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| Author(s): |
Bruna Palodetto
Total Authors: 1
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| Document type: | Doctoral Thesis |
| Press: | Campinas, SP. |
| Institution: | Universidade Estadual de Campinas (UNICAMP). Faculdade de Ciências Médicas |
| Defense date: | 2016-03-06 |
| Examining board members: |
Sara Teresinha Olalla Saad;
Claudia Bincoletto Trindade;
Lorena Lobo de Figueiredo Pontes;
Cristiane Okuda Torello;
Mariana Lazarini
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| Advisor: | Sara Teresinha Olalla Saad |
| Abstract | |
The cross-talk between hematopoietic stem cells (HSCs) and the bone marrow (BM) microenvironment is essential for hematopoiesis regulation; and the cross-talk between leukemic stem cells (LSCs) and the bone marrow (BM) microenvironment is essential for leukemogenesis. The BM microenvironment compromises several cellular types such as endothelial cells, osteoblasts and mesenchymal stromal cells (MSCs). LSCs are capable of modifying the interaction with MSCs and promoting leukemogenesis. Our group performed a microarray assay and identified VEGFA overexpression in CD34+ cells and SEMA3A overexpression in MSCs from bone marrow of myelodysplastic syndrome RARS patients. VEGFA is the most studied angiogenic factor; is increased in acute myeloid leukemia (AML) patients, and correlates with a worse prognosis. SEMA3A is a secreted protein that appears to have antiangiogenic effects in solid tumors. A competition between VEGFA and SEMA3A for the neuropilin 1 (NRP1) receptor binding has been reported. The aim of this study was to reach a better understanding of VEGFA and SEMA3A interaction in the bone marrow of MDS and AML patients and the role of this interaction in pathogenesis. To investigate VEGFA expression in CD34+ cells, we recruited 18 MDS and 15 AML patients and described, by RT-PCR, increased VEGFA expression in de novo AML patients compared to the control group (5.06 [0.42¿57.52] vs 1.00 [0.8¿2.19], P=0.0073). To investigate SEMA3A expression in MSCs, we collected 25 MDS and 17 AML bone marrow samples and we found increased SEMA3A expression in all samples compared to the control group: low-risk MDS (2.97 [0.64¿24.39] vs 0.83 [0.43¿2.86], P=0,028); High-risk MDS (9.50 [2.34¿22.60] vs 0.83 [0.43¿2.86], P=0.005); secondary AML (3.07 [0.97¿6.97] vs 0.83 [0.43¿2.86], P=0.05) and de novo AML (5.73 [1.10¿28.21] vs 0.83 [0.43¿2.86], P=0.007). In order to investigate VEGFA effects in AML cells and CD34+ normal cells, we overexpressed VEGFA in KG1 leukemic cells and in CD34+ from umbilical cord blood. The VEGFA overexpression increased KG1 (124.4± 23.03% vs 100± 0.62%; P=0.045) and CD34+ cells (159.6± 41.2% vs 102.5 ± 16.93%; P=0.042) viability; and KG1 (107.7± 2.82%; P=0.042) and CD34+ cells (134.0± 7.68%; P=0.047) proliferation compared to control cells. In order to investigate the interaction between SEMA3A and VEGFA, we overexpressed SEMA3A in HS5 stromal cell line and performed co-culture assays. The co-culture of KG1 or CD34+ cells overexpressing VEGFA with HS5 cells showed increased KG1 proliferation (176.9± 46.34%; P=0.045) and CD34+ proliferation (131.8± 7.9%; P=0.02). The co-culture of KG1 or CD34+ cells with HS5 cells overexpressing SEMA3A showed decreased KG1 proliferation (93.15± 0.79%; P=0.004) and CD34+ proliferation (72.73± 4.72%; P=0.009). When we combined VEGFA and SEMA3A overexpression, the effects of SEMA3A were dominant over VEGFA. To investigate whether the dominant effect of SEMA3A could be due to NRP1 competition, we performed immunoprecipitation assay using KG1 and HS5 treated with VEGFA and SEMA3A recombinant proteins. The combined treatment of VEGFA and SEMA3A proteins induced increased complex formation between NRP1 and Plexin A4 receptors, favoring SEMA3A signaling. Ours results suggest that VEGFA overexpression confer AML cells advantages by increasing proliferation. SEMA3A seems to reverse the effects of VEGFA. We suggested that the use of SEMA3A protein combined with VEGFA inhibitors could be beneficial to AML patient¿s treatment (AU) | |
| FAPESP's process: | 12/00529-9 - Investigation of the role of SEMA3A and VEGFA in myelodysplasia and acute leukemia deregulated pathways |
| Grantee: | Bruna Palodetto |
| Support Opportunities: | Scholarships in Brazil - Doctorate |