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von Hippel–Lindau (VHL) disease is an autosomal dominant, inherited cancer syndrome that is characterized by extensively vascularized tumors, such as hemangioblastomas of the retina and central nervous system, renal cell carcinomas (RCCs), and pheochromocytomas (1). The VHL tumor suppressor gene was cloned in 1993 and is located at chromosome 3p25–3p26 (2). According to the two-hit hypothesis for tumor suppressor genes, inactivation of both alleles of the VHL gene leads to tumor formation. One copy of the VHL gene is mutated by inheritance, and the second hit causes inactivation of the remaining wild-type VHL gene (3). Although the VHL protein is widely expressed in different organs and cell types, its expression could not be demonstrated in endothelial cells of normal tissue (4). Strong expression of the VHL protein, however, was observed in proliferating endothelial cells from placenta, lung metastases of pancreatic adenocarcinoma, the base of an esophageal ulcer, and sprouting microvessels of atherosclerotic lesions [(5); Los M: unpublished observations]. In human umbilical vein endothelial cells and in dermal microvascular endothelial cells, expression of VHL messenger RNA was detected by northern blot analysis (Los M: unpublished observations). VHL disease-related tumors are highly vascularized and are composed predominantly of endothelial and tumor cells. To explain the extensive vascularization, numerous studies [(1,6) and references therein] have shown increased production of growth factors in VHL disease tumors. In RCCs and hemangioblastomas from VHL disease patients, a dramatic increase in the expression of vascular endothelial growth factor (VEGF) has been observed (7). VEGF is a potent mitogen for endothelial cells, and its expression is regulated by the VHL gene. However, in a recent study (8), we could not demonstrate an association between VEGF levels in ocular fluid and the presence of retinal angiomas. We, therefore, hypothesized that endothelial cells may also be prone to additional mutations that result in uncontrolled proliferation. By use of tissue microdissection and techniques of molecular biology, loss of heterozygosity (LOH) has been found in the clear-cell renal lesions of VHL disease-related RCCs and in the stromal cell component of VHL disease-related cerebellar and retinal hemangioblastomas (9–11). The isolation of endothelial cells that are depleted of tumor and stromal cells is virtually impossible; thus, LOH in the vascular component of the tumor could not be studied with this method. In this study, we used a different method to examine if LOH for the VHL gene occurs in endothelial cells of VHL disease tumors. We collected tissue samples of RCCs from surgical pathology specimens from four VHL patients (patients 1–4) who underwent nephronsparing surgery. We also obtained normal kidney tissue adjacent to the tumor from patients 1 and 2. In addition, a mutational analysis on DNA samples from leukocytes of affected family members was performed. All experiments were performed according to the guidelines of the Medical Ethics Committee of the University Medical Center Utrecht. From the RCCs, we obtained singlecell suspensions of tumor-derived endothelial cells and the residual tumor cells as described earlier (12). Briefly, surgical tissue was minced and incubated for 1 hour at 37 °C with 1 mg/mL collagenase and 1 mg/mL dispase (Sigma Chemical Co., St. Louis, MO) in RPMI1640 medium (Life Technologies, Inc. [GIBCO BRL], Rockville, MD) containing 20% human pooled serum. The tissue was sieved to remove large cell clumps and cell debris, and the singlecell suspension was washed and resuspended in the same medium. Cells were either stained directly after isolation with the endothelial cell-specific marker Ulex-biotin (ICN Pharmaceuticals, Inc., Costa Mesa, CA) and streptavidin-Rphycoerythrin conjugate (DAKO, Glostrup, Denmark) or stained after overnight culture in RPMI-1640 medium in a gelatin-coated culture flask for endothelial cells with diiodoacetylated low-density lipoprotein (DiI-Ac-LDL; Biomedical Technologies, Stoughton, MA). Subsequently, DiI-Ac-LDLpositive or Ulex-positive cells were sorted in a cell sorter (Becton Dickinson Immunocytometry Systems, San Jose, CA). DNA was isolated from the cell suspensions after cell sorting by resuspending the cells in a DNA lysis buffer (i.e., 0.1 M Tris–HCl [pH 8.5], 5 mM EDTA, 0.2 M NaCl, 0.2% sodium dodecyl sulfate, and 100 g/mL proteinase K). The DNA was amplified by the polymerase chain reaction (PCR) by use of primers for the three exons of the VHL gene (13). The PCR-amplified fragment was subsequently sequenced directly by use of the forward primers for the three exons. The germline mutations could be detected in the DNA samples of the normal kidney tissue (Table 1; patients 1 and 2). The germline mutation in patient 2 is a deletion of exons 1 and 2; as a result, we detected only the normal sequence of one allele in normal kidney tissue. The mutations of patients 1 and 2 were identical to the sequence of the DNA isolated from leukocytes of affected family members. In all four patients, the only mutations that we detected in the three exons of the entire VHL gene analyzed in DNA obtained from tumor-derived endothelial cells were the germline mutations detected in DNA obtained from the adjacent normal kidney (patients 1

Mutational Analysis of Endothelial Cells Derived From von Hippel-Lindau-Related Renal Cancer