Vascular smooth muscle cell-derived hydrogen sulfide promotes atherosclerotic plaque stability via TFEB (transcription factor EB)-mediated autophagy

Abstract

Vascular smooth muscle cells (VSMCs) contribute to plaque stability. VSMCs are also a major source of CTH (cystathionine gamma-lyase)-hydrogen sulfide (H₂S), a protective gasotransmitter in atherosclerosis. However, the role of VSMC endogenous CTH-H₂S in pathogenesis of plaque stability and the mechanism are unknown. In human carotid plaques, CTH expression in ACTA2⁺ cells was dramatically downregulated in lesion areas in comparison to non-lesion areas. Intraplaque CTH expression was positively correlated with collagen content, whereas there was a negative correlation with CD68⁺ and necrotic core area, resulting in a rigorous correlation with vulnerability index (r = -0.9033). Deletion of Cth in VSMCs exacerbated plaque vulnerability, and were associated with VSMC autophagy decline, all of which were rescued by H₂S donor. In ox-LDL treated VSMCs, cth deletion reduced collagen and heightened apoptosis association with autophagy reduction, and vice versa. For the mechanism, CTH-H₂S mediated VSMC autophagosome formation, autolysosome formation and lysosome function, in part by activation of TFEB, a master regulator for autophagy. Interference with TFEB blocked CTH-H₂S effects on VSMCs collagen and apoptosis. Next, we demonstrated that CTH-H₂S sulfhydrated TFEB at Cys212 site, facilitating its nuclear translocation, and then promoting transcription of its target genes such as ATG9A, LAPTM5 or LDLRAP1. Conclusively, CTH-H₂S increases VSMC autophagy by sulfhydration and activation of TFEB, promotes collagen secretion and inhibits apoptosis, thereby attenuating atherogenesis and plaque vulnerability. CTH-H₂S may act as a warning biomarker for vulnerable plaque.Abbreviations ATG9A: autophagy related 9A; CTH: cystathionine gamma-lyase; CQ: chloroquine; HASMCs: human aortic smooth muscle cells; H₂S: hydrogen sulfide; LAMP1: lysosomal associated membrane protein 1; LAPTM5: lysosomal protein transmembrane 5; NaHS: sodium hydrosulfide hydrate; ox-LDL: oxidized-low density lipoprotein; PPG: DL- propagylglycine; TFEB: transcription factor EB; 3-MA: 3-methyladenine; VSMCs: vascular smooth muscle cells.

Keywords: Autophagy; cystathionine gamma lyase; hydrogen sulfide; plaque stability; transcription factor EB; vascular smooth muscle cell.

Figure 1.

Patient’s intraplaque CTH expression negatively correlated with plaque vulnerability. Immunofluorescent staining for CTH (green) and ACTA2 (red) in non-lesion area and lesion area of patient’s carotid plaques (A), IgG as negative control. Scale bar: 50 μm. Immunofluorescent staining of CTH, immunohistochemical staining of CD68, ACTA2 and Masson staining in stable plaque and vulnerable plaque of human (B), the yellow continuous line indicates the area of necrotic core, NC: necrotic core. IgG as negative control. Scale bar from left to right: 50 μm, 50 μm, 50 μm and 500 μm. Pearson correlations between CTH expression and ACTA2 (% area) (C), collagen volume fraction (D), CD68 (% area) (E), necrotic core (% area) (F) and vulnerable index (G). Vulnerable index was counted by (CD68⁺ area + necrotic core area)/(ACTA2⁺ area + collagen volume). N = 18/group.

Figure 2.

VSMC-specific cth deletion exacerbated plaque vulnerability. Using loxp-cre recombinase system, we generated a SMC-specific cth knockout mouse. Atherosclerosis mouse model was generated using PCSK9 overexpression in liver by injection adeno-associated virus rAAV8-D377Y-mPCSK9 then feeding Paigen diet for 16 weeks. While sacrifice, aortic en face Oil-red O staining was performed (A), and plaque area in different location was presented as a percentage of total area. AA: abdominal aorta; TA: thoracic aorta (B). Plaque size in aortic root was also evaluated by Oil-red O staining, Scale bar: 200 μm (C). The yellow line showed the size of necrotic core of aortic root plaque by H&E staining, Scale bar: 200 μm (D). In aortic root plaque, immunohistochemical staining CD68 and ACTA2 (IgG as negative control), Masson staining for collagen fraction. FC: fibrous cap; NC: necrotic core. Scale bar: 50 μm; *P < 0.05; **P < 0.01 (E). The vulnerable index changes in aortic root plaque (F). In this figure, black circle presented Cth^flox/flox group; red circle presented cth^SMC-/- group; blue circle present cth^SMC-/-+NaHS group. Cth^flox/flox group: N = 13; cth^SMC-/- group: N = 14; cth^SMC-/-+NaHS group: N = 13.

Figure 3.

Autophagy changes in intraplaque ACTA2 cells of patients and mouse model. LC3 immunofluorescent staining (green) was used for evaluation autophagy, ACTA2 (red) as reference. Nuclei stained by DAPI. The carotid plaque (lesion) and non-plaque area (non-lesion) of patients, scale bar: 75 μm, N = 7 (A). normal and atherosclerotic plaque of aortic root of mice, scale bar: 25 μm, N = 8 (B). Intraplaque autophagy changes in Cth^flox/flox, cth^SMC-/- and cth^SMC-/-+NaHS groups, scale bar: 25 μm, N = 6 (C). All IgG as negative controls.

Figure 4.

CTH-H₂S-modulated VSMC apoptosis, collagen secretion and phage-like phenotype associated with autophagy. Exposed to ox-LDL (150 µg/ml) for 24 h, the effect of H₂S donor NaHS (100 μM, 2 h) and CTH inhibitor PPG (100 μM, 24 h) on lipid droplet accumulation in HAMSCs (the upper panel) (N = 12); in isolated mouse VSMCs, the effect of cth knockout on lipid droplet accumulation both with and without ox-LDL (150 µg/ml, 24 h) in contrast with WT VSMCs (the lower panel) (N = 24). scale bar: 25 μm (A). ox-LDL induced apoptosis was analyzed by flow cytometry when pharmacological interference CTH-H₂S in HASMCs (N = 6) (B), or cth knockout in mouse primary VSMCs (N = 6) (C). ox-LDL impaired VSMC COL1 expression was measured by Western blot in HASMCs (D) or Cth-deficient VSMCs (E). Autophagy makers-LAMP1, LC3-I:LC3-II protein expression were assayed after NaHS treatment (F), Cth overexpression (G) and Cth knockdown (H) response to ox-LDL. mRFP-GFP-LC3 adenovirus were transfected to monitor autophagy flux in HASMCs while pharmacological interference CTH-H₂S. Yellow puncta presented autophagosomes numbers, red puncta presented autolysosomes formation, scale bar: 25 μm (I). Transmission electron microscope showed the autophagosomes in wild type or cth knockout VSMCs response to ox-LDL (150 µg/ml, 24 h) stimulation, red arrows indicate autophagosome, scale bar: 1 μm (J).

Figure 5.

CTH-H₂S regulated autophagosome, autolysosome formation and lysosome function. 3-methyladenine (3-MA)-a selective autophagosome formation inhibitor, and chloroquine (CQ)-a autophagosome-lysosome fusion inhibitor were used for blocking autophagosome or autolysosome formation. After AdCth transfection for 24 h, HASMCs were pre-treated with 5 mM 3-MA for 1 h or treated with 200 μM CQ for 2 h. 3-MA blocking and CQ heightening the overexpressed CTH-induced LC3 protein accumulation (A) and autophagy flux elevation, scale bar: 25 μm (B). Effect of 3-MA and CQ on cth-deletion induced LC3 protein reduction (C) and autophagy flux blocking (D), scale bar: 10 μm. After ox-LDL (150 µg/ml, 24 h) treatment in primary mouse VSMCs, LAMP1 immunofluorescence staining (E), scale bar: 25 μm; or LysoTracker Red showed the numbers and location of lysosomes in VSMCs, scale bar: 10 μm (F). IgG as negative control. HASMCs (N = 8) (G) or mouse primary VSMCs (N = 8) (H) were labeled with LysoSensor, pH values were calculated by pH standard calibration while pharmacological or genomic modification CTH-H₂S.

Figure 6.

CTH-H₂S regulated VSMC autophagy by activating TFEB. TFEB immunofluorescence staining in intraplaque ACTA2-positive cells of Cth^flox/flox (N = 4), cth^SMC-/- (N = 6) and cth^SMC-/- +NaHS mice (N = 5) (A). IgG as negative control. Exposed to ox-LDL (150 µg/ml) for 24 h, the effect of H₂S donor NaHS (100 μM, 2 h) and CTH inhibitor PPG (100 μM, 24 h) on TFEB expression and nuclear translocation in ox-LDL-stimulated HASMCs (B). IgG as negative control. In HASMCs, Knockdown TFEB by siRNA transfection (100 nM, 24 h) effects on NaHS (100 μM, 2 h) modulating VSMC phage-like phenotype and autophagy flux changes (C), COL1 and autophagy markers expression (D), and VSMC apoptosis (N = 6) (E). On the contrary, overexpressed TFEB by plasmid transfection (2 μg, 24 h) partly rescued PPG-induced (100 μM, 24 h) phage-like phenotype, autophagy flux decrease (F), COL1 and autophagy markers expression decrease (N = 5) (G), and VSMC apoptosis increase (N = 6) (H). *P < 0.05, ** P < 0.01. All scale bar: 25 μm.

Figure 7.

H₂S sulfhydrated TFEB and facilitated TFEB activity. Modified biotin switch assay measured sulfhydrated-TFEB (SHY-TFEB) (N = 3) (A). In HAMSCs, NaHS (100 μM, 2 h) increased but DTT (200 μM, 2 h) removed TFEB sulfhydration on its nuclear translocation. IgG as negative control, N = 7 (B). The effect of Cth overexpression by AdCth transfection (25 MOI, 24 h) (N = 5) or cth knockout (N = 4) on TFEB sulfhydration (C), association with TFEB nuclear translocation (D). N = 7, IgG as negative control. The C212S mutation TFEB plasmid (2 μg, 24 h) transfected into HASMCs, the NaHS-induced SHY-TFEB (N = 5) (E) and its nuclear translocation (N = 7) (F) were measured. IgG as negative control. All scale bar: 25 μm.

Figure 8.

Sulfhydrated TFEB promoted autophagy, lysosome biogenesis and lipid metabolism related gene expression. Heat map of TFEB-occupied genes based on TFEB signal around TFEB peak center, in C212S mutation TFEB (could not be sulfhydrated) cells (A) and sulfhydrated TFEB reduced VSMCs (cth knockout) (B). Venn diagram to show peak numbers in different groups (upper panel), and modification sulfhydrated-TFEB (cth knockout and C121S mutation) changed genes, analysis overlapped genes suggesting conserved sulfhydrated-TFEB occupied genes (lower panel) (C). Gene Ontology (GO) analysis of sulfhydrated-TFEB target genes in C212 mutation cells (D) and cth knockout VSMCs (E). Visualization of ChIP-Seq results for three representative TFEB occupied genes (ATG9A, LAPTM5 and LDLRAP1) by Integrative Genomics Viewer (IGV) in C212 mutation cells (F) and cth knockout VSMCs (G). Knockdown TFEB by siRNA transfection (100 nM, 24 h) effects on NaHS (100 μM, 2 h) modulating mRNA changes of ATG9A, LAPTM5 and LDLRAP1 in HASMCs. N = 6 (H). The effect of transfection WT or C212S mutation TFEB (2 μg, 24 h) on PPG-regulated mRNA levels of ATG9A, LAPTM5 and LDLRAP1 in 293A cells. N = 6 (I).