Figure 5.
Figure 5. aPC restricts inflammasome activation via PAR-1 after myocardial IRI. (A-C) The effect of aPC on inflammasome activation was analyzed after receptor inhibition. BMDMs (A), neonatal cardiomyocytes (B), or neonatal cardiac fibroblasts (C) were isolated from wild-type (WT), PAR-1−/−, PAR-2−/−, or PAR-3−/− mice. aPC fails to supress LPS/ATP- (A,C) or hypoxia/reoxygenation-induced (B) Nlrp3 expression and cl-Casp1 and cl–IL-1β in the absence of PAR-1 in all cell types, whereas loss of other receptors had no effect; representative immunoblots, with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as loading control (A-C). (D) Experimental design. (E-F) Treatment of mice with aPC-HAPC1573 complex or 3K3A-aPC reduces the infarct size as efficiently as aPC, and blocking PAR-1 signaling (pepducin P1pal-12S) abolishes the inhibitory effect of aPC. Representative heart sections showing infarcted area detected by triphenyl tetrazolium chloride staining (area encircled by dashed line; size bar, 20 µm) (E) and dot plots summarizing data (F). (G-J) Treatment of mice with aPC-HAPC1573 complex or 3K3A-aPC reduces markers of inflammasome activation as efficiently as aPC, and blocking PAR-1 signaling (pepducin P1pal-12S) abolishes the inhibitory effect of aPC. (G) Representative immunoblots of cardiac Nlrp3 expression and cl-Casp1 and cl–IL-1β, with GAPDH as loading control (cont); arrowheads indicate inactive (white) and active (black) forms of caspase-1 or IL-1β. (H) Representative images of active caspase-1 within the infarcted tissue (frozen sections incubated with FLICA-Casp1 probes; size bar, 20 µm). Dot plots summarizing plasma IL-1β (I) and IL-18 (J) levels. Sham-operated (sham) or mice with myocardial IRI without (PBS; cont), with aPC (aPC), with aPC-HAPC1573 complex (aPC+HAPC1573), with an aPC variant specifically lacking anticoagulant function (3K3A-aPC), or with aPC and PAR-1 pepducin P1pal-12S (aPC+P1pal-12S) pretreatment. Data shown represent mean ± SEM. Data obtained from at least 3 independent experiments each with at least 2 technical replicates (A-C) or from at least 6 mice per group (D-J). **P < .01; analysis of variance (F,I-J).

aPC restricts inflammasome activation via PAR-1 after myocardial IRI. (A-C) The effect of aPC on inflammasome activation was analyzed after receptor inhibition. BMDMs (A), neonatal cardiomyocytes (B), or neonatal cardiac fibroblasts (C) were isolated from wild-type (WT), PAR-1−/−, PAR-2−/−, or PAR-3−/− mice. aPC fails to supress LPS/ATP- (A,C) or hypoxia/reoxygenation-induced (B) Nlrp3 expression and cl-Casp1 and cl–IL-1β in the absence of PAR-1 in all cell types, whereas loss of other receptors had no effect; representative immunoblots, with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as loading control (A-C). (D) Experimental design. (E-F) Treatment of mice with aPC-HAPC1573 complex or 3K3A-aPC reduces the infarct size as efficiently as aPC, and blocking PAR-1 signaling (pepducin P1pal-12S) abolishes the inhibitory effect of aPC. Representative heart sections showing infarcted area detected by triphenyl tetrazolium chloride staining (area encircled by dashed line; size bar, 20 µm) (E) and dot plots summarizing data (F). (G-J) Treatment of mice with aPC-HAPC1573 complex or 3K3A-aPC reduces markers of inflammasome activation as efficiently as aPC, and blocking PAR-1 signaling (pepducin P1pal-12S) abolishes the inhibitory effect of aPC. (G) Representative immunoblots of cardiac Nlrp3 expression and cl-Casp1 and cl–IL-1β, with GAPDH as loading control (cont); arrowheads indicate inactive (white) and active (black) forms of caspase-1 or IL-1β. (H) Representative images of active caspase-1 within the infarcted tissue (frozen sections incubated with FLICA-Casp1 probes; size bar, 20 µm). Dot plots summarizing plasma IL-1β (I) and IL-18 (J) levels. Sham-operated (sham) or mice with myocardial IRI without (PBS; cont), with aPC (aPC), with aPC-HAPC1573 complex (aPC+HAPC1573), with an aPC variant specifically lacking anticoagulant function (3K3A-aPC), or with aPC and PAR-1 pepducin P1pal-12S (aPC+P1pal-12S) pretreatment. Data shown represent mean ± SEM. Data obtained from at least 3 independent experiments each with at least 2 technical replicates (A-C) or from at least 6 mice per group (D-J). **P < .01; analysis of variance (F,I-J).

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