Abstract
Angiogenesis is a highly ordered physiological process regulated by the interaction of endothelial cells with an extensive variety of growth factors, extracellular matrix components and mechanical stimuli. One of the most important challenges in tissue engineering is the rapid neovascularization of constructs to ensure their survival after transplantation. To achieve this, the use of pro-angiogenic agents is a widely accepted approach. The study of angiogenesis has gained momentum over the last two decades. Although there are various in vitro, ex vivo, and in vivo angiogenesis models that enable testing of newly discovered pro-angiogenic agents, the problem with researching angiogenesis is the choice of the most appropriate assay. In vivo assays are the most representative and reliable models, but they are expensive, time-consuming and can cause ethical concerns whereas in vitro assays are relatively inexpensive, practical, and reproducible, but they are usually lack of enabling the study of more than one aspect of angiogenesis, and they do not fully represent the complexity of physiological angiogenesis. Therefore, there is a need for the development of an angiogenesis model that allows the study of angiogenesis under physiologically more relevant, dynamic conditions without causing ethical concerns. Accordingly, in this study, we developed 3D in vitro dynamic angiogenesis model, and we tested the angiogenic potential of 2-deoxy-D-ribose (2dDR) in comparison with vascular endothelial growth factor (VEGF) using newly developed in vitro 3D dynamic model and well-established in vitro models. Our results obtained using conventional in vitro assays demonstrated that 2dDR promoted proliferation, migration and tube formation of human aortic endothelial cells (HAECs) in a dose-dependent manner. Then, the angiogenic activity of 2dDR was further assessed using the newly developed 3D in vitro model, which enabled the monitoring of cell proliferation and infiltration simultaneously under dynamic conditions. Our results showed that the administration of 2dDR and VEGF significantly enhanced the outgrowth of HAECs and the cellular density under either static or dynamic conditions.
Highlights
Angiogenesis is a complex process driven by the interactions of endothelial cells (ECs) with growth factors, extracellular matrix, and mechanical stimuli (Risau, 1997)
Preparation of Pro-angiogenic Agents Working solutions of all substances were prepared fresh at the beginning of each experiment. 10 mM 2dDR stock solution was prepared by dissolving in low serum (2% fetal calf serum (FCS)) EC Growth Medium (EC growth medium (GM)) and filtered using a 0.2 μm syringe filter. 1, 10, 100 μM, and 1 mM concentrations of 2dDR were prepared by diluting the 10 mM 2dDR stock solution in low serum EC GM. 80 ng/mL Vascular endothelial growth factor (VEGF) solution was prepared in EC GM and used as a positive control
2dDR Improves the Metabolic Activity and Proliferation of Human Aortic Endothelial Cells (HAECs) in a Dose-Dependent Manner The results of metabolic activity assays showed that 80 ng/ml of VEGF increased metabolic activities and the total number of cells as expected
Summary
Angiogenesis is a complex process driven by the interactions of endothelial cells (ECs) with growth factors, extracellular matrix, and mechanical stimuli (Risau, 1997). Delayed angiogenesis of tissue-engineered (TE) constructs is a major challenge for their translation to the clinic (Novosel et al, 2011). One approach is the use of pro-angiogenic agents to circumvent slow neovascularization. Researchers have been exploring pro-angiogenic agents to ensure rapid neovascularization in tissue engineering constructs using well-established in vitro and in vivo models. Vascular endothelial growth factor (VEGF) is a well-known stimulator of angiogenesis and recognized as the most effective pro-angiogenic factor both in vitro and in vivo (Ferrara, 2009). Translation of the exogenous use of VEGF into the clinic is difficult (Ferrara and Alitalo, 1999). Exploring alternatives to VEGF is crucial for ensuring rapid and safe neovascularization in TE constructs
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