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. 2019 Oct:27:101170.
doi: 10.1016/j.redox.2019.101170. Epub 2019 Mar 21.

Remote transplantation of human adipose-derived stem cells induces regression of cardiac hypertrophy by regulating the macrophage polarization in spontaneously hypertensive rats

Tsung-Ming Lee  1 , Horng-Jyh Harn  2 , Tzyy-Wen Chiou  3 , Ming-Hsi Chuang  4 , Chun-Hung Chen  5 , Chi-Hsuan Chuang  6 , Po-Cheng Lin  5 , Shinn-Zong Lin  7
Affiliations

Remote transplantation of human adipose-derived stem cells induces regression of cardiac hypertrophy by regulating the macrophage polarization in spontaneously hypertensive rats

Tsung-Ming Lee et al. Redox Biol. 2019 Oct.

Abstract

Left ventricular hypertrophy (LVH) in hypertension has prognostic significance on cardiovascular mortality and morbidity. Recently, we have shown that n-butylidenephthalide (BP) improves human adipose-derived stem cell (hADSC) engraftment via attenuated reactive oxygen species (ROS) production. This prompted us to investigate whether remote transplantation of BP-pretreated hADSCs confers attenuated LVH at an established phase of hypertension. Male spontaneously hypertensive rats (SHRs) aged 12 weeks were randomly allocated to receive right hamstring injection of vehicle, clinical-grade hADSCs, and BP-preconditioned hADSCs for 8 weeks. As compared with untreated SHRs, naïve hADSCs decreased the ratio of LV weight to tibia, cardiomyocyte cell size, and collagen deposition independent of hemodynamic changes. These changes were accompanied by attenuated myocardial ROS production and increased p-STAT3 levels. Compared with naïve hADSCs, BP-preconditioned hADSCs provided a further decrease of ROS and LVH and an increase of local hADSC engraftment, STAT3 phosphorylation, STAT3 activity, STAT3 nuclear translocation, myocardial IL-10 levels, and the percentage of M2 macrophage infiltration. SIN-1 or S3I-201 reversed the effects of BP-preconditioned ADSCs increase on myocardial IL-10 levels. Furthermore, SIN-1 abolished the phosphorylation of STAT3, whereas superoxide levels were not affected following the inhibition of STAT3. Our results highlighted the feasibility of remote transplantation of hADSCs can be considered as an alternative procedure to reverse cardiac hypertrophy even at an established phase of hypertension. BP-pretreated hADSCs polarize macrophages into M2 immunoregulatory cells more efficiently than naïve hADSCs via ROS/STAT3 pathway.

Keywords: Adipose-derived stem cell; Butylidenephthalide; Left ventricular hypertrophy; M2 macrophage; Signal transducer and activator of transcription 3; Spontaneously hypertensive rats.

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Figures

Fig. 1
Fig. 1
Acute stage at day 3. (A) Superoxide in the right hamstring muscle by chemiluminescence. (B) Superoxide in the hamstring muscle by DHE staining and quantitative analysis. DHE (red fluorescent) staining showed more intense signals in untreated SHR compared with WKY. (C) Superoxide in the myocardium by chemiluminescence. (D) Superoxide in the myocardium by DHE staining and quantitative analysis. (E) Plasma superoxide levels. (F) The transplanted hADSCs in the hamstring muscle by immunostaining with anti-human mitochondrial antibodies. The grafted cells were identified and their human nature was established by positive immunostaining with anti-human mitochondrial antibodies (brown). Human stem cells by quantitative analysis were observed at local implanted area of transplantation groups but not the vehicle. More grafted cells were identified in the SHR group pretreated with BP compared with naïve hADSCs. (G) The transplanted hADSCs in the myocardium by RT-PCR. For further analysis of hADSCs engraftment, RT-PCR for human Alu was performed. Alu-DNA index (amount of Alu-amplified DNA in hADSCs recipient rats relative to that detected in vehicle-treated rats). The number of animals in each group is indicated in parentheses. Bar = 50 μm *P < 0.05 versus WKY; †P < 0.05 versus vehicle(untreated)-treated SHR group; ‡P < 0.05 versus naïve hADSC-treated group.
Fig. 2
Fig. 2
Myocardial ROS levels at day 56. (A) Superoxide by chemiluminescence, and (B) DHE staining and quantitative analysis. The number of animals in each group is indicated in parentheses. Bar = 50 μm *P < 0.05 versus WKY; †P < 0.05 versus vehicle(untreated)-treated SHR group; ‡P < 0.05 versus naïve hADSC-treated group.
Fig. 3
Fig. 3
Myocardial STAT 3 activity at day 56. (A) Western analysis of STAT3. Relative abundance was obtained by normalizing the protein density against that of β-actin. Each column and bar represents mean ± SD. Each point is an average of 3 separate experiments. (B) DNA-binding activity of STAT3 was measured by ELISA with heart homogenates. (C) Representative immunostaining micrographs show STAT3 nuclear translocation (brown) and quantitative analysis. hADSCs increased the nuclear translocation of STAT3 in SHR. The value was expressed as the ratio of STAT3-stained area to total area. The number of animals in each group is indicated in parentheses. Bar = 50 μm *P < 0.05 versus WKY; †P < 0.05 versus vehicle(untreated)-treated SHR group; ‡P < 0.05 versus naïve hADSC-treated group.
Fig. 4
Fig. 4
Immunohistochemical staining of myocardial M1 and M2 macrophage phenotype at day 56. iNOS-expressing CD68 (+) M1 macrophages were observed in myocardium treated with vehicle (Veh), but were significantly reduced by hADSC administration (A). IL-10-expressing CD68 (+) M2 macrophages were predominant in hADSC-administered myocardium (B). The iNOS-expressing CD68 (+) or IL-10-expressing CD68 (+) macrophages were calculated and expressed as bar graphs. The values are mean ± SD of 5 animals from each group. The line length corresponds to 20 μm. M, merged. *P < 0.001 versus vehicle.
Fig. 5
Fig. 5
Expression of gene markers for myocardial M1 and M2 macrophages at day 56. There is an obvious shift towards M2 macrophage phenotype in the groups treated with naïve and BP-primed hADSCs as shown by changed expressions of reduced M1-related genes (IL-6, IL-1β, iNOS) and increased M2 genes (CD206, IL-10). *P < 0.05 versus WKY; †P < 0.05 versus vehicle(untreated)-treated SHR group; ‡P < 0.05 versus naïve hADSC-treated group.
Fig. 6
Fig. 6
Myocardial hypertrophy and fibrosis at day 56. (A) Morphometric analyses of left ventricular (LV) sections (magnification: 400 × ). LV cardiomyocyte cross-sectional areas in rats treated with the indicated agents were examined by hematoxylin-eosin staining. The relative myocyte cross-sectional area was normalized to the mean value of WKY. (B) RT-PCR for BNP (C) Representative sections with Sirius Red staining (red, magnification 400 × ). (D) Hydroxyproline content was measured as quantitative amount of fibrosis. The line length corresponds to 50 μm. The values are mean ± SD of 5 animals from each group. *P < 0.05 versus WKY; †P < 0.05 versus vehicle(untreated)-treated SHR group; ‡P < 0.05 versus naïve hADSC-treated group.
Fig. 7
Fig. 7
Experiment 2. In a rat isolated heart model, effect of superoxide and STAT3 on IL-10 levels. STAT3 activity was determined by Western blot analysis using the phospho-STAT3 antibody. SIN-1 significantly decreased STAT3 activity and IL-10 levels compared with BP/hADSCs alone. Besides, the administration of S3I-201, a STAT3 inhibitor, significantly decreased IL-10 levels compared with BP/hADSCs alone. *P < 0.05 versus naïve hADSC; †P < 0.05 versus BP/hADSC-treated group; ‡P < 0.05 versus BP/hADSC/SIN-treated group.
Fig. 8
Fig. 8
Proposed schematic representation illustrates the involvement of remotely transplanted hADSCs in macrophage polarization-mediated cardiac hypertrophy. It is characterized by inflammatory M1 phenotype macrophages in untreated SHR, however, after administering hADSCs, M2 phenotype would become dominant in which BP-primed hADSCs further attenuate ROS production and increased STAT3 activity. M1 and M2 phenotype macrophages can be converted into each other. Inhibition of these signaling pathways is indicated by the vertical lines.
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