by immunohistochemistry using a panel of antibodies against SDF, CXCR4, vascular endothelial growth factor receptor 2 (VEGFR-2), CD34 (endothelial cells), CD68 (macrophages) and cytokeratins (retinal pigment epithelium; RPE). SDF was detected in 2 cases of CNV in AMD, 1 inflammatory CNV, 3 idiopathic CNVs and in 3 cases of CNV associated with melanoma. A significant association was found between SDF and VEGFR-2 immunostaining in individual membranes (p<0.001). Localisation of SDF immunostaining to the presumed RPE was also significant (p<0.05). CXCR4 immunostaining was widespread in all membranes in keeping with the published work of other investigators. Our study suggests that SDF, which may be produced by the RPE, could play a role in CNV.