Functional association of a CD40 gene single-nucleotide polymorphism with the pathogenesis of coronary heart disease.

AIMS: Endothelial dysfunction is a major contributor to the pathogenesis of atherosclerosis. CD40-CD40 ligand interactions confer a pro-inflammatory phenotype to endothelial cells (ECs). Recently, a thymine to cytosine transition (-1T>C) in the Kozak sequence of the CD40 gene (rs1883832) has been associated with coronary heart disease (CHD) in an Asian population. As there are no reports yet regarding its role in other ethnic groups, this study determines if the -1T>C single-nucleotide polymorphism (SNP) could be a risk factor for CHD in Caucasians by performing an association study and elucidates its functional consequence in cultured ECs. METHODS AND RESULTS: Molecular and biochemical techniques, cell adhesion assays were used for genotype-stratified human EC characterization. SNP distribution in Caucasians was examined in a hospital-based case-control CHD study and serum levels of soluble CD40 (sCD40) were quantified by ELISA. The SNP in the CD40 gene affected baseline CD40 protein abundance on ECs. There was a genotype-dependent difference in CD40-mediated expression of pro-inflammatory genes. Monocyte adhesion was highest on the surface of cells homozygous for the C allele. Homozygosity for the C allele was associated with significant 2.32-fold higher odds of developing CHD as compared to TT genotype carriers. sCD40 plasma levels were genotype-dependently elevated in CHD patients, indicating a possible prognostic value. CONCLUSION: The C allele of the CD40 SNP provokes a pro-inflammatory EC phenotype, compensated by an enhanced CD40 shedding to neutralize excess CD40 ligand. Homozygosity for the C allele is the cause for a genetic susceptibility to atherosclerosis and its sequelae.

Impact of carbonylation on glutathione peroxidase-1 activity in human hyperglycemic endothelial cells.

AIMS: High levels of glucose and reactive carbonyl intermediates of its degradation pathway such as methylglyoxal (MG) may contribute to diabetic complications partly via increased generation of reactive oxygen species (ROS). This study focused on glutathione peroxidase-1 (GPx1) expression and the impact of carbonylation as an oxidative protein modification on GPx1 abundance and activity in human umbilical vein endothelial cells (HUVEC) under conditions of mild to moderate oxidative stress. RESULTS: High extracellular glucose and MG enhanced intracellular ROS formation in HUVECs. Protein carbonylation was only transiently augmented pointing to an effective antioxidant defense in these cells. Nitric oxide synthase expression was decreased under hyperglycemic conditions but increased upon exposure to MG, whereas superoxide dismutase expression was not significantly affected. Increased glutathione peroxidase (GPx) activity seemed to compensate for a decrease in GPx1 protein due to enhanced degradation via the proteasome. Mass spectrometry analysis identified Lys-114 as a possible carbonylation target which provides a vestibule for the substrate H2O2 and thus enhances the enzymatic reaction. INNOVATION: Oxidative protein carbonylation has so far been associated with functional inactivation of modified target proteins mainly contributing to aging and age-related diseases. Here, we demonstrate that mild oxidative stress and subsequent carbonylation seem to activate protective cellular redox signaling pathways whereas severe oxidative stress overwhelms the cellular antioxidant defense leading to cell damage. CONCLUSIONS: This study may contribute to a better understanding of redox homeostasis and its role in the development of diabetes and related vascular complications.

SLAP/SLAP2 prevent excessive platelet (hem)ITAM signaling in thrombosis and ischemic stroke in mice.

Glycoprotein VI and C-type lectin-like receptor 2 are essential platelet activating receptors in hemostasis and thrombo-inflammatory disease, which signal through a (hem)immunoreceptor tyrosine-based activation motif (ITAM)-dependent pathway. The adapter molecules Src-like adapter proteins (SLAP and SLAP2) are involved in the regulation of immune cell surface expression and signaling, but their function in platelets is unknown. In this study, we show that platelets expressed both SLAP isoforms and that overexpression of either protein in a heterologous cell line almost completely inhibited glycoprotein VI and C-type lectin-like receptor 2 signaling. In mice, single deficiency of SLAP or SLAP2 had only moderate effects on platelet function, whereas double deficiency of both adapters resulted in markedly increased signal transduction, integrin activation, granule release, aggregation, procoagulant activity, and thrombin generation in response to (hem)ITAM-coupled, but not G protein-coupled, receptor activation. In vivo, constitutive SLAP/SLAP2 knockout mice displayed accelerated occlusive arterial thrombus formation and a dramatically worsened outcome after focal cerebral ischemia. This was attributed to the absence of both adapter proteins in platelets, as demonstrated by adoptive transfer of Slap(-/-)/Slap2(-/-) platelets into wild-type mice. Our results establish SLAP and SLAP2 as critical inhibitors of platelet (hem)ITAM signaling in the setting of arterial thrombosis and ischemic stroke.