We hypothesized that transgenic mice overexpressing the p22phox subunit of the NADPH oxidase selectively in smooth muscle (Tgp22smc) would exhibit an exacerbated response to transluminal carotid injury compared to wild-type mice. To examine the role of reactive oxygen species (ROS) as a mediator of vascular injury, the injury response was quantified by measuring wall thickness (WT) and cross-sectional wall area (CSWA) of the injured and noninjured arteries in both Tgp22smc and wild-type animals at days 3, 7, and 14 after injury. Akt, p38 MAPK, and Src activation were evaluated at the same time points using Western blotting. WT and CSWA following injury were significantly greater in Tgp22smc mice at both 7 and 14 days after injury while noninjured contralateral carotids were similar between groups. Apocynin treatment attenuated the injury response in both groups and rendered the response similar between Tgp22smc mice and wild-type mice. Following injury, carotid arteries from Tgp22smc mice demonstrated elevated activation of Akt at day 3, while p38 MAPK and Src activation was elevated at day 7 compared to wild-type mice. Both increased activation and temporal regulation of these signaling pathways may contribute to enhanced vascular growth in response to injury in this transgenic model of elevated vascular ROS.

No abstract

Following percutanous transluminal coronary angioplasty (PTCA) restenosis is markedly increased in metabolic syndrome patients. We hypothesized that increased restenosis in the metabolic syndrome was due to enhanced activation of p38 MAPK (p38) and subsequent p38‐dependent activation of MMP2 and 9. Carotid arteries were injured with a Fogarty 2F catheter in a rat model mimicking the human metabolic syndrome (JCR rat) and normal, healthy controls (SD rats) and neointima formation was quantified. p38 phosphorylation and MMP2 and 9 expression were assessed by immunoblotting and MMP2 and 9 activity by zymography in injured and contralateral control carotids at 4, 7, 10 and 14 days post injury. No basal differences were observed between JCR and SD rats. Maximal increase in p38 activation of similar magnitude occurred at 4 days post injury in both rat phenotypes. p38 activation rapidly returned to baseline in SD but remained sustained in JCR rats. MMP2 and 9 expression and activation mirrored that of p38. Thus, while the absolute magnitude of activation in response to vascular injury is similar, our findings suggest that the sustained activation of p38 and MMP2 and 9 in the metabolic syndrome contribute to increased neointima formation and may provide targets for pharmacological therapies devised for prevention of restenosis following PTCA. Support: NIH HL 084159 and HL 093052

We hypothesized that increased 3‐phosphoinositide‐dependent kinase 1 (PDK1) activation contributes to arterial wall thickening following vascular injury. Vascular smooth muscle cells (VSMCs) were pretreated with BX‐912, a small molecule inhibitor of PDK1, prior to stimulation with PDGF. Western analysis confirmed inhibition of PDK1 activity in the presence of BX‐912 by decreased phosphorylation of PDK1 (ser241) by ~60% and Akt (thr308) by ~ 70%. Conversely, Akt phosphorylation at ser473 was only mildly affected. To examine VSMC migration, both scratch wound assays and modified Boyden chamber studies were completed. In both instances, wound‐ and PDGF‐induced chemotaxis was attenuated following BX‐912 treatment. Following wire injury of the carotid artery in C57BL6/J mice, in vivo PDK1 phosphorylation (ser 241) was assessed by immunoblotting in both injured and contralateral control carotids at 3, 7, and 14 days post injury. PDK1 phosphorylation was markedly increased at day 3 (~5 fold vs. non‐injured), remained slightly elevated at day 7 (~2 fold) and returned close to baseline by later timepoints following injury. Our findings indicate that BX‐912 is effective in attenuating PDK1 activity and chemotaxis in VSMCs and that increased PDK1 activity occurs following vascular injury. Thus, PDK1 inhibition, via BX‐912 treatment, could potentially be utilized to attenuate wall remodeling in response to injury. Support: NIH HL084159