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J. M. Barnett, H. S. Toma, G. W. McCollum, J. S. Penn; In vitro Parallel Plate Flow Chamber Assessment of Endothelial Progenitor Cell Homing. Invest. Ophthalmol. Vis. Sci. 2010;51(13):2641.
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Bone marrow-derived endothelial progenitor cells (EPCs) have been shown to play a significant role in the neovascularization of vascular diseases. In many of these diseases, including diabetic retinopathy and age-related macular degeneration, elevated stromal derived factor-1 (SDF-1) is observed in the neovascular tissue, and this is believed to play an important role in EPC homing function. Little is known about other factors that might contribute. This study sought to develop an in vitro system of evaluating EPC homing capacity in conditions relevant to ocular disease states.
Bone marrow was isolated from 4-6 week old Brown Norway rats and a population of EPCs was identified using the markers CD34, CD133 and CXCR4 in fluorescence-activated cell sorting experiments. EPCs were labeled with quantum dot conjugated acetylated LDL. Labeled EPCs and mature endothelial cells (ECs) were placed into a parallel plate flow chamber (PPFC) [Glycotech Corporation] in separate experiments. In the first, cells were allowed to flow at 15 dynes/cm2 shear stress over exposed extracellular matrix (ECM) and in the second, over conditioned endothelial monolayer (EML). The ECM conditions tested were hyaluronic acid (HA) or chondroitin sulfate (CS) with and without SDF-1. EMLs were conditioned by a 24-hour pretreatment in either hypoxia (0% oxygen) or normoxia (20.9% oxygen).
In the ECM studies, EPCs were found to be 3-fold (p<0.001) more adherent to HA than CS, and this adherence was increased 67% (p<0.01) with the addition of SDF-1 to HA. EPC adherence to HA + SDF-1 was reduced by 56% (p<0.01) by incubating the EPCs with anti-CXCR4 for 10 minutes prior to their addition to the PPFC. Compared to the ECs, EPCs were 59% (p<0.01) more adherent to HA alone, and 2.6-fold (p<0.001) more adherent to HA + SDF-1. In the EML studies, EPC adherence was increased by 33% (p<0.05) when EML were treated with hypoxia relative to normoxia.
The in vitro system has demonstrated adherence of EPCs and ECs to surfaces chosen to model damaged blood vessel walls found in ocular neovascular diseases. This high throughput system can now be used to further characterize homing in more specific EPC subpopulations and to define the roles of EPCs in pathological disorders of the eye.
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