Effects Of Aspirin On Number, Activity And INOS Of Endothelial Progenitor Cells From Peripheral Blood

Objective:Vascular endothelial progenitor cells (EPCs) are the precursor of endothelial cells. Increasing evidence suggests that circulating progenitor cells contribute to postnatal neovascularization. These cells home to sites of ischemia, adopt an endothelial phenotype, and contribute to new blood vessel formation, the identity of the circulating cells that contribute to neovascularization is not entirely clear. Bone-marrow derived hematopoietic progenitor cells can give rise to endothelial progenitor cells and contribute to endothelial recovery and new capillary formation after ischemia.Aspirin (acetylsalicylic acid) is widely used in the primary and secondary prevention of vascular diseases. The anti-inflammatory effect of aspirin is believed to complement its platelet inhibitory effect and is believed to be due to the inhibition of cyclooxygenase, resulting in decreased thromboxane A₂ production. Although the major beneficial effect of aspirin is due to its inhibitory action on platelet aggregation, there is emerging evidence that other effects of aspirin on cells other than platelets maybe equally important. So we investigated the effects of aspirin on number and activity of endothelial progenitor cells from peripheral blood. Nitric oxide(NO) synthesized from L-arginine by inducible nitric oxide synthase(iNOS) is a veryimportant message of signal pathway in human endothelial cells. We also detected iNOS by western blot and discussed the mechanism of iNOS in these effects. Methods:Total mononuclear cells (MNCs) were isolated from peripheral blood by Ficoll density gradient centrifugation, and then the cells were plated on fibronectin-coated culture dishs. After 7 days cultured, attached cells were stimulated with aspirin 0,l,2,5,10mmol/L for 24 hours, and aspirin 5mmol/L for 0,3,6,12,24 hours. EPCs were characterized as adherent cells double positive for DiLDL-uptake and lectin binding by direct fluorescent staining. EPCs proliferation, migration were assayed with MTT assay and modified Boyden chamber assay, respectively. EPCs adhesion assay was performed by replating them on fibronectin-coated dishes, and then adherent cells were counted. In vitro vasculogenesis activity was assayed by in vitro vasculogenesis kit. iNOS was assayed by western blot. Results: 1. Characteristics of human EPCs:Total MNCs isolated and cultured for 7 days resulted in a spindle-shaped, endothelial cells like morphology. EPCs were characterized as adherent cells double positive for DiLDL-uptake and lectin binding under a laser scanning confocal microscope. We and other investigators have previously demonstrated that endothelial progenitor cells isolated in this fashion also exhibit many other endothelial characteristics, including expression of CD31, Von Willebrand factor(vWF), and vascular endothelial growth factor receptor 2 (VEGFR-2), and so on.2. Effect of aspirin on EPCs numbers:Incubation of isolated human MNCs with aspirin decreased the number of EPCs in a concentration and time dependent manner.3. Effects of aspirin on the proliferative capacity of isolated EPCs:Incubation of isolated human MNCs with aspirin decreased EPCs proliferative capacity in a concentration and time dependent manner.4. Effects of aspirin on the migratory capacity of isolated EPCs:Incubation of isolated human MNCs with aspirin decreased EPCs migratory capacity in a concentration and time dependent manner.5. Effect of aspirin on the adhesive capacity of isolated EPCs:Incubation of isolated human MNCs with aspirin decreased EPCs adhesive capacity in a concentration and time dependent manner.6. Effects of aspirin on EPCs in vitro vasculogenesis:Incubation of isolated human MNCs with aspirin decreased EPCs in vitro vasculogenesis capacity in a concentration dependent manner.7. Effects of aspirin on EPCs iNOS:Western blot analysis showed that the expression of iNOS was significantly decreased in the cells treated with aspirin in a concentration dependent manner, strongly suggesting that the inhibitory effect of aspirin is mediated through decreasing NO.Conclusions: 1. EPCs can be isolated and cultured from mononuclear cells from peripheral blood.2. Aspirin decrease the number, proliferative, migratory, adhesive and in vitro vasculogenesis capacity of EPCs.3. Aspirin also decrease iNOS in EPCs.