Notch receptor and effector expression in von Hippel-Lindau disease–associated central nervous system hemangioblastomas

Laboratory investigation

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Central nervous system hemangioblastomas are the most common manifestation of von Hippel-Lindau (VHL) disease, an autosomal dominant tumor suppressor syndrome that results in loss of VHL protein function and continuous upregulation of hypoxia-inducible factors. These tumors are composed of neoplastic stromal cells and abundant vasculature. Stromal cells express markers consistent with multipotent embryonically arrested hemangioblasts, which are precursors for hematopoietic and vascular lineages. Notch receptors are transmembrane signaling molecules that regulate multiple developmental processes including hematopoiesis and vasculogenesis. To investigate the importance of notch signaling in the development of VHL disease–associated CNS hemangioblastomas, the authors examined the presence of the four notch receptors and downstream notch effectors in this setting.


The authors used surgical specimens obtained from confirmed VHL-associated hemangioblastomas. Immunohistochemical analysis for the four notch receptors and the downstream effectors was performed on formalin-fixed paraffin-embedded sections. Western blot analysis for HES1 was performed on frozen specimens.


All four notch receptors are present in hemangioblastomas. NOTCH1 and NOTCH4 receptors were widely and prominently expressed in both the stromal and vascular cells, NOTCH2 receptor expression was limited to primarily stromal cells, and NOTCH3 receptor expression was limited to vascular cells. All 4 receptors displayed a nuclear presence. Immunohistochemical analysis also demonstrated that downstream notch effectors, HES1 and HES5, were uniformly expressed in tumor stromal and vascular cells, but HES3, HEY1, and HEY2 were not. Strong HES1 expression was confirmed by Western blot analysis.


The presence of all four notch receptors and downstream effector molecules suggests that the notch signaling pathway plays a critical role in the maintenance of the undifferentiated pluripotent phenotype of these tumors and in the associated vascular response. Moreover, the prominent expression of notch receptors in VHL-associated CNS hemangioblastomas reveals a new and possibly potent therapeutic target.

Abbreviations used in this paper: ABC/DAB = avidin-biotin complex/3,3′-diaminobenzidine; HIF = hypoxia-inducible factor; ICD = intracellular domain; VEGF = vascular endothelial growth factor; VHL = von Hippel-Lindau.

Article Information

Address correspondence to: Marsha J. Merrill, Ph.D., 10 Center Drive, MSC 1414, Building 10, Room 3D-20, Bethesda, Maryland 20892-1414. email:

Please include this information when citing this paper: published online June 10, 2011; DOI: 10.3171/2011.5.JNS11271.

© AANS, except where prohibited by US copyright law.



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    Immunohistochemical analysis of notch receptors 1 through 4 in a representative CNS hemangioblastoma. A: NOTCH1 immunoreactivity is distributed uniformly, with cytoplasmic and nuclear staining observed in both the stromal and vascular cells. B: NOTCH2 immunoreactivity is confined to the nucleus. C: NOTCH3 immunoreactivity is strongly expressed in the vascular component of the tumor. D: NOTCH4 immunoreactivity is similar to NOTCH1, with cytoplasmic and nuclear staining observed in both the stromal and vascular cells. E: Nonimmune rabbit IgG control. F: Hematoxylin & eosin staining demonstrates the typical hemangioblastoma appearance with “foamy” stromal cells and abundant vasculature. All antibodies were raised against the C-terminus of the molecule and do not distinguish between the full-length and the cleaved forms. Staining for all four Notch receptors was performed at the same time in the same tumor. Photomicrographs were taken from the same area of the tumor with the same exposure settings, original magnification × 40. Bar = 100 μm.

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    Cellular localization of notch receptors. A: NOTCH1 antibody to the C-terminus recognizes both full-length and cleaved NOTCH1 and reacts with both cytoplasmic and nuclear Notch. B: Antibody against NOTCH ICD recognizes only the signaling portion of notch and localizes to the nucleus. The same area of the same tumor is presented in A and B. C: NOTCH2 displays a nuclear localization in stromal cells with only occasional reactivity in vascular cells. D: NOTCH3 staining from the same area presented in panel C reveals an exclusively vascular localization. E: Low-magnification image of NOTCH3 immunoreactivity displaying abundant vasculature. NOTCH3-positive vasculature of multiple morphologies, including large thick- and thin-walled vessels as well as microvessels, is observed. F: NOTCH4 reactivity is seen in the cytoplasm and nuclei of both stromal and vascular cells. Asterisks indicate stromal cells. v = blood vessel. Bar = 20 μm (A–D, and F); bar = 100 μm (E).

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    Immunohistochemical staining of Notch downstream effectors in a representative CNS hemangioblastoma. A: HES1 was observed in the nuclei of both stromal cells and blood vessels. B: Western blot analysis of 4 hemangioblastomas also demonstrating the presence of HES1. C: Scattered, faint HES3 immunoreactivity observed in the nuclei of some cells. D: HES5 displaying a pattern similar to HES1 although not as intense. E: HEY1 staining is not distinguishable from the negative IgG control (not shown). F: Scattered, faint HEY2 immunoreactivity observed in the nuclei of some cells. Staining for all 5 effector molecules was performed in the same tumor. Original magnification × 40. Bar = 100 μM.



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