Kinetic Analysis of Vasculogenesis Quantifies Dynamics of Vasculogenesis and Angiogenesis <em>In Vitro</em>

Abstract

Vasculogenesis is a complex process by which endothelial stem and progenitor cells undergo de novo vessel formation. Quantitative assessment of vasculogenesis has become a central readout of endothelial progenitor cell functionality, and therefore, several attempts have been made to improve both in vitro and in vivo vasculogenesis models. However, standard methods are limited in scope, with static measurements failing to capture many aspects of this highly dynamic process. Therefore, the goal of developing this novel protocol was to assess the kinetics of in vitro vasculogenesis in order to quantitate rates of network formation and stabilization, as well as provide insight into potential mechanisms underlying vascular dysfunction. Application of this protocol is demonstrated using fetal endothelial colony forming cells (ECFCs) exposed to maternal diabetes mellitus. Fetal ECFCs were derived from umbilical cord blood following birth, cultured, and plated in slides containing basement membrane matrix, where they underwent vasculogenesis. Images of the entire slide wells were acquired using time-lapse phase contrast microscopy over 15 hours. Images were analyzed for derivation of quantitative data using an analysis software called Kinetic Analysis of Vasculogenesis (KAV). KAV uses image segmentation followed by skeletonization to analyze network components from stacks of multi-time point phase contrast images to derive ten parameters (9 measured, 1 calculated) of network structure including: closed networks, network areas, nodes, branches, total branch length, average branch length, triple-branched nodes, quad-branched nodes, network structures, and the branch to node ratio. Application of this protocol identified altered rates of vasculogenesis in ECFCs obtained from pregnancies complicated by diabetes mellitus. However, this technique has broad implications beyond the scope reported here. Implementation of this approach will enhance mechanistic assessment and improve functional readouts of vasculogenesis and other biologically important branching processes in numerous cell types or disease states.

Transgelin induces dysfunction of fetal endothelial colony-forming cells from gestational diabetic pregnancies

“…The number of closed networks per well was quantified, and average values are reported. For kinetic, multi-time point experiments, the Matrigel slide was placed in a Nikon TiE microscope stage-top incubator to maintain temperature, CO 2, and humidity for overnight live cell imaging, similar to our recent studies (38,39). Images of entire wells were collected with the ORCA-ER every 15 min for 10 h for a total of 40 data points per ECFC sample in each experiment.…”

“…All time-lapse imaging experiments were conducted on a fully automated Nikon TiE microscope equipped with a ProScan II motorized stage (Prior Scientific, Rockland, MA), xenon lamp source, Lambda LS, and Lambda 10 -3 filter wheel controller (Sutter Instruments, Novato, CA), fitted with an ORCA-ER or ORCA-Flash 4.0 (Hamamatsu, Japan) controlled by Elements 4.20 software (Nikon Instruments, Melville, NY). Imaging was performed as previously described (38,39). Phase contrast images were acquired with a CFI PlanFluor DLL ϫ10 objective (Nikon Instruments).…”

“…Kinetic analysis of vasculogenesis. Phase contrast images were compiled, processed, and analyzed using the FIJI plugin called kinetic analysis of vasculogenesis (KAV) (38,39). KAV is reliant on the Skeletonize 2D/3D and Analyze Skeleton plugins in FIJI (1).…”

“…The graphs represent the means Ϯ SE at each time point. To statistically analyze the differences between the entire kinetic curves, all individual time point data were pooled to produce estimated mean difference curves and the corresponding pointwise 95% confidence intervals as previously described (38,39). These curves were generated using penalized spline techniques, and the analysis was performed using the MGCV package in R (33, 41).…”

“…To obtain a kinetic readout of ECFC network formation, KAV methods were applied to quantitate large time-lapse image data sets and provide high-throughput vasculogenic analyses (38,39). Skeleton and mask renditions of representative network structures enable visual confirmation of the quantitative analysis generated by the software (Fig.…”