A. D. Morris1, H. Li1, K. Kuo1, S. Dalal1, L. P. Brewster1,2 1Emory University School Of Medicine,Division Of Vascular Surgery,Atlanta, GA, USA 2Atlanta VA Medical Center,Division of Vascular Surgery,Atlanta, GA, USA
Introduction:
Diabetes increases the risk of cardiovascular disease and major amputation, but the aggressive control of blood sugar levels in recent clinical trials did not correspond to better clinical outcomes. Endothelial cell regeneration is critical to preventing thrombotic and myointimal complications after vascular injuries and interventions, but the impact of hyperglycemia on this process is not well understood. Since hyperglycemia directly inhibits cellular homeostasis, we propose that it negatively impacts endothelial cell regeneration.
Methods:
Proliferation Protocol:
Human aortic endothelial cells(HAEC) were grown in standard culture conditions, and then plated in a 96-well plate at 5000 cells per well. After reaching ~70% confluence, the cells were quiesced with serum starvation for 24 hours. The cells were restimulated with growth media (positive control) or quiescent media (negative control) with 40mM glucose or 40mM mannose (oncotic control) added to both groups. After 72 hours, HAEC proliferation was collected by commercially available cell viability assay using absorbance.
3D Angiogenesis Assay:
HAECs were formed into pellets (40,000 cells) using mechanical rotation. The pellets were suspended in a fibrin hydrogel. The cell pellets were exposed to either 40mM glucose in growth media, 40mM mannose in growth media, or growth media for 3 days. HAEC invasion was captured by daily microscopy and analyzed as average sprout length at 10-degree intervals through 360 degrees by Matlab programming.
Results:
Contrary to our hypothesis, HAECs had greater proliferation in a 72-hour high glucose and high mannose environment compared to normal growth media (p<0.0001, p=0.030). Glucose media had additional proliferation potential compared to mannose media (p=0.012). High glucose and mannose conditions did not inhibit HAEC sprout length at any time point in the 3D angiogenesis assay.
Conclusion:
While there is literature supporting an inhibitory effect of high glucose on endothelial cells, it is related to a combination of concentration and time of exposure to high glucose conditions. In this work, we are surprised to find not only a lack of inhibition of EC sprout formation, but also an increase in EC proliferation with both high glucose and high mannose conditions. Perhaps, the 24 hours of quiescence positively affects proliferation in functionally starving cells. We are actively testing this by including the high glucose levels from the beginning of the experiment. The lack of inhibition on EC sprouting suggests that acute hyperglycemia may not inhibit EC angiogenic activity in a meaningful manner. The duration and severity of hyperglycemia that must be avoided remains a clinically important idea that must be further pursued.
