N. Kociok

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.

Modulation of Hypoxia-Induced Neovascularization by JSM6427, an Integrin α5β 1 Inhibiting Molecule

Purpose: Integrin α 5β 1, a fibronectin receptor, is involved in endothelial cell migration and proliferation. Here we investigate the effect of JSM6427, an integrin α 5β 1 inhibiting molecule, on the development of retinal vascular system using the mouse model of oxygen-induced retinopathy (OIR). Methods: Endothelial cell migration and sprouting was analyzed in vitro using a 2D migration assay and a 3D sprouting/angiogenesis assay in fibrin gel. C57BL/C6 mice were exposed to 75% oxygen from postnatal day 7 (P7) to P12 and returned to room air thereafter. Intravitreal injection of 40 μ g JSM6427 was performed in each one eye on P14. On P17, vascular area, avascularized area, and neovascular blood vessel tufts were quantified after perfusion with fluorescein-coupled concanavalin A. The number of retinal neovascular cell nuclei was determined in hematoxylin-stained cross sections of the eyes. Integrin α 5 expression was determined by immunohistochemistry. Results: In vitro, JSM6427 inhibits the migration of HUVEC and the tube formation induced by both bFGF and VEGF. In vivo, integrin α 5 expression was detectable in neovascular retinal blood vessels. Oxygen treatment (positive control) in comparison with no oxygen treatment (negative control) reduced significantly the vascularized area and increased the avascularized area. A single intravitreal injection of 40 μ g JSM6427 resulted in a significant reduction of the vascularized area and the number of preretinal nuclei in comparison with the intravitreal injection of the vehicle while the avascularized area increased significantly. Conclusions: These results imply an essential role of integrin α 5β 1 in the refining of the retinal vasculature in OIR and suggest JSM6427 may have a possible therapeutic function for neovascular disease.

DNA Fingerprint Analysis Reveals Differences in Mutational Patterns in Experimentally Induced Rat Peritoneal Tumors, Depending on the Type of Environmental Mutagen

We performed tumor DNA fingerprint analysis using the synthetic minisatellite probe S3315x2 based on the 33.15-repeat unit. The aim of the study was to investigate fingerprinting patterns of peritoneal tumors induced experimentally in Wistar rats by two carcinogens with unknown mechanism of action (crocidolite asbestos and nickel powder) and, as a positive control, benzo[a]pyrene. The carcinogens were administered intraperitoneally into rats. The banding patterns obtained with DNA from 71 peritoneal tumors were compared to the corresponding normal tissues. DNA derived from peritoneal tumors induced by the three carcinogens differed with respect to mutation frequencies and mutation patterns. The mutation frequencies in these tumors, revealed by DNA fingerprinting, were 18.2% for benzo[a]pyrene, 14.8% for crocidolite asbestos, and 40.9% for nickel powder. The alterations detected in the banding pattern of benzo[a]pyrene-induced peritoneal tumors were exclusively additional bands. On the contrary, in the DNA from asbestos-induced peritoneal tumors, only deletions of bands were observed on the autoradiographs. In the DNA from nickel-induced peritoneal tumors, both types of mutations occurred. The different mutation frequencies and mutation patterns appear to discriminate between benzo[a]pyrene, crocidolite asbestos, and nickel powder, and may be related to the mechanisms of action of these compounds.