Improved Graft Mesenchymal Stem Cell Survival in Ischemic Heart With a Hypoxia-Regulated Heme Oxygenase-1 Vector
Yao Liang Tang, MD, PhD*,
Yi Tang, MD, PhD ,
Y. Clare Zhang, PhD , ,
Keping Qian, PhD,,
Leping Shen, MS, and
M. Ian Phillips, PhD, DSc*,*
* Department of Physiology and Biophysics, University of South Florida, St. Petersburg, Florida
Department of Pediatrics, College of Medicine, University of South Florida, St. Petersburg, Florida
All Children’s Hospital Research Institute, University of South Florida, St. Petersburg, Florida
Department of Surgery, University of Stanford, Stanford, California
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Figure 1 Diagram of hypoxia-regulated plasmid system, which can amplify the power of a promoter based on the strong transcription activity of the GAL4/p65 fusion protein. The ischemic biosensor is composed of an oxygen-sensing toggle (OST), which is a GAL4DNA-binding domain, ODD of the hypoxia-inducible factor-1-alpha along with p65 activation domain. The effector plasmid contains GAL4 upstream activation sequence (UAS) in front of an adenovirus E1b TATA box and the hHO-1-6xHis or LacZ fusion gene. Hypoxia inducible LacZ system is replaced hHO-1 gene with LacZ.
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Figure 2 Effect of treating mesenchymal stem cells (MSCs) with hypoxia-inducible hHO-1 vector on grafted cell survival in ischemic myocardium. (A and B) Quantification of intramyocardial terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate in situ nick end labeling-positive graft cells two weeks after MSC injection. TUNEL-positive implanted cells were significantly less in MSCHO-1 group compared with the MSCLacZ and MSCs group (p < 0.01, n = 6/group). (C) Immunofluorescent staining of hHO-1 to detect the expression of hypoxia-regulated gene in ischemic myocardium four days after implantation. The number of HO-1-positive graft cell was higher in the MSCHO-1 group than the MSCLacZ groups. (D) At the peri-infarct area, donor MSCLacZ stained positively for LacZ marker gene, indicating the ischemia-activated LacZ gene in MSCLacZ. Arrows indicate the ß-gal nuclei.
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Figure 3 Injection of mesenchymal stem cells (MSCs) inhibits left ventricular (LV) remodeling and improves LV function. Masson trichrome staining showed infarct wall was significantly thicker in MSCHO-1 group in compared with other control groups (A and C). The percent of fibrotic area in total LV area was significantly reduced in MSCHO-1 group compared with other control groups (B and D). Left ventricular systolic performance and diastolic performance, as assessed by maximum dP/dt and minimum dP/dt, were best in the MSCHO-1 group, indicating that survival MSCHO-1 contribute to preserve systolic and diastolic functions of infarct heart. Moreover, the MSCLacZ and MSCs demonstrated less LV remodeling and better hemodynamic function in comparison with the medium group (E and F).
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Figure 4 Hypoxia-induced overexpression of reporter gene in MSCHO-1 and MSCLacZ. (A) Immunostaining for human HO-1 shows that 24 h of hypoxia increased the expression of hHO-1 expression compared with normoxia (1 and 2). Beta-gal staining in vitro demonstrated that higher level expression of LacZ in hypoxia treated MSCLacZ compared with normoxia (3 and 4). Almost 80% transfected MSCs were positively stained for LacZ after hypoxic treatment. (B) Western blot demonstrated that the abundances of hHO-1 fusion protein were lower in MSCHO-1 at normoxia in comparison with hypoxia with 6xHis antibody for detection. (C) The hHO-1 fusion protein level of MSCHO-1 in hypoxia was 5.18-fold more abundant than in normoxia (mean ± SEM; p < 0.001; n = 3).
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Figure 5 Effect of hypoxia-inducible hHO-1 treatment on protecting mesenchymal stem cell (MSC) against hypoxia/reoxygen damage in vitro. (A) Hypoxia-induced hHO-1 overexpression reduces MSC apoptosis in vitro by the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate in situ nick end labeling assay. After hypoxia/reoxygen/hypoxia treatment, most of MSCHO-1 expressed human HO-1 in immunostaining whereas human HO-1 expression was low in MSCLacZ or MSCs. The increase in human HO-1 expression in MSCHO-1 was accompanied by reduction in the cell apoptosis. (B) A down-regulation in a proapoptotic gene Bax level in the cell lysate of MSCHO-1 was confirmed by Western blot in comparison with MSCLacZ and MSCs after hypoxia/reoxygen/hypoxia in vitro.
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Figure 6 Detection of inflammatory cytokine by Western blot and inflammatory cell infiltration by CD45 staining in ischemic myocardium. (A) and (B) Down-regulation of interleukin-1ß was detected in heme oxygenase-1 plasmid-treated ischemic myocardium one day after treatment, whereas an obvious elevation of interleukin-1b in LacZ plasmid-treated hearts was found. Data were shown as mean ± SE after normalization with glyseraldehyde-3-phosphate dehydrogenase and compared by Student t test, *p = 0.045 compared with LacZ. (C) and (D) Immunohistochemical staining to detect the CD45 cells in ischemic myocardium. The number of CD45-positive cells was decreased in HO-1 plasmid-treated myocardium four days after treatment. Arrows indicate the groups of CD45-positive cells.
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Figure 7 Differentiation of graft mesenchymal stem cell (MSC)HO-1 in ischemic myocardium at 14 days after transplantation. 1) Cardiac muscle differentiation. (A) DAPI-labeled nuclei is shown in blue; (B) muscle marker  -actin in green; (C) cardiac marker cardiac troponin T in red; (D) Merged image of (A), (B), and (C) indicating that some graft cells adopt cardiac phenotype. Arrows point to the graft cell express cardiac mark and arrowhead indicates to host ischemic myocardium. 2: Endothelial cell differentiation. (E) DAPI-labeled nuclear; (F) endothelial marker CD31 in green; (G) Merged image of (E) and (F) indicating some graft cells participate in new microvessel formation.
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Figure 8 Quantifying graft male mesenchymal stem cells (MSCs) in the female ischemic myocardium by real-time polymerase chain reaction. (A) Real-time amplification plot showing change in normalized reporter dye fluorescence (Rn) versus number of amplification cycles in sample containing serially diluted male MSC genomic DNA. (B) Standard curve generated from data in (a) showing relationship between threshold cycle (Ct) and number of male MSCs. (C) Time course of graft MSC survival (%). The survival for MSCHO-1 was approximately 68% at 4 h and further decreased to 18% at seven days; however, the survival for MSCLacZ decreased from 21% to 3.6% for this time period.
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