Poster T16 - β1 Integrin expression controls vasculogenesis in endothelial cells

Introduction: Endothelial cells contain many integrin receptors on their surface that can interact with their surrounding environment. The binding of these receptors is known to regulate the ability of endothelial cells to self-assemble into 3D vasculature via the process of vasculogenesis. Researchers are investigating how endothelial cells can be isolated from a patient, cultured, and combined with a biomaterial to form vasculature networks in vitro. However, the isolated and cultured endothelial cells are highly heterogeneous on a single-cell level in their ability to form vasculature. We hypothesize that subpopulations of endothelial cells of varying vasculogenic capacity can be identified and sorted by the concentration of their β1 integrin surface proteins.

Materials and

Methods: Passage 2-4 human umbilical vein endothelial cells (HuVECs) were cultured and expanded on 0.1% gelatin-treated tissue culture plastic for 1 week. The cells were then collected and stained with a PE-conjugated β1-integrin antibody (anti-CD29). The stained cells were analyzed by flow cytometry and sorted with fluorescence-activated cell sorting into 2 subpopulations based on the concentration of their surface proteins (β1 Low, and β1 High). These groups constituted the bottom 30% and top 30% of the population fluorescence respectively. The sorted cells and unsorted cells (not stained with the β1-integrin antibody) were cultured for 6 days in a 0.6% (wt/v) collagen hydrogel with EGM-2 media containing PMA, which stimulates the endothelial cells' formation of 3D blood vessel structures. The hydrogels were fixed with 4% PFA, stained against F-actin/DAPI, and imaged using confocal microscopy. The resulting images of the vascular networks were analyzed using AngioTool, yielding parameters that characterize the geometry of the vascular network. The parameters were then subjected to statistical analysis using python.

Results, Conclusions, and Discussions: All cells were strongly 100% positive for β1 integrin, including the β1-Low and β1-High sorted cells. The results show that β1 integrin plays a key role in the early vasculogenic process. For almost all measured parameters there was a significant difference between the β1-Low and β1-High groups. The β1-Low condition had significantly reduced vessel length, the area covered by the vessel network, and junction density relative to the β1-high group. The β1-low group also had significantly higher lacunarity, where a higher value corresponds to a more disorganized network. No statistical significance was observed between the unsorted and β1-High group.(FIGURE 1)
Since β1 integrin is integral for a cell’s interaction with the extracellular matrix around it, specifically collagen matrices, we hypothesize that cells containing lower concentration of β1 integrin receptors had reduced ability to engage with and remodel the surrounding collagen hydrogel relative to the cells with high concentration of β1 integrins. This reduced remodeling capacity leads to reduced vasculogenesis in the cells. Furthermore, this reduced capacity is also observed in cells that had their β1 Integrin receptors blocked. The lack of a significant structural difference between the control group and the β1- High condition could be explained by the fact that the Β1 High subpopulation may be the primary contributor to vasculogenesis in a mixed population (FIGURE 2.) It is also possible that the higher β1 concentration plays a time-dependent role in vascularization. Of note, the β1 High population created networks at Day 6 similar to an earlier time point (Day 4) compared to the β1 Low and Unstained groups, suggesting that using the β1 population can kickstart the process faster. It is unknown how much the labeling antibodies affect the angiogenic process, care was taken to choose antibodies with low blocking coefficients, but further experimentation is required to understand this effect.
In summary, there are clear structural differences in vascular networks created from endothelial cells with different surface β1 Integrin concentrations and unsorted populations. Controlling these structural differences on a cellular level will be key in future studies on in vivo vascular regeneration.

Acknowledgements (Optional):