ELEVATED CIRCULATING ENDOTHELIAL CELLS AND SUCCESS IN ENDOTHELIAL COLONY-FORMING CELLS ISOLATION

Abstract

Circulating endothelial cells (CEC) have been associated with vascular injury and are described as potential biomarkers for cardiovascular disease (1). Besides, current optimized methodologies enable the isolation of a well-characterized subtype of endothelial progenitor known as Endothelial colony-forming cells (ECFCs) (2,3). Although ECFC isolation methodologies are well described; some discrepancies remain in relation to their isolation efficiency. To evaluate the isolation efficacy ECFCs and CEC frequency in human peripheral blood. The Ethics Research Committee of the University of Campinas approved the experimental procedures and all donors signed the informed consent form. CEC enumeration was assessed by flow cytometry in the peripheral blood of 18 donors. 100 μL of blood (with a leukocyte concentration between 5 and 10 ×10³/μL) was incubated with monoclonal anti-human antibodies (CD45 PerCp, CD133 APC, CD31 FITC, and CD146 PE). FACS lysing solution was used for red blood cell removal. 300,000 events or the total volume of the tube were acquired using a FACSCalibur® flow cytometer (4). To determine the number of cells/μL, the percentage of events was multiplied per number of leucocytes and divided per 100. Cell culture from venous blood was used to isolate ECFCs which were confirmed by their cobblestone morphology and immunophenotyping for endothelial markers (CD31, CD146, CD309, and CD144). ECFC isolation was successful in 9 out of 18 donors (1 male, 8 females; mean age: 35.8 ±11.1 years), and not successful in the other half of donors (2 male, 7 females; mean age: 37.7 ±9.7 years). CEC frequency was significantly higher (p=0.004) in the group with successful ECFC isolation when compared to the group that did not yield ECFCs (2.9 ±1.2 CECs/μL against 1.2 ±0,8 CECs/μL). This suggests that the success in ECFC isolation might be related to the number of CECs, and therefore ECFC mobilization into circulation may also be linked to vascular injury. Increased number of CECs might be associated with the success of ECFCs isolation.