The infiltration of red blood cells into atheromatous plaques is implicated in atherogenesis. Inside the lesion, hemoglobin (Hb) is oxidized to ferri- and ferrylHb which exhibit prooxidant and proinflammatory activities. Cystathione gamma-lyase- (CSE-) derived H2S has been suggested to possess various antiatherogenic actions. Expression of CSE was upregulated predominantly in macrophages, foam cells, and myofibroblasts of human atherosclerotic lesions derived from carotid artery specimens of patients. A similar pattern was observed in aortic lesions of apolipoprotein E-deficient mice on high-fat diet. We identified several triggers for inducing CSE expression in macrophages and vascular smooth muscle cells including heme, ferrylHb, plaque lipids, oxidized low-density lipoprotein, tumor necrosis factor-α, and interleukin-1β. In the interplay between hemoglobin and atheroma lipids, H2S significantly mitigated oxidation of Hb preventing the formation of ferrylHb derivatives, therefore providing a novel function as a heme-redox-intermediate-scavenging antioxidant. By inhibiting Hb-lipid interactions, sulfide lowered oxidized Hb-mediated induction of adhesion molecules in endothelium and disruption of endothelial integrity. Exogenous H2S inhibited heme and Hb-mediated lipid oxidation of human atheroma-derived lipid and human complicated lesion. Our study suggests that the CSE/H2S system represents an atheroprotective pathway for removing or limiting the formation of oxidized Hb and lipid derivatives in the atherosclerotic plaque.

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-1β and nuclear translocation of the NF-κβ 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.

Background and Purpose Calcification of heart valves is a frequent pathological finding in chronic kidney disease and in elderly patients. Hydrogen sulfide (H2S) may exert anti‐calcific actions. Here we investigated H2S as an inhibitor of valvular calcification and to identify its targets in the pathogenesis. Experimental Approach Effects of H2S 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, H2S from donor compounds (NaSH, Na2S, 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 H2S by concomitant silencing of cystathionine γ‐lyase (CSE) and cystathionine β‐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 H2S generation was lower in AS valves compared to AI valves. Valvular calcification in ApoE−/− mice on a high‐fat diet was inhibited by H2S. Conclusions and Implications The endogenous CSE‐CBS/H2S 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

Graphical abstract

Accumulation of damaged or misfolded proteins resulted from oxidative protein modification induces endoplasmic reticulum (ER) stress by activating the pathways of unfolded protein response. In pathologic hemolytic conditions, extracellular free hemoglobin is submitted to rapid oxidation causing heme release. Resident cells of atherosclerotic lesions, after intraplaque hemorrhage, are exposed to heme leading to oxidative injury. Therefore, we raised the question whether heme can also provoke ER stress. Smooth muscle cells are one of the key players of atherogenesis; thus, human aortic smooth muscle cells (HAoSMCs) were selected as a model cell to reveal the possible link between heme and ER stress. Using immunoblotting, quantitative polymerase chain reaction and immunocytochemistry, we quantitated the markers of ER stress. These were: phosphorylated eIF2α, Activating transcription factor-4 (ATF4), DNA-damage-inducible transcript 3 (also known as C/EBP homology protein, termed CHOP), X-box binding protein-1 (XBP1), Activating transcription factor-6 (ATF6), GRP78 (glucose-regulated protein, 78kDa) and heme responsive genes heme oxygenase-1 and ferritin. In addition, immunohistochemistry was performed on human carotid artery specimens from patients who had undergone carotid endarterectomy. We demonstrate that heme increases the phosphorylation of eiF2α in HAoSMCs and the expression of ATF4. Heme also enhances the splicing of XBP1 and the proteolytic cleavage of ATF6. Consequently, there is up-regulation of target genes increasing both mRNA and protein levels of CHOP and GRP78. However, TGFβ and collagen type I decreased. When the heme binding proteins, alpha-1-microglobulin (A1M) and hemopexin (Hpx) are present in cell media, the ER stress provoked by heme is inhibited. ER stress pathways are also retarded by the antioxidant N-acetyl cysteine (NAC) indicating that reactive oxygen species are involved in heme-induced ER stress. Consistent with these findings, elevated expression of the ER stress marker GRP78 and CHOP were observed in smooth muscle cells of complicated lesions with hemorrhage compared to either atheromas or healthy arteries. In conclusion, heme triggers ER stress in a time- and dose-dependent manner in HAoSMCs. A1M and Hpx as well as NAC effectively hamper heme-induced ER stress, supporting their use as a potential therapeutic approach to reverse such a deleterious effects of heme toxicity.

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). 3 H-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-1β (interleukin-1β) 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.

A. Renin overexpression leads to increased titin-based stiffness contributing to diastolic dysfunction in hypertensive mRen2 rats. Am J Physiol Heart Circ Physiol 310: H1671-H1682, 2016. First published April 8, 2016; doi:10.1152/ajpheart.00842.2015.-Hypertension (HTN) is a major risk factor for heart failure. We investigated the influence of HTN on cardiac contraction and relaxation in transgenic renin overexpressing rats (carrying mouse Ren-2 renin gene, mRen2, n ϭ 6). Blood pressure (BP) was measured. Cardiac contractility was characterized by echocardiography, cellular force measurements, and biochemical assays were applied to reveal molecular mechanisms. Sprague-Dawley (SD) rats (n ϭ 6) were used as controls. Transgenic rats had higher circulating renin activity and lower cardiac angiotensin-converting enzyme two levels. Systolic BP was elevated in mRen2 rats (235.11 Ϯ 5.32 vs. 127.03 Ϯ 7.56 mmHg in SD, P Ͻ 0.05), resulting in increased left ventricular (LV) weight/body weight ratio (4.05 Ϯ 0.09 vs. 2.77 Ϯ 0.08 mg/g in SD, P Ͻ 0.05). Transgenic renin expression had no effect on the systolic parameters, such as LV ejection fraction, cardiomyocyte Ca 2ϩ -activated force, and Ca 2ϩ sensitivity of force production. In contrast, diastolic dysfunction was observed in mRen2 compared with SD rats: early and late LV diastolic filling ratio (E/A) was lower (1.14 Ϯ 0.04 vs. 1.87 Ϯ 0.08, P Ͻ 0.05), LV isovolumetric relaxation time was longer (43.85 Ϯ 0.89 vs. 28.55 Ϯ 1.33 ms, P Ͻ 0.05), cardiomyocyte passive tension was higher (1.74 Ϯ 0.06 vs. 1.28 Ϯ 0.18 kN/m 2 , P Ͻ 0.05), and lung weight/body weight ratio was increased (6.47 Ϯ 0.24 vs. 5.78 Ϯ 0.19 mg/g, P Ͻ 0.05), as was left atrial weight/body weight ratio (0.21 Ϯ 0.03 vs. 0.14 Ϯ 0.03 mg/g, P Ͻ 0.05). Hyperphosphorylation of titin at Ser-12742 within the PEVK domain and a twofold overexpression of protein kinase C-␣ in mRen2 rats were detected. Our data suggest a link between the activation of renin-angiotensin-aldosterone system and increased titin-based stiffness through phosphorylation of titin's PEVK element, contributing to diastolic dysfunction.Listen to this article's corresponding podcast at http://ajpheart. podbean.com/e/diastolic-dysfunction-in-the-hypertensive-mren2-rat/.renin-angiotensin-aldosterone system; RAAS; hypertension; diastolic dysfunction; passive stiffness; titin phosphorylation NEW & NOTEWORTHYIt is shown that activation of the renin-angiotensin-aldosterone system leads to hypertension and impaired cardiac relaxation associated with increased cardiomyocyte stiffness and hyperphosphorylation of the PEVK region of titin. Moreover, diastolic dysfunction in mRen2 rats occurs without changes in systolic parameters, similarly to human heart failure with preserved ejection fraction.

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 (β1Cys93; β1Cys112; β2Cys112) 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 receptormediated endocytosis then transported to lysosomes involving actin polymerization.Internalization of ferrylHb was accompanied by up-regulation of heme oxygenase-1 and Hferritin and accumulation of iron within lysosomes as a result of heme/iron uptake.Importantly, macrophages exposed to ferrylHb in atherosclerotic plaques exhibited proinflammatory phenotype, as reflected by elevated levels of IL-1β and TNFα. 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 which was different from arteries and atheromatous plaques. Pathways affected in complicated lesions included gene changes associated with 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 to 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 the progression of atherosclerosis via polarizing macrophages into a pro-atherogenic phenotype.