Reactive oxygen species are critical in the oleic acid-mediated mitogenic signaling pathway in vascular smooth muscle cells

Obese hypertensive patients with cardiovascular risk factor clustering have increased plasma nonesterified fatty acid levels and are at high risk for atherosclerotic events. Our previous studies demonstrated that oleic acid i nduces a mitogenic response in rat aortic smooth muscle cells (RASMCs) thro ugh protein kinase C (PKC)- and extracellular signal-regulated kinase (ERK) -dependent pathways. In the present study we investigated the possibility t hat the generation of reactive oxygen species (ROS) constitutes a critical component of the oleic acid-induced mitogenic signaling pathway in RASMCs. We studied the effect(s) of oleic acid on the generation of ROS using the o xidant-sensitive fluoroprobe 2',7'-dichlorofluorescin diacetate. Relative f luorescence intensity and fluorescent images were obtained with laser confo cal scanning microscopy from 1 to 5 minutes, since preliminary studies demo nstrated that the peak fluorescence intensity occurred within 5 minutes. Ol eic acid (100 mu mol/L) induced a time-dependent increase of cell fluoresce nce that was >8-fold of that seen in control cells at 5 minutes. This was b locked by catalase, which suggests that H2O2 was the principal ROS. The ole ic acid-induced increases in H2O2 were blocked when PKC was inhibited with the use of bisindolylmaleimide and when PKC activity was downregulated by e xposing RASMCs to phorbol 12-myristate 13-acetate for 24 hours. Stearic and elaidic acids, which are weak PKC activators, did not significantly increa se H2O2 production. The increase of H2O2 in response to oleic acid was inhi bited by the antioxidant N-acetylcysteine. N-Acetylcysteine also completely blocked ERK activation and the increase of thymidine incorporation in resp onse to oleic acid. The data suggest that generation of H2O2 in RASMCs expo sed to oleic acid is PKC dependent. Moreover, H2O2 production emerges as a critical intermediary event in the oleic acid-mediated mitogenic signaling pathway between the activation of PKC and ERK, These observations raise the possibility that the elevated plasma nonesterified fatty acids, including oleic acid, in obese hypertensive patients contribute to vascular growth an d remodeling by a PKC-dependent mechanism to generate ROS that subsequently activate ERK.