Palodetto Bruna, da Silva Santos Duarte Adriana, Rodrigues Lopes Matheus, Adolfo Corrocher Flavia, Roversi Fernanda Marconi, Soares Niemann Fernanda, Priscila Vieira Ferro Karla, Leda Figueiredo Longhini Ana, Melo Campos Paula, Favaro Patricia, Teresinha Olalla Saad Sara
Hematology and Blood Transfusion Center-University of Campinas/Hemocentro-Unicamp, Instituto Nacional de Ciência e Tecnologia do Sangue, Campinas, Brazil.
Hematology and Blood Transfusion Center-University of Campinas/Hemocentro-Unicamp, Instituto Nacional de Ciência e Tecnologia do Sangue, Campinas, Brazil; Department of Biological Sciences, Federal University of São Paulo, Diadema, Brazil.
Stem Cell Res. 2017 Jul;22:70-78. doi: 10.1016/j.scr.2017.05.012. Epub 2017 Jun 3.
Cross-talk between hematopoietic stem cells (HSCs) and bone marrow stromal cells (BMSCs) is essential for HSCs regulation and leukemogenesis. Studying bone marrow of myelodysplasia patients, a pre-leukemic condition, we found mRNA overexpression of vascular endothelial growth factor A (VEGFA) in CD34 HSCs and semaphorin 3A (SEMA3A) in BMSCs. To better understand the role of VEGFA and SEMA3A in leukemogenesis, we recruited 30 myelodysplastic syndrome (MDS) patients, 29 acute myeloid leukemia (6 secondary to MDS) patients and 12 controls. We found higher VEGFA expression in de novo AML patients (without prior MDS) group (p=0.0073) and higher SEMA3A expression in all BMSCs patient's samples compared to control group. We then overexpressed VEGFA in an acute myelogenous leukemia cell line, KG1 cells, and in normal CD34 cells. This overexpression increased KG1 (p=0.045) and CD34 cell (p=0.042) viability and KG1 (p=0.042) and CD34 cell (p=0.047) proliferation. Moreover, KG1 and CD34 cells overexpressing VEGFA also had increased proliferation when co-cultured with human marrow stromal HS5 cells (p=0.045 and p=0.02, respectively). However, co-culture of these transformed cells with HS5 cells overexpressing SEMA3A reduced KG1 (p=0.004) and CD34 (p=0.009) proliferation. Co-culture of KG1 transformed cells with HS27 cells overexpressing SEMA3A reduced KG1 proliferation as well (p=0.01). To investigate whether the dominant SEMA3A effect over VEGFA could be due to competition for neuropilin1 receptor (NRP1), we performed immunoprecipitation with anti-NRP1 antibody of cell extracts of co-cultured KG1 and HS5 cells, induced or not by VEGFA and SEMA3A recombinant proteins. Results showed a preferential association of NRP1 with SEMA3A, suggesting that SEMA3A can partially reverse the effects caused by the VEGFA preventing its binding with the NRP1 receptor. Since both hematopoietic cells, leukemic and normal, showed similar behavior, we suppose that the attempt to reversion of VEGF effects by SEMA3A is a homeostatic phenomenon in the hematopoietic niche. Finally, we conclude that VEGFA overexpression confers AML cell advantages and SEMA3A may partially reverse this effect; thus, SEMA3A protein combined with VEGFA inhibitors could be beneficial for AML treatment.
造血干细胞（HSCs）与骨髓基质细胞（BMSCs）之间的相互作用对于HSCs的调节和白血病发生至关重要。在研究骨髓增生异常综合征患者（一种白血病前期病症）的骨髓时，我们发现CD34+HSCs中血管内皮生长因子A（VEGFA）的mRNA表达上调，而BMSCs中分泌性蛋白3A（SEMA3A）的mRNA表达上调。为了更好地理解VEGFA和SEMA3A在白血病发生中的作用，我们招募了30例骨髓增生异常综合征（MDS）患者、29例急性髓系白血病（其中6例继发于MDS）患者和12例对照。我们发现，与对照组相比，初发急性髓系白血病患者（无先前MDS病史）组中VEGFA表达更高（p=0.0073），所有BMSCs患者样本中SEMA3A表达更高。然后，我们在急性髓系白血病细胞系KG1细胞和正常CD34细胞中过表达VEGFA。这种过表达增加了KG1细胞（p=0.045）和CD34细胞（p=0.042）的活力以及KG1细胞（p=0.042）和CD34细胞（p=0.047）的增殖。此外，过表达VEGFA的KG1细胞和CD34细胞在与人骨髓基质HS5细胞共培养时增殖也增加（分别为p=0.045和p=0.02）。然而，这些转化细胞与过表达SEMA3A的HS5细胞共培养会降低KG1细胞（p=0.004）和CD34细胞（p=0.009）的增殖。KG1转化细胞与过表达SEMA3A的HS27细胞共培养也会降低KG1细胞的增殖（p=0.01）。为了研究SEMA3A对VEGFA的主导作用是否可能是由于对神经纤毛蛋白1受体（NRP1）的竞争，我们用抗NRP1抗体对共培养的KG1细胞和HS5细胞的细胞提取物进行免疫沉淀，这些细胞提取物由VEGFA和SEMA3A重组蛋白诱导或未诱导。结果显示NRP1与SEMA3A优先结合，表明SEMA3A可以部分逆转VEGFA引起的效应，阻止其与NRP1受体结合。由于白血病细胞和正常造血细胞都表现出相似的行为，我们推测SEMA3A对VEGF效应的逆转尝试是造血微环境中的一种稳态现象。最后，我们得出结论，VEGFA过表达赋予急性髓系白血病细胞优势，而SEMA3A可能部分逆转这种效应；因此，SEMA3A蛋白与VEGFA抑制剂联合使用可能对急性髓系白血病治疗有益。
