Hydrogen sulfide as an anti-calcification stratagem in human aortic valve: Altered biogenesis and mitochondrial metabolism of H2S lead to H2S deficiency in calcific aortic valve disease

Abstract

Hydrogen sulfide (H₂S) was previously revealed to inhibit osteoblastic differentiation of valvular interstitial cells (VICs), a pathological feature in calcific aortic valve disease (CAVD). This study aimed to explore the metabolic control of H₂S levels in human aortic valves. Lower levels of bioavailable H₂S and higher levels of interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) were detected in aortic valves of CAVD patients compared to healthy individuals, accompanied by higher expression of cystathionine γ-lyase (CSE) and same expression of cystathionine β-synthase (CBS). Increased biogenesis of H₂S by CSE was found in the aortic valves of CAVD patients which is supported by increased production of lanthionine. In accordance, healthy human aortic VICs mimic human pathology under calcifying conditions, as elevated CSE expression is associated with low levels of H₂S. The expression of mitochondrial enzymes involved in H₂S catabolism including sulfide quinone oxidoreductase (SQR), the key enzyme in mitochondrial H₂S oxidation, persulfide dioxygenase (ETHE1), sulfite oxidase (SO) and thiosulfate sulfurtransferase (TST) were up-regulated in calcific aortic valve tissues, and a similar expression pattern was observed in response to high phosphate levels in VICs. AP39, a mitochondria-targeting H₂S donor, rescued VICs from an osteoblastic phenotype switch and reduced the expression of IL-1β and TNF-α in VICs. Both pro-inflammatory cytokines aggravated calcification and osteoblastic differentiation of VICs derived from the calcific aortic valves. In contrast, IL-1β and TNF-α provided an early and transient inhibition of VICs calcification and osteoblastic differentiation in healthy cells and that effect was lost as H₂S levels decreased. The benefit was mediated via CSE induction and H₂S generation. We conclude that decreased levels of bioavailable H₂S in human calcific aortic valves result from an increased H₂S metabolism that facilitates the development of CAVD. CSE/H₂S represent a pathway that reverses the action of calcifying stimuli.

Keywords: Arteriosclerosis; Chronic kidney disease; Hydrogen sulfide; Mitochondrial H(2)S catabolism; Phosphate; Valvular inflammation; Vascular calcification.

Graphical abstract

Fig. 1

Decreased levels of bioavailable hydrogen sulfide and high expression of inflammatory cytokines are characteristics of human calcific aortic valves (A) Hematoxylin and eosin, TNF-α, IL-1β, and CSE immunohistochemical (IHC) stainings were performed on healthy aortic valves (HAVs) derived from the Department of Forensic Medicine, University of Debrecen (N = 5) and on calcific aortic valves (CAVs) of patients diagnosed with CAVD undergoing total aortic valve replacement (N = 5). Scale bars shown in the images represent 200 μm or 50 μm. (B) Quantitative analysis of TNF-α, and IL-1β IHC staining of tissue sections were calculated using ImageJ software (N = 5) (C, left panel). Quantitative analysis of CSE IHC staining of tissue sections was calculated using ImageJ software (N = 5) (C, right panel). Levels of bioavailable H₂S of healthy aortic valves and calcific aortic valves measured with LC-MS/MS are shown (N = 5). Results were analyzed by unpaired t-test and are shown as mean values ± SEM of five independent experiments. **p < 0.01; ***p < 0.001.

Fig. 2

Expression of cystathionine beta-synthase doesn’t change in cavd (A) Hematoxylin and eosin and CBS immunohistochemical (IHC) stainings were performed on healthy aortic valves (HAVs) derived from the Department of Forensic Medicine, University of Debrecen (N = 5) and on calcific aortic valves (CAVs) of patients diagnosed with CAVD undergoing total aortic valve replacement (N = 5). Scale bars shown in the images represent 100 μm or 50 μm. (B) HAVs (N = 4) and CAVs (N = 5) were cryogenically pulverized and taken up with cell lysis buffer followed by 5-s sonication on ice three times. CBS protein expression normalized to GAPDH of healthy human aortic valves (N = 4) and calcified human aortic valves (N = 5) were measured with western blot. Quantitative analysis of the CBS western blot was calculated using ImageJ software. Results were analyzed by unpaired t-test and were shown as mean values ± SEM. Ns: not significant; *p < 0.05; ***p < 0.001.

Fig. 3

Mineralization potential and inflammatory response of valvular interstitial cells to high levels of phosphate Valvular interstitial cells (VICs) isolated from calcific aortic valves (CAV-VICs) or healthy aortic valves (HAV-VICs) were exposed to calcification medium supplemented with 2.5 mmol/L inorganic phosphate and 1.8 mmol/L calcium chloride every other day for 5 days. (A) Calcium contents of extracellular matrix normalized to a protein of VICs are shown (n = 3). (B) TNF-α levels normalized to GAPDH of VICs cultured in calcification medium or culture medium are shown (n = 3). (C) IL-1β levels normalized to GAPDH of VICs cultured in calcification medium or culture medium are shown (n = 3). Red lines indicate CAV-VICs cultured in calcification medium. Black lines indicate HAV-VICs cultured in calcification medium. Blue lines show the HAV-VICs maintained in culture medium. Results were analyzed by unpaired t-test and are shown as mean values ± SEM of three independent experiments. *p < 0.05; **p < 0.01. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 4

Distinct calcification response of valvular interstitial cells to pro-inflammatory cytokines depending upon disease state Valvular interstitial cells (VICs) isolated from calcific aortic valves (CAV-VICs) or healthy aortic valves (HAV-VICs) were cultured in growth medium or calcification medium, containing 2.5 mmol/L inorganic phosphate and 1.8 mmol/L calcium chloride every other day, for 5 days. VICs maintained in calcification media were exposed to TNF-α (10 nmol/L) or IL-1β (10 nmol/L) for 5 days. (A) Alizarin Red S staining of CAV-VICs (upper panels) and HAV-VICs (lower panels) are shown. Quantitative analysis of Alizarin Red S staining of CAV-VICs and HAV-VICs was calculated using ImageJ software (n = 5). (B) Extracellular calcium contents normalized to protein of CAV-VICs and HAV-VICs are shown (n = 5). (C) ALP protein expressions normalized to GAPDH of CAV-VICs (left panels) and HAV-VICs (right panels) were measured with western blot (n = 3). Quantitative analysis of the ALP western blots were calculated using ImageJ software. (D) ALP staining of CAV-VICs (upper panels) and HAV-VICs (lower panels) are shown. Quantitative analysis of ALP staining of CAV-VICs and HAV-VICs was calculated using ImageJ software (n = 5). Results were analyzed by one-way ANOVA, Bonferroni's Multiple Comparison Test, and are shown as mean values ± SEM of three (D and E) or five (A–C) independent experiments. IL-1β and TNF-α treatment were compared to the minus condition (calcification medium). Ns: not significant; *p < 0.05; **p < 0.01; ***p < 0.001. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 5

Transient inhibition of valvular interstitial cell calcification provoked by pro-inflammatory cytokines is associated with elevated cystathionine-γ-lyase expression and H₂S generation. Valvular interstitial cells isolated from healthy aortic valves (HAV-VICs) were cultured in calcification medium, containing 2.5 mmol/L inorganic phosphate and 1.8 mmol/L calcium chloride every other day, for 5 and 14 days. VICs maintained in calcification media were exposed to TNF-α (10 nmol/L) or IL-1β (10 nmol/L) for 5 and 14 days. (A) Calcium contents of extracellular matrix normalized to protein of HAV-VICs are shown (n = 3). (B) CSE protein expression normalized to GAPDH in HAV-VICs maintained in calcification media with or without TNF-α (10 nmol/L) and IL-1β (10 nmol/L) for 5 and 14 days are shown (n = 3). (C) H₂S generation measured with modified methylene blue method and normalized to protein of HAV-VICs are shown (n = 3). Results were analyzed by one-way ANOVA, Bonferroni's Multiple Comparison Test, and are shown as mean values ± SEM of three independent experiments. Ns: not significant; *p < 0.05; **p < 0.01; ***p < 0.001. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 6

Cystathionine-γ-lyase derived hydrogen sulfide mediates the transient inhibition of calcification in healthy valvular interstitial cells triggered by pro-inflammatory cytokines CSE and CBS genes were silenced in valvular interstitial cells isolated from healthy aortic valves (HAV-VICs). After the silencing, cells were cultured in a growth medium or exposed to a calcification medium, containing 2.5 mmol/L inorganic phosphate and 1.8 mmol/L calcium chloride every other day, in the presence or absence of TNF-α (10 nmol/L) or IL-1β (10 nmol/L) for 5 days. (A) Alizarin Red S staining of HAV-VICs is shown. Quantitative analysis of Alizarin Red S staining of HAV-VICs was calculated using ImageJ software (n = 5). (A) Calcium contents of extracellular matrix normalized to protein of HAV-VICs are shown (n = 3). (B) CSE protein levels normalized to GAPDH in valvular interstitial cells isolated from calcific aortic valves (CAV-VICs) were measured with western blot (n = 3). Quantitative analysis of the CSE western blots was calculated using ImageJ software (n = 3). (C) H₂S generation measured with modified methylene blue method and normalized to protein of CAV-VICs is shown (n = 5). Results were analyzed by one-way ANOVA, Bonferroni's Multiple Comparison Test, and are shown as mean values ± SEM. Ns: not significant; *p < 0.05; **p < 0.01; ***p < 0.001. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 7

Cystathionine-γ-lyase is functioning in calcific human aortic valves Healthy human aortic valves (HAVs) (N = 5) and calcified human aortic valves (CAVs) (N = 5) were cryogenically pulverized and resuspended in CHAPS or RIPA (CysSH/Cys ratios) buffer followed by 10-s sonication on ice. Cys, CSSC, HCys, cystathionine, lanthionine contents and protein CysSH/Cys ratios of the healthy aortic valves (N = 5) and calcified aortic valves (N = 5) are shown. Results were analyzed by unpaired t-test, and are shown as mean values ± SEM. Ns: not significant; *p < 0.05.

Fig. 8

Mitochondrial enzymes involved in H₂S metabolism are upregulated in calcific human aortic valves Healthy human aortic valves (HAVs) (N = 4) and calcific human aortic valves (CAVs) (N = 5) were cryogenically pulverized and taken up with cell lysis buffer followed by 5-s sonication on ice three times. SQR, ETHE1, SO and TST protein expression normalized to GAPDH of healthy human aortic valves (N = 4) and calcified human aortic valves (N = 5) were measured with western blot. Quantitative analyses of the SQR, ETHE1, SO and TST western blots were calculated using ImageJ software. Results were analyzed by unpaired t-test and were shown as mean values ± SEM. *p < 0.05; **p < 0.01.

Fig. 9

Valvular interstitial cells of healthy aortic valves exposed to calcifying stimuli up-regulate the expression of SQR, ETHE, SO and TST Valvular interstitial cells isolated from healthy aortic valves (HAV-VICs) were cultured in a growth medium or exposed to calcification medium, containing 2.5 mmol/L inorganic phosphate and 1.8 mmol/L calcium chloride every other day for 5 days. SQR, ETHE1, SO and TST protein levels normalized to GAPDH were measured with western blot (N = 4). Quantitative analyses of the SQR, ETHE1, SO and TST western blots were calculated using ImageJ software (N = 4). Results were analyzed by unpaired t-test and were shown as mean values ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001.

Fig. 10

Mitochondria-targeting H₂S donor prevents calcification of valvular interstitial cells derived from calcific human aortic valves Valvular interstitial cells isolated from calcific aortic valves (CAV-VICs) were cultured in a growth medium or exposed to calcification medium containing 2.5 mmol/L inorganic phosphate and 1.8 mmol/L calcium chloride every other day in the presence or absence of [10-oxo-10-[4-(3-thioxo-3H-1,2-dithiol-5-yl)phenoxy]decyl]triphenyl-phosphonium (AP39) (5 nmol/L) or sodium hydrogen sulfide (NaSH) (5 nmol/L; 25 μmol/L) for 5 days. (A and B) Alizarin Red S staining and extracellular calcium content normalized to protein of CAV-VICs are shown (n = 5). Quantitative analysis of Alizarin Red S staining of CAV-VICs was performed using ImageJ software (n = 5). Results were analyzed by one-way ANOVA, Bonferroni's Multiple Comparison Test, and were shown as mean values ± SEM. Ns: not significant; **p < 0.01; ***p < 0.001. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 11

Mitochondria-targeting H₂S donor inhibits inflammation of valvular interstitial cells Valvular interstitial cells (VICs) isolated from healthy aortic valves (HAV-VICs) were cultured in growth medium or calcification medium, containing 2.5 mmol/L inorganic phosphate and 1.8 mmol/L calcium chloride every other day, in the presence or absence of [10-oxo-10-[4-(3-thioxo-3H-1,2-dithiol-5-yl)phenoxy]decyl]triphenyl-phosphonium (AP39) (5 nmol/L) for 5 days. (A) Relative expression of IL-1β (left panel) and TNF-α (right panel) were analyzed by Real-Time qPCR (n = 3) in HAV-VICs are shown. (B) Protein expression of IL-1β, TNF-α, and GAPDH were determined by western blots in HAV-VICs are shown. Quantification of IL-1β and TNF-α optical density was calculated by ImageJ software. (C) HAV-VICs grown on coverslips and cultured in growth medium or calcification medium, containing 2.5 mmol/L inorganic phosphate and 1.8 mmol/L calcium chloride every other day, in the presence or absence of AP39 (5 nmol/L) for 5 days. Cells were stained with Hoechst 33258 for DNA (blue), an anti-IL-1β antibody with Alexa Fluor 488 secondary antibody for IL-1β (green), an anti-TNF-α antibody with Alexa Fluor 488 secondary antibody for TNF-α (green), and with Osteosense Fluorescent Probe (red). Images were taken with Leica TCS SP8 gated STED-CW nanoscopy. Images were deconvolved using Huygens Professional software. The color intensity of IL-1β, TNF-α, and Osteosense stainings was calculated using ImageJ software. Representative image, n = 5. Scale bars shown in the images represent 50 μm. Results were analyzed by one-way ANOVA, Bonferroni's Multiple Comparison Test, and were shown as mean values ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)