Concomitant overexpression of IGF1 and BMP2 in mesenchymal stem cells mediates cytoprotection through both autocrine and paracrine activation of Akt, Erk1/2 and SMAD1/5/8 pathways.

Background. Bone marrow mesenchymal stem cells (BM-MSC) are valuable tools for cardiac repair, acting mainly through release of paracrine factors. However, the effects of BM-MSC are limited by poor engraftment and low rate of differentiation events. To overcome these limitations, we genetically engineered BM-MSC with a novel bicistronic lentivirus co-expressing IGF1 and BMP2 (IB), two factors known to be involved in both cardiac differentiation and cytoprotection. Methods. Rat BM-MSC were transduced with a control virus (GFP-MSC) or IB virus (IB-MSC). Autocrine and paracrine cytoprotection was evaluated in transduced MSC or in H9c2 cells treated with unconditioned (CTRL-M) or conditioned media (GFP-CM or IB-CM), after 24h of hypoxia. Cell viability was measured by MTS assay. Apoptosis was evaluated through caspase-3 activation. Transcriptional levels of pro and anti-apoptotic genes in H9c2 were measured by RT-PCR. Activation of IGF1 and BMP2 pro-survival pathways (Akt, ERK1/2, and SMAD1/5/8) in both MSC and H9c2 were assessed by western blot. Results. IB-MSC showed a marked reduction of apoptosis (-50% p<0.001) vs GFP-MSC after 24h of hypoxia. IB-CM increased H9c2 viability (+32,1% p<0.001) compared with CTRL-M, while GFP-CM had no effect. Caspase-3 activation was reduced in the presence of IB-CM of 63,9% vs CTRL-M (p<0.001) and of 49,7 % vs GFP-MSC (p<0.05). H9c2 treated with IB-CM showed enhanced expression of Bcl-2 and Stat3 pro-survival genes, and inhibition of FasL and TNFalpha pro-apoptotic genes. Both IB-MSC or IB-CM treated-H9c2 showed a strong activation of Akt, ERK1/2 and SMAD1/5/8 pathways, confirming that IGF1 and BMP2 transgenes are acting both in autocrine and paracrine manner. Conclusions. IGF1 and BMP2 transgene overexpression in MSC increases cell survival and cytoprotective paracrine properties. In particular, these effects are mediated by the activation of pathways known to be involved in cell survival.