Adenoviral transfer of hemopexin gene attenuates oxidative stress and apoptosis in cultured primary cortical neuron cell exposed to blood clot.

BACKGROUND: A growing body of experimental evidence suggests that hemin released from heme is a potent oxidant and accumulates in intracranial hematomas. Hemopexin (Hpx) decreases hemin accumulation and catabolism by nerve cells. In previous study, we observed that Hpx gene knockout aggravated striatal injury and worsened behavioral deficits of mice subjected to intracerebral hemorrhage. AIM: To examine the effect of Hpx on oxidative damage and apoptosis in cultured nerve cells with blood clot. METHODS: Neuron and glial cells were transfected with adenoviral Hpx gene. Transfected primary neuron-glial cells were co-cultured with 50 µl of arterial blood clot using insert transwells. The sham group was co-coulture with 50 µl of DMEM/F12, which contained 28 µl of serum; the control group was transfected with adenoviral vector. At 12 and 24 h, the level of malonaldehyde (MDA), surperoxide dismutase (SOD) concentration, glutathione (GSH), apoptosis, expression of HO-1 and caspase-3 were detected. RESULTS: MDA level was decreased (P < 0.01) whereas SOD and GSH concentration were increased in the Hpx group (P < 0.05 and P < 0.01, respectively). Results of flow cytometry revealed no significant difference in apoptosis between the Hpx group and model group at 12 h. However, the percentage of cells undergoing apoptosis in the Hpx group was decreased at 24 h compared with the model group (P < 0.01). HO-1 expression decreased in the Hpx group at 24 h (P < 0.01) while caspase-3 expression decreased at both 12 and 24 h (P < 0.011 and P < 0.05, respectively) compared with the model group. CONCLUSION: Hpx protected nerve cells exposed to blood from injury by anti-oxidation and a decrease in the expression of HO-1 and caspase-3.

Temporal and age-dependent effects of haptoglobin deletion on intracerebral hemorrhage-induced brain damage and neurobehavioral outcomes.

Intracerebral hemorrhage (ICH) is a devastating stroke subtype and the presence of extracorpuscular hemoglobin (Hb) exacerbates brain damage. Haptoglobin (Hp) binds Hb, which prevents its oxidation and participation in neurotoxic reactions. Multiple studies have investigated the role of Hp under conditions of intravascular hemolysis, but little is known about its role in the brain and following ICH where extravascular hemolysis is rampant. Young and aged wildtype and Hp-/- mice underwent the autologous blood or collagenase ICH model. Early after ICH, Hp-/- mice display 58.0 ±â€¯5.6% and 36.7 ±â€¯6.9% less brain damage in the autologous blood and collagenase ICH models, respectively. In line with these findings, Hp-/- mice display less neurological deficits on several neurobehavioral tests. Hp-/- mice have less Perl's iron content, HO1 expression, and blood brain barrier dysfunction, but no difference in brain Hb content, astrogliosis and angiogenesis/neovascularization. At the later endpoint, the young cohort displays 27.8 ±â€¯9.3% less brain damage, while no difference is seen with the aged cohort. For both cohorts, no differences are seen in HO1 levels or iron accumulation, but young Hp-/- mice display less thalamic astrogliosis and striatal microgliosis. This study reveals that the presence or absence of Hp exerts important time- and age-dependent influences on ICH outcomes.

Expression of hemopexin in acute rejection of rat liver allograft identified by serum proteomic analysis.

Acute rejection (AR) and acceptance of allograft after liver transplantation (LTx) remain critical issues that need addressing to improve prognosis. We therefore performed rat orthotopic LTx and proteomic analyses to screen for immune response-related biomarkers in sera. Markers identified were validated at the mRNA and/or protein levels, and the molecules of interest were functionally explored. Compared with syngeneic controls, signs of AR as well as spontaneous acceptance were observed in hematoxylin and eosin-stained sections of liver allografts. In accordance with the severity of AR, 30 protein spots displaying significant changes in abundance were identified using two-dimensional differential gel electrophoresis. Ultimately, 14 serum proteins were sequenced and five spots of interest were identified as hemopexin (HPX). Expression of HPX was significantly and inversely associated with the severity of AR at both the mRNA and protein levels. In vitro, Mt-1, Ho-1, Fth, Ifn-γ, and Il-17 transcripts were significantly upregulated in lysates of lymphocytes stimulated with HPX, whereas Il-10 markedly was remarkably downregulated. Interferon-γ, IL-10, and IL-17 proteins in the supernatant of HPX-stimulated lymphocytes were significantly altered in keeping with the mRNA level. Our data facilitated the generation of a proteomic profile to enhance the understanding of rat liver AR. In view of finding that the HPX serum level is negatively associated with the severity of AR of rat liver allograft, we propose that in vitro treatment with HPX regulates cytokine expression in rat lymphocytes.

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.

Regulation of hemopexin synthesis in degenerating and regenerating rat sciatic nerve.

In injured peripheral nerves, hemopexin mRNA is expressed by fibroblasts, Schwann cells, and invading blood macrophages, and the protein accumulates in the extracellular matrix. This and its absence of regulation in injured central optic nerve suggest that hemopexin could play a positive role in peripheral nerve repair. Here, we studied the regulation of hemopexin expression in degenerating and regenerating nerves. After a sciatic nerve injury, both the synthesis of hemopexin and the level of its mRNA increase sharply during the first 2 days, leading to an accumulation of hemopexin in the nerve. Afterward, hemopexin expression decreases progressively in regenerating nerves. In permanently degenerated nerves, it is again transiently increased and then strongly decreased, whereas hemopexin from blood origin is accumulating. As part of the elucidation of the complex regulation of hemopexin expression in injured nerves, we demonstrate that interleukin-6 increases hemopexin synthesis in intact nerves, whereas adult rat serum, but not purified hemopexin, inhibits it in degenerated nerves. Hemopexin, known as acute-phase protein, is therefore one of the molecules rapidly and specifically up-regulated in injured peripheral nerves. More generally, our findings suggest that the acute phase could be not only a systemic liver-specific response but also a reaction of injured tissues themselves.

Changes in expression and localization of hemopexin and its transcripts in injured nervous system: a comparison of central and peripheral tissues.

The recent demonstration of hemopexin synthesis in the adult rat sciatic nerve and its accumulation after injury has raised the question of the possible role of this acute phase protein during the process of nerve repair. To gain insight into its function, we have compared the distribution of both hemopexin and its messenger RNA in the peripheral and the central nervous systems. We find that hemopexin is present in all types of peripheral nerves and ganglia, confined to the extracellular matrix and basement membranes of the endoneurium, blood vessels and connective tissues. After injury, hemopexin messenger RNA is overexpressed by Schwann cells, fibroblasts and invading macrophages. The content in hemopexin protein increases in all nerves studied, without changes in localization. Therefore, hemopexin does not appear to be associated with the fate of myelin or with the regeneration of a particular type of nerve fibre. In the central nervous system, hemopexin messenger RNA cannot be detected and the protein is only found in basement membranes of the vascular system (capillaries, meninges and choroid plexus). Furthermore, hemopexin and its messenger RNA remain absent from the distal part of the injured optic nerves. Our results further support the idea that hemopexin plays specific roles during nerve repair, and that it may be associated with the endoneurial extracellular matrix.

The type II hemopexin interleukin-6 response element predominates the transcriptional regulation of the hemopexin acute phase responsiveness.

Hemopexin (Hx) is induced during the acute phase response (APR) by the cytokine interleukin (IL)-6. A type II IL-6 response element (RE) of the Hx gene has been characterized recently (J. Biol. Chem. (1994); 269, 12654-12661). To assess Hx gene regulation by other agents, various cytokines and growth factors were tested for their ability to induce Hx in rat hepatoma H-35 cells. IL-6-type cytokines, IL-1 beta and TNF-alpha, in contrast to transforming growth factor-beta (TGF-beta), hepatocyte growth factor and insulin significantly increased Hx gene expression. Chloramphenicol acetyltransferase (CAT) activity in H-35 cells transfected with constructs that contained the 5'-flanking Hx promoter region or multiple copies of the Hx IL-6-RE fused to the CAT gene was upregulated only by IL-6-type cytokines, although to varying degrees. These data indicate that signal transduction pathways mediated by IL-6-type cytokines but not those by IL-1 beta and TNF-alpha converge on the common Hx IL-6-RE.

Long-lasting accumulation of hemopexin in permanently transected peripheral nerves and its down-regulation during regeneration.

We have previously demonstrated that hemopexin is present in the intact sciatic nerve and is overproduced in the distal stump after nerve transection (Swerts et al.: J Biol Chem 267:10596-10600, 1992). To get further insight into the function of this hemoprotein in nervous tissue, we have documented long-term changes in hemopexin levels in permanently degenerated (transected) and regenerating (crush-lesioned) sciatic nerves of adult rats, using immunochemical techniques. As early as a couple of days after nerve transection, the amount of hemopexin was raised in the distal stump and at the end of the proximal stump. Similarly, after a crush lesion hemopexin was rapidly increased at the injury site and in the distal part of the nerve. Subsequently, in transected nerves the level of hemopexin rose steadily and remained elevated, representing, three months after injury, over 20 times the amount found in intact contralateral nerves. In contrast, in crush-lesioned nerves, hemopexin level declined progressively in a proximodistal direction and returned to basal values 2 months after injury, together with axonal regeneration. This long-term increase in hemopexin in permanently degenerated nerves and its progressive return to normal levels during nerve regeneration suggests that hemopexin content could be regulated negatively, directly or indirectly, by growing axons. In turn, these results support the idea that hemopexin could be involved in the process of Wallerian degeneration and/or in nerve repair.

Identification of a liver preference enhancer element of the rat hemopexin gene and its interaction with nuclear factors.

Transcription of hemopexin (Hx) occurs predominantly in the liver. To investigate the contribution of the cis-acting enhancer element to the hepatocyte preferential expression, we performed chloramphenicol acetyl-transferase (CAT) assays in HepG2 cells. A strong enhancer element was identified by successive truncation to reside in the region -157/-104 from the cap site. The Hx region -145/-125 interacts with rat liver nuclear proteins, as shown by standard DNA-protein binding assays. The nucleotide sequence -141AGACTTTGACCT-130 includes, in reverse orientation, a direct repeat of the imperfect AGGTCA sequence, one of the recognition motifs of the steroid-thyroid hormone receptor superfamily. That this AGGTCA repeat is an enhancer core of the Hx element was affirmed by mutational analyses. In electrophoretic mobility shift assays, oligonucleotides, corresponding to binding regions of chicken ovalbumin upstream promoter transcription factor (COUP-TF) and apolipoprotein AI regulatory protein 1 (ARP-1) but not of hepatocyte nuclear factor-4 (HNF-4), competed with the Hx element for binding sites. Co-transfection analyses indicated that HNF-4 does not affect CAT expression whereas ARP-1 and COUP-TF repress it. Antibody supershift analyses suggested that HNF-4 and COUP-TF may not be major factors binding to the Hx element.

Hemopexin is synthesized in peripheral nerves but not in central nervous system and accumulates after axotomy.

In adult mammals, injured axons regrow over long distances in peripheral nerves but fail to do so in the central nervous system. Analysis of molecular components of tissue environments that allow axonal regrowth revealed a dramatic increase in the level of hemopexin, a heme-transporting protein, in long-term axotomized peripheral nerve. In contrast, hemopexin did not accumulate in lesioned optic nerve. Sciatic nerve and skeletal muscle, but not brain, were shown to be sites of synthesis of hemopexin. Thus, hemopexin expression, which can no longer be considered to be liver-specific, correlates with tissular permissivity for axonal regeneration.

Rat hemopexin. Molecular cloning, primary structural characterization, and analysis of gene expression.

A full-length hemopexin cDNA was isolated from a rat liver cDNA library and the derived amino acid sequence was obtained. Rat hemopexin shows a 76% amino acid homology with human hemopexin. The amino-terminal domain of rat hemopexin contains two histidine residues that are conserved in the human and rat sequences and are the most likely heme axial ligands. Analogous to human hemopexin, the rat hemopexin consists of 10 internal repeating peptide motifs characteristic of the pexin gene family. A complete conservation of cysteine residues is seen between the human and rat sequences suggesting an identical disulfide bridge structure in both proteins. Our analysis of the primary structure of rat hemopexin reveals characteristics typical for members of the pexin gene family and suggests a conserved evolutionary role for the C-terminal (non-heme-binding) domain of this protein. The full-length rat hemopexin cDNA was used to analyze changes in hemopexin gene expression during development and experimental inflammation. RNA blot analysis showed a single 2.0-kb hemopexin mRNA present in fetal liver at day 14. Hemopexin-specific mRNA was not detected in embryonic or fetal tissues at earlier stages of development and was confined to the liver throughout fetal, newborn, and adult life. The abundance of hemopexin mRNA was found to increase throughout gestation, with a sharp increase in the first postnatal weeks, reaching maximum levels in adult animals. Endotoxin-induced inflammation resulted in a 5-fold increase in hepatic hemopexin mRNA content within 48 h without associated changes in hemopexin transcript size. Adult animals exposed to hyperoxia (95% oxygen) showed a 3-fold increase in hepatic hemopexin mRNA content.(ABSTRACT TRUNCATED AT 250 WORDS)

Genetic control of the expression of two biological activities of an antitumor polysaccharide, lentinan.

In order to make clear whether the expression of biological activities and antitumor polysaccharides are under genetic control, the responses of mice to lentinan, a beta-1,6;1,3-glucan, in the induction of several acute phase proteins (APPs) and T-cell-mediated vascular dilation and hemorrhage (VDH) were investigated. Twenty inbred strains of mice were divided into two groups according to their phenotypes in the induction of APPs when they were administered lentinan i.p. at a dose of 10 mg/kg; sensitive strains showed a marked increase in levels of APPs and resistant strains showed as low a level of APPs as non-treated control mice. No sex-related differences and no relation with H-2 halotypes were found in the responses. Only low-level responses were observed in F1 hybrid mice obtained by crosses between a sensitive and a resistant strain, indicating that the low APP response to lentinan is dominant. The N2 progeny between the F1 and a high responder segregated into high and low responders at a ratio of almost 1:1. These results suggest that a single major gene on an autosome is responsible for the induction of APPs. The induction of VDH also depended on the strains of mice. However, the strain distribution pattern of the VDH phenotype was distinct from that of the APP phenotype, indicating that the VDH-controlling gene was different from the APP-controlling gene. Further analyses with F1 hybrid and backcross progeny mice suggested that the high VDH response was dominant, and that the phenotype was determined by a single major gene.

Expression of the haemopexin-transport system in cultured mouse hepatoma cells. Links between haemopexin and iron metabolism.

Minimal deviation hepatoma (Hepa) cells, from the mouse hepatoma B7756, synthesize and secrete haemopexin and express both the haemopexin receptor and the membrane haem-binding protein (MHBP) associated with the receptor, making this cell line the first available for detailed study of both haemopexin metabolism and hepatic transport. The 17.5 kDa MHBP was detected in Triton X-100 extracts of Hepa cells by immunoblotting with goat anti-rabbit MHBP. Scatchard-type analysis of haem-125I-haemopexin binding at 4 degrees C revealed 35,000 receptors per cell of high affinity (Kd 17 nM). Haemopexin-mediated haem transport at 37 degrees C is saturable, having an apparent Km of 160 nM and a Vmax. of 7.5 pmol of haem/10(6) cells per h during exponential growth. Haem-transport capacity is highest in the period just before the cells enter their exponential phase of growth and slowest in stationary phase. Interestingly, haem-haemopexin serves as effectively as iron-transferrin as the sole source of iron for cell growth by Hepa cells. Furthermore, depriving Hepa cells of iron by treatment with desferrioxamine (DF) increases the number of cell-surface haemopexin receptors to 65,000 per cell and consequently increases haemopexin-mediated haem transport. The effects of DF do not appear to require protein synthesis since they are not prevented by cycloheximide. Treatment of Hepa cells with hydroxyurea, an inhibitor of the iron-requiring enzyme ribonucleotide reductase that is obligatory for DNA synthesis, enhanced haemopexin-mediated haem transport. Thus, these studies provide the first evidence for regulation of haem transport by the iron status of cells and suggest a linkage between haemopexin, iron homeostasis and cell growth.

Attachment and multiplication, morphology and protein production of human fetal primary liver cells cultured in hormonally defined media.

We established for human fetal liver cells (cultured for 2 wk) in a hormonally defined medium, optimal conditions for attachment, multiplication, and preservation of epithelial morphology as well as production and secretion of serum proteins characteristic of fetal (alpha l-fetoprotein, AFP) and adult (albumin and hemopexin) life. Conditions were considered optimal when cell number, albumin, and hemopexin levels were maintained throughout the 2-wk culture period. However, the decrease in AFP concentration, which occurred after a few days of culture, could not be reversed. The culture system developed is a suitable model for studying regulatory mechanisms governing structure and function during differentiation and may prove useful for testing the effect of toxic agents during fetal development of the human liver.

Synthesis of hemopexin and cysteine protease inhibitor is coordinately regulated by HSF-II and interferon-beta 2 in rat hepatoma cells.

Rat hepatoma (H-35) cells respond to hepatocyte-stimulating factors by increased expression of major acute phase plasma proteins. The synthesis of hemopexin is stimulated 10-fold by either hepatocyte-stimulating factor-II of human squamous carcinoma cells or hepatocyte-stimulating factor/interferon-beta 2 of activated human blood monocytes. The hormone specificity, time course and dose-dependence of hemopexin regulation is closely correlated with that of cysteine protease inhibitor. The coordinate expression of hemopexin and other type II acute phase proteins suggests the existence of common molecular regulatory mechanisms.

Differential transcriptional pattern of the hemopexin gene.

In this paper we have analyzed the expression of the human hemopexin gene in different human tissues and cell lines by using the specific cDNA probe previously isolated. The results show that this gene is expressed in liver and, in lower amount, in hepatoma cell lines but not in kidney, spleen, placental cells, and in HeLa, fibroblast cell lines. We suggest that there must be cell-specific control mechanisms responsible for the specificity of expression. We have also determined, by S1 mapping, that the transcription initiation site in hepatic cells is 28 base pairs upstream from the AUG initiation codon of the hemopexin gene.

Synthesis and secretion of hemopexin in primary cultures of rat hepatocytes. Demonstration of an intracellular precursor of hemopexin.

Secretion of hemopexin (20% carbohydrate) and its dependence on glycosylation was studied in primary rat hepatocyte cultures in comparison to the secretion of transferrin (5% carbohydrate). In pulse-chase experiments with [35S]methionine half of the labeled hemopexin was secreted in 30 min. By contrast, it took approximately 50 min for secretion of half of the transferrin. Tunicamycin treatment of cultures significantly delayed the secretion of hemopexin but not that of transferrin. During the pulse period a prominent intracellular precursor of hemopexin, smaller than the mature protein, was evident. It is concluded that the extent of glycosylation of a secretory protein is not necessarily a determinant of the transit time required for intracellular processing and secretion. In the case of hemopexin the glycosylation apparently facilitates the secretion although it is not an absolute prerequisite for the exocytosis of this protein.

Long term production of acute-phase proteins by adult rat hepatocytes co-cultured with another liver cell type in serum-free medium.

Three acute-phase proteins, haptoglobin, alpha 2-macroglobulin and hemopexin, as well as albumin, have been measured daily in the hydrocortisone-supplemented serum-free medium of pure and mixed cultures of adult rat hepatocytes for 5 and 20 days respectively. Whereas plasma protein production rapidly declined in pure culture, it remained relatively stable when hepatocytes were co-cultured with rat liver epithelial cells. In the latter cultures, an early stimulation of albumin and alpha 2-macroglobulin secretion was observed. In addition, four other plasma proteins, fibrinogen, alpha 1-acute-phase protein, alpha 1-acid glycoprotein and alpha 1-antitrypsin were shown by immunodiffusion to still be produced by day 20 of co-culture. These results suggest that hepatocyte co-cultures represent a suitable model for studying the mechanism which controls synthesis of plasma proteins, including acute-phase proteins by liver cells.

Synthesis and regulation of acute phase plasma proteins in primary cultures of mouse hepatocytes.

Adult mouse hepatocytes respond in vivo to experimentally induced acute inflammation by an increased synthesis and secretion of alpha 1-acid glycoprotein, haptoglobin, hemopexin, and serum amyloid A. Concurrently, the production of albumin and apolipoprotein A-1 is reduced. To define possible mediators of this response and to study their action in tissue culture, we established primary cultures of hepatocytes. Various hormones and factors that have been proposed to regulate the hepatic acute phase reaction were tested for their ability to modulate the expression of plasma proteins in these cells. Acute phase plasma and conditioned medium from activated monocytes influenced the production of most acute phase plasma proteins, and the regulation appears to occur at the level of functional mRNA. Purified hormones produced a significant anabolic response in only a few cases: dexamethasone was found to be effective in maintaining differentiated expression of the cells; and glucagon produced a specific inhibition of haptoglobin synthesis. When cells were treated with a combination of conditioned monocyte medium and dexamethasone, secretion of proteins was markedly reduced. The carbohydrate moieties of all plasma glycoproteins were incompletely modified, apparently as a result of decreased intracellular transport of newly synthesized plasma proteins. Although primary hepatocytes were not phenotypically stable in tissue culture, the cells nevertheless retained a broad response spectrum to exogenous signals. We propose this as a useful system to study the production of plasma proteins and thereby pinpoint the nature and activity of effectors mediating the hepatic acute phase reaction.

Translational competition by mRNA species encoding albumin, ferritin, haemopexin and globin.

Messenger RNA from rat liver was translated in a micrococcal-nuclease-treated reticulocyte lysate supplemented with liver tRNA. Synthesis of the liver proteins haemopexin, ferritin and albumin was analyzed by quantitative immunoprecipitation. The relative translation yield of these proteins changed as a function of the amount of mRNA present during protein synthesis, revealing the existence of translational competition between individual species of mRNA from the liver. The results show that the mRNA species encoding haemopexin, ferritin and albumin possess distinctly different abilities to compete for one or more critical components in translation, with competitive strength increasing in this order. Although on a weight basis total liver mRNA is apparently as effective a template for protein synthesis as is globin mRNA, the latter displays a greater resistance to inhibition of its translation by KCl. In analogy with the translation properties of alpha-globin and beta-globin mRNA [Di Segni, G., Rosen, H. and Kaempfer, R. (1979) Biochemistry, 18, 2847-2854], this finding suggests that globin mRNA possesses greater competitive strength than does total liver mRNA. Increasing amounts of globin mRNA competitively inhibit the translation of albumin and ferritin mRNA present in total liver mRNA. The competition is relieved by the addition of eukaryotic initiation factor eIF-2. Translation of ferritin mRNA responds more vigorously to relief by eIF-2 than does translation of albumin mRNA, a finding consistent with the observation that albumin mRNA competes more effectively than ferritin mRNA in translation. The results support the assumption that albumin mRNA possesses a greater affinity for eIF-2 than does ferritin mRNA.