o-(chloroacetylcarbamoyl)fumagillol and Corneal-Neovascularization

To determine the efficacy of the angiogenic inhibitor TNP-470 on inflammatory corneal neovascularization. Topical and systemic delivery of the drug were investigated in a murine model as well as inhibition of endothelial cell proliferation in vitro and in vivo.. The effect of TNP-470 on VEGF- and bFGF-stimulated bovine capillary endothelial (BCE) cell proliferation was evaluated in vitro. Corneal neovascularization was induced in vivo by mechanical debridement of the corneal and limbal epithelium with 0.15 M NaOH on C57BL6 mice. TNP-470 was administered systemically at 30 mg/kg body weight (BW) every other day or topically three times daily in a concentration of 5 ng/ml dissolved in methylcellulose. Vessel length was investigated on day 7. VEGF protein content in murine corneas was analyzed by ELISA on days 2, 4, and 7 of treatment. A modified bromouridine (BrdU) ELISA was used to quantify endothelial cell proliferation.. TNP-470 exerted a dose-dependent inhibition of bFGF- and VEGF-induced endothelial cell proliferation in vitro. Both systemic and topical application of TNP-470 led to a significant reduction of inflammatory corneal neovascularization (P < 1 x 10(-5)). BrdU labeling showed that TNP-470 inhibited endothelial cell proliferation. VEGF protein levels were reduced by systemic TNP-470 treatment.. These results suggest that TNP-470 reduces inflammatory corneal angiogenesis by directly inhibiting endothelial cell proliferation. Topical and systemic treatment with TNP-470 reduces VEGF levels that are responsible for vessel growth during the neovascularization process.

Topics: Administration, Topical; Angiogenesis Inhibitors; Animals; Bromodeoxyuridine; Cell Division; Corneal Neovascularization; Cyclohexanes; DNA Replication; Dose-Response Relationship, Drug; Endothelial Growth Factors; Endothelium, Corneal; Endothelium, Vascular; Enzyme-Linked Immunosorbent Assay; Fibroblast Growth Factor 2; Injections, Subcutaneous; Lymphokines; Mice; Mice, Inbred C57BL; O-(Chloroacetylcarbamoyl)fumagillol; Sesquiterpenes; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factors

Targeting the endothelial cell cycle as an antiangiogenic strategy has been difficult given the ubiquitous expression of critical cell cycle regulators. Here, we show that the antiangiogenic drug TNP-470 displays striking cell-type specificity insofar as it induces the expression of p21(CIP/WAF), a cyclin-dependent kinase inhibitor, in endothelial cells but not in embryonic or adult fibroblasts. Moreover, primary endothelial cells isolated from p53(-/-) and p21(CIP/WAF-/-) mice are resistant to the cytostatic activity of TNP-470. We also demonstrate that p21(CIP/WAF-/-) mice are resistant to the antiangiogenic activity of TNP-470 in the basic fibroblast growth factor corneal micropocket angiogenesis assay. We conclude that TNP-470 induces p53 activation through a unique mechanism in endothelial cells leading to p21(CIP/WAF) expression and subsequent growth arrest.

Topics: Adult; Angiogenesis Inhibitors; Animals; Cell Cycle; Cell Division; Cells, Cultured; Corneal Neovascularization; Cyclin-Dependent Kinase Inhibitor p21; Cyclin-Dependent Kinases; Cyclins; Cyclohexanes; Endothelium, Vascular; Gene Expression; Humans; Mice; Mice, Knockout; Nuclear Proteins; O-(Chloroacetylcarbamoyl)fumagillol; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-mdm2; Sesquiterpenes; Tumor Suppressor Protein p53