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

Abstract

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.

Myocardial transfection of hypoxia-inducible factor-1α and co-transplantation of mesenchymal stem cells enhance cardiac repair in rats with experimental myocardial infarction

“…Decreased tissue oxygen causes nuclear accumulation of HIF-1α protein and enhancement of its transcriptional activity through binding to enhancer elements in target genes, including vascular endothelial growth factor (VEGF) [18], angiopoietin-1 (Ang-1) , angiopoietin-2 (Ang-2) , platelet-derived growth factor beta [19], nitric oxide synthase (iNOS) [20], erythropoietin [21], phosphoglycerate kinase [22] and stromal-derived factor-1(SDF-1) . Specifically, the up-regulation of CXCR4 expression in mesenchymal stem cells (MSCs) mediates a broad range of biological processes including cell proliferation, survival, migration, adhesion, differentiation, as well as pro-angiogenesis [23-27]. …”

The role of the microenvironment on the fate of adult stem cells

“…Hypoxic preconditioning of the hMSCs can effectively restore osteogenic differentiation, benefitting transplantation therapy for bone regeneration [85]. HIF-1a regulates transcriptional genes involved in the differentiation of bone marrow SCs into cardiomyocytes [86]. Natural and synthetic biomaterials provide three-dimensional differentiation niches to guide the differentiation of SCs towards specific lineages.…”

Engineering Stem Cells for Biomedical Applications

“…To this end, Wang and co-workers investigated the ability of MSCs, which were engineered to secrete hypoxia-inducible factor 1 α (HIF-1 α ), to differentiate toward cardiomyocytes. [220] Typically, HIF-1 α regulates the transcription of genes that are involved in cell proliferation, survival, and differentiation. Owing to its central role in the oxygen-sensitive signaling pathway and previous findings that suggest a relationship between hypoxic microenvironments and the ability of MSCs to acquire a cardiomyocyte phenotype, [221] Wang et al hypothesized that HIF-1 α may play a key role in guiding this differentiation process.…”