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Abstract

Atherosclerosis is a multifactorial disease, in which LDL oxidation, nitric oxide unbalance and smooth muscle cell (SMC) proliferation play essential roles. In order to find a novel multiple approach to control or to decrease atherogenesis, we synthesized NO-donor molecules (furoxans) differently substituted at positions 3 and 4 of the heteroring, able in relaxing rat aorta strips. The involvement of NO was evident by the coadministration of ODQ, a well-known inhibitor of guanilate ciclase, that abolished the vasodilating effect. Then, we joined furoxans with different antioxidants to obtain NO-donor and antioxidant hybrids. These compounds, albeit with different potency, inhibited SMC proliferation. To understand the antiproliferative mechanism of these hybrids, we split them in their native pharmacophores and we found that furoxans, but not the antioxidant moieties, decreased SMC proliferation. Blocking the position 4 of the furoxan ring by a phenyl group, the inhibitory potency on SMC proliferation and vasodilation paralleled with the electron-attractor capacity of the group in 3. Extending the study to 4-R, 3-Ph furoxans (groups in 3 and 4 are interchanged) and to their related furazans (unable to release NO), the 4-Ph-3-R furoxans were the most potent inhibitor of SMC proliferation, followed by 3-Ph-4-R furoxans. Furazans were not effective, supporting the fact that the opening of the ring is essential for growth-inhibition. To understand the molecular basis of this effect, we demonstrated that the mechanism is neither cGMP- nor polyamine-dependent, the two main NO-mediated pathways involved in SMC proliferation. The effect is possibly due to a thiol-mediated S-nitrosylation of a protein involved in the growth process (a mechanism proposed to explain the inhibitory activity of furoxans on thioredoxin glutathione reductase, the enzyme responsible of maintaining the redox balance inSchistosoma). We are attempting to verify this mechanism by in vitro and ex vivo approaches and to test these hybrids in an animal model of atherosclerosis.