Heme Carrier Protein Research Articles

The heme binding protein ChuX is a regulator of heme degradation by the ChuS protein in Escherichia coli O157:H7

Escherichia coli O157:H7 possesses an 8-gene cluster (chu genes) that contains genes involved in heme transport and processing from the human host. Among the chu genes, four encode cytoplasmic proteins (ChuS, ChuX, ChuY and ChuW). ChuX was previously shown to be a heme binding protein and to assist ChuW in heme degradation under anaerobic conditions. The purpose of this work was to investigate if ChuX works in concert with ChuS, which is a protein able to degrade heme by a non-canonical mechanism and release the iron from the porphyrin under aerobic conditions using hydrogen peroxide as the oxidant. We showed that when the heme-bound ChuX and apo-ChuS protein are mixed, heme is efficiently transferred from ChuX to ChuS. Heme-bound ChuX displayed a peroxidase activity with ABTS and H2O2 but not heme-bound ChuS, which is an efficient test to determine the protein to which heme is bound in the ChuS-ChuX complex. We found that ChuX protects heme from chemical oxidation and that it has no heme degradation activity by itself. Unexpectedly, we found that ChuX inhibits heme degradation by ChuS and stops the reaction at an early intermediate. We determined using surface plasmon resonance that ChuX interacts with ChuS and that it forms a relatively stable complex. These results indicate that ChuX in addition to its heme transfer activity is a regulator of ChuS activity, a function that was not described before for any of the heme carrier protein that delivers heme to heme degradation enzymes.

Reactive oxygen species induced by indomethacin enhance accumulation of heme carrier protein 1 and hematoporphyrin accumulation in vitro and in vivo in a brain tumor model.

Photodynamic therapy (PDT) is useful for various cancers such as high-grade glioma and cancers of other organs. However, the mechanism of tumor-specific accumulation of porphyrin is not clear. The authors previously reported that heme carrier protein 1 (HCP1) contributes to the transport of porphyrins; specifically, we showed that the production of cancer-specific reactive oxygen species from mitochondria (mitROS) leads in turn to enhanced HCP1 expression. Indomethacin (IND), a non-steroidal anti-inflammatory drug, increases ROS production by affecting mitochondrial electron transfer system. In the present work, the authors investigated the effect of pretreatment with IND on cancer-specific porphyrin accumulation, using both a glioma cell line and a rat brain tumor model. This work demonstrated that exposure of a rat glioma cell to IND results in increased generation of cancer-specific mitROS and accumulation of HCP1 expression and porphyrin concentration. Additionally, systemic dosing of a brain tumor animal model with IND resulted in elevated cellular accumulation of porphyrin in tumor cell. This is an effect not seen with normal brain tissue. Thus, the administration of IND increases intracellular porphyrin concentrations in tumor cell without exerting harmful effects on normal brain tissue, and increased porphyrin concentration in tumor cell may lead to improved PDT effect.

Intracellular glutathione levels affect the outcomes of verteporfin-mediated photodynamic therapy in esophageal cancer cells

Photodynamic therapy (PDT) induces cancer cell death by generating reactive oxygen species (ROS). In this process, photosensitizers accumulate in cancer cells irradiated by laser light of a specific wavelength, leading to ROS generation. Verteporfin (VP), a second-generation photosensitizer, is used in PDT for age-related macular degeneration. However, the antitumor effects of VP-PDT remain poorly defined. This study investigated the antitumor effects of VP-PDT on esophageal cancer (EC) cell lines in vitro. Two types of EC cell lines, the KYSE30 cell line, derived from highly differentiated esophageal carcinoma, and the KYSE170 cell line, derived from moderately differentiated carcinoma, were used in this study. VP-PDT exerted effective anticancer effects in both cell lines. Our results revealed that the low-density lipoprotein receptor, albumin receptor, and heme carrier protein-1 in VP uptake were not involved in VP uptake. However, cells rich in intracellular glutathione were resistant to VP-PDT. Our study outcomes suggest that lowering intracellular glutathione via a glutathione synthesis inhibitor or sulfasalazine can increase the effectiveness of VP-PDT-mediated anticancer effects.

Acetic acid enhances the effect of photodynamic therapy in gastric cancer cells via the production of reactive oxygen species.

Acetic acid is a major component of vinegar and is reported to have beneficial health effects. Notably, it causes oxidative stress and enhances the production of reactive oxygen species (ROS) in gastric cancer cells. ROS play important roles in cellular signal transduction, resulting in the regulation of protein expression and apoptosis. We previously reported that ROS upregulate heme carrier protein 1 (HCP1). Moreover, ROS increase the cellular uptake of porphyrins, which are precursors of heme and substrates for uptake by HCP1. Therefore, we hypothesized that photodynamic therapy (PDT) for cancer treatment using laser irradiation and photosensitizers, such as porphyrin, is enhanced via ROS produced by acetic acid. Herein, we used the rat gastric mucosal cells, RGM1, its cancer-like mutated cells, RGK1, and a manganese superoxide dismutase (MnSOD)-overexpressing RGK cell line, RGK-MnSOD. We confirmed that cancer-specific cellular uptake of porphyrin is increased upon acetic acid treatment and enhances the PDT cytotoxicity in RGK-1, not in RGM-1 and RGK-MnSOD. We believe that this occurs because of the overproduction of ROS and subsequent upregulation of HCP1 in cancerous cells. In conclusion, acetic acid can elevate the effect of PDT by inducing cancer-specific HCP1 expression via ROS production.

Peroxynitrite Production Induced by LPS and X-ray Treatment Enhances Cellular Incorporation of Porphyrin in Mouse RAW264 Macrophages

Photodynamic therapy (PDT) is a minimally invasive cancer therapy that combines the accumulation of photosensitizers such as porphyrins in cancer cells with laser irradiation. I have previously reported that mitochondrially derived reactive oxygen species (ROS) regulate the expression of a porphyrin transporter, heme carrier protein 1 (HCP1), and increase porphyrin accumulation in cancer cells. Tumors that contain activated macrophages, referred to as tumor-associated macrophages (TAMs), have been reported to have increased malignancy. TAMs produce nitric oxide (NO), via the expression of inducible NO synthase (iNOS), and the highly reactive nitrogen species, peroxynitrite, which is produced by the reaction of NO with superoxide. Here, I examined the relationship between peroxynitrite, HCP1 expression, and intracellular porphyrin uptake in the murine macrophage cell line RAW264. RAW264 cells were activated by lipopolysaccharide (LPS) treatment which resulted in increased iNOS expression and NO production. Additional X-ray irradiation resulted in the generation of ROS and the subsequent generation of peroxynitrite. Importantly, LPS and X-ray co-treatment significantly enhanced HCP1 expression and porphyrin accumulation in cells, suggesting that the peroxynitrite upregulates the porphyrin transporter, HCP1. Therefore, TAMs may be effectively targeted with PDT, and tumor progression may be suppressed in general by agents that target the activation of macrophages.

Early progression of pulmonary hypertension in the monocrotaline model in males is associated with increased lung permeability

BackgroundThe mechanisms involved in pulmonary hypertension (PH) development in patients and pre-clinical models are poorly understood. PH has a well-established sex dimorphism in patients with increased frequency of PH in females, and more severe disease with poor survival prognosis in males. Previously, we found that heme signaling plays an essential role in the development phase of the Sugen/Hypoxia (SU/Hx) model. This study is focused on the elucidation of sex differences in mechanisms of PH development related to heme action at the early stage of the monocrotaline (MCT) PH model.MethodsRats received MCT injection (60 mg/kg, i.p.) and followed for 14 days to investigate early disease changes. Hemodynamic parameters were recorded at the end of the study; plasma, lung homogenates, and nuclear fractions were used for the evaluation of protein levels.ResultsOur data indicate that on day 14, rats did not show any significant increase in the Fulton index due to the early disease phase. However, the right ventricular systolic pressure was significantly increased in male rats, while female rats showed only a trend. Interestingly, only males demonstrated an increased lung-to-bodyweight ratio that indicated lung edema. Indeed, lung histology confirmed severe perivascular edema in males. Previously, we have reported that the increased perivascular edema in SU/Hx model correlated with intravascular hemolysis and activated heme signaling. Here, we found that elevated free hemoglobin levels and perivascular edema were increased, specifically in males showing more rapid progress of PH. A high level of heme carrier protein 1 (HCP-1), which is involved in heme uptake from the bloodstream into the cells, was also found elevated in the lungs of males. The upregulation of heme oxygenase in males indicated increased intracellular heme catabolism. Increased heme signaling resulted in the activation of heme-mediated barrier-disruptive mechanisms. Thus, hemolysis in males can be responsible for increased permeability of the lungs and early disease development.ConclusionsOur study indicates the importance of barrier-disruptive mechanisms as an earlier event in the induction of pulmonary hypertension. Importantly, males are more susceptible to hemolysis and develop PH earlier than females.

Nitric oxide regulates the expression of heme carrier protein-1 via hypoxia inducible factor-1α stabilization.

Photodynamic therapy (PDT) is a cancer therapy that capitalizes on cancer-specific porphyrin accumulation. We have investigated this phenomenon to propose the following three conclusions: 1) the mechanism underlying this phenomenon is closely related to both nitric oxide (NO) and heme carrier protein-1 (HCP-1), 2) NO inactivates ferrochelatase, and thus, the intracellular porphyrin levels in the cells are increased by the administration of an NO donor after 5-aminolevulinic acid treatment, 3) HCP-1 transports not only heme but also other porphyrins. Since NO stabilizes hypoxia-inducible factor (HIF)-1α, resulting in the upregulation of heme biosynthesis, HCP-1 expression can be increased by HIF-1α stabilization. In this study, we determined whether NO regulates HCP-1 expression by stabilizing HIF-1α expression. For this purpose, rat gastric cancer cell line RGK36 was treated with L-arginine or N6-(1-iminoethyl)-L-lysine (L-NIL). L-arginine treatment increased the intracellular NO concentration, and both HCP-1 and HIF-1α expression, while L-NIL treatment decreased them. Cytotoxicity of PDT was enhanced by L-arginine, following intracellular hemato-porphyrin dihydrochloride (HpD) accumulation. Both Cytotoxicity of PDT and HpD accumulation were decreased by L-NIL. The HCP-1 and HIF-1α expression, intracellular HpD accumulation and PDT cytotoxicity were decreased by 2-methoxyestradiol, which is a HIF-1α inhibitor. Moreover, these phenomena were not increased by a combination of both L-arginine and 2-Me. Thus, HCP-1 can be a downstream target of HIF-1α. These effects were also induced in the human gastric cancer cell line MKN45. Taken together, we conclude that HCP-1 expression is regulated by NO via HIF-1α stabilization.

Hyperthermia enhances photodynamic therapy by regulation of HCP1 and ABCG2 expressions via high level ROS generation

Photodynamic therapy (PDT) is a cancer treatment that make use of the cancer-specific accumulation of porphyrins. We have reported that mitochondrial reactive oxygen species (mitROS) upregulate uptake transporter of porphyrins, heme carrier protein-1 (HCP-1). The accumulation of cancer-specific porphyrins was increased by mitROS production, thereby the cancer-specific PDT cytotoxicity was enhanced. Thus we investigated whether mitROS production by hyperthermia can enhanced the cytotoxicity of PDT or not. In this study, 1 h of hyperthermia at 42 °C increased the mitROS production, and both the accumulation of cancer-specific porphyrins and the PDT cytotoxicity increased. Moreover, the authors treated cells with N-acetyl-L-cysteine (NAC) to examine the effect of mitROS. NAC inhibited the increasing ROS production after hyperthermia to restrain the post-treatment increase of cancer-specific porphyrins accumulation. Moreover, the increase of ROS production in cancer cells after hyperthermia upregulated HCP-1 expression and downregulated ABCG2 expression. These regulation were inhibited by NAC. These results suggest that hyperthermia treatment increased mitROS production, which involved HpD accumulation and enhanced PDT effects in cancer cells. The mechanism of this phenomenon was most likely to be due to both the upregulation of HCP-1 and the downregulation of ABCG2 by mitROS.

Abstract 386: Heme function in promoting tumorigenic potential of lung cancer cells

Abstract Heme, also known as iron protoporphyrin IX, is an essential molecule for living organisms. Heme serves as a prosthetic group in enzymes, and acts as a signaling molecule in diverse cellular processes. Mammalian cells can synthesize heme endogenously, or they can import heme from circulation via heme transporters. The synthesis of heme is carried out by eight enzymes, the first of which is the rate-limiting enzyme 5-aminolevulinate (ALA) synthase. Heme can also be transported intracellularly by transporters, such as HCP1 (heme carrier protein 1) and HRG1 (heme responsive gene 1). Finally, levels of heme are also maintained by the degrading enzyme, heme oxygenase-1 (HO-1). HO-1 is localized to the endoplasmic reticulum and is the rate-limiting enzyme in the catabolism of heme. HO-1 cleaves heme to biliverdin and carbon monoxide. Biliverdin is converted to bilirubin, which is an antioxidant and scavenger of ROS. Heme also serves as a catalytic site for various proteins that are involved in aerobic metabolism, such as respiratory chain cytochromes. For example, complex II, III, and IV of the mitochondria require heme for proper function. Importantly, epidemiologic studies have shown the association of dietary heme intake, mainly from red meat, with different cancers, including lung cancer. Therefore, we have analyzed the levels of heme synthesis, uptake, degradation, oxygen consumption and mitochondrial proteins in an immortalized, nontumorigenic human bronchial epithelial cell line (NL20) compared to the tumorigenic counterpart (NL20-TA). These studies showed that the tumorigenic cell line expressed significantly higher levels of heme flux compared to the nontumorigenic cell line. This led us to alter heme availability by overexpressing the rate-limiting enzyme of heme synthesis as well as the rate-limiting enzyme of heme degradation. Experiments are under way to characterize their effects on tumor growth and metastasis. Citation Format: Chantal Vidal, Sagar Sohoni, Li Zhang. Heme function in promoting tumorigenic potential of lung cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 386.

Physiological stress-induced corticosterone increases heme uptake via KLF4-HCP1 signaling pathway in hippocampus neurons

Iron overload has attracted much attention because of its adverse effect in increasing the risk of developing several neurodegenerative disorders. Under various pathologic conditions, a lot of heme are released. The aggregation of heme is more neurotoxic than that of iron released from the heme breakdown. Our previous studies demonstrated that psychological stress (PS) is a risk factor of cerebral iron metabolism disorders, thus causing iron accumulation in rat brains. In the present study, we found PS could increase heme uptake via heme carrier protein 1 (HCP1) in rat brains. We demonstrated that Glucocorticoid (GC), which is largely secreted under stress, could up-regulate HCP1 expression, thus promoting heme uptake in neurons. We also ascertained that HCP1 expression can be induced by GC through a transcription factor, Krüppel-like factor 4 (KLF4). These results may gain new insights into the etiology of heme uptake and iron accumulation in PS rats, and find new therapeutic targets of iron accumulation in Parkinson’s disease or Alzheimer’s disease.

Cancer cell-specific mitochondrial reactive oxygen species promote non-heme iron uptake and enhance the proliferation of gastric epithelial cancer cell.

Iron is an essential nutrient for life and is involved in many important processes such as oxygen transport and DNA synthesis. However, excess amounts of iron can cause carcinogenesis by producing reactive oxygen species. Thus, the cellular transport of iron must be tightly regulated. In the human body, iron may be present as heme or non-heme iron. The mechanisms governing the cellular transport of these forms have not been clearly elucidated. We previously reported that the expression of an important heme transporter, heme carrier protein 1 was regulated by cancer-specific reactive oxygen species derived from mitochondria. In this study, we have asked if mitochondrial reactive oxygen species may also be related with non-heme iron transport. In order to address this question, we have investigated the relationship between mitochondrial reactive oxygen species and accumulation of cellular non-heme iron in a rat gastric normal, cancer and manganese superoxide dismutase-overexpressing cancer cell line, in which reactive oxygen species from mitochondria are specifically scavenged. We have also analyzed the expression of divalent metal transporter 1 and ferroprotin, involved in the incorporation and excretion of non-heme iron, respectively, as well as a hypoxia-related transcription factor HIF-1α, to elucidate the molecular mechanism of non-heme iron accumulation.

352 - Heme-Driven ROS Production Leads to Increased P38 MAP Kinase Activity and Endothelial Barrier Disruption

Heme is involved in a wide number of biological processes including gas exchange, antioxidant defense, signal transduction, as well as energy production within the mitochondria. It’s concentration within the cell is tightly regulated, and the consequences of underproduction of heme (anemia) are widely recognized. However, the pathophysiological role of high heme concentrations is less studied, but is already implicated in a number of cardiopulmonary diseases. An overabundance of intracellular heme precedes an over production of mitochondrial reactive oxygen species (ROS). ROS imbalance in the cell overwhelms basal antioxidant defenses and leads to various stress responses. Primarily, in this study, we focus on heme-driven effects of cellular signaling on human lung micro vascular endothelial cells (HLMVECs) permeability. We show that the p38 mitogen activated protein kinase (MAP kinase) pathway is activated with a specific response over a time course of heme treatment and that heme driven ROS production and cellular responses leads to endothelial barrier disruptions. In addition, we focus on a few therapeutic targets of heme pathway with potential to alleviate the harmful and cascading effects of ROS: 1) Inhibition of Heme Carrier Protein 1 (HCP-1) that mediates free heme transition into the cell; 2) Inhibition of heme mediated mitochondrial ROS production to study the downstream effects of lowering mitochondrial ROS on HLMVEC permeability; 3) Inhibition of p38 MAP Kinase to study the downstream effects of lowering activity of this pathway counteracting the response of ROS activity. These potential strategies in combating the pathophysiology of high levels of free heme are preventive and solidify our understanding of intracellular heme activity. However, the specific mechanism by which heme/ROS activates stress signaling pathways is a topic for future study and of high therapeutic value.

Intestinal expression of genes implicated in iron absorption and their regulation by hepcidin

Through inhibition of iron absorption and iron mobilization from tissue stores, hepcidin exerts a negative control on iron homeostasis. Hepcidin, in fact, promotes the degradation of ferroportin (Fpn1), the iron exporter molecule expressed on the membrane of hepatocytes and macrophages, thus preventing iron release from cells to plasma. Hepcidin effects on enterocytes, however, are less clear. Aim of the present study was to further investigate the regulation of iron absorption by hepcidin. The transcriptional response of human duodenal mucosa to hepcidin was investigated using organ cultures of duodenal biopsies perendoscopically collected from healthy controls. Biopsies were cultured for 4h with or without hepcidin-25 and were then assayed for the expression of iron-related genes. In samples that had not been exposed to hepcidin, correlations were found between the expression of genes involved in iron absorption: DMT1, Fpn1, Dcytb and HCP1. In exvivo experiments hepcidin down-regulated mRNA levels of the iron transporters Fpn1, and DMT1, of the ferric reductase Dcytb, of the ferroxidase hephaestin, and of the putative heme carrier protein HCP1. Through the reported transcriptional changes hepcidin can modulate several steps of the iron absorption process, including the reduction of dietary iron by Dcytb, its uptake by enterocytes through DMT1, the mucosal uptake of heme iron by HCP1, and enterocyte iron release to plasma by Fpn1 in conjunction with hephaestin.

Development of hematin conjugated PLGA nanoparticle for selective cancer targeting

Targeted nanomedicine for cancer therapy has gained widespread popularity and is being extensively explored. Porphyrins have intrinsic tumor localizing ability and have been studied for photodynamic therapy. However, they have not been used as cancer targeting agents for nanomedicines. In this study, PLGA nanoparticles were formulated and an iron-containing blood porphyrin, hematin was conjugated to the surface of the nanoparticles to investigate selectivity towards cancer cell and cellular internalization. Hematin was previously shown to facilitate growth and proliferation of cancer cells. PLGA nanoparticles were characterized by FE-SEM, AFM, DLS, and Zeta potential analyzer. The conjugation of hematin was confirmed by FTIR. HeLa cells were used to study tumor selectivity and uptake. Hematin conjugated particles (ζ potential: −15.19mV) showed higher affinity towards the cancer cells than the control particles. The result indicated that the particles were internalized by heme carrier protein-1. Together these data suggest that hematin is a promising cancer targeting material for nanotherapeutics.

Quantum dots-hemin: Preparation and application in the absorption of heme iron

The absorption mechanism of heme iron remains unclear due to the limit of labeling techniques. Quantum dots (QDs) are powerful fluorescent probes resistant to photobleaching, however, there is no data about the application of QDs in heme iron absorption. Herein, we prepared hemin-coated CdSe/ZnS (QDs-hemin), and studied their absorption in vitro and in vivo. Results showed that QDs-hemin had uniform particle sizes, physiological stability and high joint efficiency. Moreover, QDs-hemin could be successfully absorbed gradually into the duodenum with the time using synchrotron radiation micro X-ray fluorescence and confocal laser scanning microscopy. Furthermore, QDs-hemin were observed to degrade in lysosomes, and their absorption was blocked by Heme Carrier Protein 1 (HCP1) antibody and HCP1 siRNA. All the results demonstrate that QDs can be a good tracer for heme iron and that HCP1 pathway is critical and predominant over the endocytosis pathway in the absorption mechanism.

Enhancement of photodynamic therapy effects with mitochondrial reactive oxygen species

Enhancement of photodynamic therapy effects with mitochondrial reactive oxygen species

Synthesis and characterization of novel symmetrical and asymmetrical carboxyphthalocyanines with potential application in photodynamic therapy

Synthesis and characterization of novel symmetrical and asymmetrical carboxyphthalocyanines with potential application in photodynamic therapy

Duodenal expression of heme and non‐heme iron transporters and tissue partitioning of dietary heme and non‐heme iron in a rat mode of iron overload (246.8)

Dietary heme is an important Fe source yet the regulation of heme absorption and tissue partitioning of absorbed heme remain undefined. In a rat model of Fe overload, we aimed to 1) examine duodenal heme and non‐heme Fe transporters in relation to hepcidin, and 2) to compare utilization of absorbed heme and non‐heme Fe by erythroid (RBCs) and Fe storage tissues (liver and spleen). Twelve male Sprague Dawley rats were randomized to receive injections of PBS (control) or Fe dextran providing a total of 16 mg (Moderate Fe) or 48 mg Fe (High Fe) over 2 weeks. After Fe loading, rats were gavaged with 57Fe‐FeSO4 and 58Fe‐porcine RBC. Duodenal Fe transporter expression and tissue isotopic enrichment were assessed 10‐days post‐dosing. Fe injection in the High Fe group suppressed duodenal transcript expression of divalent metal transporter 1 (Dmt1) and duodenal cytochrome b (Dcytb) by 70% but did not impact heme carrier protein 1 (Hcp1) or Ferroportin. Liver hepcidin expression correlated with Dmt1, Dcytb and Ferroportin but not with Hcp1. Tissue isotopic analysis found greater non‐heme than heme Fe incorporation into RBC (80.5% vs. 71.3%, p = 0.02) while this relationship was reversed in the spleen (2.7% vs. 7.5%, p = 0.01). Fe status and hepcidin had no impact on duodenal Hcp1 expression. Differential tissue utilization of absorbed heme vs. non‐heme Fe suggests that heme Fe is exported in an alternate form from non‐heme Fe.Grant Funding Source: Supported by USDA 2008‐0857

Mitochondrial reactive oxygen species accelerate the expression of heme carrier protein 1 and enhance photodynamic cancer therapy effect.

Photodynamic therapy using hematoporphyrin and its derivatives is clinically useful for cancer treatments. It has been reported that cancer cells incorporate hematoporphyrin and its derivatives via heme carrier protein 1, which is a proton-coupled folate transporter. However, the mechanism of this protein expression has not been elucidated. In general, the concentration of reactive oxygen species in cancer cells is higher than that in normal cells. We previously reported that reactive oxygen species from mitochondria involved in the expression of peptide transporter 1 and accelerate the uptake of 5-aminolevulinic acid, which is a precursor of protoporphyrin IX. We suggested mitochondrial reactive oxygen species also regulated the expression of heme carrier protein 1. In this study, we used a rat gastric mucosal cell line RGM1 and its cancer-like mutated cell line RGK1. We clarified the expression of heme carrier protein 1 increased in cancer cells and it decreased in manganese superoxide dismutase expressed cancer cells. In addition, the uptake level of hematoporphyrin and photodynamic therapeutic effect were also decreased in manganese superoxide dismutase expressed cancer cells in comparison with cancer cells. Thus, we concluded that mitochondrial reactive oxygen species regulated heme carrier protein 1 expression and photodynamic therapeutic effect.

Identification and expression of iron regulators in human synovium: evidence for upregulation in haemophilic arthropathy compared to rheumatoid arthritis, osteoarthritis, and healthy controls

Recurrent joint bleeding is the most common manifestation of severe haemophilia resulting in haemophilic arthropathy (HA). Iron plays a central role in the pathogenesis of the two main features of HA: synovitis and cartilage destruction. The aim of this study was to investigate the synovial presence of the iron regulator proteins ferroportin (FPN), hepcidin, haemoglobin scavenger receptor CD163 (CD163), feline leukaemia virus subgroup C (FLVCR), and heme carrier protein 1 (HCP-1). A comparison of the expression in HA with rheumatoid arthritis (RA), osteoarthritis (OA), and healthy controls (HC) is made. Synovial expression of iron regulators was investigated by immunohistochemistry in human synovial tissue and in a murine haemophilia model. We demonstrate for the first time the synovial presence of the investigated iron regulator proteins. Expression of the iron regulator proteins FPN, CD163, FLVCR, and HCP-1 was enhanced in HA in comparison to RA, OA, and HC synovium. In addition, in a murine haemophilia model of acute joint bleeding, synovial expression of FPN, CD163, and HCP-1 was increased. In both human and murine experiment, synovial expression of hepcidin was not altered. These findings indicate the presence of iron regulator proteins in the synovium, demonstrate an enhanced expression of FPN, CD163, FLVCR, and HCP-1 in HA, and suggest a synovial adaptation mechanism to maintain synovial iron homeostasis in HA.