Heme-Scavenging Role of alpha1-Microglobulin in Chronic Ulcers.

Chronic venous ulcers are characterized by chronic inflammation. Heme and iron, originating from blood cell hemolysis as well as extravascular necrosis, have been implicated as important pathogenic factors due to their promotion of oxidative stress. It was recently reported that the plasma and tissue protein alpha1-microglobulin is involved in heme metabolism. The protein binds heme, and a carboxy-terminally processed form, truncated alpha1-microglobulin, also degrades heme. Here, we show the presence of micromolar levels of heme and free iron in chronic leg ulcer fluids. Micromolar amounts of alpha1-microglobulin was also present in the ulcer fluids and bound to added radiolabeled heme. Truncated alpha1-microglobulin was found in the ulcer fluids and exogenously added alpha1-microglobulin was processed into the truncated alpha1-microglobulin form. Histochemical analysis of chronic wound tissue showed the presence of iron deposits, heme/porphyrins in infiltrating cells basement membranes and fibrin cuffs around vessels, and alpha1-microglobulin ubiquitously distributed but especially abundant in basement membranes around vessels and at fibrin cuffs. Our results suggest that alpha1-microglobulin constitutes a previously unknown defense mechanism against high heme and iron levels during skin wound healing. Excessive heme and iron, which are not buffered by alpha1-microglobulin, may underlie the chronic inflammation in chronic ulcers

Characterization of heme binding to recombinant α1-microglobulin.

Alpha-1-microglobulin (A1M), a small lipocalin protein found in plasma and tissues, has been identified as a heme and radical scavenger that may participate in the mitigation of toxicities caused by degradation of hemoglobin. The objective of this work was to investigate heme interactions with A1M in vitro using various analytical techniques and to optimize analytical methodology suitable for rapid evaluation of the ligand binding properties of recombinant A1M versions

Plasma Heme Scavengers Alpha-l-Microglobulin and Hemopexin as Biomarkers in High-Risk Pregnancies

Women with established preeclampsia (PE) have increased plasma concentration of free fetal hemoglobin. We measured two hemoglobin scavenger system proteins, hemopexin (Hpx) and alpha-1 -microglobulin (A1M) in maternal plasma using enzyme-linked immunosorbent assay during the late second trimester of pregnancy in women with high and low risk of developing PE. In total 142 women were included in nested case-control study: 42 women diagnosed with PE and 100 controls (49 randomly selected high-risk and 51 low-risk controls). The concentration of plasma A1M in high-risk controls was higher compared to low-risk controls. Women with severe PE had higher plasma A1M levels compared to women with non-severe PE. In conclusion, the concentration of plasma A1M is increased in the late second trimester in high-risk controls, suggesting activation of endogenous protective system against oxidative stress.Peer reviewe

Hemopexin and α1-microglobulin heme scavengers with differential involvement in preeclampsia and fetal growth restriction

Hemopexin and α1-microglobulin act as scavengers to eliminate free heme-groups responsible for hemoglobin-induced oxidative stress. The present study evaluated maternal and fetal plasma concentrations of these scavengers in the different phenotypes of placenta-mediated disorders. Singleton pregnancies with normotensive fetal growth restriction [FGR] (n = 47), preeclampsia without FGR (n = 45) and preeclampsia with FGR (n = 51) were included prospectively as well as uncomplicated pregnancies (n = 49). Samples were collected at delivery and ELISA analysis was applied to measure the hemopexin and α1-microglobulin concentrations. In maternal blood in preeclampsia with and without FGR, hemopexin was significantly lower (p = 0.003 and p<0.001, respectively) and α1-microglobulin was significantly higher (p<0.001 in both) whereas no difference existed in normotensive FGR mothers compared to controls. In contrast, in fetal blood in growth restricted fetuses with and without preeclampsia, both hemopexin and α1-microglobulin were significantly lower (p<0.001 and p = 0.001 for hemopexin, p = 0.016 and p = 0.013 for α1-microglobulin, respectively) with no difference in fetuses from preeclampsia without FGR in comparison to controls. Thus, hemopexin and α1-microglobulin present significantly altered concentrations in maternal blood in the maternal disease -preeclampsia- and in cord blood in the fetal disease -FGR-, which supports their differential role in placenta-mediated disorders in accordance with the clinical presentation of these disorders

Up-Regulation of A1M/α1-Microglobulin in Skin by Heme and Reactive Oxygen Species Gives Protection from Oxidative Damage

During bleeding the skin is subjected to oxidative insults from free heme and radicals, generated from extracellular hemoglobin. The lipocalin α1-microglobulin (A1M) was recently shown to have reductase properties, reducing heme-proteins and other substrates, and to scavenge heme and radicals. We investigated the expression and localization of A1M in skin and the possible role of A1M in the protection of skin tissue from damage induced by heme and reactive oxygen species. Skin explants, keratinocyte cultures and purified collagen I were exposed to heme, reactive oxygen species, and/or A1M and investigated by biochemical methods and electron microscopy. The results demonstrate that A1M is localized ubiquitously in the dermal and epidermal layers, and that the A1M-gene is expressed in keratinocytes and up-regulated after exposure to heme and reactive oxygen species. A1M inhibited the heme- and reactive oxygen species-induced ultrastructural damage, up-regulation of antioxidation and cell cycle regulatory genes, and protein carbonyl formation in skin and keratinocytes. Finally, A1M bound to purified collagen I (Kd = 0.96×10−6 M) and could inhibit and repair the destruction of collagen fibrils by heme and reactive oxygen species. The results suggest that A1M may have a physiological role in protection of skin cells and matrix against oxidative damage following bleeding

Longitudinal changes in plasma hemopexin and alpha-1-microglobulin concentrations in women with and without clinical risk factors for pre-eclampsia

Recent studies have shown increased concentration of fetal hemoglobin (HbF) in pre-eclamptic women. Plasma hemopexin (Hpx) and alpha-1-microglobulin (A1M) are hemoglobin scavenger proteins that protect against toxic effects of free heme released in the hemoglobin degradation process. We used an enzyme-linked immunosorbent assay to analyze maternal plasma Hpx and A1M concentrations at 12–14, 18–20 and 26–28 weeks of gestation in three groups: 1) 51 women with a low risk for pre-eclampsia (LRW), 2) 49 women with a high risk for pre-eclampsia (PE) who did not develop PE (HRW) and 3) 42 women with a high risk for PE who developed PE (HRPE). The study had three aims: 1) to investigate whether longitudinal differences exist between study groups, 2) to examine if Hpx and A1M concentrations develop differently in pre-eclamptic women with small for gestational age (SGA) fetuses vs. pre-eclamptic women with appropriate for gestational age fetuses, and 3) to examine if longitudinal Hpx and A1M profiles differ by PE subtype (early-onset vs. late-onset and severe vs. non-severe PE). Repeated measures analysis of variance was used to analyze differences in Hpx and A1M concentrations between the groups. We found that the differences in longitudinal plasma Hpx and A1M concentrations in HRW compared to HRPE and to LRW may be associated with reduced risk of PE regardless of clinical risk factors. In women who developed PE, a high A1M concentration from midgestation to late second trimester was associated with SGA. There were no differences in longitudinal Hpx and A1M concentrations from first to late second trimester in high-risk women who developed early-onset or. late-onset PE or in women who developed severe or. non-severe PE.Peer reviewe

Longitudinal changes in plasma hemopexin and alpha-1-microglobulin concentrations in women with and without clinical risk factors for pre-eclampsia

Recent studies have shown increased concentration of fetal hemoglobin (HbF) in pre-eclamptic women. Plasma hemopexin (Hpx) and alpha-1-microglobulin (A1M) are hemoglobin scavenger proteins that protect against toxic effects of free heme released in the hemoglobin degradation process. We used an enzyme-linked immunosorbent assay to analyze maternal plasma Hpx and A1M concentrations at 12–14, 18–20 and 26–28 weeks of gestation in three groups: 1) 51 women with a low risk for pre-eclampsia (LRW), 2) 49 women with a high risk for pre-eclampsia (PE) who did not develop PE (HRW) and 3) 42 women with a high risk for PE who developed PE (HRPE). The study had three aims: 1) to investigate whether longitudinal differences exist between study groups, 2) to examine if Hpx and A1M concentrations develop differently in pre-eclamptic women with small for gestational age (SGA) fetuses vs. pre-eclamptic women with appropriate for gestational age fetuses, and 3) to examine if longitudinal Hpx and A1M profiles differ by PE subtype (early-onset vs. late-onset and severe vs. non-severe PE). Repeated measures analysis of variance was used to analyze differences in Hpx and A1M concentrations between the groups. We found that the differences in longitudinal plasma Hpx and A1M concentrations in HRW compared to HRPE and to LRW may be associated with reduced risk of PE regardless of clinical risk factors. In women who developed PE, a high A1M concentration from midgestation to late second trimester was associated with SGA. There were no differences in longitudinal Hpx and A1M concentrations from first to late second trimester in high-risk women who developed early-onset or. late-onset PE or in women who developed severe or. non-severe PE.</p

Synthesis of α1-microglobulin in cultured rat hepatocytes is stimulated by interleukin-6, leukemia inhibitory factor, dexamethasone and retinoic acid

AbstractThe secretion or α1-microglobulin by primary cultures of rat hepatocytes was found to increase upon the addition of interleukin-6 or leukemia inhibitory factor, two mediators of acute phase response. This stimulatory effect was further enhanced by dexamethasone. α1-Microglobulin is synthesized as a precursor also containing bikunin, and the precursor protein is cleaved shortly berore secretion. Our results therefore suggest that both α1-microglobulin and bikunin are acute phase reactants in rat hepatocytes. Furthermore, we found that retinoic acid, previously shown to be involved in the regulation of cell differentiation and development, also stimulated α1-microglobulin synthesis. Only free, uncomplexed α1-microglobulin (28,000 Da) was detected in the hopatocyte media, suggesting that the complex between α1-microglobulin and α1-inhibitor 3, found in rat serum, is formed outside the hepatocyte

Heme Induces Endoplasmic Reticulum Stress (HIER Stress) in Human Aortic Smooth Muscle Cells

Accumulation of damaged or misfolded proteins resulted from oxidative protein modification induces endoplasmic reticulum (ER) stress by activating the pathways of unfolded protein response. In pathologic hemolytic conditions, extracellular free hemoglobin is submitted to rapid oxidation causing heme release. Resident cells of atherosclerotic lesions, after intraplaque hemorrhage, are exposed to heme leading to oxidative injury. Therefore, we raised the question whether heme can also provoke ER stress. Smooth muscle cells are one of the key players of atherogenesis; thus, human aortic smooth muscle cells (HAoSMCs) were selected as a model cell to reveal the possible link between heme and ER stress. Using immunoblotting, quantitative polymerase chain reaction and immunocytochemistry, we quantitated the markers of ER stress. These were: phosphorylated eIF2α, Activating transcription factor-4 (ATF4), DNA-damage-inducible transcript 3 (also known as C/EBP homology protein, termed CHOP), X-box binding protein-1 (XBP1), Activating transcription factor-6 (ATF6), GRP78 (glucose-regulated protein, 78kDa) and heme responsive genes heme oxygenase-1 and ferritin. In addition, immunohistochemistry was performed on human carotid artery specimens from patients who had undergone carotid endarterectomy. We demonstrate that heme increases the phosphorylation of eiF2α in HAoSMCs and the expression of ATF4. Heme also enhances the splicing of XBP1 and the proteolytic cleavage of ATF6. Consequently, there is up-regulation of target genes increasing both mRNA and protein levels of CHOP and GRP78. However, TGFβ and collagen type I decreased. When the heme binding proteins, alpha-1-microglobulin (A1M) and hemopexin (Hpx) are present in cell media, the ER stress provoked by heme is inhibited. ER stress pathways are also retarded by the antioxidant N-acetyl cysteine (NAC) indicating that reactive oxygen species are involved in heme-induced ER stress. Consistent with these findings, elevated expression of the ER stress marker GRP78 and CHOP were observed in smooth muscle cells of complicated lesions with hemorrhage compared to either atheromas or healthy arteries. In conclusion, heme triggers ER stress in a time- and dose-dependent manner in HAoSMCs. A1M and Hpx as well as NAC effectively hamper heme-induced ER stress, supporting their use as a potential therapeutic approach to reverse such a deleterious effects of heme toxicity