Nalini Santanam

Irbesartan, an angiotensin type 1 receptor inhibitor, regulates markers of inflammation in patients with premature atherosclerosis

OBJECTIVESnThis study assessed the role of angiotensin II type 1 (AT1) receptor antagonists on inflammatory mechanisms involved in atherogenesis. Specific inflammatory markers included solubilized tumor necrosis factor-alpha receptor II (sTNF-alphaRII), vascular cell adhesion molecule-1 (VCAM-1) and superoxide. In addition, the AT1 receptor blocker irbesartan was evaluated for its ability to suppress these markers in individuals with atherosclerosis.nnnBACKGROUNDnMechanisms involved in the complex process of atherogenesis include alterations in the inflammatory responses. The use of compounds that suppress these responses may reduce the degree of damage seen in atherosclerosis.nnnMETHODSnWith a cross-sectional study design, 33 normotensive patients with stable coronary artery disease (CAD) were treated with irbesartan for a 24-week period. These patients were compared against a control population with no known coronary atherosclerosis. Marker levels were measured by enzyme-linked immunosorbent assay technique and lucigenin chemiluminescence assay and statistically evaluated by two-way repeated measures analysis of variance.nnnRESULTSnAll patients with coronary artery disease had increased levels of inflammatory molecules over those of control patients. Treatment with irbesartan in these patients significantly reduced levels of inflammatory molecules measured. Soluble VCAM-1 levels were reduced by 36%; soluble TNF-alpha levels were reduced by 54% and superoxide level decreased by 52%. Maximal suppression of inflammatory markers by irbesartan therapy in patients with CAD was seen at 12 weeks.nnnCONCLUSIONSnThe effect of irbesartan on each inflammatory marker is significant. Our results show that use of irbesartan may retard the inflammatory process seen in premature forms of atherosclerosis.

Cholesterol Depletion Inhibits Epidermal Growth Factor Receptor Transactivation by Angiotensin II in Vascular Smooth Muscle Cells ROLE OF CHOLESTEROL-RICH MICRODOMAINS AND FOCAL ADHESIONS IN ANGIOTENSIN II SIGNALING

Masuko Ushio-Fukai, Lula Hilenski, Nalini Santanam, Peter L. Becker, Yuxian Ma, Kathy K. Griendling, and R. Wayne Alexander This article has been retracted by the publisher. The Emory University Investigation Committee found evidence of inappropriate imagemanipulation in Figs. 2A and 3 that causedmisrepresentation of the data. THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 288, NO. 45, p. 32637, November 8, 2013

Peroxidase Properties of Extracellular Superoxide Dismutase: Role of Uric Acid in Modulating In Vivo Activity

Objective—The cytosolic form of Cu/Zn-containing superoxide dismutase (SOD1) has peroxidase activity, with H2O2 used as a substrate to oxidize other molecules. We examined peroxidase properties of the extracellular form of SOD (SOD3), a major isoform of SOD in the vessel wall, by using recombinant SOD3 and an in vivo model of atherosclerosis. Methods and Results—In the presence of HCO3−, SOD3 reacted with H2O2 to produce a hydroxyl radical adduct of the spin trap 5-diethoxyphosphoryl-5methyl-1-pyrroline N-oxide (DEMPO). SOD1 and SOD3 were inactivated by H2O2 in a dose- and time-dependent fashion, and this was prevented by physiological levels of uric acid. To examine the in vivo role of uric acid on SOD1 and SOD3, control and apolipoprotein E-deficient (ApoE−/−) mice were treated with oxonic acid, which inhibits urate metabolism. This treatment increased plasma levels of uric acid in control and ApoE−/− mice by ≈3-fold. Although increasing uric acid levels did not alter aortic SOD1 and SOD3 protein expression, aortic SOD1 and SOD3 activities were increased by 2- to 3-fold in aortas from ApoE−/− mice but not in aortas from control mice. Conclusions—These studies show that SOD1 and SOD3 are partially inactivated in atherosclerotic vessels of ApoE−/− mice and that levels of uric acid commonly encountered in vivo may regulate vascular redox state by preserving the activity of these enzymes.

Exercise and Cardiovascular Disease: A New Perspective

The oxidation of low density lipoprotein (LDL) has been suggested as a key event in atherogenesis. Paradoxically, exercise, which imposes an oxidative stress, is an important deterrent of cardiovascular disease. In study 1 the oxidizability of LDL was enhanced in exercisers compared with sedentary controls. The lag time of isolated LDL subjected to copper-induced in vitro oxidation was significantly shortened in the exercisers compared with sedentary subjects. This increased sensitivity was not due to a decreased presence of vitamin E. Instead, these findings suggested that the LDL of exercisers may contain increased amounts of preformed lipid peroxides, which account for the increased oxidizability. In study 2, a group x sex ANOVA revealed that male exercisers had a significantly longer mean lag time than male sedentary subjects and that females had similar mean lag times regardless of exercise group. This remained the case when statistical adjustment was made for age, body mass index, blood lipid levels, LDL, and plasma alpha-tocopherol levels. Study 1 exercisers had been in training for a shorter time (< 1 year) than study 2 exercisers (> 2 years). These findings suggest that truly chronic exercise (aerobic intensity over several months) decreases the susceptibility of a male exercisers LDL to undergo oxidation. Conversely, regular aerobic stress during an overall shorter time span creates a more oxidative environment in the body, thus increasing the susceptibility of LDL to undergo oxidation. The oxidative stress of aerobic exercise does not appear to adversely affect the oxidizability of LDL in women.

Autoantibodies to markers of oxidative stress are elevated in women with endometriosis

OBJECTIVEnTo measure autoantibodies that recognize oxidatively modified proteins in the sera of women with surgically proven endometriosis.nnnDESIGNnProspective study.nnnSETTINGnTertiary care academic medical center.nnnPATIENT(S)nWomen undergoing surgery for endometriosis or tubal ligation.nnnINTERVENTION(S)nNone.nnnMAIN OUTCOME MEASURE(S)nSerum and peritoneal fluid autoantibody titers to malondialdehyde-modified low-density lipoprotein, oxidized low-density lipoprotein, and lipid peroxide-modified rabbit serum albumin determined by ELISA. Correlation of autoantibody titers with revised American Fertility Society staging classification, symptoms, and morphologic type of endometriosis.nnnRESULT(S)nMean (+/-SEM) serum autoantibody titers (in optical density units) to the three antigens were as follows: [1] lipid peroxide-modified rabbit serum albumin, 0.49 +/- 0.12 units in the patients with endometriosis and 0.2 +/- 0.02 units in the controls; [2] oxidized low-density lipoprotein, 0.22 +/- 0.005 units in the patients with endometriosis and 0.18 +/- 0.006 units in the controls; and [3] malondialdehyde-modified low-density lipoprotein, 0.21 +/- 0.005 units in the patients with endometriosis and 0.16 +/- 0.003 units in the controls. There was no correlation between autoantibody titers and revised American Fertility Society stage, symptoms, or morphologic type of endometriosis. Peritoneal fluid did not contain autoantibodies to any of the three antigens.nnnCONCLUSION(S)nAutoantibodies to markers of oxidative stress were significantly increased in women with endometriosis. These findings strongly support our data demonstrating that women with endometriosis have enhanced oxidative stress.

Macrophages, Oxidation, and Endometriosis

Abstract: Retrograde menstruation has been suggested to be the cause for the presence of endometrial cells in the peritoneal cavity. However, little is known about the events that lead to the adhesion and growth of these cells that ultimately result in endometriosis, considering the fact that the disease occurs only in certain women despite the common occurrence of retrograde menstruation in most women. We postulate that, in normal women, the endometrial cells and tissue that arrive in the peritoneal cavity during menstruation are effectively removed by macrophages that are chemoattracted and become resident tissue macrophages in the peritoneal cavity. In contrast, the peritoneal macrophages in women with endometriosis are nonadherent and ineffectively scavenged, resulting in the sustained presence and growth of the endometrial cells. We also postulate that the peritoneal fluid is not a passive reservoir of the factors secreted by cells of the peritoneum, but actively promotes endometriosis. The peritoneal fluid is rich in lipoproteins, particularly low‐density lipoprotein, which generates oxidized lipid components in a macrophage‐rich inflammatory milieu. The oxidants exacerbate the growth of endometriosis by inducing chemoattractants such as MCP‐1 and endometrial cell growth‐promoting activity. We provide evidence for the presence of oxidative milieu in the peritoneal cavity of women with endometriosis, the nonscavenging properties of macrophages that are nonadherent, and the synergistic interaction between macrophages, oxidative stress, and the endometrial cells. For example, the peritoneal fluid lipoproteins of subjects with endometriosis have increased the propensity to undergo oxidation as compared with plasma lipoproteins, and the subjects also have increased titer of autoantibodies to oxidatively modified proteins. If the oxidative proinflammatory nature of the peritoneal fluid is an important mediator of endometriosis growth, anti‐inflammatory agents and antioxidants might afford protection against endometriosis.

Potential role of oxidized lipids and lipoproteins in antioxidant defense.

The atherogenic oxidative modification of low-density lipoprotein is suggested to occur in the aortic intima. There is reasonable evidence to suggest that antioxidants might be beneficial in preventing or retarding the progression of atherosclerosis. Exercise, estrogens, and substitution of polyunsaturated fat for saturated fat are beneficial in the prevention of atherosclerosis. Yet, paradoxically, they are capable of inducing an oxidative stress. To reconcile with this paradox, we postulate that under certain conditions an oxidative stress might be beneficial by inducing antioxidant enzymes in arterial cells. However, those with genetic deficiency in antioxidant enzymes or those who poorly respond to oxidative stress or those with overwhelming plasma oxidative stress might need additional antioxidant protection.

Does Acute Exercise Affect the Susceptibility of Low Density Lipoprotein to Oxidation

This study describes the effect of an acute exercise bout on the susceptibility of isolated low density lipoprotein (LDL) to in vitro oxidation. LDL was isolated from 23 subjects (exercisers, n = 11; sedentary, n = 12) immediately before and after a single bout of exercise (30 min of treadmill work at 55% & 70% peak oxygen consumption (VO2 peak) for exercisers and sedentary, respectively). A statistically significant decrease in lag time for LDL oxidation was observed following exercise compared to baseline (96.1+/-23.5 min vs. 92.1+/-23.3 minutes; n = 23, p < or = .03) using a 5 microM copper system. There was a statistically significant increase in plasma myeloperoxidase (MPO) levels following exercise compared to baseline values ( 1.58+/-.91 ng/dl versus 2.08+/-1.2 ng/dl; n = 12, p < or = .03). These results suggest that the 30 min exercise bout at a moderate intensity and duration was a sufficient oxidative stress to increase the susceptibility of LDL to in vitro oxidation. Additionally, the exercise bout appeared to activate neutrophils, subsequently releasing MPO protein.

Generation of a Polyclonal Antibody Against Lipid Peroxide-Modified Proteins

A specific polyclonal antibody against the lipid peroxide (LOOH)-modified rabbit serum albumin (RSA) was generated in rabbits. The antibody selectively recognized the modified protein in a concentration-dependent manner and did not cross react with aldehyde-modified proteins or proteins directly oxidized with the free radical generator 2,2-azobis (2-amidinopropane) hydrochloride (AAPH). Oxidized low-density lipoprotein (Ox-LDL), but not native LDL, was also recognized by the antibody in a concentration-dependent manner. The antibody also cross reacted with several other proteins modified by LOOH suggesting that the antibody is directed towards a common epitope and not towards the protein sequence. Western blot analysis of normal human plasma showed that at least three different proteins are recognized by the antibody. RAW cells, preincubated with LOOH, were immunostained with the antibody and the antigenic epitopes were present intracellularly, while controls lacking in the primary antibodies failed to show immunoreactivity. Atherosclerotic arteries from cholesterol-fed monkeys and human atherosclerotic lesions were also immunostained by the antibody. The immunoreactivity was co-localized in areas rich in foam cell macrophages. These results suggest that LOOH-modified proteins present an unique antigenic epitope that may represent a primary product of interaction of LOOH with proteins.

RU486-induced growth inhibition of human endometrial cells.

OBJECTIVEnTo determine the direct action of RU486 on endometrial cell proliferation and to differentiate whether the antioxidant or the antiprogesterone property of RU486 is predominately responsible for its effect on cell growth.nnnDESIGNnIn vitro study comparing the effects of RU486 (antiprogesterone and antioxidant), reduced RU486 (antioxidant), ZK112,993 (antiprogesterone), and lazaroid U74,500A (antioxidant) on endometrial cell growth. The human endometrial cell line EM42 was used in transient transfection assays to confirm the relative antiprogesterone potency of the various compounds.nnnSETTINGnAcademic medical centernnnPATIENT(S)nWomen presenting with pelvic pain or infertility and diagnosed with endometriosis at time of surgery or women desiring tubal ligation with a normal pelvis (controls).nnnINTERVENTION(S)nEndometrial cell cultures were treated with RU486, reduced RU486, lazaroid U74,500A, and ZK112,993.nnnMAIN OUTCOME MEASURE(S)nTritiated thymidine incorporation was used to assess cell growth. Inhibition of progesterone induction of transiently transfected reporter plasmids was used to measure antiprogesterone activity of compounds studied.nnnRESULT(S)nRU486 reduced cell growth in a dose-dependent fashion of the endometrial cell lines EM42 and RL95-2 and of endometrial and endometriosis cells from primary culture. After being reduced, RU486 lost most of its antiprogesterone activity but retained its antiproliferative properties. ZK112,993 was similar in potency to RU486 as a progesterone antagonist but did not significantly modify endometrial cell growth. Lazaroid U74,500A was devoid of antiprogesterone activity but was shown to be a potent antiproliferative agent.nnnCONCLUSION(S)nRU486 has a direct inhibitory effect on human endometrial cell growth. This activity appears to be at least partly mediated through its antioxidant property.