Capturing Membrane Trafficking Events During 3D Angiogenic Development in Vitro

ABSTRACT Objectives Mechanisms that govern angiogenesis are paramount to our understanding of how blood vessels are formed embryonically, maintained in adulthood and manifest disease. Akin to transcriptional regulation of endothelial-specific genes, vesicular trafficking events dictate protein localization, functional activity, and half-life, providing a critically important regulatory step. However, there is little information detailing endothelial-specific trafficking signatures. This is due, in part, by limitations in visualizing trafficking events in endothelial tissues. Our aim in this investigation was to explore the use of a 3-dimensional (3D) in vitro sprouting model to image and evaluate membrane trafficking events compared to the conventional 2-dimensional (2D)-based culture method. Methods Primary Human endothelial cells were challenged to make multicellular sprouts using a fibrin-bead assay. An assortment of cell polarity and Rab proteins were quantified via immunocytochemistry and live-imaging to compare their localization between 3D sprouts and 2D culture. Results Our results show that sprouts generated from the fibrin-bead assay grow close to the imaging plane allowing for an orthogonal view of apical and basal membrane domains. Compared with 2D culture in which the apical and basal domains are in the axial orientation, limiting resolution, 3D sprouts are acquired in the X-Y plane providing high-resolution for viewing trafficking events. Second, we demonstrate that fibrin-bead generated sprouts have a strong apicobasal polarity axis. Third, we directly compare imaging of trafficking mediators podocalyxin and Rab35 between 3D sprouts and 2D culture. Here, we show that 3D sprouting structures are well-suited to capture trafficking events that are not present in 2D culture due to the lack of a defined apical domain. Lastly, we compared exocytic events of von Willebrand Factor between 3D sprouting and 2D culture. Our results demonstrate a distinct imaging advantage for monitoring these trafficking programs in 3D sprouts as compared with conventional 2D culture. Conclusions In general, our results establish that the fibrin-bead sprouting assay is well-suited for sub-cellular imaging of trafficking events during angiogenic growth. Additionally, the 2D endothelial culture does not enforce the formation of an apicobasal polarity axis.