sr-59230a and Inflammation

This protocol describes microsphere-based protease assays for use in flow cytometry and high-throughput screening. This platform measures a loss of fluorescence from the surface of a microsphere due to the cleavage of an attached fluorescent protease substrate by a suitable protease enzyme. The assay format can be adapted to any site or protein-specific protease of interest and results can be measured in both real time and as endpoint fluorescence assays on a flow cytometer. Endpoint assays are easily adapted to microplate format for flow cytometry high-throughput analysis and inhibitor screening.

Topics: Animals; Biotinylation; Flow Cytometry; Fluorescence Resonance Energy Transfer; Green Fluorescent Proteins; High-Throughput Screening Assays; Humans; Inflammation; Kinetics; Microspheres; Peptide Hydrolases; Peptides; Reproducibility of Results; Temperature

Obesity-associated cardiovascular disease exerts profound human and monetary costs, creating a mounting need for cost-effective and relevant in vivo models of the complex metabolic and vascular interrelationships of obesity. Obesity is associated with endothelial dysfunction and inflammation. Free fatty acids (FFA), generated partly through beta-adrenergic receptor-mediated lipolysis, may impair endothelium-dependent vasodilation (EDV) by proinflammatory mechanisms. beta-Adrenergic antagonists protect against cardiovascular events by mechanisms not fully defined. We hypothesized that beta antagonists may exert beneficial effects, in part, by inhibiting lipolysis and reducing FFA. Further, we sought to evaluate the fat-fed rat as an in vivo model of obesity-induced inflammation and EDV. Control and fat-fed rats were given vehicle or beta antagonist for 28 d. Serum FFA were measured to determine the association to serum IL6, TNFalpha, and C-reactive protein and to femoral artery EDV. Compared with controls, fat-fed rats weighed more and had higher FFA, triglyceride, leptin, and insulin levels. Unexpectedly, in control and fat-fed rats, beta antagonism increased FFA, yet inflammatory cytokines were reduced and EDV was preserved. Therefore, reduction of FFA is unlikely to be the mechanism by which beta antagonists protect the endothelium. These results reflect the need for validation of ex vivo models of obesity-induced inflammation and endothelial dysfunction, concurrent with careful control of dietary fat composition and treatment duration.

Topics: Acetylcholine; Adrenergic beta-Antagonists; Animals; Blood Flow Velocity; Body Weight; Cytokines; Dietary Fats; Disease Models, Animal; Endothelium, Vascular; Inflammation; Male; Obesity; Propanolamines; Propranolol; Rats; Rats, Sprague-Dawley; Receptors, Adrenergic, beta; Vasodilation