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

Abstract

Introduction: Mesenchymal stem cells (MSCs) have potential for the treatment of myocardial infarction. However, several meta-analyses revealed that the outcome of stem cell transplantation is dissatisfactory. A series of studies demonstrated that the combination of cell and gene therapy was a promising strategy to enhance therapeutic efficiency. The aim of this research is to investigate whether and how the combination of overexpression of hypoxia-inducible factor-1α (HIF-1α) and co-transplantation of mesenchymal stem cells can enhance cardiac repair in myocardial infarction.

Methods: We investigated the therapeutic effects of myocardial transfection of HIF-1α and co-transplantation of MSCs on cardiac repair in myocardial infarction by using myocardial transfection of HIF-1α via an adenoviral vector. Myocardial infarction was produced by coronary ligation in Sprague-Dawley (SD) rats. Animals were divided randomly into six groups: (1) HIF-1α+MSCs group: Ad-HIF-1α (6×10⁹ plate forming unit) and MSCs (1×10⁶) were intramyocardially injected into the border zone simultaneously; (2) HIF-1α group: Ad-HIF-1α (6×10⁹ plate forming unit) was injected into the border zone; (3) HIF-1α-MSCs group: Ad-HIF-1α transfected MSCs (1×10⁶) were injected into the border zone; (4) MSCs group: MSCs (1×10⁶) were injected into the border zone; (5) CONTROL GROUP: same volume of DMEM was injected; (6) SHAM group. Cardiac performance was then quantified by echocardiography as well as molecular and pathologic analysis of heart samples in the peri-infarcted region and the infarcted region at serial time points. The survival and engraftment of transplanted MSCs were also assessed.

Results: Myocardial transfection of HIF-1α combined with MSC transplantation in the peri-infarcted region improved cardiac function four weeks after myocardial infarction. Significant increases in vascular endothelial growth factor (VEGF) and stromal cell-derived factor-1α (SDF-1α) expression, angiogenesis and MSC engraftment, as well as decreased cardiomyocyte apoptosis in peri-infarcted regions in the hearts of the HIF-1α+MSCs group were detected compared to the MSCs group and Control group.

Conclusions: These findings suggest that myocardial transfection of HIF-1α and co-transplantation of mesenchymal stem cells enhance cardiac repair in myocardial infarction, indicating the feasibility and preliminary safety of a combination of myocardial transfection of HIF-1α and MSC transplantation to treat myocardial infarction.

Figure 1

Effects of HIF-1α on MSCs in vitro. A) Hypoxia-inducible factor-1α (HIF-1α) improved the viability and proliferation of mesenchymal stem cells (MSCs). (I) Phase contrast image of MSCs, (II) ^Ad-nullMSCs, (III) ^Ad-HIF-1αMSCs, (IV) A Cell Counting Kit-8-based colorimetric assay to quantify the MSCs viability and proliferation. B) MSCs were stained with anti-HIF-1α antibody (red) and 4, 6-diamino-2-phenylindole (DAPI) (blue). No HIF-1α was detected in MSCs group and ^Ad-nullMSCs. C) MSC migration assay showed HIF-1α improved the motility of MSCs. □P <0.01 vs. ^Ad-nullMSCs, ■P <0.01 vs. MSCs.

Figure 2

The quantitative analysis of mRNA expression of HIF-1α, SDF-1α and VEGF. A1, A2, A3: mRNA expression levels of hypoxia-inducible factor-1α (HIF-1α) in peri-infarcted region at one week, two weeks and four weeks after operation in each group, respectively. B1, B2, B3: mRNA expression levels of stromal cell-derived factor-1α (SDF-1α) in the peri-infarcted region at one week, two weeks and four weeks after operation in each group, respectively. C1,C2, C3: mRNA expression levels of vascular endothelial growth factor (VEGF) in the peri-infarcted region at one week, two weeks and four weeks after operation in each group, respectively. The expression levels were normalized to the Control group. ☆P <0.01 vs. HIF-1α- mesenchymal stem cells (MSCs) group, △P <0.01 vs. MSCs group, *P <0.01 vs. Control group, ※P <0.01 vs. SHAM group, ▲P <0.05 vs. MSCs group, #P <0.05 vs. Control group, §P <0.05 vs. SHAM group.

Figure 3

Effects of HIF-1α on MSC engraftment in the border zone. A) Engrafted mesenchymal stem cells (MSCs) stained with DiR (red) were detected one week, two weeks and three weeks after cell injection. The samples were stained with anti-α-SA antibody (green) and 4, 6-diamino-2-phenylindole (DAPI) (blue). B, C) Three weeks after cell injection, five hearts from three groups were harvested and imaged for detection of red fluorescence. The optical density (photon/s/mm²) from a fixed region of interest (ROI) was measured. D) Donor male cells persistent in the female hearts were detected by quantitative PCR for the SRY gene one week and three weeks after cell injection. ▲P <0.05 vs. MSCs group, △P <0.01 vs. MSCs group. HIF-1α, hypoxia-inducible factor-1α.

Figure 4

Migration and differentiation of engrafted MSCs after cell injection. A) Migration of mesenchymal stem cells (MSCs) at four weeks after cell injection from the border zone to infarcted region. Engrafted MSCs stained with DiR, which were injected originally in the peri-infarcted region, were detected in the infarcted region. The figures demonstrated MSCs engrafted in the infarcted region. B) Engrafted MSCs contribute to the angiogenesis three weeks after cell injection, in the peri-infarcted region of hearts of hypoxia-inducible factor-1α (HIF-1α) + MSCs group. Engrafted MSCs were stained with DiR (red) before cell injection. The arrow indicates CD31+ engrafted MSCs stained with DiR. Samples were stained with anti-CD31 antibody (green) and 4, 6-diamino-2-phenylindole (DAPI) (blue).

Figure 5

Capillary densities at the peri-infarcted border zone in each group. A) More capillary densities were detected in the hypoxia-inducible factor-1α (HIF-1α) + mesenchymal stem cells (MSCs) group than other groups. (I) HIF-1α + MSCs group, (II) HIF-1α group, (III) HIF-1α-MSCs group, (IV) MSCs group, (V) Control group. B) The number of CD-31-stained capillary was expressed as the number/mm². &P <0.05 vs. HIF-1α group, ☆P <0.01 vs. HIF-1α-MSCs group, △P <0.01 vs. MSCs group, *P <0.01 vs. Control group, ▲P <0.05 vs. MSCs group.

Figure 6

Capillary densities in the infarction region in each group. A) More capillary densities were detected in the hypoxia-inducible factor-1α (HIF-1α) + mesenchymal stem cells (MSCs) group than other groups. (I) HIF-1α + MSCs group, (II) HIF-1α group, (III) HIF-1α-MSCs group, (IV) MSCs group, (V) Control group. B) The number of the CD-31-stained capillary was expressed as the number/mm². &P <0.01 vs. HIF-1α group, ☆P <0.01 vs. HIF-1α-MSCs group, △P <0.01 vs. MSCs group, *P <0.01 vs. Control group, #P <0.05 vs. Control group.

Figure 7

Cardiomyocyte apoptosis in each group. A) Hypoxia-inducible factor-1α (HIF-1α) and mesenchymal stem cell (MSC) intramyocardial injection decreased cardiomyocytes apoptosis one week after infarction. Apoptosis nuclei stained by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) (red), total nuclei were labeled with 4, 6-diamino-2-phenylindole (DAPI) (blue). Cardiomyocytes were stained with anti-α-SA antibody (green). (I) HIF-1α + MSCs group, (II) HIF-1α group, (III) HIF-1α-MSCs group, (IV) MSCs group, (V) Control group, VI. SHAM. B) Apoptosis Index. ★P <0.05 vs. HIF-1α-MSCs group, △P <0.01 vs. MSCs group, *P <0.01 vs. Control group, ※P <0.01 vs. SHAM group, ▲P <0.05 vs. MSCs group, #P <0.05 vs. Control group.