Selectin/glycoconjugate binding assays for the identification and optimization of selectin antagonists.

In this study we describe ELISA-type P- and L-selectin binding assays for the analysis of selectin antagonists. A biotinylated polyacrylamide-type glycoconjugate containing sialyl Lewis A (sLe(a)-polymer) is utilized as a synthetic ligand for both selectins analogous to the E-selectin assay we have developed recently. Following precomplexation of sLe(a)-polymer with streptavidin-peroxidase, the complex is added to microtiter plates coated with the recombinant selectins. Binding of sLe(a)-polymer to the immobilized selectins is measured by the peroxidase reaction. SLe(a)-polymer was found to bind to P- and L-selectin in a cation-dependent manner. The interaction of the polymer was blocked by neutralizing anti-P- and anti-L-selectin antibody, respectively. The reference compounds heparin and fucoidan inhibited in both assays. Sialyl Lewis X (sLe(x)) blocked binding to L-selectin by 46% at 3 mM, whereas no inhibition was observed in the P-selectin assay up to 3 mM. Control polymers containing sialic acid or beta-d-glucose instead of sLe(a) weakly bound or failed to bind to the selectins. Both assays are rapid to perform and of low variability. The P-selectin assay was successfully employed to identify and optimize novel carbohydrate-based P-selectin antagonists. The P-, L-, and E-selectin assays were used to determine the fine selectivity of several sLe(x)-related selectin antagonists. These studies together suggest that sLe(a)-polymer-based selectin assays are well suited for primary screening and the characterization of selectin antagonists.

Effect of active oxygen species on intimal proliferation in rat aorta after arterial injury.

Proliferation and migration of vascular smooth-muscle cells (VSMCs) are essential events in neointimal hyperplasia. Recent findings that active oxygen species induce pro-oncogene expression, and stimulate VSMC DNA synthesis and cell division, suggest that active oxygen species may play an important role in intimal proliferation after arterial injury. To determine how the redox state of the artery was altered by injury, the levels of thiobarbituric-acid-reactive substances (TBARs, markers of lipid oxidation) and glutathione peroxidase (GSH-PX, the enzyme responsible for the production of glutathione, a major intracellular antioxidant) were measured in the rat aorta after balloon injury. There was an inverse relationship between the level of TBARs, which increased significantly to a maximum 17% greater than normal at 10 days after injury (p<0.05, n=7), and the activity of GSH-PX, which decreased significantly to a minimum 36.5% less than normal at 10 days after injury (p<0.05, n=7). To determine whether maintaining a more reduced vessel environment would inhibit intimal proliferation, L-cysteine was administered by intraperitoneal injection from 3 days before to 14 days after balloon injury. At 14 days after the arterial injury, the aorta was harvested for histological and morphometric measurements of TBARs and GSH-PX. At 7 and 14 days after balloon injury, the aorta was harvested for [3H]thymidine incorporation studies. Efficacy of therapy was demonstrated by a significant decrease in the level of TBARs and an increase in the activity of GSH-PX (p<0.05 vs. the control group, n=7). In the L-cysteine group, all parameters of intimal proliferation were significantly reduced compared to the controls, including the intima:media ratio (0.091 vs. 0.253, p<0.05); the cross-sectional area of the neointima (0.0563 compared to 0.140 mm2, p<0.05), coverage of the internal elastic lamina by the neointima (23.44 compared to 43.27%, p<0.05), and [3H]thymidine incorporation (at 7 days 240.58 vs 350.68 cpm/mg tissue, p<0.05; at 14 days 181.71 vs. 275.30 cpm/mg tissue, p<0.05). These results demonstrate dynamic alterations in the vessel redox state after arterial injury, and suggest that maintaining a more reduced environment (e.g. administration of L-cysteine) will reduce intimal proliferation after arterial injury.