O-Glycosylation Regulates Ubiquitination and Degradation of the Anti-Inflammatory Protein A20 to Accelerate Atherosclerosis in Diabetic ApoE-Null Mice

Background: Accelerated atherosclerosis is the leading cause of morbidity and mortality in diabetic patients. Hyperglycemia is a recognized independent risk factor for heightened atherogenesis in diabetes mellitus (DM). However, our understanding of the mechanisms underlying glucose damage to the vasculature remains incomplete. Methodology/Principal Findings: High glucose and hyperglycemia reduced upregulation of the NF-κB inhibitory and atheroprotective protein A20 in human coronary endothelial (EC) and smooth muscle cell (SMC) cultures challenged with Tumor Necrosis Factor alpha (TNF), aortae of diabetic mice following Lipopolysaccharide (LPS) injection used as an inflammatory insult and in failed vein-grafts of diabetic patients. Decreased vascular expression of A20 did not relate to defective transcription, as A20 mRNA levels were similar or even higher in EC/SMC cultured in high glucose, in vessels of diabetic C57BL/6 and FBV/N mice, and in failed vein grafts of diabetic patients, when compared to controls. Rather, decreased A20 expression correlated with post-translational O-Glucosamine-N-Acetylation (O-GlcNAcylation) and ubiquitination of A20, targeting it for proteasomal degradation. Restoring A20 levels by inhibiting O-GlcNAcylation, blocking proteasome activity, or overexpressing A20, blocked upregulation of the receptor for advanced glycation end-products (RAGE) and phosphorylation of PKCβII, two prime atherogenic signals triggered by high glucose in EC/SMC. A20 gene transfer to the aortic arch of diabetic ApoE null mice that develop accelerated atherosclerosis, attenuated vascular expression of RAGE and phospho-PKCβII, significantly reducing atherosclerosis. Conclusions: High glucose/hyperglycemia regulate vascular A20 expression via O-GlcNAcylation-dependent ubiquitination and proteasomal degradation. This could be key to the pathogenesis of accelerated atherosclerosis in diabetes

Interactions entre endothélium vasculaire et microparticules d'apoptose ou d'activation cellulaire (étude des conséquences fonctionnelles et des mécanismes impliqués)

PARIS7-Bibliothèque centrale (751132105) / SudocSudocFranceF

Prevalence of uncontrolled blood pressure in Meknes, Morocco, and its associated risk factors in 2017.

BackgroundUncontrolled high blood pressure (UBP) can lead to various cardiovascular complications causing an estimated nine million deaths per year worldwide. In Meknes, epidemiologic data on UBP are scarce, depriving programs from evidence-based information that would allow a better management of hypertension. Hence, we aimed to assess UBP prevalence in hypertensive patients treated in Meknes and identify UBP-associated risk factors.MethodsBetween November and December 2017, we conducted a cross-sectional study enrolling 922 hypertensive patients managed at Meknes's primary health care facilities using the multistage sampling method. We interviewed patients face to face to collect their socio-demographic-characteristics, lifestyle behaviours, clinical parameters and the triad care system-patient-physician. Another questionnaire was self-administered by physicians to characterize therapeutic inertia. A multivariate logistic regression analysis highlighted the risk factors associated with UBP.ResultsUBP prevalence was 73% with a mean age of 61±11 years (mean±standard deviation) and a male/female ratio of 1/3. Risk factors associated with UBP were: therapeutic inertia (adjusted odds ratio to other variables (AOR) = 18.2, 95% CI [8.35-39.84]), drug non-adherence (AOR = 1.8, 95% CI [1.07-3.04]), obesity/overweight (AOR = 1.6, 95% CI [1.03-2.58]), unemployment (AOR = 1.9, 95% CI [1.09-3.01]), low income (AOR = 2.6, 95% CI [1.01-6.86]), family history of hypertension (AOR = 1.5, 95% CI [1.07-2.08]) and male sex (AOR = 1.6, 95% CI [1.04-2.58]).ConclusionUBP prevalence is high in Meknes. Prevention should firstly focus on raised awareness of hypertensive patients' self-care management. Secondly, health professionals should better comply to the guidelines of anti-hypertensive treatments. Lastly, health professionals should frequently be reminded to reach therapeutic goals to overcome therapeutic inertia

Microparticles from apoptotic monocytes induce transient platelet recruitment and tissue factor expression by cultured human vascular endothelial cells via a redox-sensitive mechanism.

International audienceCirculating microparticles derived from different types of blood cells have been reported to impair endothelial function and to induce pro-inflammatory and prothrombotic endothelial phenotypes. Although the number of monocyte-derived microparticles (M-MPs) is elevated in the blood of patients with various inflammatory conditions, their interaction with endothelial cells has been poorly investigated so far. In this study, we produced microparticles in vitro from apoptotic human monocytes and examined the effects of their interaction with cultured human umbilical vascular endothelial cells (HUVECs). We found that low concentrations of M-MPs induced the production of reactive oxygen species (ROS), mainly anion superoxide, by the endothelial cells. At sub-toxic concentrations, M-MPs induced a rapid expression of von Willebrand factor at the cell surface, which mediated the transient attachment of non-activated platelets to the endothelium in flow conditions. In parallel, M-MPs up-regulated the expression of functional tissue factor by the endothelial cells. ROS controlled these two major changes and the process involved the phosphorylation of p38 mitogen activated protein kinase. We conclude that M-MPs may contribute to thrombotic events by producing redox signalling in endothelial cells

High glucose promotes A20 O-glycosylation, ubiquitination and proteasomal degradation in EC and SMC.

<p>High glucose increases protein O-GlcNAcylation, including that of A20 and possibly other E3 Ubiquitin ligases. This leads to increased A20 ubiquitination either through auto-ubiquitination or increased activity of other O-GlcNAcylated E3 ubiquitin ligases. This targets A20 for degradation in the proteasome. Blockade of O-GlcNAcylation using DON, upstream of A20 Ubiquitination, or inhibition of proteasome activity, using MG132, downstream of A20 ubiquitination would inhibit its proteasomal degradation, restoring its expected protein levels.</p

LPS-mediated upregulation of A20 protein expression is blunted in aortae of diabetic, as compared to non-diabetic mice.

<p>(A) WB of A20 in abdominal aortae of diabetic and non-diabetic atherosclerosis-prone C57BL/6 and atherosclerosis-resistant FVB/N mice, 8 h after LPS treatment. GAPDH and βactin were used to correct for loading and quantify relative A20 expression by densitometry, as reported below the WB. Data shown are representative of 3 (non-diabetic) and 4 (diabetic) mice per time-point and illustrate the loss of LPS-induced A20 protein in diabetic mice, regardless of strain. The cuts between samples reflect the fact that these samples, while on the same gel and same experiment, were not contiguous. (B) A20 mRNA levels analyzed by real-time PCR 3 to 8 h after LPS injection in mouse abdominal aortae (n = 5 non-diabetic and 7 diabetic mice in C57BL/6 and 3 non-diabetic and 4 diabetic mice in FVB/N). Data shown demonstrates that LPS increases A20 mRNA levels in aortae of diabetic and non-diabetic C57BL/6 and FVB/N, albeit at a greater levels in diabetic mice. Expression of 18S ribosomal RNA was used to normalize expression of A20 mRNA, and the results were presented as mean±SEM of mRNA. Each sample was measured in duplicate.</p

Increasing glucose (D-Glu) concentrations decreases TNF-mediated A20 protein up-regulation without affecting its transcriptional activation in SMC.

<p>(A) Analysis of A20 expression by WB. SMC were cultured in 5, or 30 mM D-Glu, or the osmotic control (25 mM L-Glu +5 mM D-Glu), and treated with TNF for 6 h. βactin was checked as a loading control and used to quantify relative A20 expression by densitometry, as reported beneath the WB. Densitometry of the bands of interest and was determined as the mean intensity of the areas delineated by Image J, then corrected by the main intensity of the corresponding housekeeping gene band. Fold induction was determined using the non-treated 5 mM D-glucose condition sample as one (1). Data are representative of 3 independent experiments. The cuts between samples reflect the fact that these samples, while on the same gel and same experiment, were not contiguous. (B) Analysis of A20 mRNA levels by real-time PCR. SMC were cultured in 5 or 30 mM D-Glu or mannitol (25 mM Mannitol +5 mM D-GLu), as an osmotic control, and treated with TNF for 1 or 3 h. 18S ribosomal RNA was used to normalize the data. Natural log transformed data (ln) are presented as mean±SEM of 3 independent experiments performed in duplicate. No significant differences (P>0.05) were noted between all groups and at all time-points.</p

Restoring A20 levels reverts glucose-mediated upregulation of RAGE and phosphorylation of PKCβII.

<p>(A) WB analysis for RAGE and A20 expression in SMC cultured in 5 or 30 mM D-Glu for 24 h and treated with TNF in the presence or absence of 20 mM of Azaserine (prior to TNF) or 10 mM of MG132 (following TNF). Corrected RAGE fold-inductions are listed below the WB. The RAGE protein is detected as a doublet as a result of pre and post-N-glycosylated form of the protein. Both bands were used for densitometry evaluation. (B) WB analysis of phospho-PKCβII (pPKCβII) and total (c) PKCβII in NT SMC, and in SMC transduced with rAd.A20 or rAd.βgal, and treated with PMA or challenged with 30 mM D-Glu for 1 h. Data shown in A and B are representative of 3 independent experiments. NT = non-transduced cells. GAPDH was used as loading control to quantify the relative expression of RAGE and pPKCβII by densitometry.</p