title: Kinetic analyses of vasculogenesis inform mechanistic studies.
abstract: Vasculogenesis is a complex process by which endothelial stem and progenitor cells undergo de novo vessel formation. Quantitative assessment of vasculogenesis is a central readout of endothelial progenitor cell functionality. However, current assays lack kinetic measurements. To address this issue, new approaches were developed to quantitatively assess in vitro endothelial colony-forming cell (ECFC) network formation in real time. Eight parameters of network structure were quantified using novel Kinetic Analysis of Vasculogenesis (KAV) software. KAV assessment of structure complexity identified two phases of network formation. This observation guided the development of additional vasculogenic readouts. A tissue cytometry approach was established to quantify the frequency and localization of dividing ECFCs. Additionally, Fiji TrackMate was used to quantify ECFC displacement and speed at the single-cell level during network formation. These novel approaches were then implemented to identify how intrauterine exposure to maternal diabetes mellitus (DM) impairs fetal ECFC vasculogenesis. Fetal ECFCs exposed to maternal DM form fewer initial network structures, which are not stable over time. Correlation analyses demonstrated that ECFC samples with greater division in branches form fewer closed network structures. Additionally, reductions in average ECFC movement over time decrease structural connectivity. Identification of these novel phenotypes utilizing the newly established methodologies provides evidence for the cellular mechanisms contributing to aberrant ECFC vasculogenesis.
authors.affiliation: Department of Cellular and Integrative Physiology, Indiana University School of Medicine, Indianapolis, Indiana.|Herman B. Wells Center for Pediatric Research, Indianapolis, Indiana.
authors.forename: Kaela M
authors.identifier: http://orcid.org/0000-0001-9672-4158
authors.initials: KM
authors.lastname: Varberg
authors.affiliation: Indiana Center for Biological Microscopy, Indianapolis, Indiana.|Department of Medicine, Indiana University School of Medicine, Indianapolis, Indiana.
authors.forename: Seth
authors.initials: S
authors.lastname: Winfree
authors.affiliation: Department of Biostatistics, Indiana University School of Medicine, Indianapolis, Indiana.
authors.forename: Chenghao
authors.initials: C
authors.lastname: Chu
authors.affiliation: Department of Biostatistics, Indiana University School of Medicine, Indianapolis, Indiana.
authors.forename: Wanzhu
authors.initials: W
authors.lastname: Tu
authors.affiliation: Herman B. Wells Center for Pediatric Research, Indianapolis, Indiana.|Department of Pediatrics, Indiana University School of Medicine, Indianapolis, Indiana.
authors.forename: Emily K
authors.initials: EK
authors.lastname: Blue
authors.affiliation: Department of Cellular and Integrative Physiology, Indiana University School of Medicine, Indianapolis, Indiana.|Herman B. Wells Center for Pediatric Research, Indianapolis, Indiana.
authors.forename: Cassandra R
authors.initials: CR
authors.lastname: Gohn
authors.affiliation: Indiana Center for Biological Microscopy, Indianapolis, Indiana.|Department of Medicine, Indiana University School of Medicine, Indianapolis, Indiana.
authors.forename: Kenneth W
authors.initials: KW
authors.lastname: Dunn
authors.affiliation: Department of Cellular and Integrative Physiology, Indiana University School of Medicine, Indianapolis, Indiana; lhanelin@iu.edu.|Herman B. Wells Center for Pediatric Research, Indianapolis, Indiana.|Department of Pediatrics, Indiana University School of Medicine, Indianapolis, Indiana.|Department of Microbiology and Immunology, Indiana University School of Medicine, Indianapolis, Indiana; and.|Indiana University Simon Cancer Center, Indiana University School of Medicine, Indianapolis, Indiana.
authors.forename: Laura S
authors.initials: LS
authors.lastname: Haneline
mesh_terms: D000818:Animals; D002454:Cell Differentiation / Q000502:physiology; D003198:Computer Simulation; D042783:Endothelial Cells / Q000166:cytology / Q000502:physiology*; D006801:Humans; D007700:Kinetics; D008955:Models, Cardiovascular; D009389:Neovascularization, Pathologic / Q000503:physiopathology*; D018919:Neovascularization, Physiologic / Q000502:physiology*; D013234:Stem Cells / Q000166:cytology / Q000502:physiology*
keywords: diabetes; endothelial; migration; proliferation; vasculogenesis