Ben J. Wu

Kynurenine is an endothelium-derived relaxing factor produced during inflammation.

Control of blood vessel tone is central to vascular homeostasis. Here we show that metabolism of tryptophan to kynurenine by indoleamine 2,3-dioxygenase (Ido) expressed in endothelial cells contributes to arterial vessel relaxation and the control of blood pressure. Infection of mice with malarial parasites (Plasmodium berghei) or induction of endotoxemia in mice led to endothelial expression of Ido, decreased plasma tryptophan concentration, increased kynurenine concentration and hypotension. Pharmacological inhibition of Ido increased blood pressure in systemically inflamed mice but not in mice deficient in Ido or interferon-γ, which is required for Ido induction. Both tryptophan and kynurenine dilated preconstricted porcine coronary arteries; the dilating effect of tryptophan required the presence of active Ido and an intact endothelium, whereas the effect of kynurenine was endothelium independent. The arterial relaxation induced by kynurenine was mediated by activation of the adenylate and soluble guanylate cyclase pathways. Kynurenine administration decreased blood pressure in a dose-dependent manner in spontaneously hypertensive rats. Our results identify tryptophan metabolism by Ido as a new pathway contributing to the regulation of vascular tone.

Antioxidants protect from atherosclerosis by a heme oxygenase-1 pathway that is independent of free radical scavenging

Oxidative stress is implicated in atherogenesis, yet most clinical trials with antioxidants, particularly vitamin E, have failed to protect against atherosclerotic diseases. A striking exception is probucol, which retards atherosclerosis in carotid arteries and restenosis of coronary arteries after angioplasty. Because probucol has in vitro cellular-protective effects independent of inhibiting lipid oxidation, we investigated the mode of action of probucol in vivo. We used three models of vascular disease: apolipoprotein E–deficient mice, a model of atherosclerosis; rabbit aortic balloon injury, a model of restenosis; and carotid injury in obese Zucker rats, a model of type 2 diabetes. Unexpectedly, we observed that the phenol moieties of probucol were insufficient, whereas its sulphur atoms were required for protection. Probucol and its sulphur-containing metabolite, but not a sulphur-free phenolic analogue, protected via cell-specific effects on inhibiting macrophage accumulation, stimulating reendothelialization, and inhibiting vascular smooth muscle cell proliferation. These processes were mediated via induction of heme oxygenase-1 (HO-1), an activity not shared by vitamin E. Our findings identify HO-1 as the molecular target of probucol. They indicate 2-electron rather than radical (1-electron) oxidants as important contributors to atherogenesis, and point to novel lead compounds for therapeutic intervention against atherosclerotic diseases.

Probucol Protects Against Smooth Muscle Cell Proliferation by Upregulating Heme Oxygenase-1

Background—Evidence suggests that induction of heme oxygenase-1 (HO-1) inhibits proliferation of vascular smooth muscle cells and intimal thickening after arterial injury, and therapeutic molecules induce HO-1. Probucol is the only oral drug that inhibits restenosis in humans and intimal thickening in animals, although its underlying mechanism remains unclear. Methods and Results—Aortas were harvested from New Zealand White rabbits fed normal or 0.75% (wt/wt) probucol-fortified chow, with or without endothelial denudation of the abdominal aorta on day 21, and analyzed for heme oxygenase and apoptosis. Uninjured aortas were harvested on day 21 and balloon-injured aortas on days 22 and 25. Probucol significantly increased mRNA of HO-1 assessed by real-time PCR and HO activity in aortas at all time points. Probucol also enhanced apoptosis of medial cells in the injured aorta, as evidenced by the TUNEL assay. Furthermore, probucol (100 &mgr;mol/L) increased HO-1 mRNA and HO activity when added to rabbit aortic smooth muscle cells (RASMCs) cultured in serum-free medium for 24 hours. Induction of HO-1 mRNA was inhibited by actinomycin D and was associated with inhibition of RASMC proliferation. This probucol-induced increase in HO-1 mRNA and inhibition of RASMC proliferation was prevented by the HO inhibitor Sn(IV) protoporphyrin or transfection with small interference RNA (siRNA) to knockdown HO-1, but not by inactive Cu(II) protoporphyrin or scrambled siRNA. Conclusions—Probucol induces HO-1, and this contributes to the inhibition of vascular SMC proliferation. This novel finding may explain how probucol inhibits restenosis and highlights HO-1 as a target for therapeutic intervention against occlusive vascular disease.

Evidence That Niacin Inhibits Acute Vascular Inflammation and Improves Endothelial Dysfunction Independent of Changes in Plasma Lipids

Objective—To determine if niacin can confer cardiovascular benefit by inhibiting vascular inflammation and improving endothelial function independent of changes in plasma lipid and lipoprotein levels. Methods and Results—New Zealand white rabbits received normal chow or chow supplemented with 0.6% or 1.2% (wt/wt) niacin. This regimen had no effect on plasma cholesterol, triglyceride, or high-density lipoprotein levels. Acute vascular inflammation and endothelial dysfunction were induced in the animals with a periarterial carotid collar. At the 24-hour postcollar implantation, the endothelial expression of vascular cell adhesion molecule-1, intercellular adhesion molecule-1, and monocyte chemotactic protein-1 was markedly decreased in the niacin-supplemented animals compared with controls. Niacin also inhibited intima-media neutrophil recruitment and myeloperoxidase accumulation, enhanced endothelial-dependent vasorelaxation and cyclic guanosine monophosphate production, increased vascular reduced glutathione content, and protected against hypochlorous acid–induced endothelial dysfunction and tumor necrosis factor &agr;–induced vascular inflammation. Conclusion—Previous human intervention studies have demonstrated that niacin inhibits coronary artery disease. This benefit is thought to be because of its ability to reduce low-density lipoprotein and plasma triglyceride levels and increase high-density lipoprotein levels. The present study showed that niacin inhibits vascular inflammation and protects against endothelial dysfunction independent of these changes in plasma lipid levels.

What role for membranes in determining the higher sodium pump molecular activity of mammals compared to ectotherms

Abstract The major body organs of mammals have sodium pumps that turn over energy (ATP) three to four times faster than those of ectotherms, at the same temperature. To examine if membranes play a role in these differences in molecular activity, membrane cross-over experiments were performed using two representative species, Rattus norvegicus and Bufo marinus. Microsomal membrane of kidney and brain displayed characteristic molecular activity differences (three- to four-fold) between the species. These molecular activity differences could be removed by delipidation. Pre-existing molecular activities and differences could be restored when reconstituted with original membrane. Using the same reconstitution method, species membrane cross-over experiments resulted in toad sodium pumps in rat membrane significantly increasing (≈30–40%) and rat sodium pumps in toad membrane significantly decreasing (≈40%) activities in both kidney and brain. Analysis of membrane composition showed reduced cholesterol content and differences in the fatty acids of phospholipids with higher overall unsaturation in the mammal. The scope for membranes to determine protein performance and its broader implications for metabolism are discussed.

Relationships between the fatty acid composition of muscle and erythrocyte membrane phospholipid in young children and the effect of type of infant feeding.

Muscle membrane fatty acid (FA) composition is linked to insulin action. The aims of this study were to compare the FA composition of muscle and erythrocyte membrane phospholipid in young children; to investigate the effect of diet on these lipid compositions; and to investigate differential incorporation of FA into muscle, erythrocyte and adipose tissue membrane phospholipid, and adipose tissue triglyceride. Skeletal muscle biopsies and fasting blood samples were taken from 61 normally nourished children (15 males and 16 females), less than 2 yr old (means ±SE, 0.80±0.06 yr), undergoing elective surgery. Adipose tissue samples were taken from 15 children. There were significant positive correlations between muscle and erythrocyte docosahexaenoic acid (DHA) (r=0.44, P<0.0001), total n−3 polyunsaturated fatty acids (PUFA) (r=0.39, P=0.002), and the n−6/n−3 PUFA ratio (r=0.39, P=0.002). Adipose tissue triglyceride had lower levels of long-chain PUFA, especially DHA, than muscle and erythrocytes (0.46±0.18% vs. 2.44±0.26% and 3.17±0.27%). Breast-fed infants had higher levels of DHA than an age-matched group of formulafed infants in both muscle (3.91±0.21% vs. 1.94±0.18%) and erythrocytes (3.81±0.10% vs. 2.65±0.23%). The results of this study show that (i) erythrocyte FA composition is a reasonable index of muscle DHA, total n−3 PUFA, and the n−6/n−3 PUFA ratio; (ii) breast feeding has a potent effect on the FA composition of all these tissues; and (iii) there is a wide range in long-chain PUFA levels in muscle, erythrocytes, and adipose tissue.

Niacin Inhibits Vascular Inflammation via the Induction of Heme Oxygenase-1

Background— Heme oxygenase-1 (HO-1) is a cytoprotective protein whose expression is consistently associated with therapeutic benefits in a number of pathological conditions such as atherosclerotic vascular disease and inflammation. Niacin is a pleiotropic drug that slows the progression of coronary artery disease and increases serum levels of the HO-1 enzymatic product bilirubin. This study asks if the cardioprotective properties of niacin involve the induction of HO-1. Methods and Results— New Zealand White rabbits received chow or chow supplemented with 0.6% (wt/wt) niacin for 2 weeks. Acute vascular inflammation was induced in the animals by placing a nonocclusive silastic collar around the left common carotid artery. At 24 hours after collar implantation, serum bilirubin and vascular, liver, and spleen HO-1 messenger RNA levels were significantly increased. Vascular inflammation was decreased in the niacin-supplemented animals compared with control. Treatment of the animals with tin protoporphyrin-IX, a global HO inhibitor, or HO-1 small interfering RNA to knock down carotid artery HO-1 attenuated the ability of niacin to inhibit vascular inflammation. Treatment of cultured human coronary artery endothelial cells with niacin increased HO-1 expression by activating the nuclear factor-E2–related factor 2/p38 mitogen-activated protein kinase signaling pathway and inhibiting tumor necrosis factor &agr;–induced endothelial inflammation. The antiinflammatory effects of niacin in human coronary artery endothelial cells were mimicked by bilirubin and abolished by incubation with tin protoporphyrin-IX and knock down of nuclear factor-E2–related factor 2. Conclusions— Niacin activates HO-1 in vivo and in vitro. Induction of HO-1 may be partly responsible for the vascular protective properties of niacin.

Probucol Protects against Hypochlorite-induced Endothelial Dysfunction IDENTIFICATION OF A NOVEL PATHWAY OF PROBUCOL OXIDATION TO A BIOLOGICALLY ACTIVE INTERMEDIATE

Atherosclerosis is associated with endothelial dysfunction and a heightened state of inflammation characterized, in part, by an increase in vascular myeloperoxidase and proteins modified by its principal oxidant, hypochlorous acid (HOCl). Here we examined whether probucol could protect against endothelial dysfunction induced by the two-electron oxidant HOCl. Hypochlorous acid eliminated endothelium-dependent relaxation of rabbit aorta, whereas endothelial function and tissue cGMP was preserved and elevated, respectively, in animals pretreated with probucol. Exogenously added probucol also protected against HOCl-induced endothelial dysfunction. In vitro, HOCl oxidized probucol in a two-phase process with rate constants k1 = 2.7 ± 0.3 × 102 and k2 = 0.7 ± 0.2 × 102 m–1 s–1 that resulted in a dose- and time-dependent accumulation of probucol-derived disulfoxide, 4,4′-dithiobis(2,6-di-tert-butyl-phenol) (DTBP), DTBP-derived thiosulfonate, disulfone, and sulfonic acid, together with 3,3′,5,5′-tetra-tert-butyl-4,4′-diphenoquinone (DPQ) as determined by high performance liquid chromatography and mass spectrometry. Like HOCl, selected one-electron oxidants converted probucol into DTBP and DPQ. Also, dietary and in vitro added DTBP protected aortic rings from HOCl-induced endothelial dysfunction and in vitro oxidation by HOCl gave rise to the thiosulfonate, disulfone, and sulfonic acid intermediates and DPQ. However, the product profiles of the in vitro oxidation systems were different from those in aortas of rabbits receiving dietary probucol or DTBP ± HOCl treatment. Together, the results show that both probucol and DTBP react with HOCl and protect against HOCl-induced endothelial dysfunction, although direct scavenging of HOCl is unlikely to be responsible for the vascular protection by the two compounds.

Maternal undernutrition reduces aortic wall thickness and elastin content in offspring rats without altering endothelial function

Epidemiological studies suggest a link between fetal/early infant nutrition and adult coronary artery disease. In the present study, we examined the effects of altering nutrition during gestation, lactation and juvenile life on aortic structure and function in rats. Wistar rat dams were fed either a control or low-protein diet throughout pregnancy, or a low-protein diet for the final 7 days of gestation only. At 21 days post-partum, male pups were weaned on to a control, low-protein or high-fat diet. At 12 weeks, the offspring rats were killed. In 46 rats, aortic sections were mounted and stained to assess media thickness and elastin content. In a further 38 rats, aortic rings were suspended in an organ bath and vascular reactivity was tested with dose-response curves to the endothelium-dependent dilator acetylcholine and the endothelium-independent dilator sodium nitroprusside. Rats exposed to maternal protein restriction while in utero had a significantly decreased aortic wall thickness compared with control rats (P=0.005). Total elastin content of the aorta was also decreased by both maternal low-protein (P=0.02) and early postnatal low-protein (P=0.01) diets. Neither maternal nor postnatal low-protein or high-fat diets, however, resulted in any significant changes in arterial dilator function. In conclusion, fetal undernutrition in rats, induced via a maternal low-protein diet, causes a decrease in aortic wall thickness and elastin content without altering aortic dilator function. These changes in vascular structure may amplify aging-related changes to the vasculature and contribute to the pathophysiology of the putative link between impaired fetal growth and adult cardiovascular disease.

Increased expression of the TGF-b superfamily cytokine MIC-1/GDF15 protects ApoE / mice from the development of atherosclerosis

AIM MIC-1/GDF15 is a member of the TGF-b superfamily, which is thought to have pleiotropic roles in stress responses, inflammation, tissue injury and repair, energy homeostasis, and malignancy. MIC-1/GDF15 was recently identified as a new biomarker for the development of cardiovascular events and the outcome of atherosclerotic disease therapy. The aim of our study was to determine if MIC-1 also directly exerts pro- or antiatherogenic properties during the development of atherosclerosis. METHODS AND RESULTS We investigated the effect of transgenic overexpression of MIC-1 in macrophages in the ApoE(-/-) mouse model of atherosclerosis. After 6 months of high-fat diet, MIC-1/GDF15 transgenic ApoE(-/-) mice had smaller atherosclerotic lesions; however, no differences in lesion composition, pro- or anti-inflammatory cytokine production, or serum levels of lipids or cytokines were detected. CONCLUSIONS Our results suggest that MIC-1 has an overall protective effect on the disease process, but further studies will be required to define its mechanism of action.