Quantification of anti-angiogenesis using the capillaries of the chick chorioallantoic membrane demonstrates that the effect of human angiostatin is age-dependent.

We present a method whereby en face estimation of the chorionic capillary plexus can be generated in the living chick chorioallantoic membrane (CAM) and confirmed by post-fixation cross section analysis. This value does not alter significantly with age and provides a reliable and simple method to evaluate anti-angiogenesis. Anti-angiogenesis may be induced by an intervention, such as a pharmacological agent, applied to the surface of the CAM. We describe the use of silastic rings that are associated with minimal inflammatory reaction, in this process. By estimating changes in the chorionic capillary plexus to quantify anti-angiogenesis, together with silastic rings, we examined the anti-angiogenic effect of human angiostatin and demonstrated that although there is a significant loss of capillaries en face after exposure from days 7 to 9 of incubation, in contrast there is no significant inhibition after exposure to a similar dose of angiostatin from days 11 to 13 of incubation. This not only demonstrates the important effects on neo-angiogenesis compared to mature vessels, but also illustrates the potential of the CAM to readily provide a means for such a comparison.

Development of a monoclonal antibody-based enzyme-linked immunosorbent assay to quantify soluble beta-glucans in oats and barley.

A set of 31 murine monoclonal antibodies was produced against (1-->3,1-->4)beta-d-glucan from oats (Avena sativa L.) chemically cross-linked to keyhole limpet hemocyanin. Monoclonal antibodies were tested for their cross-reactivity to related and unrelated polysaccharides. The antibodies reacted strongly to unmodified beta-glucan from oats and barley (Hordeum vulgare L.) and to lichenan from Icelandic moss, a polysaccharide with a structure similar to that of beta-glucan but which is not encountered in cereals. Cross-reaction to other polysaccharides tested was minimal at physiological levels. An enzyme-linked immunosorbent assay (ELISA) that could routinely detect and quantify nanogram levels of soluble beta-glucan extracted from the flour of oats or barley was designed with one of these monoclonal antibodies. The beta-glucan extraction procedure from ground oat and barley samples and the ELISA were both optimized for reproducibility, accuracy, and throughput, and results were compared to values obtained from an established, commercially available enzyme-based assay. Correlations between the two assays were consistently high (r (2) > 0.9), indicating that the ELISA presented in this paper is a valuable alternative for assaying beta-glucan levels in cereals and cereal products, both routinely and in preparations in which beta-glucans are present in nanogram amounts. Development of the extraction procedure for ELISA is discussed.

Angiostatin II is the predominant glycoform in pleural effusates of rabbit VX-2 lung tumors.

Angiostatin (AST), a polypeptide with potent antiangiogenic properties, is released proteolytically from plasminogen in vivo. Plasminogen exists naturally in plasma as two glycoforms (PLGs), I and II. Recently it was shown with the use of a chick-embryo chorioallantoic membrane (CAM) assay that rabbit PLG-I and -II yield distinct ASTs-AST-I and -II, respectively-with different antiangiogenic activities. AST glycoforms were of similar molecular weight, approximately 30 to 32,000 kD, and probably consisted of kringles 1 to 3 only. AST has now been identified in the interpleural effusate released from VX-2 lung tumors in rabbits. Effusate was collected from six rabbits with high tumor burdens and fractionated by means of lysine-Sepharose chromatography. The epsilon-aminohexanoic acid-eluted protein of all effusates contained AST (kringles 1-3) at a mean concentration of 1.2 microg/mL of effusate; with regard to AST content, 97% was AST-II. A CAM assay revealed that the lysine-Sepharose-bound fraction from all interpleural effusates contained potent antiangiogenic activity. Blood and urine from rabbits with high burdens of VX-2 contained essentially only AST-II, at mean concentrations of 145 and 4 ng/mL, respectively. AST was absent from the blood of control rabbits. In an attempt to compare their uptake by VX-2, iodine 125-labeled AST-I and iodine 131-labeled AST-II were injected intravenously into tumor-bearing rabbits. AST-I entered the tumor 1.6 times faster than AST-II. As a means of accounting for the preponderance of AST-II in the interpleural effusate, we postulate that VX-2 cells release proteolytic activity to activate plasminogen but that of the two PLGs, PLG-II may be the preferred substrate for AST formation in vivo.