Expansion of necrotic core and shedding of Mertk receptor in human carotid plaques: a role for oxidized polyunsaturated fatty acids?

AIMS: Expansion of necrotic core (NC), a major feature responsible for plaque disruption, is likely the consequence of accelerated macrophage apoptosis coupled with defective phagocytic clearance (efferocytosis). The cleavage of the extracellular domain of Mer tyrosine kinase (Mertk) by metallopeptidase domain17 (Adam17) has been shown to produce a soluble Mertk protein (sMer), which can inhibit efferocytosis. Herein, we analysed the expression and localization of Mertk and Adam17 in the tissue around the necrotic core (TANC) and in the periphery (P) of human carotid plaques. Then we studied the mechanisms of NC expansion by evaluating which components of TANC induce Adam17 and the related cleavage of the extracellular domain of Mertk. METHODS AND RESULTS: We studied 97 human carotid plaques. The expression of Mertk and Adam17 was found to be higher in TANC than in P (P < 0.001). By immunohistochemistry, Mertk was higher than Adam17 in the area of TANC near to the lumen (P < 0.01) but much lower in the area close to NC (P < 0.01). The extract of this portion of TANC increased the expression (mRNA) of Adam17 and Mertk (P < 0.01) in macrophage-like THP-1 cells but it also induced the cleavage of the extracellular domain of Mertk, generating sMer in the medium (P < 0.01). This effect of TANC extract was most evoked by its content in F(2)-isoprostanes, hydroxyoctadecadienoic acids, and hydroxytetraenoic acids. CONCLUSION: Some oxidized derivatives of polyunsaturated fatty acids contained in TANC of human carotid plaques are strong inducers of Adam17, which in turn leads to the generation of sMer, which can inhibit efferocytosis.

Role of ox-PAPCs in the differentiation of mesenchymal stem cells (MSCs) and Runx2 and PPARγ2 expression in MSCs-like of osteoporotic patients.

BACKGROUND: Mesenchymal stem cells (MSCs) can differentiate into osteoblasts and adipocytes and conditions causing bone loss may induce a switch from the osteoblast to adipocyte lineage. In addition, the expression of Runx2 and the PPARγ2 transcription factor genes is essential for cellular commitment to an osteogenic and adipogenic differentiation, respectively. Modified lipoproteins derived from the oxidation of arachidonate-containing phospholipids (ox-PAPCs: POVPC, PGPC and PEIPC) are considered important factors in atherogenesis. METHODOLOGY: We investigated the effect of ox-PAPCs on osteogenesis and adipogenesis in human mesenchymal stem cells (hMSCs). In particular, we analyzed the transcription factor Runx2 and the PPARγ2 gene expression during osteogenic and adipogenic differentiation in absence and in presence of ox-PAPCs. We also analyzed gene expression level in a panel of osteoblastic and adipogenic differentiation markers. In addition, as circulating blood cells can be used as a "sentinel" that responds to changes in the macro- or micro-environment, we analyzed the Runx2 and the PPARγ2 gene expression in MSCs-like and ox-PAPC levels in serum of osteoporotic patients (OPs). Finally, we examined the effects of sera obtained from OPs in hMSCs comparing the results with age-matched normal donors (NDs). PRINCIPAL FINDINGS: Quantitative RT-PCR demonstrated that ox-PAPCs enhanced PPARγ2 and adipogenic gene expression and reduced Runx2 and osteoblast differentiation marker gene expression in differentiating hMSCs. In OPs, ox-PAPC levels and PPARγ2 expression were higher than in NDs, whereas Runx2 was lower than in ND circulant MSCs-like. CONCLUSIONS: Ox-PAPCs affect the osteogenic differentiation by promoting adipogenic differentiation and this effect may appear involved in bone loss in OPs.

Cigarette smoking blocks the protective expression of Nrf2/ARE pathway in peripheral mononuclear cells of young heavy smokers favouring inflammation.

Cigarette smoking is an important risk factor for atherosclerosis, a chronic inflammatory disease. However the underlying factors of this effect are unclear. It has been hypothesized that water-soluble components of cigarette smoke can directly promote oxidative stress in vasculature and blood cells. Aim of this study was to study the relationship between oxidative stress and inflammation in a group of young smokers. To do this we evaluated: 1) the oxidation products of phospholipids (oxPAPC) in peripheral blood mononuclear cells (PBMC); 2) their role in causing PBMC reactive oxygen species (ROS) generation and changes in GSH; 3) the expression of the transcription factor NF-E2-related factor 2 (Nrf2) and of related antioxidant genes (ARE); 4) the activation of NF-kB and C-reactive protein (CRP) values. We studied 90 healthy volunteers: 32 non-smokers, 32 moderate smokers (5-10 cigarettes/day) and 26 heavy smokers (25-40 cigarettes/day). OxPAPC and p47phox expression, that reasonably reflects NADPH oxidase activity, were higher in moderate smokers and heavy smokers than in non-smokers (p<0.01), the highest values being in heavy smokers (p<0.01). In in vitro studies oxPAPC increased ROS generation via NADPH oxidase activation. GSH in PBMC and plasma was lower in moderate smokers and heavy smokers than in non-smokers (p<0.01), the lowest values being in heavy smokers (p<0.01). Nrf2 expression in PBMC was higher in moderate smokers than in non-smokers (p<0.01), but not in heavy smokers, who had the highest levels of NF-kB and CRP (p<0.01). In in vitro studies oxPAPC dose-dependently increased NF-kB activation, whereas at the highest concentrations Nrf2 expression was repressed. The small interference (si) RNA-mediated knockdown of NF-kappaB/p65 increased about three times the expression of Nrf2 stimulated with oxPAPC. Cigarette smoke promotes oxPAPC formation and oxidative stress in PBMC. This may cause the activation of NF-kB that in turn may participate in the negative regulation of Nrf2/ARE pathway favouring inflammation.

Nebivolol treatment reduces serum levels of asymmetric dimethylarginine and improves endothelial dysfunction in essential hypertensive patients.

BACKGROUND: This study was conducted to evaluate (i) the effect of nebivolol, a selective beta1-adrenergic receptor antagonist, on plasma concentration of asymmetric dimethylarginine (ADMA) and on flow-mediated dilation (FMD) in essential hypertensive patients; (ii) the effect of serum derived from the treated hypertensive patients on ADMA and on dimethylarginine dimethylaminohydrolase 2 (DDAH2), the enzyme that selectively degrades ADMA, in human umbilical vein endothelial cells (HUVECs). METHODS: Forty healthy subjects and 40 matched essential hypertensive patients treated with atenolol and nebivolol according to a double-blind, randomized design participated in the study. Evaluation of brachial artery (BA) reactivity was performed by a longitudinal B-mode scan of the right BA. ADMA and L-arginine were measured by high-performance liquid chromatography. DDAH2 expression and endothelial nitric oxide synthase activity (eNOS) were also evaluated in HUVECs. RESULTS: ADMA levels were significantly decreased and FMD increased only in patients receiving nebivolol (P < 0.01). Furthermore, in nebivolol group, we found a significant correlation between changes in ADMA levels and changes in FMD (P < 0.01). Sera derived from patients treated with nebivolol but not with atenolol decreased ADMA and increased DDAH2 expression and eNOS activity (P < 0.001) in HUVECs. CONCLUSIONS: The results of this study demonstrate that the improvement of endothelial dysfunction induced by nebivolol in hypertensive patients may be related to its effect on circulating ADMA levels. Although the mechanism by which nebivolol reduces circulating ADMA in hypertensive patients remains unclear, our ex vivo results suggest that the upregulation of DDAH2 expression may have a role.

Effects of nebivolol on endothelial gene expression during oxidative stress in human umbilical vein endothelial cells.

The endothelium plays a key role in the development of atherogenesis and its inflammatory and proliferative status influences the progression of atherosclerosis. The aim of this study is to compare the effects of two beta blockers such as nebivolol and atenolol on gene expression in human umbilical vein endothelial cells (HUVECs) following an oxidant stimulus. HUVECs were incubated with nebivolol or atenolol (10 micromol/L) for 24 hours and oxidative stress was induced by the addition of oxidized (ox)-LDL. Ox-LDL upregulated adhesion molecules (ICAM-1, ICAM-2, ICAM-3, E-selectin, and P-selectin); proteins linked to inflammation (IL-6 and TNFalpha), thrombotic state (tissue factor, PAI-1 and uPA), hypertension such as endothelin-1 (ET-1), and vascular remodeling such as metalloproteinases (MMP-2, MMP-9) and protease inhibitor (TIMP-1). The exposure of HUVECs to nebivolol, but not to atenolol, reduced these genes upregulated by oxidative stress both in terms of protein and RNA expression. The known antioxidant properties of the third generation beta blocker nebivolol seem to account to the observed differences seen when compared to atenolol and support the specific potential protective role of this beta blocker on the expression of a number of genes involved in the initiation and progression of atherosclerosis.

Nebivolol reduces asymmetric dimethylarginine in endothelial cells by increasing dimethylarginine dimethylaminohydrolase 2 (DDAH2) expression and activity.

Asymmetric dimethylarginine (ADMA) has been reported to affect the synthesis of nitric oxide (NO) in endothelial cells by inhibiting endothelial NO synthase (eNOS) activity and to cause endothelial dysfunction in humans. This study was conducted in human umbilical vein endothelial cells (HUVECs) to evaluate the effect of nebivolol, a selective beta1-adrenergic receptor antagonist, on ADMA concentration and on dimethylarginine dimethylaminohydrolase (DDAH2), the enzyme that regulates ADMA catabolism. Nebivolol dose-dependently decreased ADMA/symmetric dimethylarginine (SDMA) ratio (p from <0.01 to <0.001). This was parallelled by a dose-dependent increase in DDAH2 mRNA (p from <0.01 to <0.001) and protein expression (p from <0.01 to <0.001) and activity (p from <0.01 to <0.001). The small interference RNA (siRNA)-mediated knockdown of DDAH2 abolished the modification of DDAH2 expression (p<0.001) and ADMA/SDMA ratio (p<0.001) induced by nebivolol. In conclusion, the results of this study demonstrate that nebivolol reduces ADMA concentration by increasing DDAH2 expression and activity. Our in vitro findings describe a novel vascular effect of nebivolol and clearly identify this compound as the first antihypertensive agent that modulates DDAH2 in endothelial cells.

Usefulness of a molecular strategy for the detection of bacterial DNA in patients with severe sepsis undergoing continuous renal replacement therapy.

INTRODUCTION: Sepsis is a major cause of morbidity and mortality in critically ill patients. Sepsis is associated with cell necrosis and apoptosis. Circulating plasma levels of DNA have been found in conditions associated with cell death, including sepsis, pregnancy, stroke, myocardial infarction and trauma. Plasma DNA can also derive from bacteria. We have recently implemented a method to detect bacterial DNA and, in the present study, we validated this technique comparing it to standard blood culture in terms of diagnostic efficacy. METHODS: We examined a cohort of 9 critically ill patients with a diagnosis of severe sepsis and acute renal failure requiring continuous renal replacement therapy (CRRT). We analyzed bacterial DNA in blood, hemofilters, and ultrafiltrate (UF) by polymerase chain reaction amplification of 16S rRNA gene sequence analysis. Standard blood cultures were performed for all patients. RESULTS: The blood cultures from 2 of the 9 (22%) patients were positive. However, bacterial DNA was identified in the blood of 6 patients (67%), including the 2 septic patients with positive blood cultures. In 9 (100%) patients bacterial DNA was found on the filter blood side, whereas in 7 (78%) subjects it was found in the dialysate compartment of the hemofilters. Bacterial DNA was never detected in the UF. CONCLUSIONS: Using the 16S rRNA gene, the detection of bacterial DNA in blood and adsorbed within the filter could be a useful screening tool in clinically septic, blood culture-negative patients undergoing CRRT. However, the identification of the etiologic agent is not feasible with this technique because specific primers for the defined bacteria must be used to further identify the suspected pathogenic organisms.