Hypoxia, Hypoxia-Inducible Transcription Factor, and Macrophages in Human Atherosclerotic Plaques Are Correlated With Intraplaque Angiogenesis
Judith C. Sluimer, MSc*,
Jean-Marie Gasc, PhD ,
Job L. van Wanroij, MD ,
Natasja Kisters, BSc*,
Mathijs Groeneweg, MSc*,
Maarten D. Sollewijn Gelpke, MSc||,
Jack P. Cleutjens, PhD*,
Luc H. van den Akker, MD¶,
Pierre Corvol, MD, PhD,
Bradly G. Wouters, PhD ,
Mat J. Daemen, MD, PhD*,* and
Ann-Pascale J. Bijnens, PhD*
* Department of Pathology, Cardiovascular Research Institute Maastricht (CARIM), University of Maastricht, Maastricht, the Netherlands
Department of Surgery, University of Maastricht, Maastricht, the Netherlands
Maastricht Radiation Oncology Lab (Maastro), Research Institute Growth and Development (GROW), University of Maastricht, Maastricht, the Netherlands
Collège-de-France, INSERM U36, Paris, France
|| Department of Molecular Design and Informatics, NV Organon, Oss, the Netherlands
¶ Department of Surgery, Maasland Hospital, Sittard, the Netherlands.
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Figure 1 Hypoxia Detection With Pimonidazole in Human Carotid Endarterectomy
(A) Hypoxia (pimonidazole immunoreactivity) is present in the center of an advanced human carotid atherosclerotic plaque, but not in the media. Inset shows hematoxylin and eosin staining. (B) A serial section of panel A shows that CD68-positive macrophages colocalize with hypoxia. (C) Macrophage regions of the lesion from panel A show extensive hypoxia, whereas the cap shows mild or no hypoxia. (D) Hypoxia is present in CD68-positive macrophages (inset) at 20 to 30 µm from the lumen. (E) Hypoxia is absent in CD68-positive macrophages (inset) of a plaque shoulder segment (pathological intimal thickening). (F) Hypoxia is present in an atherosclerotic plaque segment with intimal thickening, more specifically in a few CD68-positive macrophages (inset). (G) The immunoreactivity score of hypoxia (open bars) and CD68 (solid bars) is increased in stable and ruptured atherosclerotic lesions versus early lesions (*p < 0.05 vs. early; stable vs. ruptured is not significant). L = lumen.
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Figure 2 Hypoxia Colocalizes With CD68, HIF, and VEGF
(A) Hypoxia detected by pimonidazole immunoreactivity in human carotid atherosclerosis. Serial sections show colocalization with CD68-positive macrophages (B), HIF1 (C), and VEGF (D). (E) The immunoreactivity score of CD68, HIF1, and VEGF is increased when hypoxia is present (solid bars) versus absent (open bars) in human carotid atherosclerosis (*p < 0.05). HIF = hypoxia-inducible transcription factor; VEGF = vascular endothelial growth factor.
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Figure 3 Schematic Illustration of Hypoxia in Atherosclerosis
Illustration of the presence of hypoxia (blue) in a longitudinal and cross-sectional representation of atherogenesis. a.wall = arterial wall; nc = necrotic core; th = thrombus.
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Figure 4 Hypoxia Detection in Arterial Wall and Human THP-1 Macrophages After Hypoxic and/or H2O2 Exposure
(A) Hypoxia (pimonidazole immunoreactivity) is almost absent in the arterial wall collected right after carotid incision, as well as right after excision of the atherosclerotic plaque (B). Insets show origin of magnification. (C) Pimonidazole was detected in human THP-1 macrophages in 0.2% O2 with fluorescence-activated cell sorting analysis (green bars), but was undetectable in 21% O2
(open bars) or after H2O2 stimulus (black bars). No significant differences were found between single O2 and O2 + H2O2 exposure (blue bars). *p < 0.05 versus 21% O2. Pimo-FITC = fluorescein isothiocyanate-conjugated antipimonidazole.
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Figure 7 Relative Expression Levels of HIF Pathway mRNA
(A) The expression ratio determined between 9 early and 6 stable lesions by microarray analysis was significantly different for HIF1, VEGF, GLUT3, and HK2. *p < 0.01; **p < 0.001; ***p < 0.00001. (B) Quantitative reverse transcription polymerase chain reaction was used to compare the expression ratio between 5 early and 5 stable lesions collected at autopsy (*p < 0.05) and (C) between 4 stable and 5 thrombus-containing lesions collected at surgery. GLUT = glucose transporter; HK = hexokinase; mRNA = messenger ribonucleic acid; other abbreviations as in Figure 2.
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Figure 8 Semiquantification of HIF Pathway Immunoreactivity in Carotid Atherosclerosis
(A) Immunoreactivity of HIF pathway proteins was significantly increased from early (green bars, n = 5) to stable lesions (white bars, n = 5) for HIF1, HIF2, VEGF, GLUT1, and GLUT3 but similar in stable and thrombus-containing lesions (blue bars, n = 5). *p < 0.05 versus early; **p < 0.01 versus early. (B) Macrophages in advanced lesions demonstrated strong immunoreactivity of HIF-responsive genes: VEGF (B), GLUT3 (C), and HK1 (D). (E) Microvessel density was determined in 6 human carotid arteries with early (intimal thickening), 6 stable, or 5 thrombus-containing atherosclerotic lesions. *p < 0.01 versus early. L = lumen; other abbreviations as in Figures 2 and 7.
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