Hydrodynamic conditions induce changes in secretion level and glycosylation patterns of Von Willebrand factor (vWF) in endothelial cells Maritza Morga-Ramírez, 1 María Teresa Collados-Larumbe, 1 Keith E. Johnson, 2 María José Rivas-Arreola, 1 Leydi Maribel Carrillo-Cocom, 1 and Mario Moisés Álvarez 1, ⁎ Centro de Biotecnología-FEMSA, Tecnológico de Monterrey, Ave. Eugenio Garza Sada 2501 sur. Monterrey, N.L. C.P. 64849, México 1 and Independent Consultant, 2884 Sutherland Circle N.W., North Canton, OH 44790, USA 2 Received 17 March 2009; accepted 2 October 2009 Available online 6 November 2009 The effect of low-speed agitation on the level of secretion and glycosylation patterns of the Von Willebrand factor in endothelial cells cultured in 6-cm Petri dishes located in a rotating platform was studied. In this simple system, complex and non-intuitive velocity fields arise. As revealed by simple calculations of tangential velocities and CFD analysis, flow behavior strongly varies as a function of radial distance and rotational velocity. We found significant differences in both secretion level and glycosylation extent and completeness, as a result of relatively small changes in rotational speed. Results obtained support the hypothesis that differences in hydrodynamic conditions may induce important changes in the secretion and assembly of glycoproteins in mammalian cell cultures. © 2009, The Society for Biotechnology, Japan. All rights reserved. [Key words: Endothelial cells; Mammalian cells; Mixing; Glycosylation patterns; Von Willebrand factor; Shear stress; Computational fluid dynamics] Endothelial cells have been focus of intense research. According to the Citation Index of the ISI Web of Knowledge, the phrase “endothelial cell” has appeared within the title of more than 12,000 contributions since 1994. From the same source, 17% of these articles correspond to the subject area of cell biology, 17% to hematology, 16% to peripheral vascular disease, 16% to biochemistry and molecular biology, and 10% to cardiovascular systems. More than 36% of these articles have been published in the last 5 years, with its highest in 2007. Endothelial cells are cultured recurrently in laboratories around the world to learn how to engineer tissues (1–4), to study cardiovascular disease and thrombosis (5–7), to understand wound healing (8) and coagulation processes (9), to better understand biochemical cascades related to endothelial cell function (10, 11), to build platforms for drug screening and drug testing (12), among other aspects. In this contribution, we report significant changes on the level of secretion of the Von Willebrand factor (vWF) and in two particular indicators of its glycosylation extent and completeness (trimannose and sialic acid concentration) when endothelial cells are exposed to laminar rotational fields in Petri dishes. Reports on the effect of hydrodynamics on the protein expression (13) and glycosylation patterns (14) of mammalian cells in relevant culture geometries are still scarce. To our knowledge, no previous studies have addressed the effect of agitation on mammalian cell growth in Petri dishes, despite their importance as one of the most widely used culture devices for anchorage-dependent cells. The vWF is a well-characterized plasma circulating glycoprotein produced and secreted by endothelial cells (6, 15) that plays essential roles in hemostasis. It mediates platelet adhesion to the vascular wall, platelet aggregation, and serves as a plasma carrier and stabilizer for factor VIII. It has been proposed as a prognosis marker for primary pulmonary hypertension (7), thrombosis, and cardiovascular disease (6). The over-secretion of vWF has been associated to the complex biochemical cascade implicated in thrombotic events (16). The role of vWF in platelet adhesion is particularly important under conditions of high shear stress. It is well known that certain mammalian cells (i.e., endothelial vascular cells) are particularly responsive to mechanical stresses (16,17). Typical local velocity values of arteries and veins promote cell alignment to the flow field and secretion of vasodilatant and anti-coagulant compounds (18). However, when abnormal flow conditions occur in arterial flow, endothelial cells might also produce substances that promote vasoconstriction, coagulation, and platelet aggregation (19–21). In fact, some crucial metabolic processes related to the normal coagulant cascade or cardiovascular disorders are triggered or modulated by the hydrody- namics of blood in veins and arteries and its effect on the endothelial cell layer (22, 23). MATERIALS AND METHODS Isolation, cell culture, and propagation of endothelial cells Human umbilical vein endothelial cells (HUVEC) were isolated from umbilical human cords following protocols detailed elsewhere (24) (Rivas-Arreola, J.M.: Master Degree Thesis, Journal of Bioscience and Bioengineering VOL. 109 No. 4, 400 – 406, 2010 www.elsevier.com/locate/jbiosc ⁎ Corresponding author. Tel.: +52 8183 28 4131; fax: +52 8183 28 4136. E-mail address: mario.alvarez@itesm.mx (M.M. Álvarez). 1389-1723/$ - see front matter © 2009, The Society for Biotechnology, Japan. All rights reserved. doi:10.1016/j.jbiosc.2009.10.002