The role of reactive oxygen and nitrogen intermediates in endothelial cell pathophysiology

Wayne Oren Carter, Purdue University

Abstract

Endothelial cells comprise the largest tissue in the body and are an integral component of many homeostatic functions. A crucial interaction exists between the neutrophil and endothelial cell because the neutrophil depends on adhesion to the endothelial cell to exit the circulation and emigrate to the tissues. In this interaction, the neutrophil is capable of releasing reactive oxygen and nitrogen intermediates which may damage the endothelial cell and alter its normal cellular physiology. Rat pulmonary artery endothelial cells were isolated and subcultured by an enzyme-free methodology to preserve cell surface receptors and long-term culture viability. To remove contaminating pericytes, endothelial cells were purified by fluorescence-activated cell sorting. The cells demonstrated excellent viability and relatively little transformation in extended cultures. Endothelial cells demonstrated definitive signs of transformation at passage number 140 as evidenced by growth in soft agar and aneuploidy. Subtle changes in growth rate and surface epitope expression were identified after 70 to 80 cell passages. To assist studies involving neutrophil-endothelial interactions, mouse anti-rat neutrophil monoclonal antibodies were produced. We utilized two fluorochromes, hydroethidine and 2$\sp\prime,7\sp\prime$-dichlorofluorescin diacetate to measure intracellular superoxide anion and hydrogen peroxide respectively in neutrophils and endothelial cells. Utilizing the fluorochrome hydroethidine, we demonstrated that endothelial cells produce superoxide anion through the xanthine oxidase pathway after stimulation with hydrogen peroxide. Additionally hydrogen peroxide appeared to activate a detoxification mechanism within the endothelial cell. We also demonstrated that nitric oxide was capable of reducing intracellular superoxide anion in neutrophils during low activation states of the oxidative burst. In endotoxemic rats, nitric oxide inhibition resulted in increased pulmonary neutrophil accumulation and $\rm N\sp{G}$-nitro-L-arginine methyl ester resulted in lethal renal tubular necrosis. In conclusion, rat pulmonary artery endothelial cells isolated by an enzyme-free methodology and purified to remove pericytes, provided an excellent research tool to evaluate the effects of reactive oxygen and nitrogen intermediates on these cells. Superoxide anion is produced by endothelial cells by xanthine oxidase and other undefined pathways after stimulation with hydrogen peroxide. The role of nitric oxide in tissue damage associated with reactive oxygen intermediates and cell adhesion is discussed in detail.

Degree

Ph.D.

Advisors

Robinson, Purdue University.

Subject Area

Veterinary services|Animal diseases

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