A small molecule CXCR4 antagonist inhibits neointima formation and smooth muscle progenitor cell mobilization after arterial injury

Restenosis is a major problem after coronary revascularization necessitating repeated interventions [1]. Coating of stents with antiproliferative drugs has been shown to dramatically reduce the rate of restenosis [2]. As incomplete endothelial coverage of the drug-eluting stents has been reported, which may account for the increased rate of late stent thrombosis [3,4], alternative compounds specifically preventing the neointimal accumulation of smooth muscle cells (SMCs) would be preferable. In animal models circulating smooth muscle progenitor cells (SPCs) have been shown to be an important source for neointimal SMCs after vascular injury [5,6]. Mobilization and recruitment of SPCs to the injury site is regulated by the CXC chemokine stromal cell-derived factor (SDF)-1a and its receptor CXCR4 [5,6]. In patients with coronary atherosclerosis, the development of in-stent restenosis afterwards was closely correlated with a postinterventional increase of circulating CD34 cells [7]. Therefore, disruption of the SDF-1a/CXCR4 axis appears to be an attractive therapeutic modality to reduce neointimal SMC accumulation in restenosis. Small molecule CXCR4 antagonists, such as AMD3465 or AMD3100, have been initially developed as anti-HIV drugs to prevent cell entry of X4 HIV strains [8]. We studied the effect of AMD3465 on neointima formation after carotid injury in apoE mice to establish the potential of small molecule CXCR4 antagonists in the treatment of restenosis. Female, 8-week-old apoE mice (Charles River, Sulzfeld, Germany) on a high cholesterol diet were treated with 120 lg AMD3465 (AnorMed. Inc, Langley, Canada) per day for 4 weeks (6 mg kg day), a dose that has been shown to be effective in vivo [9,10], via mini-osmotic pumps (ALZET , pump rate 0.25 lL h, Charles River). The solvent phosphatebuffered saline (PBS) was used as control. One day after pump implantation, wire-injury of the common carotid artery was performed as previously described [5]. After 28 days, se rial sections (10 per mouse) of carotid arteries were stained with Movat s pentachrome and neointimal and medial areas quantified by morphometry [5]. Treatment with AMD3465 resulted in a 59% reduction of neointimal area compared with PBStreated mice (0.02 ± 0.006 vs. 0.05 ± 0.006 mm, n = 4, *P < 0.05, Fig. 1A and Fig S1A, B in Supplementary Material). Medial area tended to decrease after AMD3465 treatment, but this effect did not reach statistical significance. (0.04 ± 0.005 vs. 0.064 ± 0.01 mm, n = 4, P = NS). Accordingly, the intima/media ratio was diminished in mice treated with AMD3465 (0.85 ± 0.13 vs. 0.42 ± 0.1, n = 4, P £ 0.05, Fig. 1B). To further characterize the effect of AMD3465 on the cellular composition of the neointima, quantitative immunostaining for SMC-specific a-smooth muscle actin (a-SMA; clone 1A4, Dako, Hamburg, Germany), macrophage-specific Mac-2 (cloneM3/38, Cedarlane) and endothelial VE-Cadherin (polyclonal antibody, Santa Cruz Biotech. Inc., Heidelberg, Germany) was performed. Planimetric analysis of positive immunostaining in the neointima revealed a significant decrease of the a-SMA-positive neointimal area by 57% in AMD3465-treated mice (0.16 ± 0.03 vs. 0.37 ± 0.06 aSMA/neointima area, n = 4, *P < 0.05 vs. PBS, Fig. 1C and Fig. S1C, D), which indicates a major reduction of the neointimal SMC content. In contrast, neointimal macrophage content (0.32 ± 0.07 vs. 0.28 ± 0.01 Mac-2/neointimal area, n = 4, P = NS) was unaffected by AMD3465 (Fig. S1E, F). Endothelial recovery as determined by luminal VE-cadherin staining increased significantly between 7 days Correspondence: Andreas Schober, Cardiology Unit, Medizinische Poliklinik, University of Munich, Pettenkoferstraße 8a, 80336 Munich, Germany. Tel. : +49 89 51602111; fax: +49 89 51604483. E-mail: andreas.schober@med.uni-muenchen.de