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. 2020 Oct 23:8:583700.
doi: 10.3389/fcell.2020.583700. eCollection 2020.

Inflammatory Cytokines Alter Mesenchymal Stem Cell Mechanosensing and Adhesion on Stiffened Infarct Heart Tissue After Myocardial Infarction

Dan Zhu  1 , Peng Wu  1   2 , Changchen Xiao  1 , Wei Hu  3 , Tongtong Zhang  3 , Xinyang Hu  1 , Wei Chen  1   3 , Jian'an Wang  1
Affiliations

Inflammatory Cytokines Alter Mesenchymal Stem Cell Mechanosensing and Adhesion on Stiffened Infarct Heart Tissue After Myocardial Infarction

Dan Zhu et al. Front Cell Dev Biol. .

Abstract

Mesenchymal stem cell (MSC) transplantation has demonstrated its potential in repairing infarct heart tissue and recovering heart function after myocardial infarction (MI). However, its therapeutic effect is still limited due to poor MSC engraftment at the injury site whose tissue stiffness and local inflammation both dynamically and rapidly change after MI. Whether and how inflammatory cytokines could couple with stiffness change to affect MSC engraftment in the infarct zone still remain unclear. In this study, we characterized dynamic stiffness changes of and inflammatory cytokine expression in the infarct region of rat heart within a month after MI. We found that the tissue stiffness of the heart tissue gradually increased and peaked 21 days after MI along with the rapid upregulation of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and interleukin-1β (IL-1β) in the first 3 days, followed by a sharp decline. We further demonstrated in vitro that immobilized inflammatory cytokine IL-6 performed better than the soluble form in enhancing MSC adhesion to stiffened substrate through IL-6/src homology 2 (SH2) domain-containing tyrosine phosphatase-2 (SHP2)/integrin signaling axis. We also confirmed such mechano-immune coupling of tissue stiffness and inflammatory cytokines in modulating MSC engraftment in the rat heart after MI in vivo. Our study provides new mechanistic insights of mechanical-inflammation coupling to improve MSC mechanosensing and adhesion, potentially benefiting MSC engraftment and its clinical therapy for MI.

Keywords: adhesion; cytokine; mechanosensing; mesenchymal stem cell; myocardial infarction.

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Figures

FIGURE 1
FIGURE 1
Tissue stiffness of the infarct region of the rat heart increases after myocardial infarction (MI) and promotes mesenchymal stem cell (MSC) adhesion. (A–D) Schematics of the measurement time course of rat heart tissue stiffness by atomic force microscopy (AFM) (A) and the illustration of AFM micro-indentation to measure rat heart tissue stiffness by AFM (B) after MI, of which are the representative force-indentation curves (D) and means of tissue stiffness (C). Tissue from the infarct region was cut into small blocks for AFM probing. At least 10 varied positions over the tissue block surface were measured, and 10–15 indentations were made on each position. White dotted circles and black dots in panel (B) indicate randomly selected positions and different indentations, respectively. Dashed lines in panel (D) are the best fit to corresponding stiffness measurements by AFM with the Hertz Cone model. (E,F) Representative images (E) and average numbers (F) of the attached MSCs after wash assay on PAA gel substrates with different stiffnesses that mimic infarct regions after MI. Scale bar in panel (E) refers to 200 μm. ** and * in panels (C,F) refer to p < 0.01 and p < 0.05, respectively. Error bars in panels (C,F) represent SEM of three repeats.
FIGURE 2
FIGURE 2
Upregulation of inflammatory cytokines in the infarct region of the rat heart early after myocardial infarction (MI). Representative images of immunohistochemical staining of interleukin-1β (IL-1β) (A), interleukin-6 (IL-6) (D), and tumor necrosis factor-α (TNF-α) (G) in the infarct region of the rat heart at different times after MI. Comparisons of the numbers of IL-1β+CD68+ (B), IL-6+CD68+ (E), and TNF-α+CD68+ (H) cells per field, respectively, and the percentage of the surface area within the infarct region that was stained positively for IL-1β (C), IL-6 (F), and TNF-α (I), respectively. In panels (A,D,G), scale bars refer to 40 μm. **, *, and NS refer to p < 0.01, p < 0.05, and no significance, respectively. N = 3 rats for each time point. Error bars in panels (B,C,E,F,H,I) represent SEM of three repeats.
FIGURE 3
FIGURE 3
Immobilized interleukin-6 (IL-6) promotes mesenchymal stem cell (MSC) adhesion under shear flow. (A) Schematics of flow chamber assay to qualitatively measure shear force-dependent adhesion strength of MSCs to microfluidic channel’s substrate coated with fibronectin (FN) in the absence or presence of immobilized IL-6. (B–E) Representative images of attached MSCs after perfusion over microfluidic channels coated with increasing concentrations of FN (B) or IL-6 (D) at shear stress of 10 dyn/cm2. Quantitative results of average numbers of adherent MSCs per field are shown in panels (C,E), respectively. Scale bars in panels (B,D) refer to 200 μm. **, *, and NS in panels (D,E) refer to p < 0.01, p < 0.05, and no significance, respectively. Error bars in panels (D,E) represent SEM of three repeats.
FIGURE 4
FIGURE 4
The interleukin-6 (IL-6)-dependent adhesion enhancement on mesenchymal stem cell (MSC) adhesion under shear is mediated through SHP2–integrinα5β1 signaling axis. Schematics (A) of MSC perfusion over microfluidic channels coated with 50 μg/ml of fibronectin (FN) in the absence (first and second rows in B–D) or in the presence of 1 μg/ml IL-6 (third and fourth rows in B–D), of which are their representative images of attached MSCs pretreated with α5β1 blocking mAb (B), Gpen (C), or shp099 (D) for 30 min before perfusion and are corresponding average numbers of adherent MSCs per field (E–G), respectively. Scale bars in panels (B–D) refer to 200 μm. **, *, and NS in panels (E–G) refer to p < 0.01, p < 0.05, and no significance, respectively. Error bars in panels (E–G) represent SEM of three repeats.
FIGURE 5
FIGURE 5
The enhancement of mesenchymal stem cell (MSC) engraftment in vivo after myocardial infarction (MI) temporally coincides with cytokine upregulation and tissue stiffness increase. (A–C) The illustration of experimental procedure of MSC transplantation, tissue harvest, and imaging of MSC engraftment (A), of which are the representative fluorescence images of engrafted ZsGreen-labeled MSCs in the infarct region (B) and average numbers of engrafted MSCs per field (C). qPCR measurements of ZsGreen gene level were used to confirm fluorescence microscopy examination, and the percentage of MSC engraftment (D) is calculated by dividing the number of adherent MSCs with total cell number administered in tissue samples from heart apex. (E–G) Correlation analyses of the number of IL-6+CD68+ cells with tissue stiffness of infarct region after MI (E) or of the number of IL-6+CD68+ cells with MSC engraftment (F) or of MSC engraftment with tissue stiffness of infarct region after MI (G). Scale bars in panel (B) refer to 40 μm. **, *, and NS in panels (C,D) refer to p < 0.01, p < 0.05, and no significance, respectively. N = 3 rats for each time point. Error bars in panels (C,D) represent SEM of three repeats.
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