Lung alveoli are lined by alveolar type (AT) 1 cells and cuboidal AT2 cells. The AT1 cells are likely to be exposed to cell-free hemoglobin (Hb) in multiple lung diseases; however, the role of Hb redox (reduction–oxidation) reactions and their precise contributions to AT1 cell injury are not well understood. Using mouse lung epithelial cells (E10) as an AT1 cell model, we demonstrate here that higher Hb oxidation states, ferric Hb (HbFe3+) and ferryl Hb (HbFe4+) and subsequent heme loss play a central role in the genesis of injury. Exposures to HbFe2+ and HbFe3+ for 24 hours induced expression of heme oxygenase (HO)-1 protein in E10 cells and HO-1 translocation in the purified mitochondrial fractions. Both of these effects were intensified with increasing oxidation states of Hb. Next, we examined the effects of Hb oxidation and free heme on mitochondrial bioenergetic function by measuring changes in the mitochondrial transmembrane potential and oxygen consumption rate. In contrast to HbFe2+, HbFe3+ reduced basal oxygen consumption rate, indicating compromised mitochondrial activity. However, HbFe4+ exposure not only induced early expression of HO-1 but also caused mitochondrial dysfunction within 12 hours when compared with HbFe2+ and HbFe3+. Exposure to HbFe4+ for 24 hours also caused mitochondrial depolarization in E10 cells. The deleterious effects of HbFe3+ and HbFe4+ were reversed by the addition of scavenger proteins, haptoglobin and hemopexin. Collectively, these data establish, for the first time, a central role for cell-free Hb in lung epithelial injury, and that these effects are mediated through the redox transition of Hb to higher oxidation states.
We describe a novel oxidative pathway by which ferric and ferryl hemoglobin (Hb) oxidation products induce cellular injury and mitochondrial dysfunction, which may contribute to inflammatory responses and vascular toxicity. Intervention with endogenous plasma–derived antioxidants may control Hb oxidative side reactions and offer potential therapeutic remedies in hemolytic anemias and when free Hb is used as oxygen therapeutics.
Red blood cells are highly enriched in hemoglobin (Hb), a tetrameric protein consisting of two α and two β subunits (α2β2). Each subunit has a single, iron-containing protoporphyrin (heme) moiety. The highly reducing environment within red blood cells (RBCs) prevent the oxidation of Hb and maintains the functional ferrous form (HbFe2+) (1). Small amounts of Hb are released to circulation due to aging and during the process of RBC renewal. The released Hb tetramers dissociate rapidly into αβ dimers, which can be cleared by binding to haptoglobin (Hp) to form a complex of Hp–Hb (2, 3). The complex then binds to CD163 receptor for endocytosis and degradation in the lysosomes (4). Heme oxygenases (HOs) catalyze the degradation of heme to generate carbon monoxide, free heme iron, and biliverdin. Finally, biliverdin reductase further catalyzes the conversion of biliverdin to bilirubin. Both ferritin, which sequesters the free iron, and HO-1 are induced by multiple stimuli, including the presence of acellular Hb and heme (5).
Acellular Hb concentration increases after excessive hemolysis in malaria, sickle cell disease, blood transfusion, and infusion of Hb-based oxygen carriers. Severe vasoconstriction due to nitric oxide sequestration by Hb is thought to be a cause of Hb-induced toxicity in hemolytic anemias and in therapeutic uses of Hb (6, 7). It is increasingly becoming evident that heme loss after Hb oxidation may also play a significant role in the toxicity associated with acellular Hb. During auto-oxidation, pro-oxidants, such as the superoxide ion (O2−) and hydrogen peroxide (H2O2) drive a catalytic pseudoperoxidase cycle that oxidize HbFe2+ into HbFe3+, HbFe4+, and the ferryl radical (⋅HbFe4+). These oxidatively unstable Hb intermediates oxidize residues within the Hb globin chains (and other proteins within proximity), ultimately leading to Hb degradation and heme loss (8). Our recent study indicated that heme derived from the hemolysis of sickle RBCs acts as a damage-associated molecular pattern molecule (DAMP) (9). A number of highly efficient endogenous scavenging mechanisms exist to sequester acellular Hb and heme, including Hp, hemopexin (Hpx), albumin, and α-Hb stabilizing protein (10). Hp, an acute-phase protein is predominantly expressed in the liver, lung, skin, and spleen (11). Hp attenuated Hb-induced vascular, cardiac, and renal injury after infusion of free Hb and aged RBCs in dogs, guinea pigs, and in a mouse model of sickle cell disease, mainly by preventing extravasation of Hb through the kidney. Moreover, Hp–Hb complex was also shown to diffuse the damaging ferryl radicals associated with the vicious ferric/ferryl cycle (12–15).
Diffuse intra-alveolar hemorrhage, a life-threatening condition, occurs due to accumulation of RBCs in the lung alveoli (16). Extravasation of blood into air spaces is also common in multiple other lung diseases, such as chronic bronchitis, bronchiectasis, cystic fibrosis, lung cancer, pulmonary embolism, pneumonia, tuberculosis, and traumatic injury (17). Subsequent RBC lysis leads to release of acellular Hb, which, in turn, damages the alveolar epithelial cells. Pulmonary complication of hemolytic diseases, such as sickle cell disease, have been reported (18, 19). Lung alveoli are the functional units of respiration. Lung alveoli are lined by squamous alveolar type (AT) 1 and cuboidal AT2 cells. The AT1 cells are also likely to be exposed to very high concentrations of acellular Hb, due to their anatomical predisposition (occupy >95% of lung alveolar surface area and close apposition to endothelium) and inherent sensitivity. However, the mechanisms of acellular Hb-induced effects of lung epithelial cells, particularly on AT1 cells, remain unknown.
Mitochondria are considered to be the most critical regulators of cell survival and death, due to their roles in the maintenance of cellular energy homeostasis and regulation of redox-dependent signaling pathways (20). Functional mitochondrial bioenergetics have recently been advocated for use as a bioenergetic health index in humans (21). However, mitochondrial bioenergetic analyses have not been explored extensively in the context of Hb cytotoxicity. Recently, however, heme and cell-free Hb have been shown to disrupt mitochondrial bioenergetic function in different cell types (22–24). Using lung epithelial cells (E10) as an AT1 cell model, we investigated the differential toxicity of acellular Hb and its oxidized species on the mitochondrial transmembrane potential and also on oxygen consumption rate (OCR). Furthermore, the roles of Hp (Hb scavenger) and Hpx (heme scavenger) were also investigated in attenuating Hb-induced injury.
Adult HbFe2+ and HbFe3+ were prepared as previously described (25). HbFe4+ was produced by addition of 10-fold molar excess of H2O2 to endotoxin-free HbFe3+ in potassium phosphate buffer (pH 7.4). Spectral identification and quantification of HbFe2+ and HbFe3+ were performed based on published extinction coefficients (25). The HbFe4+ was quantified using sodium sulfide (NasS) derivatization, as previously described (26). The concentrations of HbFe2+, HbFe3+, and HbFe4+ were expressed as heme equivalents.
Mouse E10 cells were cultured in CMRL-1066 supplemented with 10% FBS and antibiotics. The cells were used between passages 8 and 20. The media were changed every 48 hours (for detailed protocols, see the online supplement).
E10 cells were grown to 80–90% confluency in complete media. Before exposures, the cells were serum starved overnight. The cells were then exposed to HbFe2+, HbFe3+, or HbFe4+ for varying periods of time (12 or 24 h). For studying the role of Hp, human plasma–derived unfractionated Hp (50 μM) was added to the media before the addition of Hb. HbFe2+, HbFe3+, and HbFe4+ (in equimolar ratio to Hp) were then added immediately. After exposure to Hb proteins for specified time periods, cells were washed extensively in ice-cold PBS and cell lysates were prepared for further studies.
Mitochondria were isolated from cultured E10 cells using a mitochondria isolation kit for cultured cells (Pierce Biotechnology, Rockford, IL).
Immunoblotting was done as previously reported (27). The primary antibodies used were in 1:2,500 dilutions. The proteins were visualized using enhanced chemiluminescence kit (GE Healthcare, Piscataway, NJ). The expression of HO-1 and ferritin, α, and β subunits of Hb was not detectable in unexposed cells. For quantification, the density of HO-1 was normalized with density of β-actin. Fold expression was obtained by comparing the normalized expression in unexposed cells.
The cells were exposed to the indicated concentrations of HbFe2+, HbFe3+, and HbFe4+. Immunocytochemistry was performed as described previously (27). The colocalization of the HO-1 protein with Mito Tracker Red CMXRos (Thermo Fisher Scientific, Waltham, MA) was visualized using an LSM710 Meta Laser-scanning confocal microscope (Zeiss, Thornwood, NY).
Loss of mitochondrial transmembrane potential was assessed in E10 cells using a cationic lipophilic dye, tetraethyl-benzimidazolyl carbocyanine iodide (JC-1). The cells were exposed to the Hb proteins, as indicated earlier for 24 hours. The cells were thoroughly washed four to five times in prewarmed PBS to remove excess unbound Hb, then loaded with JC-1 dye (8 μM) for 30 minutes as described previously (24). After removal of excess dye, cells were detached using 0.025% trypsin-EDTA and washed in PBS. Cell suspensions were analyzed for red fluorescence (λex 530 nm, λem 590 nm) for J-aggregates (indicative of hyperpolarization) and green fluorescence (λex 490 nm, λem 530nm) from JC-1 monomer (indicative of low mitochondrial transmembrane potential or depolarization) in a Synergy HTX multi-mode plate reader (Biotek Instruments, Inc. Winooski, VT). The ratios were then plotted on a percentage scale in which the ratio from oligomycin (1 μM)-treated cells indicates 100% hyperpolarization (maximum mitochondrial transmembrane potential) and ratio from uncoupler carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone (FCCP 1 μM)-treated cells indicates 0% or complete depolarization (22). The percentage values thus obtained from Hb-treated cells were represented as the percent of hyperpolarized cells. The emission fluorescence ratio of 590 nm versus 530 nm from Hb-treated cells that were not incubated with JC-1 was also monitored to eliminate any Hb interference (see Figure E2 in the online supplement).
Mitochondrial bioenergetic function and the glycolytic flux were simultaneously monitored in intact E10 cells using an XF24 extracellular flux analyzer (Seahorse Bioscience, Billerica, MA). Briefly, E10 cells were seeded (20,000 cells/well) and cultured for 24 hours in a specialized 24-well XF cell culture plate (V7) obtained from Seahorse Bioscience. Before exposure, the cells were cultured in serum-free media overnight. They were then exposed to either HbFe2+ or HbFe3+ or HbFe4+ for various time periods up to 24 hours. The cells were washed thoroughly in unbuffered XF-assay medium (pH 7.4). Mitochondrial OCR and extracellular acidification rate (ECAR) were assessed as described previously (24). The OCR values from individual wells were recorded and plotted using XF24 software, version 1.8. The values were normalized with total protein in individual wells after the completion of the assay. Blank wells with Hb variants were also run to eliminate any background OCR. Different Hb species were also injected directly to the cells grown on V7 plates before the addition of oligomycin to assess Hb interference in the XF assay. However, in our experimental setup, Hb species showed no noticeable interference on OCR or ECAR. Various cellular bioenergetic parameters, such as basal respiration, ATP-linked respiration, proton leak, maximal OCR, and reserve respiratory capacity, were calculated as described previously (28).
All values are expressed as mean (±SEM). A value of P less than 0.05 was considered significant. The different treatments were compared using unpaired Student’s t test.
Highly purified human HbFe2+ and HbFe3+ were used in this study. The catalase activity was reduced by over 98%. The levels of HbFe2+ and HbFe3+ in these preparations contained greater than 95% ferrous or ferric heme, respectively. These preparations were further cleared of contaminating endotoxin using polymyxin B immobilized agarose gel columns. Endotoxin levels were confirmed to be lower than 5 EU/ml in all stock solutions. Similarly, final HbFe4+ solutions contained over 95% ferryl heme. Typical ultraviolet-visible absorbance spectroscopy of the main redox states of Hb is shown in Figure 1. Ferrous/oxy Hb (red) is characterized by two major absorption peaks at 541 and 576 nm, respectively. The oxidized form of Hb (ferric; brown) absorbs strongly at 500 and 630 nm, respectively. The spectrum of ferryl Hb (HbFe4+; green) in the visible region is characterized by two peaks at 544 and 585 nm, and a flattened region between 600 and 700 nm. To confirm the redox identity of the ferryl state intermediate, sodium sulfite was added to convert it to a sulfhemoglobin (purple), which absorbs very strongly at 620 nm.
Mouse E10 cells were exposed to increasing concentration of HbFe2+ and HbFe3+ for 24 hours. Hb is known to dimerize at lower concentration, while it remains a tetramer at higher concentration (29). We observed a dose-dependent increase in HO-1 after exposure to HbFe2+ and HbFe3+, whereas the untreated control cells did not express HO-1 protein (Figure 2A). We monitored the expression of P2X7 receptor protein, a phenotypic marker of AT1 cells, after exposure to HbFe2+ and HbFe3+ and, as can be seen in Figure 2A, there were no alterations in the expression of P2X7 receptor protein. Exposure to Hb and its oxidized species did not result in cell death. The cell viability was over 90% after exposure to different exposure regimens, as shown by trypan blue and propidium iodide/annexin V staining. We may therefore rule out the induction of apoptosis as described previously (30). Similarly, exposure to HbFe2+ and HbFe3+ did not induce or alter the expression of heat shock protein of 70 kD (Hsp70) (a stress marker protein) or proliferation cell nuclear antigen (proliferation marker) in E10 cells. Thus, our exposure regimens did not adversely affect the phenotype, cell viability, or differentiation in E10 cells. We also found that HbFe2+ and HbFe3+ activate NF-κB and mitogen-activated protein kinase pathways, as shown by phosphorylation of NF-κB p65 subunit and p44/42, respectively (Figure E3) as noted previously (31).
Hp is a potent scavenger of acellular Hb, and is known to reduce Hb-induced injury. Hp attenuated HbFe2+- and HbFe3+-induced up-regulation of HO-1 and H-ferritin proteins (Figure 2B). Exposure to ferric Hb (HbFe3+) induced a significant expression in HO-1 protein– (15.17 ± 1.04-fold) when compared with HbFe2+ (9.32 ± 0.76-fold)-induced expression (Figure 2C). Similarly, HbFe3+ induced a significant expression H-ferritin protein (60.40 ± 2.76-fold) when compared with a 25.25 (±1.91)-fold induction by HbFe2+ (Figure 2D). Western blotting of cell lysates indicated the presence of significant amounts of Hb α and β subunits. Furthermore, mass spectrometric analysis of lysates unequivocally confirmed the identity of human Hb α and β subunits in the cell lysates (data not shown). Hp reduced the uptake of Hb, as indicated by reduced Hb α and β subunits in the cell lysates (Figure 2E).
Previous studies have shown hemin, cigarette smoke, and indomethacin induced the translocation of HO-1 into the mitochondria (32–34). We reasoned that the increased HO-1 expression in E10 cells might also be associated with its translocation into the mitochondria. The mitochondrial and cytosolic fractions were isolated, purified, and then probed for the expression of HO-1 protein. We found a significant enrichment of HO-1 in the mitochondrial, but not in the cytosolic fractions after exposure to HbFe2+ and HbFe3+ (Figures 3A and 3B). Isolation and equal loading of the mitochondrial proteins were confirmed by probing the fractions with cytochrome c oxidase IV, a mitochondrial respiratory chain complex protein. Exposure to HbFe2+ and HbFe3+ did not alter the expression of cytochrome c oxidase IV protein (Figure 3A). Interestingly, Hp attenuated the HbFe2+- and HbFe3+-induced HO-1 expression in the mitochondria (Figures 3A and 3B).
Extracellular flux analyzer was used to assess the mitochondrial bioenergetics and glycolytic proton flux in real time. The OCR and ECAR serve as indicators of mitochondrial respiration and cellular glycolytic flux, respectively. E10 cells were exposed to HbFe2+ and HbFe3+ for 24 hours, as described earlier. The changes in OCR and ECAR were then examined. Exposure to HbFe3+, but not HbFe2+, caused a significant decrease in basal OCR in the E10 cells. This effect indicates a loss of mitochondrial bioenergetic function after exposure to HbFe3+ (Figures 3C and 3D). However, coincubation with Hp completely restored the HbFe3+-induced decrease in OCR (Figure 3C). No significant changes in the ECAR after exposure to HbFe2+ and HbFe3+ for 24 hours were observed in E10 cells (data not shown).
HbFe4+ is a highly reactive and proinflammatory species produced during the oxidation of HbFe2+ or HbFe3+ with H2O2. E10 cells were exposed to freshly prepared HbFe4+ for 12–24 hours. Exposure to HbFe4+ caused a significant up-regulation of HO-1 within 12 hours when compared with HbFe2+ and HbFe3+ (Figures 4A and 4B). We found significant enrichment of HO-1 in mitochondrial fraction after exposure to HbFe3+ and HbFe4+ (Figure 4C). To support this finding, we also employed immunocytochemical staining using laser confocal microscopy to confirm colocalization of HO-1 protein within the mitochondria in Hb-treated cells. Mitochondria were stained with a highly selective fluorescent probe, Mito Tracker Red CMXRos (Figure 4D). We found a significant colocalization of HO-1 protein within the mitochondria, as indicated by the appearance of strong merged (yellow) fluorescence after exposures to either HbFe3+ or HbFe4+ (Figure 4D).
Mitochondrial activity is routinely assessed by monitoring mitochondrial transmembrane potential (Δψm) using JC-1, a potentiometric dye (35, 36). The dye preferentially accumulates in the mitochondria, as indicated by the high 590:530 nm emission fluorescence ratio. The ratio decreases drastically with a loss of mitochondrial bioenergetic function (23). We monitored the changes in the red:green ratio and expressed them on a percent scale drawn from the ratio obtained from oligomycin (100% hyperpolarized cells) and from FCCP (0% or completely depolarized cells). Interestingly, incubation of E10 cells with HbFe2+ for 24 hours resulted in a significant rise in the Δψm. However, incubation with HbFe3+ showed no hyperpolarization over untreated control (Figure 5A). Exposure to HbFe4+ resulted in a drop in hyperpolarized cell percentage over untreated control, thus indicating a significant mitochondrial depolarization or compromised mitochondrial respiration. We assumed that this HbFe4+-mediated loss of Δψm is due to its oxidative reactivity. We confirmed heme-mediated loss of Δψm after exposure of E10 cells to hemin (10 μM) for 24 hours. Hemin drastically reduced Δψm, as indicated by a greater fall of 590:530 nm emission ratio, thus explaining the HbFe4+-mediated loss of Δψm. Hpx, an endogenous scavenger of heme restored heme-induced decrease in Δψm (Figure 5A). The Hb-treated cells that were not incubated with JC-1 showed no significant fluorescence at 590 nm compared with the JC-1–incubated cells (Figure E2).
Interestingly, HbFe4+ also caused a significant loss in basal cellular oxygen consumption, indicating an impaired mitochondrial respiration (Figures 5B and 5C). ATP linked respiration, an important bioenergetic parameter, was also significantly altered by HbFe4+ without affecting maximal OCR and reserve respiratory capacity. Exposure to HbFe2+ and HbFe3+ did not affect mitochondrial respiration within short-time exposure (12 h). However, we have seen a potentiation of reserve respiratory capacity by HbFe2+, probably due to an additional nonspecific uncoupling effect by HbFe2+. Hp, when incubated with HbFe4+, partially reversed the inhibitory effects of HbFe4+ on OCR (Figures 5B and 5C). However, HbFe4+ did not cause any alteration in the ECAR (data not shown).
E10 cells, where exposed to 25 μM (heme) HbFe2+, HbFe3+, or HbFe4+ separately in the presence and absence of equimolar amount of Hpx. We found that Hpx effectively attenuated HO-1 induction by all redox forms of Hb. Slightly less inhibition was seen in the presence of HbFe4+, however, possibly due to a slower rate of heme release from the ferryl protein (unpublished observations; Figure E1).
Several recent cell culture experiments in which Hb toxicity were investigated have shown conflicting outcomes as to the precise contribution of Hb oxidation states. For example, HbFe4+ (20 μM), but not HbFe3+ or HbFe2+, induced cytoskeletal reorganization, inflammation and disrupted barrier integrity in endothelial cells after 8 hours of incubation with the proteins. Furthermore, the toxicity of HbFe3+ was attributed to its contamination with LPS (37). In the same study, it was reported that HbFe4+ did not elicit any inflammatory response in macrophages/monocytes (37). Exposure to a higher concentration of HbFe4+ (100 μM) increased endothelial cell permeability and, in the case of a cross-linked Hb (α-bis[3,5-dibromosalicyl] fumarate), HbFe4+ caused cell death (38, 39). A recent study ruled out any significant contribution of metHb-iron and redox-mediated reactions in human lung AT2 cells (31). It is thus evident that Hb toxicity depends on the cell type, duration of exposure, and, more importantly, on the nature of heme/iron redox states used in these experiments. Because Hb undergoes auto-oxidation (Fe2+→Fe3+) and autoreduction (Fe4+→Fe3+) spontaneously, controlling Hb redox transition becomes an important element in the validity of these studies.
Highly purified and well characterized redox forms of human Hb (50 μM, heme) were used in this study. Although the concentrations of Hb or its oxidized species in lung air spaces are not known, AT1 cells might be exposed to chronically low levels of acellular Hb in patients with acute lung injury, hemolytic disorders, and very high acute concentrations after massive blood and Hb-based oxygen carrier transfusion (>500 μM plasma heme ).
The exposure of AT1 (E10) cells to HbFe2+ and HbFe3+ for 24 hours under our experimental conditions did not affect proliferation, fibrosis, apoptosis, and loss of AT1 cell phenotype. Hb α and β subunits, however, were detected in the cell lysates after exposures to HbFe2+ and HbFe3+. Earlier studies have shown that endothelial cells endocytose Hb even when present in high–molecular weight complexes (41, 42). Although it is possible that some Hb is bound to cell membrane, up-regulation of HO-1 is a strong indication of the internalization of the protein in our experiment.
Oxidized Hbs are generally less stable than the ferrous forms, due to unfolding of the proteins and subsequent heme loss (25). Exposure of E10 cells under our experimental conditions to HbFe3+ resulted in a higher expression of HO-1 and H-ferritin when compared with HbFe2+, which may be due to the reported differences in the rates of heme release from the two proteins. Exposure of these cells to HbFe4+, on the other hand, elicited a robust increase in HO-1 expression when compared with HbFe2+, but these expressions were lower in intensity than HO-1 content induced by the ferric form. The persistence of the ferryl iron in solutions and its radical protein together are, however, more damaging to biological molecules and tissues than the HbFe3+ (24). Regardless of differences in the rates of heme release from these proteins, confocal microscopy images and immunoblotting of purified mitochondrial fractions confirmed that a portion of expressed HO-1 translocated into the mitochondria. HOs are predominantly localized in the microsomes, but translocation into the mitochondria under certain stressful conditions has been reported (32, 34). Mitochondrial translocation of HO-1 might enable detoxification of accumulated heme as a result of oxidation and unfolding of Hb. In the absence of such a phenomenon, the bioenergetics might be more severely compromised. A recent study indicated that expression of HO-1 targeted to mitochondria attenuated oxidative stress (43). These observations, taken together, suggest that mitochondrial translocation of HO-1 could serve as a cytoprotective mechanism against possible mitochondrial dysfunction induced by oxidative or metabolic insults (32). However, hemin or LPS-induced mitochondrial accumulation of HO-1 was associated with decreased mitochondrial heme content and reduced expression of heme-sensitive subunit I of complex IV with loss of activity (33). Moreover, mitochondrial translocation of HO-1 can also lead to localized CO production as a result of heme degradation, thus inhibiting electron flow though mitochondrial electron transport chain complex IV (44).
HbFe2+ did not have any significant effect on the mitochondrial OCR, even after 24-hour exposure. However, a significant rise in cellular respiration was observed when E10 cells were exposed to HbFe2+ for a shorter incubation period (12 h). This could be also due to its slower rate of auto-oxidation that maintained higher oxygen supply to cells, apart from a possible uncoupling effect. In contrast, HbFe3+ produced significant inhibition in basal OCR respiration within 24 hours. We also compared the effects of all three redox forms of Hb on mitochondrial dysfunction for a shorter time period (12 h). HbFe4+ at an equivalent heme concentration produced significant changes in the basal, ATP-linked respiration in E10 cells within a shorter exposure. However, respiratory reserve capacity was not affected by HbFe4+. Interestingly, we did not observe any significant effect by HbFe2+ or HbFe3+ on these bioenergetic parameters of cells when compared with the ferryl species. It is thus clear that the higher oxidation state of Hb and duration of exposure determined the toxicity in our cell culture system. The inhibitory effect of cell-free Hb on human platelet mitochondrial function and cellular bioenergetics in sickle cell disease has recently been shown (22). Reduction in complex V activity was proposed to be a possible mechanism behind the compromised bioenergetic profile. Previous reports from our laboratory and others also found that cell-free Hb reduced (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide) (MTT) reduction capacity, indicating a loss of mitochondrial activity (45, 46). It has also been shown that heme induces significant mitochondrial dysfunction in endothelial cells (23). Similarly, our results showing a loss of mitochondrial function with a concomitant increase in mitochondrial HO-1 colocalization can be correlated with a possible loss mitochondrial respiratory chain activity (35).
Hp attenuated the HbFe2+-, HbFe3+-, and HbFe4+-induced increase in HO-1 and H-ferritin. Moreover, Hp also prevented heme loss from HbFe3+ and attenuated the HbFe3+- and HbFe4+-induced mitochondrial dysfunction (14, 25). Our results also indicate that Hp reduced Hb uptake, as shown by the absence of Hb α and β subunits in the cell lysates. Thus, Hp not only reduces heme overload externally, but also internally by reducing endocytosis. We have in recent years outlined the mechanistic basis of Hp-mediated Hb protection (14, 25, 47). The binding of Hp to Hb (known to be one of the strongest in biological systems) effectively diffuses Hb-mediated radicals, even when challenged with excess H2O2. Intriguingly, although H2O2 and other ligands were found to have direct access to the heme pocket and unhindered pseudoperoxidase activity, this complex prevented heme loss, even at longer time period of incubation (25). In our current investigation, Hp might have also prevented the transport of Hb across E10 cell monolayer, possibly due to the large size of the Hb-Hp complex and/or altered surface charge (48).
In summary, we propose a likely mechanism that can explain Hb-induced injury reported here (see Figure 6). Exposure to Hb and its oxidized products increases heme overload on the AT1 cells. Heme overload induces the expression of HO-1 and iron-sequestering proteins, such as ferritin. HO-1 translocates to the mitochondria, which leads to mitochondrial dysfunction. Alternatively, increased HO-1 activity in the cytosol might lead to excess production of CO, which could also reduce mitochondrial function. Hp effectively attenuates mitochondrial dysfunction. Our results also indicate that the higher oxidation species, HbFe3+ and HbFe4+, elicit more pronounced effects when compared with HbFe2+, due to their higher redox activities and rates of heme release.
The authors thank Drs. Mary C. Williams and Maria I. Ramirez, (Boston University, Boston, MA) for providing the mouse E10 cells, and Bio Products Laboratory (Hertfordshire, UK) for providing the unfractionated human haptoglobin. They also thank Francine Wood, Dr. Tigist Kassa, and Dr. Fantao Meng for providing different oxidized Hb products, and Dr. Michael Brad Strader for mass spectrometric analysis.
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*These authors contributed equally to this work.
This work was supported by National Institutes of Health grant P01-HL110900 from the National Heart, Lung, and Blood Institute (A.I.A.) and the Food and Drug Administration Modernizing Science grant (A.I.A.).
Author Contributions: Conception and design—N.R.C., S.J., and A.I.A.; experiments—N.R.C. and S.J.; data analysis—N.R.C. and S.J.; drafting manuscript—N.R.C., S.J., and A.I.A.
This article has an online supplement, which is accessible from this issue’s table of contents at www.atsjournals.org
Originally Published in Press as DOI: 10.1165/rcmb.2015-0197OC on March 14, 2016