Key Points Heme, released from hemoglobin, elicits vaso-occlusion in transgenic sickle mice via endothelial TLR4 signaling. Heme/TLR4 signaling activates NF-κB and triggers vaso-occlusion through Weibel-Palade body degranulation and adhesion molecule expression.

Low-grade polymicrobial infection induced by cecal ligation and puncture is lethal in heme oxygenase-1-deficient mice (Hmox1(-/-)), but not in wild-type (Hmox1(+/+)) mice. Here we demonstrate that the protective effect of this heme-catabolizing enzyme relies on its ability to prevent tissue damage caused by the circulating free heme released from hemoglobin during infection. Heme administration after low-grade infection in mice promoted tissue damage and severe sepsis. Free heme contributed to the pathogenesis of severe sepsis irrespective of pathogen load, revealing that it compromised host tolerance to infection. Development of lethal forms of severe sepsis after high-grade infection was associated with reduced serum concentrations of the heme sequestering protein hemopexin (HPX), whereas HPX administration after high-grade infection prevented tissue damage and lethality. Finally, the lethal outcome of septic shock in patients was also associated with reduced HPX serum concentrations. We propose that targeting free heme by HPX might be used therapeutically to treat severe sepsis.

Heme oxygenase-1 (HO-1), the rate-limiting enzyme in heme degradation, is an integral membrane protein of the smooth endoplasmic reticulum. However, we detected an HO-1 immunoreactive signal in the nucleus of cultured cells after exposure to hypoxia and heme or heme/hemopexin. Under these conditions, a faster migrating HO-1 immunoreactive band was enriched in nuclear extracts, suggesting that HO-1 was cleaved to allow nuclear entry. This was confirmed by the absence of immunoreactive signal with an antibody against the C terminus and the lack of a C-terminal sequence by gas chromatographymass spectrometry. Incubation with leptomycin B prior to hypoxia abolished nuclear HO-1 and the faster migrating band on Western analysis, suggesting that this process was facilitated by CRM1. Furthermore, preincubation with a cysteine protease inhibitor prevented nuclear entry of green fluorescent proteinlabeled HO-1, demonstrating that protease-mediated C-terminal cleavage was also necessary for nuclear transport of HO-1. Nuclear localization was also associated with reduction of HO activity. HO-1 protein, whether it was enzymatically active or not, mediated activation of oxidant-responsive transcription factors, including activator protein-1. Nevertheless, nuclear HO-1 protected cells against hydrogen peroxide-mediated injury equally as well as cytoplasmic HO-1. We speculate that nuclear localization of HO-1 protein may serve to up-regulate genes that promote cytoprotection against oxidative stress.Heme oxygenase (HO) 3 catalyzes the degradation of heme and the formation of biliverdin and carbon monoxide. It is highly inducible in response to various stimuli, including oxidative stress, heavy metals, UV radiation, and inflammation (1-4). Cytoprotective roles for HO have been demonstrated in many models; however, the mechanisms by which this occurs are still under intensive study. Many have speculated that either heme catabolites, such as biliverdin, or its derivative, bilirubin, and carbon monoxide or the degradation of the pro-oxidant heme results in cytoprotection against oxidative stress (5-7). Nevertheless, all of the by-products of the HO reaction, despite being potentially cytoprotective, are also cytotoxic. Bilirubin is a potent neurotoxin (8), as is carbon monoxide (9). Furthermore, the HO reaction releases iron, which could interact with cellular oxidants to generate the hydroxyl radical (10). Transfection with an inactive HO-1 mutant protein results in cytoprotection against chemically induced oxidative stress (11). Because this effect of the mutant HO-1 could not be attributable to changes in heme catabolites, it alludes to a role for the HO-1 protein itself. Furthermore, the inactive form of HO-1 increased catalase and glutathione content (11). This suggests that the HO-1 protein itself may play a role in cellular signaling. If this were true, HO-1 would need to migrate to the nucleus or produce nuclear changes that affect transcription. There are several examples of cytoplasmic enzymes serving in nuclear functi...

Objective-We investigated whether red cell infiltration of atheromatous lesions promotes the later stages of atherosclerosis. Methods and Results-We find that oxidation of ferro (FeII) hemoglobin in ruptured advanced lesions occurs generating ferri (FeIII) hemoglobin and via more extensive oxidation ferrylhemoglobin (FeIII/FeIVϭO). The protein oxidation marker dityrosine accumulates in complicated lesions, accompanied by the formation of cross-linked hemoglobin, a hallmark of ferrylhemoglobin. Exposure of normal red cells to lipids derived from atheromatous lesions causes hemolysis and oxidation of liberated hemoglobin. In the interactions between hemoglobin and atheroma lipids, hemoglobin and heme promote further lipid oxidation and subsequently endothelial reactions such as upregulation of heme oxygenase-1 and cytotoxicity to endothelium. Oxidative scission of heme leads to release of iron and a feed-forward process of iron-driven plaque lipid oxidation. The inhibition of heme release from globin by haptoglobin and sequestration of heme by hemopexin suppress hemoglobin-mediated oxidation of lipids of atheromatous lesions and attenuate endothelial cytotoxicity. Conclusion-The interior of advanced atheromatous lesions is a prooxidant environment in which erythrocytes lyse, hemoglobin is oxidized to ferri-and ferrylhemoglobin, and released heme and iron promote further oxidation of lipids. Oxysterols and oxidation products of polyunsaturated fatty acids are present in human atheromatous lesions. 4,5 Atherosclerotic lesions are hazardous regions for nucleated cells, both endothelial cells and, quite probably, incoming macrophages. 6 The major cytotoxic species may be oxidation products of lipids, particularly lipid hydroperoxides (LOOHs), aldehydes, and carbonyls. 6,7 In artificial systems, oxidation of polyunsaturated fatty acids requires reactive transition metals such as iron and copper. Based on our earlier work, 6,8,9 the metal in atheromatous lesions might be iron derived from heme. Nonprotein-bound heme is a particularly deleterious species inasmuch as it is hydrophobic and easily able to enter cell membranes. 10 In previous studies, we found that endothelial cells exposed to oxidized low-density lipoprotein (LDL) upregulated both heme oxygenase-1 (HO-1) and ferritin, 8,9 presumably as a defense mechanism. 6,11-14 Upregulation of HO-1 15 and ferritin H chain 16 in endothelial cells has been reported in the early phase of progression of atherosclerotic lesions. Expression of HO-1 provides protection against atherosclerosis in several experimental models, 17,18 and HO-1 deficiency in humans has been associated with the appearance of vasculature fatty streaks and atheromatous plaques at the age of 6. 19 We tested the hypothesis that heme-iron may accumulate in atherosclerotic lesions by intrusion and lysis of erythrocytes. Liberated hemoglobin is oxidized, and released hemeiron-dependent oxidation of lipids is strongly favored, contributing to further plaque development. Methods Tissue SamplesSpecimens of ...