P58: Secondary amines containing one aromatic nitro group: Preparation, nitrosation, sustained nitric oxide release, and the synergistic effects of released nitric oxide and an arginase inhibitor on vascular smooth muscle cell proliferation

Abstract

Background Nitric oxide (NO) is synthesized by vascular endothelial cells in vivo from l -arginine by nitric oxide synthases (NOS). Initial events in the onset of atherosclerosis lead to endothelial dysfunction and subsequent decrease of endogenous NO. Within the vasculature NO inhibits smooth muscle cell proliferation (SMC), therefore decreased concentrations lead to rapid SMC division, a key event in the formation of atherosclerotic plaques. Reestablishing physiological levels exogenously with NO-donors, aims to stop uncontrolled SMC proliferation and the progression of atherosclerosis. However current NO donors rapidly release NO in highly concentrated, single bursts, leading to cellular oxidative stress. In contrast, N-nitroso (N–NO) NO donors synthesized in our lab through the nitrosation of secondary amines, release NO in a slow, sustained manner, establishing and maintaining low NO concentrations that effectively reduce SMC proliferation, without onset of oxidative stress.Additionally, NOS competes for l -arginine with arginase, which catalyzes its conversion to l -ornithine, the precursor to various polyamines-known stimulators of SMC proliferation. Arginase inhibition would reduce SMC proliferation by decreasing l -ornithine concentrations and providing increased l -arginine for endogenous NO production. Combining arginase inhibition with NO-donor application may prevent SMC proliferation more effectively than either method individually, and may have a synergistic effect. Methods Two isomeric families of NO-donors, prepared in our laboratory, were applied to SMC cultures at various concentrations. Each secondary amine precursor was also tested for any toxic or anti-proliferative effects. Likewise the potent arginase inhibitor, ABH was tested to determine the most effective concentration for proliferation reduction. Finally, optimized concentrations of NO-donors and ABH were added in tandem. Proliferation and toxicity were monitored using a cell viability and LIVE/DEAD assay. A third assay was implemented to monitor changes in intracellular NO concentrations due to the addition of each compound. Results Cellular viability assays showed eight of ten amines exerted no toxic or anti-proliferative effects in the range tested (0.1–20 μM), nor did they increase intracellular NO concentrations. These compounds were nitrosated and tested in same concentration range. All donors exhibited proliferation inhibition without inducing cell death between 5% and 20 μM in a concentration dependent manner. At 20 μM, the decrease in viable cells ranged from 5% to 22%. Likewise, ABH decreased the number of viable cells in a concentration dependent manner from 0.001 to 5.0 mM by 9.9–30.0%, respectively. Both the NO-donors and ABH increased intracellular NO with increasing concentrations. Finally, the most effective NO-donor from each family (20 μM) in combination with ABH (5 mM) inhibited SMC proliferation in a synergistic manner, reducing the number of viable cells by an additional 2% and 16% compared to the sum of individual components alone. These results were supported by the synergistic increase of intracellular NO. Conclusion Inhibition of SMC proliferation after endothelial dysfunction could slow down the establishment of arterial plaques, reducing the occurrence of cardiovascular diseases including atherosclerosis. Reestablishing NO concentrations both exogenously with NO-donors and endogenously by arginase inhibition with ABH simultaneously has shown to more effectively reduce SMC proliferation than either method individually, while avoiding onset of oxidative stress. Disclosure NIH, Award # R15HL 106600.