Defective WNT Signaling and Genetic Profile Of Endothelial Cells In Patients With Low Risk Myelodysplastic Syndromes Suggest a Contribution Of Vascular Niches 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