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.

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-1ß (IL-1ß) 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 ß-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-1ß 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-1ß 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.

Oxidation of Hemoglobin Drives a Proatherogenic Polarization of Macrophages in Human Atherosclerosis.

Aim: The aim of our study was to explore the pathophysiologic role of oxidation of hemoglobin (Hb) to ferrylHb in human atherosclerosis. Results: We observed a severe oxidation of Hb to ferrylHb in complicated atherosclerotic lesions of carotid arteries with oxidative changes of the globin moieties, detected previously described oxidation hotspots in Hb (ß1Cys93; ß1Cys112; ß2Cys112) and identified a novel oxidation hotspot (α1Cys104). After producing a monoclonal anti-ferrylHb antibody, ferrylHb was revealed to be localized extracellularly and also internalized by macrophages in the human hemorrhagic complicated lesions. We demonstrated that ferrylHb is taken up via phagocytosis as well as CD163 receptor-mediated endocytosis and then transported to lysosomes involving actin polymerization. Internalization of ferrylHb was accompanied by upregulation of heme oxygenase-1 and H-ferritin and accumulation of iron within lysosomes as a result of heme/iron uptake. Importantly, macrophages exposed to ferrylHb in atherosclerotic plaques exhibited a proinflammatory phenotype, as reflected by elevated levels of IL-1ß and TNF-α. To find further signatures of ferrylHb in complicated lesions, we performed RNA-seq analysis on biopsies from patients who underwent endarterectomies. RNA-seq analysis demonstrated that human complicated lesions had a unique transcriptomic profile different from arteries and atheromatous plaques. Pathways affected in complicated lesions included gene changes associated with phosphoinositide 3-kinase (PI3K) signaling, lipid transport, tissue remodeling, and vascularization. Targeted analysis of gene expression associated with calcification, apoptosis, and hemolytic-specific clusters indicated an increase in the severity of complicated lesions compared with atheroma. A 39% overlap in the differential gene expression profiles of human macrophages exposed to ferrylHb and the complicated lesion profiles was uncovered. Among these 547 genes, we found inflammatory, angiogenesis, and iron metabolism gene clusters regulated in macrophages. Innovation and Conclusion: We conclude that oxidation of Hb to ferrylHb contributes to the progression of atherosclerosis via polarizing macrophages into a proatherogenic phenotype. Antioxid. Redox Signal. 35, 917-950.

Hemoglobin oxidation generates globin-derived peptides in atherosclerotic lesions and intraventricular hemorrhage of the brain, provoking endothelial dysfunction.

The lysis of red blood cells was shown to occur in human ruptured atherosclerotic lesions and intraventricular hemorrhage (IVH) of the brain. Liberated cell-free hemoglobin was found to undergo oxidation in both pathologies. We hypothesize that hemoglobin-derived peptides are generated during hemoglobin oxidation both in complicated atherosclerotic lesions and IVH of the brain, triggering endothelial cell dysfunction. Oxidized hemoglobin and its products were followed with spectrophotometry, LC-MS/MS analysis and detection of the cross-linking of globin chains in complicated atherosclerotic lesions of the human carotid artery and the hemorrhaged cerebrospinal liquid of preterm infants. The vascular pathophysiologic role of oxidized hemoglobin and the resultant peptides was assessed by measuring endothelial integrity, the activation of endothelial cells and the induction of proinflammatory genes. Peptide fragments of hemoglobin (VNVDEVGGEALGRLLVVYPWTQR, LLVVYPWTQR, MFLSFPTTK, VGAHAGEYGAELERMFLSFPTTK, and FLASVSTVLTSKYR) were identified in ruptured atherosclerotic lesions and in IVH of the human brain. Fragments resulting from the oxidation of hemoglobin were accompanied by the accumulation of ferryl hemoglobin. Similar to complicated atherosclerotic lesions of the human carotid artery, a high level of oxidized and cross-linked hemoglobin was observed in the cerebrospinal fluid after IVH. Haptoglobin inhibited hemoglobin fragmentation provoked by peroxide. The resultant peptides failed to bind haptoglobin or albumin. Peptides derived from hemoglobin oxidation and ferryl hemoglobin induced intercellular gap formation, decreased junctional resistance in the endothelium, and enhanced monocyte adhesion to endothelial cells. Enhanced expression of TNF and the activation of NLRP3 and CASP1 followed by the increased generation of IL-1ß and nuclear translocation of the NF-κß transcription factor occurred in response to hemoglobin-derived peptides, and ferryl hemoglobin in endothelium was upregulated in both pathologies. We conclude that the oxidation of hemoglobin in complicated atherosclerotic lesions and intraventricular hemorrhage of the brain generates peptide fragments and ferryl hemoglobin with the potential to trigger endothelial cell dysfunction.

Hydrogen sulfide inhibits calcification of heart valves; implications for calcific aortic valve disease.

BACKGROUND AND PURPOSE: Calcification of heart valves is a frequent pathological finding in chronic kidney disease and in elderly patients. Hydrogen sulfide (H2 S) may exert anti-calcific actions. Here we investigated H2 S as an inhibitor of valvular calcification and to identify its targets in the pathogenesis. EXPERIMENTAL APPROACH: Effects of H2 S on osteoblastic transdifferentiation of valvular interstitial cells (VIC) isolated from samples of human aortic valves were studied using immunohistochemistry and western blots. We also assessed H2S on valvular calcification in apolipoprotein E-deficient (ApoE-/- ) mice. KEY RESULTS: In human VIC, H2 S from donor compounds (NaSH, Na2 S, GYY4137, AP67, and AP72) inhibited mineralization/osteoblastic transdifferentiation, dose-dependently in response to phosphate. Accumulation of calcium in the extracellular matrix and expression of osteocalcin and alkaline phosphatase was also inhibited. RUNX2 was not translocated to the nucleus and phosphate uptake was decreased. Pyrophosphate generation was increased via up-regulating ENPP2 and ANK1. Lowering endogenous production of H2 S by concomitant silencing of cystathionine γ-lyase (CSE) and cystathionine ß-synthase (CBS) favoured VIC calcification. analysis of human specimens revealed higher Expression of CSE in aorta stenosis valves with calcification (AS) was higher than in valves of aortic insufficiency (AI). In contrast, tissue H2 S generation was lower in AS valves compared to AI valves. Valvular calcification in ApoE-/- mice on a high-fat diet was inhibited by H2 S. CONCLUSIONS AND IMPLICATIONS: The endogenous CSE-CBS/H2 S system exerts anti-calcification effects in heart valves providing a novel therapeutic approach to prevent hardening of valves. LINKED ARTICLES: This article is part of a themed section on Hydrogen Sulfide in Biology & Medicine. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v177.4/issuetoc.

Potential Role of H-Ferritin in Mitigating Valvular Mineralization.

Objective- Calcific aortic valve disease is a prominent finding in elderly and in patients with chronic kidney disease. We investigated the potential role of iron metabolism in the pathogenesis of calcific aortic valve disease. Approach and Results- Cultured valvular interstitial cells of stenotic aortic valve with calcification from patients undergoing valve replacement exhibited significant susceptibility to mineralization/osteoblastic transdifferentiation in response to phosphate. This process was abrogated by iron via induction of H-ferritin as reflected by lowering ALP and osteocalcin secretion and preventing extracellular calcium deposition. Cellular phosphate uptake and accumulation of lysosomal phosphate were decreased. Accordingly, expression of phosphate transporters Pit1 and Pit2 were repressed. Translocation of ferritin into lysosomes occurred with high phosphate-binding capacity. Importantly, ferritin reduced nuclear accumulation of RUNX2 (Runt-related transcription factor 2), and as a reciprocal effect, it enhanced nuclear localization of transcription factor Sox9 (SRY [sex-determining region Y]-box 9). Pyrophosphate generation was also increased via upregulation of ENPP2 (ectonucleotide pyrophosphatase/phosphodiesterase-2). 3H-1, 2-dithiole-3-thione mimicked these beneficial effects in valvular interstitial cell via induction of H-ferritin. Ferroxidase activity of H-ferritin was essential for this function, as ceruloplasmin exhibited similar inhibitory functions. Histological analysis of stenotic aortic valve revealed high expression of H-ferritin without iron accumulation and its relative dominance over ALP in noncalcified regions. Increased expression of H-ferritin accompanied by elevation of TNF-α (tumor necrosis factor-α) and IL-1ß (interleukin-1ß) levels, inducers of H-ferritin, corroborates the essential role of ferritin/ferroxidase via attenuating inflammation in calcific aortic valve disease. Conclusions- Our results indicate that H-ferritin is a stratagem in mitigating valvular mineralization/osteoblastic differentiation. Utilization of 3H-1, 2-dithiole-3-thione to induce ferritin expression may prove a novel therapeutic potential in valvular mineralization.