Plaque area in aortic roots from granzyme B or perforin deficient apolipoprotein E knockout mice.

<p>(A) Representative images of aortic root cross sections from high fat diet-fed wild type (WT), apolipoprotein E knockout (ApoE KO), granzyme B (GzmB)/ApoE double knockout (DKO) and perforin (Prf1)/ApoE DKO mice stained with Movat’spentachrome. Scale bars = 500 µm. No significant difference in the size of plaque was observed in GzmB/ApoE DKO (n = 9) or Prf1/ApoE DKO mice (n = 8) compared to ApoE KO mice (n = 10). (B) Example images of plaques from aortic roots stained with Movat’spentachrome. The same number of animals were used for these measurements as in panel A. Arrows indicate boundaries of the intimal plaque. Scale bars = 100 µm. No significant difference was detected in the ratio of intimal/medial thickness. ns = not significant (One-way ANOVA with bonferronipost test). Error bars represent SEM.</p

Granzyme B and perforin contribute to plaque development in the descending aorta of apolipoprotein Eknockout mice.

<p>(A) Representative images on the descending aorta from high fat diet-fed apolipoprotein E knockout (ApoE KO), granzyme B (GzmB)/ApoE double knockout (DKO) and perforin (Prf1)/ApoE DKO mice stained en face with sudan IV. (B) When plaque area was quantified, GzmB/ApoE DKO mice (n = 22) had significantly reduced plaque area compared to ApoE KO mice (n = 16). Prf1/ApoE DKO mice (n = 14) had significantly less plaque than both the ApoE KO mice and the GzmB/ApoE DKO mice. *<i>P</i><0.05, ***<i>P</i><0.005 (One-way ANOVA with bonferonnipost test). Error bars represent SEM.</p

Increased decorin in plaques from granzyme B and perforin deficient apolipoprotein Eknockout mice.

<p>Representative images of aortic root sections fromapolipoprotein E knockout (ApoE KO), granzyme B (GzmB)/ApoE double knockout (DKO) and perforin (Prf1)/ApoE DKO mice stained for decorin. Decorin in the GzmB deficient animals was observed near the surface of the plaque in concentrated pockets (black arrowheads) while decorin in Prf1 deficient animals stained more diffusely throughout the plaque (white arrowheads). White scale bars = 50 µm, black scale bars = 500 µm.</p

Granzyme B and perforin are present in atherosclerotic plaques from apolipoprotein E knockout mice.

<p>(A)Representative images of aorta cross sections from high fat diet-fed wild type (WT),apolipoprotein E knockout(ApoE KO), Granzyme B (GzmB)/ApoE double knockout (DKO) and perforin (Prf1)/ApoE DKOmice stained for GzmB and Prf1. Black scale bars = 400 µm, white scale bars = 100 µm.(B)Neither GzmB nor Prf1 deficiency resulted in a significant difference in circulating levels of cholesterol (n = 4) and triglycerides (n = 7) in ApoE KO mice when fed a high fat diet for 30 weeks.</p

Expression of granzyme A, T cells and macrophages in atherosclerotic plaques.

<p>Representative images of wild type (WT), apolipoprotein E knockout (ApoE KO), granzyme B (GzmB)/ApoE double knockout (DKO) and perforin(Prf1)/ApoE DKO mouse aortas stained for (A) granzyme A, (B) CD3 and (C) F4/80. Scale bars = 100 µm.</p

Granzyme B, but not perforin deficiency results in increased collagen content in atherosclerotic plaques.

<p>(A) Representative images of aortic root sections stained for collagen using picrosirius red and visualized under bright field or polarized light. Bright field images were used to define the area of plaque and collagen was quantified using the images taken under polarized light. Scale bars = 500 µm. (B) Compared to the high fat diet-fed apolipoprotein E knockout (ApoE KO) mice (n = 4), granzyme B (GzmB)/ApoE double knockout (DKO) mice (n = 6) exhibited increased collagen content in atherosclerotic plaques of the aortic root. Perforin (Prf1)/ApoE DKO mice (n = 5) on the other hand, showed no difference in collagen content compared to ApoE KO mice and significantly less collagen compared to GzmB/ApoE DKO mice. *P<0.05, ***P<0.005 (One-way ANOVA with bonferronipost test). Error bars represent SEM.</p

GrB-mediated cleavage of decorin, biglycan and betaglycan.

<p>Increasing concentrations of GrB (25, 50, 100 and 200 nM) were incubated with decorin (a), biglycan (b), and betaglycan (c) for 24 h at RT. * denotes full length protein, arrows indicate cleavage fragments and ∧ indicates GrB.</p

GrB cleaves native smooth muscle cell-derived decorin and biglycan.

<p>HCASMCs were incubated at confluency for adequate ECM synthesis. Cells were removed, GrB was incubated with the ECM and decorin and biglycan cleavage fragments were detected by western immunoblotting.</p

GrB-mediated cleavage of decorin, biglycan and betaglycan results in the release of active TGF-β1.

<p>Decorin, biglycan and betaglycan complexed with TGF-β1 were treated with GrB. Supernatants (containing released TGF-β1), were collected and released TGF-β1 was detected by Western blotting. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033163#s3" target="_blank">Results</a> shown are representative western blots from at least 3 separate experiments for each PG (a). As endogenous SMC-derived ECM only contains latent TGF-β (as shown in (b)), GrB-mediated release from active TGF-β1 supplemented ECM was also examined (c).</p

GrB-mediated PG cleavage is inhibited by DCI and cleavage site identification.

<p>GrB was incubated with decorin (a), biglycan (b) and betaglycan (c), +/− DCI and the solvent control DMSO, for 4 h and 24 h. Cleavage sites in biglycan and betaglycan were identified by N-terminal Edman degradation. * denotes full length protein, arrows indicate cleavage fragments, and cleavage sites are displayed on the right.</p