Trymethylamine-N-oxide, a gut-derived metabolite, induces myofibroblastic activation of valvular interstitial cells through endoplasmic reticulum stress

Figures

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  Figure 1.. TMAO triggers qVIC activation. (A) Schematic illustration of the experimental design. Primary VICs were isolated from porcine aortic valves and cultured to generate quiescent VICs (qVICs). qVICs were treated with TMAO for 3 days. (B-C) Quantification of myofibroblastic gene expression levels for (B) ACTA2 and (C) SM22 via qRT-PCR. (D) Representative images of immunocytochemistry staining for αSMA (red). Cell nuclei are stained in blue. (E) Quantification of αSMA staining intensity in (D). (F) Representative images of immunocytochemistry staining for transgelin (green). Cell nuclei are stained in blue. (G) Quantification of transgelin staining intensity in (E). (H) EdU staining to identify proliferating cells after 8 hours of incubation with EdU. (I) Quantification of the percentage of EdU-positive proliferating cells in (H). Scale bars represent 200 μm. n = 3-5 replicates per condition. One-way ANOVA followed by Tukey’s multiple comparisons test. **p<0.005, ***p<0.001 compared with the control. #p<0.01 compared with aVICs.

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  Figure 2.. TMAO increases extracellular matrix production by qVICs. (A-B) Gene expression analysis of (A) COL1A1 and (B) FN after 3 days of TMAO treatment via qRT-PCR. (C-D) Quantification of (C) collagen and (D) fibronectin secretion via sandwich ELISAs. n = 3-5 replicates per condition. **p<0.005, ***p<0.001 compared with the control, #p<0.05 compared to aVICs.

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  Figure 3.. Effects of TMAO on conventionally cultured aVICs. (A) Schematic illustration describing the generation of activated VICs (aVICs) from primary porcine cells through conventional culture. aVIC were treated with TMAO for 3 days. (B-C) Gene expression of (B) ACT2 (C) SM22 via qRT-PCR. (D) Immunocytochemistry staining for αSMA. The scale bar represents 200 μm. Cell nuclei are stained blue. (E) Quantification of αSMA staining intensity in (D). (F-G) Analysis of (F) COL1A1 and (G) FN gene expression via qRT-PCR. n = 3-5 replicates per condition. One-way ANOVA followed by Tukey’s multiple comparisons test. **p<0.005, ***p<0.001 compared with the control. #p<0.01 compared to treatment with TGF-ß1.

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  Figure 4.. The activation of qVICs by TMAO does not depend on sex. Female and male qVICs were generated and treated with TMAO (25-150μM) for 3 days. (A) Representative images of immunocytochemistry staining for αSMA (red). Cell nuclei are stained blue. Scale bars represent 200 μm. (B) Quantification of αSMA staining intensity in (A). (C) Analysis of fibronectin deposition via in situ ELISA. n = 3-5 replicates per condition. Two-way ANOVA was performed, followed by Tukey’s multiple comparisons test. **p < 0.005, *p < 0.001 compared to the untreated control for the corresponding sex. n.s. denotes no statistically significant difference between male and female VICs for that condition. #p<0.01 for comparison shown.

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  Figure 5.. TMAO induces ROS production and ER stress. Female qVICs were treated with TMAO (25-150 μM) for 3 days. (A) Representative fluorescent images of intracellular 2’,7’-dichlorofluorescin diacetate (green), indicative of ROS. Scale bars represent 200 μm. (B) Quantification of the ROS-Glo™ luminescent substrate in the culture media normalized to cell number. (C) Representative fluorescence images of the ER-ID Red dye, indicative of ER stress. Cell nuclei are stained blue. Scale bars represent 100 μm. (D) Quantification of fluorescence intensity in (C). n = 3-5 replicates per condition. One-way ANOVA followed by Tukey’s multiple comparisons test. **p<0.005, ***p<0.001 compared with the control. #p<0.05 compared to treatment with TGF-ß1.

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  Figure 6.. PERK inhibition mitigates the effects of TMAO. Female qVICs were pre-treated with GSK2656157 a PERK inhibitor, for 1 hour prior to treatment with TMAO. (A) Representative fluorescence images of the ER-ID Red dye, indicative of ER stress, at day 3. Cell nuclei are stained blue. Scale bars represent 100 μm. (B) Quantification of fluorescence intensity in (A). (C) Representative images of immunocytochemistry staining for αSMA (red) after 3 days of TMAO treatment. Cell nuclei are stained blue. Scale bars represent 200 μm. (D) Quantification of αSMA staining intensity in (B). (E) Quantification of the percentage of EdU-positive proliferating cells at day 3. (D) Quantification of ROS in the culture media through the ROS-Glo™ luminescent substrate normalized to cell number. (G-H) Gene expression analysis of (G) COL1A1 and (H) FN. n = 3-5 replicates per condition. Two-way ANOVA, followed by Tukey’s multiple comparisons test. **p<0.005, ***p<0.001 compared with the control. n.s. denotes no statistically significant differences found between samples treated with and without the PERK inhibitor. #p<0.001 indicates a significant difference between samples treated with the PERK inhibitor and…