NADPH oxidase is a major enzymatic source of oxygen free radicals in stimulated endothelial cells (ECs). The ortho-methoxy-substituted catechol, apocynin (4-hydroxy-3-methoxyacetophenone), isolated from the traditional medicinal plant Picrorhiza kurroa, inhibits the release of superoxide anion (O2*-) by this enzyme. The compound acts by blocking the assembly of a functional NADPH oxidase complex. The underlying chemistry of this inhibitory activity, and its physiological significance to EC proliferation, have been investigated. A critical event is the reaction of ortho-methoxy-substituted catechols with reactive oxygen species (ROS) and peroxidase. Analysis of this reaction reveals that apocynin is converted to a symmetrical dimer through the formation of a 5,5' carbon-carbon bond. Both reduced glutathione and L-cysteine inhibit this dimerization process. Catechols without the ortho-methoxy-substituted group do not undergo this chemical reaction. Superoxide production by an endothelial cell-free system incubated with apocynin was nearly completely inhibited after a lagtime for inhibition of ca. 2 min. Conversely, O2*- production was nearly completely inhibited, without a lagtime, by incubation with the dimeric form of apocynin. The apocynin dimer undergoes a two-electron transfer reaction with standard redox potentials of -0.75 and -1.34 V as determined by cyclic voltammetry. Inhibition of endothelial NADPH oxidase by apocynin caused a dose-dependent inhibition of cell proliferation. These findings identify a metabolite of an ortho-methoxy-substituted catechol, which may be the active compound formed within stimulated ECs that prevents NADPH oxidase complex assembly and activation.

In order to study the major cellular source of reactive oxygen species (ROS) in perturbed human endothelial cells (EC), the effect of thrombin, a phospholipase A2 activator, on cultured EC ROS generation has been investigated. EC were incubated with 0.1-1 unit/ml thrombin and cellular superoxide anion (O(-)2) release and hydrogen peroxide (H2O2) production measured. Thrombin exposure caused an elevation in EC O(-)2 release and H2O2 production. The effects of protein kinase C, arachidonic acid metabolism, NADPH oxidase, and phospholipase A2 inhibitors on thrombin-induced EC H2O2 production were examined. EC were exposed to 0.5 unit/ml thrombin and cellular H2O2 production measured in the presence and absence of the protein kinase C inhibitor, H-7; arachidonic acid metabolism inhibitors, indomethacin, nordihydroguaiaretic acid, and SKF525A; NADPH oxidase inhibitor, apocynin; and phospholipase A2 inhibitor, 4-bromophenacyl bromide. All inhibitors, with the exception of H-7 and indomethacin, suppressed thrombin-induced EC H2O2 production. The pattern of effects of these metabolic antagonists on thrombin-induced EC ROS production is similar to that previously reported on ROS production in EC exposed to high low-density lipoprotein levels, and in stimulated leukocytes. These findings further implicate NADPH oxidase as a major ROS source in EC.

Eighteen patients with metastatic renal cell carcinoma, who were treated by vaccines for active specific immunotherapy, also completed skin testing with autologous tumor cells, both prior to and following vaccine treatment. All patients have now been followed for more than 5 years. Ten patients who remained skin-test-negative following treatment had no clinical responses, and all had expired by 22 months. Eight patients became skin-test-positive; three of these had clinical regressions and three remain alive after more than 69 months. The survival times of the skin-test-positive group were significantly superior to those of the skin-test-negative group. The results suggest that skin testing with autologous tumor cells may accurately identify those patients who have acquired antigen-specific cell-mediated antitumor immunity.

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The effects of known leukocyte NADPH oxidase inhibitors on general cellular oxidant production in cultured human endothelial cells (EC) has been investigated. EC were stimulated with 10 nM phorbol 12-myristate 13-acetate and cellular oxidant production measured in the presence and absence of inhibitors that act on various substituents of the oxidase complex and its activation pathways. The effects of the cytosolic oxidase subunit translocation inhibitors, catechols (3,4-dihydroxybenzaldehyde, caffeic acid, and protocatechuic acid), ortho-methoxy-substituted catechols (apocynin, vanillin, and 4-nitroguaiacol), and quinone, 1,4-naphthoquinone; flavoprotein inhibitors, diphenylene iodonium and quinacrine; haem ligands, imidazole and pyridine; directly acting thiol reagents, disulfiram and penicillamine; NADPH analogue, Cibacron Blue; redox active inhibitors, quercetin and esculetin; intracellular calcium antagonist, TMB-8; and calmodulin antagonists, W-7 and trifluoperazine, were determined. All compounds reduced oxidant production in stimulated EC. These findings add to previous observations suggesting the presence of a functionally active NADPH oxidase in EC. Identifying the major cellular reactive oxygen species source in perturbed EC will provide new insights into our understanding of endothelial dysfunction, which has been hypothesized to be a major contributing factor in the pathogenesis of atherosclerosis.