Iron distribution and histopathological characterization of the liver and heart of β-thalassemic mice with parenteral iron overload: Effects of deferoxamine and deferiprone

The liver and heart are the major target organs for iron accumulation and iron toxicity in β-thalassemia. To mimic the phenomenon of heavy iron overload resulting from repeated blood transfusions, a total of 180 mg of iron dextran was intraperitoneally injected into C57BL/6J mice (WT) and heterozygous β-globin knockout mice (^muβ^th-3/+, BKO). The effects of deferiprone and deferoxamine in this model were investigated. The iron was distributed homogenously throughout the 4 liver lobes (left, caudate, right and median) and was present in hepatocytes, Kupffer cells and the sinusoidal space. Iron accumulation in phagocytic macrophages, recruitment of hepatic lymphocytes and nucleus membrane degeneration were observed as a result of iron overload in the WT and BKO mice. However, the expansion of hepatic extramedullary hematopoiesis was observed only in the BKO mice with iron overload. In the heart, the iron accumulated in the cardiac interstitium and myocytes, and moderate hypertrophy of the myocardial fibers and cardiac myocyte degeneration were observed. Although the total liver iron was not significantly altered by iron chelation therapy, image analysis demonstrated a difference in the efficacies of two iron chelators. The major site of chelation was the extracellular compartment, but treatment with deferiprone also resulted in intracellular iron chelation. Interestingly, iron chelators reversed the pathological changes resulting from iron overload in WT and BKO mice despite being used for only a short treatment period. We suggest that some of these effects may be secondary to the anti-inflammatory activity of the chelators.     

Effect of green tea on iron status and oxidative stress in iron-loaded rats

Plasma non-transferrin bound iron (NTBI) is potentially toxic and contributes to the generation of reactive oxygen species (ROS), consequently leading to tissue damage and organ dysfunction. Iron chelators and antioxidants are used for treatment of thalassemia patients. Green tea (GT) contains catechins derivatives that have many biological activities. The purpose of this study was to investigate the iron-chelating and free-radical scavenging capacities of green tea extract in vivo. Rats were injected ip with ferric citrate together with orally administered GT extract (GTE) for 4 months. Blood was collected monthly for measurement of iron overload and oxidative stress indicators. Plasma iron (PI) and total iron-binding capacity (TIBC) were quantified using bathophenanthroline method. Plasma NTBI was assayed with NTA chelation/HPLC. Plasma malonyldialdehyde (MDA) was determined by using the TBARS method. Erythrocyte oxidative stress was assessed using flow cytometry. Levels of PI, TIBC, NTBI and MDA, and erythrocyte ROS increased in the iron-loaded rats. Intervention with GT extract markedly decreased the PI and TIBC concentrations. It also lowered the transferrin saturation and effectively inhibited formation of NTBI. It also decreased the levels of erythrocyte ROS in week 4, 12 and 16. Therefore, green tea extract can decrease iron in plasma as well as eliminate lipid peroxidation in plasma, and destroy formation of erythrocyte ROS in the rats challenged with iron. The bifunctional effects could be beneficial in alleviating the iron and oxidative stress toxicity. In prospective, these GTE activities should be further examined in thalassemic animals or humans.     

Curcumin contributes to in vitro removal of non-transferrin bound iron by deferiprone and desferrioxamine in thalassemic plasma

Non-transferrin-bound iron (NTBI) is detectable in plasma of beta-thalassemia patients with transfusional iron overload. This form of iron may cause oxidative tissue damage and increased iron uptake, into several vital organs. Removal of NTBI species is incomplete and transient using standard intermittent desferrioxamine (DFO) or deferiprone (DFP) monotherapy. Combinations of these or other chelators may improve the protection time from NTBI and increase removal of harmful NTBI species. Curcuminoids from Curcuma longa L. is a naturally occurring phytochemical which shows a wide range of pharmacological properties including anti-oxidative, anti-inflammatory, anti-cancer and iron-chelating activities. In this study, the curcuminoids was investigated for NTBI chelation in thalassemic plasma in vitro and for the potential to improve NTBI removal when used with other chelators. Curcumin bound Fe(3+) to form a Fe(3+)-curcumin complex with a predominant absorption at 500 nm. The chemical binding of curcumin was dose- and time-dependent and more specific for Fe(3+) than Fe(2+). Using a HPLC-based NTBI assay without an aluminium blocking step, curcumin shuttled the iron from Fe(3+)-NTA complex, giving underestimated NTBI values. At equivalent concentrations DFO, DFP and curcumin decreased plasma NTBI with the order of DFP>DFO>curcumin. None of these chelators removed NTBI completely, but curcumin appeared to increase the rate of NTBI removal when added to DFP. It is proposed that the beta-diketo moiety of curcumin participates in the NTBI chelation.     

铁螯合剂治疗高量输血重型β地中海贫血患者铁过载的疗效观察

目的探讨铁螯合剂治疗高量输血重型β地中海贫血铁过载的临床疗效。方法选择长期维持高量输血的重型β地中海贫血铁过载患者84例,其中不足量使用铁螯合剂患者36例,足量使用铁螯合剂患者48例。不足量使用铁螯合剂患者分为去铁胺（DFO）组、去铁酮（DFP）组、DFO联合DFP组;足量使用铁螯合剂患者分为DFO组、DFP组、地拉罗司（DFX）组、DFO联合DFP组。观察1年后血清铁蛋白（SF）的变化。结果不足量DFO、DFP组,SF均较1年前升高,DFO联合DFP组SF无明显变化;足量DFO、DFP组SF变化差异无统计学意义（P〉0.05）,DFX组、DFO联合DFP组SF下降差异均有统计学意义（P〈0.05）,其中DFX组疗效最佳,其次为DFO联合DFP组。结论高量输血重型β地中海贫血患者铁过载的治疗,应长期坚持使用足量的铁螯合剂,DFX以及DFO联合DFP祛铁效果明显。不良反应以骨关节疼痛及胃肠道反应为主。     

Association of renal injury with increased oxygen free radical activity and altered nitric oxide metabolism in chronic experimental hemosiderosis

Chronic iron (Fe) overload is associated with a marked increase in renal tissue iron content and injury. It is estimated that 10% of the American population carry the gene for hemochromatosis and 1% actually suffer from iron overload. The mechanism of iron overload-associated renal damage has not been fully elucidated. Iron can accelerate lipid peroxidation leading to organelle membrane dysfunction and subsequent cell injury/death. Iron-catalyzed generation of reactive oxygen species (ROS) is responsible for initiating the peroxidatic reaction. We investigated the possible association of oxidative stress and its impact on nitric oxide (NO) metabolism in iron-overload-associated renal injury. Rats were randomized into Fe-loaded (given 0.5 g elemental iron/kg body weight as iron dextran; i.v.), Fe-depleted (given an iron-free diet for 20 weeks), and control groups. Renal histology, tissue expression of endothelial and inducible nitric oxide synthases (eNOS and iNOS), renal tissue expression of nitrotyrosine, plasma, and renal tissue lipid peroxidation product, malondialdehyde (MDA), and plasma and urinary NO metabolites (NOx) were examined. Iron overload was associated with mild proteinuria, tissue iron deposition together with significant glomerulosclerosis, tubular atrophy, and interstitial fibrosis. Rare focal glomerulosclerosis and tubulointerstitial changes were noted in normal controls. No renal lesions were observed in Fe-depleted rats. Iron deposits were seen in glomeruli, proximal tubules, and interstitium. The iron staining in the distal tubules was negligible. Both plasma and renal tissue MDA and renal tissue nitrotyrosine were increased significantly in Fe-loaded rats compared with control rats. In contrast, Fe-depleted animals showed a marked reduction in plasma and renal tissue MDA and nitrotyrosine together with significant elevation of urinary NOx excretion. In addition, iron-overload was associated with up-regulation of renal eNOS and iNOS expressions when compared with the control and Fe-depleted rats that showed comparable values. In conclusion, chronic iron overload resulted in iron deposition in the glomeruli and proximal tubules with various renal lesions and evidence of increased ROS activity, enhanced ROS-mediated inactivation, and sequestration of NO and compensatory up-regulation of renal eNOS and iNOS expressions. However, iron depletion was associated with reduced MDA and tissue nitrotyrosine abundance, increased urinary NOx excretion, normal nitric oxide synthase (NOS) expression, and absence of renal injury. These findings point to the possible role of ROS in chronic iron overload-induced renal injury.     

Desferri-Exochelin, a lipid-soluble, hexadentate iron chelator, effectively removes tissue iron

Chronic iron-overload is damaging to the heart, liver, and other organs. Better iron chelators are needed to treat this serious medical condition. The uptake and distribution of the lipid-soluble, hexadentate iron chelator desferri-Exochelin 772SM (D-Exo) is studied and its efficacy in removing iron from tissue in rodent models is evaluated. After an intravenous bolus of tritiated D-Exo to rats, counts rapidly disappeared from the blood and rapidly appeared in 15 organs studied, usually peaking within 15 min. There was considerable uptake in the heart and liver, 2 organs especially susceptible to damage from clinical iron overload. To assess actual decreases in cardiac and hepatic iron in response to D-Exo, mice loaded with 42 mg of iron dextran (2100 mg/kg) were studied. Untreated, iron-loaded mice sacrificed 9 weeks later had a 4-fold increase in cardiac iron and a 20-fold increase in hepatic iron compared with controls that were not iron-loaded. In iron-loaded mice treated with 7 mg of D-Exo intraperitoneally (i.p.) 4 days/week for 8 weeks (total 224 mg), tissue iron, measured by atomic absorption, was reduced by 20% in the liver and 25% in the heart (P < 0.01 for each organ). During the first 8 h after a D-Exo dose, iron was excreted in the urine. Mice treated with D-Exo gained weight normally and showed no evidence of toxicity. In conclusion, in this iron-overload mouse model, D-Exo administered intravenously or i.p. rapidly diffuses into multiple organs, including the heart and liver, and effectively removes iron without apparent toxicity.     

BACH1 regulates erythrophagocytosis and iron-recycling in β-thalassemia

Macrophages play essential roles in erythrophagocytosis and iron recycling. β-thalassemia is characterized by a genetic defect in hemoglobin synthesis, which increases the rate of iron recycling. We previously showed that reduced expression of the BTB and CNC homolog 1 (BACH1) gene leads to increased phagocytosis of abnormal RBCs by activated monocytes. However, the mechanisms underlying this abnormal RBC clearance remained unclear. Herein, the spleen and bone marrow cells of β-thalassemic mice were examined for erythrophagocytosis CD markers and iron-recycling genes. Higher expression levels of CD47 and CD163 on RBCs and macrophages, respectively, were observed in β-thalassemic mice than in wild-type cells. The decreased expression of BACH1 caused an increase in Nrf2, Spic, Slc40a1, and HMOX1 expression in splenic red pulp macrophages of thalassemic mice. To investigate BACH1 regulation, a macrophage cell line was transfected with BACH1-siRNA. Decreased BACH1 expression caused an increase in CD163 expression; however, the expression levels were lower when the cells were cultured in media supplemented with β-thalassemia/HbE patient plasma. Additionally, the iron recycling-related genes SPIC, SLC40A1, and HMOX1 were significantly upregulated in BACH1-suppressed macrophages. Our findings provide insights into BACH1 regulation, which plays an important role in erythrophagocytosis and iron recycling in thalassemic macrophages.     

Epigallocatechin-3-gallate and epicatechin-3-gallate from green tea decrease plasma non-transferrin bound iron and erythrocyte oxidative stress

Beta-thalassemia patients suffer from secondary iron overload caused by increased iron absorption and multiple blood transfusions. Excessive iron catalyzes free-radical formation, causing oxidative tissue damage. Non-transferrin bound iron (NTBI) detected in thalassemic plasma is highly toxic and chelatable. Desferrioxamine and deferiprone are used to treat the iron overload, but many side effects are found. Epigallocatechin gallate (EGCG) and epicatechin gallate (ECG) in green tea (GT) show strong antioxidant properties. We separated the EGCG and ECG from GT extract using an HPLC, and examined their iron-binding and free-radical scavenging activities. They bound Fe(3+) rapidly to form a complex with a predominant absorption at 560 nm. EGCG and ECG bound chemical Fe(3+) and chelated the NTBI in a time- and dose dependent manner. They also decreased oxidative stress in iron-treated erythrocytes. In conclusion, EGCG and ECG could be natural iron chelators that efficiently decrease the levels of NTBI and free radicals in iron overload.     

Effect of iron stores and hysterectomy on iron absorption and distribution in pregnant mice

Intestinal uptake (U) and transport (T) of 50Fe from a 1-mug oral dose was studied in the mouse. U increased to a similar degree in both pregnant and iron-deficient animals. The increase in T was greater in pregnant than in iron-deficient mice. In iron-loaded pregnant mice, U increased to levels found in pregnant animals, whereas T increased, but by a lesser amount. Termination of pregnancy by delivery or hysterectomy at days 20-21 resulted in a fall in U and T to normal levels within 24 h. Eighty percent of iron transported across the intestine in pregnancy was recovered from the fetuses, compared to 6.7% recovered in the maternal liver. Hepatic uptake was lower in pregnant than in iron-deficient mice or in postpartum mice with persisting iron deficiency. Hepatic uptake by the maternal liver after an intravenous dose of 2 mug 50Fe increased to levels in iron-deficient mice within 5 min of hysterectomy. The results demonstrate an effect of pregnancy on intestinal iron transport and its distribution that was dependent on the presence of fetuses and placentas.     

Impact of iron loading and iron chelation on murine tuberculosis

Objective: To demonstrate in a mouse model of tuberculosis that excess iron load enhances the growth of Mycobacterium tuberculosis and to assess whether or not iron chelation may abrogate the effect of iron loading on Mycobacterium tuberculosis growth in the mouse model.
Methods: In the first experiment, female BALB/C mice were infected intravenously with 5.4 × 10⁴ CPU of M. tuberculosis H37Rv per mouse. Before infection, half of them were treated for 2 weeks with 50 mg/kg polymaltose ferric hydroxide, a source of iron. In a second experiment, female BALB/C mice were infected intravenously with 9 × 10⁴ CPU per mouse and half of them were iron loaded for 2 weeks before infection. Both iron-loaded and non-iron-loaded mice were treated with desferrioxamine (DFO), an iron chelator, or isoniazid. At each sacrifice, mice and their spleens were weighed, lung lesions were noted, and the number of M. tuberculosis CFU determined by quantitative cultures of spleen and lungs.
Results: In the first experiment, the number of CFU was significantly higher in the spleen of iron-treated mice than in non-iron-loaded mice at days 14 and 28 after infection. In the second experiment, iron loading enhanced the multiplication of M. tuberculosis in the spleen but not in the lungs, DFO displayed a modest but significant effect on the multiplication of M. tuberculosis in iron-loaded mice, and isoniazid therapy was effective in both iron-loaded and non-iron-loaded mice.
Conclusions: Iron loading of BALB/C mice enhanced the multiplication of M. tuberculosis in the spleens but not in the lungs. DFO exhibited significant activity against M. tuberculosis in iron-loaded mice, and isoniazid therapy was strongly bactericidal in both iron-loaded and non-iron-loaded mice.     

Accumulation of iron by primary rat hepatocytes in long-term culture: changes in nuclear shape mediated by non-transferrin-bound forms of iron.

We have previously shown that hepatocytes in long-term dimethylsulfoxide (DMSO) culture, fed a chemically defined medium, are highly differentiated and an excellent in vitro model of adult liver. Hepatocytes in long-term DMSO culture can be iron loaded by exposure to non-transferrin-bound iron (NTBI) in the form of ferrous sulfate (FeSO4), ferric nitrilotriacetate, or trimethylhexanoyl (TMH)-ferrocene. Holotransferrin, at equivalent times and concentrations, was unable to load hepatocytes. Of the iron compounds tested, TMH-ferrocene most accurately simulated the morphological features of iron-loaded hepatocytes in vivo. When exposed to 25 micromol/L TMH-ferrocene, hepatocytes loaded increasing amounts of iron for 2 months before the cells died. When exposed to lower concentrations of TMH-ferrocene (as low as 2.5 micromol/L), hepatocytes continuously loaded iron and remained viable for more than 2 months. The cellular deposition of iron was different in hepatocytes exposed to TMH-ferrocene compared with those exposed to FeSO4; exposure to TMH-ferrocene resulted in the presence of more ferritin cores within lysosomes than were seen with FeSO4. When the concentration of TMH-ferrocene was increased, a greater number of ferritin cores were observed within the lysosome, and total cellular ferritin, as assessed by Western blot, increased. The formation of hemosiderin was also observed. Furthermore, nuclear shape was distorted in iron-loaded hepatocytes. The extent of deviation from circularity in the nucleus correlated with increasing concentrations of TMH-ferrocene and was greater in hepatocytes exposed to FeSO4 than an equivalent concentration of TMH-ferrocene. The deviation from circularity was smallest in hepatocytes that contained well formed ferritin cores and increased in hepatocytes that contained greater amounts of hemosiderin. Furthermore, in hepatocytes treated with FeSO4, a large amount of cell-associated iron was detected but without a significant increase in the total amount of ferritin. The deviation from circularity was the largest in FeSO4-treated hepatocytes, indicating that iron not properly incorporated into ferritin caused more cellular damage. We conclude that iron-loaded hepatocytes in long-term DMSO culture represent a flexible system for studying the effects of chronic iron loading on hepatocytes.     

Iron metabolism in hemodialyzed patients – a story half told?

Introduction

All living organisms have evolved sophisticated mechanisms to maintain appropriate iron levels in their cells and within their body. Recently our understanding of iron metabolism has dramatically increased. Overt labile plasma iron (LPI) represents a component of non-transferrin bound iron (NTBI) that is both redox active and chelatable, capable of permeating into organs and inducing tissue iron overload. The LPI measures the iron-specific capacity of a given sample to produce reactive oxygen species. We studied for the first time NTBI correlations with markers of iron status and inflammation in prevalent hemodialyzed patients.

Material and methods

Complete blood count, urea, serum lipids, fasting glucose, creatinine, ferritin, serum iron, total iron binding capacity (TIBC) were studied by standard laboratory method. The NTBI was assessed commercially available kits from Aferrix Ltd in Tel Aviv, Israel. A test result of 0.6 units of LPI or more indicates a potential for iron-mediated production of reactive oxygen species in the sample.

Results

Patients with LPI units ≥ 0.6 had higher serum iron, erythropoiesis stimulating agents (ESA) dose, ferritin, high-sensitivity C-reactive protein (hsCRP), hepcidin and lower hemojuvelin. In hemodialyzed patients NTBI correlated with hsCRP (r = 0.37, p < 0.01), ferritin (r = 0.41, p < 0.001), IL-6 (r = 0.43, p < 0.001). In multivariate analysis predictors of NTBI were hemoglobin and alkaline phosphatase, explaining 58% of the variability

Conclusions

Elevated NTBI in HD may be due to disturbed iron metabolism. Anemia and liver function might also contribute to the presence of NTBI in this population.     

Characterization of hepatic iron overload following dietary administration of dicyclopentadienyl iron (Ferrocene) to mice: cellular, biochemical, and molecular aspects

A unique organic form of iron (dicyclopentadienyl iron; ferrocene) has been used to further elucidate specific hepatic histopathologic, biochemical, and molecular parameters associated with dietary iron overload. Male C57BL/6Ibg mice fed a diet containing 0.04-0.2% w/w ferrocene for 115 days displayed severe hepatic siderosis of hepatocytes accompanied by a 15-fold induction of nonheme iron content compared to control mice receiving a diet with normal amounts of iron. The ferrocene treatment led to significant increases in hepatocellular necrosis as measured by plasma alanine aminotransferase activity. Histological assessment of hepatic fibrosis revealed mild increases in collagen deposition localized with accumulations of hemosiderin primarily in centrilobular hepatocytes. Hepatic fibrosis was confirmed by measurement of hepatic hydroxyproline content that was increased 4-fold in ferrocene-fed animals compared to control animals not ingesting ferrocene. Hepatic siderosis was accompanied by significant increases in hepatic malondialdehyde content suggesting the ferrocene-induced iron burden initiated lipid peroxidation in vivo. Expression of the heavy-chain isoform of ferritin mRNA and protein measured in liver after ferrocene feeding was increased approximately 8- and 2-fold, respectively, compared to the appropriate controls. These results, using an organic form of iron fed to genetically well-characterized inbred mice, provide new additional insight into the specific molecular and biochemical events that occur in association with histopathologic changes initiated by iron-induced liver injury. These data support the hypothesis that peroxidation of cellular membrane lipids is an important mechanism involved in the toxicity of excess hepatic iron and possibly the initiation of liver fibrogenesis. The results presented here also provide novel in vivo evidence documenting the cellular modulation of ferritin in response to the toxic effects of hepatic iron overloading and iron-mediated oxidative stress.     

Genetic differences in hepatic lipid peroxidation potential and iron levels in mice.

Oxidative damage to macromolecules, including lipids, has been hypothesized as a mechanism of aging. One end product of lipid peroxidation, malondialdehyde (MDA), is often quantified as a measure of oxidative damage to lipids. We used a commercial colorimetric assay for MDA (Bioxytech LPO-586, Oxis International, Portland, OR) to measure lipid peroxidation potential in liver tissue from young (2 month) male mice from recombinant inbred (RI) mouse strains from the C57BL/6J (B6)xDBA/2J (D2) series (BXD). The LPO-586 assay (LPO) reliably detected significant differences (P<0.0001) in lipid peroxidation potential between the B6 and D2 parental strains, and yielded a more than two-fold variation across the BXD RI strains. In both B6 and D2 mice, LPO results were greater in old (23 month) mice, with a larger age-related increase in the D2 strain. As the level of iron can influence lipid peroxidation, we also measured hepatic non-heme iron levels in the same strains. Although iron level exhibited a slightly negative overall correlation (r(2)=0.119) with LPO results among the entire group of BXD RI strains, a sub-group with lower LPO values were highly correlated (r(2)=0.704). LPO results were also positively correlated with iron levels from a group of 8 other inbred mouse strains (r(2)=0.563). The BXD RI LPO data were statistically analyzed to nominate quantitaive trait loci (QTL). A single marker, Zfp4, which maps to 55.2 cM on chromosome 8, achieved a significance level of P<0.0006. At least two potentially relevant candidate genes reside close to this chromosomal position. Hepatic lipid peroxidation potential appears to be a strain related trait in mice that is amenable to QTL analysis.     

NADPH oxidase in bone marrow-derived cells mediates pulmonary ischemia-reperfusion injury

Reactive oxygen species (ROS) play a crucial role in ischemia-reperfusion (IR) injury after lung transplantation. We hypothesized that NADPH oxidase derived from bone marrow (BM) cells contributes importantly to lung IR injury. An in vivo mouse model of lung IR injury was employed. Wild-type C57BL/6 (WT) mice, p47(phox) knockout (p47(phox)-/-) mice, or chimeras created by BM transplantation between WT and p47(phox)-/- mice were assigned to either Sham (left thoracotomy) or six study groups that underwent IR (1 h left hilar occlusion and 2 h reperfusion). After reperfusion, pulmonary function was assessed using an isolated, buffer-perfused lung system. Lung injury was assessed by measuring vascular permeability (via Evans blue dye), edema, neutrophil infiltration (via myeloperoxidase [MPO]), lipid peroxidation (via malondialdyhyde [MDA]), and expression of proinflammatory cytokines. Lung IR resulted in significantly increased MDA in WT mice, indicative of oxidative stress. WT mice treated with apocynin (an NADPH oxidase inhibitor) and p47(phox)-/- mice displayed significantly reduced pulmonary dysfunction and injury (vascular permeability, edema, MPO, and MDA). In BM chimeras, significantly reduced pulmonary dysfunction and injury occurred after IR in p47(phox)-/--->WT chimeras (donor-->recipient) but not WT-->p47(phox)-/- chimeras. Induction of TNF-alpha, IL-17, IL-6, RANTES (CCL5), KC (CXCL1), MIP-2 (CXCL2), and MCP-1 (CCL2) was significantly reduced after IR in NADPH oxidase-deficient mice and p47(phox)-/--->WT chimeras but not WT-->p47(phox)-/- chimeras. These results indicate that NADPH oxidase-generated ROS specifically from BM-derived cells contributes importantly to lung IR injury. NADPH oxidase may represent a novel therapeutic target for the treatment of IR injury after lung transplantation.     

Lack of ataxia telangiectasia mutated kinase induces structural and functional changes in the heart: role in β-adrenergic receptor-stimulated apoptosis

Ataxia telangiectasia mutated kinase (ATM) is involved in cell cycle checkpoints, DNA repair and apoptosis. β-Adrenergic receptor (β-AR) stimulation induces cardiac myocyte apoptosis. Here we analysed basal myocardial structure and function in ATM knockout (KO) mice and tested the hypothesis that ATM modulates β-AR-stimulated myocyte apoptosis. Left ventricular (LV) structure and function, myocyte apoptosis, fibrosis and expression of fibrosis-, hypertrophy- and apoptosis-related proteins were examined in wild-type (WT) and KO mice with or without l-isoprenaline treatment for 24 h. Body and heart weights were lower in KO mice. M-Mode echocardiography showed reduced septal wall thicknesses and LV diameters in KO mice. Doppler echocardiography showed an increased ratio of early peak velocity (E wave) to that of the late LV filling (A wave) in KO mice. Basal fibrosis and myocyte cross-sectional area were greater in KO hearts. Expression of fibrosis-related genes (connective tissue growth factor and plasminogen activator inhibitor-1) and hypertrophy-related gene (atrial natriuretic peptide) was higher in KO hearts. β-Adrenergic receptor stimulation increased myocyte apoptosis to a similar extent in both groups. Activation of c-Jun N-terminal kinases and expression and phosphorylation of p53 in response to β-AR stimulation were only observed in the WT group. Akt phosphorylation was lower in KO sham-treated animals and remained lower following β-AR stimulation in the KO group. β-Adrenergic receptor stimulation activated glycogen synthase kinase-3β to a similar extent in both groups. Thus, lack of ATM induces structural and functional changes in the heart, with enhanced myocardial fibrosis and myocyte hypertrophy. β-Adrenergic receptor-stimulated apoptosis in WT hearts is associated with a p53- and JNKs-dependent mechanism, while decreased Akt activity may play a role in increased myocyte apoptosis in the absence of ATM.     

Adiponectin and skeletal muscle: pathophysiological implications in metabolic stress

Upregulation of muscular adiponectin could act as a local protective mechanism to counteract cellular damage in obesity by weakening inflammation, oxidative stress, and apoptosis. To test this hypothesis, adiponectin-knockout (KO) and wild-type (WT) mice were fed a Western diet (WD). WT mice under WD conditions displayed 63% higher adiponectin expression in myocytes than those under standard laboratory diet (SLD) conditions (P = 0.011). WD-fed KO mice exhibited approximately threefold larger myocyte degeneration than WT mice (P = 0.003). Even under SLD conditions, myotubes of KO mice displayed already moderate immunolabeling for markers of oxidative stress (peroxiredoxin-3/5) and for a lipid peroxidation product (hydroxynonenal). Expression of tumor necrosis factor-α (TNF-α) and caspase-6, a marker of apoptosis, was also present. After WD challenge, immunoreactivity for these markers was strong in muscle of KO mice, although it was detected to a lesser extent in WT mice. Activation of NF-κB and caspase-6 doubled in myocytes of WD-fed KO mice when compared to WT mice (P < 0.001). Furthermore, muscle electrotransfer of the adiponectin gene prevented these abnormalities in WD-fed KO mice. Finally, gene abrogation of the adiponectin receptor 1 (AdipoR1) by siRNA recapitulated a pro-inflammatory state in C2C12 myotubes. Thus, upregulation of muscular adiponectin may be triggered by obesity and be crucial locally to counteract oxidative stress, inflammation, and apoptosis. These effects operate in an autocrine/paracrine manner via AdipoR1 and down-regulation of NF-κB signaling.     

Interferon-γ deficiency reduces neointimal formation in a model of endoluminal endothelial injury combined with atherogenic diet

Interferon (IFN)-γ has been implicated in restenosis, however its precise role in the pathophysiology of neointimal formation following angioplasty is unclear, as it has been shown to both promote and inhibit neointimal formation. Dietary-induced hypercholesterolemia enhances injury-mediated neointimal formation, associated with increased systemic inflammation and serum IFN-γ. This study examined the effect of IFN-γ gene deficiency ((-/-)) on neointimal formation in a mouse model of endothelial injury combined with an atherogenic diet. Neointimal formation was induced via endoluminal endothelial injury of the common iliac arteries of IFN-γ(-/-) and wild-type (WT) C57Bl/6 mice. Histopathological analysis of the arteries was performed at 3 and 6 weeks post-surgery. IFN-γ(-/-) mice demonstrated a significant reduction in neointimal formation at the 3-week time point, compared to their WT counterpart. No significant differences in plasma lipid profile and the extent of re-endothelialization were detected between IFN-γ(-/-) and WT mice, suggesting that the effect of IFN-γ on neointimal formation is due to injury-mediated vessel neointimal responses. In support of the histopathological findings, immunohistochemical analysis revealed a significant reduction in vessel infiltrating macrophages, and neointimal PDGF-B expression, vascular smooth muscle cell composition and cellular proliferation in the IFN-γ(-/-) mice, in comparison to their corresponding WT group at the 3-week time point. In conclusion, the IFN-γ-mediated pathway plays an important role in inflammatory responses and proliferative effects following injury, suggesting that modulation of the IFN-γ pathway would be beneficial in controlling neointimal formation and restenosis.     

Absence of the β1 subunit of AMP‐activated protein kinase reduces myofibroblast infiltration of the kidneys in early diabetes

Activation of the heterotrimeric energy‐sensing kinase AMP‐activated protein kinase (AMPK) has been reported to improve experimental diabetic kidney disease. We examined the effect of type 1 diabetes in wild‐type (WT) mice and mice lacking the β1 subunit of AMPK (AMPK β1^?/? mice), which have reduced AMPK activity in kidneys and other organs. Diabetes was induced using streptozotocin (STZ) and the animals followed up for 4 weeks. Hyperglycaemia was more severe in diabetic AMPK β1^?/? mice, despite the absence of any difference in serum levels of insulin, adiponectin and leptin. There was no change in AMPK activity in the kidneys of diabetic WT mice by AMPK activity assay, or phosphorylation of either the αT172 activation site on the α catalytic subunit of AMPK or the AMPK‐specific phosphosite S79 on acetyl CoA carboxylase 1 (ACC1). Phosphorylation of the inhibitory αS485 site on the α subunit of AMPK was significantly increased in the WT diabetic mice compared to non‐diabetic controls. Despite increased plasma glucose levels in the diabetic AMPK β1^?/? mice, there were fewer myofibroblasts in the kidneys compared to diabetic WT mice, as evidenced by reduced α‐smooth muscle actin (α‐SMA) protein by Western blot, mRNA by qRT‐PCR and fewer α‐SMA‐positive cells by immunohistochemical staining. Albuminuria was also reduced in the AMPK β1^?/? mice. In contrast to previous studies, therefore, myofibroblasts were reduced in the kidneys of AMPK β1^?/? diabetic mice compared to diabetic WT mice, despite increased circulating glucose, suggesting that AMPK can worsen renal fibrosis in type 1 diabetes.