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

International audienceSolar 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

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. Keywords: NO, ENOS, S-glutathionylation, Glutathione, Oxidative stress, O2, Pregnancy, Trophoblast, Migration, Preeclampsi

Proatherogenic effects of 4-hydroxynonenal

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

International audiencePreeclampsia (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 (O 2 •−), 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 Larginine 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 O 2 •− , 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

4-Hydroxynonenal Contributes to Angiogenesis through a Redox-Dependent Sphingolipid Pathway: Prevention by Hydralazine Derivatives

The neovascularization of atherosclerotic lesions is involved in plaque development and may contribute to intraplaque hemorrhage and plaque fragilization and rupture. Among the various proangiogenic agents involved in the neovascularization process, proatherogenic oxidized LDLs (oxLDLs) contribute to the formation of tubes via the generation of sphingosine 1-phosphate (S1P), a major mitogenic and proangiogenic sphingolipid mediator. In this study, we investigated whether 4-hydroxynonenal (4-HNE), an aldehydic lipid oxidation product abundantly present in oxLDLs, contributes to their proangiogenic properties. Immunofluorescence analysis of human atherosclerotic lesions from carotid endarterectomy showed the colocalization of HNE-adducts with CD31, a marker of endothelial cells, suggesting a close relationship between 4-HNE and neovessel formation. In vitro, low 4-HNE concentration (0.5–1 µM) elicited the formation of tubes by human microvascular endothelial cells (HMEC-1), whereas higher concentrations were not angiogenic. The formation of tubes by 4-HNE involved the generation of reactive oxygen species and the activation of the sphingolipid pathway, namely, the neutral type 2 sphingomyelinase and sphingosine kinase-1 (nSMase2/SK-1) pathway, indicating a role for S1P in the angiogenic signaling of 4-HNE. Carbonyl scavengers hydralazine and bisvanillyl-hydralazone inhibited the nSMase2/SK1 pathway activation and the formation of tubes on Matrigel® evoked by 4-HNE. Altogether, these results emphasize the role of 4-HNE in the angiogenic effect of oxLDLs and point out the potential interest of pharmacological carbonyl scavengers to prevent the neovascularization process

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

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

International audienceObjective: 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

Leucine-Rich Alpha-2 Glycoprotein 1 Accumulates in Complicated Atherosclerosis and Promotes Calcification

Atherosclerosis is the primary cause of cardiovascular disease. The development of plaque complications, such as calcification and neo-angiogenesis, strongly impacts plaque stability and is a good predictor of mortality in patients with atherosclerosis. Despite well-known risk factors of plaque complications, such as diabetes mellitus and chronic kidney disease, the mechanisms involved are not fully understood. We and others have identified that the concentration of circulating leucine-rich α-2 glycoprotein 1 (LRG1) was increased in diabetic and chronic kidney disease patients. Using apolipoprotein E knockout mice (ApoE−/−) (fed with Western diet) that developed advanced atherosclerosis and using human carotid endarterectomy, we showed that LRG1 accumulated into an atherosclerotic plaque, preferentially in calcified areas. We then investigated the possible origin of LRG1 and its functions on vascular cells and found that LRG1 expression was specifically enhanced in endothelial cells via inflammatory mediators and not in vascular smooth muscle cells (VSMC). Moreover, we identified that LRG1 was able to induce calcification and SMAD1/5-signaling pathways in VSMC. In conclusion, our results identified for the first time that LRG1 is a direct contributor to vascular calcification and suggest a role of this molecule in the development of plaque complications in patients with atherosclerosis

Temporal Arteritis Revealing Antineutrophil Cytoplasmic Antibody-Associated Vasculitides: Case-Control Study of 50 Cases.

BACKGROUND: Temporal arteritis (TA) is a typical manifestation of giant cell arteritis (GCA). Rarely, ANCA-associated vasculitides (AAV) can be revealed by TA, leading to misdiagnoses of GCA and inappropriate treatments. METHODS: We retrospective included patients with TA revealing AAV (TA-AAV), and compared them with classical GCA patients. RESULTS: Fifty patients with TA-AAV (median age 70 years) were included. Thirty-three (66%) patients presented atypical symptoms for GCA (ENT, renal, pulmonary and neurological involvement in 32%, 26%, 20% and 16%, respectively). ANCA were screened at disease onset in 33 cases, and were positive in 88%, leading to diagnosis of early TA-AAV in 20 cases. AAV diagnosis was delayed in 30 cases after a median interval of 15 months. Compared to GCA, TA-AAV patients were younger (70 vs. 74 years), more frequently men (48 vs. 30%), had high frequency of atypical manifestations and higher CRP levels (10.8 vs. 7.0 mg/dL). Temporal artery biopsy (TAB) from TA-AAV showed fibrinoid necrosis and small branch vasculitis in 23% each, whereas it did not in GCA. Treatment failure-free survival was comparable between early TA-AAV and GCA, whereas those with delayed TA-AAV patients had a significant higher risk of treatment failure compared to GCA [HR 3.85 (1.97-7.51), P<0.0001)]. CONCLUSION: TA revealing AAV should be considered in case of atypical manifestations for GCA, GCs refractoriness or early relapse. Analysis of TAB for small branch vasculitis and/or fibrinoid necrosis could be useful. Detection of ANCA should be performed in case of suspected GCA with atypical clinical features and/or TAB.status: Published onlin