Alteration of EPC number has been described in a wide range of conditions, such as cardiovascular, inflammatory, immune, and infectious diseases [21, 44]. A decreased in vitro growth capacity of bone marrow-derived progenitors in DS mouse model Ts65Dn  and a reduced number of CD34+ in DS fetuses and children were also reported . More recently, Diller et al. (2008) reported an impairment of CD34+/AC133+/KDR+ cells in a small subset of DS individuals affected by Eisenmenger syndrome . Despite the use of different experimental approaches and the limited number of individuals, the reduced number of circulating progenitors is an important common finding. These evidences may possibly account for the differences in angiogenesis, inflammatory and immune response reported in DS .
Here, we have shown that DS patients exhibit a marked reduction of ≈ 40% in the number of EPCs, also displaying a significant increase in cell size and major detrimental morphological changes (i.e., cell vacuolization and high number of active lysosomes). The reduced number of progenitors could be associated with alterations in the cell cycle; however, we did not find any significant difference between the groups [Additional file 3: Supplemental Figure S1C]. Thus, we investigated other possible mechanisms responsible for the observed EPCs impairment, such as mobilization/homing and oxidative stress susceptibility.
Growing evidences indicate that chemokines and cytokines, such as SDF-1α and its receptor CXCR4, play a crucial role in the mobilization and homing of EPCs from bone marrow [45, 46], also affecting cell proliferation [41, 42]. Our findings of a significant decrease in SDF-1α plasma levels in young DS - compared to age-matched euploids - and a strong decrease of CXCL12 and CXCR4 gene transcription in their EPCs, suggest a link with the reduced number of circulating progenitors and the angiogenesis suppression observed in DS.
These results are strengthened by microarray analysis, indicating that DS progenitors have a pronounced perturbation, at least at transcriptional level, in the angiogenesis and cell cycle pathways. Indeed, by using this approach we demonstrated the transcriptional deregulation of CXCR7, IL8 and RCAN1 genes, crucial factors involved in endothelial cells' migration, homing and angiogenesis [47–49]. Moreover, we found a down-regulation of CASP8, which has been demonstrated to have a novel apoptosis-unrelated role in proangiogenic cells , although this gene was found to be associated with breast cancer by gene-based association study, and its down-regulation has also been reported in breast cancer .
We recently demonstrated the relevance of oxidative stress on the number and function of progenitor cells . Besides, it is known that oxidative stress is a crucial issue in the pathogenesis of DS, especially due to the high incidence of Alzheimer-like disease at young age . It has been also suggested that constitutive expression of trisomic genes, S100B and SOD1, is likely to represent a leading cause of ROS generation and increased oxidation in DS neurons [39, 40]. Oxidative stress relevance in DS fetuses was also highlighted by microarray analysis of uncultured amniotic fluid .
Here, we have assessed, by both experimental evidences and microarray analysis, that DS progenitors are more susceptible to hydroperoxide-induced LP and significantly over-express S100B and SOD1 genes. These findings strengthen the hypothesis of their involvement in the susceptibility to oxidation observed in DS endothelial progenitors.
In our study we have infected DS progenitors with a human pathogen, B. henselae, responsible of vasoproliferative diseases such as bacillary angiomatosis and peliosis in immunocompromised patients [27, 28], previously demonstrated to adhere to and invade EPCs . Particularly, we investigated the effect of infection on DS progenitors' number, morphology and oxidative stress response. After hydroperoxide treatment, we observed a significant LP decrease in Bartonella-infected DS progenitors compared to uninfected DS cells. This finding confirms the beneficial effect of B. henselae infection at low MOI on mature endothelial cells' survival [37, 38].
In contrast, a reduced cell number in both DS and euploid groups was observed after Bartonella infection at higher MOIs; however, detrimental changes were visible only in DS EPCs, displaying a higher number of invasomes and infected cells compared to euploid cells.
The molecular basis of such Bartonella-induced detrimental effect on endothelial progenitors of DS was investigated at a transcriptional level by microarray. Significant up-regulation of the Jak/STAT pathway was observed only within infected DS progenitors, whereas, on the opposite, infected euploid cells displayed a significant down-regulation. These findings strengthen the hypothesis that transcriptional analysis of EPCs is clearly of major interest in the context of this syndrome. Indeed, the activation of ISGs, involved in the immune response against infections and in tumor surveillance , also inhibits angiogenesis by decreasing the production of angiogenic factors such as VEGF and IL8 . Our results clearly show that a similar ISGs activation occurs in infected DS progenitor cells, and, albeit at lower levels, in uninfected DS progenitors (data not shown).
Pathologic immune and inflammatory responses are regulated by the cross-talk between interferons and TNFα , as well as deregulation of chemokines/cytokines greatly affects the mobilization and recruitment of endothelial progenitors. The imbalance between ISGs and other molecules might be of great immunological relevance concerning the well-known DS haematological defects.