Eugenia Rosa Nuzzolo

Endothelial progenitor cells are clonal and exhibit the JAK2 V617F mutation in a subset of thrombotic patients with Ph-negative myeloproliferative neoplasms

In this study we investigated whether neoplastic transformation occurring in Philadelphia (Ph)-negative myeloproliferative neoplasms (MPNs) could involve also the endothelial cell compartment. We evaluated the level of endothelial colony-forming cells (E-CFCs) in 42 patients (15 with polycythemia vera, 12 with essential thrombocythemia, and 15 with primary myelofibrosis). All patients had 1 molecular abnormality (JAK2(V617F) or MPL(W515K) mutations, SOCS gene hypermethylation, clonal pattern of growth) detectable in their granulocytes. The growth of colonies was obtained in 22 patients and, among them, patients with primary myelofibrosis exhibited the highest level of E-CFCs. We found that E-CFCs exhibited no molecular abnormalities in12 patients, had SOCS gene hypermethylation, were polyclonal at human androgen receptor analysis in 5 patients, and resulted in JAK2(V617F) mutated and clonal in 5 additional patients, all experiencing thrombotic complications. On the whole, patients with altered E-CFCs required antiproliferative therapy more frequently than patients with normal E-CFCs. Moreover JAK2(V617F)-positive E-CFCs showed signal transducer and activator of transcription 5 and 3 phosphorylation rates higher than E-CFCs isolated from healthy persons and patients with MPN without molecular abnormalities. Finally, JAK2(V617F)-positive E-CFCs exhibited a high proficiency to adhere to normal mononuclear cells. This study highlights a novel mechanism underlying the thrombophilia observed in MPN.

Endothelial progenitor cell trafficking in human immunodeficiency virus-infected persons.

Objective:Human immunodeficiency virus (HIV)-infected people exhibit a high incidence of vascular diseases. Since in the general population the high cardiovascular risk has been associated with an impaired endothelial cell function, we investigated circulating endothelial progenitor cells in HIV-positive patients. Design:We evaluated circulating colony-forming unit–endothelial cell (CFU-EC) and endothelial colony-forming cell (ECFC) progenitors in 14 antiviral therapy-naive HIV-positive patients, in comparison with 15 normal controls. Methods:CFU-EC and ECFC derived from peripheral blood mononuclear cells from HIV-infected and HIV-uninfected individuals were recovered and evaluated for HIV genome presence by PCR. Vascular endothelial growth factor (VEGF) and apolipoprotein B mRNA-editing enzyme catalytic polypeptide like (APOBEC) subunits expression were evaluated in infected colonies by real-time PCR. Results:We found that circulating CFU-EC but not ECFC were significantly reduced in HIV-positive patients and that proviral HIV DNA was detectable only in CFU-EC but not in ECFC. Furthermore, the expression of APOBEC subunits was significantly lower in CFU-EC than in circulating monocytes. Accordingly, the CFU-EC displayed a high content of proviral DNA copies, suggesting that these cells have a high sensitivity to the HIV infection. Conclusions:Although HIV does not affect the ‘true endothelial progenitor’ compartment, it infects and strongly depletes circulating endothelial progenitors with hematopoietic signature. We unravel a novel pathogenetic mechanism by which HIV infection might cause vascular diseases.

Cyclooxygenase-2 (COX-2) inhibition constrains indoleamine 2,3-dioxygenase 1 (IDO1) activity in acute myeloid leukaemia cells

Indoleamine 2,3-dioxygenase 1 (IDO1) metabolizes L-tryptophan to kynurenines (KYN), inducing T-cell suppression either directly or by altering antigen-presenting-cell function. Cyclooxygenase (COX)-2, the rate-limiting enzyme in the synthesis of prostaglandins, is over-expressed by several tumours. We aimed at determining whether COX-2 inhibitors down-regulate the IFN-γ-induced expression of IDO1 in acute myeloid leukaemia (AML) cells. IFN-γ at 100 ng/mL up-regulated COX-2 and IDO1 in HL-60 AML cells, both at mRNA and protein level. The increased COX-2 and IDO1 expression correlated with heightened production of prostaglandin (PG)E2 and kynurenines, respectively. Nimesulide, a preferential COX-2 inhibitor, down-regulated IDO1 mRNA/protein and attenuated kynurenine synthesis, suggesting that overall IDO inhibition resulted both from reduced IDO1 gene transcription and from inhibited IDO1 catalytic activity. From a functional standpoint, IFN-γ-challenged HL-60 cells promoted the in vitro conversion of allogeneic CD4+CD25− T cells into bona fide CD4+CD25+FoxP3+ regulatory T cells, an effect that was significantly reduced by treatment of IFN-γ-activated HL-60 cells with nimesulide. Overall, these data point to COX-2 inhibition as a potential strategy to be pursued with the aim at circumventing leukaemia-induced, IDO-mediated immune dysfunction.

Human cord blood endothelial progenitors promote post-ischemic angiogenesis in immunocompetent mouse model

BACKGROUNDnHuman cord blood (CB) endothelial colony forming cells (ECFCs) are endowed with high vascular regenerative ability in immunodeficient mice, but their immunogenicity and susceptibility to rejection in immunocompetent models has yet to be explored.nnnMETHODSnWe injected CB ECFCs in non-immuno-suppressed C57BL/6J mice after having induced the hindlimb ischemia and we investigated their contribution to the recovery from the ischemic injury. Human ECFCs (hECFCs) were administered by intramuscular injection and hindlimb blood perfusion was measured by laser Doppler analysis at 7-day intervals for 28days after treatment. Mice were sacrificed after 7 and 28days and immunohistochemistry for specific human (CD31) and mouse (von Willebrand factor) endothelial antigens was carried out. Before euthanasia, blood samples to assess cytokines and angiogenic growth factor levels were collected.nnnRESULTSnMice injected with hECFCs showed a prompter and greater recovery of blood flow than controls. Several endothelial cells of human origin were detected at day7 after injection and their number declined progressively. Likewise, a progressive increase of mouse-derived vascular structures were observed, paralleled by the amplified endogenous production of various soluble mediators of angiogenesis, including Vascular Endothelial Growth Factor and Fibroblast Growth Factor.nnnCONCLUSIONSnOverall, our findings are consistent with the hypothesis that human ECFCs might expand the endogenous vascular repair potential of recipients and support their possible HLA-independent unconventional use.

Endothelial progenitor cell dysfunction in myelodysplastic syndromes: possible contribution of a defective vascular niche to myelodysplasia.

We set a model to replicate the vascular bone marrow niche by using endothelial colony forming cells (ECFCs), and we used it to explore the vascular niche function in patients with low-risk myelodysplastic syndromes (MDS). Overall, we investigated 56 patients and we observed higher levels of ECFCs in MDS than in healthy controls; moreover, MDS ECFCs were found variably hypermethylated for p15INK4b DAPK1, CDH1, or SOCS1. MDS ECFCs exhibited a marked adhesive capacity to normal mononuclear cells. When normal CD34 + cells were co-cultured with MDS ECFCs, they generated significant lower amounts of CD11b + and CD41 + cells than in co-culture with normal ECFCs. At gene expression profile, several genes involved in cell adhesion were upregulated in MDS ECFCs, while several members of the Wingless and int (Wnt) pathways were underexpressed. Furthermore, at miRNA expression profile, MDS ECFCs hypo-expressed various miRNAs involved in Wnt pathway regulation. The addition of Wnt3A reduced the expression of intercellular cell adhesion molecule-1 on MDS ECFCs and restored the defective expression of markers of differentiation. Overall, our data demonstrate that in low-risk MDS, ECFCs exhibit various primary abnormalities, including putative MDS signatures, and suggest the possible contribution of the vascular niche dysfunction to myelodysplasia.

Adult and cord blood endothelial progenitor cells have different gene expression profiles and immunogenic potential

BACKGROUNDnEndothelial colony-forming cells (ECFC) are endowed with vascular regenerative ability in vivo and in vitro. In this study we compared the genotypic profile and the immunogenic potential of adult and cord blood ECFC, in order to explore the feasibility of using them as a cell therapy product.nnnMATERIALS AND METHODSnECFC were obtained from cord blood samples not suitable for haematopoietic stem cell transplantation and from adult healthy blood donors after informed consent. Genotypes were analysed by commercially available microarray assays and results were confirmed by real-time polymerase chain reaction analysis. HLA antigen expression was evaluated by flow-cytometry. Immunogenic capacity was investigated by evaluating the activation of allogeneic lymphocytes and monocytes in co-cultures with ECFC.nnnRESULTSnMicroarray assays revealed that the genetic profile of cord blood and adult ECFC differed in about 20% of examined genes. We found that cord blood ECFC were characterised by lower pro-inflammatory and pro-thrombotic gene expression as compared to adult ECFC. Furthermore, whereas cord blood and adult ECFCs expressed similar amount of HLA molecules both at baseline and after incubation with γ-interferon, cord blood ECFC elicited a weaker expression of pro-inflammatory cytokine genes. Finally, we observed no differences in the amount of HLA antigens expressed among cord blood ECFC, adult ECFC and mesenchymal cells.nnnCONCLUSIONSnOur observations suggest that cord blood ECFC have a lower pro-inflammatory and pro-thrombotic profile than adult ECFC. These preliminary data offer level-headed evidence to use cord blood ECFC as a cell therapy product in vascular diseases.

Hypoxia-inducible factor-1α(Pro-582-Ser) polymorphism prevents iron deprivation in healthy blood donors.

BACKGROUNDnFrequent blood loss induces progressive depletion of iron stores, leading to iron deficiency and, ultimately, to overt iron-deficient anaemia. The erythropoietin-mediated bone marrow response to anaemia is under the control of hypoxia-inducible factors (HIF), the master regulators of oxygen and iron homeostasis. Since the HIF-1α(Pro-582-Ser) variant is associated with elevated trans-activation capacity of hypoxia responsive elements of target genes, we investigated whether the HIF-1α(Pro-582-Ser) polymorphism might influence the response to repeated blood withdrawals.nnnMATERIALS AND METHODSnUsing polymerase chain reaction analysis and DNA sequencing, we retrospectively investigated the presence of HIF-1α(Pro-582-Ser) in a series of 163 blood donors. Haematological findings, serum ferritin levels and frequency of donations were compared according to the mutational status of the HIF-1α gene.nnnRESULTSnWe found that male carriers of the HIF-1α(Pro-582-Ser) polymorphism had higher haemoglobin and ferritin levels than individuals homozygous for the wild-type allele. Moreover, the HIF-1α(Pro-582-Ser) polymorphism protected regular blood donors from developing iron deficiency and anaemia and predicted uninterrupted donation activity.nnnDISCUSSIONnThese findings show for the first time that the HIF-1α(Pro-582-Ser) polymorphism significantly affects red blood cell and iron homeostasis after blood loss, conferring to male carriers a resistance to anaemia. Regarding the female gender, large series of individuals should be investigated to establish whether there is an effect of the HIF-1α(Pro-582-Ser) polymorphism in this population. Although these data need to be confirmed in prospective studies, they could have important implications in blood donor selection and donation procedures.

Endothelial progenitor cells and thrombosis

The remodelling of existing vessels (i.e. angiogenesis) and the de novo vessel formation (i.e. vasculogenesis) occur not only during the embryonic development but also over the entire postnatal life. In 1997, the Asahara group first reported that endothelial progenitor cells circulate in peripheral blood and that they are recruited at sites of ischemia, thus proving that these cells are able to promote vasculogenesis. Since then, several different approaches have been set up to investigate endothelial progenitor cells. This review summarizes the different modalities utilized to enumerate these cells, delineates their involvement in the haemostatic pathways, and depicts their altered trafficking during cardiovascular diseases. Finally, recent observations suggesting a primary role for endothelial progenitors in particular situations such as pulmonary arterial hypertension or Philadelphia negative-myeloproliferative neoplasms are discussed.

Effect of antiviral therapy on pro-angiogenic hematopoietic and endothelial progenitor cells in HIV-infected people

BACKGROUNDnHIV infection is an independent risk factor for cardiovascular disease. HIV-sustained impairment of endothelial progenitor cells (EPCs) could contribute to this process, so that it is important to assess whether antiviral therapy (ART) is able to revert these abnormalities.nnnMETHODSnWe quantified in 21 naïves and 34 treated patients two functionally distinct clonogenic progenitors which have been acknowledged important for vascular repair: the hematopoietic progenitor colony forming unit - endothelial cells (CFU-EC) and the true endothelial progenitor, the endothelial colony forming cells (ECFC). We correlated results obtained with conventional vascular risk factors and with HIV-related parameters.nnnRESULTSnWe found that these progenitors behaved differently in naive and treated patients. In particular, CFU-EC level was significantly low in all naive patients and slowly recovered during ART. In contrast, the ECFC level was abnormally high in naive patients while it decreased upon ART. The CFU-EC level was related to conventional cardiovascular risk factors, as reported in general population, but also to inflammatory indexes and CD4 cell count. In contrast, the ECFC number was exclusively related to viral replication activity and to CD4 cell count.nnnCONCLUSIONSnIn HIV-infected people, the levels of CFU-EC and ECFC are related to classical cardiovascular risk factors but, in addition, they are also significantly influenced by the infection itself and by antiviral therapy.

Endothelial progenitor cells in HIV-positive patients: what are we talking about?

To the Editors: Gómez-Garre et al 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 HIVinfected patients. Indeed, the authors concluded that their findings can explain the proatherogenic effect of ART. 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 studies 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 al observed that naive HIVinfected patients have EPC levels lower than general people. On the opposite, Costiniuk et al found that naive HIVpositive patients have similar EPC levels as healthy controls, whereas Papasavvas et al reached further different conclusions demonstrating that naive HIV-positive patients have EPC levels higher than HIVnegative individuals (Table 1). Moreover, in striking contrast with the aforementioned study of Gómez-Garre et al, Papasavvas et al argued that, similarly to naive patients, patients receiving ART have a persistent increase of EPC output. These discordances led to unavoidable controversies, 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, 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. 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. Basically, 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 al and Yoder et al, 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. 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 al 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. 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. 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). 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.