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Biochim Biophys Acta. 2015 Feb;1853(2):396-408. doi: 10.1016/j.bbamcr.2014.11.012. Epub 2014 Nov 15.

Chen Y1, Li X1, Boini KM1, Pitzer AL1, Gulbins E2, Zhang Y3, Li PL4.

Author information

  • 1Department of Pharmacology & Toxicology, School of Medicine, Virginia Commonwealth University, Richmond, VA 23298, USA.
  • 2Department of Molecular Biology, University of Duisburg-Essen, Essen, Germany.
  • 3Department of Pharmacology & Toxicology, School of Medicine, Virginia Commonwealth University, Richmond, VA 23298, USA. Electronic address: .
  • 4Department of Pharmacology & Toxicology, School of Medicine, Virginia Commonwealth University, Richmond, VA 23298, USA. Electronic address: .

Abstract

Inflammasomes play a critical role in the development of vascular diseases. However, the molecular mechanisms activating the inflammasome in endothelial cells and the relevance of this inflammasome activation is far from clear. Here, we investigated the mechanisms by which an Nlrp3 inflammasome is activated to result in endothelial dysfunction during coronary arteritis by Lactobacillus casei (L. casei) cell wall fragments (LCWE) in a mouse model for Kawasaki disease. Endothelial dysfunction associated with increased vascular cell adhesion protein 1 (VCAM-1) expression and endothelial-leukocyte adhesion was observed during coronary arteritis in mice treated with LCWE. Accompanied with these changes, the inflammasome activation was also shown in coronary arterial endothelium, which was characterized by a marked increase in caspase-1 activity and IL-1β production. In cultured endothelial cells, LCWE induced Nlrp3 inflammasome formation, caspase-1 activation and IL-1β production, which were blocked by Nlrp3 gene silencing or lysosome membrane stabilizing agents such as colchicine, dexamethasone, and ceramide. However, a potassium channel blocker glibenclamide or an oxygen free radical scavenger N-acetyl-l-cysteine had no effects on LCWE-induced inflammasome activation. LCWE also increased endothelial cell lysosomal membrane permeability and triggered lysosomal cathepsin B release into cytosol. Silencing cathepsin B blocked LCWE-induced Nlrp3 inflammasome formation and activation in endothelial cells. In vivo, treatment of mice with cathepsin B inhibitor also abolished LCWE-induced inflammasome activation in coronary arterial endothelium. It is concluded that LCWE enhanced lysosomal membrane permeabilization and consequent release of lysosomal cathepsin B, resulting in activation of the endothelial Nlrp3 inflammasome, which may contribute to the development of coronary arteritis.

Copyright © 2014 Elsevier B.V. All rights reserved.

KEYWORDS:

Cathepsin B; Endothelium; LCWE; Lysosomal membrane permeabilization; Nlrp3 inflammasome

PMID: 25450976 [PubMed - indexed for MEDLINE] PMCID: PMC4289419 Free PMC Article

Images from this publication.See all images (9)Free text

Fig. 1

Increased VCAM-1 expression and leukocyte adhesion in coronary arterial endothelium of mice treated with LCWE

Mice were intraperitoneally treated with either saline control (Ctrl) or LCWE (500 μg, 2 weeks). (A) IHC staining showing the expression of VCAM-1 in the endothelium of coronary arteries. Summarized data showing area percentage of the endothelium positive for VCAM-1 staining in coronary arteries. (B) IHC staining showing the expression of neutrophil marker in the endothelium of coronary arteries. Summarized data showing area percentage of the endothelium positive for neutrophil marker in coronary arteries. (C) IHC staining showing the expression of T cell marker CD43 in the endothelium of coronary arteries. Summarized data showing area percentage of the endothelium positive for CD43 in coronary arteries. (D) IHC staining showing the expression of macrophage marker F4/80 in the endothelium of coronary arteries. Summarized data showing area percentage of the endothelium positive for F4/80 in coronary arteries. Enlarged images of area of interest (AOI) are indicated with an arrow. Scale bar: 50 μm. N = 6 mice per group. P < 0.05 vs. LCWE on WT.

Endothelial Nlrp3 Inflammasome Activation Associated with Lysosomal Destabilization during Coronary Arteritis

Biochim Biophys Acta. ;1853(2):396-408.

Fig. 2

Activation of endothelial inflammasomes in coronary arteries of mice treated with LCWE

Mice were intraperitoneally treated with saline control (Ctrl) or LCWE (500 μg, 2 weeks). (A) Frozen sections of mouse hearts were stained with FLICA, a green fluorescent probe specific for active caspase-1, and Alexa555-conjugated antibodies against an endothelial cell marker vWF in coronary arteries. The merged images displayed yellow dots or patches indicating the colocalization of FLICA (green) with vWF (red). Summarized data showing the colocalization coefficient of FLICA with vWF (n = 5). (B) IHC staining showing the expression of IL-1β in the endothelium of coronary arteries. Summarized data showing area percentage of the endothelium positive for IL-1β in coronary arteries. High-power magnification of AOI is indicated with an arrow. Scale bar = 50 μm. n = 6 mice per group. P < 0.05 vs. control.

Endothelial Nlrp3 Inflammasome Activation Associated with Lysosomal Destabilization during Coronary Arteritis

Biochim Biophys Acta. ;1853(2):396-408.

Fig. 3

Formation of Nlrp3 inflammasome in MVECs upon LCWE stimulation

(A, B) MVECs were stained with Alexa488-conjugated anti-Nlrp3 and Alexa555-conjugated anti-ASC or anti-caspase1 antibodies. Representative images in panel A show the colocalization (yellow) of Nlrp3 (green) with ASC (red) or Nlrp3 (green) with caspase-1 (red) under stimulation of LCWE (10 μg/ml) for 0–24 hours. Summarized data in panel B show the colocalization coefficiency of Nlrp3 with ASC or Nlrp3 with caspase-1 (n = 5). P < 0.05 vs. LCWE 0 h. (C) MVECs were stimulated without (Ctrl) or with LCWE (10 μg/ml) for different time points (2, 4, 8, 16, and 24 hours) and then lysed. Cell lysates were used for size-exclusion chromatography (SEC) followed by Western blot analysis of eluted fractions using antibodies against pro-caspase-1. Summarized data show the percentage of pro-caspase-1 expression in the high molecular weight fractions (#12–14) compared to total expression of pro-caspase-1 (#1–14) (n = 3). P < 0.05 vs. Ctrl.

Endothelial Nlrp3 Inflammasome Activation Associated with Lysosomal Destabilization during Coronary Arteritis

Biochim Biophys Acta. ;1853(2):396-408.

Fig. 4

LCWE increased Nlrp3 inflammasome activity in MVECs

(A) Representative immunoblotting documents and summarized data showing the effects of LCWE (10 μg/ml for 0–24 hours) on the expression of pro-caspase-1, cleaved caspase-1 and β-actin in MVECs (n = 4). P < 0.05 vs. LCWE 0 h. (B) Effects of LCWE (10 μg/ml for 0–8 hour) on IL-1β production in MVECs (n = 6) P < 0.05 vs. LCWE 0 h. (C) Representative Western blot documents and summarized data showing the effect of LCWE (10 μg/ml, 8 h) on the expression of pro-caspase-1, cleaved caspase-1 and β-actin expression in MVECs transfected with scrambled shRNA (Scr) or Nlrp3 shRNA plasmids (n = 4). P < 0.05 vs. scrambled control; # P < 0.05 vs. scramble+LCWE.

Endothelial Nlrp3 Inflammasome Activation Associated with Lysosomal Destabilization during Coronary Arteritis

Biochim Biophys Acta. ;1853(2):396-408.

Fig. 5

Effects of potassium channel blockade or ROS scavenging on LCWE-induced activation of Nlrp3 inflammasomes in MVECs

MVECs were stimulated with or without LCWE (10 μg/ml, 8 h) in the presence of PBS (Vehl: vehicle), potassium channel blocker glibenclamide (GLY, 10 μM, Sigma) or ROS scavenger N-acetyl-L-cysteine (NAC, 10 μM, Sigma). (A) Representative Western blot documents and summarized data showing the effects of glibenclamide or N-acetyl-L-cysteine on the expression of pro-caspase-1, cleaved caspase-1 and β-actin (n = 3). (B) IL-1β production measurement in MVECs by ELISA (n=6). P < 0.05, LCWE vs. control; # P < 0.05 vs. vehicle+LCWE.

Endothelial Nlrp3 Inflammasome Activation Associated with Lysosomal Destabilization during Coronary Arteritis

Biochim Biophys Acta. ;1853(2):396-408.

Fig. 6

LCWE-induced activation of Nlrp3 inflammasomes is associated with increased lysosome membrane permeability

MVECs were stimulated without (control: Ctrl) or with LCWE (10 μg/ml, 8 hour) in either the absence or presence of lysosomal function stabilization reagents (Vehl: vehicle only; Col: colchicine, 10 μM; Cer: C2-ceramide, 20 μM, Enzolife science; and Dex: dexamethasone, 100 μM, Sigma). (A) and (B) MVECs were stained with Alexa488-conjugated anti-Nlrp3 and Alexa555-conjugated anti-ASC or anti-caspase1 antibodies. Representative images show the colocalization (yellow) of Nlrp3 (green) with ASC (red) or Nlrp3 (green) with caspase-1 (red). Summarized data in panels A and B show the colocalization coefficiency of Nlrp3 with ASC or Nlrp3 with caspase-1 (n=5). (C) Western blot analysis of cleaved caspase-1 and pro-caspase-1 in MVECs (n = 6). (D) IL-1β production in MVECs by ELISA (n = 6). P < 0.05 vs. vehicle control; # P < 0.05 vs. vehicle+LCWE.

Endothelial Nlrp3 Inflammasome Activation Associated with Lysosomal Destabilization during Coronary Arteritis

Biochim Biophys Acta. ;1853(2):396-408.

Fig. 7

LCWE increased lysosome membrane permeability and cathepsin B release in MVECs

(A) MVECs were incubated with either LCWE (10 μg/ml) for 0, 2, 4 and 8 hours or GPN (100 μM) for 4 h. Lysosome membrane permeability was detected by acridine orange staining and visualization by fluorescence microscopy. (B) Summarized data show the red-to-green fluorescence ratio of acridine orange staining in MVECs by flow cytometry analysis (n = 3). P < 0.05 vs. LCWE 0 hour. (C) Western blot analysis of cathepsin B expression in cytosolic fractions of MVECs with or without LCWE (10 μg/ml, 8 h) (n = 5). P < 0.05 vs. control.

Endothelial Nlrp3 Inflammasome Activation Associated with Lysosomal Destabilization during Coronary Arteritis

Biochim Biophys Acta. ;1853(2):396-408.

Fig. 8

Effects of cathepsin B gene silencing on LCWE-induced Nlrp3 inflammasome activation

MVECs were transfected with scramble (Scr) or cathepsin B siRNA (CatBsi) for 24 hours and then stimulated with or without LCWE (10 μg/ml, 8 h). (A) Confocal fluorescent images and summarized co-localization coefficient show the co-localization (yellow) between Nlrp3 (green) and caspase-1 (red) (n = 4). (B) Western blot analysis of cleaved caspase-1 and pro-caspase-1 (n = 4). (C) IL-1β production (n = 6). (D) Western blot analysis of cathepsin B expression in cell lysates of MVECs transfected with scramble or cathepsin B siRNA (n = 4). P < 0.05 vs. scramble control; # P < 0.05 vs. scramble+LCWE.

Endothelial Nlrp3 Inflammasome Activation Associated with Lysosomal Destabilization during Coronary Arteritis

Biochim Biophys Acta. ;1853(2):396-408.

Fig. 9

Effects of cathepsin B inhibition on LCWE-induced inflammasome activity in coronary arterial endothelium of mice

Mice were intraperitoneally treated with saline control (Ctrl), LCWE (500 μg, 2 weeks) with or without cathepsin B inhibitor Ca-074Me (5mg/kg). (A) and (B) Frozen sections of mouse hearts were stained with FLICA, a green fluorescent probe specific for active caspase-1, and Alexa555-conjugated antibodies against an endothelial cell marker vWF in coronary arteries. The merged images displayed yellow dots or patches indicating the colocalization of FLICA (green) with vWF (red). Summarized data showing the colocalization coefficient of FLICA with vWF. (C) and (D) IHC staining showing the expression of macrophage marker F4/80 in the endothelium of coronary arteries. Summarized data showing area percentage of the endothelium positive for F4/80 in coronary arteries. (E) and (F) IHC staining showing the expression of neutrophil marker in the endothelium of coronary arteries. Summarized data showing area percentage of the endothelium positive for neutrophil marker in coronary arteries. Enlarged images of area of interest (AOI) are indicated with an arrow. Scale bar: 50 μm. N = 5 mice per group. P < 0.05 vs. control; # P<0.05 vs. LCWE only.

Endothelial Nlrp3 Inflammasome Activation Associated with Lysosomal Destabilization during Coronary Arteritis

Biochim Biophys Acta. ;1853(2):396-408.

Publication Types, MeSH Terms, Substances, Grant Support

Publication Types

  • Research Support, N.I.H., Extramural

MeSH Terms

  • Animals
  • Arteritis/metabolism
  • Arteritis/pathology
  • Carrier Proteins/metabolism
  • Cathepsin B/antagonists & inhibitors
  • Cathepsin B/metabolism
  • Cell Wall/chemistry
  • Coronary Vessels/metabolism
  • Coronary Vessels/pathology
  • Endothelial Cells/drug effects
  • Endothelial Cells/metabolism
  • Endothelial Cells/pathology
  • Free Radical Scavengers/metabolism
  • Gene Silencing/drug effects
  • Inflammasomes/metabolism
  • Inflammation/pathology
  • Lactobacillus casei
  • Lysosomes/drug effects
  • Lysosomes/metabolism
  • Mice, Inbred C57BL
  • Potassium Channel Blockers/pharmacology
  • Reactive Oxygen Species/metabolism
  • Vascular Cell Adhesion Molecule-1/metabolism

Substances

  • CIAS1 protein, mouse
  • Carrier Proteins
  • Free Radical Scavengers
  • Inflammasomes
  • Potassium Channel Blockers
  • Reactive Oxygen Species
  • Vascular Cell Adhesion Molecule-1
  • Cathepsin B

Grant Support

  • HL057244/HL/NHLBI NIH HHS/United States
  • HL075316/HL/NHLBI NIH HHS/United States
  • HL091464/HL/NHLBI NIH HHS/United States
  • HL122769/HL/NHLBI NIH HHS/United States
  • R01 DK104031/DK/NIDDK NIH HHS/United States
  • R01 HL057244/HL/NHLBI NIH HHS/United States
  • R01 HL075316/HL/NHLBI NIH HHS/United States
  • R01 HL122769/HL/NHLBI NIH HHS/United States
  • R01 HL122937/HL/NHLBI NIH HHS/United States

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