Elimination of macrophages reduces glutaraldehyde‐fixed porcine heart valve degeneration in mice subdermal model

Figures

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  FIGURE 1. Experimental protocol. For details, see the Methods section. Groups of mice were either treated with PBS or clodronate liposomes. Surgical operations were conducted one day after the first administration of clodronate liposomes (or PBS liposomes). The administration of clodronate liposomes (or PBS liposomes) was done at day 2, day 5, day 8 and day 11 after subcutaneous implant. After 4 weeks, glutaraldehyde‐fixed porcine heart valve (GPHV) and spleen tissue were collected for further analysis (Lipclod = clodronate liposomes, PBS = phosphate buffered saline)

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  FIGURE 2. Macrophages were removed from spleen under clodronate liposome treatment. (A) Flow cytometric analysis shows the percentage of F4/80+ macrophages in the spleen of blank (non‐staining), PBS, and Lipclod groups at 7 days after surgical operations. (B) Bar graph shows the quantification of the percentage of F4/80+ macrophages in the spleen of blank, PBS, and Lipclod groups. (C, D) Representative images of F4/80+ immunohistochemical staining and quantification data showing that liposome treatment significantly decreases the infiltration of macrophages in mice spleens compared with PBS liposomes (data are mean ± standard deviation (SD), n = 5 per group, *p < .05; **p < .01; ***p < .001)

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  FIGURE 3. Histological characterization of host response tendency of GPHV in vivo implantation in a mouse subdermal model for a period of 4 weeks. (A, B) Representative images of H&E and Masson trichrome staining of the implanted valve leaflets and the surrounding tissues. (C) Thickness of the fibrous capsule formed around the implanted valve leaflets. (D) Number of inflammatory cells observed in the implanted valve leaflets after retrieval (data are mean ± SD n = 5 per group, *p < .05; **p < .01; ***p < .001)

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  FIGURE 4. Clodronate liposome treatment reduced the number of F4/80+ macrophages, CD3+ T‐cell infiltration, and the production of inflammatory factor in the GPHVs after surgical operations. (A) Representative images of F4/80+ macrophages (green) of the GPHV in PBS and Lipclod groups 28 days after surgical operations. (B) Quantification data show that the liposomal treatment significantly decreased the macrophage infiltration of the GPHV Lipclod groups compared with the PBS groups. (C) Representative images of CD3+ T cell (red) of the GPHV+PBS and the GPHV+Lipclod groups at 28 days after surgical operations. (D) Quantification data show that liposomal treatment significantly decreased the CD3+ T‐cell infiltration of the GPHV+Lipclod groups compared with the PBS groups. (E) Quantification data show that liposomal treatment significantly decreases the infiltration of inflammatory factors in the case of the GPHV+Lipclod group compared with the GPHV+PBS group (data are mean ± SD, n = 5 per group; *p < .05; **p < .01; ***p < .001)

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  FIGURE 5. In situ zymography shows the activities of metalloproteinases (MMPs) in GPHV. (A) In situ zymography of GPHV showing decreased MMP activity in the GPHV+Lipclod group that was mitigated by the Lipclod treatment. Green fluorescence indicates active MMP. (B) Graphical presentation of MMP activity. (C) Messenger ribonucleic acid (mRNA) expression levels of MMP‐2 and (D) MMP‐9 that varied between groups (data are mean ± SD, n = 5 per group, *p < .05; **p < .01; ***p < .001)

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  FIGURE 6. Histological characterization of calcification of GPHV in an in vivo implantation subdermal mouse model over a four‐week period. (A, B) Representative images of the immunohistochemical staining and quantification data show that liposomal treatment significantly decreased Runx2 expression in GPHV compared with PBS liposomes. (C) Polymerase chain reaction (PCR) expression levels of Runx2 varied between groups (n = 5 per group, *p < .05; ***p < .001, Student's t test). (D) Quantitative calcium content comparisons between studied groups

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  FIGURE 7. Macrophages can induce valve degradation and calcification by secreting cytokines and MMPs, and can also increase CD3+ T‐cell activation, thus increasing the infiltration of T cells into GBHV. Once activated, stationary T cells can differentiate into cytotoxic T cells and secrete cytokines mediating valve degradation