John D. Belcher

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.

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.

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.

Association of Serum Vitamin Levels, LDL Susceptibility to Oxidation, and Autoantibodies Against MDA-LDL With Carotid Atherosclerosis

Oxidative modification of LDL is believed to be a crucial step in atherosclerosis. Thus, antioxidant vitamins may have a role in the prevention of coronary disease. We examined the cross-sectional association of serum vitamin levels, the susceptibility of LDL to hemin-induced oxidation (lag phase to conjugated diene formation), and the malondialdehyde-LDL (MDA-LDL) to native LDL radioactivity binding ratio with carotid intima-media thickness (IMT), a measure of asymptomatic early atherosclerosis. The participants in this observational study were 231 asymptomatic age-, sex-, race-, and field center-matched case-control pairs selected from the Atherosclerosis Risk in Communities (ARIC) study cohort on the basis of B-mode carotid artery ultrasonograms obtained from 1986 through 1989. Cases exceeded the 90th percentile of IMT, and control subjects were below the 75th percentile of IMT for all arterial segments. Biochemical analyses were performed on fasting frozen (-70 degrees C) serum specimens collected from 1990 through 1992. In conditional logistic regression adjusting for age, blood storage time, total cholesterol, and log-triglyceride concentrations, serum beta-cryptoxanthin and lutein plus zeaxanthin levels were inversely related to the extent of atherosclerosis (odds ratio [OR] per 1-SD increase: 0.75, 95% confidence interval [CI]: 0.59-0.94; and OR per 1-SD increase: 0.76, 95% CI: 0.59-0.95, respectively). Increases in alpha-carotene and lycopene were associated with nonsignificantly lower odds of being a case, whereas beta-carotene, retinol, and alpha-tocopherol were unrelated to IMT. Although not reaching statistical significance, the lag phase and autoantibodies against MDA-LDL were positively associated with asymptomatic atherosclerosis. After adjustment for potential confounders, only the inverse association of lutein plus zeaxanthin with asymptomatic atherosclerosis was maintained. This study supports a modest inverse association between circulating levels of some carotenoids, particularly lutein plus zeaxanthin, and carotid IMT. These findings suggest that these carotenoid compounds (regarded as biomarkers of fruit and vegetable intake) may be important in early stages of atherosclerosis.

Lowering of body iron stores by blood letting and oxidation resistance of serum lipoproteins: a randomized cross-over trial in male smokers

Abstract. Objectives. The purpose of this study was to test the hypothesis that the reduction of body iron stores by venesection (blood letting) would reduce the susceptibility to oxidation of atherogenic serum lipoproteins.

Vitamin E, LDL, and endothelium. Brief oral vitamin supplementation prevents oxidized LDL-mediated vascular injury in vitro.

In previously reported in vitro studies, we found that heme, a physiologically widespread hydrophobic iron compound, can rapidly generate oxidized low-density lipoprotein (LDL), which then becomes cytotoxic to cultured vascular endothelial cells; both LDL oxidation and endothelial cytotoxicity were inhibited by incubation with exogenous alpha-tocopherol (vitamin E) or ascorbic acid (vitamin C). Seeking relevance to in vivo conditions, we performed a study in which 10 human volunteers were given daily antioxidant supplements of 800 IU of DL-alpha-tocopherol acetate alone or in combination with 1000 mg of ascorbic acid for 2 weeks. LDL resistance to heme oxidation ex vivo, as measured by the lag time for conjugated-diene formation, increased by as much as threefold from a mean +/- SD of 58 +/- 11 to 104 +/- 18 minutes (P < .001); LDL alpha-tocopherol increased from 11 +/- 2 to 26 +/- 6 molecules per LDL particle (P < .001); and most impressively, cytotoxicity to porcine aortic endothelial cells incubated with LDL conditioned with heme plus H2O2 or with copper was completely prevented (cytotoxicity before supplementation was 42 +/- 12%, decreasing after supplementation to 3 +/- 2%, P < .001). These measurements reverted to their presupplement levels within 2 weeks after participants stopped taking antioxidant supplements and were reproduced in 4 subjects taking 800 IU of DL-alpha-tocopherol acetate supplements alone but not in the same subjects taking 1000 mg ascorbic acid supplements alone. In conclusion, oral vitamin E supplementation increases LDL alpha-tocopherol content, increases LDL resistance to oxidation, and decreases the cytotoxicity of oxidized LDL to cultured vascular endothelial cells.

Carbon Monoxide Expedites Metabolic Exhaustion to Inhibit Tumor Growth

One classical feature of cancer cells is their metabolic acquisition of a highly glycolytic phenotype. Carbon monoxide (CO), one of the products of the cytoprotective molecule heme oxygenase-1 (HO-1) in cancer cells, has been implicated in carcinogenesis and therapeutic resistance. However, the functional contributions of CO and HO-1 to these processes are poorly defined. In human prostate cancers, we found that HO-1 was nuclear localized in malignant cells, with low enzymatic activity in moderately differentiated tumors correlating with relatively worse clinical outcomes. Exposure to CO sensitized prostate cancer cells but not normal cells to chemotherapy, with growth arrest and apoptosis induced in vivo in part through mitotic catastrophe. CO targeted mitochondria activity in cancer cells as evidenced by higher oxygen consumption, free radical generation, and mitochondrial collapse. Collectively, our findings indicated that CO transiently induces an anti-Warburg effect by rapidly fueling cancer cell bioenergetics, ultimately resulting in metabolic exhaustion.

Association of serum vitamin levels, LDL susceptibility to oxidation, and autoantibodies against MDA-LDL with carotid atherosclerosis. A case-control study. The ARIC Study Investigators. Atherosclerosis Risk in Communities.

Oxidative modification of LDL is believed to be a crucial step in atherosclerosis. Thus, antioxidant vitamins may have a role in the prevention of coronary disease. We examined the cross-sectional association of serum vitamin levels, the susceptibility of LDL to hemin-induced oxidation (lag phase to conjugated diene formation), and the malondialdehyde-LDL (MDA-LDL) to native LDL radioactivity binding ratio with carotid intima-media thickness (IMT), a measure of asymptomatic early atherosclerosis. The participants in this observational study were 231 asymptomatic age-, sex-, race-, and field center-matched case-control pairs selected from the Atherosclerosis Risk in Communities (ARIC) study cohort on the basis of B-mode carotid artery ultrasonograms obtained from 1986 through 1989. Cases exceeded the 90th percentile of IMT, and control subjects were below the 75th percentile of IMT for all arterial segments. Biochemical analyses were performed on fasting frozen (-70 degrees C) serum specimens collected from 1990 through 1992. In conditional logistic regression adjusting for age, blood storage time, total cholesterol, and log-triglyceride concentrations, serum beta-cryptoxanthin and lutein plus zeaxanthin levels were inversely related to the extent of atherosclerosis (odds ratio [OR] per 1-SD increase: 0.75, 95% confidence interval [CI]: 0.59-0.94; and OR per 1-SD increase: 0.76, 95% CI: 0.59-0.95, respectively). Increases in alpha-carotene and lycopene were associated with nonsignificantly lower odds of being a case, whereas beta-carotene, retinol, and alpha-tocopherol were unrelated to IMT. Although not reaching statistical significance, the lag phase and autoantibodies against MDA-LDL were positively associated with asymptomatic atherosclerosis. After adjustment for potential confounders, only the inverse association of lutein plus zeaxanthin with asymptomatic atherosclerosis was maintained. This study supports a modest inverse association between circulating levels of some carotenoids, particularly lutein plus zeaxanthin, and carotid IMT. These findings suggest that these carotenoid compounds (regarded as biomarkers of fruit and vegetable intake) may be important in early stages of atherosclerosis.

Association of Serum Vitamin Levels, LDL Susceptibility to Oxidation, and Autoantibodies Against MDA-LDL With Carotid Atherosclerosis: A Case-Control Study

Oxidative modification of LDL is believed to be a crucial step in atherosclerosis. Thus, antioxidant vitamins may have a role in the prevention of coronary disease. We examined the cross-sectional association of serum vitamin levels, the susceptibility of LDL to hemin-induced oxidation (lag phase to conjugated diene formation), and the malondialdehyde-LDL (MDA-LDL) to native LDL radioactivity binding ratio with carotid intima-media thickness (IMT), a measure of asymptomatic early atherosclerosis. The participants in this observational study were 231 asymptomatic age-, sex-, race-, and field center-matched case-control pairs selected from the Atherosclerosis Risk in Communities (ARIC) study cohort on the basis of B-mode carotid artery ultrasonograms obtained from 1986 through 1989. Cases exceeded the 90th percentile of IMT, and control subjects were below the 75th percentile of IMT for all arterial segments. Biochemical analyses were performed on fasting frozen (-70 degrees C) serum specimens collected from 1990 through 1992. In conditional logistic regression adjusting for age, blood storage time, total cholesterol, and log-triglyceride concentrations, serum beta-cryptoxanthin and lutein plus zeaxanthin levels were inversely related to the extent of atherosclerosis (odds ratio [OR] per 1-SD increase: 0.75, 95% confidence interval [CI]: 0.59-0.94; and OR per 1-SD increase: 0.76, 95% CI: 0.59-0.95, respectively). Increases in alpha-carotene and lycopene were associated with nonsignificantly lower odds of being a case, whereas beta-carotene, retinol, and alpha-tocopherol were unrelated to IMT. Although not reaching statistical significance, the lag phase and autoantibodies against MDA-LDL were positively associated with asymptomatic atherosclerosis. After adjustment for potential confounders, only the inverse association of lutein plus zeaxanthin with asymptomatic atherosclerosis was maintained. This study supports a modest inverse association between circulating levels of some carotenoids, particularly lutein plus zeaxanthin, and carotid IMT. These findings suggest that these carotenoid compounds (regarded as biomarkers of fruit and vegetable intake) may be important in early stages of atherosclerosis.