Cellular FXIII in Human Macrophage-Derived Foam Cells.

Macrophages express the A subunit of coagulation factor XIII (FXIII-A), a transglutaminase which cross-links proteins through Nε-(γ-L-glutamyl)-L-lysyl iso-peptide bonds. Macrophages are major cellular constituents of the atherosclerotic plaque; they may stabilize the plaque by cross-linking structural proteins and they may become transformed into foam cells by accumulating oxidized LDL (oxLDL). The combination of oxLDL staining by Oil Red O and immunofluorescent staining for FXIII-A demonstrated that FXIII-A is retained during the transformation of cultured human macrophages into foam cells. ELISA and Western blotting techniques revealed that the transformation of macrophages into foam cells elevated the intracellular FXIII-A content. This phenomenon seems specific for macrophage-derived foam cells; the transformation of vascular smooth muscle cells into foam cells fails to induce a similar effect. FXIII-A containing macrophages are abundant in the atherosclerotic plaque and FXIII-A is also present in the extracellular compartment. The protein cross-linking activity of FXIII-A in the plaque was demonstrated using an antibody labeling the iso-peptide bonds. Cells showing combined staining for FXIII-A and oxLDL in tissue sections demonstrated that FXIII-A-containing macrophages within the atherosclerotic plaque are also transformed into foam cells. Such cells may contribute to the formation of lipid core and the plaque structurization.

BGP-15 Inhibits Hyperglycemia-Aggravated VSMC Calcification Induced by High Phosphate.

Vascular calcification associated with high plasma phosphate (Pi) level is a frequent complication of hyperglycemia, diabetes mellitus, and chronic kidney disease. BGP-15 is an emerging anti-diabetic drug candidate. This study was aimed to explore whether BGP-15 inhibits high Pi-induced calcification of human vascular smooth muscle cells (VSMCs) under normal glucose (NG) and high glucose (HG) conditions. Exposure of VSMCs to Pi resulted in accumulation of extracellular calcium, elevated cellular Pi uptake and intracellular pyruvate dehydrogenase kinase-4 (PDK-4) level, loss of smooth muscle cell markers (ACTA, TAGLN), and enhanced osteochondrogenic gene expression (KLF-5, Msx-2, Sp7, BMP-2). Increased Annexin A2 and decreased matrix Gla protein (MGP) content were found in extracellular vesicles (EVs). The HG condition markedly aggravated Pi-induced VSMC calcification. BGP-15 inhibited Pi uptake and PDK-4 expression that was accompanied by the decreased nuclear translocation of KLF-5, Msx-2, Sp7, retained VSMC markers (ACTA, TAGLN), and decreased BMP-2 in both NG and HG conditions. EVs exhibited increased MGP content and decreased Annexin A2. Importantly, BGP-15 prevented the deposition of calcium in the extracellular matrix. In conclusion, BGP-15 inhibits Pi-induced osteochondrogenic phenotypic switch and mineralization of VSMCs in vitro that make BGP-15 an ideal candidate to attenuate both diabetic and non-diabetic vascular calcification.

Ferryl Hemoglobin and Heme Induce A1-Microglobulin in Hemorrhaged Atherosclerotic Lesions with Inhibitory Function against Hemoglobin and Lipid Oxidation.

Infiltration of red blood cells into atheromatous plaques and oxidation of hemoglobin (Hb) and lipoproteins are implicated in the pathogenesis of atherosclerosis. α1-microglobulin (A1M) is a radical-scavenging and heme-binding protein. In this work, we examined the origin and role of A1M in human atherosclerotic lesions. Using immunohistochemistry, we observed a significant A1M immunoreactivity in atheromas and hemorrhaged plaques of carotid arteries in smooth muscle cells (SMCs) and macrophages. The most prominent expression was detected in macrophages of organized hemorrhage. To reveal a possible inducer of A1M expression in ruptured lesions, we exposed aortic endothelial cells (ECs), SMCs and macrophages to heme, Oxy- and FerrylHb. Both heme and FerrylHb, but not OxyHb, upregulated A1M mRNA expression in all cell types. Importantly, only FerrylHb induced A1M protein secretion in aortic ECs, SMCs and macrophages. To assess the possible function of A1M in ruptured lesions, we analyzed Hb oxidation and heme-catalyzed lipid peroxidation in the presence of A1M. We showed that recombinant A1M markedly inhibited Hb oxidation and heme-driven oxidative modification of low-density lipoproteins as well plaque lipids derived from atheromas. These results demonstrate the presence of A1M in atherosclerotic plaques and suggest its induction by heme and FerrylHb in the resident cells.

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.

Therapeutic Potential of Carbon Monoxide (CO) and Hydrogen Sulfide (H2S) in Hemolytic and Hemorrhagic Vascular Disorders-Interaction between the Heme Oxygenase and H2S-Producing Systems.

Over the past decades, substantial work has established that hemoglobin oxidation and heme release play a pivotal role in hemolytic/hemorrhagic disorders. Recent reports have shown that oxidized hemoglobins, globin-derived peptides, and heme trigger diverse biological responses, such as toll-like receptor 4 activation with inflammatory response, reprogramming of cellular metabolism, differentiation, stress, and even death. Here, we discuss these cellular responses with particular focus on their mechanisms that are linked to the pathological consequences of hemorrhage and hemolysis. In recent years, endogenous gasotransmitters, such as carbon monoxide (CO) and hydrogen sulfide (H2S), have gained a lot of interest in connection with various human pathologies. Thus, many CO and H2S-releasing molecules have been developed and applied in various human disorders, including hemolytic and hemorrhagic diseases. Here, we discuss our current understanding of oxidized hemoglobin and heme-induced cell and tissue damage with particular focus on inflammation, cellular metabolism and differentiation, and endoplasmic reticulum stress in hemolytic/hemorrhagic human diseases, and the potential beneficial role of CO and H2S in these pathologies. More detailed mechanistic insights into the complex pathology of hemolytic/hemorrhagic diseases through heme oxygenase-1/CO as well as H2S pathways would reveal new therapeutic approaches that can be exploited for clinical benefit.

The Fungal Iron Chelator Desferricoprogen Inhibits Atherosclerotic Plaque Formation.

Hemoglobin, heme and iron are implicated in the progression of atherosclerosis. Therefore, we investigated whether the hydrophobic fungal iron chelator siderophore, desferricoprogen (DFC) inhibits atherosclerosis. DFC reduced atherosclerotic plaque formation in ApoE-/- mice on an atherogenic diet. It lowered the plasma level of oxidized LDL (oxLDL) and inhibited lipid peroxidation in aortic roots. The elevated collagen/elastin content and enhanced expression of adhesion molecule VCAM-1 were decreased. DFC diminished oxidation of Low-density Lipoprotein (LDL) and plaque lipids catalyzed by heme or hemoglobin. Formation of foam cells, uptake of oxLDL by macrophages, upregulation of CD36 and increased expression of TNF-α were reduced by DFC in macrophages. TNF-triggered endothelial cell activation (vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecules (ICAMs), E-selectin) and increased adhesion of monocytes to endothelium were attenuated. The increased endothelial permeability and intracellular gap formation provoked by TNF-α was also prevented by DFC. DFC acted as a cytoprotectant in endothelial cells and macrophages challenged with a lethal dose of oxLDL and lowered the expression of stress-responsive heme oxygenase-1 as sublethal dose was employed. Saturation of desferrisiderophore with iron led to the loss of the beneficial effects. We demonstrated that DFC accumulated within the atheromas of the aorta in ApoE-/- mice. DFC represents a novel therapeutic approach to control the progression of atherosclerosis.

Heme cytotoxicity is the consequence of endoplasmic reticulum stress in atherosclerotic plaque progression.

Hemorrhage and hemolysis with subsequent heme release are implicated in many pathologies. Endothelial cells (ECs) encounter large amount of free heme after hemolysis and are at risk of damage from exogenous heme. Here we show that hemorrhage aggravates endoplasmic reticulum (ER) stress in human carotid artery plaques compared to healthy controls or atheromas without hemorrhage as demonstrated by RNA sequencing and immunohistochemistry. In EC cultures, heme also induces ER stress. In contrast, if cultured ECs are pulsed with heme arginate, cells become resistant to heme-induced ER (HIER) stress that is associated with heme oxygenase-1 (HO-1) and ferritin induction. Knocking down HO-1, HO-2, biliverdin reductase, and ferritin show that HO-1 is the ultimate cytoprotectant in acute HIER stress. Carbon monoxide-releasing molecules (CORMs) but not bilirubin protects cultured ECs from HIER stress via HO-1 induction, at least in part. Knocking down HO-1 aggravates heme-induced cell death that cannot be counterbalanced with any known cell death inhibitors. We conclude that endothelium and perhaps other cell types can be protected from HIER stress by induction of HO-1, and heme-induced cell death occurs via HIER stress that is potentially involved in the pathogenesis of diverse pathologies with hemolysis and hemorrhage including atherosclerosis.

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.

Zinc Inhibits HIF-Prolyl Hydroxylase Inhibitor-Aggravated VSMC Calcification Induced by High Phosphate.

Vascular calcification is a life-threatening clinical condition in chronic kidney disease (CKD) and is associated with reduced zinc serum levels. Anemia is another frequent complication of CKD. Hypoxia-inducible factor (HIF) stabilizers, also known as HIF prolyl hydroxylase inhibitors (PHI), are promising candidates to treat CKD-associated anemia by increasing erythropoietin synthesis. Recent evidence suggests that HIFs play a pivotal role in vascular calcification. Our study explored feasible impacts of HIF PHI on phosphate (Pi)-induced calcification of vascular smooth muscle cells (VSMCs) and tested whether zinc might inhibit this mineralization process. Treatment of VSMCs with PHI aggravated Pi-induced calcium deposition and Pi uptake. PHI promoted Pi-induced loss of smooth muscle cell markers (ACTA-2, MYH11, SM22α) and enhanced osteochondrogenic gene expression (Msx-2, BMP-2, Sp7) triggering osteochondrogenic phenotypic switch of VSMCs. These effects of PHI paralleled with increased pyruvate dehydrogenase kinase 4 (PDK4) expression, decreased Runx2 Ser451 phosphorylation, and reduced cell viability. Zinc inhibited Pi-induced mineralization of VSMCs in a dose-dependent manner and also attenuated the pro-calcification effect of PHI in Pi-induced mineralization. Zinc inhibited osteochondrogenic phenotypic switch of VSMCs reflected by lowering Pi uptake, decreasing the expressions of Msx-2, BMP-2, and Sp7 as well as the loss of smooth muscle cell-specific markers. Zinc preserved phosphorylation state of Runx2 Ser451, decreased PDK4 level, and restored cell viability. PHI alone reduced the expression of smooth muscle markers without inducing mineralization, which was also inhibited by zinc. In addition, we observed a significantly lower serum zinc level in CKD as well as in patients undergoing carotid endarterectomy compared to healthy individuals. Conclusion - PHI promoted the loss of smooth muscle markers and augmented Pi-induced osteochondrogenic phenotypic switch leading to VSMCs calcification. This mineralization process was attenuated by zinc. Enhanced vascular calcification is a potential risk factor during PHI therapy in CKD which necessitates the strict follow up of vascular calcification and zinc supplementation.

Heme Induces Endoplasmic Reticulum Stress (HIER Stress) in Human Aortic Smooth Muscle Cells.

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, TGFß 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.

Hydrogen Sulfide Abrogates Hemoglobin-Lipid Interaction in Atherosclerotic Lesion.

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-1ß. 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.