I. Semkova

Differential role of tumor necrosis factor (TNF)-alpha receptors in the development of choroidal neovascularization.

PURPOSE Tumor necrosis factor alpha (TNF)-alpha contributes to inflammation-associated angiogenesis. This study investigates the role of TNF-alpha receptors 1a and 1b in the development of choroidal neovascularization (CNV). METHODS CNV was induced in Tnfrsf1a(-/-) and Tnfrsf1b(-/-) mice with C57Bl6/J background and their wild-type (WT) (C57Bl/6J) controls by laser damage to the Bruchs membrane. TNF-alpha expression in RPE/choroid was determined by Western blot analysis. Pathologic angiogenesis was estimated qualitatively and quantitatively by fluorescein angiography and histology on choroidal flat mounts and paraffin cross-sections. Inflammatory cell invasion was investigated by clodronic acid depletion of circulating macrophages and immunochemistry, and the apoptotic activity was investigated by TUNEL assay and by caspase-3 and caspase-8 expression. Receptor 1b-specific Bmx/Etk kinase was detected by immunochemistry. RESULTS TNF-alpha levels were elevated after laser treatment. Severe CNV lesions and increased macrophage invasion were observed in Tnfrsf1a(-/-) compared with WT and Tnfrsf1b(-/-) mice. Increased immunoreactivity for Bmx/Etk kinase corresponded to the severity of CNV formation. Reduced pathologic angiogenesis and macrophage invasion in Tnfrsf1b(-/-) mice (vs. WT and Tnfrsf1a(-/-)) was accompanied by enhanced endothelial cell apoptosis and by caspase-3 and caspase-8 activation. CONCLUSIONS Receptor 1b promotes the recruitment of inflammatory cells to the site of injury and exacerbated pathologic angiogenesis probably by way of the Bmx/Etk-kinase-dependent pathway in the absence of receptor 1a. On the other hand, receptor 1a-dependent apoptosis in the absence of receptor 1b leads to reduced inflammatory response and CNV lesions after laser treatment. This demonstrates the potential for specific targeting of TNF-alpha receptors for future therapies of inflammation-associated choroidal neovascularization.

Retinal localization of the glutamate receptor GluR2 and GluR2-regulating proteins in diabetic rats.

Impaired glutamatergic activity and synaptic dysfunction contributing to excitotoxicity and neuronal degeneration has been observed in the diabetic retina. Here we analyzed the expression changes and trafficking abnormalities of the AMPA glutamate receptor 2 subunit (GluR2) and its regulators protein kinase Calpha (PKCalpha) and PKC-interacting protein 1 (PICK1) in the rat retina during the early phases of streptozotocin-(STZ-) induced diabetes. Diabetes was induced in Long Evans rats by injection of STZ. Two and six weeks after induction of diabetes, immunohistochemistry and in situ hybridization were performed on retinal paraffin sections to investigate the expression and localization of GluR2 and its regulators PKCalpha and PICK1. The cellular distribution and trafficking of these proteins in retinae were also investigated by subcellular fractionation and western blotting. While no significant changes were observed for GluR2 transcripts, we observed a strong increase in GluR2 immunoreactivity, predominantly in the ganglion cell layer (GCL) and the inner plexiform layer (IPL), as early as two weeks of diabetes. GluR2/3 immunoreactivity was further increased from the GCL to OPL after 6 weeks of diabetes. Increased expression of a phosphorylated non-synaptic population of GluR2 was detected in the GCL, the IPL and in distinct photoreceptor cells within the outer nuclear layer (ONL) of diabetic animals. Further, the PICK1 retinal distribution was unchanged two and six weeks after onset of diabetes and in both control and diabetic rat retinae the PKCalpha immunoreactivity remained the same. However, phosphorylated PKCalpha immunoreactivity was increased in diabetic retina as compared to control and peaked after 6 weeks of diabetes. Activated PKCalpha was almost completely lost in all membrane fractions and primarily recovered in the cytosolic fraction. These results are consistent with PKCalpha being re-localized in the diabetic retina. The observations indicate a diabetes-dependent increase in the activation of PKCalpha and a disturbed GluR2 regulation by altered internalization and recycling.

Deposition of nidogens and other basement membrane proteins in the young and aging mouse retina.

Aim: To determine the distribution of major basement membrane constituents, particularly nidogen 1 and 2, in young and aging mouse retinae. Methods: The specificity of antibodies against basement membrane proteins was ascertained by immunoblotting with proteins extracted from mouse retinae. The same antibodies were used in indirect immunofluorescence microscopy to localize basement membrane proteins in paraffin sections of retinae from 1-, 12- and 18-month-old C57BL/6 mice. Results: At a young age, laminin, perlecan and collagen IV were most abundant in Bruch’s membrane. Later, the proteins were clearly detected in capillary basement membranes and the inner limiting membrane. In both of these basement membranes, a massive increase in protein amount was seen upon aging, whereas in Bruch’s membrane the staining intensity was less drastically changed. Both nidogen 1 and 2 were present in vascular basement membranes and Bruch’s membrane throughout the age periods studied. In the inner limiting membrane, the nidogens were more strongly expressed at higher ages, with an earlier and more extensive deposition of nidogen 1. Conclusions: All major basement membrane constituents are present in the mouse retina, but the onset of deposition differs among the different proteins and between the various retinal basement membranes. In general, basement membrane protein deposition increases with age.

ICAM-1 depletion does not alter retinal vascular development in a model of oxygen-mediated neovascularization.

ICAM-1 has been identified as a mediator of inflammatory and VEGF-dependent corneal neovascularization. Furthermore, ICAM-1 has been demonstrated to be involved in leukocyte-mediated endothelial injury in diabetic retinopathy. Here we investigated the role of ICAM-1 in retinal vaso-obliteration and vascularization. ICAM-1 deficient mice as well as their respective wild-type controls were exposed to 75% oxygen from postnatal day 7 to day 12. Retinal vascularization was investigated after lectin labeling of endothelial cells on day 14, 17, and 20 in flat mount preparations. Retinal mRNA expression of VEGF, Angiopoietin 1 and 2 as well as PDGFbeta was examined at day 14 and 20 by Real Time RT-PCR. ICAM-1(-/-) mice and their respective wild-type controls demonstrated similar retinal development and vascularization under normoxic conditions. Similarly, after oxygen challenge, the vascular area, the avascularized area as well as the area of neovascular tufts did not differ between ICAM-1(-/-) and the respective wild-type mice although the mRNA expression of VEGF, ang-1, ang-2, and PDGFbeta differed clearly. This study demonstrates that lack of ICAM-1 leads to an altered expression of angiogenic factors that in combination may neutralize each other and do not alter retinal development and angiogenesis in oxygen-induced retinopathy.