Early Modulation Of The Injury Response By The Short-acting NO Donor PROLI/NO Leads To Durable Inhibition Of Neointimal Hyperplasia

Abstract

Introduction: We have previously demonstrated that periadventitial delivery of the NO donor PROLI/NO provides durable inhibition of neointimal hyperplasia for up to 8 weeks in spite of its very short half-life of 2 seconds under physiologic conditions in vitro. The goal of this current study was to examine the effect of PROLI/NO on the arterial wall in the first 7 days following injury. Our hypothesis was that early modulation of cell populations in the arterial wall accounts for the durable efficacy of NO at inhibiting neointimal hyperplasia. Methods: The rat carotid injury model was performed in 10wk old male Sprague Dawley rats. Treatment groups included control, injury, or injury +PROLI/NO (10mg). Arteries were harvested at 2, 24, 72 hours and 7 days for immunohistochemistry as well as protein assessment via Western blot analysis. Total cell counts were performed on H&E stained sections. Proliferation was assessed with BrdU incorporation. Phenotypic differentiation was assessed by desmin and αSMC-actin expression. Results: Compared to uninjured arteries, overall cellularity was increased by 60% 7 days after injury (p<0.001). Increased cellularity was due to an 80% increase in cell number in the intima and adventitia, respectively (p<0.05). However, injury decreased cell number in the media at all time points by up to 85% (p<0.001). Treatment with NO prevented the increased cellularity produced with injury primarily by preventing neointima formation and repopulation of the media. The effects of NO were seen as early as 2 hours. At 7 days NO-treated arteries had cellularity similar to the uninjured artery and 69% fewer cells compared to injury alone (p≤0.009), with an 87% reduction in the intima and media respectively (p<0.05). NO treated arteries also had fewer cells in the adventitia compared to injury but to a lesser extent with a 54% reduction in cell number at 7 days. NO also inhibited proliferation up to 78%, 95% and 80% in the intima, media and adventitia, respectively (p<0.04). Compared to injury alone, NO-treated arteries inhibited the ubiquitous VSMC marker αSMC-actin at all time points with a 10-fold reduction seen at 7 days compared to injury. In contrast, there was a dramatic decrease in the VSMC specific marker desmin at 2 and 24 hours with NO compared to injury and a subsequent 2-fold increase in expression at the later time points. Conclusions: The short-acting NO-donor PROLI/NO inhibits neointimal hyperplasia via modulation of proliferation and phenotypic differentiation in the arterial wall as early as 2 hours following injury. These data suggest that the early effects of NO on the arterial injury response are ultimately responsible for the durability of this short-acting donor.