Abstract
BackgroundGuanylate binding protein-1 (GBP-1) is a large GTPase which is actively secreted by endothelial cells. It is a marker and intracellular inhibitor of endothelial cell proliferation, migration, and invasion. We previously demonstrated that stable expression of GBP-1 in murine endothelial progenitor cells (EPC) induces their premature differentiation and decreases their migration capacity in vitro and in vivo. The goal of the present study was to assess the antiangiogenic capacity of EPC expressing GBP-1 (GBP-1-EPC) and their impact on blood vessel formation in an axially vascularized 3-D bioartificial construct in vivo.ResultsFunctional in vitro testing demonstrated a significant increase in VEGF secretion by GBP-1-EPC after induction of cell differentiation. Undifferentiated GBP-1-EPC, however, did not secrete increased levels of VEGF compared to undifferentiated control EPC expressing an empty vector (EV-EPC). In our In vivo experiments, we generated axially vascularized tissue-engineered 3-D constructs. The new vascular network arises from an arterio-venous loop (AVL) embedded in a fibrin matrix inside a separation chamber. Total surface area of the construct as calculated from cross sections was larger after transplantation of GBP-1-EPC compared to control EV-EPC. This indicated reduced formation of fibrovascular tissue and less resorption of fibrin matrix compared to constructs containing EV-EPC. Most notably, the ratio of blood vessel surface area over total construct surface area in construct cross sections was significantly reduced in the presence of GBP-1-EPC. This indicates a significant reduction of blood vessel density and thereby inhibition of blood vessel formation from the AVL constructs caused by GBP-1. In addition, GBP-1 expressed from EPC significantly reduced cell apoptosis compared to GBP-1-negative controls.ConclusionTransgenic EPC expressing the proinflammatory antiangiogenic GTPase GBP-1 can reduce blood vessel density and inhibit apoptosis in a developing bioartificial vascular network and may become a new powerful tool to manipulate angiogenetic processes in tissue engineering and other pathological conditions such as tumour angiogenesis.
Highlights
Guanylate binding protein-1 (GBP-1) is a large GTPase which is actively secreted by endothelial cells
VEGF secretion in vitro We quantified the ability of EV endothelial progenitor cells (EPC) and GBP-1 EPC to secrete VEGF under incubation with basal medium as well as differentiation medium
Differentiation medium induced a significant stimulation of VEGF secretion in expressing empty vector as a control (EV EPC) and even more so in GBP-1 EPC (p
Summary
Guanylate binding protein-1 (GBP-1) is a large GTPase which is actively secreted by endothelial cells. In pathologic conditions involving tissue ischemia such as myocardial infarction, limb ischemia, and tumor angiogenesis [1,2], EPC are chemotactically recruited from bone marrow and the peripheral blood to the site of injury where they differentiate into mature endothelial cells. These cells may contribute directly to de novo microvascular blood vessel formation, a process termed vasculogenesis [1]. It has been previously shown that chronic inflammation and increased levels of proinflammatory cytokines induce the secretion of the large GTPase Guanylate binding Protein-1 (GBP-1) from endothelial cells. GBP-1 mediates the potent antiangiogenic effects of the cytokines TNF-α, IFN-γ and IL-1 in vitro and in vivo [5,6,7,8]
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