Increased angiogenic capabilities of endothelial cells from microvessels of malignant human gliomas

https://doi.org/10.1016/j.intimp.2005.08.004Get rights and content

Abstract

Vascular endothelial cells (ECs) that initiate tumor angiogenesis may acquire distinct properties after conditioning in tumor microenvironment as compared to ECs in non-malignant tissues. Thus far, most in vitro studies of angiogenesis used ECs isolated from normal tissues, which may not fully represent the nature of ECs in tumor vasculature. In this study, glioma-derived microvascular ECs (GDMEC) were purified from human glioma tissues by incubating with magnetic beads coated with anti-CD105 antibody and highly pure (98%) preparations of GDMEC were obtained. These cells exhibited typical EC phenotype, and proliferated rapidly in culture. Interestingly, GDMEC expressed higher levels of VEGF receptors, flt-1 and flk-1, as compared to an established human EC cell line ECV304 and primary human umbilical vascular EC (HUVEC). Functionally, GDMEC were capable of forming intercellular junctions and tubule-like structures (TLS) of various sizes. Stimulation by VEGF further promoted TLS formation with diverse tubular walls by GDMEC. In contrast, TLS formed by ECV304 and HUVEC showed significantly different features. We further observed that Nordy, a synthetic lipoxygenase inhibitor, potently inhibited TLS formation by GDMEC. The results suggest that isolation of highly pure ECs derived from tumor tissues is more appropriate for studies of tumor angiogenesis and for test of potential anti-cancer therapeutic targets.

Introduction

Angiogenesis is crucial for the growth of solid tumors. In the process of neovascularization of tumor tissue, new blood vessels are initiated by endothelial cells (EC) from various sources [1], [2], including EC precursors from bone marrow and ECs from the sprouts of preexisting vessels adjacent to the tumor. Both cell types are recruited by growth factors and chemokines produced by tumor and stromal cells. After entering the tumor parenchyma, EC precursors undergo maturation and in conjunction with ECs existing in surrounding tissues, they form new microvessels through proliferation, tubulogenesis and remodeling. These events constitute the process of angiogenesis.

Although ECs have been reported to be highly heterogeneous in different organs and vascular beds [3], [4], accumulating evidence suggests the presence of distinct features of ECs in malignant tumor. The unique microenvironment of tumors may cause the formation by such ECs of the diverse vessel architecture, termed tumor microvessel architecture phenotype heterogeneity (T-MAPH) [5]. Elucidation of the mechanisms that regulate T-MAPH will not only benefit the understanding of the process of angiogenesis in malignant tumors but also permit the design of anti-tumor angiogenesis agents [5], [6]. However, until recently, most in vitro studies of tumor angiogenesis used primary ECs or EC lines derived from normal blood vessels [7], [8], [9], [10], which may not fully represent the properties of ECs that have been “conditioned” by the microenvironment of the malignant tumor tissue, where aberrant growth factors and chemoattractants for ECs are produced. It is therefore desirable to utilize ECs directly isolated from malignant tumors to more precisely delineate the role of ECs in tumor progression and angiogenesis.

Malignant gliomas are characterized by their high degree of aggressiveness. Gliomas contain abundant microvessels, which are highly heterogeneous in number, density, distribution, morphology and architecture. In fact, microvessel density in gliomas has become one of the important criteria for histological grading of the malignancy and prediction of prognosis [11]. The ECs lining microvessels of gliomas are highly proliferative and potentially very useful as models for in vitro studies of tumor angiogenic processes.

The purpose of this study is to establish models of in vitro angiogenesis by using ECs directly derived from tissues of primary human glioma. Here we report the isolation and purification of malignant glioma-derived ECs (GDMEC) from surgically removed tumor tissues. We additionally characterized the phenotype of GDMEC and their unique properties in forming tubule-like structures (TLS) in a 3-dimensional (3D) matrix model. Our results showed that GDMEC differ from human umbilical cord vein ECs (HUVEC) and an established human EC cell line in their increased responsiveness to vascular endothelial cell growth factor (VEGF) and TLS formation presumably based on their higher level expression of VEGF receptors.

Section snippets

Human glioma tissues

A total of 16 cases of primary human brain glioma tissues were obtained from the Department of Neurosurgery, Southwest Hospital, Chongqing, China for diagnostic purposes. The procedures were strictly governed by institute regulations. Part of the tumor tissues was fixed in 10% neutral-formalin in PBS (pH 7.2) for conventional pathological sections and part of the tissues was placed in ice-cold M199 medium (HyClone, Logan, Utah, U.S.A.) for isolation of microvessel ECs.

Formalin-fixed tumor

Morphology and phenotype of glioma microvessels

Of the 16 surgically removed human gliomas, 6 were diffuse astrocytoma (WHO grade II), 7 were anaplastic astrocytomas (WHO grade III) and 3 were glioblastoma (WHO grade IV). All glioma specimens contained multiple microvessels (Fig. 1). In the lower grade astrocytoma specimens, most blood vessels were small and thin-walled (Fig. 1A), while the more aggressive grade III and IV gliomas contained more heterogeneous microvessels of a higher density than those in grade II tumors (Fig. 1B,C). Grade

Discussion

Malignant gliomas are the most common tumor type in the central nervous system. They are highly vascularized [11] and blood vessels in such tumors exhibit architecture distinct from normal or inflammatory tissues. Since ECs in malignant gliomas are in an actively proliferative state, using such cells to study the process of tumor angiogenesis and vascularization can yield valuable information for the design of therapeutic approaches to tumor [18], [19]. Our isolated GDMEC are more than 98% pure

Acknowledgements

The authors thank Rong Zhang of Southwest Hospital, Chongqing, P.R. China, for technical assistance and Dr. Joost J. Oppenheim of National Cancer Institute at Frederick, NIH, U.S.A., for critical review of the manuscript. This project is supported by grants #30370552 and #30270526 from National Natural Science Foundation of China, and by grant #2002AA001010 from National 863 Project, China, P.R.

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    These authors contributed equally to the study.

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