Trimethylamine-N-Oxide Induces Vascular Inflammation by Activating the NLRP3 Inflammasome Through the SIRT3-SOD2-mtROS Signaling Pathway

Abstract

Background: Trimethylamine-N-oxide (TMAO) has recently been identified as a novel and independent risk factor for promoting atherosclerosis through inducing vascular inflammation. However, the exact mechanism is currently unclear. Studies have established a central role of nucleotide-binding oligomerization domain-like receptor family pyrin domain-containing 3 (NLRP3) inflammasome in the pathogenesis of vascular inflammation. Here, we examined the potential role of the NLRP3 inflammasome in TMAO-induced vascular inflammation in vitro and in vivo and the underlying mechanisms.

Methods and results: Experiments using liquid chromatography-tandem mass spectrometry, Western blot, and fluorescent probes showed that TMAO-induced inflammation in human umbilical vein endothelial cells (HUVECs) and aortas from ApoE^-/- mice. Moreover, TMAO promoted NLRP3 and activated caspase-1 p20 expression and caspase-1 activity in vitro and in vivo. Notably, a caspase-1 inhibitor (YVAD), an NLRP3 inhibitor (MCC950), as well as NLRP3 short interfering RNA attenuated TMAO-induced activation of the NLRP3 inflammasome, subsequently leading to suppression of inflammation in HUVECs. TMAO additionally stimulated reactive oxygen species (ROS) generation, in particular, mitochondrial ROS, while inhibiting manganese superoxide dismutase 2 (SOD2) activation and sirtuin 3 (SIRT3) expression in HUVECs and aortas from ApoE^-/- mice. TMAO-induced endothelial NLRP3 inflammasome activation was ameliorated by the mitochondrial ROS scavenger Mito-TEMPO, or SIRT3 overexpression in HUVECs. Conversely, TMAO failed to further inhibit SOD2 and activate the NLRP3 inflammasome or induce inflammation in SIRT3 short interfering RNA-treated HUVECs and aortas from SIRT3^-/- mice.

Conclusions: TMAO promoted vascular inflammation by activating the NLRP3 inflammasome, and the NLRP3 inflammasome activation in part was mediated through inhibition of the SIRT3-SOD2-mitochondrial ROS signaling pathway.

Keywords: NOD‐like receptor family pyrin domain containing 3 inflammasome; atherosclerosis; sirtuin 3; trimethylamine‐N‐oxide; vascular inflammation.

Figure 1

Trimethylamine‐N‐oxide (TMAO)‐induced inflammation in endothelial cells. A, Human umbilical vein endothelial cells (HUVECs) were incubated with different concentrations of TMAO (50, 100, 200, 300, 400, 600, 800, 1000, and 2000 μmol/L) for 24 hours. Thereafter, cell viability was determined. B, Cells were treated with TMAO (600 μmol/L) for different time‐periods (4, 8, 12, 24, 36, 48, and 72 hours), and cell viability was detected. C, Cells were treated as described in A, and the expression of IL‐1β, ICAM‐1, and MMP‐9 was detected via Western blot. D, Bar charts show the quantification of the indicated proteins. E, Cells were treated as described in B, and the expression of IL‐1β, ICAM‐1, and MMP‐9 was analyzed via Western blot. F, Bar graphs show the quantification of the indicated proteins. Values are presented as means±SE (n=3); ^a P<0.05, ^b P<0.01 vs the vehicle‐treated control group; AU indicates arbitrary units; ICAM, intercellular adhesion molecule; IL, interleukin; MMP, matrix metallopeptidase.

Figure 2

Trimethylamine‐N‐oxide (TMAO)‐induced inflammation via nucleotide‐binding oligomerization domain–like receptor family pyrin domain–containing 3 (NLRP3) inflammasome activation in endothelial cells. A, Cells were treated with TMAO at a series of concentrations (150, 300, 600, and 900 μmol/L) for 24 hours, and the expression of NLRP3 and caspase‐1 p20 was detected via Western blot. B, Bar charts showing quantification of endogenous NLRP3 and caspase‐1 p20. C, Cells were incubated with 600 μmol/L TMAO for different time intervals (4, 8, 12, and 24 hours), and the expression of NLRP3 and caspase‐1 p20 was detected via Western blot. D, Bar charts showing quantification of endogenous NLRP3 and caspase‐1 p20. Cells were treated as described for A and B. Thereafter, caspase‐1 activity was measured using caspase‐1 activity kits (E and F). G, Cells were pretreated with YVAD (10 μmol/L) or MCC950 (10 μmol/L) for 2 hours, and then exposed to TMAO (600 μmol/L) for a further 24 hours. Expression of IL‐1β, ICAM‐1, MMP‐9, and caspase‐1 p20 was detected via Western blot. H, Bar charts showing quantification of the indicated proteins. I, Human umbilical vein endothelial cells (HUVECs) were transfected with NLRP3 siRNA as described in Materials and Methods. After 24 hours, cells were incubated with TMAO (600 μmol/L) for 24 hours, and the expression of IL‐1β, ICAM‐1, MMP‐9 and Casp1 p20 was detected via Western blot. J, Bar charts showing quantification of indicated proteins. Values are expressed as means±SE (n=3). ^a P<0.05, ^b P<0.01 vs vehicle‐treated control group; ^c P<0.01 vs TMAO‐treated group; AU indicates arbitrary units; ICAM, intercellular adhesion molecule; IL, interleukin; MMP, matrix metallopeptidase.

Figure 3

Mitochondrial reactive oxygen species (mtROS) played a key role in trimethylamine‐N‐oxide (TMAO)‐induced activation of the nucleotide‐binding oligomerization domain–like receptor family pyrin domain–containing 3 (NLRP3) inflammasome in endothelial cells. Cells were treated with TMAO at a series of concentrations (150, 300, 600, and 900 μmol/L) for 24 hours or 600 μmol/L TMAO for the indicated time intervals (4, 8, 12, and 24 hours). A and B, Total ROS levels were detected via DCFH‐DA. C and D, mtROS levels were detected with MitoSOX ^™ Red. Human umbilical vein endothelial cells (HUVECs) were pretreated with TEMPO (50 μmol/L) for 2 hours followed by the addition of TMAO (600 μmol/L) for a further 24 hours. E, mtROS and (F) expression of the indicated protein were detected. Values are expressed as means±SE (n=3). ^a P<0.05, ^b P<0.01 vs the vehicle‐treated control group; ^c P<0.01 vs TMAO‐treated group; AU, arbitrary units.

Figure 4

Trimethylamine‐N‐oxide (TMAO) exposure decreased superoxide dismutase (SOD)2 activity and sirtuin‐3 (SIRT3) expression. Cells were treated with TMAO at a series of concentrations (150, 300, 600, and 900 μmol/L) for 24 hours or incubated with 600 μmol/L TMAO at indicated time intervals (4, 8, 12, and 24 hours). A and B, SOD2 enzymatic activity was assayed using SOD1 and SOD2 Assay Kits with WST‐8 following the manufacturer's instructions. C and E, Western blot analysis of Ac‐SOD2 and SOD2 expression. D and F, The bar graph shows quantification of endogenous Ac‐SOD2 and SOD2. G and I, SIRT3 expression was detected via Western blot. H and J, The bar graph shows quantification of endogenous SIRT3. Values are expressed as means±SE (n=3); ^a P<0.05, ^b P<0.01 vs the vehicle‐treated control group; AU, arbitrary units.

Figure 5

Trimethylamine‐N‐oxide (TMAO) induced mitochondrial reactive oxygen species (mtROS) accumulation via the sirtuin‐3–superoxide dismutase‐2 (SIRT3‐SOD2) pathway in endothelial cells. Human umbilical vein endothelial cells (HUVECs) were transfected with SIRT3 siRNA or a plasmid overexpressing SIRT3 as described in Materials and Methods. After 24 hours, cells were incubated with 600 μmol/L TMAO for 24 hours. A, Assay of SOD2 enzymatic activity using SOD1 and SOD2 Assay Kits with WST‐8 following the manufacturer's instructions. B, Detection of mtROS levels with MitoSOX ^™ Red. C, Detection of total ROS levels using DCFH‐DA. D, Measurement of caspase‐1 activity with a caspase‐1 activity assay kit. E, Western blot analysis of caspase‐1 p20, nucleotide‐binding oligomerization domain–like receptor family pyrin domain–containing 3 (NLRP3), Ac‐SOD2, SOD2, and SIRT3 contents. F, Bar graphs showing quantification of the indicated proteins. G, Western blot analysis of IL‐1β, ICAM‐1, and MMP‐9 expression. H, Bar graphs showing quantification of the indicated proteins. Values are expressed as means±SE (n=3); ^b P<0.01 vs the vehicle‐treated control group; ^c P<0.01 vs TMAO‐treated group; AU, arbitrary units; ICAM, intercellular adhesion molecule; IL, interleukin; MMP, matrix metallopeptidase.

Figure 6

Trimethylamine‐N‐oxide (TMAO) induced vascular inflammation via nucleotide‐binding oligomerization domain–like receptor family pyrin domain–containing 3 (NLRP3) inflammasome activation in vivo. Eight‐week‐old female ApoE^−/− mice (n=10 per group) were fed chow diet or chow diet combined with choline (1%) for 4 months. Mice were killed, and their blood and aorta samples were collected immediately, snap‐frozen in liquid nitrogen, and stored at −80°C until required. A, Ultrasound B‐mode images of aortic sinus and quantification. Arrows indicate the regions of interest. B, Oil‐red O staining of whole aortas, including aortic arch and thoracic and abdominal regions, and their quantitation. C, Oil‐red O–stained aortic root (counterstained with hematoxylin) and quantification. D, Western blot analysis of caspase‐1 p20, NLRP3, IL‐1β, ICAM‐1, and MMP‐9 contents in aortas. E, Bar graphs showing quantification of the indicated proteins. F, Caspase‐1 activity in aortas. G, Measurement of plasma TMAO levels using liquid chromatography–tandem mass spectrometry. Values are expressed as means±SE (n=10). *P<0.01 vs vehicle‐treated control group; AU, arbitrary units; ICAM, intercellular adhesion molecule; IL, interleukin; MMP, matrix metallopeptidase.

Figure 7

Trimethylamine‐N‐oxide (TMAO) induced vascular nucleotide‐binding oligomerization domain–like receptor family pyrin domain–containing 3 (NLRP3) inflammasome activation in a sirtuin‐3 (SIRT3)‐dependent manner in vivo. Eight‐week‐old female ApoE^−/− mice were treated with or without 1% choline for 4 months. Mice were killed, and their aorta samples were collected immediately, snap‐frozen in liquid nitrogen, and stored at −80°C until required. A, Western blot detection of Ac‐SOD2, SOD2, and SIRT3 expression in aortas. B, Bar graphs showing quantification of the indicated proteins. Eight‐week‐old female wild type (WT) and SIRT3^−/− mice were fed with or without 1% choline for 4 months. Mice were killed, and their aorta samples were collected immediately, snap‐frozen in liquid nitrogen, and stored at −80°C until required. C, Western blot analysis of caspase‐1 p20, NLRP3, Ac‐SOD2, SOD2, and SIRT3 contents in aortas. D, Bar graphs showing quantification of the indicated proteins. E, SOD2 enzymatic activity in aortas was assayed using a SOD1 and SOD2 Assay Kit with WST‐8 following the manufacturer's instructions. F, Measurement of caspase‐1 activity in aortas. G, Western blot analysis of IL‐1β, ICAM‐1, MMP‐9 and SIRT3 expression. H, Bar graphs showing quantification of the indicated proteins. Values are expressed as means±SE (n=10); *P<0.01 vs the vehicle‐treated control group; AU, arbitrary units; ICAM, intercellular adhesion molecule; IL, interleukin; MMP, matrix metallopeptidase; SOD, superoxide dismutase.

Figure 8

Trimethylamine‐N‐oxide (TMAO) mediated vascular inflammation by activating the nucleotide‐binding oligomerization domain–like receptor family pyrin domain–containing 3 (NLRP3) inflammasome via the sirtuin‐3 (SIRT3)–supeoxide dismutase (SOD)–mitochondrial reactive oxygen species (mtROS) signaling pathway.