Research ArticleBIOENGINEERING

Injectable hydrogel with MSNs/microRNA-21-5p delivery enables both immunomodification and enhanced angiogenesis for myocardial infarction therapy in pigs

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Science Advances  24 Feb 2021:
Vol. 7, no. 9, eabd6740
DOI: 10.1126/sciadv.abd6740
  • Fig. 1 In vitro bioactivity of the MSN/miR-21-5p complex.

    (A) In vitro uptake of the MSN/miR-21-5p complex by adherent endothelial cells (ECs) and macrophages (MCs). (B) In vitro transfection efficiency of miR-21-5p was determined by quantifying the miRNA level using real-time quantitative PCR. (C) Representative flow cytometry analysis of CD31 levels in ECs and F4/80 levels in MCs. (D) In vitro uptake of the MSN/miR-21-5p complex by ECs and MCs was determined by quantifying the double-positive cells (CD31 or F4/80 and Cy3) using flow cytometric analysis. The protein expression levels of VEGFA and PDGF-BB in endothelial cells (E) and tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and IL-6 in macrophages (F) were determined by the real-time quantitative PCR and Western blot analysis. (G) The endothelial cells that formed three-dimensional (3D) capillary-like tubular structures were evaluated at indicated times (8 and 16 hours). (H) Schematic diagram of the experimental setup. TUNEL, terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate nick end labeling. (I) Apoptosis-positive cardiomyocytes from these treatment groups were further quantified. (J) Protein levels of secreted proangiogenic factors were determined by enzyme-linked immunosorbent assay (ELISA) analysis of cell supernatants from the MSN/miRNA-treated ECs (scale bars, 50 μm). *P < 0.05 and ***P < 0.01. All experiments were carried out in triplicate. n = 5 per group. The data are shown as means ± SD. Photo credit: Yan Li, Shanghai Ninth People’s Hospital, College of Stomatology, Shanghai Jiao Tong University School of Medicine, Shanghai 200011, China.

  • Fig. 2 The mechanism underlying the effects of the MSN complex on modifying the immune response.

    (A) A heatmap of selected proteins representing major altered signaling pathways in three datasets of macrophages treated with MSNs, MSN/miR-NC, or MSN/miR-21-5p complexes. Macrophages with no treatment were used as a negative control. The color bar indicates normalized z score intensity-based absolute quantification. (B) KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway analysis of both up- and down-regulated pathways in macrophages after MSN treatment. The most significant pathways in the phosphoproteome are plotted on the x axis as the −log10 of the P value, compared with the proteome. (C) KEGG pathway map of Toll-like signaling pathway. Proteins shown with red backgrounds are down-regulated in macrophages after MSN complex treatments when compared with macrophages with no treatment, as determined by pathway analysis. (D) Real-time quantitative PCR and Western blot analysis of TLR1, TLR2, TLR3, TLR8, P-NFκB, TNF-α, IL-1β, and IL-6 protein content alteration in macrophages after treatment with MSNs, MSN/miR-NC, or MSN/miR-21-5p complexes. (E) Real-time quantitative PCR and Western blot analysis of P-NFκB, TNF-α, IL-1β, and IL-6 protein content alteration in MSN/miR-21-5p complex–treated macrophages that overexpress TLR2 with the TLR2 overexpression vector. ***P < 0.01. n = 3 per group. The data are shown as means ± SD. Photo credit: Yan Li, Shanghai Ninth People’s Hospital, College of Stomatology, Shanghai Jiao Tong University School of Medicine, Shanghai 200011, China.

  • Fig. 3 The mechanism underlying how the MSN/miR-21-5p complex promotes angiogenesis.

    (A) A heatmap of selected proteins representing strongly altered signaling pathways in three datasets of endothelial cells treated with MSN/miR-NC or MSN/miR-21-5p complexes. (B) KEGG pathway analysis of both up- and down-regulated pathways in endothelial cells after MSN/miR-21-5p complex treatment. (C) Western blot analysis of changes in SPRY1, P-ERK1/2, P-FAK, P-p38, P-AKT, VEGFA, and PDGF-BB protein content alteration in endothelial cells after treatment with the MSN/miR-21-5p complex. (D) The effect of MSN/miR-21-5p or MSN/miR-NC on SPRY1 mRNA levels (left) and SPRY1 protein levels (right) in endothelial cells. (E) Schematic diagram illustrating the design of luciferase reporters with the WT SPRY1 3′ untranslated region (WT 3′UTR) or the site-directed mutant SPRY1 3′UTR (3′UTR-Mut). (F) The effect of MSN/miR-21-5p on luciferase activity in endothelial cells transfected with either the WT SPRY1 3′UTR reporter (left) or the mutant SPRY1 3′UTR reporter (right). (G) Western blot analysis of P-ERK1/2, P-FAK, P-p38, P-AKT, VEGFA, and PDGF-BB protein level alteration in MSN/miR-21-5p complex–treated endothelial cells after overexpressing SPRY1 with the SPRY1 overexpression vector. *P < 0.05 and ***P < 0.01. n = 3 per group. The data are shown as means ± SD. Photo credit: Yan Li, Shanghai Ninth People’s Hospital, College of Stomatology, Shanghai Jiao Tong University School of Medicine, Shanghai 200011, China.

  • Fig. 4 Gel@MSN/miR-21-5p attenuates adverse LV remodeling and improves pumping effectiveness of the heart after MI.

    (A) Representative echocardiography imaging by the modified Simpson method of short-axis views for each treatment group at baseline and 45 min, 14 days, and 28 days after MI. The site of the infarct zone is shown by arrows. Notable chamber dilation and wall thinning occurred at 28 days following MI, consistent with the adverse remodeling process. (B) Time course analysis of the EF, LVEDV, LVESV, and LVPWd. (C) MI caused a gradual decline in the EF over 28 days, which was notably attenuated by Gel@MSN/miR-21-5p. (D) MI caused a gradual increase in the LVEDV at day 14 and day 28. The LVEDV of the Gel@MSN/miR-21-5p treatment group was substantially attenuated compared with those of the other three treatment groups. (E) MI caused progressive thinning of the LVPWd thickness at the diastole, which was attenuated by Gel@MSN/miR-NC and agomiR-21-5p treatment and further attenuated by Gel@MSN/miR-21-5p treatment at day 14 and day 28. *P < 0.05 and ***P < 0.01. Sham, n = 3; MI/saline, n = 5; MI/agomir, n = 5; MI/Gel@MSN/miR-NC, n = 6; and MI/Gel@MSN/miR-21-5p, n = 6. The data are shown as the means ± SD. Photo credit: Yan Li, Shanghai Ninth People’s Hospital, College of Stomatology, Shanghai Jiao Tong University School of Medicine, Shanghai 200011, China.

  • Fig. 5 Delayed enhancement CT analysis of LV segmentation.

    Representative delayed enhancement CT images of cross-sectional planes of hearts from two-axis (long axis and short axis) slices at day 28 after MI are shown. (A) Bull’s eye plots display the LV wall thickness, wall motion, and regional EFs. (B) The infarct zone was characterized by wall thinning (identified by white arrows). (C) Global cardiac functional measures such as cardiac output, stroke volume, and EF are shown in the inserted table. Photo credit: Yan Li, Shanghai Ninth People’s Hospital, College of Stomatology, Shanghai Jiao Tong University School of Medicine, Shanghai 200011, China.

  • Fig. 6 Gel@MSN/miR-21-5p improved cardiac remodeling and reduced the infarct size after MI.

    A porcine model of MI was used to investigate the post-MI responsiveness of different groups to treatments. Healing at the infarct zone was analyzed after 28 days after treatment. (A) Representative image of TTC-stained hearts and morphometric measures of the infarct area from each group. White coloring in the TTC-stained sections indicates infarct zone and tissue necrosis. (B) Representative histological analysis of the infarcted myocardium among the treatment groups. H&E (left) staining, Masson’s trichrome staining (middle), and immunohistochemistry staining for cardiac troponin T (right) 28 days after MI showed a loss of cardiomyocytes and collagen deposition, and interstitial fibrosis was substantially reduced in the infarct zone after the Gel@MSN/miR-21-5p treatment (scale bars, 2000 μm in the low-magnification images and 60 μm in the high-magnification images). Quantitative analysis showing the percentage of the TTC-negative infarct area (C) and fibrotic area (D). (E) miRNA transfection efficiency was investigated using real-time quantitative PCR at 28 days following MI. *P < 0.05 and ***P < 0.01. Sham, n = 3; MI/Saline, n = 5; MI/Agomir, n = 5; MI/Gel@MSN/miR-NC, n = 6; and MI/Gel@MSN/miR-21-5p, n = 6. The data are shown as the mean ± SD. Photo credit: Yan Li, Shanghai Ninth People’s Hospital, College of Stomatology, Shanghai Jiao Tong University School of Medicine; Shanghai, 200011, China.

  • Fig. 7 Time course analysis of the transfection efficiency of Gel@MSN/miR-21-5p in vivo.

    MSNs were prelabeled with FITC (green), and miR-21-5p was prelabeled with Cy3 (red). The hydrogel (FITC-labeled Gel@MSN/miR-21-5p or Cy3-labeled Gel@MSN/miR-21-5p) was injected into the mid-myocardium of each target site in the pigs. The duration and efficiency of MSNs and miRNA delivery upon Gel@MSN/miR-21-5p injection were monitored using time course analysis at 1, 14, and 28 days after injection. (A) Histological sections of the infarct region in the Gel@MSN/miR-21-5p group were immunolabeled with the hematoxylin and eosin (H&E) macrophage marker F4/80. (B) Histological sections of the infarct region in the Gel@MSN/miR-21-5p group were immunolabeled with the endothelial marker CD31. Cell nuclei were counterstained with DAPI (blue). (C) F4/80+FITC+ and CD31+Cy3+ double-positive cells were quantified from at least eight high-resolution images acquired from at least eight different regions of each heart. (D) miR-21-5p levels were detected using real-time quantitative PCR at different time points. The transfection efficiency was determined by quantifying the miRNA level. Scale bars, 100 μm. n = 3 per group. The data are shown as means ± SD. Photo credit: Yan Li, Shanghai Ninth People’s Hospital, College of Stomatology, Shanghai Jiao Tong University School of Medicine, Shanghai 200011, China.

  • Fig. 8 The MSN/miRNA complex could only be released from Gel@MSN/miR-21-5p at the infarct region.

    For examination of on-demand delivery, the hearts were harvested at 28 days after MI for fluorescent imaging, RNA extraction, and real-time quantitative PCR analysis. (A) The fluorescent images showed that there were no transfecting cells detected in the sham group. In contrast, it showed that the area of FITC and Cy3 fluorescence exactly overlapped with the infarct region. (B) Quantification of miR-21-5p levels showed that the MSN/miRNA complex could be highly transfected into cells within the infarct region in vivo. Scale bar, 100 μm. ***P < 0.01. The data are shown as means ± SD. Photo credit: Yan Li, Shanghai Ninth People’s Hospital, College of Stomatology, Shanghai Jiao Tong University School of Medicine, Shanghai 200011, China.

  • Fig. 9 Gel@MSN/miR-21-5p promoted local neovascularization at the infarct site after MI.

    (A) Micro-CT angiography analysis of 3D vascular structures within the infarct zone 28 days after MI indicates that the vascular volume was significantly increased in the Gel@MSN/miR-21-5p treatment group. The vascular volume within the infarct zone was quantitatively analyzed. *P < 0.05 and ***P < 0.01. n = 3 per group. (B) Immunofluorescence staining for CD31 (red) identified the vascular endothelium, and staining for α-SMA (green) identified myofibroblasts and pericytes, showing that the cardiac capillary density in histological sections of the healing infarct zone was significantly higher in the Gel@MSN/miR-21-5p treatment group than in the other groups. The CD31 and α-SMA staining intensities in the above-described groups were quantitatively analyzed (scale bars, 500 mm). *P < 0.05 and ***P < 0.01. Sham, n = 3; MI/saline, n = 5; MI/agomir, n = 5; MI/Gel@MSN/miR-NC, n = 6; and MI/Gel@MSN/miR-21-5p, n = 6. The data are shown as the means ± SD. Photo credit: Yan Li, Shanghai Ninth People’s Hospital, College of Stomatology, Shanghai Jiao Tong University School of Medicine, Shanghai 200011, China.

  • Fig. 10 Immunomodulatory effects of Gel@MSN/miR-21 on the infarct region at 1 day post MI in vivo.

    Histological sections of the infarct zone (day 1 after MI) were immunolabeled with antibodies targeting TNF-α (A), IL-6 (B), or IL-1β (C) and colabeled with the macrophage marker F4/80 (green). Cell nuclei were counterstained with DAPI (blue). (D) The percentages of cells double positive for F4/80 and TNF-α, IL-1β, or IL-6 (TNF-α–, IL-1β–, or IL-6–expressing macrophages, respectively) were quantified. Quantification was performed in at least eight high-resolution images acquired from at least eight different regions of each heart. Scale bars, 100 μm. ***P < 0.01. n = 3 per group. The data are shown as the means ± SD. Photo credit: Yan Li, Shanghai Ninth People’s Hospital, College of Stomatology, Shanghai Jiao Tong University School of Medicine, Shanghai 200011, China.

Supplementary Materials

  • Supplementary Materials

    Injectable hydrogel with MSNs/microRNA-21-5p delivery enables both immunomodification and enhanced angiogenesis for myocardial infarction therapy in pigs

    Yan Li, Xin Chen, Ronghua Jin, Lu Chen, Ming Dang, Hao Cao, Yun Dong, Bolei Cai, Guo Bai, J. Justin Gooding, Shiyu Liu, Duohong Zou, Zhiyuan Zhang, Chi Yang

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