Transformation of vascular endothelial cells by a point mutation in the Tie2 gene from human intramuscular haemangioma Hua Wang 1,2 , Yan Zhang 1 , Shigeaki Toratani 1 and Tetsuji Okamoto* ,1 1 Department of Molecular Oral Medicine and Maxillofacial Surgery, Division of Frontier Medical Science, Graduate School of Biomedical Sciences, Hiroshima University, Hiroshima 734-8553, Japan; 2 Department of Oral Medicine and Maxillofacial Surgery, Shanghai Changhai Hospital, The Second Military Medical University, Shanghai 200433, China Tie2, an endothelial-cell-specific receptor tyrosine kinase, collaborates with vascular endothelial growth factor (VEGF) in regulating angiogenesis and vascular matura- tion. Here, we report a mutation of glycine to aspartic acid at the second glycine of the GXGXXG motif of Tie2 ( G833D Tie2) in human intramuscular haemangiomas (IMHs) of the capillary type. Murine endothelial cells (ECs) overexpressing this G833D Tie2 receptor exhibited an increase in cell proliferation at low serum concentrations and angiosarcomas developed in nude mice, whereas cells overexpressing either wild-type Tie2 or Q837H Tie2 failed to elicit these responses. Furthermore, the G833D Tie2 receptor increased VEGF expression in ECs. These findings provide molecular mechanisms for pathogenesis of IMH. Oncogene (2004) 23, 8700–8704. doi:10.1038/sj.onc.1208006 Published online 20 September 2004 Keywords: Tie2; VEGF; haemangioma; angiosarcoma; endothelial cell Intramuscular haemangiomas (IMHs) are benign vas- cular tumours characterized by infiltrating growth in skeletal muscle. IMHs are divided into capillary, cavernous, or mixed type depending on their gross and microscopic appearances. Capillary type is composed of a disordered proliferation of small vessels, whereas cavernous type is large, thin-walled, dilated vessels by flattened endothelial cells (ECs). The current treatment, surgical ablation of the tumour, is only partially effec- tive. More than 18% of patients suffered local recurr- ence (Enzinger and Weiss, 1995). The precise pathogenic mechanisms of IMH remain to be elucidated. Tie2 is an endothelium-specific receptor tyrosine kinase consisting of an extracellular domain, a trans- membrane domain, and a split intracellular kinase domain (Dumont et al., 1992, 1994). Mutational analyses of the Tie2 gene have demonstrated that the receptor plays a critical role in the maintenance of EC– pericyte communication during vascular morphogenesis (reviewed in Folkman and D’Amore, 1996; Hanahan, 1997). In the present study, we identified two point mutations in the Tie2 kinase domain from 37 haeman- gioma samples: a G2646A change resulting in a glycine to aspartic acid substitution at residue 833 (G833D) in eight unrelated patients and another A2659T change resulting in a glutamine to histidine substitution at residue 837 (Q837H) in one patient (Figure 1a and b). The relative of these patients did not have a history of IMH. To confirm the sequencing result, the PCR products of the patients, their relatives, and controls were analysed using restriction endonuclease assay. The two mutations were not present in any of 50 control individuals or in other probands tested. All of the patients with the G833D mutation had a large disfigur- ing lesion affecting one side of the face, and associated early postoperative recurrence, whereas the patient with the Q837H mutation had no recurrence during post- operative follow-up after 5 years. Histological sections of the G833D tumours showed a number of small capillary-sized vessels that extended between individual muscle fibres. No evidence of enlarged vessels was observed (Figure 1c). Immunohistochemical analysis indicated that the expression of Tie2 protein was moderate in G833D-tumour ECs (Figure 1d), and the expression of vascular endothelial growth factor (VEGF) protein was upregulated (Figure 1e) when compared to human normal muscular tissue (Figure 1h). However, the Q837H tumour comprised large dilated thin-walled vessels lined with flattened endothelium (Figure 1f). Although the expression of Tie2 protein was moderate in Q837H-tumour ECs, the expression of VEGF protein was unable to be detected (data not shown). Pathological evaluation revealed that the G833D tumours were IMHs of the capillary type, whereas the Q837H lesion was an IMH of the cavernous type. To characterize the biological effects of the two mutations, we generated the corresponding mutant receptor cDNAs in a Tie2 mammalian expression vector by site-directed mutagenesis (Figure 2a) and analysed them by transient transfection. G833D Tie2 showed a 2.9- fold increase in ligand-independent autophosphoryla- tion relative to wild-type Tie2, whereas Q837H Tie2 showed Received 23 January 2004; revised 18 May 2004; accepted 23 June 2004; published online 20 September 2004 *Correspondence: Dr T Okamoto, Department of Molecular Oral Medicine and Maxillofacial Surgery, Division of Frontier Medical Science, Graduate School of Biomedical Sciences, Hiroshima Uni- versity, Hiroshima 734-8553, Japan; E-mail: tetsuok@hiroshima-u.ac.jp Oncogene (2004) 23, 8700–8704 & 2004 Nature Publishing Group All rights reserved 0950-9232/04 $30.00 www.nature.com/onc ONCOGENOMICS