Instigation of endothelial Nlrp3 inflammasome by adipokine visfatin promotes inter-endothelial junction disruption: role of HMGB1 - PubMed (original) (raw)

Instigation of endothelial Nlrp3 inflammasome by adipokine visfatin promotes inter-endothelial junction disruption: role of HMGB1

Yang Chen et al. J Cell Mol Med. 2015 Dec.

Abstract

Recent studies have indicated that the inflammasome plays a critical role in the pathogenesis of vascular diseases. However, the pathological relevance of this inflammasome activation, particularly in vascular cells, remains largely unknown. Here, we investigated the role of endothelial (Nucleotide-binding Oligomerization Domain) NOD-like receptor family pyrin domain containing three (Nlrp3) inflammasomes in modulating inter-endothelial junction proteins, which are associated with endothelial barrier dysfunction, an early onset of obesity-associated endothelial injury. Our findings demonstrate that the activation of Nlrp3 inflammasome by visfatin markedly decreased the expression of inter-endothelial junction proteins including tight junction proteins ZO-1, ZO-2 and occludin, and adherens junction protein VE-cadherin in cultured mouse vascular endothelial (VE) cell monolayers. Such visfatin-induced down-regulation of junction proteins in endothelial cells was attributed to high mobility group box protein 1 (HMGB1) release derived from endothelial inflammasome-dependent caspase-1 activity. Similarly, in the coronary arteries of wild-type mice, high-fat diet (HFD) treatment caused a down-regulation of inter-endothelial junction proteins ZO-1, ZO-2, occludin and VE-cadherin, which was accompanied with enhanced inflammasome activation and HMGB1 expression in the endothelium as well as transmigration of CD43(+) T cells into the coronary arterial wall. In contrast, all these HFD-induced alterations in coronary arteries were prevented in mice with Nlrp3 gene deletion. Taken together, these data strongly suggest that the activation of endothelial Nlrp3 inflammasomes as a result of the increased actions of injurious adipokines such as visfatin produces HMGB1, which act in paracrine or autocrine fashion to disrupt inter-endothelial junctions and increase paracellular permeability of the endothelium contributing to the early onset of endothelial injury during metabolic disorders such as obesity or high-fat/cholesterol diet.

Keywords: Nlrp3 inflammasome; endothelium; obesity; tight junction proteins; visfatin.

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Figures

Figure 1

Effects of Nlrp3 gene silencing on visfatin‐induced disruption of junction proteins in mouse vascular endothelial cells (

MVEC

s).

MVEC

s were transfected scramble (Scr) or Nlrp3 sh

RNA

(Nlrp3sh) plasmids by Nucleofection and then stimulated with or without visfatin (Visf: 4 μg/ml) for 24 hrs. (A) Immunofluorescence stainings were performed with Alexa555‐conjugated antibodies against

‐1,

‐2, occludin or

‐Cadherin (

‐Cad) for determination of the expression of these junction proteins. Representative images show the cell membrane of fluorescence of

‐1,

‐2, occludin or

‐Cadherin (red) are representative of at least three independent experiments. (B–F) Representative Western blot gel document and summarized data showing the protein expression of

‐1,

‐2, occludin,

‐Cadherin and β‐actin expression in the microsomes of

MVEC

s (n = 3–4). *P < 0.05 versus Scr; # P < 0.05 versus Scr+Visf.

Figure 2

(A) Western blot documents and summarized data showing the effect of control or visfatin (0 or 4 μg/ml for 24 hrs) on the expression of high mobility group box protein 1 (

HMGB

or β‐actin in either cell culture medium (Medium) or homogenized cytoplasm (Homo) of mouse vascular endothelial cells (

MVEC

s; n = 6). *P < 0.05 versus control. (B)

MVEC

s transfected with scramble sh

RNA

(Scr) or Nlrp3 sh

RNA

(Nlrp3sh) plasmids and then stimulated with or without visfatin (Visf: 4 μg/ml) for 24 hrs. Summarized data show the concentration of

HMGB

1in culture medium of

MVEC

s as analysed by

ELISA

(n = 6). *P < 0.05 versus Scr; # P < 0.05 versus Scr+Visf. (C) Cells were incubated with fresh culture medium before addition of recombinant

HMGB

1 (15 μg/ml) for 24 hrs. Representative Western blot gel document and summarized data show the effect of recombinant

HMGB

1 (n = 3) on protein expression of

‐1,

‐2, occludin,

‐Cadherin and β‐actin in the microsomes of

MVEC

s. *P < 0.05 versus control.

Figure 3

Inhibition of high mobility group box protein 1 (

HMGB

or caspase‐1 activity prevents visfatin‐induced disruption of junction proteins in mouse vascular endothelial cells (

MVEC

s).

MVEC

s were stimulated with or without visfatin (Visf, 4 μg/ml) for 24 hrs in the presence of

PBS

(Vehl: vehicle),

HMGB

1 inhibitor glycyrrhizin (

GLY

, 130 μmol/l) or caspase‐1 inhibitor Z‐

WEHD

‐fluoromethyl ketone (

FMK

) (

WEHD

, 0.2 μg/ml). (A) Immunofluorescence stainings were performed with Alexa555‐conjugated antibodies against

‐1,

‐2, occludin or

‐Cadherin (

‐Cad) for determination of the expression of these junction proteins. Representative images show the cell membrane of fluorescence of

‐1,

‐2, occludin or

‐Cadherin (red) are representative of at least three independent experiments. (B–F) Representative Western blot gel document and summarized data showing the protein expression of

‐1,

‐2, occludin,

‐Cadherin and β‐actin expression in the microsomes of

MVEC

s (n = 4–5). *P < 0.05 versus Vehl Ctrl; # P < 0.05 versus Visf alone.

Figure 4

High mobility group box protein 1 (

HMGB

1)‐induced endothelial junction disruption effect is mediated by the

RAGE

. Mouse vascular endothelial cells (

MVEC

s) were transfected with scramble (Scr),

RAGE

RNA

(si

RAGE

) or

TLR

4 si

RNA

(si

TLR

for 4 hrs and then stimulated with or without Visfatin (4 μg/ml, 24 hrs). (A‐E) Representative Western blot gel document and summarized data showing the protein expression of

‐1,

‐2, occludin,

‐Cadherin and β‐actin expression in the microsomes of

MVEC

s (n = 4–5). *P < 0.05 versus Scr Ctrl; # P < 0.05 versus Scr+Visf.

Figure 5

Nlrp3 gene deletion inhibits high‐fat diet (

HFD

)‐induced disassembly of tight junction and adherens junction proteins in mouse coronary arterial endothelium. Wild‐type (Nlrp3 +/+) or Nlrp3 knockout (Nlrp3 −/−) Mice were fed with either a normal diet (

), or a high‐fat diet (

HFD

) for 6 weeks. (A) Frozen sections of mouse hearts were stained with Alexa555‐conjugated antibodies against

‐1,

‐2, Occludin, or

‐Cadherin; scale bar = 50 μm. (B) The summarized data show the fluorescence intensity of the endothelial layer (n = 4–6). *P < 0.05 versus

on Nlrp3 +/+; # P < 0.05 versus Nlrp3 +/+ with

HFD

Figure 6

Nlrp3 deficiency gene deletion inhibits high‐fat diet (

HFD

)‐induced inflammasome activation in mouse coronary arterial endothelium. Wild‐type (Nlrp3+/+) or Nlrp3 knockout (Nlrp3−/−) Mice were fed with normal diet (

) and high‐fat diet (

HFD

) for 6 weeks. Frozen sections of mouse hearts were stained with

FLICA

, a green fluorescent probe specific for active caspase‐1, and Alexa555‐conjugated antibodies against an endothelium marker

vWF

in coronary arteries. (A) The merged images displayed yellow dots or patches indicating the colocalization of

FLICA

(green) with

vWF

(red). Enlarged images of area of interest (

AOI

) in merged images are shown; scale bar = 50 μm. (B) The summarized data show the colocalization coefficient of

FLICA

with

vWF

(n = 5–7). *P < 0.05 versus Nlrp3 +/+ with

; # P < 0.05 versus Nlrp3 +/+ with HFD.

Figure 7

Nlrp3 deficiency inhibits high‐fat diet (

HFD

)‐induced increases of high mobility group box protein 1 (

HMGB

release in coronary arterial endothelium. Wild‐type (Nlrp3+/+) or Nlrp3 knockout (Nlrp3−/−) Mice were fed with normal diet (

) and high‐fat diet (

HFD

) for 6 weeks. Frozen sections of mouse hearts were used for confocal immunofluorescent analysis. (A) Representative confocal fluorescence images of

HMGB

1 with endothelium marker

vWF

in coronary arteries of mice. Enlarged images of area of interest (

AOI

) in merged images are shown; scale bar = 50 μm. (B) The summarized data show the colocalization coefficient (

PCC

) of

HMGB

1 with

vWF

(n = 4–8). *P < 0.05 versus Nlrp3 +/+ with

; # P < 0.05 versus Nlrp3 +/+ with HFD.

Figure 8

Nlrp3 deficiency blocks high‐fat diet (

HFD

)‐induced T cell adhesion and infiltration in coronary arterial wall. Wild‐type (Nlrp3 +/+) or Nlrp3 knockout (Nlrp3 −/−) Mice were fed with either a normal diet (

), or a high‐fat diet (

HFD

) for 6 weeks. (A) Frozen sections of mouse hearts were stained with T cell marker (

43). The fluorescence images displayed red dots indicating the

43‐positive cells infiltrated into the arterial wall. (B) Summarized data show the fluorescence intensity of the arterial wall area (n = 4). *P < 0.05 versus Nlrp3 +/+ with

; # P < 0.05 versus Nlrp3 +/+ with

HFD

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Instigation of endothelial Nlrp3 inflammasome by adipokine visfatin promotes inter-endothelial junction disruption: role of HMGB1 - PubMed (original) (raw)