Mikio Masuzawa

Establishment of a human hemangiosarcoma cell line (ISO-HAS)

A cell line (ISO‐HAS) has been established from tumor tissue of a human hemangiosarcoma arising on the scalp by the use of conditioned medium from a murine‐phenotypic angiosarcoma cell line (ISOS‐1). Cells have been cultured for more than 2 years with up to 100 passages. The cells retained endothelial‐cell properties, such as a characteristic cobblestone appearance at confluency, contact‐inhibited growth, active uptake of acetylated low‐density lipoprotein labeled with 1,1‐dioctadecyl 1,3,3,3,3‐tetramethyl‐indocarbocyanine perchlorate (Dil‐Ac‐LDL) and CD31 expression. However, they were weakly positive for von‐Willebrand‐factor (vWf) antigen and for binding of Ulex europaeus agglutinin‐I (UEA‐1) lectin, and lacked tube‐formation activity. These findings indicate that ISO‐HAS is a poorly differentiated endothelial cell line. ISO‐HAS cells showed accumulation of p53 protein in the nuclei, and a new‐typed p53‐gene point mutation was found in exon 7 at codon 240. When inoculated s.c. into severe‐combined‐immunodeficiency (SCID) mice, the cells showed solid‐tumor growth that caused death. These properties suggest that ISO‐HAS is a malignant endothelial cell line with high tumorigenicity. Int. J. Cancer 81: 305–308, 1999.

Conversion of the CD4+ T cell profile from TH2-dominant type to TH1-dominant type after varicella-zoster virus infection in atopic dermatitis☆☆☆★

Skin lesions of atopic dermatitis were examined for cytokine expression by reverse transcription-polymerase chain reaction. The profile of mRNA for various cytokines revealed that both T(H1) and T(H2) types of CD4+ T cells, probably including T(H0) type, infiltrate into the skin lesion. We observed that atopic skin lesions improved after varicella infection. In such lesions, expression of T(H1) type cytokines predominated. The peripheral blood T cells from atopic patients exhibited a differentiation into T(H2) type cells upon in vitro stimulation with mite antigen. In contrast they differentiated into T(H1) type cells upon stimulation by varicella antigen. Since IL-12 has been reported to switch the in vitro recall response of allergen-specific T cells of atopic donors from a T(H2)- to a T(H1)-like phenotype, we examined its local production in varicella lesions. IL-12 p35 and p40 mRNA were expressed in fresh lesions. Peripheral blood mononuclear cells from atopic patients expressed p40 mRNA upon in vitro stimulation with live varicella zoster virus, but they did not show p40 mRNA without stimulation. This finding suggested that in atopic skin lesions containing the virus, IL-12 was produced and the cell type was changed to T(H1) type-predominance. These results suggested that patients with atopic dermatitis always have highly reactive CD4+ T cells infiltrating into their skin, and that the switch to T(H1) or T(H2) dominance is related to whether the lesion is improved or exacerbated.

Role for connexin 26 in metastasis of human malignant melanoma: communication between melanoma and endothelial cells via connexin 26.

Connexins form the intercellular channels of the gap junction and play an integral part in a variety of biological functions, such as maintaining tissue homeostasis, cell growth control, and development. Previously it was demonstrated that the expression of connexin 26 (Cx26) can increase the metastatic potential of mouse melanoma cells. The objective of the study was to investigate the role Cx26 plays in the metastasis of human melanoma cells, focusing on the communication between melanoma cells and endothelial cells.

Drug‐induced Chronic Pigmented Purpura

A close correlation between purpuric reaction and drugs was observed in seven cases of chronic pigmented purpura. The patients developed purpuric lesions after taking certain drugs for more than 3 years, were thiamine propyldisulfide in 2 cases, and chlordiazepoxide in 1 case. The purpuric lesions stopped recurring after removal of the drugs in the rest of the cases. It is suggested that drugs are among the etiological factors in chronic pigmented purpura.

Autocrine and paracrine roles of VEGF/VEGFR-2 and VEGF-C/VEGFR-3 signaling in angiosarcomas of the scalp and face

Angiosarcoma of the skin is an extremely rare malignant tumor of vascular origin that usually arises in the scalp and face of elderly persons. To clarify its characteristic features and cell cycle kinetics, we quantitatively evaluated the expression of cell cycle-related molecules and vascular endothelial growth factors using immunohistochemical staining, for comparison with 2 benign vascular tumors of the skin, the capillary hemangioma and the cavernous hemangioma. Cell proliferation, determined with reference to the Ki-67 labeling index, was highest in angiosarcomas and lowest in cavernous hemangiomas (angiosarcomas versus capillary hemangioma, P = .014; capillary hemangioma versus cavernous hemangiomas, P = 1.4 x 10(-4)). Similar differences were also found in cyclin A, cyclin E, and p21(Waf1) expression. Expressions of cyclin D1 and p16(INK4A) were also significantly higher in angiosarcoma than in cavernous hemangioma. Expressions of these 5 proteins showed significant positive correlations with Ki-67 labeling indices (Spearman rho = 0.91-0.43). Expression levels of vascular endothelial growth factor and its receptor, VEGFR-2, were highest in angiosarcomas. VEGF-C expression in angiosarcomas was significantly higher than in cavernous hemangiomas, and its receptor VEGFR-3 expression was highest in angiosarcomas. Furthermore, significant positive correlations of these protein expression with Ki-67 labeling indices were noted (Spearman rho = 0.88-0.40). Among them, VEGFR-3 showed the highest correlation coefficient. These results suggest that not only VEGFR-2-mediated signal but also VEGFR-3-mediated signal may contribute to proliferation of vascular tumor cells as autocrine and paracrine signaling factors.

Expression of vascular endothelial growth factor in a human hemangiosarcoma cell line (ISO-HAS).

Abstract Vascular endothelial growth factor (VEGF), in addition to being a specific mitogen of endothelial cells in vitro, is also known to induce angiogenesis in vivo. These functions suggest that VEGF may play an important role in the growth of hemangiosarcomas. Previous studies have demonstrated the expression of VEGF and its receptors, flt-1 or KDR/flk-1, in hemangiosarcomas by immunohistochemical staining and in situ hybridization. In the present study, however, we demonstrated that tumor cells of the hemangiosarcoma cell line ISO-HAS express mRNA of VEGF and its two receptors, flt-1 and KDR, and secrete VEGF protein. VEGF mRNA expression and protein secretion were found to be enhanced by phorbol 12-myristate 13-acetate. In addition, we demonstrated that ISO-HAS cells respond to recombinant human VEGF 165 with a dose-dependent up-regulation of cell proliferation and growth. These results suggest that the VEGF-VEGF receptor system plays a role in proliferation and growth of hemangiosarcoma cells.

Anticardiolipin antibody in Henoch-Schönlein purpura and related vascular disorders

To clarify the exact role of these phospholipid antibodies in vascular injury, ACL titers were examined in 24 patients with Henoch-Schonlein purpura (HSP) and other forms of cutaneous vasculitis. ACL titers for HSP were lower than for cutaneous polyarteritis nodosa (PNC) or Takayasus arteritis, but were significantly higher than for normal controls, as is shown in Fig. 1. The patients with erythema nodosum or hypergammaglobulinemic purpura showed normal ACL titers. ACL titers for allergic vasculitis could not be determined in this study because of the difficulty to obtain the serum. Next, clinical analysis was performed on five HSP cases who showed elevated ACL titers (three elderly cases and two younger ones)

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

Establishment of a new murine-phenotypic angiosarcoma cell line (ISOS-1)

A cell line, designated ISOS-1, was established from a tumor formed by transplantation of a human angiosarcoma into mice with severe combined immunodeficiency (SCID). The cells showed endothelial properties, based on the uptake of Dil-Ac-LDL and binding of UEA-I/GSA-I lectins, but were negative for CD11b and Pan Cytokeratin. However, the cells lost differentiated characteristics such as expression of von Willebrand factor, contact inhibition growth and tube formation activity. These findings indicate that ISOS-1 is a poorly-differentiated endothelial cell line. At the 81st passage, all of the cells were positive for H-2Dd in various intensity, but not HLA-ABC. The metaphase chromosomes consistently showed a characteristic mouse, but not human, telocentric form. Furthermore, this cell line produced fatal tumor growth in SCID mice and also in BALB/c mice. These results suggest that ISOS-1 is a murine-phenotypic angiosarcoma cell line.

Cutaneous extramedullary hematopoiesis in a patient with idiopathic myelofibrosis

Idiopathic myelofibrosis (IM) is a chronic myeloproliferative disorder and some cases of IM have extramedullary hematopoiesis. Extramedullary hematopoiesis is commonly seen in the liver, spleen and lymph nodes, but cutaneous extramedullary hematopoiesis (CEH) is very rare in cases of IM. We report a case of CEH in a 65‐year‐old Japanese woman with IM. This patient had many hard brownish nodules on her chest, abdomen and scalp. Histopathological examination of the nodule on her chest showed the existence of various stages of immature erythrocytes, leukocytes and megakaryocytes indicating that these hard nodules showed extramedullary hematopoiesis in the dermis. The proliferation of these immature cells in the dermis plays an important role in the pathogenesis of CEH. CEH is a very rare manifestation of IM and progresses slowly.