Previous studies using PBMC have been used to identify PH-specific genes 
as well as distinguishing between IPAH and SSc-PAH 
. In general, however, these studies have shown considerable heterogeneity when examining directly the contrast in gene expression profiles in PBMC from SSc-PAH and SSc patients 
. For example, while Pendergrass et al. 
focused on the increased expression of 9 genes that distinguished SSc-PAH from SSc, Risbano et al. 
described 5 genes which when down-regulated distinguish SSc-PAH from SSc patients. In a comparison of the complete datasets from these studies in combination with results from this current study we found one gene, in particular, ALAS2, which is significantly over-expressed in SSc-PAH versus SSc in all three datasets. We present data here in support of the finding that one of the distinguishing molecular phenotypes in terms of gene expression between patients with PAH versus either healthy controls or patients with SSc alone, involves a distinctive signature uniquely associated with erythrocyte development. This signature was also present in patients with IPAH and SSc-PH-ILD in the current study and segregates with PH in multiple published clinical studies for which data is publicly available 
. We have demonstrated here that the EDS signature is quantitatively enriched and validated in at least four independently published datasets derived from microarray studies across multiple microarray platforms (Affymetrix, Illumina, and Agilent) in which the PBMCs from PH patients were tested.
In all cases (our data and others) only a subset of PH patients in the affected groups were EDS positive and in our data, at least, the presence of the EDS was associated with increasingly severe disease (as judged by hemodynamic parameters) for IPAH but not for SSc-PAH patients (, Figure S3
). The presence of the EDS signature induced by PAH may be indicative of increased red blood cell recruitment as part of a systemic response to severe chronic local hypoxia. An increase of up-regulated genes selectively expressed in erythrocytes/reticulocytes (including ALAS2 and ERAF/AHSP, and many other EDS genes) in whole blood was also noted to be consistent with previous observations of higher red blood cell counts (hematocrit) in obesity 
. An increase in RBC trafficking may constitute a useful marker of PH disease, in general, and serve as a useful marker of increased disease severity specifically in IPAH patients. The lack of correlation of the EDS genes with hemodynamic measurements in the SSc-PAH patients is puzzling, particularly, as the signature as a whole is similarly enriched in patients from both disease groups. This perhaps reflects the different etiologies of these two different types of PH and emphasizes their distinct origins. Furthermore, we have previously reported that hemodynamic alterations are distinct between IPAH and SSc-PAH patients and do not always reflect, in the latter group, the severity of PAH 
The cell specific source of the EDS remains an open question. In order to eliminate the likelihood that direct RBC contamination in the PBMC preparations used in this current study might account for the presence of the EDS, PBMC samples from both high and low EDS patients were subjected to multiple rounds of isotonic ammonium chloride hemolysis. This treatment lyses mature red blood cells with minimal effect on lymphocytes and does not appreciably affect nucleated red cells. In our test samples high EDS gene expression was not affected by this treatment (Figure S4
High levels of gene expression for ALAS2 and ERAF are found almost exclusively in CD71+ erythroid progenitor cells, and the complete EDS signature (as defined here) appears to correlate especially well with genes known to be expressed in reticulocytes and particularly well in cell populations enriched for less mature reticulocytes (as in cord blood – ). Our hypothesis is that the EDS gene expression signature is derived from a population of nucleated reticulocytes which co-sediment with lymphocytes and monocytes in the PBMC fraction of Ficoll gradients.
The identity of the EDS corresponds closely to a gene expression signature reported by Ebert et al., 
where the authors provide detailed gene expression information for an in vitro experiment in which bone marrow CD34+ cells were expanded under conditions which induced erythroid differentiation and major changes in gene expression were recorded pre- and post- induction. Our EDS gene list overlapped the induced erythroid gene list by over 50% including both the ALAS2 and ERAF genes (ALAS2, CA1, EPB42, ERAF, FECH, GLRX5, GSPT1, GYPB, GYPE, HBA2, MYL4, SELENBP1, SLC25A37, SNCA, TMCC2, and TSPAN5). Interestingly, a major difference between the Ebert induced erythroid signature and the PAH EDS is the very strong upregulation of IL8 recorded in the Ebert signature but not in the PAH-EDS (IL8 was upregulated sporadically and non-specifically across the entire PAH dataset). The reason for this discrepancy is not clear but worth noting given the otherwise very strong correlation between the two signatures.
Additional evidence for the source of the EDS in PBMCs has recently been obtained indirectly by others. Researchers from the Children's Hospital in Cincinnati reported the up-regulation of the expression of genes involved in the processes of hemoglobin synthesis and oxygen transport in the PBMCs of systemic juvenile idiopathic arthritis (sJIA) patients relative to healthy controls 
. They suggested (as we do here) that this cluster of genes might represent the signature of immature nucleated RBCs that can copurify with PBMCs isolated on Ficoll gradients. The PH and the sJIA erythropoiesis signatures are highly overlapping (Figure S5
) suggesting a common PBMC cell source. The Cincinnati team also mapped their experimentally derived EDS using bioinformatics methods to CD71+ immature erythroid precursor cells 
and, in addition, they present data showing that patients with sJIA had significantly increased proportions of immature cell populations, including CD34+ cells, correlating highly with the strength of their measurements of the erythropoiesis signature. It has been independently demonstrated by in vitro
expansion experiments that the potential, at least, for the production of large amounts of erythroid progenitor cells can be derived directly from PBMCs without additional purification 
, although whether the source of this expansion is derived from CD34+ cells remains uncertain 
. Despite the strong indirect evidence presented by ourselves and others, the direct evidence for the expansion of CD71+ erythrocyte precursors in the periphery as the source of the EDS remains to be demonstrated.
The exact role of the EDS in specific disease states remains to be determined. Clearly there are strong associations between the EDS and PH and sJIA, but also the EDS has been observed in active lupus as well, and co-segregates with multiple additional immune-related signatures in a large asthma cohort study (unpublished data). In an ongoing longitudinal PAH study that we are currently conducting, it has become clear that the EDS as well as the immune response and platelet signatures are also variable in patients over time. Unfortunately, it is still too early for more than a speculative interpretation of these results. For now, it would appear that the EDS is an important new marker in chronic disease with the distinct property that in hypertension, at least, the expansion of immature precursor cells may actually constitute an active biological response to increasingly severe disease conditions.