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

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

GrK stimulates IL-6, IL-8 and MCP-1 protein production in lung fibroblasts.

<p>(A–C) HFLs were exposed to concentrations of GrK ranging from 10–300 nM at for 24 h. Cell culture supernatants were then collected and analyzed for IL-6, IL-8 and MCP-1 production by ELISA. Cell counts were used to normalize cytokine concentrations by controlling for variability in cell numbers. Control wells include cells treated with media alone or cells treated with heat in-activated GrK (100 nM). All data are expressed as mean protein production (pg/10⁶ cells) ± SEM from three independent triplicate experiments. * p<0.05 when compared to media alone; ** p<0.01 when compared to media alone.</p

GrK induces cell proliferation in HFLs in a dose-dependent manner.

<p>(A) Cells were incubated with 100 and 200 nM of GrK for 48 h then trypsinized and counted using a hemocytometer to determine cell number. (B) PAR-1 neutralization with ATAP-2 (5 µg/ml) abolished GrK (200 nM)-induced cell proliferation. Data are expressed as mean fold change ± SEM from three independent experiments run in triplicate. * p<0.05 compared to media control.</p

Development of Autoimmune Hair Loss Disease Alopecia Areata Is Associated with Cardiac Dysfunction in C3H/HeJ Mice

<div><p>Alopecia areata (AA) is a chronic autoimmune hair loss disease that affects several million men, women and children worldwide. Previous studies have suggested a link between autoimmunity, stress hormones, and increased cardiovascular disease risk. In the current study, histology, immunohistology, quantitative PCR (qPCR) and ELISAs were used to assess heart health in the C3H/HeJ mouse model for AA and heart tissue response to adrenocorticotropic hormone (ACTH) exposure. Mice with AA exhibited both atrial and ventricular hypertrophy, and increased collagen deposition compared to normal-haired littermates. QPCR revealed significant increases in <i>Il18</i> (4.6-fold), IL18 receptor-1 (<i>Il18r1</i>; 2.8-fold) and IL18 binding protein (<i>Il18bp</i>; 5.2-fold) in AA hearts. Time course studies revealed a trend towards decreased <i>Il18</i> in acute AA compared to controls while <i>Il18r1</i>, <i>Il18bp</i> and <i>Casp1</i> showed similar trends to those of chronic AA affected mice. Immunohistochemistry showed localization of IL18 in chronic AA mouse atria. ELISA indicated cardiac troponin-I (cTnI) was elevated in the serum and significantly increased in AA heart tissue. Cultures of heart atria revealed differential gene expression between AA and control mice in response to ACTH. ACTH treatment induced significant increase in cTnI release into the culture medium in a dose-dependent manner for both AA and control mice. In conclusion, murine AA is associated with structural, biochemical, and gene expression changes consistent with cardiac hypertrophy in response to ACTH exposure.</p></div

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

qPCR analysis of selected genes in AA and sham-grafted mice in chronic stage.

<p>In an initial screen for various heart disorder related gene markers, there was a significant increase of <i>Il18, Il18r1 and Il18bp</i> gene in both skin (A) and hearts (B) of AA mice (n = 4) compared to sham-grafted control mice (n = 5). The expression of <i>Nppa</i> was also significantly increased in the hearts of AA mice. QPCR relative fold change in gene expression analyses were calculated using 2^−ΔΔCt; average fold change is presented. Error bars represent the range factor difference (2^{−ΔΔCt±ΔCtSD}). Statistical significance determined with Student’s <i>t</i>-test; *denotes p<0.05.</p

Desensitization of PAR-1 with Thrombin (2.5 U/ml) reduces GrK-induced IL-6, IL-8 and MCP-1 production in lung fibroblasts.

<p>(A–C) HFL were treated with thrombin (2.5 U/ml) for 10 min, monolayers were then rinsed three times with fresh media and incubated with GrK (200 nM) for 24 h. Supernatants were collected and IL-6, IL-8 and MCP-1 levels were analyzed by ELISA. Supernatants were normalized using cell counts. Data are expressed as % change over media control ± SEM from three separate experiments run in triplicate. (D) GrK (200 nM) and thrombin (2.5 U/ml) induced ERK1/2 phosphorylation following 10 min incubation. Pre-treatment (PT) of HFL with thrombin (2.5 U/ml) for 10 min prior to treatment with GrK (200 nM) for an additional 10 min decreased ERK1/2 phosphorylation when compared to monolayers incubated with GrK (200 nM) alone. ERK1/2 phosphorylation was assessed by Western blotting of cell lysates using anti-phospho-ERK1/2 and anti-total-ERK1/2 antibodies. Protein loading was verified and normalized using β-tubulin. Data represent the mean ± SEM of at least three independent experiments. + p<0.05 when compared to media control (1% DMSO in DMEM); ** p<0.05 compared to GrK (200 nM) treated cells.</p

Western blot analysis of effect of GrK on ERK1/2 and p38 MAPK phosphorylation.

<p>Cells were incubated with GrK (200 nM) for 10 min, 30 min, or 2 h. Cell lysates were collected and screened for (A) ERK1/2 phosphorylation using anti-phospho-ERK1/2 and anti-total-ERK1/2 antibodies and (C) p38 MAPK phosphorylation using anti-phospho-p38MAPK and total p38MAPK antibodies. Cells incubated with ERK1/2 inhibitor (U0126; 10 µM) or p38MAPK inhibitor (SB202190; 10 µM) prior to treatment with GrK (200 nM) for 10 min are included to demonstrate the activity of each inhibitor. Densitometry analysis of immunoblots was carried out using Li-COR Odyssey Infrared imaging system (Li-COR biosciences). (B) Relative levels of ERK1/2 phosphorylation (ERK1 white bars; ERK2 black bars) are expressed as a ratio of phospho-ERK1/2 to total ERK1/2. (D) Relative levels of p38 MAPK phosphorylation are expressed as a ratio of phospho-p38 MAPK to total p38 MAPK. Protein loading was normalized using β-tubulin. The values shown are mean +/− SEM from three separate experiments. * p<0.05 compared to media alone.</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