[CANCER RESEARCH 63, 7051–7055, November 1, 2003] Advances in Brief Developmental Arrest of Angioblastic Lineage Initiates Tumorigenesis in von Hippel-Lindau Disease Alexander O. Vortmeyer, 1 Stephan Frank, Seon-Yong Jeong, Kristy Yuan, Barbara Ikejiri, Youn-Soo Lee, Deb Bhowmick, Russell R. Lonser, Reginald Smith, Griffin Rodgers, Edward H. Oldfield, and Zhengping Zhuang Surgical Neurology Branch, National Institute of Neurological Disorders and Stroke [A. O. V., S. F., S-Y. J., K. Y., B. I., Y-S. L., D. B., R. L., E. H. O., Z. Z.] and Molecular and Clinical Hematology Branch, National Institutes of Diabetes, Digestive and Kidney Diseases [R. S., G. R.], NIH, Bethesda, Maryland 20892 Abstract The nature of the cell responsible for von Hippel-Lindau (VHL) dis- ease-associated tumor formation has been controversial for decades. We demonstrate that VHL disease-associated central nervous system tumors are composed of developmentally arrested angioblasts that coexpress erythropoietin (Epo) and Epo receptor. The angioblasts are capable of differentiating into RBCs via formation of blood islands with extramed- ullary hematopoiesis. Because of VHL deficiency, Epo receptor-express- ing, developmentally arrested angioblasts simultaneously coexpress Epo, which may represent a crucial pathogenetic step in tumor formation. Introduction VHL 2 disease is a tumor syndrome caused by germ-line mutation of the VHL gene (1). Tumors most frequently develop in the retina, cerebellum, spinal cord, and kidneys. Central nervous system tumors, hemangioblastomas, are characterized by complex morphological fea- tures and endocrine activity, and hemangioblastoma histogenesis is considered uncertain (2, 3). According to the hypothesis of Knudson, tumor formation is initiated by inactivation of the wild-type VHL allele (4). Subsequently, lack of VHL protein induces profound intra- cellular metabolic changes that closely resemble, if not mimic, changes observed in oxidative stress. In wild-type cells, the  subunit of HIF is controlled by the pVHL-elongin-cullin-2 complex (5, 6). In VHL protein-defective cells, however, HIF is up-regulated and further mediates the up-regulation of hypoxia-inducible genes, including Epo and VEGF (5, 6). It remains unclear, however, how these general and ubiquitous mechanisms of hypoxic response can be implicated in the tumorigenesis of specific subsets of cells. In 1928, Cushing and Bailey (7) divided vascular malformations of the nervous system into two major groups, the angiomatous malfor- mations and the hemangioblastomas. In 1931, Arvid Lindau (8) based the distinctness of central nervous system hemangioblastoma on a series of criteria including “unmistakable neoplasticity” with “com- position of blood vessel elements” and a “tendency toward cyst formation.” He speculated that hemangioblastoma tissue may be de- rived from a “congenital anlage” and that the histological picture revealed an “. . . embryological type of the tumor cells.” As further evidence for an embryological origin, Lindau cites a paper by Roussy and Oberling (9), who “. . . have found evidence of hemopoiesis, a circumstance that Cushing and Bailey, as well as myself [Arvid Lindau], have sought for in vain.” In 1960, Stein et al. (10) suggested an angiomesenchymal origin of hemangioblastoma, based on original observations by Florence Sabin (11). On morphological grounds, Stein et al. (10) described embryonic blood and vessel formation in hemangioblastoma tissue and speculated the presence of “an arrest or defect in maturation of one or more tissues at a particular time in embryonic development.” During the last decades, however, the his- togenesis of hemangioblastomas has been highly controversial, and contemporary texts characterize these tumors as “neoplasms of un- certain histogenesis” constituted of vascular and so-called “stromal” cells (3, 12). Recently, we have provided evidence in VHL disease- associated hemangioblastomas that these “stromal” cells are VHL- defective neoplastic cells (13). We here report that the “stromal” cells are the morphological correlate of neoplastic angiomesenchyme and capable of blood island formation. We thus confirm the original hypotheses of Lindau and Stein and provide new insight into the histogenesis and pathogenetic progression of hemangioblastoma and, possibly, other VHL disease-associated tumors. Materials and Methods Immunohistochemistry. Immunohistochemistry was performed on both paraffin-embedded and frozen sections. Routinely, multiple serial sections were taken from frozen and paraffin-embedded tissues to facilitate comparison of immunohistochemistry results with morphological phenotype. Frozen tissue sections were used after fixation in 80% ethanol. Sections from formalin-fixed paraffin-embedded tissue were first soaked in xylene and then washed in decreasing concentrations of ethanol. For antigen retrieval, sections were treated with DAKO Target Retrieval Solution (DAKO, Carpinteria, CA) and incubated at 95°C for 20 –30 min. Sections were cooled at room temperature and washed three times in PBS. Both paraffinized and frozen sections were then quenched for 20 min in a solution of 3 ml of H 2 O 2 and 180 ml of methanol. After three washes in PBS, sections were drenched in 10% horse serum for 1 h. The primary antibody was diluted in 2% horse serum, and the sections were incubated in a humidified chamber at 4°C overnight. Primary antibodies used were as follows: mouse ascites antihuman HIF-1a, 1:200; rabbit polyclonal antihuman Epo, 1:100; sheep polyclonal antihuman EpoR, 1:100; rabbit polyclonal antihuman neuron-specific enolase, 1:200; and mono- clonal CD 34, 1:400. The sections were then incubated with secondary anti- body and avidin-biotin complex for 1 h each. Diaminobenzidine was left on the sections for only the amount of time it took for the reactivity to become apparent; this time ranged from 20 s to 4 min. Tissue reaction with diamino- benzidine was stopped by dipping sections in tap water. A 10-min counter- staining with Mayer’s hematoxylin followed. The sections were then dehy- drated by graded ethanol washes and xylene wash before being mounted. The presence and intensity of antibody expression were examined in conjunction with the various morphological features of hemangioblastoma. Western Blot. For Western blotting, 20 l of cell lysate were separated by electrophoresis on 10 –20% gradient Tris-glycine gels (Novex, San Diego, CA). Proteins were electrotransferred onto Immobilon-P membranes (Milli- pore Corp., Milford, MA). Blots were blocked in PBS/0.05% Tween 20 containing 5% fetal bovine serum and incubated with anti-Epo antibodies Received 7/16/03; revised 9/2/03; accepted 9/4/03. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 To whom requests for reprints should be addressed, at Surgical Neurology Branch, National Institute of Neurological Disorders and Stroke, 10 Center Drive, NIH, Bethesda, MD 20892. 2 The abbreviations used are: VHL, von Hippel-Lindau; HIF, hypoxia-inducible factor; Epo, erythropoietin; VEGF, vascular endothelial growth factor; RT, reverse transcription; FAM, 6-carboxyfluorescein; TAMRA, 6-carboxytetramethylrhodamine; EMH, extramed- ullary hematopoiesis; EpoR, erythropoietin receptor. 7051 Research. on January 11, 2016. © 2003 American Association for Cancer cancerres.aacrjournals.org Downloaded from