Abstract
Purpose :
Endothelial progenitor cells (EPCs) have been shown to play a vital role in angiogenesis and in the repair of damaged blood vessels. EPC levels have been found to be decreased in conditions that have microvascular abnormalities such as Alzheimer’s disease and primary open-angle glaucoma (POAG). Previous studies have shown that autologous EPC transplant is an effective treatment method for advanced cardiovascular disease. In this study, we tested varying concentrations of toll-4 receptor antagonists to determine their ability to increase the number of EPCs in a peripheral blood sample using existing EPC culture methods.
Methods :
Whole blood was collected from control subjects (n=4) via venipuncture, and separated via density gradient centrifugation using Lymphoprep (Stemcell Technologies). The peripheral blood mononuclear cell layer was then collected, resuspended in CFU-Hill Media (Stemcell Technologies), and plated onto a fibronectin-coated 6 well plate (Corning). After two days, the non-adherent cells were removed and re-plated onto an 8 well chamber slide. The cells were treated with low, medium, or high doses of resveratrol (R), quercetin (Q), and naltrexone (N) on days 0 and 2. On the 5th day of culture, the plates were viewed for the presence of colony forming units (CFU). EPCs were identified in culture using dil-acetylated-LDL (endothelial cell marker) and FITC-ulex (fucose-residue marker) and through flow cytometry as CD34+/CD309+/CD133+ cells. A two tailed t-test was used to determined statistical significance.
Results :
In untreated controls, an average of 10 CFU’s per well were observed. Low, medium, and high doses of RQN showed an average of 11.0 (p=0.6), 12.2 (p=0.09), and 13.1 (p=0.02) CFU’s, corresponding to a change of +10%, +22%, and +31%, respectively.
Conclusions :
Toll-4 receptors antagonists—RQN--increased functional CFUs and EPC levels in culture in a dose dependent manner, with the most marked growth at the high dose (10 μM resveratrol, 10 μM quercetin, 50 μM naltrexone). Although EPC are relatively scarce in whole blood, functional CFUs and a marked increase in EPC numbers were observed using this culture method. While further trials are needed to determine the feasibility of autologous transplant of EPCs for POAG treatment, these results provide a promising first step.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.