Endothelial Progenitor Cells in HIV-Positive Patients (2012)

To the Editors: Gómez-Garre et al1 recently reported that HIV-infected patients receiving antiretroviral therapy (ART) have endothelial progenitor cell (EPC) levels lower than naive patients and that ART exposure is the main predictor for EPC of in HIV-infected patients. Indeed, the authors concluded that their findings can explain the proatherogenic effect of ART.1 Actually, the contribution of EPC dysfunction to the pathogenesis of HIV-related cardiovascular diseases is an important topic, considering that both the virus and ART can affect this cell population. Among various studies1–5 investigating EPC levels in HIV-infected patients, most studies quantified EPCs by analyzing different antigen combinations through flow cytometry (Table 1). Unfortunately, the data reported are often conflicting each other. In example, Lopez et al2 observed that naive HIV-infected patients have EPC levels lower than general people. On the opposite, Costiniuk et al3 found that naive HIV-positive patients have similar EPC levels as healthy controls, whereas Papasavvas et al4 reached further different conclusions demonstrating that naive HIV-positive patients have EPC levels higher than HIV-negative individuals (Table 1). Moreover, in striking contrast with the aforementioned study of Gómez-Garre et al,1 Papasavvas et al4 argued that, similarly to naive patients, patients receiving ART have a persistent increase of EPC output. These discordances led to unavoidable controversies,6,7 which are the tip of the iceberg of a wider debate occurring in the vascular cell community on the most appropriate phenotype combination for defining EPC. Unfortunately, this challenge remained unsolved, so far. Actually, after the seminal study of Asahara et al,8 it became evident that cells contributing to the new vessel formation (ie, angiogenic cells) are not a homogeneous population but rather they include various cell types with different lineage derivation, proliferative capacity, and functions.9 This cell pool expresses such a large spectrum of antigens so that it is nearly impossible to figure out an univocal phenotype marker. However, despite the drawback of immunophenotype identification of EPCs, the majority of studies performed in general people and in HIV-infected persons do not carry out functional assays to understand if what they have named EPCs are really immature progenitors, able to proliferate and produce a mature endothelial cell progeny, which directly and definitely takes part in the new vessel building, or in contrast, if they analyzed a heterogeneous population, including rare immature endothelial progenitors among numerous mature endothelial cells. As a consequence, the lack of functional correlates of immunophenotype-defined EPCs led to a controversial interpretation of the results of several clinical trials.10TABLE 1: Studies on EPCs in HIV-Positive PatientsBasically, we believe that this debate clearly demonstrates that there is one important issue to address: do really immunophenotype-defined cells accurately reflect the number of EPCs? Ingram et al11 and Yoder et al12, by using a rigorous and extensive laboratory work based on cell cultures techniques, distinguished at least two clonogenic progenitors with endothelial phenotype: the “colony forming unit–endothelial cells” (CFU-ECs) and the “endothelial colony forming cells” (ECFCs). The CFU-ECs derive from the hematopoietic lineage and promote neoangiogenesis mainly by local secretion of growth factors. In contrast, the ECFCs lack myeloid markers, have an extensive proliferative potential, and they do produce functional blood vessels. Up to date, the ECFC is the only EPC unequivocally identified for its ability to originate a progeny of mature endothelial cells.11,12 Overall, these findings provided the rationale for restricting the flow cytometry detection of EPC within the CD45− gated cell population (ie, non-hematopoietic cells); nonetheless, the problem of discriminating, rare circulating endothelial progenitors from circulating endothelial cells still persists. In a recent study, Mund et al13 attempted to definitely address this issue, by using a complex approach including polychromatic flow cytometry incorporating the DNA staining, immunomagnetic cell sorting, electron microscopy, and functional assays. As expected, both progenitors and mature cells express CD34 and other endothelial markers (CD31, CD105, and CD146), whereas they lack CD45 and CD133. More strikingly, the authors demonstrated that conventional flow cytometry leads to the misidentification of endothelial cells (both progenitors and mature cells) for the presence of a masking cell population constituted by erythrocytes, monocytes, dead/apoptotic cells, and circulating platelet and endothelial extracellular vesicles.13 The unavoidable consequence is that it will be necessary to thoroughly reexamine past conclusions about EPC frequencies in different diseases and patient groups because almost all evidences from the literature in the past 15 years have been based on a misidentified cell population.14 As far as the issue if and how HIV affects EPC is concerned, we first demonstrated in naive patients that HIV infects and destroys angiogenic cells with hematological signature (ie, CFU-EC) but not the true endothelial progenitor (ie, ECFC).5 It remains to address if this impairment contributes to the development of HIV-related cardiovascular diseases and the eventual effect of antiviral therapy on angiogenic cells. We believe, therefore, that before giving any specific message, all researchers involved in this field should raise the level of awareness by using functional assays in addition to flow cytometry quantification, to avoid possible misleading interpretations, and shed more clarity on what we are really talking about.