Indiana University-Purdue University Indianapolis (IUPUI) / 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, including a tissue cytometry approach to quantify the frequency
and localization of dividing ECFCs within cell networks. Additionally, FIJI TrackMate was
used to quantify ECFC displacement and speed at the single cell level during network
formation. These novel approaches were then applied to determine how intrauterine
exposure to maternal type 2 diabetes mellitus (T2DM) impairs fetal ECFC vasculogenesis,
and whether increased Transgelin 1 (TAGLN) expression in ECFCs from pregnancies
complicated by gestational diabetes (GDM) was sufficient to impair vasculogenesis. Fetal
ECFCs exposed to maternal T2DM formed fewer initial network structures, which were
not stable over time. Correlation analyses identified that ECFC samples with greater
division in branches formed fewer closed network structures and that reductions in ECFC
movement decreased structural connectivity. To identify specific cellular mechanisms and
signaling pathways altered in ECFCs following intrauterine GDM exposure, these new
techniques were also applied in TAGLN expression studies. Similarly, ECFCs from GDM pregnancies and ECFCs overexpressing TAGLN exhibited impaired vasculogenesis and
decreased migration. Both ECFCs from GDM pregnancies as well as ECFCs over
expressing TAGLN exhibited increased phosphorylation of myosin light chain. Reduction
of myosin light chain phosphorylation via Rho kinase inhibition increased ECFC migration;
therefore, increased TAGLN was sufficient to impair ECFC vasculogenic function. Overall,
identification of these novel phenotypes provides evidence for the molecular mechanisms
contributing to aberrant ECFC vasculogenesis. Determining how intrauterine exposure to
maternal T2DM and GDM alters fetal ECFC function will enable greater understanding of
the chronic vascular pathologies observed in children from pregnancies complicated by
diabetes mellitus.
| Identifer | oai:union.ndltd.org:IUPUI/oai:scholarworks.iupui.edu:1805/13765 |
| Date | 11 May 2017 |
| Creators | Varberg, Kaela Margaret |
| Contributors | Haneline, Laura S., Clauss, Matthias A., Day, Richard N., Harrington, Maureen A., Srour, Edward F. |
| Source Sets | Indiana University-Purdue University Indianapolis |
| Language | en_US |
| Detected Language | English |
| Type | Dissertation |
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