Hypoxia accelerates intraplaque neovascularization derived from endothelial progenitor cells in carotid stenosis

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OBJECTIVE

The relationship between intraplaque hypoxia and intraplaque hemorrhage (IPH) has been reported, but the details remain obscure. In this study, the authors aimed to clarify the relationship among intraplaque hypoxia, endothelial progenitor cells (EPCs), and neovascularization, which causes IPH. The histological findings of specimens obtained from carotid endarterectomy were assessed.

METHODS

This study included 49 patients who underwent carotid endarterectomy. Magnetic resonance plaque imaging was performed to analyze the components of the carotid plaques, and surgical specimens were subjected to immunohistochemical analysis. The numbers of hypoxia-inducible factor-1 alpha (HIF-1α)–, CD34-, CD133-, and vascular endothelial growth factor receptor-2 (VEGFR-2)–positive cells in the carotid plaques were precisely quantified, as were the number and maximum diameter of CD31-positive microvessels.

RESULTS

Plaque components were judged as fibrous in 7 samples, lipid-rich in 22, and IPH in 20. The number of CD34-, VEGFR-2–, and CD133-positive cells as an EPC-specific marker was significantly correlated with the number of HIF-1α–positive cells (r = 0.9, r = 0.82, and r = 0.81, respectively). These numbers varied among the 3 plaque components (IPH > lipid-rich > fibrous). The number and maximum luminal diameter of CD31-positive microvessels were also significantly correlated with the number of HIF-1α–positive cells (r = 0.85 and r = 0.89, respectively) and varied among the 3 plaque components (IPH > lipid-rich > fibrous).

CONCLUSIONS

The present findings suggest that intraplaque hypoxia may accelerate abnormal microvessel formation derived from EPCs, which in turn promotes IPH. The results also suggest that microvessel enlargement is a pivotal characteristic of IPH and these enlarged microvessels are immature endothelial tubes with disorganized branching and are fragile and prone to rupture.

ABBREVIATIONS AB = diaminobenzidine tetrahydrochloride; EPC = endothelial progenitor cell; HIF = hypoxia-inducible factor; IPH = intraplaque hemorrhage; TOF = time of flight; VEGF = vascular endothelial growth factor; VEGFR = VEGF receptor.

Article Information

Correspondence Daina Kashiwazaki: Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, Toyama, Japan. dkashiwa@med.u-toyama.ac.jp.

INCLUDE WHEN CITING Published online October 5, 2018; DOI: 10.3171/2018.4.JNS172876.

Disclosures The authors report no conflict of interest concerning the materials or methods used in this study or the findings specified in this paper.

© AANS, except where prohibited by US copyright law.

Headings

Figures

  • View in gallery

    Immunohistochemical analysis revealed that the number of HIF-1α–positive cells was higher in plaques with IPH (A) compared with lipid-rich plaques (B) and fibrous plaques (C). The number of HIF-1α–positive cells varied among the 3 plaque components and differed significantly (D). **p < 0.01, bar = 50 µm. Figure is available in color online only.

  • View in gallery

    Scatterplots demonstrate the significant correlations between HIF-1α cells and CD34-, VEGFR-2–, and CD133-positive cells as well as CD31-positive microvessels (r = 0.9, p < 0.01; r = 0.82, p < 0.01; r = 0.81, p < 0.01; and r = 0.85, p < 0.01, respectively).

  • View in gallery

    The results of double-fluorescence immunohistochemistry demonstrated that CD34-positive cells (arrows) were also positive for HIF-1α, which suggests that the CD34-positive cells were hypoxic. Bar = 100 µm. DAPI = 4′,6-diamidino-2-phenylindole. Figure is available in color online only.

  • View in gallery

    Immunohistochemical analysis revealed that CD31-positive microvessels (arrows) in plaques with IPH (A) had diameters larger than those in lipid-rich plaques (B) and fibrous plaques (C). The diameter of CD31-positive vessels varied among the 3 plaque components and differed significantly (D). **p < 0.01. Figure is available in color online only.

  • View in gallery

    A: A scatterplot demonstrates the significant correlations between HIF-1α–positive cells and the maximum diameter of CD31-positive vessels (r = 0.89, p < 0.01). B: Immunohistochemical analysis showed that the endothelial cells of enlarged microvessels (arrows) were strongly positive against VEGFR-2 antibody. C: In contrast, endothelial cells of microvessels without enlargement (arrows) were negative against VEGFR-2 antibody. Figure is available in color online only.

  • View in gallery

    A schematic representation of the key factors involved in plaque instability. Hypoxia accelerates microvessel formation derived from EPCs, and clinical features of unstable plaque, such as enlarged vessels with increased permeability, are prone to rupture. Figure is available in color online only.

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