Observations on angiopoietin 2 in patients with angiosarcoma

SIR, Vascular remodelling in host tissues surrounding growing tumours is implicated in the successful development of tumour neovasculature. Cooperation between vascular endothelial growth factor (VEGF) and angiopoietins (Angs) is considered to be critical in this context. VEGF is a prime regulator of endothelial cell proliferation, angiogenesis, vasculogenesis and vascular permeability. We have recently established a human angiosarcoma cell line, ISO-HAS. We have previously demonstrated that tumour cells of the angiosarcoma cell line ISO-HAS secrete VEGF-A protein. Tie2 is an endothelium-specific receptor tyrosine kinase known to play a role in tumour angiogenesis. Modulation of Tie2 receptor activity by its Ang ligands is crucial for angiogenesis, blood vessel maturation and integrity of the vascular endothelium. Ang1 and Ang2 are respectively proangiogenic and antiangiogenic owing to their respective agonist and antagonist signalling action through the Tie2 receptor. It has recently been reported that in the presence of endogenous VEGF-A, Ang2 promotes a rapid increase in capillary diameter, remodelling of the basal lamina and proliferation and migration of endothelial cells, and stimulates sprouting of new blood vessels in vivo. By contrast, Ang2 promotes endothelial cell death and vessel regression if the activity of endogenous VEGF is inhibited. These observations support a model for regulation of vascularity where VEGF can convert the consequence of Ang2 stimulation from antiangiogenic to proangiogenic. In the present study, we have demonstrated that tumour cells of the angiosarcoma cell line ISO-HAS express mRNA of Ang2 and its receptor, Tie2, and secrete the Ang2 protein, and that serum levels of Ang2 increase with advancing stages of the tumour in patients with angiosarcoma. A human angiosarcoma cell line (ISO-HAS) and a murine phenotypic angiosarcoma cell line (ISO-S1) maintained in our laboratory and normal human endothelial cells (HMvEC) (Morinaga, Yokohama, Japan) were used in this study. ISO-HAS cells were derived from the periauricular metastatic tissue of an 84-year-old Japanese man. ISO-S1 cells were cultured in a complete medium, and ISO-HAS cells were cultured in a complete medium mixed with 50% (v ⁄ v) of the conditioned medium of ISO-S1. Complete medium consisted of high-glucose Dulbecco’s modified Eagle’s medium (Gibco-BRL, Gaithersburg, MD, U.S.A.) supplemented with 15% (v ⁄ v) heat-inactivated fetal calf serum (JRH Biosciences, Lanexa, KS, U.S.A.). HMvEC were cultured in the medium provided by the manufacturer. Polymerase chain reaction (PCR) was performed with primers specific to Ang1, Ang2 and Tie2 (which have been reported by Zhang et al.), and to b-actin as a control. For PCR, 1 lL of cDNA was added to a 25-lL reaction mixture containing 10 mmol L Tris–HCl pH 9Æ0, 50 mmol L KCl, 1Æ5 mmol L MgCl2, 0Æ1% (w ⁄ v) gelatin, 0Æ2 mmol L deoxyribonucleoside triphosphates, 25 pmol L 5¢ and 3¢ oligonucleotide primers, and 2Æ5 U of Taq polymerase (Takara Shuzo Co., Kyoto, Japan). A DNA thermocycler 480 (PerkinElmer Cetus, Norwalk, CT, U.S.A.) was used for one cycle of 95 C for 9 min, followed by 35 cycles of denaturation at 95 C for 30 s, annealing at 58 C for 30 s, and a final cycle of 72 C for 7 min. The PCR product was subjected to electrophoresis in 1Æ5% agarose gel and was visualized by staining with ethidium bromide. We investigated 11 elderly patients (seven men and four women; mean age 75Æ3 years, range 67–84) with definite angiosarcoma of the face and scalp. The serum level of VEGF was measured using a human Ang2 enzyme-linked immunosorbent assay (ELISA) kit (R&D Systems, Minneapolis, MN, U.S.A.) according to the manufacturer’s protocol. Normal control sera were obtained from 18 healthy volunteers (10 men and eight women; mean age 70Æ3 years, range 62–81). We also analysed Ang2 protein levels by ELISA in the conditioned media of ISO-HAS cells and HMvEC. ISO-HAS cells and HMvEC were treated with trypsin, plated out at a density of 5Æ0 · 10 cells per well in 24-multiwell plates, and allowed to attach overnight. After 24 h, cell-free culture supernatants were removed and assayed for VEGF protein levels by ELISA. We have previously demonstrated that tumour cells of the angiosarcoma cell line ISO-HAS secrete VEGF-A protein. In addition, we have observed serum VEGF protein levels to increase with advancing tumour stage in the patient from whom the ISO-HAS cells were derived. In the present