A Vaccine Against Fibroblast Activation Protein Improves Murine Cardiac Fibrosis by Preventing the Accumulation of Myofibroblasts

Abstract

Background: Myofibroblasts are primary cells involved in chronic response-induced cardiac fibrosis. Fibroblast activation protein (FAP) is a relatively specific marker of activated myofibroblasts and a potential target molecule. This study aimed to clarify whether a vaccine targeting FAP could eliminate myofibroblasts in chronic cardiac stress model mice and reduce cardiac fibrosis.

Methods: We coadministered a FAP peptide vaccine with a cytosine-phosphate-guanine (CpG) K3 oligonucleotide adjuvant to male C57/BL6J mice and confirmed an elevation in the anti-FAP antibody titer. After continuous angiotensin II and phenylephrine administration for 28 days, we evaluated the degree of cardiac fibrosis and the number of myofibroblasts in cardiac tissues.

Results: We found that cardiac fibrosis was significantly decreased in the FAP-vaccinated mice compared with the angiotensin II and phenylephrine control mice (3.45±1.11% versus 8.62±4.79%; P=4.59×10^-3) and that the accumulation of FAP-positive cells was also significantly decreased, as indicated by FAP immunohistochemical staining (4077±1746 versus 7327±1741 cells/mm²; FAP vaccine versus angiotensin II and phenylephrine control; P=6.67×10^-3). No systemic or organ-specific inflammation due to antibody-dependent cell cytotoxicity induced by the FAP vaccine was observed. Although the transient activation of myofibroblasts has an important role in maintaining the structural robustness in the process of tissue repair, the FAP vaccine showed no adverse effects in myocardial infarction and skin injury models.

Conclusions: Our study demonstrates the FAP vaccine can be a therapeutic tool for cardiac fibrosis.

Keywords: fibrosis; myofibroblasts; vaccines.

Figure 1.

Evaluation of FAP (fibroblast activation protein) vaccine-induced antibody production. C57BL/6J mice were immunized with a FAP vaccine (FAP#1, FAP#2, or FAP#3) or the keyhole limpet hemocyanin (KLH) vaccine at the ages of 10 and 12 weeks. n=4, 5, and 5 for FAP#1, FAP#2, and FAP#3, respectively; KLH, n=5. A, Three-dimensional structure of FAP. Red: candidate FAP B-cell epitopes. Green: cytoplasmic motif. Blue: transmembrane domain. Yellow: extracellular domain. The structure was made using Swiss PDB Viewer, version 4.1.0. B, The serum titer of antibodies against FAP-BSA in mice immunized with a FAP vaccine, expressed as the half-maximal binding (OD 50%). n=4, 5, and 5 for FAP#1, FAP#2, and FAP#3, respectively. C, The serum titer of antibodies against a recombinant mouse FAP protein at 28 days postimmunization, expressed as the OD at 450 nm. D, The antibody induced by a FAP (FAP#3) vaccine recognizes the rmFAP (recombinant mouse FAP) protein, as shown by western blot analysis. As a negative control, 100 ng of recombinant mouse DPPIV (rmDPPIV) was run in lane 1, while 100 and 30 ng of rmFAP were run in lanes 2 and 3, respectively. Anti-FAP Ab (as a positive control), 0.05 µg/mL; KLH vaccine serum, 1:200; FAP vaccine serum, 1:200. E, Evaluation of T-cell activation in FAP-vaccinated mice at 28 days postimmunization by enzyme-linked immunospot. Splenocytes (2×10⁵ cells per well) from FAP-vaccinated mice were stimulated with 1 µg/mL FAP peptide (FAP#1, FAP#2, or FAP#3), 1 µg/mL KLH, or 0.1 µg/mL phorbol myristate acetate (PMA) + ionomycin (as a positive control). The number of IFN-γ- (interferon-gamma) or IL-4 (interleukin-4)-producing cells was quantified in triplicate wells for each group. All data are expressed as the mean±SD. Statistical analyses were 2-sided. Statistical significance was assessed with the Kruskal-Wallis test and Dunn multiple comparisons test for post hoc analysis vs KLH (C, 3 comparisons) or with 1-way ANOVA and Bonferroni correction for post hoc analysis vs nonstimulated (E, 3 comparisons each group). Aa indicates amino acid.

Figure 2.

Effects of FAP (fibroblast activation protein) vaccine on angiotensin II and phenylephrine (AngII/PE) infusion model mice. A, Time course of AngII/PE loading and injection of vaccines. Mice immunized with the FAP vaccine (n=15) or keyhole limpet hemocyanin (KLH) vaccine (n=20) were vaccinated at the ages of 8, 10, and 12 weeks. After all vaccinations, the mice were continuously administered AngII/PE via an osmotic minipump for 28 days to induce cardiac fibrosis. The mice in the saline control group (n=10) and mice in the AngII/PE control group (n=20) were unvaccinated and continuously administered saline or AngII/PE via an osmotic minipump. B, Kaplan-Meier survival curves of mice continuously administered AngII/PE with an osmotic minipump for 28 days. n=10, 20, 19, and 15 for the saline, AngII/PE, KLH vaccine, and FAP vaccine groups, respectively. Median survival time: saline, KLH vaccine, and FAP vaccine, not reached; AngII/PE, 10 days. One mouse in the KLH vaccine group died from the anesthesia procedure before continuous AngII/PE administration. C, The body weight changes of the surviving mice in B. n=10, 10, 10, and 13 for the saline, AngII/PE, KLH vaccine, and FAP vaccine groups, respectively. D, Quantification of murine organ (heart, kidney, and lung) weight-to-body weight (OW/BW) ratios 28 days after continuous AngII/PE administration. n=10, 10, 10, and 13 for the saline, AngII/PE, KLH vaccine, and FAP vaccine groups, respectively. E, Serum levels of anti-cardiac auto-antibodies against mouse cardiac lysate in the serum as tested by ELISA using serum of 28 days after continuous AngII/PE administration mice. n=5, each group. F, Serum levels of cardiac troponin I (cTnI) in mice after continuous AngII/PE administration for 28 days. n=10, each group. All the data are expressed as the mean±SD. Statistical analyses were 2-sided. Statistical significance was assessed with 1-way ANOVA and Bonferroni correction for post hoc analysis, vs saline (B and C, 10, 12, 14, and 15 weeks, 3 comparisons), vs FAP vaccine (E, 3 comparisons), with the Kruskal-Wallis test and Dunn multiple comparisons test for post hoc analysis (F, 6 comparisons), vs saline (C, 13 and 16 weeks, 3 comparisons), or with 1-way ANOVA and Tukey multiple comparison test for post hoc analysis (D, 6 comparisons). ns indicates not significant.

Figure 3.

FAP (fibroblast activation protein) vaccine can improve cardiac fibrosis. A, Top, Picro-Sirius Red staining of heart coronal sections. Fibrotic areas were stained red. Bottom, magnified images of left ventricular fibrosis. Scale bars=100 µm. B, Quantification of cardiac fibrosis areas. n=8, 8, 8, and 9 for the saline, angiotensin II and phenylephrine (AngII/PE), keyhole limpet hemocyanin (KLH) vaccine, and FAP vaccine groups, respectively. C Echocardiography 28 days after continuous AngII/PE administration. M-mode echocardiography was performed from a short- or long-axis view. n=5, 4, 5, and 5 for the saline, AngII/PE, KLH vaccine, and FAP vaccine groups, respectively. D, The levels of IgG1 and IgG2 (IgG2b+IgG2c) in FAP-vaccinated mice (n=10) after continuous AngII/PE administration for 28 days. E and F, The antibody-dependent cell cytotoxicity activity and the complement-dependent cytotoxicity (CDC) activity of FAP vaccine-induced antibodies were assessed (n=3, each sample) using 1.5×10⁴ human embryonic kidney (HEK) 293 cells overexpressing FAP. Sera were collected after continuous saline or AngII/PE administration for 28 days and were serially diluted from 10-fold to 6250-fold. The relative value of the CDC assay was defined as 100% for the absorbance of the positive control group, in which the cells were completely lysed, and 0% for the absorbance of the negative control group, in which only the solution without cells was used. G, FAP staining of heart coronal sections. Left, FAP-positive fibroblasts were stained brown in fibrotic areas. Scale bars= 500 µm (top) and 100 µm (bottom). Right, quantification of FAP-positive fibroblasts in the fibrotic areas. The saline group values were used as background and subtracted from the obtained values in the other groups. n=5, 5, and 6 for the AngII/PE, KLH vaccine, and FAP vaccine groups, respectively. All the data are expressed as the mean±SD. Statistical analyses were 2-sided. Statistical significance was assessed with 1-way ANOVA and Bonferroni correction for post hoc analysis vs saline (E and F, 2 comparisons each dilution), with 1-way ANOVA and Tukey multiple comparison test for post hoc analysis (B and C, 6 comparisons; and G, 3 comparisons) or with unpaired 2-tailed t test (D). EF indicates ejection fraction; E/E’, ratio of mitral peak velocity of early filling (E) to early diastolic mitral annular velocity (E’); LVIDd, left ventricular internal diameter end diastole; LVIDs, left ventricular internal diameter end systole; and ns, not significant.

Figure 4.

Efficacy of the FAP (fibroblast activation protein) vaccine in cardiac tissues. A, F4/80 staining of heart coronal sections. Left, F4/80-positive macrophages were stained brown. Scale bars=100 µm. Right, quantification of F4/80-positive macrophages. n=5, 5, 5, and 6 for the saline, angiotensin II and phenylephrine (AngII/PE), keyhole limpet hemocyanin (KLH) vaccine, and FAP vaccine groups, respectively. B, IgG staining of heart coronal sections. Left, IgG-positive cells were stained brown. Scale bars=100 µm. Right, quantification of IgG-positive cells. n=5, 5, 5, and 6 for the saline, AngII/PE, KLH vaccine, and FAP vaccine groups, respectively. C, Representative western blots of the FAP protein in cardiac tissues. D, Western blot analysis of the FAP protein in cardiac tissues. The results are presented as the fold change in the levels of the FAP protein normalized to the level of GAPDH. The average fold change in the saline group was set to 1 for normalization. n=5, 6, 6, and 6 for the saline, AngII/PE, KLH vaccine, and FAP vaccine groups, respectively. E, Relative Acta2, Cilp, Col1a1, Fap, Postn, and Tgfb1 expression levels in cardiac tissues. The average expression levels in the saline group were set to 1 for normalization. n=5, 6, 6, and 6 for the saline, AngII/PE, KLH vaccine, and FAP vaccine groups, respectively. All the data are expressed as the mean±SD. Statistical analyses were 2-sided. Statistical significance was assessed with Kruskal-Wallis test and Dunn multiple comparisons test for post hoc analysis (A, B, and E, Clip, Col1a1, and Postn, 6 comparisons) or with 1-way ANOVA and Tukey multiple comparison test for post hoc analysis (D and E, Acta2, Fap, and Tgfb1, 6 comparisons).

Figure 5.

Evaluation of the safety of the FAP (fibroblast activation protein) vaccine. A, Serum levels of cytokines (IFN-γ [interferon-gamma], IL [interleukin]-6, IL-10, IL-12p70, MCP-1 [monocyte chemoattractant protein 1], and TNF [tumor necrosis factor]) in FAP-vaccinated mice after continuous angiotensin II and phenylephrine (AngII/PE) administration for 28 days. n=10, 10, 10, and 13 for the saline, AngII/PE, keyhole limpet hemocyanin (KLH) vaccine, and FAP vaccine groups, respectively. B Serum levels of pancreatic amylase to test pancreatic toxicity. n=10, 10, 10, and 13 for the saline, AngII/PE, KLH vaccine, and FAP vaccine groups, respectively. C, IgG staining of heart coronal sections, kidney tissues, and lung tissues from FAP-vaccinated mice at 28 days postimmunization. Representative images of n=3 independent mice per group. IgG-positive cells were stained brown. Scale bars=100 µm. D, Hematoxylin and eosin staining of heart coronal sections, kidney tissues, and lung tissues from FAP-vaccinated mice after continuous AngII/PE administration for 28 days. Representative images of n=3 independent mice per group. Scale bars=100 µm. E, Quantification of wound area by circular biopsy punch in the FAP-vaccinated mice. Mice immunized with the saline (used as a control), KLH vaccine, or FAP vaccine were vaccinated at the ages of 8, 10, and 12 weeks, followed by skin biopsy. The relative value of the wound area was defined as 100% for the wound area of each sample on day 0. n=5, each group. F, Photographs of 0 days, 8 days, and 14 days after wounding. All the data are expressed as the mean±SD. Statistical analyses were 2-sided. Statistical significance was assessed with Kruskal-Wallis test and Dunn multiple comparisons test for post hoc analysis vs saline (A, 3 comparisons; and E, day 0, 8 and 11, 2 comparisons), with 1-way ANOVA and Tukey multiple comparison test for post hoc analysis (B, 6 comparisons) or with 1-way ANOVA and Bonferroni correction for post hoc analysis vs saline (E, day 3, 6, and 14, 2 comparisons).

Figure 6.

Efficacy of the FAP (fibroblast activation protein) vaccine in the acute phase of myocardial infarction. A, Probability of survival 28 days after myocardial infarction (MI). n=8, 11, 11, and 12 for the sham control, MI control, keyhole limpet hemocyanin (KLH) vaccine, and FAP vaccine groups, respectively. B, Echocardiography 7 days after MI. M-mode echocardiography was performed from a short- or long-axis view. n=8, 7, 10, and 8 for the sham control, MI control, KLH vaccine, and FAP vaccine groups, respectively. C, Echocardiography 28 days after MI. M-mode echocardiography was performed from a short- or long-axis view. n=8, 6, 10, and 8 for the sham control, MI control, KLH vaccine, and FAP vaccine groups, respectively. D, Masson trichrome and immunohistochemical staining (FAP, α-SMA [α-smooth muscle actin], and troponin I) of heart coronal sections from FAP-vaccinated mice at 28 days post MI. Scale bars: whole image, 500 µm; Masson trichrome, FAP, α-SMA, and troponin I, 50 µm. E, Quantification of cardiac fibrosis areas. n=6, 5, and 8 for the MI, KLH vaccine, and FAP vaccine groups, respectively. Fibrosis was evaluated after trimming 100 µm of the outer frame to exclude the effect of the MI site. F, Quantification of FAP-positive fibroblast area. n=4, 3, and 5 for the angiotensin II and phenylephrine (AngII/PE), KLH vaccine, and FAP vaccine groups, respectively. All the data are expressed as the mean±SD. Statistical analyses were 2-sided. Statistical significance was assessed with χ² test (A) or with 1-way ANOVA and Tukey multiple comparison test for post hoc analysis (B and C, 6 comparisons; and E and F, 3 comparisons). EF indicates ejection fraction; LVIDd, left ventricular internal diameter end diastole; LVIDs, left ventricular internal diameter end systole; and ns, not significant.