Cancer Letters

Cancer Letters

Volume 278, Issue 1, 8 June 2009, Pages 113-121
Cancer Letters

Upregulation of HAb18G/CD147 in activated human umbilical vein endothelial cells enhances the angiogenesis

https://doi.org/10.1016/j.canlet.2009.01.004Get rights and content

Abstract

Previous studies demonstrated that CD147 molecule, highly expressed on the surface of various malignant tumor cells, significantly correlated with the malignancy of these cancers; however, the role of HAb18G/CD147 in endothelial cells has yet to be established. In this study, we found that the expression of HAb18G/CD147 was significantly upregulated in activated HUVECs. The inhibition of HAb18G/CD147 expression by specific siRNA led to significantly decreased angiogenesis in vitro. Our data indicate that HAb18G/CD147 may regulate angiogenesis via several mechanisms including proliferation, survival, migration, MMPs secretion, and PI3K/Akt activation. Our findings for the first time suggest that upregulation of HAb18G/CD147 in activated HUVECs might play an important role in angiogenesis.

Introduction

HAb18G /CD147, which was found on the surface of human hepatoma cells, is a highly glycosylated transmembrane protein that contains two extracellular immunoglobulin domains (C and V domains) and belongs to the immunoglobulin superfamily [1]. Various independent laboratories have discovered the CD147 protein in different origins of human cells and tissues, designating it extracellular matrix metalloproteinase inducer (EMMPRIN) [2], basigin [3] or M6 antigen [4]. Several proteins with high levels of homology to HAb18G/CD147, i.e. neurothelin, HT7, OX47, and gp42, have also been characterized in other species [5].

Previous studies demonstrated that CD147 molecule is highly expressed on the surface of various malignant tumor cells, including cancers of liver [6], skin [7], lung [8], breast [9], bladder [10], and brain [11]. Elevated CD147 expression is significantly correlated with the malignancy of these cancers. The biological implication of increased CD147 in tumor cells has been investigated by in vitro studies using recombinant CD147 or native CD147 purified from tumor cells, indicating that CD147 mainly functions as an inducer of MMPs production in tumor local environment [12], [13]. CD147-positive tumor cells stimulate adjacent fibroblast cells to secrete MMPs (mainly including MMP-2 and MMP-9) and thus promote tumor invasion and metastasis [14].

Except for tumor cells, CD147 is also expressed at varying levels in many other cell types, including activated T cells [15], differentiated macrophage [16], epithelial and endothelial cells [17], and normal human keratinocytes [18]. The expression of CD147 in non-tumor tissues suggests that this molecule may be also involved in other physiological and/or pathological processes that may be associated with increased MMPs expression. For example, its presence in the epidermis and several embryonic epithelia suggests that CD147 may participate in epithelial-mesenchymal interactions, leading to changes in tissue architecture during embryonic development [19] and wound healing [20]. Also, CD147 on the surface of activated lymphocytes and monocytes may result in elevated MMPs levels and therefore contribute to progression of chronic inflammation [21], [22].

Most recently, the biological activity of CD147 has been linked to the tumor angiogenesis. Tumors depend upon angiogenesis for growth and the development of metastases. Tumor angiogenesis is a complex and multi-step process requiring the sequential activation of various factors. Tang et al. [23] found that CD147 stimulates production of VEGF in tumor cells, therefore indicating its involvement in regulating tumor angiogenesis. Millimaggi et al. [24] reported that CD147 is expressed in microvesicles derived from epithelial ovarian cancer cells and CD147-positive vesicles promote an angiogenic phenotype in endothelial cells in vitro. In studies mentioned above, the study of CD147 in tumor angiogenesis was mainly focused on its tumor cell-derived expression. A previous report has shown that CD147 is also expressed in HUVECs [25], suggesting that this molecule may play a role in angiogenic function of HUVECs. However, no data are available on the functional role of HAb18G/CD147 in endothelial cells.

A recent study by Tang et al. [12] reported a positive feedback regulation model of CD147 expression in which tumor cell-associated CD147 stimulates its own expression in tumor stroma, consequently contributing to tumor angiogenesis, tumor growth, and metastasis. Therefore, we hypothesize that HAb18G/CD147 expression in endothelial cells around tumor cells can be up-regulated and thus contribute to the tumor angiogenesis. In the present study, we investigated the expression of HAb18G/CD147 in activated HUVECs and its effect on angiogenic phenotype of HUVECs. To the best of our knowledge, this is the first study to examine the functional role of HAb18G/CD147 in HUVECs.

Section snippets

Cell cultures

HUVECs was isolated and cultured as previously described [26]. In brief, human umbilical cord veins were digested with collagenase (Roche Diagnostics). Cells were routinely cultured in flasks coated with 0.2% gelatin in endothelial basal medium (EBM-2) (Lonza, Walkersville, MD) supplemented with 10% fetal bovine serum (FBS). To activate the quiescent HUVECs, cells were then grown in endothelial cell growth medium (EGM-2) (Lonza, Walkersville, MD) which is composed of EBM-2 and supplements, such

HAb18G/CD147 was upregulated in activated HUVECs

The expression level of HAb18G/CD147 was evaluated in quiescent and activated HUVECs. Flow cytometry analysis showed that the HAb18G/CD147 was significantly upregulated in activated HUVECs, compared with quiescent cells. The mean fluorescence intensities were 321 ± 7.8 in activated HUVECs and 106 ± 5.1 in quiescent cells, respectively (Fig. 1A). Similar result was also observed in Western blot analysis, indicating that the expression of HAb18G/CD147 is notably higher in activated HUVECs than

Discussion

In our study, we evaluated the expression and angiogenic function of HAb18G/CD147 in activated HUVECs. We found that HAb18G/CD147 was significantly upregulated in activated HUVECs, compared with quiescent cells. More importantly, our data demonstrated that the upregulated HAb18G/CD147 enhances angiogenesis through promoting in vitro proliferation, migration and MMPs secretion of activated HUVECs and protecting these cells from apoptosis. In addition, the enhancement of angiogenesis was noted to

Acknowledgements

This work was supported by Grants 2006CB708504 and 2009CB521704 from the National Basic Research Program of China and Grant 30530720 from National Natural Science Foundation of China.

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    Yanke Chen and Hongxin Zhang equally contributed to this work.

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