Background: Recently, notch signaling has been investigated in relation to the mechanism of endothelial sprouting, elongation and vascular maturation. Although it is recognized that diabetes mellitus (DM) elicits many cellular and molecular abnormalities, the relation between notch signaling and vascular morphogenetic changes in diabetes mellitus remains to be elucidated.
Objective: The aim of this study is to investigate the role of notch signaling pathway in hyperglycemic condition.
Methods: We evaluated the direct effects of high concentration of glucose on notch signaling in both cultured HUVEC and a model of angiogenesis, a 3-dimensional (3D) spheroid co-culture model. The changes of notch signaling were evaluated by RT-PCR and Western blot with or without signal-modulating chemicals. Confocal microscopy was used to visualizing the endothelial sprouts in 3D collagen gel which were similar to growing capillaries, thus mimicked the physiological blood vessel formation. Endothelial cells were labeled with GFP-or YFP expressing lentiviral vectors.
Results: One of the Notch ligands, Jaggged-1, is upregulated in response to high glucose. Analysis of relative expression results using real-time PCR show both dose- and time-dependent effects (3.5-fold increase at 25.6mM, 7-fold increase after 6days) of glucose on Jagged-1 expression in HUVECs. High glucose-induced Jagged-1 expression was completely inhibited by addition of each inhibitor of NF-κB and PKC (91.7±1.7%, p<0.001; 48.6±9.5, p=0.003, respectively). We also examined high glucose effects on 3D-cultured HUVEC spheroids. Confocal image analysis revealed that prolonged exposure of high glucose decreased total tube length (1.68±0.27㎜ at 25.6mM) than in control cultures (3.37±1.15㎜) (p=0.035) as well as maximum tube length (0.86±0.08 versus 0.63±0.04, p=0.009). Interestingly, the number of sprouts per spheroid circumference (㎜) was increased by 24h high glucose exposure (51.9±5.8) than in control cultures (38.5±2.6) (p=0.04) and it was markedly reversed by shRNA-mediated suppression of Jagged-1 expression (30.2±4.7) (p=0.020). A similar increase in sprout number was observed by Jagged-1 over-expression using lentiviral vector (74.7±5.0, p=0.001) or DAPT, Notch signaling inhibitor, treatment (70.2±6.2, p=0.003) than control (48.6±5.9).
Conclusion: We found that high glucose induces Jagged-1 expression through the PKC and NF-κB which disturbs the Notch signaling-controlled tip cells formation. Better study is warranted to understand and treat diabetes-associated vascular diseases by modulating notch signaling.
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