178. Effect of Heme Oxygenase-1 Overexpression on DNA Synthesis of Adult Cardiac Progenitor Cells Under Hypoxia

Abstract

Although the studies in animals and humans have shown that the beneficial effects of adult c-kit+ cardiac progenitor cells (CPCs) on left ventricle function and remodeling post-myocardial infarction persist for several weeks and up to 1 year after CPC administration, the vast majority (>95%) of transplanted CPCs in animal studies die or vanish shortly after cell administration. Additionally, the less blood flow in the scar tissue after heart attack results in the oxygen reduced to 1 - 2% (hypoxia) which makes the cardiac microenvironment inadequate for cell DNA synthesis, thereby implying that gene manipulations on the transplanted CPCs that limit cell death under hypoxia may enhance the efficacy (and thus clinical applicability) of cell therapy. One of the most powerful cytoprotective proteins known is heme oxygenase-1 (HO-1). To test the hypothesis that HO-1 gene may improve newly synthesized DNA level in CPCs under hypoxia, adult CPCs were isolated and sorted from wild-type (WT), HO-1 transgenic (TG) or knockout (KO) mouse hearts. The c-kit positivity in all live CPCs was 60 - 65% detected by FACS with specific monoclonal anti-c-kit antibody in which Mast cells were used as positive control and CHO cells as negative control. HO-1 protein overexpression in HO-1TG CPCs was confirmed by FACS and immunoblotting (2.8-fold greater than that in WT CPCs); in contrast, HO-1KO CPCs had no HO-1. A hypoxia chamber system containing 1% of O2, 5% of CO2, and 94% of nitrogen was employed to mimic the damaged environment in the cardiac scar tissue. Furthermore, Bromodeoxyuridine (BrdU), an analog of thymidine that can be incorporated into the newly synthesized DNA of replicating cells in S-phase, was used to determine DNA synthesis levels in CPCs. Cells were cultured for 16 h and then labeled with BrdU for 30 min before BrdU immunofluorescent staining. Under normal oxygen tension (21% of O2), HO-1TG CPCs exhibited an significant increase in nuclear BrdU intensity relative to either WT or HO-1KO CPCs (+88% vs WT group, and +63% vs HO-1KO group), indicating that the new DNA synthesis in HO-1TG CPCs are very active during cell mitosis as compared to WT or HO-1KO CPCs. Under hypoxic condition (1% of O2), although new DNA synthesis in S-phase in all of the groups was much less active, HO-1TG CPCs showed even greater differences (+2.4-fold vs WT, +2.0-fold vs HO-1KO) in the extent of BrdU nuclear incorporation as compared with WT or HO-1KO CPC group (FigFig), providing a strong evidence that HO-1 gene plays an important role in protecting CPCs under hypoxia via promotion of new DNA synthesis in BrdU-labeled cell mitosis. These results demonstrate that HO-1 gene promotes new DNA synthesis during mitoses in adult CPCs not only under normal oxygen condition but also in the hypoxia environment, therefore HO-1TG CPCs may provide higher survival rate than WT and HO-1KO CPCs. The data support the hypothesis that HO-1 gene improves the survival and reparative ability of adult CPCs via enhancing newly synthesized DNA in replicating cells in S-phase under hypoxia. Thus, the adult CPCs carried HO-1 gene as a new approach may have potentially clinical application to increase the efficacy of cell therapy for patients with heart attack.View Large Image | Download PowerPoint Slide