The Effects of Different Densities in the Extracellular Matrix on Notch Activation
If Notch is controlled by the extracellular matrix (ECM) then ECM stiffness should have different effects on Notch activity. To test this hypothesis we created polyacrylamide hydrogels representing the stiffness of various physiological tissues. Human microvascular endothelial cells (HMEC) were transfected with a Notch responsive reporter plasmid called 12X-CSL GFP. This reporter plasmid illuminates upon Notch signaling. The plasmid, pEYFP C1, was used as a positive control and will illuminate if transfection worked. The hydrogels were coated with collagen 1 to promote cell adhesion. Human microvascular HMEC endothelial cells were then transfected with the control pEYFP C1, or Notch reporter 12X-CSL GFP. These cells were placed onto collagen-coated hydrogels and non-collagen coated glass coverslips and incubated for 24 hours. The hydrogels and glass cover slips are viewed using fluorescent microscopy and fluorescence intensity was quantified and averaged. It can be concluded that Notch activation was inversely proportional as the “stiffness” of the ECM was increased. This result supports the hypothesis that integrin stimulation controls Notch activation. In soon to follow studies, we will compare Notch activation on other substrates such as collagen 4, laminin, and vitronectin on different stiffness of hydrogels.
Thomson, Nick and Albig, Allan, "The Effects of Different Densities in the Extracellular Matrix on Notch Activation" (2015). College of Health Sciences Presentations. 1.