Gregory M. Vercellotti

Induction of heme oxygenase is a rapid, protective response in rhabdomyolysis in the rat.

Heme proteins such as myoglobin or hemoglobin, when released into the extracellular space, can instigate tissue toxicity. Myoglobin is directly implicated in the pathogenesis of renal failure in rhabdomyolysis. In the glycerol model of this syndrome, we demonstrate that the kidney responds to such inordinate amounts of heme proteins by inducing the heme-degradative enzyme, heme oxygenase, as well as increasing the synthesis of ferritin, the major cellular repository for iron. Prior recruitment of this response with a single preinfusion of hemoglobin prevents kidney failure and drastically reduces mortality (from 100% to 14%). Conversely, ablating this response with a competitive inhibitor of heme oxygenase exacerbates kidney dysfunction. We provide the first in vivo evidence that induction of heme oxygenase coupled to ferritin synthesis is a rapid, protective antioxidant response. Our findings suggest a therapeutic strategy for populations at a high risk for rhabdomyolysis.

Hemin: a possible physiological mediator of low density lipoprotein oxidation and endothelial injury.

Oxidized low density lipoprotein (LDL), formed in vivo from presently unknown reactions, may play a role in atherogenesis. In vitro, transition metals such as iron and copper will facilitate LDL oxidation, but these metals are unlikely to exist in free form in normal body fluids. We have explored the possibility that LDL oxidation may be promoted by heme, a physiologically ubiquitous, hydrophobic, iron-containing compound. Indeed, during several-hour incubation, heme caused extensive oxidative modification of LDL; however, such modification requires only minutes in the presence of small amounts of H2O2 or preformed lipid hydroperoxides within the LDL. Oxidative interactions between heme, LDL, and peroxides lead to degradation of the heme ring and consequent release of heme iron, which further accelerates heme degradation. Coupled (evidently iron-catalyzed) heme degradation and LDL oxidation are both effectively inhibited by hydrophobic antioxidants and iron chelators. That such hemin-induced LDL oxidation may be involved in atherogenesis is supported by the finding that LDL oxidized by hemin is extremely cytotoxic to cultured aortic endothelial cells. Overall, these investigations not only lend support to the idea that LDL oxidation by physiological substances such as heme may play a role in the process of atherogenesis but also may have broader implications, as similar oxidative reactions between heme and unsaturated fatty acids may occur consequent to hemorrhagic injury.

Hemolysis and free hemoglobin revisited: exploring hemoglobin and hemin scavengers as a novel class of therapeutic proteins

Hemolysis occurs in many hematologic and nonhematologic diseases. Extracellular hemoglobin (Hb) has been found to trigger specific pathophysiologies that are associated with adverse clinical outcomes in patients with hemolysis, such as acute and chronic vascular disease, inflammation, thrombosis, and renal impairment. Among the molecular characteristics of extracellular Hb, translocation of the molecule into the extravascular space, oxidative and nitric oxide reactions, hemin release, and molecular signaling effects of hemin appear to be the most critical. Limited clinical experience with a plasma-derived haptoglobin (Hp) product in Japan and more recent preclinical animal studies suggest that the natural Hb and the hemin-scavenger proteins Hp and hemopexin have a strong potential to neutralize the adverse physiologic effects of Hb and hemin. This includes conditions that are as diverse as RBC transfusion, sickle cell disease, sepsis, and extracorporeal circulation. This perspective reviews the principal mechanisms of Hb and hemin toxicity in different disease states, updates how the natural scavengers efficiently control these toxic moieties, and explores critical issues in the development of human plasma-derived Hp and hemopexin as therapeutics for patients with excessive intravascular hemolysis.

Inhibition of complement-mediated endothelial cell cytotoxicity by decay-accelerating factor: Potential for prevention of xenograft hyperacute rejection

Complement plays a major role in hyperacute rejection of discordant xenografts. In immediately vascularized xenografts, such as porcine organs to humans, C activation contributes to triggering of endothelial cell activation and adhesion of leukocytes and platelets to the endothelial cells, which is followed by thrombosis and tissue necrosis. We investigated the potential utility of the membrane-associated inhibitor of C, decay accelerating factor (DAF), in the prevention of C-mediated tissue injury. We used an in vitro model of xenotransplantation consisting of porcine aortic endothelial cells incubated with human serum as the source of xenogeneic natural antibodies and C. Because C inhibitors such as DAF may be relatively species-specific, we tested whether human DAF would incorporate into porcine endothelial cells and function to inhibit cytotoxicity of such cells by human C. We found that purified radiolabeled human DAF incorporated into porcine endothelial cells in a dose-dependent manner and that human DAF very significantly protected the endothelial cells from the cytotoxic effect of human C.

Heme triggers TLR4 signaling leading to endothelial cell activation and vaso-occlusion in murine sickle cell disease

Treatment of sickle cell disease (SCD) is hampered by incomplete understanding of pathways linking hemolysis to vaso-occlusion. We investigated these pathways in transgenic sickle mice. Infusion of hemoglobin or heme triggered vaso-occlusion in sickle, but not normal, mice. Methemoglobin, but not heme-stabilized cyanomethemoglobin, induced vaso-occlusion, indicating heme liberation is necessary. In corroboration, hemoglobin-induced vaso-occlusion was blocked by the methemoglobin reducing agent methylene blue, haptoglobin, or the heme-binding protein hemopexin. Untreated HbSS mice, but not HbAA mice, exhibited ∼10% vaso-occlusion in steady state that was inhibited by haptoglobin or hemopexin infusion. Antibody blockade of adhesion molecules P-selectin, von Willebrand factor (VWF), E-selectin, vascular cell adhesion molecule 1, intercellular adhesion molecule 1, platelet endothelial cell (EC) adhesion molecule 1, α4β1, or αVβ3 integrin prevented vaso-occlusion. Heme rapidly (5 minutes) mobilized Weibel-Palade body (WPB) P-selectin and VWF onto EC and vessel wall surfaces and activated EC nuclear factor κB (NF-κB). This was mediated by TLR4 as TAK-242 blocked WPB degranulation, NF-κB activation, vaso-occlusion, leukocyte rolling/adhesion, and heme lethality. TLR4(-/-) mice transplanted with TLR4(+/+) sickle bone marrow exhibited no heme-induced vaso-occlusion. The TLR4 agonist lipopolysaccharide (LPS) activated ECs and triggered vaso-occlusion that was inhibited by TAK-242, linking hemolysis- and infection-induced vaso-occlusive crises to TLR4 signaling. Heme and LPS failed to activate VWF and NF-κB in TLR4(-/-) ECs. Anti-LPS immunoglobulin G blocked LPS-induced, but not heme-induced, vaso-occlusion, illustrating LPS-independent TLR4 signaling by heme. Inhibition of protein kinase C, NADPH oxidase, or antioxidant treatment blocked heme-mediated stasis, WPB degranulation, and oxidant production. We conclude that intravascular hemolysis in SCD releases heme that activates endothelial TLR4 signaling leading to WPB degranulation, NF-κB activation, and vaso-occlusion.

Infection and Atherosclerosis Potential Roles of Pathogen Burden and Molecular Mimicry

Infection has been implicated as a cause of atherosclerosis since the first half of the 19th century. Over the years, sporadic publications have appeared in the literature reflecting a persistent but relatively low level of research activity in this area. In the last decade, however, publications relating to this topic have increased markedly. And very recently, new epidemiological and mechanistic data relating infection to several different diseases, including atherosclerosis, have appeared, stimulating the emergence of important paradigm shifts in how we think about the causes of chronic disease. The following article reviews some of these newer concepts as they relate to a possible role of infection in atherosclerosis.

Heme oxygenase-1 is a modulator of inflammation and vaso-occlusion in transgenic sickle mice

Transgenic sickle mice expressing betaS hemoglobin have activated vascular endothelium that exhibits enhanced expression of NF-kappaB and adhesion molecules that promote vascular stasis in sickle, but not in normal, mice in response to hypoxia/reoxygenation. Sickle mice hemolyze rbcs in vivo as demonstrated by increased reticulocyte counts, plasma hemoglobin and bilirubin, and reduced plasma haptoglobin. The heme content is elevated in sickle organs, which promotes vascular inflammation and heme oxygenase-1 expression. Treatment of sickle mice with hemin further increases heme oxygenase-1 expression and inhibits hypoxia/reoxygenation-induced stasis, leukocyte-endothelium interactions, and NF-kappaB, VCAM-1, and ICAM-1 expression. Heme oxygenase inhibition by tin protoporphyrin exacerbates stasis in sickle mice. Furthermore, treatment of sickle mice with the heme oxygenase enzymatic product carbon monoxide or biliverdin inhibits stasis and NF-kappaB, VCAM-1, and ICAM-1 expression. Local administration of heme oxygenase-1 adenovirus to subcutaneous skin increases heme oxygenase-1 and inhibits hypoxia/reoxygenation-induced stasis in the skin of sickle mice. Heme oxygenase-1 plays a vital role in the inhibition of vaso-occlusion in transgenic sickle mice.

Paraneoplastic autoimmune phenomena in patients with myelodysplastic syndromes: response to immunosuppressive therapy

Summary. We analysed the clinical features, course and response to immunosuppressive therapy in 30 patients with autoimmune disorders associated with myelodysplastic syndromes (MDS). 18 patients with MDS developed acute systemic autoimmune disorders. Common manifestations were skin vasculitis (n=15) and arthritis (n=11). Seven patients had an acute clinical syndrome of vasculitic skin rash, fever and arthritis with peripheral oedema in three and pulmonary infiltrates in five of these seven patients. Other acute manifestations included pericarditis, pleural effusions, skin ulceration, seizures, myositis and peripheral neuropathy. Chronic or isolated autoimmune manifestations (n=11) included glomerulonephritis, polyneuropathy, pyoderma gangrenosum, ulcerative colitis and polyarthritis. Classic connective tissue disorders recognized included relapsing polychondritis, polymyalgia rheumatica, Raynauds syndrome and Sjogrens syndrome. Autoimmune manifestations responded to immunosuppressive therapy (primarily prednisone) in 26/27 patients treated. Furthermore, cytopenias improved substantially in six patients, including complete normalization of peripheral blood counts in two patients with cytogenetic remission in one. Patients with a haematological response to immunosuppressive therapy had improved survival compared with non‐responding patients. The autoimmune syndrome was implicated as a primary cause of death in 8/17 patients who died. Autoimmune manifestations may be more common than previously recognized in patients with MDS. Aggressive therapy with immunosuppressive agents in selected patients often controls autoimmune phenomena associated with MDS and may lead to haematological responses in some patients.

A histopathologic study of gastric and small intestinal graft-versus-host disease following allogeneic bone marrow transplantation

Twenty-two stomach and 14 small intestinal biopsy specimens from 24 allogeneic bone marrow transplant recipients were reviewed to evaluate the histopathologic changes of graft-versus-host disease (GVHD) in these organs. Associations between these results and clinical symptoms and other biopsy results were sought. In both organs, single epithelial cell necrosis was found to correlate with GVHD. Gastric GVHD was diagnosed in eight patients and small intestinal GVHD in four. Gastric GVHD was characterized by nausea, vomiting, and upper abdominal pain without diarrhea (the latter being present in only two patients), while all four of the patients with small intestinal GVHD had upper gastrointestinal symptoms and diarrhea. These symptoms correlated with concurrent rectal biopsy findings; pathologic alterations were seen in only one of six specimens from patients with gastric GVHD but in three of four with small intestinal GVHD. These findings suggest that stomach biopsy may be necessary to diagnose GVHD in patients with upper gastrointestinal symptoms but no diarrhea and normal rectal biopsy specimens. Diagnostic problems may arise in the early posttransplantation period, when the effects of cytoreductive therapy may simulate GVHD, and in patients with gastrointestinal cytomegalovirus infection, which may also produce changes identical to those of GVHD.

Red Cells, Hemoglobin, Heme, Iron, and Atherogenesis

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/FeIV=O). 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. These events amplify the endothelial cell cytotoxicity of plaque components.