Meta-iodobenzylguanidine, an inhibitor of arginine-dependent mono(ADP-ribosyl)ation, prevents neointimal hyperplasia.

The association of ADP-ribosylation with cell proliferation and ischemia-reperfusion injury suggests that it may be a suitable target for therapeutic control of revascularization-induced injury. The purpose of this study was to investigate the inhibitory actions of ADP-ribosylation inhibitors on restenosis. In organ culture, the poly(ADP-ribose) polymerase (PARP) inhibitor 3,4-dihydro-5-methylisoquinolinone (PD128763) was unable to prevent neointimal hyperplasia, whereas the arginine-dependent mono(ADP-ribosyl)transferase (ART) inhibitor meta-iodobenzylguanidine (MIBG) was highly effective (EC(50) 21 microM). Treatment with 3-aminobenzamide (3AB), a less potent ART inhibitor, also produced a significant reduction in neointimal hyperplasia. Single doses (25 mM) of MIBG and 3AB were also applied within a fibrin coagulum directly to the adventitial surface of the porcine femoral artery after balloon catheter injury in vivo. MIBG reduced the neointimal index, measured 14 days after angioplasty, by 82%, whereas 3AB was ineffective. However, when extended to 45 days, the neointimal index was not significantly decreased by MIBG treatment relative to control. Assessment of MIBG release from the fibrin glue showed that the bulk of the compound was eluted within 3 days, suggesting that the vehicle was not suitable for long-term delivery. On the other hand, direct infusion of MIBG into vessels was able to reduce neointimal hyperplasia over 14 days in organ culture. These data support the conclusion that the cellular retention characteristics of MIBG contribute significantly to the efficacy of this compound. Based on these results, ART, but not PARP, may be a credible target for therapeutic treatment of restenosis.

Activation of MMP-2 in response to vascular injury is mediated by phosphatidylinositol 3-kinase-dependent expression of MT1-MMP.

Phosphatidylinositol 3-kinase (PI3K) is required for smooth muscle cell (SMC) proliferation. This study reports that inhibitors of PI3K also prevent SMC migration and block neointimal hyperplasia in an organ culture model of restenosis. Inhibition of neointimal formation by LY-294002 was concentration and time dependent, with 10 muM yielding the maximal effect. Continuous exposure for at least the first 4-7 days of culture was essential for significant inhibition. To assess the role of matrix metalloproteinases (MMPs) in this process, we monitored MMP secretion by injured vessels in culture. Treatment with LY-294002 selectively reduced active MMP-2 in media samples according to zymography and Western blot analysis without concomitant changes in latent MMP-2. Parallel results with wortmannin indicate that MMP-2 activation is PI3K dependent. Previous research has shown a role for both furin and membrane-type 1 (MT1)-MMP (MMP-14) in the activation of MMP-2. The furin inhibitor decanoyl-Arg-Val-Lys-Arg-chloromethylketone did not prevent MMP-2 activation after balloon angioplasty. In contrast, balloon angioplasty induced a significant increase in the levels of MT1-MMP, which was suppressed by LY-294002. No change in MT1-MMP mRNA was observed with LY-294002, because equivalent amounts of this mRNA were present in both injured and noninjured vessels. These results implicate PI3K-dependent regulation of MT1-MMP protein synthesis and subsequent activation of latent MMP-2 as critical events in neointimal hyperplasia after vascular injury.

NF-kappaB activation is essential for angiotensin II-dependent proliferation and migration of vascular smooth muscle cells.

Angiotensin II (AngII) functions as a stress hormone under conditions of stretch, pressure and injury to stimulate smooth muscle cell migration and proliferation. Since the cellular response to stress is mediated in part by the transcription factor NF-kappaB, the relationship between AngII and NF-kappaB was investigated. Our study revealed that AngII promoted a dose-dependent and transient phosphorylation of the regulatory IkappaBalpha protein in smooth muscle cells from porcine coronary artery, with concomitant nuclear translocation of NF-kappaB and increased binding to a kappaB promoter element. Both nuclear translocation and kappaB-element binding were prevented by the AT(1) receptor antagonist losartan. The role of NF-kappaB in AngII-dependent smooth muscle cell migration and proliferation was then assessed. Inhibitors of NF-kappaB nuclear translocation (phenethyl caffeiate) and IkappaB phosphorylation (Bay 11-7085) effectively arrested both AngII-dependent DNA synthesis and migration. These results were confirmed with SN50, a highly selective peptide inhibitor of NF-kappaB activation. Phenethyl caffeiate also prevented the phosphorylation of cdk2 and Rb, indicating NF-kappaB was required for G1/S transition. The target of NF-kappaB inhibition was identified as cyclin E, since induction of this gene, but not cyclin D1, was suppressed by phenethyl caffeiate. We subsequently examined the relationship between NF-kappaB and neointimal formation in response to angioplasty-induced injury, a process susceptible to inhibition by losartan. Both phenethyl caffeiate and Bay 11-7085 blocked neointimal hyperplasia in organ culture following balloon angioplasty. These data indicate NF-kappaB is an important mediator of intracellular signalling by AngII under normal physiological conditions, and following vascular injury.