A. E. Leake1,2, G. Hong1, B. S. Zuckerbraun3, E. Tzeng1,2 3University of Pittsburgh,Department Of Surgery,Pittsburgh, PA, USA 1VA Pittsburgh Healthcare System,Vascular Surgery,Pittsburgh, PA, USA 2University Of Pittsburgh,Department Of Surgery, Division Of Vascular Surgery,Pittsburgh, PA, USA
Introduction: Carbon Monoxide (CO) is a potent anti-inflammatory molecule and dramatically attenuates neointimal hyperplasia (NIH) after carotid injury with one hour of inhaled CO at 250 parts per million (PPM) just prior to injury. The mechanism of how this brief treatment of inhalation of CO leads to profound and prolonged anti-inflammatory effects is still poorly understood. We have shown marked and prolonged changes in macrophage phenotype following inhaled CO, and the responsiveness of these cells to inflammatory stimuli is significantly altered. Based on these findings, we hypothesize that monocyte/macrophages mediate the vasoprotective effects of CO.
Methods: Sprague-Dawley rats (6-7/group) received Clodronate liposomes (5mg/mL IP) at day 0 for transient macrophage depletion and empty liposomes for control. On day 3, rats were treated with inhaled CO (1-hr at 250ppm) or maintained in room air and then underwent left carotid artery balloon injury. At the time of injury, a manual differential cell count of whole blood was used to confirm macrophage/monocyte depletion. Two-weeks after injury, rats were sacrificed and carotid arteries were collected for histologic analysis for intimal hyperplasia (IH) by determination of intima/media ratio (I/M). Statistical analysis was performed with ANOVA.
Results:IP administration of clodronate liposome resulted in a complete depletion of circulating monocytes by day 3 with a differential count of zero. Control liposome treated rats receiving CO demonstrated a 50% reduction in IH compared to the air group (Figure; I/M = 0.38 ± .09 vs. 0.75 ± .08, respectively; P=.01). After macrophage depletion, there was no difference in the amount of IH in the air treated rats compared to rats treated with control liposomes (I/M = 0.78 ± .09 vs. 0.75± .08, respectively; P=NS). The protective effect of inhaled CO was completely reversed in the macrophage-depleted rats compared with the CO treated control rats (I/M = 0.83 ± .11 vs. 0.38 ± .09, respectively; P = .01).
Conclusion: CO significantly decreases IH formation in the rat carotid injury model. This protective effect of CO is lost after macrophage depletion. Macrophage depletion itself did not affect IH. These findings support that the vasoprotective properties of inhaled CO are mediated through monocytes/macrophages. This regulation of the innate immune system may explain the profound anti-inflammatory actions of inhaled CO and future studies will focus on deciphering the signaling pathways that link inhaled CO and the changes in macrophage phenotype.
