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The hypoxia-inducible factor 1alpha (HIF-1alpha) regulates transcriptional genes involved in cell proliferation, survival, and differentiation. Under normoxia, HIF-1alpha has a short half-life (t((1/2)) approximately 5 min) and low transcriptional activity. An HIF-1alpha mutant, produced by substitution of alanine (Ala) for proline (Pro) at position 564 and asparagine (Asp) at position 803, can prevent HIF-1alpha hydroxylation and results in a highly active form of HIF-1alpha (HIF-1alpha-Ala564-Ala803). We hypothesized that adenovirus (Ad)-mediated transfer of the active form of HIF-1alpha (pAd-HIF-1alpha-Ala564-Ala803) could effectively occur in bone marrow stem cells (MSCs) and promote MSC differentiation under normoxia. PCR-based site-specific mutagenesis was used to construct the Ad vector expressing HIF-1alpha-Ala564-Ala803. RT-PCR and immunostaining were used to study whether pAd-HIF-1alpha-Ala564-Ala803 affected MSC differentiation to cardiomyocyte (CMC). pAd-HIF-1alpha-Ala564-Ala803 exhibited higher transcriptional activity and stable HIF-1alpha protein expression. Under normoxia, an MSC-CMC co-culture treated with pAd-HIF1a-Ala564-Ala803 augmented TGF-beta(1), Smad4, NKx2.5, and GATA4 expression. Higher expression of cTnT and alpha-actinin was observed by immunostaining in MSCs, compared with the control and contrast groups. Adenovirus-mediated hypoxia-inducible factor 1alpha double-mutant, pAd-HIF-1alpha-Ala564-Ala803, can stably express HIF-1alpha and promote its downstream genes and MSC differentiation to CMC in the MSC-CMC co-culture system under normoxia.

Adenovirus-mediated hypoxia-inducible factor 1α double-mutant promotes differentiation of bone marrow stem cells to cardiomyocytes