Cell Hemangioma Research Articles

Identification of Nucleolin as a Binding Protein for Midkine (MK) and Heparin-Binding Growth Associated Molecule (HB-GAM)1

Midkine (MK) is a heparin-binding growth/differentiation factor with a molecular weight of 13 kDa, and is structurally unrelated to fibroblast growth factors (FGF). We studied MK-binding proteins in order to clarify the action mechanism of MK. A 100-kDa protein was identified in PYS-2, 3T3, and L cells as an MK-binding protein by a ligand blot experiment. This MK-binding protein was purified by affinity chromatography on an MK-agarose column followed by SDS polyacrylamide gel electrophoresis. Sequence determination of N-terminal 23 amino acid residues revealed that the MK-binding protein was nucleolin, a major nucleolar protein, which functions as a shuttle protein between the nucleus and cytoplasm and is located also on the cell surface. Heparin-binding growth associated molecule (HB-GAM), which has 50% sequence identity with MK, fused to maltose-binding protein also bound to nucleolin. On the other hand, basic FGF (bFGF) scarcely bound to nucleolin in the absence of heparin, while both MK and bFGF bound weakly to nucleolin in the presence of heparin. Nuclear localization of MK was shown in hemangioma cells by immunohistochemical staining. These findings supported the hypothesis that parts of the MK and HB-GAM are translocated to the nucleus after binding with nucleolin.

A case of pulmonary sclerosing hemangioma

A case of pulmonary sclerosing hemangioma was studied using light microscopic, immunohistochemical and electron microscopic methods. The cytoplasm of the sclerosing hemangioma cells was positive to the anti-lung surfactant apoprotein monoclonal antibody (PE-10). These cells included pale cells of the solid areas, cells covering papillary projections, and cells lining cleft-like spaces. Electron microscopic study showed that the predominant cells included poorly-differentiated pneumocytes and normal Type 2 pneumocytes. We concluded that the sclerosing hemangioma was an epithelial tumor with differentiation towards Type 2 pneumocytes.

Mast cells in angiolipomas and hemangiomas of human skin: are they important for angiogenesis?

To characterize the potential role of mast cells (MC) in angiogenesis, this study tests the hypothesis that MC may be more abundant in angiolipomas than in classic lipomas. MC counts were compared in 13 subcutaneous angiolipomas and 15 subcutaneous classic lipomas stained with Giemsa. Angiolipomas had ten times as many MC as did classic lipomas (25.34 +/- 2.83 versus 2.41 +/- 0.37 per mm2, mean +/- SE). To clarify whether this difference was primary (angiogenic activity) or secondary to the increased vascularity, MC were counted in 8 longstanding cutaneous capillary hemangiomas versus 13 cutaneous capillary hemangiomas of recent onset (pyogenic granulomas). If MC were mediating primary angiogenesis, one would expect them to be present in greater numbers in early than in late hemangiomas. To the contrary, however, long-standing hemangiomas were found to have significantly more MC than had those of recent onset (52.48 +/- 14.99 versus 6.59 +/- 3.37 per mm2, mean +/- SE). These results suggest that MC may not play an essential, early role in the proliferation of blood vessels in angiolipomas and hemangiomas, but rather may be related to maturation of blood vessels in these tumors.

Characterization of thrombomodulin expression in response to retinoic acid and identification of a retinoic acid response element in the human thrombomodulin gene

Thrombomodulin (TM) is an essential cofactor for the physiologic activation of the anticoagulant protein C by thrombin. We have observed that the expression of TM mRNA in response to retinoic acid was markedly increased in human U937 monoblast-like cells, and human MEG01 megakaryocyte-like cells, but not in human umbilical vein cells, murine hemangioma cells, human K562 erythroblast-like cells, and murine HSD fibroblast-like cells. TM activity in U937 cells and MEG01 cells was not detectable in untreated cells, but developed rapidly after treatment with retinoic acid. In endothelial cells there was minimal change in TM activity in response to retinoic acid treatment. We have isolated clones for the genes for murine and human TM and have identified potential retinoic acid response elements in the 5'-flanking region of the human gene. In U937 cells the increase in mRNA levels was associated with increased transcription, and transient transfection studies with reporter plasmids demonstrate functional retinoic acid response elements present in the 5'-flanking region of the gene. Deletion of, and mutations introduced into, the potential retinoic acid response element confirm the functional response in transient transfections.

Cutaneous vascular neoplasia in 15 cats: clinical, morphologic, and immunohistochemical studies.

Seven cases of cutaneous hemangioma and nine of cutaneous hemangiosarcoma were diagnosed from biopsy specimens of 15 Domestic Shorthairs of 5,091 cats that were examined by necropsy or biopsy during the 5-year period from 1 January 1986 through 31 December 1990. All but three cats were male. Tumor cells of both hemangiomas and hemangiosarcomas were immunoreactive for factor VIII-related antigen and for vimentin by the avidin biotin peroxidase complex method. In cats with a median age of 10 years, hemangiomas occurred in skin with pigmented hair in six of seven cases without apparent site predilection. These solitary tumors did not recur after excision although one cat (No. 3) subsequently developed cutaneous hemangiosarcoma at another site. Seven of nine hemangiosarcomas occurred in dermis and subcutis of the head, usually on the pinna. All five hemangiosarcomas of the head, for which cutaneous pigmentation could be determined, occurred in unpigmented skin. Cats with hemangiosarcoma had a median age of 12.5 years at the time of diagnosis. Metastasis has not been documented, but hemangiosarcoma has recurred, from 1 month to 2 years after excision, in 6/7 cats that were studied.

Acquired Benign and “Borderline” Vascular Lesions

In recent years, the classification of vascular lesions has been expanded and modified with the addition of several newly described entities, the redefinition of others, the recognition of lesions of borderline biologic behavior, and the need to avoid misdiagnosis with early Kaposi's sarcoma. This review clarifies the nomenclature, updates information on previously known lesions, and summarizes data on several recently discovered, lesser-known entities such as glomeruloid hemangioma, microvenular hemangioma, and multinucleate cell angiohistiocytoma. Clinicopathologic features and differential diagnosis are emphasized.

Increased expression of thrombomodulin on the cultured human umbilical vein endothelial cells and mouse hemangioma cells by cyclic AMP

We previously reported that the expression of thrombomodulin on the MEG-01, a cell line from human megakaryoblastic leukemia, was increased by agents that increase intracellular cAMP. In this paper we examine the effect of these agents on cultured human umbilical vein endothelial cells (HUVEC) and mouse hemangioma cells. Incubation of the cells with 3 mM dibutyryl cAMP (dbcAMP) increased functionally active thrombomodulin by about 2-fold on HUVEC and 4-fold on hemangioma cells. This effect was observed from 1 hour after the incubation and continued up to 24 hours. Dot hybridization of mRNA demonstrated a dose dependent increase in thrombomodulin mRNA in response to dbcAMP. Treatment of HUVEC with 20 μM forskolin or 100 uM isobutylmethylxanthine (IBMX) also increased cell-surface thrombomodulin on HUVEC. These agents prevented the interleukin I(IL-I) or tumor necrosis factor (TNF)-induced decrease in thrombomodulin on HUVEC. These data suggest that the expression of thrombomodulin on HUVEC and mouse hemangioma cells may be regulated by intracellular cAMP level.

Phenotypic Characterization of Normal and Neoplastic Canine Endothelial Cells by Lectin Histochemistry

Cell surface glycoconjugate expression of endothelial cells in canine cutaneous hemangiomas and hemangiosarcomas was compared to normal cutaneous endothelial cells using eight different lectins (with and without neuraminidase pretreatment) in an indirect immunoperoxidase technique. Direct comparison of lectin binding pattern of neoplastic endothelial cells with adjacent normal endothelial cells revealed minor changes in the binding intensity of several lectins (enhanced: Wheat germ agglutinin [WGA]; reduced: Griffonia simplicifolia-I [GS-I], Ricinus communis agglutinin-I [RCA-I], Soybean agglutinin after neuraminidase pretreatment [Neu-SBA], and Wheat germ agglutinin after neuraminidase treatment [Neu-WGA]). Neoplastic endothelial cells in some tumors exhibited varying binding of Ulex europaeus agglutinin-I (UEA-I; not binding to normal canine endothelial cells) and no Soybean agglutinin (SBA) binding (variably binding to normal endothelial cells in small cutaneous vessels). Lectin binding of neoplastic cells was rather heterogenous within one tumor compared to the uniform binding pattern of normal endothelial cells. These lectin binding studies demonstrate the phenotypic heterogeneity of neoplastic endothelial cells, indicating changes of cell surface glycosylation during neoplastic transformation.

Transcription of thrombomodulin mRNA in mouse hemangioma cells is increased by cycloheximide and thrombin.

We have measured mRNA levels for thrombomodulin, an endothelial membrane cofactor for the activation of protein C by thrombin, in a mouse hemangioma cell line. Cycloheximide, an inhibitor of protein synthesis, increased levels of thrombomodulin mRNA, as measured in an S1 nuclease protection assay, to 2.5-4.0 times control levels. Thrombomodulin transcription in response to cycloheximide treatment, as determined by nuclear run-on analysis, was 3.9 +/- 1.3 (mean +/- SD) times that found in untreated cells. Thrombin also increased thrombomodulin mRNA levels to 151 +/- 21% (mean +/- SD) of control levels after 2 hr. Transcription increased in response to thrombin by 2.1- to 7.3-fold. The combination of thrombin and cycloheximide had no additive effect on thrombomodulin mRNA levels. Thrombin treatment of hemangioma cells also caused an increase in thrombomodulin protein synthesis to 142 +/- 17% (mean +/- SD) of control levels as determined by immunoprecipitation of [32S]methionine-labeled thrombomodulin. We conclude that thrombomodulin expression is determined in part by the rate of transcription and that thrombomodulin mRNA levels in hemangioma cells are increased by treatment with cycloheximide or thrombin. The increased transcription in response to cycloheximide suggests the existence of a labile protein repressor of thrombomodulin transcription.

The structure and function of mouse thrombomodulin. Phorbol myristate acetate stimulates degradation and synthesis of thrombomodulin without affecting mRNA levels in hemangioma cells.

Thrombomodulin is an endothelial membrane anticoagulant protein that is a cofactor for protein C activation. We have evaluated the expression of thrombomodulin in cultured mouse hemangioma cells before and after treatment with phorbol myristate acetate (PMA), an agent that stimulates protein kinase C. We also isolated a cDNA encoding 481 amino acids of mouse thrombomodulin and the entire 3'-untranslated portion of its mRNA. The deduced amino acid sequence of mouse thrombomodulin is similar to those determined for human and bovine thrombomodulin. An S1 nuclease protection assay was used to measure thrombomodulin mRNA in hemangioma cells. The half-life for thrombomodulin mRNA was 8.9 +/- 1.8 h (S.D.) in cells treated with actinomycin D. Treatment with PMA had no effect on thrombomodulin mRNA levels. Thrombomodulin turnover was evaluated by immunoprecipitation of [35S]methionine-labeled thrombomodulin. The t1/2 was 19.8 +/- 3.9 h (S.D.); PMA treatment decreased the t1/2 to 10.9 +/- 1.1 h (S.D.) while increasing the rate of synthesis to a maximum of 190% of control. Protein C cofactor activity on hemangioma cells was reduced 35 +/- 4% by treatment with PMA within 30 min. This decrease was associated with a parallel decline in cell surface thrombomodulin antigen and with enhanced phosphorylation of thrombomodulin on serine residues. We conclude that thrombomodulin is phosphorylated in response to treatment of hemangioma cells with PMA which leads to decreased protein C cofactor activity and both increased degradation and synthesis of thrombomodulin.

Mast Cells in Surface Hemangiomas

SummaryThirty five patients of surface hemangioma within the age group one month to sixty-five years were studied for the presence of mast cells in them by using special stains. It was found that lesions with an increase in the number of mast cells showed a tendency for spontaneous involution.

Soft-tissue abnormalities of the external auditory canal: subject review of CT findings.

We review the normal anatomy and discuss characteristic findings of soft-tissue abnormalities of the external auditory canal (EAC). The indications for computed tomography (CT) of the temporal bone have been significantly expanded with the inclusion of soft-tissue abnormalities of the external ear and the auditory canal. Soft-tissue abnormalities of the EAC that can be evaluated with CT include atresia, edema, hemorrhage, fracture, posttraumatic or infection-caused keloid, malignant external otitis, hemangioma, lymphangioma, papilloma, keratosis obturans, acquired cholesteatoma, adenoma, ceruminoma, fibroma, mixed tumor, sarcoma, and basal cell, squamous cell, or adenocystic carcinoma. CT scans of 25 patients who had soft-tissue abnormalities of the EAC were reviewed. The clinical data were correlated with the radiographic findings. We conclude that CT is the best overall radiographic modality for evaluating the extent and character of soft-tissue abnormalities of the EAC. Significant clinical information that is helpful in patient management decisions is added by this technique.

Antibodies to Intermediate Filament Proteins

One hundred cutaneous tumors were investigated immunohistopathologically for the expression of intermediate filament (IF) proteins. Epithelial tumors, such as basocellular and squamous cell carcinomas, cutaneous adnexal tumors, and metastatic carcinomas showed keratin positivity in a varying number of tumor cells with two keratin antibodies with different specificities. Neoplastic cells of fibrohistiocytic tumors, pigmented nevi, melanomas, hemangiomas, glomus tumors, and lymphomas were positive for vimentin, but not for keratin or desmin. Cutaneous leiomyomas and leiomyosarcomas, on the other hand, were positive for desmin. The results show that the typing of IFs enables the differential diagnosis between carcinomas and sarcomas or melanomas, epidermal appendage tumors, and mesenchymal tumors, and between fibrohistiocytic and leiomyocytic tumors, and therefore are of diagnostic value in histopathologic problems of the skin.

Coagulation factor Va binds to human umbilical vein endothelial cells and accelerates protein C activation.

In vitro the rate of protein C activation by thrombin is significantly accelerated by two distinct cofactors (a) the endothelial cell surface protein, thrombomodulin, and (b) human coagulation Factor Va. We have recently reported that the activity of Factor Va is contained in the 78,000-D light chain. In this study we have investigated the effects of Factor Va and its light chain on the activation of protein C in the presence of cultured endothelial cells. Thrombin-catalyzed protein C activation on human umbilical vein endothelial cells was enhanced by Factor Va. The ability of Factor Va to stimulate protein C activation on these cells was saturated at 50 nM Factor Va and was observed at several protein C concentrations. Isolated Factor Va light chain in concentrations up to 50 nM also accelerated protein C activation on endothelial cells, but higher concentrations inhibited the reaction. The effects of Factor Va or its light chain on protein C activation were also shown on a mouse hemangioma cell line but not on human fibroblasts nor on a human amelanotic melanoma cell line. Protein C activation on endothelial cells was partially inhibited by a goat anti-thrombomodulin antibody and further addition of a polyclonal rabbit anti-Factor V(Va) antibody resulted in additional inhibition. Endothelial cells grown in medium supplemented with human serum, devoid of Factor V coagulant activity, contained cell surface Factor V(Va) (approximately 15,000 molecules/cell) as measured by the binding of a monoclonal IgG directed against Factor V(Va). These cells also bound an additional 6,000-10,000 molecules Factor Va per cell as determined by direct binding studies using 125I-Factor Va. We suggest that thrombomodulin and Factor Va act in concert to regulate protein C activation on the surface of endothelial cells.

Crystalloid inclusions in endothelial cells of cellular and capillary hemangiomas. A possible sign of cellular immaturity

Crystalloid inclusions in endothelial cells of cellular and capillary hemangiomas. A possible sign of cellular immaturity

Mast Cells in Hemangiomas and Vascular Malformations

Common pediatric vascular birthmarks, classified as hemangiomas or malformations, were analyzed for the presence of mast cells. Hemangiomas in the proliferative phase contained large numbers of mast cells (27 +/- 15 cells/high-power field [HPF]) in comparison with hemangiomas in the involuting phase (2.6 +/- 2.9), vascular malformations (1.7 +/- 3.2), and normal skin (5.0 +/- 1.0). Inasmuch as hemangiomas are characterized by endothelial proliferation and increased numbers of mast cells, these data raise the possibility that mast cells may have an important role in the formation and/or maintenance of these lesions.

Functional properties of an endothelial cell cofactor for thrombin-catalyzed activation of protein C.

Thrombin-catalyzed activation of Protein C is accelerated by a human endothelial cell surface cofactor. The cofactor occurs also on mouse hemangioma cells (a transformed endothelial cell line), but not on cultured human smooth muscle cells or fibroblasts. The cofactor remains bound to the cell surface during Protein C activation. The cofactor is saturable with respect to both Protein C (Km = 0.72 +/- 0.07 microM) and thrombin (Km = 0.48 +/- 0.05 nM). Diisopropylphosphoryl-thrombin is a competitive inhibitor of the cofactor-dependent reaction with Ki = 0.56 +/- 0.1 nM. Prothrombin Fragment 1, the peptide derived from prothrombin that retains phospholipid binding capacity, does not inhibit activation of Protein C when present in a 7:1 molar excess over Protein C. Platelet Factor 4 (20 microgram/ml) also fails to inhibit Protein C activation. It is concluded that the endothelial cell provides a surface on which Protein C can be activated under physiological conditions.

Immunohistochemical identification of factor VIII-related antigen in endothelial cells of cutaneous lesions of alleged vascular nature.

Immunohistochemical staining for factor VIII-related antigen (FVIII-RAG) with the peroxidase-antiperoxidase technic was used as a marker for endothelial cells in a variety of nevoid, reactive, and malignant vascular cutaneous proliferations. The endothelial cells of small normal cutaneous vessels gave the strongest reaction. The cells of hemangioma and angiokeratoma generally were stained, but with less consistency. In lymphangioma, pyogenic granuloma, and pigmented purpuric dermatosis, positivity was either patchy or of lesser intensity. Numerous strongly positive endothelial cells lining well-formed blood vessels were present in lesions of Kaposi's sarcoma, but the proliferating spindle cells forming "vascular slits" were uniformly negative. The cells of angiosarcoma were essentially negative, except for isolated elements in the better-differentiated areas. Plasma was also strongly positive; this may aid in distinguishing vascular from lymphatic channels. Anti-FVIII-RAG immunoperoxidase staining is a helpful aid in evaluating cutaneous vascular proliferations.