The roles of free iron, heme, haemoglobin, and the scavenger proteins haemopexin and alpha-1-microglobulin in preeclampsia and fetal growth restriction.

BACKGROUND: Preeclampsia (PE) is a complex pregnancy syndrome characterised by maternal hypertension and organ damage after 20 weeks of gestation and is associated with an increased risk of cardiovascular disease later in life. Extracellular haemoglobin (Hb) and its metabolites heme and iron are highly toxic molecules and several defence mechanisms have evolved to protect the tissue. OBJECTIVES: We will discuss the roles of free iron, heme, Hb, and the scavenger proteins haemopexin and alpha-1-microglobulin in pregnancies complicated by PE and fetal growth restriction (FGR). CONCLUSION: In PE, oxidative stress causes syncytiotrophoblast (STB) stress and increased shedding of placental STB-derived extracellular vesicles (STBEV). The level in maternal circulation correlates with the severity of hypertension and supports the involvement of STBEVs in causing maternal symptoms in PE. In PE and FGR, iron homeostasis is changed, and iron levels significantly correlate with the severity of the disease. The normal increase in plasma volume taking place during pregnancy is less for PE and FGR and therefore have a different impact on, for example, iron concentration, compared to normal pregnancy. Excess iron promotes ferroptosis is suggested to play a role in trophoblast stress and lipotoxicity. Non-erythroid α-globin regulates vasodilation through the endothelial nitric oxide synthase pathway, and hypoxia-induced α-globin expression in STBs in PE placentas is suggested to contribute to hypertension in PE. Underlying placental pathology in PE with and without FGR might be amplified by iron and heme overload causing oxidative stress and ferroptosis. As the placenta becomes stressed, the release of STBEVs increases and affects the maternal vasculature.

Role of hemoglobin/heme scavenger protein hemopexin in atherosclerosis and inflammatory diseases.

PURPOSE OF REVIEW: Hemoglobin and its scavenger proteins haptoglobin and hemopexin (Hx) associate with HDL and influence the inflammatory properties of HDL. Moreover, HDL from Hx-null mice is proinflammatory. In addition, Hx deficiency is implicated in a number of other inflammatory diseases such as septic shock and experimental autoimmune encephalomyelitis. This article highlights studies that demonstrate novel insights into the physiological protective role of Hx in inflammatory diseases. RECENT FINDINGS: Recent studies demonstrate that Hx-dependent uptake of extracellular heme leads to the deactivation of Bach1 repression leading to the transcriptional activation of antioxidant heme oxygenase-1 gene. Levels of circulating Hx have been implicated in the prognosis for patients with septic shock. In addition, Hx therapy has been shown to be beneficial in cardiovascular disease, cerebral ischemic injury, and experimental autoimmune encephalomyelitis. SUMMARY: These studies suggest that heme scavenging is a major mechanism by which Hx defends against oxidative stress and related inflammatory disorders. Hx therapy may provide a novel protective role against heme and oxidative stress-mediated inflammatory conditions including atherosclerosis.

A structurally novel hemopexin fold protein of rice plays role in chlorophyll degradation.

Proteins containing hemopexin fold domain are suggested to have diverse functions in various living organisms. In order to investigate the structure and function of this type of protein in rice plant (Oryza sativa), the gene encoding a hemopexin fold protein (OsHFP) was cloned, analyzed in silico and characterized. Molecular modeling revealed that the OsHFP is closely related to other hemopexin fold proteins, but is unique with a cylindrical central tunnel as well as extended N- and C-terminal domains. The recombinant OsHFP was found to bind hemin, the oxidized form of heme in vitro. The expression of the single copy OsHFP gene was detected in rice flower buds. Heterologous expression of OsHFP in green leaf tissues resulted in chlorophyll degradation; however, stable expression of OsHFP was observed in transgenic hairy roots, a non-green tissue. The possible role of OsHFP in regulating programmed cell death in anther green tissues of rice is proposed.

Hemopexin domains as multifunctional liganding modules in matrix metalloproteinases and other proteins.

The heme-binding hemopexin consists of two, four-bladed propeller domains connected by a linker region. Hemopexin domains are found in different species on the phylogenetic tree and in the human species represented in hemopexin, matrix metalloproteinases (MMPs), vitronectin, and products of the proteoglycan 4 gene. Hemopexin and hemopexin domains of human proteins fulfill functions in activation of MMPs, inhibition of MMPs, dimerization, binding of substrates or ligands, cleavage of substrates, and endocytosis by low-density lipoprotein receptor-related protein-1 (LRP-1; CD91) and LRP-2 (megalin, GP330). Insights into the structures and functions of hemopexin (domains) form the basis for positive or negative interference with the formation of molecular complexes and hence, might be exploited therapeutically in inflammation, cancer, and wound healing.

Depletion of serum hemopexin in fulminant rhabdomyolysis. Evidence for an interaction of hemopexin with myoglobin-derived heme.

Hemopexin is a normal serum glycoprotein that functions as a carrier for intravascularly liberated free heme. Although its role is well established in the reutilization of hemoglobin-derived heme, there have been no previous clinical data to support its suspected interaction with heme released in the degradation of myoglobin. In a patient with fulminant rhabdomyolysis, we found depletion of serum hemopexin in the presence of high serum levels of myoglobin with normal levels of haptoglobin and hemoglobin. This combination of laboratory findings is evidence for an interaction between myoglobin-derived heme and hemopexin and implies a role for hemopexin in the catabolism of myoglobin. These findings support the proposed induction mechanism for observed increases of serum hemopexin in Duchenne muscular dystrophy and in polymyositis.

Hemopexin: a review of biological aspects and the role in laboratory medicine.

BACKGROUND: Hemopexin is a heme-binding plasma glycoprotein which, after haptoglobin, forms the second line of defense against hemoglobin-mediated oxidative damage during intravascular hemolysis. A decrease in plasma hemopexin concentration reflects a recent release of heme compounds in the extracellular compartment. Heme-hemopexin complexes are delivered to hepatocytes by receptor-mediated endocytosis after which hemopexin is recycled to the circulation. METHODS OF ANALYSIS: Immunonephelometric and -turbidimetric hemopexin assays are available as more precise and rapid alternatives to the radial immunodiffusion technique. INTERPRETATIONS: Hemopexin determinations are not subject to interference by in vitro hemolysis. Altered serum or plasma concentrations of hemopexin are found not only in hemolytic anemias but also in other conditions such as chronic neuromuscular diseases and acute intermittent porphyria. In laboratory medicine, while hemopexin determination in tandem with haptoglobin has potential applications in the assessment of intravascular hemolysis and allows for the monitoring of the severity of hemolysis after depletion of haptoglobin, its diagnostic utility is less clear in other pathological conditions. Further studies are necessary to fully establish the clinical significance of hemopexin determination.

Regulation of plasma hemopexin activity by stimulated endothelial or mesangial cells.

The pathogenesis of glomerular alterations and proteinuria in corticosteroid-responsive nephrotic syndrome (CRNS) is unknown. As an isoform of plasma hemopexin (Hx) with protease activity may be implicated in this disease, we have studied the inhibition of Hx by ADP and reactivation to its active form by endothelial or mesangial cells in vitro. We hypothesized that these cells might potentially be able to convert the inactivated form of Hx (Hxi) to active Hx (Hxa) in vitro, mediated by cellular ecto-ADPase. Since ecto-ADPase (CD39) is upregulated after stimulation of these cells with lipopolysaccharide (LPS) or certain cytokines, we postulated that this conversion might occur specifically after inflammatory stimulation of these cells. Human endothelial or mesangial cell cultures were incubated overnight with or without LPS (10.0 ng/ml) or TNFalpha (10.0 ng/ml), washed and subsequently incubated with Hxi (1.5 mg/ml) in serum-free conditions (Hxi was prepared by treatment of Hxa with ADP or ADP-beta-S). After 60 min, supernatants were tested for their capacity to alter glomerular extracellular matrix molecules (i.e. ecto-apyrase) in vitro using standard methods, and compared with Hxi that had not been incubated with cells. Supernatants containing Hxa (1.5 mg/ml) served as positive control. The results show significant activity in supernatants with Hxi (prepared using native ADP). However, Hxi inactivated by ADP-beta-S (which is non-hydrolyzable) could not be reactivated after contact with LPS-stimulated or unstimulated cells in vitro. As ecto-ADPase of these cells is upregulated by LPS, we believe that reactivation of Hxi to Hxa is mediated by cellular ecto-ADPase. Although the relevance of this inflammation-mediated activation mechanism of Hx in patients with CRNS requires further experimentation, our preliminary observations suggesting that this mechanism is corticosteroid dependent may support a potential role of Hxa in CRNS.

[Serum hemopexin: suppressive effect on neutrophil functions and prospect of clinical application to autoimmune diseases].

Hemopexin is a serum glycoprotein with a high binding affinity for heme, and is known as a scavenger/transporter of heme. Recent studies indicated that purified hemopexin suppresses neutrophil adhesion and phagocytosis by a mechanism unrelated to heme-binding, suggesting a novel activity of hemopexin. Unidentified serum factor in combination with Ca2+ dampens the effect of hemopexin. So, hemopexin in peripheral blood may not act as an inhibitor of neutrophil function. However, because hemopexin is synthesized in injured peripheral nerves, it may be hypothesized that hemopexin has an anti-inflammatory role in nerve repair by suppressing phagocyte accumulation/phagocytosis. Further studies of hemopexin may provide new therapeutic strategies aimed at suppressing neutrophil functions to control inflammation and tissue injury, especially in autoimmune diseases such as rheumatoid arthritis.

Hemopexin activity is associated with angiotensin II responsiveness in humans.

BACKGROUND: Hemopexin, an acute phase protein, can downregulate the angiotensin (ang) II type 1 receptor (AT1-R) in vitro. Whether hemopexin is involved in the responsiveness to ang II in vivo is unknown. Therefore, we tested whether variations in endogenous hemopexin activity are associated with the responsiveness of blood pressure to ang II in healthy volunteers. METHOD: Healthy men (n = 33, age 26 ±â€Š9) were studied in balance on low sodium (50 mmol Na per 24 h) and high sodium (200 mmol Na per 24 h) diet, respectively. After baseline measurements of blood pressure, ang II was infused at 0.3, 1 and 3 ng/kg per min for 1 h per dose. Hemopexin activity was measured at baseline in EDTA-plasma samples by an amidolytic assay with a chromogenic substrate suitable for hemopexin activity evaluation. RESULTS: During high sodium the hemopexin activity was lower; 1.6 × 10 (0.6 × 10 - 4.7 × 10) versus 2.8 × 10 (1.1 × 10 - 5.1 × 10) arbitrary units (P < 0.01) and the pressor response to 3 ng ang II/kg per minute larger than during low sodium (17.6 ±â€Š6.5 versus 14.6 ±â€Š6.9 mmHg, P < 0.01). Hemopexin activity negatively correlated with the pressor response to ang II during either type of sodium intake (high sodium: r = 0.42, P < 0.05; low sodium: r = 0.35, P < 0.05). CONCLUSION: These in-vivo data obtained in healthy individuals support recent in-vitro data showing that active hemopexin downregulates the availability of the AT1-R. Therefore, activated hemopexin might be considered as a factor mediating ang II effects upon blood pressure by modulating AT1-R availability.

Plasma hemopexin as a potential regulator of vascular responsiveness to angiotensin II.

This brief review focuses on the functional activities of plasma hemopexin recently recognized by several authors. In particular, the protease-like activity of hemopexin in vitro is linked with downregulation of the vascular angiotensin II receptor in vivo, leading to vascular expansion. Also a potential mechanism of inhibition of hemopexin activity by extracellular adenosine triphosphate is considered.

Hemopexin decreases hemin accumulation and catabolism by neural cells.

Hemopexin is a serum, CSF, and neuronal protein that is protective after experimental stroke. Its efficacy in the latter has been linked to increased expression and activity of heme oxygenase (HO)-1, suggesting that it facilitates heme degradation and subsequent release of cytoprotective biliverdin and carbon monoxide. In this study, the effect of hemopexin on the rate of hemin breakdown by CNS cells was investigated in established in vitro models. Equimolar hemopexin decreased hemin breakdown, as assessed by gas chromatography, by 60-75% in primary cultures of murine neurons and glia. Extracellular hemopexin reduced cell accumulation of 55Fe-hemin by over 90%, while increasing hemin export or extraction from membranes by fourfold. This was associated with significant reduction in HO-1 expression and neuroprotection. In a cell-free system, hemin breakdown by recombinant HO-1 was reduced over 80% by hemopexin; in contrast, albumin and two other heme-binding proteins had no effect. Although hemopexin was detected on immunoblots of cortical lysates from adult mice, hemopexin knockout per se did not alter HO activity in cortical cells treated with hemin. These results demonstrate that hemopexin decreases the accumulation and catabolism of exogenous hemin by neural cells. Its beneficial effect in stroke models is unlikely to be mediated by increased production of cytoprotective heme breakdown products.

Increased striatal injury and behavioral deficits after intracerebral hemorrhage in hemopexin knockout mice.

OBJECT: Heme toxicity may contribute to the pathogenesis of intracerebral hemorrhage (ICH). The primary defense against extracellular heme is provided by hemopexin, a serum and neuronal glycoprotein that binds it with very high affinity and mitigates its prooxidant effect. In the present study, the authors tested the hypothesis that hemopexin knockout mice would sustain more injury after experimental ICH than their wild-type counterparts. METHODS: Striatal ICH was induced by the stereotactic injection of bacterial collagenase or autologous blood. Three days later, striatal protein oxidation was assessed via carbonyl assay. Cell viability was quantified at 8-9 days by using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Behavioral deficits were detected with high-resolution digital analysis of 6-hour home cage video recordings and standard testing. RESULTS: Perihematomal protein oxidation was increased in wild-type collagenase-injected striata by approximately 2.1-fold, as compared with contralateral striata; protein carbonyls were increased 3-fold in knockout mice. Striatal cell viability was reduced by collagenase injection in wild-type mice to 52.9 ± 6.5% of that in the contralateral striata, and to 31.1 ± 3.7% of that in the contralateral striata in knockout mice; similar results were obtained after blood injection. Digital analysis of 6-hour video recordings demonstrated an activity deficit in both models that was significantly exacerbated at 8 days in knockout mice. Striatal heme content 9 days after blood injection was increased approximately 2.7-fold in knockouts as compared with wild-type mice. CONCLUSIONS: These results suggest that hemopexin has a protective effect against hemorrhagic CNS injuries. Hemopexin deficiency, which is often associated with sickle cell disease, may worsen outcome after ICH.

Heme scavenging and the other facets of hemopexin.

Hemopexin is an acute-phase plasma glycoprotein, produced mainly by the liver and released into plasma, where it binds heme with high affinity. Other sites of hemopexin synthesis are the nervous system, skeletal muscle, retina, and kidney. The only known receptor for the heme-hemopexin complex is the scavenger receptor, LDL receptor-related protein (LRP)1, which is expressed in most cell types, thus indicating multiple sites of heme-hemopexin complex recovery. The better-characterized function of hemopexin is heme scavenging at the systemic level, consisting of the transport of heme to the liver, where it is catabolyzed or used for the synthesis of hemoproteins or exported to bile canaliculi. This is important both in physiologic heme management for heme-iron recycling and in pathologic conditions associated with intravascular hemolysis to prevent the prooxidant and proinflammatory effects of heme. Other than scavenging heme, the heme-hemopexin complex has been shown to be able to activate signaling pathways, thus promoting cell survival, and to modulate gene expression. In this review, the importance of heme scavenging by hemopexin, as well as the other emerging functions of this protein, are discussed.

Heme-hemopexin complex attenuates neuronal cell death and stroke damage.

Hemoproteins undergo degradation during hypoxic/ischemic conditions, but the pro-oxidant free heme that is released cannot be recycled and must be degraded. The extracellular heme associates with its high-affinity binding protein, hemopexin (HPX). Hemopexin is shown here to be expressed by cortical neurons and it is present in mouse cerebellum, cortex, hippocampus, and striatum. Using the transient ischemia model (90-min middle cerebral artery occlusion followed by 96-h survival), we provide evidence that HPX is protective in the brain, as neurologic deficits and infarct volumes were significantly greater in HPX(-/-) than in wild-type mice. Addressing the potential protective HPX cellular pathway, we observed that exogenous free heme decreased cell survival in primary mouse cortical neuron cultures, whereas the heme bound to HPX was not toxic. Heme-HPX complexes induce HO1 and, consequently, protect primary neurons against the toxicity of both heme and pro-oxidant tert-butyl hydroperoxide; such protection was decreased in HO1(-/-) neuronal cultures. Taken together, these data show that HPX protects against heme-induced toxicity and oxidative stress and that HO1 is required. We propose that the heme-HPX system protects against stroke-related damage by maintaining a tight balance between free and bound heme. Thus, regulating extracellular free heme levels, such as with HPX, could be neuroprotective.