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Platelet-rich plasma (PRP) is an inexpensive and safe substitute of recombinant growth factors in Vitro and in vivo. Due to its putative effect on tissue repair, the use of autologous PRP has become largely popular. Recently, a jellified PRP derivative obtained from umbilical cord blood (CB) has been utilized in vivo to treat mucosal and cutaneous lesions. Nevertheless, whether PRP derived from CB and adult blood display different potency in promoting cell growth in Vitro has been rarely investigated. In this study, we compared cytokine profile and mesenchymal cell growth supporting the ability of platelet lysate obtained from adult and cord blood. Our in vitro results strongly back the utilization of CB platelet lysate in vivo, as an efficacious, safe and inexpensive alternative to promote damaged tissue healing when the autologous PRP is contraindicated. Moreover, the policy of manufacturing CB platelet lysate can limit the current disposal of many collected CB units not suitable for transplant due to their low nucleated cell count.

Background Human 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. Methods We injected CB ECFCs in non-immuno-suppressed C57BL/6 J 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 28 days after treatment. Mice were sacrificed after 7 and 28 days 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. Results Mice 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. Conclusions Overall, 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.

Background HIV 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. Methods We quantified in 21 naives 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. Results We 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. Conclusions In 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.

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.

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.

Endothelial cells are relevant to normal hematopoiesis. Vascular niches within the bone marrow allow hematopoietic stem cell proliferation and differentiation. In vivo, the contemporary administration of endothelial progenitor cells in BALB/c mice fosters the homing of transplanted hematopoietic cells (Salter et al. Blood 2009; 113:2104). In vitro, the differentiation of CD34+ progenitors is facilitated by the presence of an underlying confluent layer of endothelial colony forming cells (ECFCs Ingram et al. Blood. 2004;104: 2752), even though this effect is highly dependent from the physical contact between hematopoietic and endothelial cells (Teofili et al, Blood , ASH Annual Meeting, 2012;120:1718). In Myelodysplastic Syndromes (MDS) the dysfunction of vascular niches might contribute to cause the ineffective hematopoiesis typical of the disease. Accordingly, we found that normal CD34+ cells cultured over ECFCs isolated from patients with low risk MDS exhibit a deep perturbation of the expression of lineage-specific genes, with higher expression of genes involved in the early differentiation (such as PU.1 and RUNX1) and lower expression of genes usually up-regulated during the late differentiation (such as MPO and GP1b) (Teofili et al, Blood, ASH Annual Meeting, 2012; 120:1718). In order to figure out the underlying pathogenesis, we compared the cytokine milieu in the media of MDS and normal ECFCs cultures (Bio-Plex pro-human cytokine 27-plex assay, (Bio-Rad). We found that in MDS cultures, significant higher amounts of IL13, IL1b, PDGFb and VEGF were secreted, whilst the levels of IL6, IL7, IL10. G-CSF, CXCL-10, CCL-2 and CCL-3 were significantly lower than in normal ECFC cultures (Teofili et al. Leukemia Research 2013; 37, Supp.1, S129-S130). Moreover, we evaluate through PCR arrays the gene expression profiles of normal and MDS ECFCs, focusing on genes involved in both endothelial cell biology and WNT signaling (QIAGEN, Milan, Italy). In comparison with normal ECFCs, MDS ECFCs overexpressed various adhesive molecules such as ICAM-1, L-selectin and V-CAM. This over-expression resulted in the more pronounced capacity of MDS ECFCs to adhere to hematopoietic cells in comparison with normal ECFCs, as demonstrated by the significant higher proportion of CFSE-labeled mononuclear cells trapped within the different adherent layer after 6 hours-incubation (40+11 % versus 14+4% in MDS and normal ECFC, respectively). Furthermore, at PCR arrays, we found that MDS ECFCs showed the down-regulation of several members of the canonical WNT pathway, including WNT3a, WNT5a and WNT5b. Basically, the WNT/bcatenin pathway is the main regulator of the microenvironment dependent hematopoietic stem cell fate, both in steady state and in regenerative processes. Therefore we investigated if the addition of WNT3a and WNT5a to MDS ECFC cultures affected their over-expression of adhesion molecules. To this purpose, we added to the culture medium 100 ng/ml of purified WNT3a and WNT5a (both purchased from R&D). We found that both WNT3a and WNT5a lowered the expression of several adhesion molecules in MDS ECFCs, including L-selectin, ICAM and VCAM, with WNT3a showing more pronounced and durable effects. On the whole our findings suggest that the ineffective hematopoiesis typical of MDS may be partly due to the abnormal entrapping of hematopoietic progenitors within the vascular niche, in the presence of a deeply perturbed surrounding cytokine milieu. Since our findings allege the defective WNT pathway as putative responsible defect, restoring the adequate WNT signaling could represent a potential therapeutic approach to MDS. The study was supported by the Grant AIRC 2012 n.11799 Disclosures: No relevant conflicts of interest to declare.

Chronic HIV infection induces significant changes in the trafficking of circulating endothelial progenitor cells (EPCs). Specifically, it causes marked depletion of proangiogenic hematopoietic cells, the so-called colony-forming unit-endothelial cells (CFU-ECs). In this study we evaluated CFU-ECs in two subjects with acute HIV infection. We found that both patients already had a low CFU-EC level at the time of diagnosis. Nevertheless, after 6 months of antiretroviral therapy, the CFU-EC concentration reverted to normal values in both cases. HIV significantly depletes the CFU-EC compartment even in the early phase of infection, while 6-month therapy appears to be able to restore it.

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.

MicroRNAs (miRNAs or miRs) regulate diverse normal and abnormal cell functions. We have identified a regulatory pathway in normal megakaryopoiesis, involving the PLZF transcription factor, miR-146a and the SDF-1 receptor CXCR4. In leukaemic cell lines PLZF overexpression downmodulated miR-146a and upregulated CXCR4 protein, whereas PLZF knockdown induced the opposite effects. In vitro assays showed that PLZF interacts with and inhibits the miR-146a promoter, and that miR-146a targets CXCR4 mRNA, impeding its translation. In megakaryopoietic cultures of CD34(+) progenitors, PLZF was upregulated, whereas miR-146a expression decreased and CXCR4 protein increased. MiR-146a overexpression and PLZF or CXCR4 silencing impaired megakaryocytic (Mk) proliferation, differentiation and maturation, as well as Mk colony formation. Mir-146a knockdown induced the opposite effects. Rescue experiments indicated that the effects of PLZF and miR-146a are mediated by miR-146a and CXCR4, respectively. Our data indicate that megakaryopoiesis is controlled by a cascade pathway, in which PLZF suppresses miR-146a transcription and thereby activates CXCR4 translation.

An abnormal secretion of soluble mediators contributes to the hematopoietic-niche dysfunction in low-risk myelodysplastic syndrome

The bone marrow (BM) vascular niche has an important role in supporting adult hematopoietic stem cells (HSC) proliferation and differentiation. Using expanded circulating endothelial colony forming cells (ECFCs) of patients with low risk MDS patients and healthy donors as a surrogate of the vascular niche, we previously demonstrated in MDS patients an impaired talking between endothelial and hematopoietic cells. Molecular studies have revealed that MDS ECFCs show the promoter hypermethylation of p15INK4b, DAPK1, CDH1 and SOCS1 genes and have an altered gene expression profile, including adhesion, angiogenesis, cell activation, cell survival and apoptosis pathways. We also demonstrated that the Wingless and int (Wnt) pathway, one of the main regulators of cell-to-cell interactions in hematopoietic tissues (in particular Wnt3 and Wnt5) was down regulated at multiple levels in MDS ECFCs in comparison to the normal counterpart. As normally ECFCs regulate hematopoiesis by autocrine/paracrine secretion of various soluble mediators such as growth factors, cytokines and chemokines, we also investigated their production by MDS ECFCs. Moreover, since the release of angiogenic factors from BM microvasculature is regulated by specific microRNA (miRNA), we investigated the expression profile of a set of 84 miRNAs including those with predictable effects on the Wnt pathway using the miScript miRNA PCR array (Qiagen, Milan, Italy). We cultured normal CD34+ over a layer of MDS and normal ECFCs for 8 days in different cytokine-containing media able to induce selective granulocytic-macrophage, erythroid or megakaryocyte differentiation. At baseline and after 5 and 8 days of co-culture, we collected supernatants that were analyzed by Bio-Plex Pro™ human cytokine 27-plex immunoassay using Bio-Plex 200 system (Bio-Rad, Milan, Italy). We observed that in co-cultures with normal hematopoietic cells and MDS ECFCs, the level of several soluble mediators was significantly different in normal or MDS ECFC co-coltures. In particular, MDS co-cultures produced significant higher amounts of IL-1b, IL5, IP10, IL13 and VEGF (p≤0.05). Conversely, IL6, IL10, IL8 and RANTES were produced in lower quantities in MDS co-cultures (p≤0.05). These soluble mediators have a pleiotropic role and affect, directly or indirectly, different cellular pathways such as cell adhesion, hematopoietic differentiation, cell survival and angiogenesis. We also observed that their alterations were present in all the three lineages of differentiation but were more marked in megakaryocytic and myeloid than in erythroid differentiation. Moreover, the miRNA array revealed that MDS ECFCs contained significant lower amounts of miR-142-3p, miR-424-5p and miR-196b-5p (p=0.003, p=0.001 and p=0.033, respectively). These miRNAs are all variously involved in hematopoiesis or angiogenesis; in particular, miR-142-3p has been demonstrated to modulate the Wnt pathway and to down-regulate the expression of BMP4, a powerful inducer of erythroid differentiation which was highly expressed by MDS ECFCs. Considering that the addition of Wnt3 and Wnt5 to cultures restored only partially the normal differentiation of hematopietic cells, the abnormal secretion of soluble mediators as well as the altered expression of miRNAs might contribute to the dysfunction of vascular niches leading to myelodysplasia. This study was supported by AIRC grant to LML. Disclosures No relevant conflicts of interest to declare.

Avoidance of immunosurveillance is presently considered as a hallmark of cancer. Indoleamine 2,3-dioxygenase (IDO) is a cytosolic enzyme which metabolizes L-tryptophan to kynurenines and induces T cell suppression either directly on T cells, or by altering antigen presenting cell function and generating regulatory T cells (Treg). Recently, IDO expression by cancer cells and/or cancer-associated stromal cells has been correlated with the induction of immune dysfunction by human solid tumors, such as ovarian and colon cancer. It has been previously shown that IDO may be expressed by human acute myeloid leukemia, where it promotes the differentiation of Treg cells (Curti A et al. Blood2007;109:2871–7). Cyclooxygenase (COX) is the rate-limiting enzyme in the synthesis of prostaglandins and exists in two isoforms, COX-1 and COX-2. COX-2 is over-expressed by several tumor types and reportedly affects multiple pathways involved in tumorigenesis, including angiogenesis, invasion, and tumor-induced immune suppression. The present study aimed to determine whether COX-2 inhibitors may interfere with the IFN-γ-induced expression of IDO-1 in leukemia cells. The IDO-negative HL-60 acute myeloid leukemia cells, characterized by a CD34− CD33+CD119+ phenotype, were challenged with 100 ng/ml IFN-γ, a well-known inducer of IDO. Cytokine-treated HL-60 cells markedly up-regulated IDO-1 mRNA (~ 66-fold after 96 hours of culture) and protein and released a significant amount of kynurenines (12.58±2.66 μM after 72 hours and 19.21±9.8 μM after 96 hours of culture compared with 1.35±0.36 μM and 1.31±0.42 μM from untreated HL-60 cells; p =0.0004). This was associated with an average 35% depletion of tryptophan from culture supernatants. Not unexpectedly, IFN-γ-treated HL-60 cells promoted the in vitro conversion of naïve allogeneic CD4+CD25− T cells into bona fide CD4+CD25+FoxP3+ Treg cells. Phosphorylated signal transducer and activator of transcription (STAT)-1 could be detected in HL-60 cells starting from 48 hours of IFN-γ treatment, but not in untreated HL-60 cells, and its expression kinetics and relative amount closely paralleled those of IDO-1. IFN-γ strongly induced COX-2 mRNA (average fold-induction equal to 17.7±1.37 compared with untreated HL-60 cells) and protein. Conversely, IFN-γ exerted no appreciable effects on the expression levels of the tryptophan transporter molecule (LAT-1/CD98 receptor complex) on HL-60 leukemia cells. The addition of 100 μM nimesulide, a preferential COX-2 inhibitor, to HL-60 cells that were pre-treated with IFN-γ as above detailed translated into an average 54% reduction of kynurenines in culture supernatants (18.78±4.02 μM in the absence of nimesulide compared with 8.75±3.26 μM in cultures established with the drug; p = 0.0082). Superimposable results in terms of kynurenine production and tryptophan depletion were recorded in cultures of HL-60 cells that were simultaneously established with IFN-γ and nimesulide. Exposure to nimesulide attenuated mRNA signals for IDO-1, suggesting that the overall inhibition of IDO activity leading to suppressed kynurenine synthesis might also be attributed to the reduction of IDO-1 gene transcription. Interestingly, nimesulide diminished the expression of phosphorylated STAT-1 in HL-60 cells, indicating the involvement of this signaling pathway in the regulation of IDO-1 expression. In a further set of experiments, aspirin was added for 24 hours at 1 mM (final concentration) to IFN-γ-treated HL-60 cells in order to investigate its effects on IDO expression and function. In good agreement with the above results with nimesulide, aspirin promoted an average 65% inhibition of kynurenine release from IFN-γ-treated HL-60 cells and abrogated tryptophan consumption from culture supernatants. Collectively, these data suggest that COX-2 inhibition might be exploited as a valuable strategy to interfere with IDO-mediated tryptophan catabolism and hopefully to interfere with the tumor-induced immune dysfunction.

Abstract 1718 In the embryo, hematopoiesis and angiogenesis occur concurrently, since endothelial and hematopoietic cells derive from a common cell precursor, the hemangioblast. In the post natal life, the bone marrow (BM) vascular niche has an important role in supporting hematopoietic stem cells (HSC) proliferation and differentiation. Moreover, during the processes of homing and mobilization, BM vascular niche regulates HSC migration. In BM failure, these functions are promoted by vascular niches located outside of the BM, such as in the spleen, thus confirming that endothelial cells are relevant to the normal hematopoietic differentiation. The dominant feature of myelodyspslatic syndromes (MDS) is the impaired capacity of HSC to give rise to a terminally differentiated normal cell population in one or more lineages. We hypothesized that in MDS patients the vascular niches within and eventually outside of the BM might be altered and that they do not support adequately the physiologic hematopoietic differentiation, thus contributing to the pathogenesis of the disease. To investigate this hypothesis, we isolated and expanded endothelial colony forming cells (ECFCs) (Ingram et al. Blood. 2004;104: 2752) from the peripheral blood of patients with MDS (RCMD) and we used ECFCs as a surrogate of the vascular niche to differentiate hematopoietic cells. Normal CD34+ were seeded over a layer of endothelial cells and cultured for 8 days in different cytokine- containing media able to induce selective granulocytic-macrophage, erythroid or megakaryocyte differentiation. At baseline and after 5 and 8 days of co-culture, hematopoietic cells were recovered by gentle repeated washings, and then they were analyzed with RT-PCR for the expression of lineage specific genes. For each lineage, two genes, mainly involved either in the early or in the late phase of the differentiation, respectively, were analyzed. Basically, we found that co-culturing CD34+ cells in contact with a normal endothelial layer the gene expression observed in the corresponding no-layer cultures was amplified. This effect was strictly dependent on the physical contact between hematopoietic and endothelial cells, since it was abolished when cells were grown separated from a porous membrane. We observed that in co-cultures with normal hematopoietic cells and MDS ECFCs, the modulatory effect exerted by the endothelial layer was partially or completely lost. For example, CD34+ cells undergoing granulocyte differentiation showed a progressive decline of PU.1 gene expression, followed by the rise of the expression of MPO gene; these effects were much more evident if cells were cultured over normal ECFCs. In contrast, this pattern of gene expression was deeply perturbed when normal CD34+ cells were cultured with MDS ECFCs: actually, at day 7, the PU.1 RNA level was six folds higher and the MPO RNA level was three folds lower than that observed in co-cultures with normal ECFCs. Similar figures were observed for RUNX1 and GP1b gene expression in cells undergoing megakaryocyte differentiation. Moreover, CD34+ cells undergoing erythroid differentiation on normal ECFCs exhibited the progressive up-regulation of FOG-1 and of transferrin receptor (TFR) gene expression: interestingly, the co-culture with MDS ECFCs resulted in a lower expression of FOG -1 gene but also in a surprisingly higher expression of the TFR gene. Overall, these data suggest that in MDS does exist an impairment of talking between endothelial and hematopoietic cells. To understand which molecular pathways are involved could represent the basis for a novel therapy approach. Disclosures: No relevant conflicts of interest to declare.