Background. Bone marrow mesenchymal stem cells (BM-MSC) repair infarcted hearts mainly through paracrine mechanisms. However, donor age negatively influences the production of paracrine factors from BM-MSC. In particular, we have shown that in BM-MSC from elderly patients the expression of TGFbeta1 (T) and FGF2 (F) is decreased. We hypothesized that the overexpression of these two factors involved in cytoprotection and angiogenesis may improve MSC repair capacity. Methods. Rat BM-MSC were transduced with control virus (GFP-MSC) or TF virus (TF-MSC). To study cytoprotective paracrine properties H9c2 cells were exposed to 24h of hypoxia in the presence of control medium (CTRL-M) or media conditioned by GFP- (GFP-CM) or TF-MSC (TF-CM). Cell viability was measured by MTS assay. Apoptosis was evaluated through caspase-3 activation (luminometric assay and western blot). Angiogenesis was assessed by quantifying HUVEC tube formation on Matrigel. Transcriptional levels of known survival genes in H9c2 cells and of soluble factors other than transgenes in MSC were measured by RT-PCR. Activation of FGF2 and TGF1 pathways (Akt, ERK1/2, and SMAD2) was evaluated by western blot. Results. Compared with CTRL-M, TF-CM increased H9c2 viability (+46,3% p<0.001), while GFP-CM had no effect. Caspase-3 activation was reduced by TF-CM of 60,3% (p<0.001) vs CTRL-M and of 44,7 % (p<0.05) vs GFP-MSC. H9c2 treated with TF-CM showed a strong activation of Akt, ERK1/2 and SMAD2 anti-apoptotic pathways, enhanced expression of Bcl-2 and Stat3 pro-survival genes, and inhibition of FasL and TNF pro-apoptotic genes. HUVEC tube formation were enhanced by TF-CM of 70% (p<0.01) vs CTRL-M and of 59% (p<0.05) vs GFP-CM. Finally, we documented that TF-MSC upregulated transcriptional levels of other soluble factors like IGF1, PDGF-β, BMP2, VEGF and IL11. Conclusions. Simultaneous overexpression of TF transgenes improves cytoprotective and pro-angiogenic paracrine properties and enhances expression of soluble factors in BM-MSC.
IRES-based lentivirus co-espressing TGFbeta1 and FGF2 improves cell survival and angiogenesis in bone marrow-derived mesenchymal stem cells.
PISANO, FEDERICA;DANIELI, PATRIZIA;CERVIO, ELISABETTA;GNECCHI, MASSIMILIANO
2012-01-01
Abstract
Background. Bone marrow mesenchymal stem cells (BM-MSC) repair infarcted hearts mainly through paracrine mechanisms. However, donor age negatively influences the production of paracrine factors from BM-MSC. In particular, we have shown that in BM-MSC from elderly patients the expression of TGFbeta1 (T) and FGF2 (F) is decreased. We hypothesized that the overexpression of these two factors involved in cytoprotection and angiogenesis may improve MSC repair capacity. Methods. Rat BM-MSC were transduced with control virus (GFP-MSC) or TF virus (TF-MSC). To study cytoprotective paracrine properties H9c2 cells were exposed to 24h of hypoxia in the presence of control medium (CTRL-M) or media conditioned by GFP- (GFP-CM) or TF-MSC (TF-CM). Cell viability was measured by MTS assay. Apoptosis was evaluated through caspase-3 activation (luminometric assay and western blot). Angiogenesis was assessed by quantifying HUVEC tube formation on Matrigel. Transcriptional levels of known survival genes in H9c2 cells and of soluble factors other than transgenes in MSC were measured by RT-PCR. Activation of FGF2 and TGF1 pathways (Akt, ERK1/2, and SMAD2) was evaluated by western blot. Results. Compared with CTRL-M, TF-CM increased H9c2 viability (+46,3% p<0.001), while GFP-CM had no effect. Caspase-3 activation was reduced by TF-CM of 60,3% (p<0.001) vs CTRL-M and of 44,7 % (p<0.05) vs GFP-MSC. H9c2 treated with TF-CM showed a strong activation of Akt, ERK1/2 and SMAD2 anti-apoptotic pathways, enhanced expression of Bcl-2 and Stat3 pro-survival genes, and inhibition of FasL and TNF pro-apoptotic genes. HUVEC tube formation were enhanced by TF-CM of 70% (p<0.01) vs CTRL-M and of 59% (p<0.05) vs GFP-CM. Finally, we documented that TF-MSC upregulated transcriptional levels of other soluble factors like IGF1, PDGF-β, BMP2, VEGF and IL11. Conclusions. Simultaneous overexpression of TF transgenes improves cytoprotective and pro-angiogenic paracrine properties and enhances expression of soluble factors in BM-MSC.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
