Reactive Carbonyl Species and Protein Lipoxidation in Atherogenesis.

Atherosclerosis is a multifactorial disease of medium and large arteries, characterized by the presence of lipid-rich plaques lining the intima over time. It is the main cause of cardiovascular diseases and death worldwide. Redox imbalance and lipid peroxidation could play key roles in atherosclerosis by promoting a bundle of responses, including endothelial activation, inflammation, and foam cell formation. The oxidation of polyunsaturated fatty acids generates various lipid oxidation products such as reactive carbonyl species (RCS), including 4-hydroxy alkenals, malondialdehyde, and acrolein. RCS covalently bind to nucleophilic groups of nucleic acids, phospholipids, and proteins, modifying their structure and activity and leading to their progressive dysfunction. Protein lipoxidation is the non-enzymatic post-translational modification of proteins by RCS. Low-density lipoprotein (LDL) oxidation and apolipoprotein B (apoB) modification by RCS play a major role in foam cell formation. Moreover, oxidized LDLs are a source of RCS, which form adducts on a huge number of proteins, depending on oxidative stress intensity, the nature of targets, and the availability of detoxifying systems. Many systems are affected by lipoxidation, including extracellular matrix components, membranes, cytoplasmic and cytoskeletal proteins, transcription factors, and other components. The mechanisms involved in lipoxidation-induced vascular dysfunction are not fully elucidated. In this review, we focus on protein lipoxidation during atherogenesis.

PI3KCIIα-Dependent Autophagy Program Protects From Endothelial Dysfunction and Atherosclerosis in Response to Low Shear Stress in Mice.

BACKGROUND: The ability to respond to mechanical forces is a basic requirement for maintaining endothelial cell (ECs) homeostasis, which is continuously subjected to low shear stress (LSS) and high shear stress (HSS). In arteries, LSS and HSS have a differential impact on EC autophagy processes. However, it is still unclear whether LSS and HSS differently tune unique autophagic machinery or trigger specific autophagic responses in ECs. METHODS: Using fluid flow system to generate forces on EC and multiscale imaging analyses on ApoE-/- mice whole arteries, we studied the cellular and molecular mechanism involved in autophagic response to LSS or HSS on the endothelium. RESULTS: We found that LSS and HSS trigger autophagy activation by mobilizing specific autophagic signaling modules. Indeed, LSS-induced autophagy in endothelium was independent of the class III PI3K (phosphoinositide 3-kinase) VPS34 (vacuolar sorting protein 34) but controlled by the α isoform of class II PI3K (phosphoinositide 3-kinase class II α [PI3KCIIα]). Accordingly, reduced PI3KCIIα expression in ApoE-/- mice (ApoE-/-PI3KCIIα+/-) led to EC dysfunctions associated with increased plaque deposition in the LSS regions. Mechanistically, we revealed that PI3KCIIα inhibits mTORC1 (mammalian target of rapamycin complex 1) activation and that rapamycin treatment in ApoE-/-PI3KCIIα+/- mice specifically rescue autophagy in arterial LSS regions. Finally, we demonstrated that absence of PI3KCIIα led to decreased endothelial primary cilium biogenesis in response to LSS and that ablation of primary cilium mimics PI3KCIIα-decreased expression in EC dysfunction, suggesting that this organelle could be the mechanosensor linking PI3KCIIα and EC homeostasis. CONCLUSIONS: Our data reveal that mechanical forces variability within the arterial system determines EC autophagic response and supports a central role of PI3KCIIα/mTORC1 axis to prevent EC dysfunction in LSS regions.

Lipidome Investigation of Carnosine Effect on Nude Mice Skin to Prevent UV-A Damage.

The lipid profile of skin is fundamental in the maintenance of the protective barrier against the external environment. Signaling and constitutive lipids of this large organ are involved in inflammation, metabolism, aging, and wound healing, such as phospholipids, triglycerides, FFA, and sphingomyelin. Skin exposure to ultraviolet (UV) radiation results in a photoaging process that is an accelerated form of aging. UV-A radiation deeply penetrates the dermis and promotes damage to DNA, lipids, and proteins by increasing the generation of reactive oxygen species (ROS). Carnosine, an endogenous ß-alanyl-L-histidine dipeptide, demonstrated antioxidant properties that prevent photoaging and modification of skin protein profiling, making carnosine a compelling ingredient to consider for use in dermatology. The aim of this research was to investigate the modification of skin lipidome after UV-A treatment in presence or not of topic administration of carnosine. Quantitative analyses based on high-resolution mass spectrometry of nude mice skin-extracted lipids resulted in several modifications of barrier composition after UV-A radiation, with or without carnosine treatment. In total, 328 out of 683 molecules showed significant alteration-262 after UV-A radiation and 126 after UV-A and carnosine treatment versus controls. Importantly, the increased oxidized TGs after UV-A radiation, responsible of dermis photoaging, were completely reverted by carnosine application to prevent the UV-A damage. Network analyses also showed that the production of ROS and the calcium and TNF signaling were modulated by UV-A and carnosine. In conclusion, lipidome analyses attested the carnosine activity to prevent the UV-A damage, reducing the lipid oxidation, the inflammation, and the dysregulation of lipid skin barrier.

Post-Translational Modifications Evoked by Reactive Carbonyl Species in Ultraviolet-A-Exposed Skin: Implication in Fibroblast Senescence and Skin Photoaging.

Photoaging is an accelerated form of aging resulting from skin exposure to ultraviolet (UV) radiation. UV-A radiation deeply penetrates the dermis and triggers the generation of reactive oxygen species (ROS) which promotes damage to DNA, lipids and proteins. Lipid peroxidation results from the oxidative attack of polyunsaturated fatty acids which generate a huge amount of lipid peroxidation products, among them reactive carbonyl species (RCS) such as α, ß-unsaturated hydroxyalkenals (e.g., 4-hydroxynonenal), acrolein or malondialdehyde. These highly reactive agents form adducts on free NH2 groups and thiol residues on amino acids in proteins and can also modify DNA and phospholipids. The accumulation of RCS-adducts leads to carbonyl stress characterized by progressive cellular and tissular dysfunction, inflammation and toxicity. RCS-adducts are formed in the dermis of skin exposed to UV-A radiation. Several RCS targets have been identified in the dermis, such as collagen and elastin in the extracellular matrix, whose modification could contribute to actinic elastosis lesions. RCS-adducts may play a role in fibroblast senescence via the modification of histones, and the sirtuin SIRT1, leading to an accumulation of acetylated proteins. The cytoskeleton protein vimentin is modified by RCS, which could impair fibroblast motility. A better identification of protein modification and carbonyl stress in the dermis may help to develop new treatment approaches for preventing photoaging.

Oxidative stress, lipid peroxidation and premature placental senescence in preeclampsia.

Accelerated placental senescence is associated with preeclampsia (PE) and other pregnancy complications. It is characterized by an accelerated decline in placental function due to the accumulation of senescence patterns such as telomere shortening, mitochondrial dysfunction, oxidative damages, increased expression of phosphorylated (serine-139) histone γ-H2AX, a sensitive marker of double-stranded DNA breaks, accumulation of cross-linked ubiquitinated proteins and sirtuin inhibition. Among the lipid oxidation products generated by the peroxidation of polyunsaturated fatty acids, aldehydes such as acrolein, 4-hydroxy-2-nonenal, 4-oxo-2-nonenal, are present in the blood and placenta from PE-affected women and could contribute to PE pathogenesis and accelerated placental aging. In this review we summarize the current knowledge on premature placental senescence and the role of oxidative stress and lipid oxidation-derived aldehydes in this process, as well as their links with PE pathogenesis. The interest of developing (or not) new therapeutic strategies targeting lipid peroxidation is discussed, the objective being a better understanding of accelerated placental aging in PE pathophysiology, and the prevention of PE bad outcomes.

Study of Carnosine's effect on nude mice skin to prevent UV-A damage.

The skin is an important barrier against external attacks from bacteria, radicals, or radiations. UV-A radiations cause significant impairment of this barrier, inducing inflammation, oxidative stress, and wrinkle formation, thereby promoting photoaging. Previous studies reported that carnosine, a potent antioxidant, and carbonyl scavenger agent, may prevent photoaging features in the skin of hairless mice exposed to UV-A radiations. In the present study, we used a quantitative proteomic approach to analyze the changes evoked by carnosine in the skin proteome of hairless mice exposed to UV-A. This approach allowed to quantify more than 2480 proteins, among them consistent differences were observed for 89 proteins in UV-A exposed vs control unexposed skins, and 252 proteins in UV-A-exposed skin preventively treated by carnosine (UVAC) vs UV-A. Several functional pathways were altered in the skins of UV-A exposed hairless mice, including the integrin-linked kinase, calcium signaling, fibrogenesis, cell migration and filament formation. An impairment of mitochondrial function and metabolism was observed, with an up-regulation of cytochrome C oxidase 6B1 and NADH: ubiquinone oxidoreductase S8. Skins pre-treated by carnosine were prevented from UV-A induced proteome alterations. In conclusion, our study emphasizes the potency of a proteomic approach to identify the consequences of UV radiations in the skins, and points out the capacity of carnosine to prevent the alterations of skin proteome evoked by UV-A.

4-Hydroxynonenal Contributes to Fibroblast Senescence in Skin Photoaging Evoked by UV-A Radiation.

Solar ultraviolet A (UV-A) radiation promotes a huge variety of damages on connective tissues and dermal fibroblasts, including cellular senescence, a major contributor of skin photoaging. The mechanisms of skin photoaging evoked by UV-A partly involve the generation of reactive oxygen species and lipid peroxidation. We previously reported that 4-hydroxynonenal (HNE), a lipid peroxidation-derived aldehyde, forms adducts on elastin in the skins of UV-A irradiated hairless mice, possibly contributing to actinic elastosis. In the present study, we investigated whether and how HNE promotes fibroblast senescence in skin photoaging. Dermal fibroblasts of skins from UV-A-exposed hairless mice exhibited an increased number of γH2AX foci characteristic of cell senescence, together with an accumulation of HNE adducts partly colocalizing with the cytoskeletal protein vimentin. Murine fibroblasts exposed to UV-A radiation (two cycles of 15 J/cm2), or HNE (30 µM, 4 h), exhibited senescence patterns characterized by an increased γH2AX foci expression, an accumulation of acetylated proteins, and a decreased expression of the sirtuin SIRT1. HNE adducts were detected on vimentin in cultured fibroblasts irradiated by UV-A or incubated with HNE. The HNE scavenger carnosine prevented both vimentin modification and fibroblast senescence evoked by HNE in vitro and in the skins of UV-A-exposed mice. Altogether, these data emphasize the role of HNE and lipid peroxidation-derived aldehydes in fibroblast senescence, and confirm the protective effect of carnosine in skin photoaging.

Role of oxidative stress in the dysfunction of the placental endothelial nitric oxide synthase in preeclampsia.

Preeclampsia (PE) is a multifactorial pregnancy disease, characterized by new-onset gestational hypertension with (or without) proteinuria or end-organ failure, exclusively observed in humans. It is a leading cause of maternal morbidity affecting 3-7% of pregnant women worldwide. PE pathophysiology could result from abnormal placentation due to a defective trophoblastic invasion and an impaired remodeling of uterine spiral arteries, leading to a poor adaptation of utero-placental circulation. This would be associated with hypoxia/reoxygenation phenomena, oxygen gradient fluctuations, altered antioxidant capacity, oxidative stress, and reduced nitric oxide (NO) bioavailability. This results in part from the reaction of NO with the radical anion superoxide (O2•-), which produces peroxynitrite ONOO-, a powerful pro-oxidant and inflammatory agent. Another mechanism is the progressive inhibition of the placental endothelial nitric oxide synthase (eNOS) by oxidative stress, which results in eNOS uncoupling via several events such as a depletion of the eNOS substrate L-arginine due to increased arginase activity, an oxidation of the eNOS cofactor tetrahydrobiopterin (BH4), or eNOS post-translational modifications (for instance by S-glutathionylation). The uncoupling of eNOS triggers a switch of its activity from a NO-producing enzyme to a NADPH oxidase-like system generating O2•-, thereby potentiating ROS production and oxidative stress. Moreover, in PE placentas, eNOS could be post-translationally modified by lipid peroxidation-derived aldehydes such as 4-oxononenal (ONE) a highly bioreactive agent, able to inhibit eNOS activity and NO production. This review summarizes the dysfunction of placental eNOS evoked by oxidative stress and lipid peroxidation products, and the potential consequences on PE pathogenesis.

A role for 4-hydroxy-2-nonenal in premature placental senescence in preeclampsia and intrauterine growth restriction.

Premature placental senescence is a hallmark of pregnancy-related disorders such as intrauterine growth restriction (IUGR) and preeclampsia (PE), two major cause of maternal and neonatal morbidity and mortality. Oxidative stress and lipid peroxidation are involved in the pathogenesis of PE and IUGR, and may play a role in placental aging. In this study, we investigated whether 4-hydroxy-2-nonenal (HNE), a lipid peroxidation-derived aldehyde present in preeclamptic placentas, may contribute to premature senescence in placenta-related complications. Placentas from PE-affected women, exhibited several senescence patterns, such as an increased expression of phosphorylated (serine-139) histone γH2AX, a sensitive marker of double-stranded DNA breaks, the presence of lipofuscin granules, and an accumulation of high molecular weight cross-linked and ubiquitinated proteins. PE placentas showed an accumulation of acetylated proteins consistent with the presence of HNE-adducts on sirtuin 1 (SIRT1). Likewise, oxidative stress and senescence markers together with SIRT1 modification by HNE, were observed in murine placentas from mice treated with lipopolysaccharide during gestation and used as models of IUGR. The addition of HNE and ONE (4-oxo-2-nonenal), to cultured HTR-8/SVneo human trophoblasts activated the senescence-associated- ß-galactosidase, and generated an accumulation of acetylated proteins, consistent with a modification of SIRT1 by HNE. Altogether, these data emphasize the role of HNE and lipid peroxidation-derived aldehydes in premature placental senescence in PE and IUGR, and more generally in pathological pregnancies.

Impact of Thoracic Endografting on the Hemodynamics of the Native Aorta: Pre- and Postoperative Assessments of Wall Shear Stress and Vorticity Using Computational Fluid Dynamics.

PURPOSE: To quantify the hemodynamic consequences of thoracic endovascular aortic repair (TEVAR) by comparing the preoperative and postoperative wall shear stress (WSS) and vorticity profiles on computational fluid dynamics (CFD) simulations. MATERIALS AND METHODS: The pre- and postoperative computed tomography (CT) scans from 20 consecutive patients (median age 69 years, range 20-87) treated for different thoracic aortic pathologies (11 aneurysms, 5 false aneurysms, 3 penetrating ulcers, and 1 traumatic aortic rupture) were segmented to construct patient-specific CFD models using a meshless code. The simulations were run over the cardiac cycle, and the WSS and vorticity values measured at the proximal and distal landing zones were compared. RESULTS: The CFD runs provided 4-dimensional simulations of blood flow in all patients. WSS and vorticity profiles at the proximal landing zone (located in zones 0-3 in 15 patients) varied in 18 and 20 of the cases, respectively; WSS was increased in 11 cases and the vorticity in 9. Pre- and postoperative WSS median values were 4.19 and 4.90 Pa, respectively. Vorticity median values were 40.38 and 39.17 Hz, respectively. CONCLUSION: TEVAR induces functional alterations in the native thoracic aorta, though the prognostic significance of these changes is still unknown. CFD appears to be a valuable tool to explore aortic hemodynamics, and its application in a larger series would help define a predictive role for these hemodynamic assessments.

Combining Volumetric and Wall Shear Stress Analysis from CT to Assess Risk of Abdominal Aortic Aneurysm Progression.

Background Despite known limitations, the decision to operate on abdominal aortic aneurysm (AAA) is primarily on the basis of measurement of maximal aneurysm diameter. Purpose To identify volumetric and computational fluid dynamics parameters to predict AAAs that are likely to progress in size. Materials and Methods This study, part of a multicenter prospective registry (NCT01599533), included 126 patients with AAA. Patients were sorted into stable (≤10-mL increase in aneurysm volume) and progression (>10-mL increase in aneurysm volume) groups. Initial AAA characteristics of the derivation cohort were analyzed (maximal diameter and surface, thrombus and lumen volumes, maximal wall pressure, and wall shear stress [WSS]) to identify relevant parameters for a logistic regression model. Model and maximal diameter diagnostic performances were assessed in both cohorts and for AAAs smaller than 50 mm by using area under the receiver operating characteristic curve (AUC). Results Eighty-one patients were included (mean age, 73 years ± 7 years [standard deviation]; 78 men). The derivation and validation cohorts included, respectively, 50 and 31 participants. In the derivation cohort, there was higher mean lumen volume and lower mean WSS in the progression group compared with the stable group (60 mL ± 14 vs 46 mL ± 18 [P = .005] and 66% ± 6 vs 53% ± 9 [P = .02], respectively). Mean lumen volume and mean WSS at baseline were correlated to total volume growth (r = 0.47 [P = .002] and -0.42 [P = .006], respectively). In the derivation cohort, a regression model including lumen volume and WSS to predict aneurysm enlargement was superior to maximal diameter alone (AUC, 0.78 vs 0.52, respectively; P = .003); although no difference was found in the validation cohort (AUC, 0.79 vs 0.71, respectively; P = .51). For AAAs smaller than 50 mm, a regression model that included both baseline WSS and lumen volume performed better than maximal diameter (AUC, 0.79 vs 0.53, respectively; P = .01). Conclusion Combined analysis of lumen volume and wall shear stress was associated with enlargement of abdominal aortic aneurysms at 1 year, particularly in aneurysms smaller than 50 mm in diameter. © RSNA, 2020 Online supplemental material is available for this article. See also the editorial by Mitsouras and Leach in this issue.

Opuntia cladode powders inhibit adipogenesis in 3 T3-F442A adipocytes and a high-fat-diet rat model by modifying metabolic parameters and favouring faecal fat excretion.

BACKGROUND: Obesity is a major public health concern worldwide. A sedentary life and a nutritional transition to processed foods and high-calorie diets are contributing factors to obesity. The demand for nutraceutical foods, such as herbal weight-loss products, which offer the potential to counteract obesity, has consequently increased. We hypothesised that Opuntia cladodes consumption could assist weight management in an obesity prevention context. METHODS: This study was designed to explore the anti-adipogenic effects of lyophilised Opuntia cladode powders (OCP) in an in vitro cellular model for adipocyte differentiation and an in vivo high-fat-diet (HFD)-induced obesity rat model. Two OCP were tested, one from wild species O. streptacantha and the second from the most known species O. ficus-indica. RESULTS: Pre-adipocytes 3 T3-F442A were treated by OCP during the differentiation process by insulin. OCP treatment impaired the differentiation in adipocytes, as supported by the decreased triglyceride content and a low glucose uptake, which remained comparable to that observed in undifferentiated controls, suggesting that an anti-adipogenic effect was exerted by OCP. Sprague-Dawley rats were fed with a normal or HFD, supplemented or not with OCP for 8 weeks. OCP treatment slightly reduced body weight gain, liver and abdominal fat weights, improved some obesity-related metabolic parameters and increased triglyceride excretion in the faeces. Taken together, these results showed that OCP might contribute to reduce adipogenesis and fat storage in a HFD context, notably by promoting the faecal excretion of fats. CONCLUSIONS: Opuntia cladodes may be used as a dietary supplement or potential therapeutic agent in diet-based therapies for weight management to prevent obesity.

Role of reactive oxygen species in atherosclerosis: Lessons from murine genetic models.

Atherosclerosis is a multifactorial chronic and inflammatory disease of medium and large arteries, and the major cause of cardiovascular morbidity and mortality worldwide. The pathogenesis of atherosclerosis involves a number of risk factors and complex events including hypercholesterolemia, endothelial dysfunction, increased permeability to low density lipoproteins (LDL) and their sequestration on extracellular matrix in the intima of lesion-prone areas. These events promote LDL modifications, particularly by oxidation, which generates acute and chronic inflammatory responses implicated in atherogenesis and lesion progression. Reactive oxygen species (ROS) (which include both free radical and non-free radical oxygen intermediates), play a key-role at each step of atherogenesis, in endothelial dysfunction, LDL oxidation, and inflammatory events involved in the initiation and development of atherosclerosis lesions. Most advanced knowledge supporting the "oxidative theory of atherosclerosis" i.e. the nature and the cellular sources of ROS and antioxidant defences, as well as the mechanisms involved in the redox balance, is based on the use of genetically engineered animals, i.e. transgenic, genetically modified, or altered for systems producing or neutralizing ROS in the vessels. This review summarizes the results obtained from animals genetically manipulated for various sources of ROS or antioxidant defences in the vascular wall, and their relevance (advance or limitation), for understanding the place and role of ROS in atherosclerosis.

Immune and Smooth Muscle Cells Interactions in Atherosclerosis: How to Target a Breaking Bad Dialogue?

Inflammation is a well-known pathophysiological factor of atherosclerosis but its therapeutic targeting has long been ignored. However, recent advances in the understanding of the immune mechanisms implicated in atherosclerosis have unveiled several therapeutic targets currently undergoing clinical trials. These studies have also shed light on a dialogue between the immune compartment and vascular smooth muscle cells (VSMCs) that plays a critical role in atherosclerotic disease initiation, progression, and stabilization. Our review focuses on the link between cellular and soluble immune effectors and VSMC behavior at different phases of the pathology. Furthermore, we discuss the potential targeting of these interactions to efficiently prevent cardiovascular diseases.

Modification of endothelial nitric oxide synthase by 4-oxo-2(E)-nonenal(ONE) in preeclamptic placentas.

Preeclampsia (PE) is a leading cause of pregnancy complications, affecting 3-7% of pregnant women worldwide. The pathophysiology of preeclampsia involves a redox imbalance, oxidative stress and a reduced nitric oxide (NO) bioavailability. The molecular and cellular mechanisms leading to the dysfunction of the placental endothelial NO synthase (eNOS) are not clarified. This study was designed to investigate whether aldehydes generated by lipid peroxidation products (LPP), may contribute to placental eNOS dysfunction in PE. The analysis of placentas from PE-affected patients and normal pregnancies, showed a significant increase in protein carbonyl content, indicative of oxidative stress-induced protein modification, as shown by the accumulation of acrolein, 4-hydroxynonenal (HNE), and 4-oxo-2(E)-nonenal (ONE) adducts in PE placentas. In contrast, the levels of these LPP-adducts were low in placentas from normal pregnancies. Immunofluorescence and confocal experiments pointed out a colocalization of eNOS with ONE-Lys adducts, whereas eNOS was not modified in normal placentas. LC-MS/MS analysis of recombinant eNOS preincubated with ONE, allowed to identify several ONE-modified Lys-containing peptides, confirming that eNOS may undergo post-translational modification by LPP. The preincubation of HTR-8/SVneo human trophoblasts (HTR8) with ONE, resulted in ONE-Lys modification of eNOS and a reduced generation of NO. ONE inhibited the migration of HTR8 trophoblasts in the wound closure model, and this was partly restored by the NO donor, NOC-18, which confirmed the important role of NO in the invasive potential of trophoblasts. In conclusion, placental eNOS is modified by ONE in PE placentas, which emphasizes the sensitivity of this protein to oxidative stress in the disturbed redox environment of preeclamptic pregnancies.

High glutathionylation of placental endothelial nitric oxide synthase in preeclampsia.

Decreased nitric oxide (NO) bioavailability plays a critical role in the pathophysiology of preeclampsia (PE). Recent evidence indicates that S-glutathionylation may occur on the endothelial nitric oxide synthase (eNOS), leading to eNOS uncoupling, characterized by a decreased NO production and an increased generation of superoxide anion (O2•-). We hypothesized that eNOS glutathionylation may occur in PE placentas and participate in eNOS dysfunction. The glutathionylation of eNOS was investigated in thirteen PE-affected patients and in nine normal pregnancies. Immunofluorescence, confocal microscopy and western-blot experiments carried out on eNOS immunoprecipitates, revealed a high level of eNOS glutathionylation in PE placentas, mostly reversed by dithiotreitol (DTT), thus indicative of S-glutathionylation. In order to investigate whether eNOS glutathionylation may alter trophoblast migration, an important event occurring during early placentation, cultured HTR-8/SVneo human trophoblasts (HTR8) were exposed either to low pO2 (O2 1%) or to pO2 changes (O2 1-20%), in order to generate oxidative stress. Trophoblasts exposed to low pO2, did not undergo oxidative stress nor eNOS S-glutathionylation, and were able to generate NO and migrate in a wound closure model. In contrast, trophoblasts submitted to low/high pO2 changes, exhibited oxidative stress and a (DTT reversible) S-glutathionylation of eNOS, associated with reduced NO production and migration. The autonomous production of NO seemed necessary for the migratory potential of HTR8, as suggested by the inhibitory effect of eNOS silencing by small interfering RNAs, and the eNOS inhibitor L-NAME, in low pO2 conditions. Finally, the addition of the NO donor, NOC-18 (5 µM), restored in part the migration of HTR8, thereby emphasizing the role of NO in trophoblast homeostasis. In conclusion, the high level of eNOS S-glutathionylation in PE placentas provides new insights in the mechanism of eNOS dysfunction in this disease.

A thioredoxin-mimetic peptide exerts potent anti-inflammatory, antioxidant, and atheroprotective effects in ApoE2.Ki mice fed high fat diet.

Aims: Oxidative stress and inflammation play a pathogenic role in atherosclerosis. Thioredoxin-1 (Trx-1) is an anti-oxidative, anti-inflammatory protein with atheroprotective effects. However, in vivo cleavage of Trx-1 generates a truncated pro-inflammatory protein, Trx-80, which compromises the therapeutic use of Trx-1. Here we analysed whether the thioredoxin-mimetic peptide (TxMP), CB3 might exert anti-oxidative, anti-inflammatory, and atheroprotective effects in ApoE2.Ki mice. Methods and results: We synthesized a small TxMP, Ac-Cys-Pro-Cys-amide, CB3 and characterized its antioxidant and anti-inflammatory effects on cultured peritoneal murine macrophages. CB3 significantly and dose-dependently reduced the level of reactive oxygen species in lipopolysaccharides (LPS)-activated macrophages. In addition, it efficiently lowered LPS-induced inflammatory process through NF-κB inhibition, as evidenced by the reduced secretion of monocyte chemoattractant protein-1, interleukin (IL)-1ß, IL-6, and tumor necrosis factor (TNF)-α by macrophages. Nevertheless, CB3 did not affect cholesterol accumulation in macrophages. A daily-administered dose of 10 µg/g body weight CB3 to ApoE2.Ki mice on high fat diet did not affect plasma of total cholesterol and triglycerides levels but significantly reduced the plasma levels of pro-inflammatory cytokines (IL-33 and TNF-α) and oxidative markers. In contrast, it significantly induced the plasma levels of anti-inflammatory proteins (adiponectin, IL-10). In addition, CB3 reduced the number of pro-inflammatory M1 macrophages in spleen and decreased the ratio of M1/M2 macrophages in atherosclerotic lesion areas. Finally, CB3 significantly reduced the surface area of aortic lesions. Conclusions: Our results clearly showed that similar to the full length Trx-1, CB3 exerts protective effects, by reducing inflammation and oxidative stress in macrophages and in ApoE2.Ki mice. The atheroprotective effect of CB3 opens promising therapeutic approaches for treatment of atherosclerosis.

nSMase2 (Type 2-Neutral Sphingomyelinase) Deficiency or Inhibition by GW4869 Reduces Inflammation and Atherosclerosis in Apoe-/- Mice.

OBJECTIVE: Atherosclerosis is a chronic multifactorial and inflammatory disease of large and medium arteries and the leading cause of cardiovascular diseases worldwide. The aim of this study was to investigate whether and how the nSMase2 (type 2-neutral sphingomyelinase), a key enzyme of sphingolipid metabolism, may contribute to the development of atherosclerotic lesions. APPROACH AND RESULTS: The role of nSMase2 in atherosclerosis was investigated in Apoe-/-;Smpd3fro/fro mice, mutant for nSMase2, and in Apoe-/-;Smpd3+/+ mice intraperitoneally injected with GW4869, a pharmacological nSMase2 inhibitor. The defect or inhibition of nSMase2 resulted in a reduction of atherosclerotic lesions and a decrease in macrophage infiltration and lipid deposition, although cholesterolemia remained unchanged. nSMase2 inhibition decreased the inflammatory response of murine endothelial cells to oxLDL (oxidized low-density lipoprotein), as assessed by the significant reduction of MCP-1 (monocyte chemoattractant protein 1), ICAM-1 (intercellular adhesion molecule-1), and VCAM-1 (vascular cell adhesion molecule-1) mRNA expressions and macrophage recruitment. Likewise, in RAW264.7 or in macrophages isolated from Apoe-/-/Smpd3fro/fro or Apoe-/-/Smpd3+/+ mice stimulated by lipopolysaccharides, nSMase2 inhibition resulted in a decrease in the expression of inflammatory molecules. Mechanistically, the anti-inflammatory response resulting from nSMase2 inhibition involves Nrf2 (nuclear factor [erythroid-derived 2]-like 2 or NF-E2-related factor-2) activation in both endothelial cells and macrophages, as assessed by the lack of protective effect of GW4869 in endothelial cells silenced for Nrf2 by small interfering RNAs, and in lipopolysaccharide-stimulated macrophages issued from Nrf2-KO mice. CONCLUSIONS: The genetic deficiency or inhibition of nSMase2 strongly decreases the development of atherosclerotic lesions in Apoe-/- mice, by reducing inflammatory responses through a mechanism involving the Nrf2 pathway. Inhibitors of nSMase2 may, therefore, constitute a novel approach to slow down atherosclerosis progression.

Small dense HDLs display potent vasorelaxing activity, reflecting their elevated content of sphingosine-1-phosphate.

The functional heterogeneity of HDL is attributed to its diverse bioactive components. We evaluated whether the vasodilatory effects of HDL differed across HDL subpopulations, reflecting their distinct molecular composition. The capacity of five major HDL subfractions to counteract the inhibitory effects of oxidized LDL on acetylcholine-induced vasodilation was tested in a rabbit aortic rings model. NO production, an essential pathway in endothelium-dependent vasorelaxation, was studied in simian vacuolating virus 40-transformed murine endothelial cells (SVECs). Small dense HDL3 subfractions displayed potent vasorelaxing activity (up to +31% vs. baseline, P < 0.05); in contrast, large light HDL2 did not induce aortic-ring relaxation when compared on a total protein basis. HDL3 particles were enriched with sphingosine-1-phosphate (S1P) (up to 3-fold vs. HDL2), with the highest content in HDL3b and -3c that concomitantly revealed the strongest vasorelaxing properties. NO generation was enhanced by HDL3c in SVECs (1.5-fold, P < 0.01), a phenomenon that was blocked by the S1P receptor antagonist, VPC 23019. S1P-enriched reconstituted HDL (rHDL) was a 1.8-fold (P < 0.01) more potent vasorelaxant than control rHDL in aortic rings. Small dense HDL3 particles displayed potent protective effects against oxidative stress-associated endothelium dysfunction, potentially reflecting their elevated content of S1P that might facilitate interaction with S1P receptors and ensuing NO generation.