15-Deoxy-Δ12,14-Prostaglandin J2 Reinforces the Anti-Inflammatory Capacity of Endothelial Cells With a Genetically Determined NO Deficit.

RATIONALE: Fluid shear stress (FSS) maintains NOS-3 (endothelial NO synthase) expression. Homozygosity for the C variant of the T-786C single-nucleotide polymorphism of the NOS3 gene, which solely exists in humans, renders the gene less sensitive to FSS, resulting in a reduced endothelial cell (EC) capacity to generate NO. Decreased bioavailability of NO in the arterial vessel wall facilitates atherosclerosis. Consequently, individuals homozygous for the C variant have an increased risk for coronary heart disease (CHD). OBJECTIVE: At least 2 compensatory mechanisms seem to minimize the deleterious effects of this single-nucleotide polymorphism in affected individuals, one of which is characterized herein. METHODS AND RESULTS: Human genotyped umbilical vein ECs and THP-1 monocytes were used to investigate the role of 15-deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2) in vitro. Its concentration in plasma samples from genotyped patients with CHD and age-matched CHD-free controls was determined using quantitative ultraperformance LC-MS/MS. Exposure of human ECs to FSS effectively reduced monocyte transmigration particularly through monolayers of CC-genotype ECs. Primarily in CC-genotype ECs, FSS elicited a marked rise in COX (cyclooxygenase)-2 and L-PGDS (lipocalin-type prostaglandin D synthase) expression, which appeared to be NO sensitive, and provoked a significant release of 15d-PGJ2 over baseline. Exogenous 15d-PGJ2 significantly reduced monocyte transmigration and exerted a pronounced anti-inflammatory effect on the transmigrated monocytes by downregulating, for example, transcription of the IL (interleukin)-1ß gene (IL1B). Reporter gene analyses verified that this effect is due to binding of Nrf2 (nuclear factor [erythroid-derived 2]-like 2) to 2 AREs (antioxidant response elements) in the proximal IL1B promoter. In patients with CHD, 15d-PGJ2 plasma levels were significantly upregulated compared with age-matched CHD-free controls, suggesting that this powerful anti-inflammatory prostanoid is part of an endogenous defence mechanism to counteract CHD. CONCLUSIONS: Despite a reduced capacity to form NO, CC-genotype ECs maintain a robust anti-inflammatory phenotype through an enhanced FSS-dependent release of 15d-PGJ2.

Endothelial Microparticles From Acute Coronary Syndrome Patients Induce Premature Coronary Artery Endothelial Cell Aging and Thrombogenicity: Role of the Ang II/AT1 Receptor/NADPH Oxidase-Mediated Activation of MAPKs and PI3-Kinase Pathways.

BACKGROUND: Microparticles (MPs) have emerged as a surrogate marker of endothelial dysfunction and cardiovascular risk. This study examined the potential of MPs from senescent endothelial cells (ECs) or from patients with acute coronary syndrome (ACS) to promote premature EC aging and thrombogenicity. METHODS: Primary porcine coronary ECs were isolated from the left circumflex coronary artery. MPs were prepared from ECs and venous blood from patients with ACS (n=30) and from healthy volunteers (n=4) by sequential centrifugation. The level of endothelial senescence was assessed as senescence-associated ß-galactosidase activity using flow cytometry, oxidative stress using the redox-sensitive probe dihydroethidium, tissue factor activity using an enzymatic Tenase assay, the level of target protein expression by Western blot analysis, platelet aggregation using an aggregometer, and shear stress using a cone-and-plate viscometer. RESULTS: Senescence, as assessed by senescence-associated ß-galactosidase activity, was induced by the passaging of porcine coronary artery ECs from passage P1 to P4, and was associated with a progressive shedding of procoagulant MPs. Exposure of P1 ECs to MPs shed from senescent P3 cells or circulating MPs from ACS patients induced increased senescence-associated ß-galactosidase activity, oxidative stress, early phosphorylation of mitogen-activated protein kinases and Akt, and upregulation of p53, p21, and p16. Ex vivo, the prosenescent effect of circulating MPs from ACS patients was evidenced only under conditions of low shear stress. Depletion of endothelial-derived MPs from ACS patients reduced the induction of senescence. Prosenescent MPs promoted EC thrombogenicity through tissue factor upregulation, shedding of procoagulant MPs, endothelial nitric oxide synthase downregulation, and reduced nitric oxide-mediated inhibition of platelet aggregation. These MPs exhibited angiotensin-converting enzyme activity and upregulated AT1 receptors and angiotensin-converting enzyme in P1 ECs. Losartan, an AT1 receptor antagonist, and inhibitors of either mitogen-activated protein kinases or phosphoinositide 3-kinase prevented the MP-induced endothelial senescence. CONCLUSIONS: These findings indicate that endothelial-derived MPs from ACS patients induce premature endothelial senescence under atheroprone low shear stress and thrombogenicity through angiotensin II-induced redox-sensitive activation of mitogen-activated protein kinases and phosphoinositide 3-kinase/Akt. They further suggest that targeting endothelial-derived MP shedding and their bioactivity may be a promising therapeutic strategy to limit the development of an endothelial dysfunction post-ACS.

Impact of the -1T>C single-nucleotide polymorphism of the CD40 gene on the development of endothelial dysfunction in a pro-diabetic microenvironment.

BACKGROUND AND AIMS: Hyperglycemia reinforces pro-inflammatory conditions that enhance CD40 expression in endothelial cells (EC). Thymine to cytosine transition (-1T > C) in the promoter of the CD40 gene (rs1883832) further increases the abundance of CD40 protein on the EC surface. This study examines potential associations of the -1T > C SNP of the CD40 gene with type 1 (T1D) or type 2 (T2D) diabetes. Moreover, it investigates the impact of a pro-inflammatory diabetic microenvironment on gene expression in human cultured umbilical vein EC (HUVEC) derived from CC- vs. TT-genotype donors. METHODS: Tetra-ARMS-PCR was used to compare genotype distribution in 252 patients with diabetes. Soluble CD40 ligand (sCD40L) and soluble CD40 receptor (sCD40) plasma levels were monitored using ELISA. RNA-sequencing was performed with sCD40L-stimulated CC- and TT-genotype HUVEC. Quantitative PCR, Western blot, multiplex-sandwich ELISA array, and immunocytochemistry were used to analyse changes in gene expression in these cells. RESULTS: Homozygosity for the C-allele was associated with a significant 4.3-fold higher odds of developing T2D as compared to individuals homozygous for the T-allele. Inflammation and endothelial-to-mesenchymal transition (EndMT) driving genes were upregulated in CC-genotype but downregulated in TT-genotype HUVEC when exposed to sCD40L. Expression of EndMT markers significantly increased while that of endothelial markers decreased in HUVEC following exposure to hyperglycemia, tumour necrosis factor-α and sCD40L. CONCLUSIONS: The -1T > C SNP of the CD40 gene is a risk factor for T2D. Depending on the genotype, it differentially affects gene expression in human cultured EC. CC-genotype HUVEC adopt a pro-inflammatory and intermediate EndMT-like phenotype in a pro-diabetic microenvironment.

A non-enzymatic function of 17beta-hydroxysteroid dehydrogenase type 10 is required for mitochondrial integrity and cell survival.

Deficiency of the mitochondrial enzyme 2-methyl-3-hydroxybutyryl-CoA dehydrogenase involved in isoleucine metabolism causes an organic aciduria with atypical neurodegenerative course. The disease-causing gene is HSD17B10 and encodes 17beta-hydroxysteroid dehydrogenase type 10 (HSD10), a protein also implicated in the pathogenesis of Alzheimer's disease. Here we show that clinical symptoms in patients are not correlated with residual enzymatic activity of mutated HSD10. Loss-of-function and rescue experiments in Xenopus embryos and cells derived from conditional Hsd17b10(-/-) mice demonstrate that a property of HSD10 independent of its enzymatic activity is essential for structural and functional integrity of mitochondria. Impairment of this function in neural cells causes apoptotic cell death whilst the enzymatic activity of HSD10 is not required for cell survival. This finding indicates that the symptoms in patients with mutations in the HSD17B10 gene are unrelated to accumulation of toxic metabolites in the isoleucine pathway and, rather, related to defects in general mitochondrial function. Therefore alternative therapeutic approaches to an isoleucine-restricted diet are required.