Mesenchymal Stem Cell-Mediated Therapy of Peripheral Artery Disease Is Stimulated by a Lamin A-Progerin Binding Inhibitor

Abstract

Objective: Human adipose tissue-derived mesenchymal stem cells (ASCs) have been reported to promote angiogenesis and tissue repair. However, poor survival and engraftment efficiency of transplanted ASCs are the major bottlenecks for therapeutic application. The present study aims to improve the therapeutic efficacy of ASCs for peripheral artery diseases.

Methods: Hydrogen peroxide (H₂O₂) was used to induce apoptotic cell death in ASCs. To measure apoptosis, we used flow cytometry-based apoptosis analysis and terminal deoxynucleotidyl transferase dUTP nick end labeling staining. A murine hindlimb ischemia model was established to measure the ASC-mediated therapeutic angiogenesis and in vivo survival ability of ASCs.

Results: We identified that the inhibitor of lamin A-progerin binding, JH4, protects ASCs against H₂O₂-induced oxidative stress and apoptosis. Co-administration of ASCs with JH4 improved ASC-mediated blood reperfusion recovery and limb salvage compared to that of the control group in a mouse hind limb ischemia model. Immunofluorescence showed that JH4 treatment potentiated ASC-mediated vascular regeneration via reducing ASC apoptosis post transplantation.

Conclusion: JH4 exerts anti-apoptotic effects in ASCs in conditions of oxidative stress, and contributes to the repair of ischemic hind limb injury by improving cell survival.

Keywords: Apoptosis; Mesenchymal stem cells; Oxidative stress; Peripheral artery disease.

Fig. 1. Effects of JH4 pretreatment on the H₂O₂-induced cell death of ASCs. (A) H₂O₂-induced cell death of ASCs. ASCs were treated with 0.5 mM H₂O₂ or vehicles for 48 hours, and cell images were taken under microscope. (B) Dose-dependent effects of H₂O₂ on cell viability of ASCs. ASCs were treated with the indicated concentrations of H₂O₂ for 48 hours, and cell viability was measured using the MTT assay. (C) Effects of JH4 on H₂O₂-induced cell death of ASCs. ASCs were pretreated with the indicated concentrations of JH4, treated with 0.5 mM H₂O₂ for 48 hours, followed by measurement of cell viability. (D) Effects of JH4 on H₂O₂-induced morphological change of ASCs. ASCs were treated with 0.5 mM H₂O₂ for 48 hours in the absence or presence of 1 μM JH4, followed by capturing cell images. (E) Effects of JH4 on cell viability of ASCs. ASCs were treated with the indicated concentrations of JH4 for 48 hours, followed by measurement of cell viability. The data represent the mean ± standard error of the mean. Scale bar, 200 μm.

H₂O₂, hydrogen peroxide; NS, not significant; ASC, adipose tissue-derived mesenchymal stem cell; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide. ^*p<0.001 vs. control; ^†p<0.05; ^‡p<0.01; ^§p<0.001 vs. H₂O₂ only by 1-way analysis of variance.

Fig. 2. Effects of JH4 on H₂O₂-induced apoptosis in ASCs. The cells were pretreated with 1 μM JH4 for 30 minutes, followed by treatment with 0.5 mM H₂O₂ for 48 hours. (A) Flow cytometric analysis of the Annexin V-FITC/PI staining in ASCs and (B) quantitative analysis of Annexin+ apoptotic cell population. (C) Flow cytometric analysis of the sub-G1 cell death population and (D) quantitative analysis of the sub-G1 cells. (E) TUNEL-positive cells were determined by calculating the percentage in high-power field. Arrows indicate the nuclei of TUNEL-positive cells. (F) Quantitative analysis of TUNEL assay. The data represent the mean ± standard error of the mean. Scale bar, 100 μm.

H₂O₂, hydrogen peroxide; ASC, adipose tissue-derived mesenchymal stem cell; TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labeling; DAPI, 4′,6-diamidino-2-phenylindole. ^*p<0.05; ^†p<0.01; ^‡p<0.001 by 1-way analysis of variance.

Fig. 3. Effects of JH4 on ASC-stimulated blood perfusion and ischemic limb salvage. The ischemic limbs were injected with ASCs in the absence or presence of JH4. For negative control, HBSS was injected into ischemic limbs. (A) Representative images of the ischemia-induced mouse hind limb on day 28. (B) Quantitative analysis of blood flow measured by laser doppler perfusion imaging analysis. (C) Analysis of the necrosis score on day 28. (B, C) The data represent the mean ± standard error of the mean.

HBSS, Hank's balanced salt solution; ASC, adipose tissue-derived mesenchymal stem cell; ANOVA, analysis of variance. ^*p<0.05 vs. HBSS; ^†p<0.05 vs. ASCs by 2-way ANOVA; ^‡p<0.05; ^§p<0.001 vs. HBSS by 2-way ANOVA (n=6).

Fig. 4. Effects of JH4 treatment on ASC-stimulated neovascularization in ischemic muscle. Tissues from the ischemic limbs on day 28 were analyzed by immunostaining. (A) Effects of JH4 treatment on ASC-stimulated angiogenesis in vivo. Sections were stained with anti-CD31 antibodies (red) and nuclei were counterstained with DAPI (blue). (B) The number of CD31+ capillaries per HPF was counted. (C) Effects of JH4 treatment on ASC-stimulated arteriogenesis in vivo. Sections were stained with anti-α-SMA antibodies (green) and nuclei were counterstained with DAPI (blue). (D) The number of α-SMA-positive arteries/arterioles per HPF was counted. The data represent the mean ± standard error of the mean. Scale bar, 100 μm.

HBSS, Hank's balanced salt solution; ASC, adipose tissue-derived mesenchymal stem cell; DAPI, 4′,6-diamidino-2-phenylindole; HPF, high power field; α-SMA, α-smooth muscle actin. ^*p<0.05; ^†p<0.01; ^‡p<0.001 by 1-way analysis of variance.

Fig. 5. Effects of JH4 treatment on in vivo survival and blood vessel forming ability of ASCs. ASCs were transduced with GFP-expressing lentivirus, followed by transplantation of the cells into ischemic limb with or without JH4. (A) Effects of JH4 treatment on in vivo survival of ASCs. Tissue section from the ischemic limbs on day 7 were analyzed by immunostaining with anti-GFP antibodies (green) and TUNEL staining kit. Nuclei were counterstained with DAPI (blue). (B) The percentage of TUNEL-positive apoptotic ASCs/GFP-positive ASCs were quantified. (C) Effects of JH4 treatment on ASC-mediated vasculogenesis in vivo. Tissue sections from the ischemic limbs on day 28 were analyzed by immunostaining with anti-GFP (green) and anti-α-SMA (red) antibodies. Nuclei were counterstained with DAPI (blue). (D) The percentage of GFP+ ASCs per α-SMA+ blood vessels was quantified. The data represent the mean ± standard error of the mean. Scale bar, 100 μm.

ASC, adipose tissue-derived mesenchymal stem cell; DAPI, 4′,6-diamidino-2-phenylindole; GFP, green fluorescent protein; TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labeling; α-SMA, α-smooth muscle actin. ^*p<0.05 by Student's t-test.