Spatiotemporal endothelial cell \u2013 pericyte association in tumors as shown by high resolution 4D intravital imaging

Ann L. B. Seynhaeve,Timo L. M. ten Hagen,Douwe Oostinga,Rien van Haperen,Susanne Adams,Ralf H. Adams,Hanna M. Eilken

doi:10.1038/s41598-018-27943-8

Abstract

Endothelial cells and pericytes are integral cellular components of the vasculature with distinct interactive functionalities. To study dynamic interactions between these two cells we created two transgenic animal lines. A truncated eNOS (endothelial nitric oxide synthase) construct was used as a GFP tag for endothelial cell evaluation and an inducible Cre-lox recombination, under control of the Pdgfrb (platelet derived growth factor receptor beta) promoter, was created for pericyte assessment. Also, eNOStag-GFP animals were crossed with the already established Cspg4-DsRed mice expressing DsRed fluorescent protein in pericytes. For intravital imaging we used tumors implanted in the dorsal skinfold of these transgenic animals. This setup allowed us to study time and space dependent complexities, such as distribution, morphology, motility, and association between both vascular cell types in all angiogenetic stages, without the need for additional labeling. Moreover, as fluorescence was still clearly detectable after fixation, it is possible to perform comparative histology following intravital evaluation. These transgenic mouse lines form an excellent model to capture collective and individual cellular and subcellular endothelial cell – pericyte dynamics and will help answer key questions on the cellular and molecular relationship between these two cells.

Highlights

Blood vessels consist of an endothelial lining surrounded by perivascular cells
DsRed fluorescence in pericytes was seen in the cellular body (Fig. 1B; asterisk) and in several processes protruding from the cellular body (Fig. 1B; arrow)
In this report we demonstrate the use of several transgenic mouse lines using a developing tumor as an angiogenic model showing the dynamic interaction between endothelial cells and pericytes in 4D high resolution followed by whole-mount re-evaluation

Summary

Introduction

Blood vessels consist of an endothelial lining surrounded by perivascular cells (i.e. pericytes and vascular smooth muscle cells). Angiogenesis, the formation of new blood vessels from a pre-existing vascular network, is a very dynamic biological process which involves a series of interdependent and multicellular processes In general it starts with sprouting of endothelial cells[18,19] from existing vessels, followed by formation of a functional tube through anastomosis[20], pruning[21,22] and re-introduction of perivascular cells[16,23]. Synthase) promoter as a tag controlling GFP expression to evaluate endothelial cells, and a line with an inducible Cre-lox recombination under control of the Pdgfrb (platelet derived growth factor receptor beta) promoter for assessment of pericytes. We used a tumor transplanted in the dorsal skinfold chamber as an angiogenic model in order to achieve high resolution 4D intravital imaging and evaluated spatial, temporal and morphological interactions between endothelial cells and pericytes

Methods

Results

Conclusion