Secretion of SPARC by endothelial cells transformed by polyoma middle T oncogene inhibits the growth of normal endothelial cells in vitro.

Endothelioma cells expressing the polyoma virus middle T oncogene induced hemangiomas in mice by the recruitment of nonproliferating endothelial cells from host blood vessels (Williams et al. 1989). I now report that SPARC, a Ca(2+)-binding glycoprotein that perturbs cell-matrix interactions and inhibits the endothelial cell cycle, is produced by endothelioma cells and is in part responsible for the alterations in the morphology and growth that occur when nontransformed bovine aortic endothelial cells are cocultured with endothelioma cells. Normal endothelial cells cocultured with two different middle T-positive endothelial cell lines, termed End cells, exhibited changes in shape that were accompanied by the formation of cell clusters. Media conditioned by End cells repressed proliferation of normal endothelial cells, but enhanced that of an established line of murine capillary endothelium. Radiolabeling studies revealed no apparent differences in the profile of proteins secreted by aortic or capillary cells cultured in End cell conditioned media. Characterization of proteins produced by End cells led to the identification of type IV collagen, laminin, entactin, and SPARC as major secreted products. Although SPARC did not affect the morphology of End or capillary cells, it was associated with overt changes in the shape of aortic endothelial cells. Moreover, SPARC and a synthetic peptide from SPARC domain II inhibited the incorporation of [3H]thymidine by aortic cells, but had minimal to no effect on the capillary endothelial cell line. The inhibition of growth exhibited by aortic endothelial cells cultured in End cell conditioned media could be partially reversed by antibodies specific for SPARC and SPARC peptides. These studies indicate a potential role for SPARC in the generation of hemangiomas by End cells in vivo, a process that requires normal (host) endothelial cells to disengage from the extracellular matrix, withdraw from the cell cycle, migrate, and reassociate into the disorganized cellular networks that comprise cavernous and capillary hemangiomas.     

Isolation and characterization of an established endothelial cell line from transgenic mouse hemangiomas

A murine endothelial cell line was isolated from hemangiomas induced by expression of the polyoma early region gene in transgenic mice. After two cell sortings using acetylated low-density lipoprotein with a fluorescent label (Dil-Ac-LDL), a pure population of endothelial cells has been carried for more than 60 passages from the animal. The cells retain endothelial cell properties such as a characteristic cobblestone appearance at confluency, contact-inhibited growth, and active uptake of Ac-LDL. Expression analysis shows that the cells express both the polyoma transgene and the von Willebrand factor, an endothelial cell marker. Subcutaneous injection of the cultured endothelial cells into nontransgenic histocompatible mice or nude mice led to hemangioma formation, and endothelial cells were re-isolated by cell sorting from these secondary hemangiomas. This cell line represents a renewable source of murine endothelial cells derived from transgenic mice that can be studied both in vitro and by reintroduction into a host.     

Polyoma virus middle-T-transformed PECAM-1 deficient mouse brain endothelial cells proliferate rapidly in culture and form hemangiomas in mice

Wild-type mouse brain endothelial (bEND) cells transformed with the polyoma virus middle-T proliferate rapidly in culture and form hemangiomas in mice. These cells express high levels of platelet/endothelial cell adhesion molecule-1 (PECAM-1), a molecule shown to be important during hemangioma formation. In this study, we have examined the ability of polyoma virus middle-T-transformed mouse bEND cells prepared from PECAM-1-/- mice to proliferate in culture and form hemangiomas in mice. We show that these cells express a number of endothelial cell markers and share a similar morphology with PECAM-1+/+ bEND cells. PECAM-1-/- bEND cells exhibit a limited ability to form tubes in Matrigel and rapidly form hemangioma when injected into nude mice, very similar to PECAM-1+/+ bEND cells. These cells, however, have increased proliferation, slower migration, altered endothelial cell adhesion molecule expression, and are less adherent when compared to PECAM-1+/+ bEND cells. Therefore, lack of PECAM-1 expression impacts polyoma middle-T-transformed endothelial cell proliferative, adhesive, and migratory properties without impacting their ability to rapidly form hemangiomas in mice or poorly organize to capillary-like structures in Matrigel.     

COSMC Is Overexpressed in Proliferating Infantile Hemangioma and Enhances Endothelial Cell Growth via VEGFR2

Infantile hemangiomas are localized lesions comprised primarily of aberrant endothelial cells. COSMC plays a crucial role in blood vessel formation and is characterized as a molecular chaperone of T-synthase which catalyzes the synthesis of T antigen (Galβ1,3GalNAc). T antigen expression is associated with tumor malignancy in many cancers. However, roles of COSMC in infantile hemangioma are still unclear. In this study, immunohistochemistry showed that COSMC was upregulated in proliferating hemangiomas compared with involuted hemangiomas. Higher levels of T antigen expression were also observed in the proliferating hemangioma. Overexpression of COSMC significantly enhanced cell growth and phosphorylation of AKT and ERK in human umbilical vein endothelial cells (HUVECs). Conversely, knockdown of COSMC with siRNA inhibited endothelial cell growth. Mechanistic investigation showed that O-glycans were present on VEGFR2 and these structures were modulated by COSMC. Furthermore, VEGFR2 degradation was delayed by COSMC overexpression and facilitated by COSMC knockdown. We also showed that COSMC was able to regulate VEGF-triggered phosphorylation of VEGFR2. Our results suggest that COSMC is a novel regulator for VEGFR2 signaling in endothelial cells and dysregulation of COSMC expression may contribute to the pathogenesis of hemangioma.     

Increased proteolytic activity is responsible for the aberrant morphogenetic behavior of endothelial cells expressing the middle T oncogene

Expression of the polyoma virus middle T (mT) oncogene in vivo is associated with a profound subversion of normal vascular development, which results in the formation of endothelial tumors (hemangiomas). In an attempt to understand the molecular mechanisms responsible for this phenomenon, we have investigated, in an in vitro system, the morphogenetic properties of endothelial cells expressing this oncogene. mT-expressing endothelioma (End) cells grown within fibrin gels formed large hemangioma-like cystic structures. All End cell lines examined expressed high levels of fibrinolytic activity resulting from increased production of urokinase-type plasminogen activator and decreased production of plasminogen activator inhibitors. Neutralization of excess proteolytic activity by exogenously added serine protease inhibitors corrected the aberrant in vitro behavior of End cells and allowed the formation of capillary-like tubules. These results suggest that tightly controlled proteolytic activity is essential for vascular morphogenesis and that physiological protease inhibitors play an important regulatory role in angiogenesis.     

Fine needle aspiration cytology of cellular hemangioma of infancy. A case report

BACKGROUND: Cellular hemangioma is a common benign vascular neoplasm of infants and children. The lesion typically occurs within the superficial dermis, where it is recognized as a strawberry nevus. Occasionally, this neoplasm is situated within deep soft tissues of the head or neck, with a particular predilection for the parotid gland region. Fine needle aspiration cytology (FNAC) of cellular hemangioma involving the parotid gland has been reported previously, but never confirmed by cytologic findings alone. We report the first case of infantile cellular hemangioma with sufficient characteristic cytologic features to be diagnosed by FNAC. CASE: A 3-month-old male presented with a rapidly enlarging, sensitive, solid, supraparotid mass. Ultrasound and computed tomography were performed but were nondiagnostic. Subsequent FNAC of the mass demonstrated a highly cellular specimen composed predominantly of elongated spindled cells arranged in three-dimensional coils and arcades. Immunohistochemistry demonstrated the endothelial origin of the spindled cells and confirmed the diagnosis of cellular hemangioma. CONCLUSION: Deeply situated cellular hemangiomas may pose a difficult diagnostic challenge to the clinician as well as to the radiologist. The infantile variant of this tumor enlarges rapidly, simulating an aggressive malignant tumor, and is occasionally accompanied by substantial compressive symptoms. Radiographic presentation of the lesion may be that of a solid tumor mass, unlike most other hemangiomas. Precise cytologic diagnosis of infantile cellular hemangioma can be rendered on aspirated material and is crucial in planning conservative medical treatment.     

Rapamycin Inhibits Proliferation of Hemangioma Endothelial Cells by Reducing HIF-1-Dependent Expression of VEGF

Hemangiomas are tumors formed by hyper-proliferation of vascular endothelial cells. This is caused by elevated vascular endothelial growth factor (VEGF) signaling through VEGF receptor 2 (VEGFR2). Here we show that elevated VEGF levels produced by hemangioma endothelial cells are reduced by the mTOR inhibitor rapamycin. mTOR activates p70S6K, which controls translation of mRNA to generate proteins such as hypoxia inducible factor-1 (HIF-1). VEGF is a known HIF-1 target gene, and our data show that VEGF levels in hemangioma endothelial cells are reduced by HIF-1α siRNA. Over-expression of HIF-1α increases VEGF levels and endothelial cell proliferation. Furthermore, both rapamycin and HIF-1α siRNA reduce proliferation of hemangioma endothelial cells. These data suggest that mTOR and HIF-1 contribute to hemangioma endothelial cell proliferation by stimulating an autocrine loop of VEGF signaling. Furthermore, mTOR and HIF-1 may be therapeutic targets for the treatment of hemangiomas.     

MiR-200c-3p increased HDMEC proliferation through the notch signaling pathway

Excessive proliferation of vascular endothelial cells can cause hemangioma. Although typically benign, hemangiomas can become life-threatening. The microRNA miR-200c-3p is abnormally expressed in some types of tumors, but its expression, biological role, and mechanism of action in infantile hemangioma remain to be fully elucidated. The expression levels of miR-200c-3p in hemangioma tissue were compared with those in adjacent healthy tissue by using bioinformatics analyses and TargetScan. Western blot, enzyme-linked immunosorbent assay, and Cell Counting Kit 8 analyses were used to determine the biological function and site of action of miR-200c-3p in human dermal microvascular endothelial cells (HDMECs). MiR-200c-3p was one of the top 10 differentially expressed genes between healthy tissue, and hemangiomas tissues, having markedly decreased expression in hemangioma tissue. Reduction of miR-200c-3p expression in HDMECs through the transfection of a miR-200c-3p inhibitor significantly increased HDMEC proliferation. The addition of the Notch signaling pathway inhibitor DAPT to HDMECs transfected with the miR-200c-3p inhibitor eliminated the inhibitor-induced enhancement of proliferation in HDMECs. These findings indicate that miR-200c-3p targets the Notch signaling pathway to promote the proliferation of vascular endothelial cells, suggesting that miR-200c-3p plays an important role in the pathogenesis of hemangioma.     

Mice transgenic with SV40-late-promoter-driven Polyomavirus Middle T oncogene exclusively develop hemangiomas

In order to develop a model system of infantile hemangioma, transgenic mice were developed carrying the Polyomavirus Middle T (PyMT) gene driven by the SV40 late promoter. From the 520 fertilized eggs surviving microinjection, there were 25 live births. Three of these showed the hemangioma phenotype and carried and expressed the PyMT gene; the remaining descendants were normal. The tumors showed abnormal vascular proliferation with cavernous hemangioma-like structures in the skin surface, tongue, ear mucosa and gastric mucosal tissue in the transgenic mice with hemangioma phenotype. Immunohistochemical staining for Ki-67 was negative, showing the tumors were hemangiomas rather than angiosarcomas. None of the PyMT transgenic mice survived beyond 4 weeks. Previously reported PyMT transgenic mice under the control of various promoters induce many tumor types including hemangiomas. PyMT driven by the SV40 late promoter is an improved model system because it only induces hemangiomas. However, it is limited by the post-natal lethality. Thus, conditional variants of this model system would be desirable.     

Tiaml和Rac1在皮肤血管瘤组织中的表达及意义

目的 探讨Tiaml和Rac1在人皮肤血管瘤组织中的表达及其临床意义.方法 收集武汉大学人民医院病理科2008年-2011年皮肤毛细血管瘤存档蜡块40例,其中男性15例,女性25例.采用免疫组织化学S-P法检测40例皮肤血管瘤增生期、退化期及正常皮肤组织Tiaml和Rac1表达水平,采用HPIAS-1000图文报告管理系统对Tiaml和Rac1的表达进行定量分析,并用SPSS11.5软件对各组免疫组织化学反应阳性颗粒的平均光密度、阳性面积率做单因素方差分析和SNK（q）检验.结果 （1）增生期血管瘤血管内皮细胞中可见密集分布的棕黄色颗粒,Tiaml呈高表达,正常皮肤组及退化组血管内皮细胞中可见少量的棕黄色颗粒,Tiaml呈低表达.增生期组Tiaml的表达明显高于退化期组和正常皮肤组（P〈0.05）,而后两组比较差异无统计学意义（P〉0.05）.（2）增生期血管瘤血管内皮细胞中可见密集分布的棕黄色颗粒,Rac1呈高表达,正常皮肤组及退化组血管内皮细胞中可见少量的棕黄色颗粒,Rac1呈低表达.增生期组Rac1的表达明显高于退化期组和正常皮肤组（P〈0.05）,而后两组比较差异无统计学意义（P〉0.05）.结论 Tiaml和Rac1在血管瘤增生期均呈高表达,表明Tiaml和Rac1在血管瘤的发生和发展中起了重要作用.     

Embryonic lethalities and endothelial tumors in chimeric mice expressing polyoma virus middle T oncogene

The effect of the middle T oncogene of polyoma virus was studied in vivo using a replication-defective selectable retrovirus. Injection of virus into newborn and adult mice resulted in the rapid appearance of cavernous hemangiomas. Infection of embryos did not yield transgenic mice; therefore, embryonal stem (ES) cells were used as an alternative system. Several infected ES cell clones were established that constitutively expressed middle T and its associated tyrosine kinase activity. Chimeric embryos obtained by blastocyst injection of individual ES cell clones were specifically arrested at midgestation, when multiple hemangiomas disrupted blood vessel formation. From these tumors endothelial cell lines were established that retained expression of von Willebrand factor yet were tumorigenic in vivo. These results suggest that middle T acts in endothelial cells as a single-step oncogene and that ES cells provide a valuable system for the study of growth control during embryogenesis.     

Role of pigment epithelium-derived factor in the involution of hemangioma: Autocrine growth inhibition of hemangioma-derived endothelial cells

Hemangioma is a benign tumor derived from abnormal blood vessel growth. Unlike other vascular tumor counterparts, a hemangioma is known to proliferate during its early stage but it is followed by a stage of involution where regression of the tumor occurs. The critical onset leading to the involution of hemangioma is currently not well understood. This study focused on the molecular identities of the involution of hemangioma. We demonstrated that a soluble factor released from the involuting phase of hemangioma-derived endothelial cells (HemECs) and identified pigment epithelium-derived factor (PEDF) as an anti-angiogenic factor that was associated with the growth inhibition of the involuting HemECs. The growth inhibition of the involuting HemECs was reversed by suppression of PEDF in the involuting HemECs. Furthermore, we found that PEDF was more up-regulated in the involuting phase of hemangioma tissues than in the proliferating or the involuted. Taken together, we propose that PEDF accelerates the involution of hemangioma by growth inhibition of HemECs in an autocrine manner. The regulatory mechanism of PEDF expression could be a potential therapeutic target to treat hemangiomas.     

Down-Regulation of Platelet Endothelial Cell Adhesion Molecule-1 Results in Thrombospondin-1 Expression and Concerted Regulation of Endothelial Cell Phenotype

bEND.3 cells are polyoma middle T-transformed mouse brain endothelial cells that express very little or no thrombospondin-1, a natural inhibitor of angiogenesis, but express high levels of platelet endothelial cell adhesion molecule-1 (PECAM-1) that localizes to sites of cell–cell contact. Here, we have examined the role of PECAM-1 in regulation of bEND.3 cell proliferation, migration, morphogenesis, and hemangioma formation. We show that down-regulating PECAM-1 expression by antisense transfection of bEND.3 cells has a dramatic effect on their morphology, proliferation, and morphogenesis on Matrigel. There is an optimal level for PECAM-1 expression such that high levels of PECAM-1 inhibit, whereas moderate levels of PECAM-1 stimulate, endothelial cell morphogenesis. The down-regulation of PECAM-1 in bEND.3 cells resulted in reexpression of endogenous thrombospondin-1 and its antiangiogenic receptor CD36. The expression of the vascular endothelial growth factor receptors flk-1 and flt-1, as well as integrins and metalloproteinases (which are involved in angiogenesis), were also affected. These observations are consistent with the changes observed in proliferation, migration, and adhesion characteristics of the antisense-transfected bEND.3 cells as well as with their lack of ability to form hemangiomas in mice. Thus, a reciprocal relationship exists between thrombospondin-1 and PECAM-1 expression, such that these two molecules appear to be constituents of a “switch” that regulates in concert many components of the angiogenic and differentiated phenotypes of endothelial cells.     

Expression of L-selectin ligands by transformed endothelial cells enhances T cell-mediated rejection

Recent immunohistochemical studies have suggested that L-selectin ligands may be implicated in the infiltration of tumors and rejected transplants by lymphocytes. In the present study, polyoma-middle T Ag-transformed endothelial cells (H.end), which typically form in vivo immunogenic vascular tumors resembling Kaposi's sarcoma, were engineered to express L-selectin ligands by stable transfection with a cDNA encoding alpha(1,3/4)-fucosyltransferase (H.endft). The ability of these cells to form tumors in the s.c. tissues of normal and immunocompromised mice was then compared with that of H.end cells transfected with the hygromycin-resistance vector only (H. endhygro). H.endhygro cells rapidly formed local and metastatic tumors in normal syngeneic mice, leading to death within 2-3 mo postinjection. By contrast, tumors derived from H.endft cells displayed a slower rate of growth, an absence of metastasis, and marked lymphocyte infiltration. Animals bearing these tumors survived for a significantly longer duration than animals injected with H.endhygro cells. Alternatively, H.endft and H.endhygro cells formed tumors with comparable aggressiveness in immunocompromised mice, resulting in animal death within 3 wk of injection. H.endft but not H.endhygro cells supported L-selectin-dependent adhesion and cytolytic T cell activity in vitro. Taken together, our observations indicate that the in situ expression of fucosyltransferase may significantly influence the cellular immune response in endothelioma tumors. These results may be relevant in understanding the development of vascular opportunistic tumors such as Kaposi's sarcoma.     

Identifying potential regulators of infantile hemangioma progression through large-scale expression analysis: a possible role for the immune system and indoleamine 2,3 dioxygenase (IDO) during involution

Hemangiomas are benign endothelial tumors. Often referred to as hemangiomas of infancy (HOI), these tumors are the most common tumor of infancy. Most of these lesions proliferate rapidly in the first months of life, and subsequently slowly involute during early childhood without significant complications. However, they often develop on the head or neck, and may pose a significant cosmetic concern for families. In addition, a fraction of these tumors can grow explosively and ulcerate, bleed, or obstruct vision or airway structures. Current treatments for these tumors are associated with significant side effects, and our knowledge of the biology of hemangiomas is limited. The natural evolution of these lesions creates a unique opportunity to study the changes in gene expression that occur as the endothelium of these tumors proliferates and then subsequently regresses. Such information may also increase our understanding of the basic principals of angiogenesis in normal and abnormal tissue. We have performed large-scale genomic analysis of hemangioma gene expression using DNA microarrays. We recently identified insulin-like growth factor 2 as a potentially important regulator of hemangioma growth using this approach. However, little is known about the mechanisms involved in hemangioma involution. Here we explore the idea that hemangioma involution might be an immune-mediated process and present data to support this concept. We also demonstrate that proliferating hemangiomas express indoleamine 2,3 dioxygenase (IDO) and discuss a possible mechanism that accounts for the often slow regression of these lesions.