Research ArticlePro-angiogenic properties of orosomucoid (ORM)
Introduction
Orosomucoid (ORM), also known as alpha1-acid glycoprotein (AGP), was first described in 1950 by Schmid [1]. It belongs to a group of acute phase proteins (APPs) and along with many other functions plays a role in the modulation of immune response to stress [2], [3]. The high serum concentration of ORM (0.5–1.4 mg/ml) in healthy humans is known to rise two- to fivefold in response to different conditions such as acute infection, inflammation and lymphoproliferative disorders and various types of cancers [3].
The structure of ORM is composed of a polypeptide chain carrying about 45% carbohydrate residues including a large amount of fucosylic and sialic acids. Thus, its proposed immunomodulatory activities have been attributed to its glycosylation pattern. It has been postulated that the strongly fucosylated and sialylated ORM glycoforms have the ability to bind to E-selectin and to inhibit complement activation [4].
ORM is synthesized in liver and various extrahepatic cell types e.g. granulocytes and endothelial cells [5]. IL-1, IL-6 and glucocorticoids are the major modulators of ORM gene expression in liver cells [6], [7], [8]. An induced expression of sialyl Lewis X (sLeX) on ORM during acute inflammation has been reported, leading to the speculation that it might influence the E- or P-selectin-mediated influx of sLeX-expressing leukocytes into inflamed areas. It has been suggested that an increased level of sLeX-expressing ORM might have a feedback inhibitory effect on the extravasation of leukocytes, by competition for E-selectin [9]. By this mechanism and by interacting with the endothelial glycocalyx ORM is thought to modify the permeability of the vascular endothelium [10], [11], [12], [13], [14], [15], [16], [17]. Thus far it has been shown that ORM binds to the vascular endothelial cell surface and subsequently causes transcytosis across the cell without passing the intercellular junction [18], [19], [20]. It has been reported that ORM inhibits the action of histamine on human endothelial cells through cAMP [21].
Angiogenesis as well as postnatal vasculogenesis are regulated by angiogenic activators and inhibitors [22], [23], [24], [25]. Among these factors VEGF is the key regulator of physiological and pathological angiogenesis and acts as a survival factor for endothelial cells (EC), both in vitro and in vivo [26].
Up to now, it is unclear which potential influence the constitutive expression of ORM or the application of ORM in vascular endothelial cells possesses during angiogenesis. Therefore, we aimed to analyze the potential role of ORM in angiogenesis. To this aim we performed endothelial overexpresion versus silencing via siRNA of ORM and tested the effect on endothelial cells in in vitro and in vivo angiogenesis assays such as endothelial tube assay, migration assay and CAM assay using recombinant ORM (ORM). Employing ORM we show here that ORM increases endothelial cell migration in a dose dependent manner and supports the VEGF-A-induced endothelial tube formation in vitro when VEGF and ORM are applied simultaneously. While in vitro ORM alone does not induce endothelial tubes it increases the number of blood vessels in vivo in CAM tissue. Also, in CAM assay ORM enhances the VEGF-A-induced new vessel formation. These data suggest for the first time that ORM is involved in angiogenesis and supports VEGF-A mediated new vessel formation.
Section snippets
Reagents
The mouse monoclonal anti-ORM antibody was purchased from Sigma (Germany). For the stimulation of cultured HDMECs human ORM protein (ORM) was purchased from Calbiochem (Germany). Recombinant human VEGF-A was obtained from Immunotools (Germany). The mouse monoclonal anti-vimentin antibody was obtained from Dako (Germany).
Cell culture
Commercial human dermal microvascular endothelial cells (HDMECs) (Promocell, Germany) were cultured on gelatine-coated dishes in endothelial cell growth medium MV (Promocell,
Secretion of ORM by ORM-overexpressing HDMECs
To address the role of ORM in angiogenesis, we first performed ORM overexpression versus ORM gene silencing in HDMECs. The efficiency of genetic manipulation was confirmed by Western blot analyses using the lysates and supernatants of transfected HDMECs (Figs. 1A, B). ORM was detected at 41 kDa using the monoclonal anti-ORM antibody. Empty vector transfected endothelial cells (Fig. 1A) produced endogenously synthesized ORM as previously described [34], [35]. However, they barely secreted ORM
Discussion
The present results demonstrate for the first time that ORM exhibits pro-angiogenic properties in vitro and in vivo. Furthermore, ORM is involved in the modulation of VEGF-A-induced endothelial migration and tube formation, two basic steps of angiogenesis. Briefly, we show here that i) the major part of ORM expressed by EC is secreted into the supernatant, ii) ORM enhances the EC migration in a dose dependent manner, iii) it supports the VEGF-A- and FGF-2-induced endothelial tube formation in
Acknowledgments
The authors thank Mrs. D. Schünke and B. Maranca-Hüwel for the excellent technical assistance.
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