Statins, inhibitors of 3-Hydroxy-3-methylglutaryl coenzyme A reductase, are known to effectively reduce plasma cholesterol and reduce the mortality and morbidity of atherosclerotic disease. Recent investigation suggests that statins influence vascular biology through mechanisms other than decreased plasma cholesterol. Statins can inhibit the activation of the beta-2 integrin CD18 by inhibiting the signaling molecule RhoA and can block LFA-1 (an integrin that pairs CD11a with CD18 on lymphocytes) from binding its ligand ICAM-1. We have previously described leukocyte (largely granulocytes) rolling on arterial thrombi and leukocyte firm adhesion to the thrombus coincident with CD18 activation. We therefore hypothesized that statins can block leukocyte firm adhesion to a thrombus by inhibiting the activation of CD18 in vivo. A placebo controlled experiment measured the leukocyte thrombus interaction in C57/Bl6 mice treated with atorvastatin (Lipitor) for 14 days prior to in vivo experimentation. The six mice in the atrovastatin group received a diet prepared by Dyets.com containing 0.2 mg of atorvastatin in each gram of food. The six mice in the control group received a normal mouse diet without the drug. All mice consumed approximately 5 g of their respective food each day. We monitored the weight of the mice and of the food to ensure that the mice consumed their diet and that they maintained a consistent weight. The mice were anesthetized via an intraperitoneal injection and the jugular vein was cannulated to maintain anesthesia throughout the procedure. Exteriorization of the cremaster muscle that surrounds the testicle allowed visualization of arterioles. A thrombus was induced with a MicroPoint LASER attached to a microscope. There appeared to be no difference in thrombus formation between the two groups. We observed rolling and firmly adhering leukocytes on the thrombus for 45 minutes after injury. In each mouse, on average three thrombi were induced in arterioles that ranged from 26 to 76 μm in diameter (18 thrombi in control group, 16 thrombi in atorvastatin group). At the end of the experiment blood was obtained from each mouse by cardiac puncture for white blood cell count by hemacytometer. There was no significant difference (P=0.37) in the white blood count for the mice in the different groups of the experiment (see Table). There was no significant difference (P=0.22) between the groups in the number of rolling leukocytes observed suggesting similar thrombus size. Leukocyte firm adhesion to the thrombus was defined as a cell that stuck to the thrombus for more than two minutes. Significantly fewer leukocytes (P=0.019) firmly adhered to a thrombus in the atorvastatin group than in the control group. Thus, atorvastatin reduced the number of firmly adherent leukocytes to a thrombus. Because granulocytes lack CD11a, we propose that atorvastatin inhibited the activation of CD18 by inhibiting the signaling molecule RhoA. The observation that statins can influence the dynamic leukocyte thrombus relationship broadens our understanding of the mechanisms by which statins prevent and treat atherosclerosis.
Control Atorvastatin Mean±SEM WBC Count 3,948,303±296,590 4,687,121±371,291 Total Rolling Leukocytes 493±104 341±56 Total Firmly Adherent Leukocytes 4.8±3.3 2.3±2.5