Investigating Pericyte Dynamics during Angiogenesis in the Mouse Mesometrium Culture Model

Angiogenesis requires multiple cells and systems working in concert to develop new blood vessels from existing ones. Hence, understanding the exact dynamics between cells and systems proves to be beneficial when developing innovative therapies. In the microcirculation, endothelial cells that line the blood vessels are supported by perivascular cells that are elongated cells called pericytes. Cell membrane proteins that modulate cellular responses of different growth factors play an important role during microvascular remodeling. For pericytes, the neural glial antigen‐2 (NG2) proteoglycan is a cell membrane protein that is exclusively expressed by mural cells in mouse microvasculature. Pericytes' multifaceted roles in angiogenesis and where they originate still requires further research. Our lab has recently introduced the mouse mesometrium culture model that allows for the investigation of cell dynamics within intact microvascular networks. The objective of this study was to demonstrate the usefulness of the mouse mesometrium culture model for investigating NG2 inhibition and cell‐lineage tracing. For the NG2 inhibition studies, female C57BL/6 (WT) and NG2 KO were euthanized to aseptically harvest mesometrium tissues and culture them in MEM + 1% PS and 20% FBS at standard incubating conditions. WT and NG2 KO tissues were cultured for 3 days before labeling with PECAM and NG2 antibodies to quantify angiogenesis. PECAM and NG2 labeling confirmed the lack of NG2‐positive pericytes on capillaries from NG2 KO tissues. The angiogenic response after culture from NG2 KO tissues was significantly decreased when compared to WT tissues (NG2 KO: 0.98 ± 0.29; WT: 2.11 ± 0.32 number of sprouts per total vessel length, p = 0.019). For cell‐lineage tracing studies, female NG2dsRed mice were given a daily IP injection of 0.1 mL of sunflower oil for 5 consecutive days to increase microvascular density. Mesometrium tissues were harvested 21 days post last injection and cultured in MEM + 1% PS and 20% FBS at standard incubating conditions for 5 days. The tissues were imaged for time‐lapse at least every 12 hours to track individual cell interactions. Time‐lapse imaging taken every 2 hours for 12 hours on days 1–3 of NG2dsRed tissues revealed that NG2‐positive pericytes migrated along pre‐existing vessels to new vessel segments. Moreover, a subpopulation of NG2‐positive interstitial cells were observed to migrate throughout the tissue. These results validate that inhibiting NG2 expression has a direct effect on angiogenesis in the mouse mesometrium culture model. Furthermore, it highlights the usage of the mouse mesometrium culture model to track individual cell‐to‐cell dynamics of pericytes in intact microvasculature during angiogenesis. Support or Funding Information NIH R01AG049821 This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .