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    • br Methods br Results br

    2018-10-20


    Methods
    Results
    Discussion Mastocytosis is a rare disease characterized by an abnormal accumulation of mast 7-Nitroindazole in a number of tissues including the marrow, liver, spleen, lymph nodes and skin. In SM, the bone marrow is consistently affected (George and Horny, 2011) and skeletal abnormalities are well described (Delsignore et al., 1996; Abramowitz and Weinerman, 2012; Hermine et al., 2008; Lim et al., 2009). Since the bone marrow is home to not only the hematopoietic stem and progenitor cells, but also the BMSCs that maintain the BM environment, we were interested in whether the disease affects these cells as well and be associated with abnormal function. In the work presented, we found that BMSCs from patients with SM have a slower growth rate and a shorter life span than those from healthy donors. Since SM is associated with somatic activating mutations in KIT (Carter et al., 2014; Horny et al., 2007; Longley et al., 1999; Pardanani, 2012), we first wanted to determine if the BMSCs also carry the KIT D816V mutation. All 5 patients\' BMSCs examined for the mutation showed no presence of a mutant KIT D816V allele. This result suggests that the slow growth rate and early senescence of the BMSCs from patients with SM is most likely due to epigenetic factors. These effects might be due to a variety of factors. For example, MCs release histamine and BMSCs bear histamine receptors. A chronically increased histamine concentration in the tissue environment might produce epigenetic changes — such as disturbances in growth properties as well as affecting their “stamens”. It is possible that as the disease progresses, increasingly more BMSC clones become abnormal, thus compounding the disease state for these patients (for detailed patients\' data see Table 2). Histamine also induces the BMSC production of IL-6 and IL-8 (Nemeth et al., 2012) and high levels of these cytokines might also affect the composition of the environment surrounding the BMSCs. In addition to histamine, mast cell secretory granules contain other mediators (proteases, proteoglycans, heparin, heparan sulfates, cytokines etc.) that are released into the environment upon cell stimulation. All of these MC derived factors might have an effect on the biology of the BMSCs in the bone marrow niche and the effect of all these factors individually and in combination will also need to be examined in the future. Our in vitro data exposing healthy BMSCs to high levels of histamine revealed an elevation of a peroxisome proliferator-activated receptor (PPAR) gamma mRNA in the cells. PPARγ is a prime inducer of adipogenesis in BMSCs and its elevation shifts the differentiation potential of BMSCs from osteogenic towards adipogenic lineage (Takada et al., 2009). Due to the exposure of high levels of histamine over a long period of time in the BM of SM patients, this shift could partially explain the poor osteogenic differentiation of the cells and could underlie some of the skeletal problems of these patients. We demonstrate that in addition to the growth deficiencies, the SM BMSCs have an altered expression of surface markers. Thus, CD 105 or endoglin is overexpressed in the abnormal SM BMSCs. The overexpression of endoglin in mouse fibroblasts resulted in a reduction in extracellular matrix components (Guerrero-Esteo et al., 1999), and a decreased cellular migration and altered cellular morphology was observed. The expression pattern of CD146 is also of interest. CD146 (also known as melanoma cell adhesion molecule) has been reported to be the most specific marker of skeletal (self renewing) stem cells in the bone marrow (Bianco et al., 2010; Sacchetti et al., 2007). Thus a lower expression of this protein suggests that fewer skeletal stem cells are present in the BM, or may reflect a change in their functionality, which might explain the skeletal problems reported in patients with SM. Since the same molecule is also characteristic of the cell that supplies hematopoietic support within the BM niche, our finding that CD34+ cells that are co-cultured with the abnormal SM colonies (i.e., CD146low expression) showed a decrease in engraftment which might also be due to differences in CD146 expression. CD 146 is an adhesion molecule, so the effect on CD34+ cells might thus relate to a decrease in homing ability following transplantation. Furthermore, we also observed significant down-regulation in SM derived BMSCs of CXCL12 (0.5-fold p<0.0003), PDGFα (0.66-fold p<0.004), VCAM1 (0.36-fold p<0.0004) and FGF7 (0.27-fold p<0.00001) — all of these factors are known to play a role in supporting hematopoiesis in the BM niche (Li and Wu, 2011; Nagasawa et al., 2011; Sugiyama et al., 2006).