Newly recognized causes of atherosclerosis: the role of microorganisms and of vascular iron overload.

We have hypothesized that shear stresses at sites of increased vascular turbulence may foster atherogenesis by two previously unknown mechanisms: The first involves Herpes virus activation, which can provoke direct or inflammatory cell-mediated endothelial damage while altering the vascular surface to a highly procoagulant entity. The second derives from red blood cell fragmentation, with resulting uptake by endothelium of released heme groups. In this instance the opening of the heme ring by induced endothelial heme oxygenase frees iron, which sensitizes cells to damage by oxidants--for instance, those generated by closely apposed inflammatory cells. An additional injurious effect of released heme results from its potent catalysis of LDL oxidation--a process specifically and rapidly inhibited by oral supplementation of vitamin E. Although heme-protein's deleterious actions can be counteracted by the plasma constituents haptoglobin and hemopexin, we suggest that these may not be sufficiently present in "sanctuary" sites of vessel walls such as in intramural hemorrhages associated with atherosclerotic intimal tears.

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.

Oxidized low-density lipoproteins and endothelium: oral vitamin E supplementation prevents oxidized low-density lipoprotein-mediated vascular injury.

Vitamin E supplements may decrease the incidence of myocardial infarction by inhibiting LDL oxidation to atherogenic moieties. We previously reported that hemin is a potent and relevant lipophilic source of iron that can rapidly intercalate into LDL, catalyzing its oxidation and promoting its cytolysis of endothelium. The effects of oral vitamin E on heme-catalyzed LDL oxidation and resulting endothelial damage were studied in 10 volunteers who received daily 800 I.U. of vitamin E with or without vitamin C (1000 mg) for 2 weeks. Prior, during, and 2 weeks after supplementation, plasma LDL was isolated and its number of alpha-tocopherol molecules, resistance to heme-catalyzed oxidation, and ability to damage porcine aortic endothelial cells were assayed. Vitamin E supplementation doubled the lag phase of LDL peroxidation as compared to control (104 +/- 18 vs. 58 +/- 11 min; p < 0.001) accompanied by an increase in alpha-tocopherol content of LDL particles (26 +/- 6 vs. 11 +/- 2 mol/mol; p < 0.001). Most intriguingly, LDL-mediated endothelial cell cytotoxicity was prevented (3 +/- 2% vs. 42 +/- 12%; p < 0.001). After a 2-week washout period, LDL alpha-tocopherol content, the lag time of LDL oxidation, and oxidized LDL-mediated cytolysis all returned to baseline levels. To determine whether supplements of vitamin E and vitamin C beneficially synergize in these effects, we monitored several volunteers on daily vitamin E alone or vitamin C alone. Vitamin E alone (at doses as low as 400 I.U./day) affected all measurements in a manner identical to that when it was taken with vitamin C. Vitamin C alone had no significant effect on these measurements. We conclude: dietary vitamin E supplementation provides cytoprotection against LDL oxidation-mediated endothelial cell injury, but this salutary effect is rapidly lost after supplementation is stopped.

Oxygen radical-induced erythrocyte hemolysis by neutrophils. Critical role of iron and lactoferrin.

Human neutrophils (PMN), when stimulated with such chemotaxins as phorbol myristate acetate (PMA), destroy erythrocytes and other targets. Cytotoxicity depends on PMN-generated reactive oxygen metabolites, yet the exact toxic specie and its mode of production is a matter of some dispute. Using 51Cr-labeled erythrocytes as targets, we compared various reactive-O2 generating systems for their abilities to lyse erythrocytes as well as to oxidize hemoglobin to methemoglobin. PMA-activated PMNs or xanthine oxidase plus acetaldehyde were added to target erythrocytes in amounts that provided similar levels of superoxide. PMNs lysed 68.3 +/- 2.9% (SEM) of targets, whereas the xanthine oxidase system was virtually impotent (2.3 +/- 0.8%). In contrast, methemoglobin formation by xanthine oxidase plus acetaldehyde was significantly greater than that caused by stimulated PMNs (P less than 0.001). A similar dichotomy was noted with added reagent H2O2 or the H2O2-generating system, glucose plus glucose oxidase; neither of these caused 51Cr release, but induced 10-70% methemoglobin formation. Thus, although O2- and H2O2 can cross the erythrocyte membrane and rapidly oxidize hemoglobin, they do so evidently without damaging the cell membrane. That a granule constituent of PMNs is required to promote target cell lysis was suggested by the fact that agranular PMN cytoplasts (neutroplasts), although added to generate equal amounts of O2- as intact PMNs, were significantly less lytic to target erythrocytes (P less than 0.01). Iron was shown to be directly involved in lytic efficiency by supplementation studies with 2 microM iron citrate; such supplementation increased PMN cytotoxicity by approximately 30%, but had much less effect on erythrocyte lysis by neutroplasts (approximately 3% increase), and no effect on lysis in the enzymatic oxygen radical-generating systems. These results suggest a critical role for an iron-liganding moiety that is abundantly present in PMN, marginally so in neutroplasts, and not at all in purified enzymatic systems--a moiety that we presume catalyzes very toxic O2 specie generation in the vicinity of juxtaposed erythrocyte targets. The obvious candidate is lactoferrin (LF), and indeed, antilactoferrin IgG, but not nonspecific IgG, reduced PMN cytotoxicity by greater than 85%. Re-adding 10(-8) M pure LF to neutroplasts increased their ability to promote hemolysis by 48.4 +/- 0.9%--to a level near that of intact PMNs. We conclude that O-2 and H2O2 are not sufficient to mediate target cell lysis, but require iron bound to LF, which, in turn, probably generates and focuses toxic O2 radicals, such as OH, to target membrane sites.

Influence of dietary omega-3 fatty acids on granulocyte function.

Manipulation of dietary fatty acid content has been shown to influence platelet aggregation responses. Because granulocytes and platelets interact in a variety of biologic systems, we wondered whether a similar effect might be observed on granulocytes. Granulocyte function was therefore studied in three donors prior to and after three weeks upon a diet supplemented with large amounts of eicosapentaenoic acid. The previously reported attenuation of platelet aggregation was observed, but no effect was seen on granulocyte aggregation, chemotaxis, or superoxide production. Although several other explanations are possible, we suggest that the most likely explanation for this dichotomy is that granulocyte aggregation and chemotaxis are not centrally dependent upon production of thromboxane A.

Activation of plasma complement by perfluorocarbon artificial blood: probable mechanism of adverse pulmonary reactions in treated patients and rationale for corticosteroids prophylaxis.

Perfluorocarbons have shown promise as clinical blood substitutes. Although early experience in Japan with one such product--Fluosol-DA--has been uncomplicated, we observed an adverse pulmonary reaction in the first American patient to receive it and know of similar reactions in two other Americans so treated. Postulating that activation of plasma complement (C) by the perfluorocarbon emulsion might have caused the reaction, we tested the product to determine if it is an activator of complement. Incubation of Fluosol with plasma led to C3 conversion, decrement in CH50, and generation of C5a-related PMN aggregating activity; EDTA prevented such activation, while EGTA did not, suggesting that it proceeded via the alternative C pathway. Infusion of Fluosol into rabbits produced hypoxemia, neutropenia, thrombocytopenia, and pulmonary leukostasis, mimicking abnormalities previously demonstrated in rabbits receiving infusions of zymosan-activated plasma C. These deleterious responses to Fluosol were diminished by premedicating rabbits with corticosteroids (which had seemed to benefit when used empirically in our patient). In vitro and in vivo, Fluosol's effects were reproduced by Pluronic F-68, the nonionic detergent used to maintain the emulsion stability of Fluosol-DA. We conclude that adverse reactions to Fluosol are probably mediated by C activation and that steroid premedication may prevent them in susceptible patients.

Corticosteroids inhibit complement-induced granulocyte aggregation. A possible mechanism for their efficacy in shock states.

Granulocyte (PMN) aggregation and embolization may underlie complement (C)-mediated organ dysfunction in such syndromes as hemodialysis neutropenia and Purtscher's ischem;c retinopathy. Because of clinical and pathologic parallels, we have further suggested a role for this phenomenon in the genesis of the adult respiratory distress syndrome (ARDS). Because corticosteroids are commonly used in immune diseases, and have particularly been claimed efficacious in shock and ARDS, we tested the capability of methylprednisolone (MP), hydrocortisone (HC), and dexamethasone (DEX) to inhibit PMN aggregation. Aggregation engendered in vitro by zymosan-activated plasma (ZAP) was inhibited by MP and HC at concentrations approximating plasma levels achieved with the large bolus (30 mg/kg i.v) therapy advocated in shock states; DEX was almost without effect. Using intravital fluorescence microscopy, we observed PMN aggregation and embolization in the mesenteric vessels of rats given intra-arterial infusions of ZAP; this was also prevented by pretreatment with 30 mg/kg MP. Steroid inhibition of aggregation seemed not to involve disruption of receptor function, because aggregation induced by alternative agents, n-formyl-Met-Leu-Phe and the ionophore A23187, was also inhibited by MP. Moreover, corticosteroid inhibition of PMN prostaglandin synthesis is also an unlikely explanation for our results, since aspirin and ibuprofen failed to block aggregation and arachidonic acid neither effected aggregation itself nor ameliorated the steroid effect. Our studies provide a plausible rationale for the empiric observation that high-dose corticosteroids may benefit patients with syndromes associated with microvascular leukostasis.

Membrane lipid depletion in hyperpermeable red blood cells: its role in the genesis of spherocytes in hereditary spherocytosis.

Hereditary spherocytosis (HS) red cells lose membrane lipids excessively during incubation in vitro. Individual phosphatides as well as cholesterol are lost in proportion to their content in membranes, suggesting that fragments of membrane are removed. Supplementation of HS red cells with glucose during incubation has no consistent protective effect, whereas diminishing the excessive sodium flux through these cells by suspending them in either sodium-free or hypertonic media prevents membrane fragmentation. The characteristic excessive increase in osmotic fragility which occurs in incubated HS red cells results both from inordinate accumulation of intracellular sodium ions which produces osmotic swelling, and from depletion of surface material which generates microspherocytosis. Inhibiting both of these processes by incubating HS red cells in sodium-free media completely prevents increases in osmotic fragility despite prolonged incubation. Normal red cells rendered hyperpermeable to cations by exposure either to n-butanol or to inhibitors of membrane sulfhydryl groups, lose membrane lipid upon incubation in a similar fashion to untreated HS red cells; perfectly smooth microspherocytes, akin to those seen in HS, are thereby generated.I conclude that depletion of membrane lipids in HS which leads to microspherocytosis is correlatable with the excessive cation flux and possibly to the stimulated metabolism of acidic phosphatides in these red cells. It is suggested that this relation is derived from the fact that these phosphatides are in some way involved in maintaining the proper alignment of repeating membrane lipoprotein units, and that this function is adversely affected when these molecules are turning over more rapidly in response to increased cation flux.