Comparative Anti-Platelet Profiling Reveals a Potent Anti-Aggregatory Effect of CD34+ Progenitor Cell-Derived Late-Outgrowth Endothelial Cells in vitro

Abstract

Background/Aims: Platelets affect endothelial progenitor cell (EPC) functionality, inducing their differentiation into mature endothelial cells. However, it remains to be established whether EPCs can influence platelet functionality. Methods: Mononuclear proangiogenic cells (MPCs) and early outgrowth cells (EOCs) were prepared from peripheral blood mononuclear cells, whereas late-outgrowth endothelial cells (OECs) were prepared from cord blood CD34+ cells. The effect of the above cells and their supernatants on washed platelet aggregation was studied. The effect of OECs and their supernatant on the adenosine diphosphate (ADP)-induced magnitude of platelet integrin receptor α_IIbβ₃ activation and on P-selectin membrane expression was investigated. The levels of nitric oxide (NO) and prostacyclin (PGI₂) that were secreted from EOCs, OECs, and human umbilical vein endothelial cells (HUVECs) were also assessed. Results: Among all progenitors, OECs and their supernatant exhibited the most potent inhibitory activity towards platelet aggregation. Furthermore, OECs and their supernatant, but not CD34+ cells, reduced α_IIbβ₃ activation and P-selectin membrane expression. Finally, OECs secreted higher NO and PGI₂ levels than EOCs did. Conclusion: The anti-platelet effect of EPCs depends highly on the degree of their endothelial phenotype, with OECs expressing the highest potency. Therefore, the induction of OEC generation and the enhancement of their functionality in vivo could be a new approach for the treatment of patients at a high thrombotic risk.

The Effect of Platelet-Rich Plasma on Endothelial Progenitor Cell Functionality

“…CD34 þ cells were isolated from human cord blood mononuclear cells using MACS MicroBeads and cultured, as previously described. 26 Briefly, cord blood was collected into a tube containing 20 mM EDTA/phosphate-buffered saline solution as an anticoagulant and mononuclear cells were separated by density gradient centrifugation. Isolation of CD34 þ progenitor cells was performed using the human CD34 MicroBead Kit according to the manufacturer's instructions.…”

“…The medium was changed daily for the first 7 to 14 days and every 2 days until the appearance of endothelial colonies, which were cultured until the formation of a confluent monolayer of highly proliferative OECs. Outgrowth endothelial cells were characterized as previously described by our group 26 and others. 27 All experiments of the present work were performed using OECs of passage 3, cultured with EGM-2 Bullet Kit supplemented with 10% FBS until confluent.…”

“…The P-selectin membrane expression was determined by flow cytometry in a FACSCalibur flow cytometer (Becton-Dickinson) using the CellQuest Software (Becton Dickenson) as we previously described. 26 Results are expressed as MFI (mean fluorescence intensity). 26 In other experiments, platelets in 500 mL PRP were incubated for 5 minutes under continuous stirring (1200 rpm), at 37 C in a Chrono-Log Model 700-4DR Whole Blood/Optical Lumi-Aggregometer equipped with the AggroLink software package.…”

“…26 Results are expressed as MFI (mean fluorescence intensity). 26 In other experiments, platelets in 500 mL PRP were incubated for 5 minutes under continuous stirring (1200 rpm), at 37 C in a Chrono-Log Model 700-4DR Whole Blood/Optical Lumi-Aggregometer equipped with the AggroLink software package. In some experiments, platelets were aggregated in the presence of 20 mm ADP; whereas in other experiments, the PRP was preincubated in the aggregometer with 4 mM ticagrelor (or its vehicle; DMSO) for 2 minutes before the addition of 20 mm ADP.…”

Interleukin-17A Triggers the Release of Platelet-Derived Factors Driving Vascular Endothelial Cells toward a Pro-Angiogenic State

“…In vivo platelet depletion ameliorates atherothrombosis [15][16][17] whilst at the same time it impairs angiogenesis [18,19] and tissue regeneration [20][21][22][23]. Platelets modulate vascular and tissue inflammatory events either through cellular interactions [24][25][26][27][28][29][30][31][32] or through differential releasing of immunomodulatory ligands indirectly regulating the function of other blood and vascular wall cells [33,34]. We have previously shown that platelet-derived stromal cell-derived factor-1 (SDF-1) is critically involved in the chemotaxis and recruitment of blood cells to vascular endothelium controlling vascular inflammation and post-injury tissue healing [35][36][37][38].…”