Authors:

Ria P. Thomas

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Angiogenesis is the process of the formation of new blood vessels from pre-existing ones. Notch signaling is a juxtacrine signaling pathway that plays a critical role in the mediation of this process. Of the four Notch proteins, Notch1 has been extensively studied, while the role of Notch4 in vascular development is less well understood. Notch1 is broadly expressed in many different cell types. Meanwhile, Notch4 is predominantly found in endothelial cells and has been less extensively studied. The murine retinal angiogenesis model has shown that the loss of endothelial Notch1 results in hypersprouting while the global loss of Notch4 is characterized by reduced radial outgrowth of vasculature. However, their similar protein structure and targets suggest that Notch1 and Notch4 may have- overlapping functions. Therefore, we hypothesize that endothelial Notch1 and Notch4 have both distinct and redundant roles in angiogenesis. To investigate this further, we used four experimental groups: control mice with only the Cre allele, an endothelial Notch4 knockout group, an endothelial Notch1 knockout group, and a double mutant endothelial Notch1; Notch4 endothelial knockout group. Mice were injected with tamoxifen at either postnatal days 1-3 or days 5-7 to knockout endothelial Notch4 and/or Notch1 at different points of vascular plexus development. Retinas were harvested from mice at postnatal day 5, day 10, and day 12 to analyze vascular growth in the superficial and deeper plexuses. Samples underwent wholemount immunofluorescence staining for Isolectin B4 to identify vasculature, lba1 to identify macrophages, and ESM1 to identify tip cells. Vascular smooth muscle cells and pericytes were also identified with alpha SMA and NG2 staining, respectively. Retinas were then mounted and imaged using widefield microscopy for vascular analysis.

Source:

University of Illinois Chicago

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Co-authors:

Ria P. Thomas