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. 2013 Aug 6;128(6):632-42.
doi: 10.1161/CIRCULATIONAHA.113.002714. Epub 2013 Jul 9.

Sterol regulatory element binding protein 2 activation of NLRP3 inflammasome in endothelium mediates hemodynamic-induced atherosclerosis susceptibility

Han Xiao  1 , Min Lu, Ting Yang Lin, Zhen Chen, Gang Chen, Wei-Chi Wang, Traci Marin, Tzu-Pin Shentu, Liang Wen, Brendan Gongol, Wei Sun, Xiao Liang, Ju Chen, Hsien-Da Huang, Joao H F Pedra, David A Johnson, John Y J Shyy
Affiliations

Sterol regulatory element binding protein 2 activation of NLRP3 inflammasome in endothelium mediates hemodynamic-induced atherosclerosis susceptibility

Han Xiao et al. Circulation. .

Abstract

Background: The molecular basis for the focal nature of atherosclerotic lesions is poorly understood. Here, we explored whether disturbed flow patterns activate an innate immune response to form the NLRP3 inflammasome scaffold in vascular endothelial cells via sterol regulatory element binding protein 2 (SREBP2).

Methods and results: Oscillatory flow activates SREBP2 and induces NLRP3 inflammasome in endothelial cells. The underlying mechanisms involve SREBP2 transactivating NADPH oxidase 2 and NLRP3. Consistently, SREBP2, NADPH oxidase 2, and NLRP3 levels were elevated in atheroprone areas of mouse aortas, suggesting that the SREBP2-activated NLRP3 inflammasome causes functionally disturbed endothelium with increased inflammation. Mimicking the effect of atheroprone flow, endothelial cell-specific overexpression of the activated form of SREBP2 synergized with hyperlipidemia to increase atherosclerosis in the atheroresistant areas of mouse aortas.

Conclusions: Atheroprone flow induces NLRP3 inflammasome in endothelium through SREBP2 activation. This increased innate immunity in endothelium synergizes with hyperlipidemia to cause topographical distribution of atherosclerotic lesions.

Keywords: NLRP3 protein, human; atherosclerosis; endothelial cell; shear stress; sterol regulatory element binding proteins.

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Conflict of interest statement

Conflict of Interest Disclosures: None.

Figures

Figure 1
Figure 1
OS Induces SREBP2 and miR-33 in HUVECs. HUVECs were exposed to a PS, (12 ± 4 dyn/cm2) or OS (1 ± 4 dyn/cm2) for 14 hr. (A) Representative immunoblot for precursor SREBP2 and mature form of SREBP2. (B) Analysis of levels of SREBP2 mRNA and miR-33 by RT-PCR. (C) The mRNA levels of HMG-CoA synthase, HMG-CoA reductase, squalene synthase, and LDLR. (D) The mRNA levels of ABCA1 and ABCG1. (E) Representative immunoblot for ABCA1 and ABCG1. (F) Total cellular cholesterol level (n = 8). (G) Representative immunoblot for precursor SREBP2 and mature form of SREBP2 in HUVECs treated with MβCD, 25-HC (1 µg/mL), or OS plus 25-HC (1 µg/mL). Data are mean ± SEM from at least 3 independent experiments. Student’s t test or Mann-Whitney U test with exact method was used. ‘*’ indicates p < 0.05.
Figure 2
Figure 2
OS Induces NLRP3 Inflammasomes in ECs via SREBP2. (A) Representative immunoblot for pro-caspase-1, caspase-1 p20, pro-IL-1β, and IL-1β p17 in HUVECs exposed to PS or OS for 14 hr and (B) in HUVECs treated with Adenovirus-null (Ad-null, 5 MOI), Adenovirus-SREBP2(N) tagged with HA [Ad-SREBP2(N), 2.5 MOI or 5 MOI]. (C) Caspase-1 activity of extracts from HUVECs treated with Ad-null, 5 MOI, Ad-SREBP2(N), 2.5 MOI or 5 MOI. Data are mean ± SEM normalized to Ad-null group for each independent experiment. Mann-Whitney U test with exact method was used. *p < 0.05 compared with Ad-null. (D) Representative immunoblot of pro-caspase-1, caspase-1 p20, pro-IL-1β, IL-1β p17, SREBP2 precursor, and mature form of SREBP2 in HUVECs transfected with 20 nM control RNA or SREBP2 siRNA for 48 hr, then exposed to static conditions or OS for 14 hr. (E) Representative immunoblot for pro-caspase-1, caspase-1 p20, pro-IL-1β, IL-1β p17, and NLPR3 in HUVECs transfected with 20 nM control RNA or NLRP3 siRNA for 48 hr, then exposed to static conditions or OS for 14 hr. (F) Representative immunoblot of pro-caspase-1, caspase-1 p20, pro-IL-1β, IL-1β p17, and ASC in HUVECs transfected with 20 nM control RNA or ASC siRNA for 48 hr, then exposed to static conditions or OS for 14 hr.
Figure 3
Figure 3
SREBP2 Up-regulates NOX2 with Attendant Increase in ROS in ECs. (A) ROS production monitored by measuring H2DCFDA fluorescence in HUVECs infected with Ad-null or Ad-SREBP2(N) (5 MOI). (B) Cytometric analysis of HUVECs infected with Ad-null or Ad-SREBP2(N) (5 MOI) for 48 hr or treated with rotenone (40 µM) for 3 hr and then stained with MitoSOX for 30 min. (C) Depiction of the 3 putative SRE binding sites, at −2381/−2367(SRE1), −867/−861(SRE2), and −674/668(SRE3) bp upstream of the transcription initiation site in the human NOX2 promoter. NOX2 mRNA levels (D) and representative immunoblot (and quantification) of NOX2 and the cleaved SREBP2 (E) in HUVECs treated with Ad-null (5 MOI) or Ad-SREBP2(N) (2.5 MOI or 5 MOI). (F) NOX2 mRNA levels in lung ECs from wild-type or EC-SREBP2(N)-Tg mice (n = 8). (G) ChIP analysis with antibodies against SREBP2 or IgG, soluble chromatin (~500 bp in length) from HUVECs infected with Ad-null or Ad-SREBP2(N), and primers targeting the region spanning the 3 SRE binding sites in the NOX2 promoter. (H) Representative immunoblot of NOX2, SREBP2 precursor and mature form of SREBP2 in HUVECs transfected with 20 nM control RNA or SREBP2 siRNA for 48 hr, then exposed to static or OS for 14 hr. (I) Representative immunoblot of caspase-1 p20, IL-1β p17, and NOX2 in HUVECs transfected with 20 nM control RNA or NOX2 siRNA for 48 hr, then exposed to static or OS for 14 hr. At least 3 independent experiments were performed and results are mean ± SEM. Mann-Whitney U test with exact method was used. ‘*’ indicates p < 0.05 vs Ad-null or wild-type. ‘#’ indicates p = 0.05.
Figure 4
Figure 4
SREBP2 Overexpression Up-regulates NLRP3 Inflammasome. (A) Depiction of the putative SRE binding site located at −1379/−1368 bp upstream of the transcription initiation site in the human NLRP3 promoter. The NLRP3 mRNA levels (B) and representative immunoblot (and quantification) of NLRP3 and the mature form of SREBP2 (C) in HUVECs treated with Ad-null (5MOI), Ad-SREBP2(N) (2.5 MOI or 5 MOI). (D) NLRP3 mRNA levels in lung ECs from wild-type or EC-SREBP2(N)-Tg mice (n=8). (E) ChIP analysis with antibodies against SREBP2 or IgG, soluble chromatin from HUVECs infected with Ad-null or Ad-SREBP2(N), and primers targeting the region spanning the SRE binding site in the NLRP3 promoter. (F) Representative immunoblot of NLRP3, precursor SREBP2, and mature form of SREBP2 in HUVECs transfected with 20 nM control RNA or SREBP2 siRNA for 48 hr, then exposed to static conditions or OS for 14 hr. Bar graphs represent mean ± SEM from at least 3 independent experiments. Mann-Whitney U test with exact method was used. ‘*’ indicates p < 0.05 vs Ad-null or wild-type whereas ‘#’ indicates p = 0.05 vs Ad-null.
Figure 5
Figure 5
Increased Level of SREBP2 Causes EC NLRP3 Inflammation. (A) Quantification of mRNA levels of MCP-1, VCAM-1, and E-selectin in HUVECs treated with Ad-null (5 MOI) or Ad-SREBP2(N) (2.5 or 5 MOI) or (B) in lung ECs from wild-type or EC-SREBP2(N)-Tg mice (n = 8). (C,D) HUVECs were infected with Ad-null or Ad-HA-SREBP2(N) for 24 hr, then treated with or without caspase-1 inhibitor Z-YVAD-FMK (2 µM) for 24 hr. In separate experiments, HUVECs were transfected with control RNA, NOX2 siRNA, or NLRP3 siRNA, then infected with Ad-null or Ad-HA-SREBP2(N) for 24 hr. The cells were incubated for 30 min with LeukoTracker™-labeled THP1 cells. For quantification, parallel batches of treated cells were lysed and the fluorescence measured. Data are mean ± SEM from 3 independent experiments performed in triplicate. Mann-Whitney U test with exact method was used and ‘*’ indicates p < 0.05 for comparisons with Ad-null or wild- type ECs.
Figure 6
Figure 6
SREBP2-NOX2-inflammasome Activation in Atheroprone Regions of Mouse Aortas. (A) Quantification of mRNA levels of SREBP2, NOX2, NLRP3, MCP-1, VCAM-1, ICAM-1, and E-selectin in the thoracic aorta (TA) and aortic arch (AA) of C57BL/6 mice (n = 7). (B) Representative immunoblot of pro-caspase-1, caspase-1 p20, pro-IL-1β, and IL-1β p17 in TA and AA from C57BL/6 mice. (C) The level of SREBP2, NOX2 and NLRP3 mRNA in aortic intima or media and adventitia. Data are mean ± SEM of the relative mRNA normalized to that of β-actin (n = 18). (D) Quantification of mRNA levels of SREBP2, NOX2, NLRP3, MCP-1, VCAM-1, ICAM-1, and E-selectin in TA from wild-type (n = 7) and EC-SREBP2(N)-Tg mice (n = 7). Mann-Whitney U test with exact method was used. ‘*’ indicates p < 0.05 for comparisons with TA or WT.
Figure 7
Figure 7
Overexpression of SREBP2 in ECs Enhances Atherosclerosis. (A) Representative Oil Red O staining of aortas from (A) Eight-week-old ApoE−/− (n = 17) and ApoE−/−/SREBP2(N) mice (n = 18) fed a high-fat diet for 8 weeks and (B) 24-week-old ApoE−/− (n = 7) and ApoE−/−/SREBP2(N) mice (n = 6) fed a normal chow. (C,D) Quantification of percentage lesion areas in the TA, AA, and whole aorta from groups in (A) and (B). Student’s t test or Mann-Whitney U test with exact method was used. ‘*’ indicates p < 0.05.
Figure 8
Figure 8
Graphic Summary for the Mechanism that SREBP2 Activation of NLRP3 Inflammasome in Endothelium Mediates Atheroprone flow-Induced Atherosclerosis.
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