Abstract
Angiogenesis defines the process of formation of new vascular structures form existing blood vessels, involved during development, repair processes like wound healing but also linked to pathological changes. During angiogenic processes, endothelial cells build a vascular network and recruit perivascular cells to form mature, stable vessels. Endothelial cells and perivascular cells secret and assemble a vascular basement membrane and interact via close cell-cell contacts. To mimic these processes in vitro we have developed a versatile three-dimensional culture system where perivascular cells (PVC) are co-cultured with human umbilical cord vascular endothelial cells (HUVEC) in a collagen type I gel. This co-culture system can be used to determine biochemical and cellular processes during neoangiogenic events with a wide range of analyses options.
Keywords: Endothelial cells, Perivascular cells, Pericytes, Angiogenesis, Co-culture, Collagen gel
Background
The coordinated interaction between endothelial and perivascular cells is essential to form a stable vascular network according to the local needs within a given tissue. Multiple molecular components contribute to the interactions but are still poorly understood. Various growth factors are needed to attract endothelial cells to sites of low oxygen concentrations and build new vessels that are then covered by perivascular cells. Both cell types interact to secrete a specialized extracellular matrix and stabilize the newly formed vessels. In the past multiple assays have been established to analyze vascular cell interaction and vessel-like network formation on two-dimensional matrigel substrates, but those are limited in providing information about initial steps of endothelial-perivascular cell interaction and vascular basement membrane formation in a three-dimensional microenvironment. In addition, well characterized perivascular cell suitable for culture experiments were missing. We have previously isolated cells with perivascular characteristics, as they express pericyte-specific markers, produce and secrete extracellular matrix proteins and stimulate angiogenic processes in vivo (Brachvogel et al., 2005 and 2007). These cells were used to establish a co-culture system with human umbilical vein endothelial cells and study critical steps in neoangiogenesis upon interaction of the two cell types in a three-dimensional microenvironment (Pitzler et al., 2016; Zhou et al., 2016).The co-cultures showed a superior activity to promote the formation endothelial tube formation and stabilization by perivascular cell recruitment and basement membrane formation. The culture system allows to monitor migration and interaction of vascular cells by time-lapse microscopy and to study the deposition of basement membrane protein by immunofluorescence analysis. To quantify and isolate endothelial and perivascular cells from co-cultures immunomagnetic or flow cytometry sorting approaches can be used and cell type-specific global changes in mRNA and miRNA expression can be analyzed by subjecting isolated RNA from the separated cell populations to microarray analysis or RNA sequencing. Moreover, the effects of anti- and pro-angiogenic substances on endothelial-perivascular cell interaction can be analyzed in vitro. Hence, the three-dimensional culture protocol allows to study cellular, biochemical and transcriptional events during neoangiogenesis and to characterize the pro- and anti-angiogenetic effects of molecules on endothelial perivascular cell interaction ex vivo.
Materials and Reagents
Equipment
Software
Procedure
Data analysis
Notes
Recipes
Acknowledgments
The three-dimensional co-culture assay was used in two recent publications (Pitzler et al., 2016; Zhou et al., 2016). The work is supported by Deutsche Forschungsgemeinschaft (DFG 2304/5-3, 2304/7-1, 2304/9-1).
References
If you have any questions/comments about this protocol, you are highly recommended to post here. We will invite the authors of this protocol as well as some of its users to address your questions/comments. To make it easier for them to help you, you are encouraged to post your data including images for the troubleshooting.