Hemolytic or hemorrhagic episodes are often associated with inflammation even when infectious agents are absent suggesting that red blood cells (RBCs) release damage‐associated molecular patterns (DAMPs). DAMPs activate immune and nonimmune cells through pattern recognition receptors. Heme, released from RBCs, is a DAMP and induces IL‐1β production through the activation of the nucleotide‐binding domain and leucine‐rich repeat‐containing family and pyrin domain containing 3 (NLRP3) in macrophages; however, other cellular targets of heme‐mediated inflammasome activation were not investigated. Because of their location, endothelial cells can be largely exposed to RBC‐derived DAMPs; therefore, we investigated whether heme and other hemoglobin‐ (Hb‐) derived species induce NLRP3 inflammasome activation in these cells. We found that heme upregulated NLRP3 expression and induced active IL‐1β production in human umbilical vein endothelial cells (HUVECs). LPS priming largely amplified the heme‐mediated production of IL‐1β. Heme administration into C57BL/6 mice induced caspase‐1 activation and cleavage of IL‐1β which was not observed in NLRP3^−/− mice. Unfettered production of reactive oxygen species played a critical role in heme‐mediated NLRP3 activation. Activation of NLRP3 by heme required structural integrity of the heme molecule, as neither protoporphyrin IX nor iron‐induced IL‐1β production. Neither naive nor oxidized forms of Hb were able to induce IL‐1β production in HUVECs. Our results identified endothelial cells as a target of heme‐mediated NLRP3 activation that can contribute to the inflammation triggered by sterile hemolysis. Thus, understanding the characteristics and cellular counterparts of RBC‐derived DAMPs might allow us to identify new therapeutic targets for hemolytic diseases.

Vascular calcification is the deposition of hydroxyapatite crystals in the medial or intimal layers of arteries that is usually associated with other pathological conditions including but not limited to chronic kidney disease, atherosclerosis and diabetes. Calcification is an active, cell-regulated process involving the phenotype transition of vascular smooth muscle cells (VSMCs) from contractile to osteoblast/chondrocyte-like cells. Diverse triggers and signal transduction pathways have been identified behind vascular calcification. In this review, we focus on the role of reactive oxygen species (ROS) in the osteochondrogenic phenotype switch of VSMCs and subsequent calcification. Vascular calcification is associated with elevated ROS production. Excessive ROS contribute to the activation of certain osteochondrogenic signal transduction pathways, thereby accelerating osteochondrogenic transdifferentiation of VSMCs. Inhibition of ROS production and ROS scavengers and activation of endogenous protective mechanisms are promising therapeutic approaches in the prevention of osteochondrogenic transdifferentiation of VSMCs and subsequent vascular calcification. The present review discusses the formation and actions of excess ROS in different experimental models of calcification, and the potential of ROS-lowering strategies in the prevention of this deleterious condition.

Calcific aortic valve stenosis (CAVS) is a heart disease characterized by the progressive fibro-calcific remodeling of the aortic valves, an actively regulated process with the involvement of the reactive oxygen species-mediated differentiation of valvular interstitial cells (VICs) into osteoblast-like cells. Nuclear factor erythroid 2-related factor 2 (Nrf2) regulates the expression of a variety of antioxidant genes, and plays a protective role in valve calcification. Heme oxygenase-1 (HO-1), an Nrf2-target gene, is upregulated in human calcified aortic valves. Therefore, we investigated the effect of Nrf2/HO-1 axis in VIC calcification. We induced osteogenic differentiation of human VICs with elevated phosphate and calcium-containing osteogenic medium (OM) in the presence of heme. Heme inhibited Ca deposition and OM-induced increase in alkaline phosphatase and osteocalcin (OCN) expression. Heme induced Nrf2 and HO-1 expression in VICs. Heme lost its anti-calcification potential when we blocked transcriptional activity Nrf2 or enzyme activity of HO-1. The heme catabolism products bilirubin, carbon monoxide, and iron, and also ferritin inhibited OM-induced Ca deposition and OCN expression in VICs. This study suggests that heme-mediated activation of the Nrf2/HO-1 pathway inhibits the calcification of VICs. The anti-calcification effect of heme is attributed to the end products of HO-1-catalyzed heme degradation and ferritin.

Following an intraventricular hemorrhage (IVH), red blood cell lysis and hemoglobin (Hb) oxidation with the release of heme can cause sterile neuroinflammation. In this study, we measured Hb derivates and cellular adhesion molecules ICAM-1 and VCAM-1 with cell-free miRNAs in cerebrospinal fluid (CSF) samples obtained from Grade-III and Grade-IV preterm IVH infants (IVH-III and IVH-IV, respectively) at multiple time points between days 0–60 after the onset of IVH. Furthermore, human choroid plexus epithelial cells (HCPEpiCs) were incubated with IVH and non-IVH CSF (10 v/v %) for 24 h in vitro to investigate the IVH-induced inflammatory response that was investigated via: (i) HMOX1, IL8, VCAM1, and ICAM1 mRNAs as well as miR-155, miR-223, and miR-181b levels by RT-qPCR; (ii) nuclear translocation of the NF-κB p65 subunit by fluorescence microscopy; and (iii) reactive oxygen species (ROS) measurement. We found a time-dependent alteration of heme, IL-8, and adhesion molecules which revealed a prolonged elevation in IVH-IV vs. IVH-III with higher miR-155 and miR-181b expression at days 41–60. Exposure of HCPEpiCs to IVH CSF samples induced HMOX1, IL8, and ICAM1 mRNA levels along with increased ROS production via the NF-κB pathway activation but without cell death, as confirmed by the cell viability assay. Additionally, the enhanced intracellular miR-155 level was accompanied by lower miR-223 and miR-181b expression in HCPEpiCs after CSF treatment. Overall, choroid plexus epithelial cells exhibit an abnormal cell phenotype after interaction with pro-inflammatory CSF of IVH origin which may contribute to the development of later clinical complications in preterm IVH.

Cataract, an opacification in the crystalline lens, is a leading cause of blindness. Deposition of hydroxyapatite occurs in a cataractous lens that could be the consequence of osteogenic differentiation of lens epithelial cells (LECs). Nuclear factor erythroid 2-related factor 2 (Nrf2) controls the transcription of a wide range of cytoprotective genes. Nrf2 upregulation attenuates cataract formation. Here we aimed to investigate the effect of Nrf2 system upregulation in LECs calcification. We induced osteogenic differentiation of human LECs (HuLECs) with increased phosphate and calcium-containing osteogenic medium (OM). OM-induced calcium and osteocalcin deposition in HuLECs. We used heme to activate Nrf2, which strongly upregulated the expression of Nrf2 and heme oxygenase-1 (HO-1). Heme-mediated Nrf2 activation was dependent on the production of reactive oxygens species. Heme inhibited Ca deposition, and the OM-induced increase of osteogenic markers, RUNX2, alkaline phosphatase, and OCN. Anti-calcification effect of heme was lost when the transcriptional activity of Nrf2 or the enzyme activity of HO-1 was blocked with pharmacological inhibitors. Among products of HO-1 catalyzed heme degradation iron mimicked the anti-calcification effect of heme. We concluded that heme-induced upregulation of the Nrf2/HO-1 system inhibits HuLECs calcification through the liberation of heme iron.

Hemolytic diseases are characterized by an accelerated breakdown of red blood cells (RBCs) and the release of hemoglobin (Hb). Following, RBC lysis Hb oxidation occurs with the formation of different redox states of Hb (metHb and ferrylHb) and the release of heme. ferrylHb is unstable and decomposes to metHb with the concomitant formation of globin radicals and eventually covalently crosslinked Hb multimers. The goal of the present study was to determine the concentrations of the different redox states of Hb in biological samples during hemolytic conditions. We used plasma and urine samples of mice with intravascular hemolysis and human cerebrospinal fluid (CSF) samples following intraventricular hemorrhage. Because ferrylHb is highly unstable, we also addressed the fate of this species. metHb and free heme time-dependently accumulate in plasma and CSF samples following intravascular hemolysis and intraventricular hemorrhage, respectively. ferrylHb is hardly detectable in the biological samples during hemolytic conditions. Underin vitroconditions, ferrylHb decomposes quickly to metHb, which process is associated with the formation of covalently crosslinked Hb multimers. We detected these covalently crosslinked Hb multimers in plasma, urine, and CSF samples during hemolytic conditions. Because globin modification is specific for these Hb forms, we propose to call this heterogeneous form of Hb produced during ferrylHb decomposition as globin-modified oxidized Hb (gmoxHb). Understanding the formation and the contribution of gmoxHb species to the pathogenesis of hemolytic conditions could have therapeutic implications in the treatment of hemolytic diseases.