An ex vivo angiogenesis assay utilizing commercial porcine carotid artery: modification of the rat aortic ring assay.

The study of angiogenesis as a therapeutic target requires a reliable, physiologically relevant, and technically straightforward assay. An ex vivo assay bridges the gap between cell-based assays, which may not realistically represent the complex process of vessel sprouting, and in vivo assays, which are time consuming and expensive. Porcine carotid arteries provide an ideal tissue source for angiogenesis inhibitor screens due to their availability, physiological relevance and large size. 1.5 mm2 fragments of porcine carotid arteries were incubated in 48-well culture plates and sandwiched between two 100 microliters layers of Matrigel. Sprouting was observed from the explants and quantitated, using a digital imaging system, after two weeks of incubation. Histological analysis using Factor VIII-related antigen (von Willebrand Factor) as an endothelial cell-specific marker identified these sprouts, which were consistent with endothelial cell morphology, supporting the system as a model of angiogenesis. Accordingly, the angiogenesis inhibitors suramin, 2-methoxyestradiol, and the matrix metalloprotease inhibitor Batimastat were shown to completely inhibit sprouting at 50, 0.5, and 5.0 micrograms/ml, respectively and to have ED50 values of 23, 0.15, and 0.14 microgram/ml. This assay shows good reproducibility and eliminates animal to animal variation. The system should prove adaptable to other forms of angiogenic stimulation, ultimately making a variety of assays for angiogenesis available to laboratories of limited resources.

Structural requirements for human inducible nitric oxide synthase substrates and substrate analogue inhibitors.

Inducible nitric oxide synthase (iNOS; EC 1.14.13.39) catalyzes the NADPH-dependent oxidation of one of the free guanidino nitrogens of L-Arg to form nitric oxide and L-citrulline. Analogues of L-Arg and the inhibitor, L-N6-(1-iminoethyl)lysine, were used to define structural elements required for the binding and catalysis of compounds. L-Arg analogues with sequentially shorter methylene spacing between the guanidino group and the amino acid portion of the molecule were not iNOS substrates but were reversible inhibitors. L-Arg analogues such as agmatine with a hydroxyl substitution at the 2-amino position were substrates. Desaminoarginine was not a substrate but a reversible inhibitor. Desaminoarginine, agmatine, and argininic acid bound to the enzyme to give type I difference spectra similar to that of L-Arg. The amidino compounds L-N6-(1-iminoethyl)lysine, L-N5-(1-iminoethyl)ornithine, and N5-(1-iminoethyl)cadaverdine, but not N6-(1-iminoethyl)-6-aminocaproic acid, were NADPH-dependent, irreversible inactivators of iNOS. For both the L-Arg and L-N6-(1-iminoethyl)lysine analogues, the 2-amino group appeared to play an important role in catalytic events leading to either substrate turnover or mechanism-based inactivation. Inactivation of iNOS by L-N6-(1-iminoethyl)lysine was NADPH- and dioxygen-dependent, but low incorporation of radiolabel with DL--4, 5-3H]-N6-(1-iminoethyl)lysine indicates that the mechanism of enzyme inactivation is not covalent modification of the protein.