Abnormal Iron Deposition Research Articles

Abnormal iron accumulation is involved in the pathogenesis of the demyelinating dmy rat but not in the hypomyelinating mv rat

In the central nervous system (CNS), iron is enriched in oligodendrocytes, the myelin forming cells. The importance of iron in CNS myelination has been demonstrated by studies using rodent models, in which iron deficiency is associated with hypomyelination. Besides iron deficiency, an abnormal iron accumulation is thought to be involved in the pathogenesis of demyelination in multiple sclerosis, an autoimmune demyelinating disease in humans. To determine the importance of iron metabolism in myelin diseases, we investigated iron metabolism using two different myelin mutant dmy (demyelination model) and mv (hypomyelination model) rats. Our results demonstrated an abnormal iron deposition, and significant upregulation of antioxidant enzyme heme oxygenase-1 (HO-1) and iron storage protein ferritin in the dmy rat, but not in the mv rat. The expression of iron transporter transferrin mRNA was significantly decreased in the mv but not dmy rat, which may reflect a part of functional abnormalities of oligodendrocytes in the mv rat. Iron accumulation and increased expression of ferritin in the dmy rat were mainly found in astrocytes, suggesting a protective role of astrocytes in iron-mediated cytotoxicity. HO-1 was predominantly induced in oligodendrocytes during the early stage of demyelination in the dmy rat, suggesting that iron-mediated oxidative stress is most likely involved in the pathogenesis of demyelination in the dmy rat.

Identifying abnormal iron deposition in chronic liver disease using a quantitative MRI technique (T2* decay): an ROC study.

Aim: To identify a cut-off value for the T2* decay of liver parenchyma in cirrhosis to identify patients with iron deposition. Methods: 76 patients (mean age: 58.6 years; 34 females) who underwent MRI of the liver were retrospectively analyzed including 46 patients with cirrhosis and 30 patients without cirrhosis. Mean T2* decay (milliseconds) of liver was determined by average of six regions of interest. Analysis between cirrhotic and non-cirrhotic patients was assessed using student’s t-test and receiver operator characteristic (ROC) curve. Results: Mean T2* value for cirrhotic patients (21.9 ms) was significantly lower than that of non-cirrhotics (27.8 ms) (p=.0015). Using ROC curve analysis, a threshold T2* value of 23 ms was optimal for identifying abnormal iron. Conclusions: Liver iron deposition in cirrhosis can be identified by MRI based on T2* decay.

PLA2G6 Mutation Underlies Infantile Neuroaxonal Dystrophy

Infantile neuroaxonal dystrophy (INAD) is an autosomal recessive progressive neurodegenerative disease that presents within the first 2 years of life and culminates in death by age 10 years. Affected individuals from two unrelated Bedouin Israeli kindreds were studied. Brain imaging demonstrated diffuse cerebellar atrophy and abnormal iron deposition in the medial and lateral globus pallidum. Progressive white-matter disease and reduction of the N-acetyl aspartate : chromium ratio were evident on magnetic resonance spectroscopy, suggesting loss of myelination. The clinical and radiological diagnosis of INAD was verified by sural nerve biopsy. The disease gene was mapped to a 1.17-Mb locus on chromosome 22q13.1 (LOD score 4.7 at recombination fraction 0 for SNP rs139897), and an underlying mutation common to both affected families was identified in PLA2G6, the gene encoding phospholipase A2 group VI (cytosolic, calcium-independent). These findings highlight a role of phospholipase in neurodegenerative disorders.

Renoprotective Properties of Angiotensin Receptor Blockers beyond Blood Pressure Lowering

Clinical studies have demonstrated that some antihypertensive agents provide renoprotection independent of BP lowering. Recent in vitro and in vivo studies evaluated the mechanisms involved in this protection. First, the in vitro effects of several angiotensin II type 1 receptor blockers (ARB), calcium channel blockers (CCB), and beta blockers (BB) on various mediators were compared: Formation of pentosidine (an advanced glycation end product), hydroxyl radical-induced formation of o-tyrosine, and transition metals-induced oxidation of ascorbic acid (the Fenton reaction). All of the six tested ARB but neither the six CCB nor the nine BB inhibited pentosidine formation. ARB, as well as BB but not CCB, inhibited hydroxyl radicals-mediated o-tyrosine formation. ARB but neither BB nor CCB inhibited efficiently transition metals-catalyzed oxidation of ascorbic acid. Second, the in vivo consequences for the kidney of these various in vitro effects were evaluated. Hypertensive, type 2 diabetic rats with nephropathy, SHR/NDmcr-cp, were given for 20 wk either olmesartan (ARB) or nifedipine (CCB), or atenolol (BB). Despite similar BP reduction, only ARB significantly reduced proteinuria and prevented glomerular and tubulointerstitial damage (mesangial activation, podocyte injury, tubulointerstitial injury, and inflammatory cell infiltration). It is interesting that only ARB prevented abnormal iron deposition in the interstitium, corrected chronic hypoxia, reduced expressions of heme oxygenase and p47phox (a subunit of NADPHoxidase), and inhibited pentosidine formation (which correlates well with proteinuria). These observations confirm unique renoprotective properties of ARB, independent of BP lowering but related to decreased oxidative stress (hydroxyl radicals scavenging and inhibition of the Fenton reaction), correction of chronic hypoxia, and inhibition of advanced glycation end product formation and of abnormal iron deposition. These benefits of ARB may contribute to the renoprotection observed beyond BP lowering.

Plasma Protein Haptoglobin Modulates Renal Iron Loading

Haptoglobin is the plasma protein with the highest binding affinity for hemoglobin. The strength of hemoglobin binding and the existence of a specific receptor for the haptoglobin-hemoglobin complex in the monocyte/macrophage system clearly suggest that haptoglobin may have a crucial role in heme-iron recovery. We used haptoglobin-null mice to evaluate the impact of haptoglobin gene inactivation on iron metabolism. Haptoglobin deficiency led to increased deposition of hemoglobin in proximal tubules of the kidney instead of the liver and the spleen as occurred in wild-type mice. This difference in organ distribution of hemoglobin in haptoglobin-deficient mice resulted in abnormal iron deposits in proximal tubules during aging. Moreover, iron also accumulated in proximal tubules after renal ischemia-reperfusion injury or after an acute plasma heme-protein overload caused by muscle injury, without affecting morphological and functional parameters of renal damage. These data demonstrate that haptoglobin crucially prevents glomerular filtration of hemoglobin and, consequently, renal iron loading during aging and following acute plasma heme-protein overload.

Iron chelation and a free radical scavenger suppress angiotensin II-induced downregulation of klotho, an anti-aging gene, in rat

Administration of angiotensin II to rats decreases renal expression of klotho, an aging-related gene, and also causes abnormal iron deposition in renal cells. Here we have examined the effects of iron overload and iron chelation on renal expression of klotho in untreated rats and rats treated with angiotensin II. Administration of iron–dextran caused a downregulation of klotho expression, and iron chelation suppressed the angiotensin II-induced downregulation of this gene. In addition, a free radical scavenger (T-0970), which effectively decreased plasma levels of 8-epi-prostaglandin F 2α (8-epi-PGF 2α), suppressed angiotensin II-induced downregulation of klotho. Collectively, these findings suggest that abnormal iron metabolism and increased oxidative stress are involved in the mechanism of angiotensin II-mediated modulation of klotho expression.

Iron overload augments angiotensin II-induced cardiac fibrosis and promotes neointima formation.

Abnormal iron deposition may cause oxidant-induced damage in various organs. We have previously reported that continuous administration of angiotensin II to rats results in an overt iron deposition in the renal tubular epithelial cells, which may have a role in angiotensin II-induced renal damage. In the present study, we investigated the role of iron in the development of cardiac injury induced by angiotensin II. Angiotensin II was continuously infused to rats at a dose of 0.7 mg/kg per day for 7 consecutive days. No iron deposits were observed in the hearts of untreated rats, whereas iron deposition was seen in the cells in the subepicardial and granulation regions after angiotensin II infusion. Concomitant administration of deferoxamine, an iron chelator, significantly reduced the extent of cardiac fibrosis, which suggests that iron deposition aggravates the cardiac fibrosis induced by angiotensin II. Iron overload caused by the administration of iron-dextran resulted in an augmentation of cardiac fibrosis and the generation of neointimal cells in the coronary artery in angiotensin II-infused rats. By contrast, neointima was not formed in the cardiac vessels in norepinephrine-infused rats with iron overload. Cardiac iron deposition may be involved in the development of cardiac fibrosis induced by angiotensin II. In addition, iron overload may enhance the formation of neointima under conditions of increased circulating angiotensin II but not catecholamines.

Abnormal Iron Deposition in Renal Cells in the Rat with Chronic Angiotensin II Administration

Acute experimental iron loading causes iron to accumulate in the renal tissue. The accumulation of iron may play a role in enhancing oxidant-induced tubular injury by producing increased amounts of reactive oxygen species. From findings in cells from heme oxygenase-1 (HO-1)-deficient mice, HO-1 is postulated to prevent abnormal intracellular iron accumulation. Recently, it has been reported that HO-1 is induced in the renal tubular epithelial cells, in which iron is deposited after iron loading, and that this HO-1 induction may be involved in ameliorating iron-induced renal toxicity. We previously showed that chronic administration of angiotensin II to rats induces HO-1 expression in the tubular epithelial cells. These observations led us to investigate whether there is a link between iron deposition and HO-1 induction in renal tubular cells in rats undergoing angiotensin II infusion. In the present study, rats were given angiotensin II for continuously 7 days. Prussian blue staining revealed the distinct deposits of iron in the proximal tubular epithelial cells after angiotensin II administration. Electron microscopy demonstrated that iron particles were present in the lysosomes of these cells. Histologic and immunohistochemical analyses showed that stainable iron and immunoreactive ferritin and HO-1 were colocalized in the tubular epithelial cells. Treatment of angiotensin II-infused rats with an iron chelator, deferoxamine, blocked the abnormal iron deposition in kidneys and also the induced expression of HO-1 and ferritin expression. Furthermore, deferoxamine treatment suppressed the angiotensin II-induced increase in the urinary excretion of protein and N-acetyl-β-d-glucosaminidase, a marker of tubular injury; however, deferoxamine did not affect the angiotensin II-induced decrease in glomerular filtration rate. These results suggest that angiotensin II causes renal injury, in part, by inducing the deposition of iron in the kidney.

Polymorphisms in the Transferrin 5′ Flanking Region Associated with Differences in Total Iron Binding Capacity: Possible Implications in Iron Homeostasis

ABSTRACTWe have identified five single nucleotide polymorphisms (SNPs) upstream (5′) of the transferrin coding region. One polymorphism is in the 5′ UTR at nt +49, and four are in the promoter region at nt −34, −551, −617, and −739, numbering from the start of transcription. The −34 and −617 SNPs are tightly but not completely linked. The −34 polymorphism lies between a conserved Sp1 site and the TATA box. The −617 polymorphism is within the DRII enhancer region. Five haplotypes have been defined from these SNPs by the identification of at least one homozygous individual, and two other haplotypes were deduced from heterozygous individuals. The total iron-binding capacity associated with each transferrin haplotype was haplotype 2 > 1 > 4 > 3. Transferrin promoter haplotype 2 had a significantly higher mean TIBC and haplotype 3 had a significantly lower mean TIBC than the more common haplotype 1. Persons with haplotype 4, which includes the −34T and −617A minor alleles, have a lower mean TIBC but the difference was not statistically significant. In normal individuals, the differences in the haplotypes were not found to be associated with differences in transferrin saturation and ferritin levels. There was no difference in the extent of increase in the mean TIBC levels in individuals with iron deficiency anemia in regard to their haplotype. Furthermore, there was no difference in the relative frequencies of the transferrin haplotypes in the iron-deficient population. In hemochromatosis patients who were homozygous for the C282Y HFE mutation, no particular haplotype was associated with a significant difference in transferrin saturation or ferritin levels. In White patients with Parkinson's disease, a disorder in which there is abnormal iron deposition in the brain, the presence of transferrin haplotype 3 was in slight excess over the normal White population.

Alternative RNA Splicing Generates a Glycosylphosphatidylinositol-anchored Form of Ceruloplasmin in Mammalian Brain

Ceruloplasmin is a copper-containing ferroxidase that is essential for normal iron homeostasis. Whereas ceruloplasmin in plasma is produced and secreted by hepatocytes, in the brain a glycosylphosphatidylinositol (GPI)-anchored form of ceruloplasmin is expressed on the surface of astrocytes. By using a cDNA cloning approach, we have now determined that the GPI-anchored form of ceruloplasmin is generated by alternative RNA splicing. The splicing occurs downstream of exon 18 and replaces the C-terminal 5 amino acids of the secreted form with an alternative 30 amino acids that signal GPI anchor addition. RNase protection analysis demonstrates that the GPI-anchored form is the major form in the brain, whereas the secreted form predominates in the liver. Individuals with aceruloplasminemia, a hereditary deficiency of ceruloplasmin, have severe iron deposition in a number of organs, including the brain where it results in neurodegeneration. Therefore, this novel GPI-anchored form of ceruloplasmin is likely to play an important role in iron metabolism in the central nervous system.

Defective Recovery and Severe Renal Damage After Acute Hemolysis in Hemopexin-Deficient Mice

Hemopexin (Hx) is a plasma glycoprotein mainly expressed in liver and, less abundantly, in the central and peripheral nervous systems. Hx has a high binding affinity with heme and is considered to be a major transport vehicle of heme into the liver, thus preventing both heme-catalyzed oxidative damage and heme-bound iron loss. To determine the physiologic relevance of heme-Hx complex formation, Hx-deficient mice were generated by homologous recombination in embryonic stem (ES) cells. The Hx-deficient mice were viable and fertile. Their plasma iron level and blood parameters were comparable to those of control mice and they showed no evidence of tissue lesions caused by oxidative damage or abnormal iron deposits. Moreover, they were sensitive to acute hemolysis, as are wild-type mice. Nevertheless, Hx-null mice recovered more slowly after hemolysis and were seen to have more severe renal damage than controls. After hemolytic stimulus, Hx-deficient mice presented prolonged hemoglobinuria with a higher kidney iron load and higher lipid peroxidation than control mice. Moreover, Hx-null mice showed altered posthemolysis haptoglobin (Hp) turnover in as much as Hp persisted in the circulation after hemolytic stimulus. These data indicate that, although Hx is not crucial either for iron metabolism or as a protection against oxidative stress under physiologic conditions, it does play an important protective role after hemolytic processes.

Defective Recovery and Severe Renal Damage After Acute Hemolysis in Hemopexin-Deficient Mice

AbstractHemopexin (Hx) is a plasma glycoprotein mainly expressed in liver and, less abundantly, in the central and peripheral nervous systems. Hx has a high binding affinity with heme and is considered to be a major transport vehicle of heme into the liver, thus preventing both heme-catalyzed oxidative damage and heme-bound iron loss. To determine the physiologic relevance of heme-Hx complex formation, Hx-deficient mice were generated by homologous recombination in embryonic stem (ES) cells. The Hx-deficient mice were viable and fertile. Their plasma iron level and blood parameters were comparable to those of control mice and they showed no evidence of tissue lesions caused by oxidative damage or abnormal iron deposits. Moreover, they were sensitive to acute hemolysis, as are wild-type mice. Nevertheless, Hx-null mice recovered more slowly after hemolysis and were seen to have more severe renal damage than controls. After hemolytic stimulus, Hx-deficient mice presented prolonged hemoglobinuria with a higher kidney iron load and higher lipid peroxidation than control mice. Moreover, Hx-null mice showed altered posthemolysis haptoglobin (Hp) turnover in as much as Hp persisted in the circulation after hemolytic stimulus. These data indicate that, although Hx is not crucial either for iron metabolism or as a protection against oxidative stress under physiologic conditions, it does play an important protective role after hemolytic processes.

Regulation of heme oxygenase-1 expression by dopamine in cultured C6 glioma and primary astrocytes

Heme oxygenase-1 (HO-1) is an inducible enzyme involved in heme catabolism, tissue iron homeostasis and the cellular response to oxidative stress. Elevated HO-1 expression in astrocytes has been observed in association with abnormal iron deposition and increased oxidative stress in Parkinson's disease (PD). Since HO-1 could contribute to these aspects of PD pathobiology we have investigated its regulation in cultured astrocytes and C6 glioma cells. Here we report that dopamine is a potent inducer of HO-1. This induction is not mediated by a classical dopamine receptor and is not mimicked by a range of catecholamines and dopamine metabolites. When the time-course of HO-1 expression was compared between dopamine and hemin, the latter induced the gene immediately while the former did so with a lag. This suggested two distinct signal transduction pathways. However, cycloheximide blocked both hemin- and dopamine-induced HO-1 expression, suggesting that both pathways may involve proteins with short half-lives. Ascorbic acid blocked dopamine induction of HO-1 but had no effect on hemin-induced expression. This suggested that dopamine may signal upstream of the unstable protein by producing pro-oxidant metabolites or byproducts. Inhibition of monoamine oxidases A or B or catechol- O-methyl transferase did not block HO-1 induction by dopamine, indicating that these enzymes were not converting dopamine to an active metabolite. These results suggest that dopamine, released or secreted from affected neurons, may trigger HO-1 expression in neighboring astrocytes. HO-1 and its metabolites could then contribute to the oxidative stress and iron deposition associated with PD.

333 Astrocyte targeted interleukin-6 expression in transgenic mice promotes severe neurodegeneration with abnormal iron deposition and oxidative stress

333 Astrocyte targeted interleukin-6 expression in transgenic mice promotes severe neurodegeneration with abnormal iron deposition and oxidative stress

Abnormal iron deposition associated with lipid peroxidation in transgenic mice expressing interleukin-6 in the brain.

Transgenic mice, named GFAP-IL6, that express interleukin-6 in astrocytes in the central nervous system (CNS) have a constitutive blood-brain barrier (BBB) defect and develop a progressive neurodegenerative disease. Based on ultrastructural observations showing electron-dense pigment in the brain of the GFAP-IL6 mice, we hypothesized that iron metabolism was altered in the brains of these animals. Enhanced histochemical methods revealed abnormal iron deposition in the cerebellum from 1 month of age that worsened with progression of the disease. Immunohistochemical analysis of iron-binding proteins (IBP) showed increased ferritin immunoreactivity and a decreased signal from the transferrin receptor in symptomatic animals. Atomic absorption spectroscopy revealed a 40% increase of total iron concentration in the cerebellum at the symptomatic stage. In order to obtain evidence that accumulation of this oxidizing metal was toxic, we looked for the presence of oxidative damage. Using the MAL-2 antibody, extensive lipid peroxidation (LP) was detected in the neocortex and the cerebellum in symptomatic animals. Ultrastructural analysis indicated lipofuscin deposition at the sites of neuro-axonal degeneration and abnormal iron deposition. These results suggest that the IL6-induced BBB defect precipitates iron accumulation in the GFAP-IL6 mouse brain and that subsequent IBP regulation mediates protective responses. As these defenses become overwhelmed, the iron overload seems to promote LP, which may contribute to the neurodegeneration that ensues. This transgenic mouse model of IL6-mediated neurodegeneration provides a unique opportunity to examine several aspects of iron metabolism in the brain, including its entry at the site of the BBB, its distribution through the IBP, and its mechanisms of toxicity.

Removal of erythrocyte membrane iron in vivo ameliorates the pathobiology of murine thalassemia.

Abnormal deposits of free iron are found on the cytoplasmic surface of red blood cell (RBC) membranes in beta-thalassemia. To test the hypothesis that this is of importance to RBC pathobiology, we administered the iron chelator deferiprone (L1) intraperitoneally to beta-thalassemic mice for 4 wk and then studied RBC survival and membrane characteristics. L1 therapy decreased membrane free iron by 50% (P = 0.04) and concomitantly improved oxidation of membrane proteins (P = 0.007), the proportion of RBC gilded with immunoglobulin (P = 0.001), RBC potassium content (P < 0.001), and mean corpuscular volume (P < 0.001). Osmotic gradient ektacytometry confirmed a trend toward improvement of RBC hydration status. As determined by clearance of RBC biotinylated in vivo, RBC survival also was significantly improved in L1-treated mice compared with controls (P = 0.007). Thus, in vivo therapy with L1 removes pathologic free iron deposits from RBC membranes in murine thalassemia, and causes improvement in membrane function and RBC survival. This result provides in vivo confirmation that abnormal membrane free iron deposits contribute to the pathobiology of thalassemic RBC.

Histological changes in rat embryonic blood cells as a possible mechanism for ventricular septal defects produced by an N-phenylimide herbicide.

An N-phenylimide herbicide, S-53482, inhibits protoporphyrinogen oxidase, an enzyme common to chlorophyll and heme biosynthesis, and produces embryolethality, teratogenicity [mainly ventricular septal defects (VSD) and wavy ribs], and growth retardation in rats. In order to elucidate the mechanism of the developmental toxicity, in particular VSD, effects of the herbicide on rat embryonic blood cells were investigated histologically at the light and electron microscopic levels at 6, 12, 24, 36, and 48 h after oral administration of the chemical to pregnant rats on day 12 of gestation, the most sensitive day for toxicity. Electron and light microscopy demonstrated mitochondrial lesions, including abnormal iron deposits that were probably due to inhibition of heme biosynthesis, in erythroblasts derived from the yolk sac. Subsequently, degeneration of these erythroblasts occurred followed by erythrophagocytosis. Histologically hearts from exposed embryos had a thin ventricular wall, which may reflect a compensatory reaction to a loss of embryonic blood cells. Thus, the herbicide may induce VSD due to hematological dysfunction caused by the inhibition of heme biosynthesis rather than by direct injurious effects on the heart.

Bilateral total knee arthroplasty for haemochromatosis arthropathy

Haemochromatosis, a hereditary disorder of iron metabolism, is associated with abnormal tissue iron deposition and polyarthropathy. Arthritis usually affects the small joints of the hand but can also occur in large weight bearing joints. A 63-year-old man who developed haemochromatosis arthropathy of both knees presented with severe joint erosions, articular deformity and soft tissue contracture. Operative treatment was successful but required surgical techniques commonly used during revision total knee arthroplasty.

Amyotrophic lateral sclerosis: T2 shortening in motor cortex at MR imaging.

To determine whether decreased signal intensity of the motor cortex (T2 shortening) at magnetic resonance (MR) imaging is a useful finding for supporting the diagnosis of amyotrophic lateral sclerosis (ALS). High-field-strength (1.5-T) MR images of 15 patients (seven men and eight women, aged 28-80 years) and 49 neurologically normal age-matched control patients were examined for T2 shortening in the motor cortex. In addition, brains of patients with ALS were examined at autopsy. The MR images of 14 of the 15 patients showed T2 shortening in precentral cortices, while the images of all but one of the control patients showed no such finding. In three of eight brains at autopsy, sections from the precentral cortex showed sparsely distributed, intensely stained astrocytes and macrophages. Abnormal iron deposition associated with the degenerative process could be the source of T2 shortening, which is a useful MR imaging finding in the diagnosis of ALS.

Nonrandom association of free iron with membranes of sickle and beta- thalassemic erythrocytes

To further define the nature of abnormal iron deposits on the membranes of pathologic red blood cells, we have used sickle cell anemia (HbSS), HbSC, and beta-thalassemic erythrocytes (RBCs) to prepare inside-out membranes (IOM) and insoluble membrane aggregates (AGGs) containing coclustered hemichrome and band 3. Study of IOM from HbSC and thalassemic patients showed that amounts of heme iron and, especially, free iron were much higher in patients who had undergone surgical splenectomy. The membrane AGGs from HbSS and beta-thalassemic RBCs contained much more globin than heme, with this discrepancy being variable from patient to patient. Although these AGGs were enriched (compared with the ghosts from which they were derived) for heme, as expected, less than 10% of total ghost heme was recovered in them. Remarkably, these AGGs also were enriched for nonheme iron, markedly so in some patients. Iron binding studies showed that the association of free iron with these hemichrome/band 3 AGGs is explained by the fact that free iron binds to denatured hemoglobin. These results document that free iron is nonrandomly associated with the membranes of sickle and beta-thalassemic RBCs. Whether this plays a causative role in the premature removal of such cells from the circulation remains to be seen.