Wolfram Eichler

Characterization of the CD55 (DAF)-binding site on the seven-span transmembrane receptor CD97

CD97 is an activation‐induced antigen on leukocytes which belongs to a new group of seven‐span transmembrane (7‐TM) molecules, designated EGF‐TM7 family. Family members, including EMR1 and F4/80, are characterized by an extended extracellular region with several N‐terminal epidermal growth factor‐like (EGF) domains. Alternative splicing of CD97 results in isoforms possessing either three (EGF1, 2, 5), four (EGF1, 2, 3, 5) or five EGF domains (EGF1, 2, 3, 4, 5). We recently identified decay accelerating factor (DAF, CD55), a regulatory protein of the complement cascade, as a cellular ligand of the smallest isoform. Employing mutants of CD97(EGF1, 2, 5) in which the EGF domains have been systematically deleted, we here demonstrate the necessity of at least three tandemly linked EGF domains for the interaction with CD55. Consistent with the involvement of different EGF domains, monoclonal antibodies directed against the first EGF domain as well as the removal of Ca2+ , for which binding sites exist in the second and fifth EGF domain, blocked binding to CD55. Compared to CD97(EGF1, 2,5) the larger isoforms CD97(EGF1, 2, 3, 5) and CD97(EGF1, 2, 3, 4, 5) have a significantly lower affinity for CD55. Thus, alternative splicing may regulate the ligand specificity of CD97 and probably other members of the EGF‐TM7 family.

VEGF release by retinal glia depends on both oxygen and glucose supply

Isolated retinae or isolated Müller cells were cultured in vitro, and vascular endothelial growth factor (VEGF) was assayed as protein (by ELISA) and as mRNA (by semi-quantitative RTPCR). In both types of cultures, hypoxia (5% O2) resulted in an upregulated VEGF release. While the unstimulated VEGF secretion was virtually independent of glucose (0.125–25 mM), elevated glucose concentrations (10–25 mM) blocked most of the stimulatory effect of hypoxia on VEGF mRNA synthesis (determined in Müller cell cultures) as well as on VEGF release (in both retina and Müller cell cultures). It is concluded that in retinal glial (Müller) cells, being responsible for retinal VEGF synthesis (and, thus, for undesirable neovascularization), the metabolic effects of hypoxia can be compensated by a surplus of glucose.

Characterization of an Early Activation‐Dependent Antigen on Lymphocytes Defined by the Monoclonal Antibody BL‐Ac(F2)

A novel activation‐dependent lymphocyte cell‐surface antigen which is recognized by a MoAb, BL Ac(F2), is described. Although not found on resting lymphocytes the antigen is induced rapidly wiihin 2–4 h following stimulation of the cells using mitogens or antibodies against the T‐eell CD3 antigen and sIgM on B cells, respectively. Immunopreeipitation and Western Blotting indicated that the MoAb recognizes a molecule in a range of 78 85 kDa. Beyond its activalion‐dependent expression on lymphocytes the antigen was detected also on myelo‐monocytic cells. Expression kinetics and cellular distribution of this molecule suggest that it is distinct from previously described activation‐dependent cell‐surface antigens sueh as CD69, CD25 and 4F2.

Pigment epithelium-derived factor released by Müller glial cells exerts neuroprotective effects on retinal ganglion cells.

Survival of retinal ganglion cells (RGC) is compromised in several vision-threatening disorders such as ischemic and hypertensive retinopathies and glaucoma. Pigment epithelium-derived factor (PEDF) is a naturally occurring pleiotropic secreted factor in the retina. PEDF produced by retinal glial (Müller) cells is suspected to be an essential component of neuron-glial interactions especially for RGC, as it can protect this neuronal type from ischemia-induced cell death. Here we show that PEDF treatment can directly affect RGC survival in vitro. Using Müller cell-RGC-co-cultures we observed that activity of Müller-cell derived soluble mediators can attenuate hypoxia-induced damage and RGC loss. Finally, neutralizing the activity of PEDF in glia-conditioned media partially abolished the neuroprotective effect of glia, leading to an increased neuronal death in hypoxic condition. Altogether our results suggest that PEDF is crucially involved in the neuroprotective process of reactive Müller cells towards RGC.

Regulation of pigment epithelium-derived factor production and release by retinal glial (Müller) cells under hypoxia.

PURPOSE To assess the regulation of pigment epithelium-derived factor (PEDF) production by retinal Müller glial cells, especially under ischemic or hypoxic conditions. METHODS PEDF was determined in surgically excised retinal tissue originating from patients with ischemic diabetic retinopathy and in primary guinea pig Müller cell cultures exposed to the protein synthesis inhibitor cycloheximide (CHX) and to hypoxia. PEDF production and secretion were studied by immunohistochemistry, quantitative RT-PCR, ELISA, fluorescence-activated cell sorter analysis, and Western blotting. RESULTS Gliotic Müller cells displayed decreased PEDF immunoreactivity in fibrovascular tissue from patients with diabetes compared with tissue from subjects with pathologic myopia. In Müller cell cultures, CHX treatment resulted in an increase, whereas mild hypoxia (2.5%-10% O(2)) induced a decrease, of PEDF mRNA and protein levels. However, strong hypoxia (0.2% O(2)) induced an upregulation of PEDF mRNA expression and resulted in only slightly reduced PEDF levels after 24 hours, detected as either a released, soluble, or cell surface-linked protein. CONCLUSIONS These results suggest that under certain conditions including mild hypoxia, Müller cells synthesize a protein factor that downregulates PEDF expression or its turnover. Generally, the cells appear to generate a biphasic response to hypoxia. In moderate hypoxia, PEDF is downregulated such that the VEGF-to-PEDF ratio increases (and angiogenesis is facilitated). During severe (or chronic) oxygen deficiency, however, the PEDF decline is arrested or even reversed; thus, the neurotrophic effects of PEDF remain available.

Retinal Endothelial Angiogenic Activity: Effects of Hypoxia and Glial (Müller) Cells

Objective: To explore the impact of retinal glial (Müller) cells on survival and neovascularization‐related activities of cultured retinal endothelial cells under normoxic and hypoxic conditions.

Pigment epithelium-derived factor acts as an opponent of growth-stimulatory factors in retinal glial-endothelial cell interactions.

Pigment epithelium‐derived factor (PEDF), a glycoprotein with pleiotropic functions, is naturally occuring in the eye and considered as crucial to prevent pathological angiogenesis. Since retinal glial (Müller) cells produce PEDF, the authors have studied its impact on glial–endothelial cellular interactions. Bovine retinal endothelial cells were cultured in the presence of culture media originating from primary Müller cells, and endothelial proliferation as well as phosphorylation of the mitogen‐activated protein kinases extracellular signal‐regulated kinases (ERK)‐1/‐2 were investigated. The concerted activity of Müller‐cell derived soluble mediators attenuated endothelial proliferation and ERK‐1/‐2 activation, regardless of whether the Müller cells were preincubated under normoxia or hypoxia, and even though the endothelial cells were stimulated by vascular endothelial growth factor‐A (VEGF). This inhibitory activity was no longer demonstrable if high levels of basic fibroblast growth factor or VEGF were supplied, suggesting that in cases of pathological neovascularization, overproduction of proangiogenic mediators overrides the “antiangiogenic background” provided by Müller cells. However, neutralizing the activity of PEDF partially restored endothelial cell proliferation and resulted in increased ERK‐1/‐2 activation, which is in concordance with findings demonstrating that exogenously applied PEDF is able to suppress VEGF‐induced ERK‐1/‐2 phosphorylation. PEDF production by Müller cells is not only regulated by retinal oxygen but also by the activity of soluble factors released from retinal endothelial cells. For instance, PEDF levels were significantly elevated in glial (Müller)–endothelial cell cocultures as compared with bovine retinal endothelial cell‐free Müller cell cultures. These results have implications for the pathogenesis of retinal neovascularization since the Müller cell may be regarded as a central control element which modulates retinal PEDF levels and, thus, is of critical importance for adjusting the balance between proangiogenic and antiangiogenic mediators.

Antineovascular agents in the treatment of eye diseases.

Neovascularization is a common and potentially visually threatening complication of eye diseases such as diabetic retinopathy (DR) and age-related macular degeneration (AMD). An antiangiogenic therapy is aimed at inhibiting the growth of new blood vessels and should prevent onset or progression of neovascularization. Accumulated evidence indicates that growth factors, endothelial cell surface receptors, and extracellular matrix (ECM) proteins are major mediators of neovascularization and appealing targets for pharmacotherapeutical intervention. Vascular endothelial growth factor (VEGF) plays a critical role in the pathogenesis of retinal neovascularization (in linking tissue ischemia to angiogenesis), and is likely to contribute also significantly to choroidal neovascularization (CNV). Several antineovascular agents antagonize the function of VEGF, by blocking its proangiogenic activity. Indeed, VEGF targeting or disruption of VEGF signalling is the most effective strategy known so far in the pharmacological treatment of ocular neovascularization. Other compounds such as pigment epithelium-derived factor (PEDF) either aim at balancing the levels of pro-angiogenic and angiostatic molecules, target inflammation (cyclooxygenase inhibitors, steroids) or comprise modifiers of the ECM such as inhibitors of matrix metalloproteinases (MMPs) and agents that block the action of integrins. Vascular targeting agents (combretastatin) promote removal of newly formed vessels. This review provides an update on recent investigations directed at the pharmacotherapeutical management of ocular neovascular diseases, placing special emphasis on the underlying target molecules and relevant intracellular signalling pathways.

Characteristics of Iris and Retinal Pigment Epithelial Cells Cultured on Collagen Type I Membranes

Purpose: Transplantation of pigment epithelial cells is a promising treatment modality to repair retinal damage in age-related macular degeneration. For this purpose, it is necessary to establish cell culture techniques that allow acquisition of proper functional and morphological characteristics by the cells to be transplanted. Methods: Primary retinal pigment epithelial (RPE) and iris pigment epithelial (IPE) cells grown to confluence on collagen membranes were examined for morphology, adhesion, proliferation, apoptosis, as well as viability after ex vivo transplantation. Results: Pigment epithelial cells adhere, proliferate, form monolayers, acquire differentiated properties, and remain viable during transplantation to the subretinal space. Conclusions: Pigment epithelial cells cultured on collagen membranes acquire differentiated characteristics and are amenable to be transplanted as cell monolayers.

Anti-angiogenic effects of the receptor tyrosine kinase inhibitor, pazopanib, on choroidal neovascularization in rats

Neovascularization in the eye is a major cause of irreversible vision loss. The present study was undertaken to determine mechanisms through which pazopanib, a drug that targets multiple receptor tyrosine kinases such as VEGF receptors, inhibits angiogenesis and experimental choroidal neovascularization (CNV). Pazopanib inhibited VEGF expression by retinal pigment epithelium (RPE) cells and choroidal endothelial cells (CEC), decreased VEGF-induced cellular migration in a dose-dependent manner and suppressed extracellular signal-regulated kinase (ERK)-1/-2 phosphorylation. To assess the impact of pazopanib in vivo, CNV was induced in rats by rupturing the Bruchs membrane by laser coagulation. These experiments demonstrated that twice-daily topical eye drop treatment significantly (P<0.001) decreased leakage from photocoagulated lesions by 89.5%. Furthermore, the thickness of the developed CNV lesions was significantly inhibited by 71.7% (P<0.001) in pazopanib-treated eyes, and immunoreactivity of VEGF was lower than in control eyes. Our data suggest that pazopanib is a promising inhibitor of angiogenesis leading to an effective inhibition of CNV development in vivo. This activity can be largely ascribed to the down-regulation of VEGF release in the retina as well as to impaired VEGF-induced signaling and chemotaxis. Using a convenient topical dosing regimen, pazopanib may prove useful for treating a variety of ocular neovascular diseases such as neovascular age-related macular degeneration.