Adventitial Microvessel Formation After Coronary Stenting and the Effects of SU11218, a Tyrosine Kinase Inhibitor
Asim N. Cheema, MD, PhD, FACC*,
Tony Hong*,
Nafiseh Nili, PhD*,
Amit Segev, MD*,
John G. Moffat, PhD ,
Kenneth E. Lipson, PhD,
Anthony R. Howlett, PhD,
David W. Holdsworth, PhD ,
Michael J. Cole, MESc,
Beiping Qiang, MD*,
Frank Kolodgie, PhD ,
Renu Virmani, MD, FACC,
Duncan J. Stewart, MD, FACC* and
Bradley H. Strauss, MD, PhD, FACC*,*
* Roy and Ann Foss Cardiovascular Research Program, Terrence Donnelly Heart Center, St. Michael’s Hospital, University of Toronto, Toronto, Ontario, Canada
SUGEN, Inc., South San Francisco, California
Robarts Research Institute, London, Ontario, Canada
Department of Cardiovascular Pathology, Armed Forces Institute of Pathology, Washington, DC
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Figure 1 Study protocol: the number of vessels analyzed and time points studied.
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Figure 2 Cross-sectional (A) and maximum intensity projection (B) images of a three-dimensional microscopic computed tomography (3D micro CT) scan of stented artery for assessment of adventitial microvessels (Ad-MV). (A), radio-opaque contrast can be seen in the main artery as well in the Ad-MV (arrow). Contrast-filled microvessels were counted in the media/adventitia, defined as a 1.0-mm annular area surrounding the stent struts. 1 = lumen cross-sectional area (CSA); 2 = intima CSA; 3 = arterial media/adventitia. (B), 3D micro CT shows detail of Ad-MV around stent struts. White bars = 2 mm.
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Figure 3 Temporal profile of Ad-MV after stenting. (A) A marked increase in the number of microvessels was present at both 1 week and 4 weeks. (B) Correlation between Ad-MV and in-stent intimal hyperplasia (IH) at 4 weeks. (C) Number of Ad-MV to intimal cross-sectional area (CSA) ratio was significantly higher at 1 week compared with 4 weeks after stenting.
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Figure 4 Tissue hypoxia in the arterial wall. (A) Hypoxia-inducible factor (HIF)-1 alpha expression was increased at all time points after stenting (upper panel). (B) Pimonidazole adducts were identified at all time points after stenting (upper panel). Protein loading was assessed by Ponceau-red staining (lower panel of A and B). (C) Immunohistochemistry showing pimonidazole adducts (stained brown) at the media/adventitia border at 1 week after stenting. Isch-m = ischemic muscle + pimonidazole; UI = uninjured artery; UI-P = uninjured + pimonidazole.
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Figure 5 In vitro effects of SU11218. A tyrosine kinase inhibitor, SU11218 inhibited platelet-derived growth factor (PDGF)-stimulated coronary artery smooth muscle cell (CA-SMC) proliferation (A), vascular endothelial growth factor (VEGF)-stimulated coronary artery endothelial cell (CA-EC) proliferation (B), PDGF-stimulated CA-SMC migration (C), and VEGF-stimulated CA-EC migration (D) in a dose-dependent manner. BrdU = bromo-dexoyuridine.
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Figure 6 Effects of SU11218 on platelet-derived growth factor receptor-beta (PDGFR-ß) phosphorylation. The SU11218 inhibited phosphorylation of the PDGFR-ß in a dose dependent manner. The lower panel shows equal protein loading of PDFGR-ß. IP = immunoprecipitation; pY = phosphorylated PDGFR-beta; WB = Western blot.
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Figure 7 In vivo effects of SU11218. (A) The SU11218 significantly inhibited the Ad-MV response after stenting at 1 week and 4 weeks. (B) The SU11218 resulted in approximately 80% reduction in the amount of IH at 4 weeks. Abbreviations as in Figures 2 and 3.
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Figure 8 Representative cross-sectional (A to C) and maximum intensity projection (MIP) (D to F) images obtained from 3D micro CT scans of uninjured (A, D), stented and placebo-treated (B, E, respectively), and stented and SU11218-treated arteries (C, F, respectively). The MIP images represent a projection of image intensity along the axial direction, obtained over a 2-mm-thick sample region. Arrow = stent. I = intima; L = lumen. Adventitial microvessels (Ad-MV) (arrowhead) appear as white dots behind the stent struts in cross-sections and as bright annulus external to the stent in MIP images. There was a significant increase in Ad-MV at 4 weeks after stenting in placebo-treated animals (B, E) compared with uninjured arteries (A, D) or SU11218-treated animals (C, F). White bar = 2 mm.
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