ReviewTechnical notes on endothelial progenitor cells: Ways to escape from the knowledge plateau
Introduction
Since 1997 [1], a huge amount of literature has described the involvement of endothelial progenitor cells (EPCs) in cardiovascular disease. EPCs were originally thought to derive exclusively from the bone marrow, but recent studies have indicated alternative sources of EPCs, including parenchymatous organs and blood vessels [2], [3]. We have learned from animal studies that EPCs participate in endothelial homeostasis and stimulate the formation of new blood vessels [4], [5]. Clinically, EPCs have been found to be reduced in peripheral blood of subjects with cardiovascular risk factors and/or established atherosclerosis [6], [7], [8], [9], [10], [11], [12]: this is why depletion of circulating EPCs is considered a marker of the ongoing vascular damage [13]. Additionally, EPC reduction is an independent predictor of future cardiovascular events [14], [15], a notion that strengthens the concepts that EPCs are at the same time markers and actors in the entire atherogenetic process. Most importantly, cell therapies that restore the EPC pool have proven beneficial in patients with coronary and peripheral artery disease [16], [17]. Really, this has been a revolution, as EPCs are now considered an integrated part of the cardiovascular system.
Unfortunately, expanding interest and lack of a consensus have multiplied the number of different methodologies to study EPCs. A myriad of small studies are now available, each using slightly or profoundly different methods. Likely, this will ingenerate a sort of “knowledge plateau”: because of mutual inconsistencies, new studies often raise more questions than they answer. Herein, we would like to focus attention on a few simple technical aspects that all researches should consider when approaching the study of EPCs.
To start, let us define what an EPC is: a cell that derives from the bone marrow and probably from other sources [18], circulates in the bloodstream, proliferates, and can differentiate into a mature endothelial cell [19]. Basically, there are two techniques to study EPCs: (i) flow cytometry of fresh samples and (ii) cell culture.
Section snippets
Flow cytometry
Cell count by flow cytometry is based on immunolabelling cells with antibodies directed against surface or intracellular antigens. This method has two important limitations: first, we do not know the precise antigenic phenotype of EPCs, mainly because it overlaps with that of other cell lineages (this is why we should always refer to as “putative EPCs”). Second, definition of EPCs by flow cytometry implies a conceptual abstraction, because a presumed function is attributed to a relatively
Cell culture
Ex vivo analysis of blood cells by flow cytometry can only count EPCs and quantify the expression of a limited number of surface or intracellular antigens. To gather qualitative data, we are compelled to isolate EPCs from the bloodstream and, given their very low frequency, to expand them in culture. The variety of culture protocols used to isolate EPCs renders this method even more intricate than flow cytometry, and caution should be paid when choosing the right protocol. We were initially
Conclusions
We have discussed in detail the technical aspects of flow cytometry and culture methods commonly used to study EPCs. It is clear that researchers should pay attention to a variety of insidious confounders and potential biases when choosing the most appropriate protocol for a given study (Table 1).
Our opinion is that, when EPCs are used as a surrogate marker of cardiovascular risk or vascular damage, pure quantitative data should be obtained with flow cytometry of fresh blood samples. The
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2020, International Journal of CardiologyAssociation between Type I interferon and depletion and dysfunction of endothelial progenitor cells in C57BL/6 mice deficient in both apolipoprotein E and Fas ligand
2018, Current Research in Translational MedicineThoughts modulate the expression of inflammatory genes and may improve the coronary blood flow in patients after a myocardial infarction
2018, Journal of Traditional and Complementary MedicineCitation Excerpt :In blood samples we assessed the following NEI molecules: stress mediators (cortisol, corticotropin (ACTH), copeptin, epinephrine, norepinephrine, insulin, thyroid-stimulating hormone (TSH), growth hormone (GH), testosterone, dehydroepiandrosterone (DEHA-S), prolactin (PRL)), inflammatory markers (erythrocyte sedimentation rate (ESR), fibrinogen, highly sensitive C-reactive protein (HS-CRP), interleukin-6 (IL-6), transforming growth factor beta-1 (TGFβ-1), galectin-3), markers of oxidative stress (malondialdehyde (MDA), asymmetrical dimethyl arginine (ADMA)), a cellular stress marker (high mobility group box1 protein (HMGB-1)) and endocannabinoids (eCBs) (N-arachidonylethanolamine (anandamide, AEA) and 2-arachidonoylglycerol (DAG)) able to counteract the stress axis activation. The dosage of endothelial progenitor cells (EPCs) was also provided according to a well known procedure.11 Moreover, we evaluated the expression of NFkB, p53 and Toll-like-receptor-4 (TLR4) genes in circulating peripheral blood mononuclear cells (PBMCs) in accordance with a procedure already described.8