br Materials and Methods br Results br

Materials and Methods
Results
Discussion The importance of bone marrow microenvironment in leukemogenesis becomes more evident each day. Kim and colleagues described the ability of leukemic stem order LY-411575 to reset the communication between hematopoietic stem cells and the bone marrow microenvironment, inducing leukemogenesis (Kim et al., 2015). With the aim of better understanding this interaction, we investigated the relationship between VEGFA and SEMA3A in AML and MDS bone marrows. We found a significant increase of VEGFA in CD34+ cells of de novo AML bone marrows. Moreover, SEMA3A was significantly increased in BMSC of AML and MDS samples, compared to healthy controls. VEGFA increase has been described in several types of cancer and correlates with worse prognosis in solid tumors (Ferrara, 2004). The increase of VEGF mRNA, plasma levels and protein expression in hematopoietic diseases has been reported and may be correlated with increased angiogenesis and microvascular density (MVD) in bone marrows of AML patients (Pruneri et al., 1999; Aguayo et al., 2000; Hussong et al., 2000; de Bont et al., 2001; Song et al., 2015). Increased VEGFA plasma levels (Aguayo et al., 2002; Aguayo et al., 2000; Lee et al., 2007; Brunner et al., 2002) and mRNA in mononuclear cells from AML bone marrows has also been described (Fiedler et al., 1997; Hussong et al., 2000; de Bont et al., 2001; Mourah et al., 2009); however, to our knowledge, no data has been reported regarding VEGFA in CD34+ cells. In our study, we found no difference between VEGFA mRNA expression in CD34+ cells from MDS patients and healthy donors. The disclosed data regarding VEGFA and MDS patients is controversial. Some authors described a gradual increase of VEGFA plasma levels and MVD according to MDS progression (Pruneri et al., 1999; Aguayo et al., 2000), however these results have not yet been confirmed (Zorat et al., 2001; Madry et al., 2007). To investigate how VEGF increase influences leukemic cells, we overexpressed VEGFA in KG1 leukemic cell line, which is CD34+ positive (Morimoto et al., 1994), and in CD34+ cells isolated from umbilical cord blood and performed proliferation, viability, apoptosis and colony formation assays. VEGFA overexpression increased KG1 and CD34+ cells viability and proliferation. Our data are in accordance with the results of List and colleagues that described increased proliferation of KG1 cells after treatment with VEGF recombinant protein (List et al., 2004). They also reported an unexplained increase in apoptosis rates of KG1 cells treated with VEGFA. However, our study did not find differences in the apoptosis rates of KG1 VEGFA and KG1 Lacz cells, confirming that the increased viability herein described may be related to increased proliferation. Further corroborating our data, Xu and colleagues described increased viability in leukemic cell line HL-60 after VEGFA overexpression, despite having also reported decreased apoptosis and increased colony formation (Xu et al., 2003). Thus, we suggest that increased VEGFA expression confers advantages for leukemic cells that become more capable of proliferating over normal hematopoietic cells. Increased VEGFA expression was found only in CD34+ cells from de novo AML patients; this result is in agreement with the “paracrine-autocrine loop” theory suggested by some authors (Dias et al., 2000; Casella et al., 2003; Kampen et al., 2013). Leukemic cells are believed to produce and secrete VEGFA and in a paracrine manner, stimulate BMSCs from bone marrow microenvironment to produce more VEGFA, other cytokines, and growth factors, therefore benefiting leukemic cells in an autocrine manner (Dias et al., 2000; Casella et al., 2003; Kampen et al., 2013). Our group investigated VEGFA gene expression in BMSCs from low and high risk MDS, secondary and de novo AML bone marrows and found increased VEGFA expression in BMSCs from de novo AML patients compared to the control group (unpublished observations) corroborating the “paracrine-autocrine loop” theory.