Abnormal Mirna Expression Profile and Cytokine Production in Myelodysplastic Vascular Niche

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.