Abstract 19437: Concomitant Overexpression of FGF2 and TGFbeta1 in Mesenchymal Stem Cells Improves Both Autocrine and Paracrine Properties Through Activation of Akt, ERK1/2 and SMAD2 Pathways and Upregulation of Soluble Factors
Background: mesenchymal stem cells (MSC) repair infarcted hearts mainly through paracrine mechanisms. However, donor age negatively influences paracrine properties of bone marrow MSC (BM-MSC). We have shown that in BM-MSC from elderly patients both TGFβ1 (T) and FGF2 (F) expression is decreased. We hypothesized that overexpression of these two factors involved in cytoprotection and angiogenesis may improve MSC repair capacity.
Methods: rat BM-MSC were transduced with control (GFP-MSC) or with a bicistronic (TF-MSC) lentivirus. Cytoprotection was evaluated in MSC (autocrine) or in H9c2 (paracrine) cells exposed to 24 hrs of hypoxia in the presence of either unconditioned (CTRL-M) or conditioned medium (GFP-CM or TF-CM). Viability was measured by MTS assay and apoptosis by caspase-3 activation. Angiogenesis was assessed by evaluating HUVEC tube formation on Matrigel. Transcriptional levels of the pro-survival gene Bcl2 and genes encoding soluble factors were measured by RT-PCR. Activation of the three main TF-related pathways (ERK1/2, Akt and SMAD2) was tested by western blot.
Results: TF-MSC showed a marked reduction of apoptosis rate (-27% p<0.001) compared with GFP-MSC after 24 hrs of hypoxia. Furthermore, TF-CM increased H9c2 viability compared with CTRL-M (+46.3% p<0.001). Caspase-3 activation in H9C2 was reduced by 60.3% in the presence of TF-CM vs CTRL-M (p<0.001) and by 44.7% vs GFP-CM (p<0.05). HUVEC tube formation was significantly enhanced by TF-CM compared to both CTRL-M (+57.5%; p<0.001) and GFP-CM (+45.2%; p<0.001). Both in TF-MSC and TF-CM treated-H9c2 we observed a strong activation of Akt, ERK1/2 and SMAD2 pathways and enhanced expression of Bcl2. Finally, in TF-MSC the transcriptional levels of soluble factors such as PDGF-β, VEGF, Ang1, IGF1, BMP2, and IL11 was upregulated.
Conclusions: TF overexpression increases both autocrine and paracrine properties of MSC through upregulation of several soluble factors and activation of pathways known to be involved in cytoprotection and angiogenesis. These results pave the way for further research on modification of MSC as effective strategy to improve efficacy of stem cell therapy.
- © 2012 by American Heart Association, Inc.