Marielle Kaplan

Aldosterone Administration to Mice Stimulates Macrophage NADPH Oxidase and Increases Atherosclerosis Development A Possible Role for Angiotensin-Converting Enzyme and the Receptors for Angiotensin II and Aldosterone

Background—The renin-angiotensin-aldosterone system is involved in the pathogenesis of atherosclerosis, partially because of its pro-oxidative properties. We questioned the effect and mechanisms of action of administration of aldosterone to apolipoprotein E–deficient (E0) mice on their macrophages and aorta oxidative status and the ability of pharmacological agents to block this effect. Methods and Results—Aldosterone (0.2 to 6 μg · mouse−1 ··d−1) was administered to E0 mice alone or in combination with eplerenone (200 mg · kg−1 ··d−1), ramipril (5 mg · kg−1 ··d−1), or losartan (25 mg · kg−1 ··d−1). Mouse aortic atherosclerotic lesion area and macrophage and aortic oxidative status were evaluated. Aldosterone administration enhanced the mouse atherosclerotic lesion area by 32%. Mouse peritoneal macrophages and aortic segments from aldosterone-treated mice exhibited increased superoxide anion formation by up to 155% and 69%, respectively, and this effect was probably mediated by NADPH oxidase activation, because increased translocation of its cytosolic component p47phox to the macrophage plasma membrane was observed. THP-1 macrophages incubated in vitro with aldosterone (10 μmol/L) exhibited a higher capacity to release superoxide ions by 110% and increased ability to oxidize LDL by 74% compared with control cells. Aldosterone administration enhanced mouse peritoneal macrophage ACE activity and mRNA expression by 2.3-fold and 2.4-fold, respectively. Only cotreatment of eplerenone with ramipril or losartan completely blocked the oxidative effects of aldosterone. Conclusions—Aldosterone administration to E0 mice increased macrophage oxidative stress and atherosclerotic lesion development. Blocking of the mineralocorticoid receptor and inhibition of tissue ACE and/or the angiotensin receptor-1 reduced aldosterone deleterious pro-oxidative and proatherogenic effects.

Angiotensin II stimulates macrophage-mediated oxidation of low density lipoproteins

Increased incidence of myocardial infarction was found in hypertensive patients with high plasma renin activity and increased susceptibility to oxidation was demonstrated in low density lipoprotein (LDL) that was obtained from hypertensive patients. As lipid peroxidation was demonstrated in areas of the atherosclerotic lesion, we sought to analyze the effect of angiotensin II (AN-II) on LDL oxidation, both in vitro and in vivo. Preincubation of J-774 A.1 macrophage-like cell line or mouse peritoneal macrophages (MPM) with AN-II (10(-7) M) for 1 h at 37 degrees C, followed by the addition of LDL for a further 18 h of incubation, resulted in a substantial increase in macrophage-mediated oxidation of LDL (by 55% and 19%, respectively). Similarly, incubation of LDL with MPM harvested from AN-II-injected mice resulted in a substantially increased oxidation of the lipoprotein by up to 90% in comparison to saline-injected mice. Analysis of cellular lipid peroxidation in the MPM themselves, in both the in vitro and the in vivo studies, revealed a 25% or 90% increased macrophage lipid peroxidation, respectively. The mechanism of AN-II-mediated cellular lipid peroxidation involved AN-II binding to its receptor on macrophages as saralasin, an AN-II receptor antagonist, completely inhibited this effect. Inhibitors of phospholipases A2, C and D substantially reduced macrophage lipid peroxidation, suggesting the involvement of phospholipases A2, C and D substantially reduced macrophage lipid peroxidation, suggesting the involvement of phospholipid metabolites in AN-II-mediated macrophage lipid peroxidation, suggesting the involvement of phospholipid metabolites in AN-II-mediated macrophage lipid peroxidation. Extracellular calcium ions, which active phospholipases, were also essential for AN-II-mediated macrophage lipid peroxidation since calcium channel blockers substantially inhibited cellular lipid peroxidation. Finally, the nature of the oxidant and oxygenase involved in AN-II-mediated cellular lipid peroxidation was studied using oxygenase inhibitors. Angiotensin II-mediated macrophage lipid peroxidation was found to involve the action of cellular NADPH oxidase as well as 15-lypoxygenase. We conclude that AN-II stimulates macrophage-mediated mediated oxidation of LDL secondary to cellular lipid peroxidation, and this may have a role in the accelerated atherosclerosis found in hypertensive patients.

Oxidized low density lipoprotein: atherogenic and proinflammatory characteristics during macrophage foam cell formation. An inhibitory role for nutritional antioxidants and serum paraoxonase.

Abstract Oxidative stress and inflammatory processes are of major importance in atherogenesis because they stimulate oxidized LDL (Ox-LDL)-induced macrophage cholesterol accumulation and foam cell formation, the hallmark of early atherosclerosis. Under oxidative stress, both blood monocytes and plasma lipoproteins invade the arterial wall, where they are exposed to atherogenic modifications. Oxidative stress stimulates endothelial secretion of monocyte chemoattractant protein 1 (MCP-1) and of macrophage colony stimulating factor (M-CSF), leading to monocyte adhesion and differentiation, respectively. LDL binds to extracellular matrix (ECM secreted by endothelial cells, smooth muscle cells and macrophages) proteoglycans, in a process that contributes to the enhanced susceptibility of the lipoprotein to oxidation by arterial wall macrophages. ECM-retained Ox-LDL is taken up by activated macrophages via their scavenger receptors. This leads to cellular cholesterol accumulation and enhanced atherogenesis. Protection of LDL against oxidation by antioxidants that can act directly on the LDL, or indirectly on the cellular oxidative machinery, or conversion of Ox-LDL to a non-atherogenic particle by HDL-associated paraoxonase (PON-1), can contribute to attenuation of atherosclerosis.

Effect of eplerenone, a selective aldosterone blocker, on blood pressure, serum and macrophage Oxidative stress, and atherosclerosis in apolipoprotein E-deficient mice

Oxidative stress is involved in the pathogenesis of atherosclerosis, and angiotensin II (AT-II) induces oxidative stress and enhances atherogenesis. Aldosterone, which has an important role in the pathology of heart failure, has recently been implicated as a mediator of AT-II biologic activities. In this study, we analyzed whether administration of the selective aldosterone blocker eplerenone to atherosclerotic apolipoprotein E–deficient (E0) mice would affect their oxidative status and atherogenesis. Apolipoprotein E–deficient mice were administered chow containing eplerenone (200 mg/kg/day) for 3 months. Blood pressure, serum and macrophage oxidative status, and aortic atherosclerotic lesion area were evaluated in mice treated with eplerenone compared with untreated mice. Eplerenone administration significantly decreased systolic and diastolic blood pressure by 12% and 11%, respectively, compared with untreated mice. Serum susceptibility to lipid peroxidation decreased by as much as 26%, and serum paraoxonase activity increased by 28% in eplerenone-treated mice compared with untreated mice. Peritoneal macrophages from eplerenone-treated mice contained reduced levels of lipid peroxides, and their macrophage oxidation of low-density lipoprotein (LDL) and superoxide ion release were significantly reduced (by 17% and 43%, respectively), compared to untreated mice. Daily injections of AT-II (0.1 mL, 10−7M) during the final 3 weeks of the study in eplerenone-treated mice substantially attenuated the eplerenone-mediated reduction in macrophage superoxide release and LDL oxidation. Finally, the atherosclerotic lesion area in aortas of eplerenone-treated mice was significantly reduced (by 35%) versus untreated mice, and this effect was reversed by AT-II. Administration of the selective aldosterone blocker eplerenone significantly reduced oxidative stress and atherosclerosis progression in E0 mice. These data suggest that aldosterone could have a significant pro-oxidative role in the pathogenesis of atherosclerosis.

Angiotensin II–Modified LDL Is Taken Up by Macrophages Via the Scavenger Receptor, Leading to Cellular Cholesterol Accumulation

The incidence of myocardial infarction is significantly higher in hypertensive patients with increased plasma concentration of angiotensin (Ang) II. Ang II was shown to bind to LDL in vitro, and in the present study we showed its binding to LDL in vivo. Ang II (10(-7) mol/L) was incubated with LDL for 3 hours at 37 degrees C, followed by reseparation of the modified lipoprotein (Ang II-LDL) and its incubation with J-774 A.1 macrophages. Binding of Ang II to LDL significantly increased the lipoprotein protein degradation (by 25%) and its cell association (by 75%) compared with nontreated LDL. Unlike Ang II-LDL, both Ang I-LDL and Ang III-LDL were taken up by macrophages similar to native LDL. The lipid composition and size of Ang II-LDL were similar to those of native LDL, and it was not aggregated. Ang II-LDL was not oxidized, as the contents of malondialdehyde and peroxides were not different from those found in native LDL. On heparin-Sepharose column chromatography, Ang II-LDL was eluted in the void volume, like acetylated LDL (Ac-LDL) and unlike native LDL, which binds to heparin. The cellular degradation of Ang II-125I-labeled LDL by J-774 A.1 macrophages of Ang II-125I-labeled LDL by J-774 A.1 macrophages was studied in the presence of a 50-fold excess of nonlabeled native LDL, Ang II-LDL, Ac-LDL, or oxidized LDL (Ox-LDL). Whereas native LDL had no effect on the degradation of Ang II-125I-LDL by the macrophages, Ac-LDL, Ox-LDL, and Ang II-LDL reduced the cellular uptake of the lipoprotein by 77%, 82%, and 87%, respectively. Similarly, fucoidin but not free Ang II reduced macrophage degradation of the labeled Ang II-LDL. We conclude that Ang II can modify LDL to a form that is not oxidized or aggregated but is still taken up at an enhanced rate by macrophages via the scavenger receptor.

Increased uptake of LDL by oxidized macrophages is the result of an initial enhanced LDL receptor activity and of a further progressive oxidation of LDL.

Iron ions were recently shown to induce cellular lipid peroxidation in macrophages, and these oxidized cells can convert native low-density lipoprotein (LDL) to oxidized LDL (Ox-LDL). The present study demonstrates that deoxycholic acid (DCA) and angiotensin II (ANG-II) can also induce oxidative modification of macrophages via metal ions independent mechanisms. Furthermore, incubation of LDL (200 micrograms of protein/ml) for 24 h at 37 degrees C with DCA, ANG-II, as well as FeSO4-induced oxidized macrophages, resulted in oxidative modification of the lipoprotein as evidenced by increased TBARS formation in LDL (by 50, 105, and 258%, respectively), decreased TNBS reactivity (by 45, 56, and 42%, respectively), and increased cellular uptake (by 60, 166, and 230%, respectively). A positive correlation (n = .88) was found between the extent of the cellular lipid peroxidation and the increment in the cellular uptake of the LDL. The oxidative modification of LDL by oxidized macrophages was found to be a progressive process. Incubation of LDL with oxidized macrophages for increasing periods of time up to 24 h resulted in progressive increment in: (1) the electrophoretic mobility of the LDL; (2) the TBARS formation in LDL; (3) the cellular uptake of LDL by the oxidized macrophages via the Ox-LDL receptor. Upon fractionation on a heparin-sepharose column of LDL that was incubated for different periods of time with oxidized macrophages, a gradual increment in the unbound LDL fraction was obtained, up to 72% after 24 h of incubation. During the first hour of LDL incubation with the oxidized macrophages a twofold increase in the cellular uptake of LDL by these cells was detected, although no significant oxidation of the lipoprotein occurred during this short time period. This effect could be attributed to an increased number of LDL receptors on the cell surface of the oxidized macrophages. In conclusion, increased uptake of LDL by oxidized macrophages results from two routes: (1) enhanced uptake via the LDL receptor due to increased LDL receptor activity; (2) lipoprotein uptake via the Ox-LDL receptors due to cellular modification of LDL. Both of these processes lead to macrophage cholesterol accumulation and foam cell formation, and thus contribute to accelerated atherosclerosis under oxidative stress.

Mineralocorticoid Receptor Blocker Increases Angiotensin-Converting Enzyme 2 Activity in Congestive Heart Failure Patients

Aldosterone plays an important role in the pathophysiology of congestive heart failure (CHF), and spironolactone improves cardiovascular function and survival rates in patients with CHF. We hypothesized that the mineralocorticoid receptor blockade (MRB) exerted its beneficial effects by reducing oxidative stress and changing the balance between the counter-acting enzymes angiotensin-converting enzyme (ACE) and ACE2. Monocyte-derived macrophages were obtained from 10 patients with CHF before and after 1 month of treatment with spironolactone (25 mg/d). Spironolactone therapy significantly (P<0.005) reduced oxidative stress, as expressed by reduced lipid peroxide content, superoxide ion release, and low-density lipoprotein oxidation by 28%, 53%, and 70%, respectively. Although spironolactone significantly (P<0.01) reduced macrophage ACE activity by 47% and mRNA expression by 53%, ACE2 activity and mRNA expression increased by 300% and 654%, respectively. In mice treated for 2 weeks with eplerenone (200 mg · kg−1 · d−1), cardiac ACE2 activity significantly (P<0.05) increased by 2-fold and was paralleled by increased ACE2 activity in macrophages. The mechanism of aldosterone antagonist action was studied in mouse peritoneal macrophages (MPMs) in vitro. Although ACE activity and mRNA were significantly increased by 250 nmol/L aldosterone, ACE2 was significantly reduced. Cotreatment with eplerenone (2 &mgr;mol/L) attenuated these effects. In MPM obtained from p47 knockout mice, where NADPH oxidase is inactive, as well as in control MPMs treated with NADPH oxidase inhibitor, aldosterone did not increase ACE or decrease ACE2. MRB reduced oxidative stress, decreased ACE activity, and increased ACE2 activity, suggesting a protective role for MRB by possibly increasing generation of angiotensin (1–7) and decreasing formation of angiotensin II. These effects are mediated, at least in part, by NADPH oxidase.

Oral Insulin Supplementation Attenuates Atherosclerosis Progression in Apolipoprotein E-Deficient Mice

Objective—The role of insulin in atherosclerosis progression in diabetes is uncertain. We examined the effects of oral insulin supplementation on atherogenesis in apolipoprotein E-deficient (E0) mice. Methods and Results—One-month-old male E0 mice were orally supplemented with human insulin (0.1, 0.5, and 1 U/mL) or placebo for 3 months. At the end of the study, serum and macrophage oxidative stress and atherosclerosis progression were studied. Insulin reduced lesion size by 22% to 37% (P <0.05) in all study groups. Lipid peroxides serum levels were 18% lower (P <0.01), and serum paraoxonase activity was 30% higher (P <0.01) in mice supplemented with 1.0 U/mL insulin compared with controls. Insulin reduced mouse peritoneal macrophage (MPM) lipid peroxides content and superoxide anion release by up to 44% and 62%, respectively (P <0.01). In addition, oral insulin reduced MPM cholesterol content and cholesterol biosynthesis by up to 36% and 53%, respectively (P <0.01). In vitro incubation of E0 mice MPM with increasing insulin concentrations (0 to 100 &mgr;U/mL) resulted in a dose-dependent reduction of cholesterol synthesis by up to 66% (P <0.05). Conclusions—In E0 mice, oral insulin supplementation attenuates the atherosclerotic process. This may be attributable to insulin-mediated reduction of oxidative stress in serum and macrophages as well as reduction in macrophage cholesterol content.

Relation between changes in red cell distribution width and clinical outcomes in acute decompensated heart failure

BACKGROUND Increased red blood cell distribution (RDW) has been associated with adverse outcomes in patients with heart failure. We studied the association between baseline RDW and changes in RDW during hospital course with clinical outcomes in acute decompensated heart failure (ADHF) patients. METHODS AND RESULTS We prospectively studied 614 patients with ADHF. Baseline RDW and RDW change during hospital course were determined. The relationship between RDW and clinical outcomes after hospital discharge was tested using Cox regression models, adjusting for clinical characteristics, echocardiographic findings and brain natriuretic peptide levels. During follow up (1 year), 286 patients (46.6%) died and 84 were readmitted for ADHF (13.7%). Median RDW was significantly higher among patients who died compared to patients who survived (15.6% interquartile range [14.5 to 17.1] vs. 14.9% mg/L interquartile range [14.1 to 16.1], P<0.0001). Compared with patients in the 1st RDW quartile, the adjusted hazard ratio [HR] for death or rehospitalization was 1.9 [95% CI 1.3-2.6] in patients in the 4th quartile. Changes in RDW during hospitalization were strongly associated with changes in mortality risk. Compared with patients with persistent normal RDW (<14.5%), the adjusted HR for mortality was 1.9 [95% CI 1.1-3.1] for patients in whom RDW increased above 14.5% during hospital course, similar to patients with persistent elevation of RDW (HR was 1.7, 95% CI 1.2-2.3). CONCLUSION In patients hospitalized with ADHF, RDW is a strong independent predictor of greater morbidity and mortality. An increase in RDW during hospitalization also portends adverse clinical outcome.

Oxidative stress and macrophage foam cell formation during diabetes mellitus‐induced atherogenesis: Role of insulin therapy

Diabetes mellitus (DM) and hyperglycemia are associated with premature and accelerated atherosclerosis. This is mediated by induction of vascular dysfunction, increased inflammatory burden and increased lipid peroxidation, all leading to enhanced macrophage foam cell formation. In DM, low density lipoprotein (LDL) oxidation by macrophages is increased due to the activation of several pro-oxidant systems, as well as the depletion of antioxidants, such as the paraoxonases (PONs). Paraoxonases protect against atherogenesis, as serum PON1 exerts a protective role against DM development by stimulating insulin secretion from β cells, and its unique antioxidant properties. Oral supplementation of insulin to mice significantly attenuates macrophage foam cell formation, reduces oxidative stress and decreases the atherosclerotic plaque area and. Insulin may directly inhibit lipid peroxidation via inhibition of NADPH oxidase expression. Insulin has additional protective effects against DM-induced macrophage cholesterol accumulation by inhibiting CD36 expression (an oxidized LDL receptor), and by inhibiting HMGCoA reductase expression (the rate limiting enzyme in cholesterol biosynthesis), through inhibition of the formation of active SREBP-1 (the transcription factor that activates HMGCoA reductase). Although insulin is mainly an anti-atherogenic agent, it also has some pro-atherosclerotic effects in insulin resistant individuals including the induction of dyslipidemia, cellular triglycerides accumulation and pro-thrombotic effects. This reviews intent is to help clarify the mechanisms underlying the protective anti-atherogenic role of insulin in DM as well as some pro-atherogenic effects. A better understanding of insulins involvement in the pathogenesis of atherosclerosis in DM could have major therapeutic implications for DM treatment and its consequent cardiovascular complications.