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Aims To define endotypes of carotid subclinical atherosclerosis. Methods and results We integrated demographic, clinical, and molecular data (n = 124) with ultrasonographic carotid measurements from study participants in the IMPROVE cohort (n = 3340). We applied a neural network algorithm and hierarchical clustering to identify carotid atherosclerosis endotypes. A measure of carotid subclinical atherosclerosis, the c-IMTmean-max, was used to extract atherosclerosis-related features and SHapley Additive exPlanations (SHAP) to reveal endotypes. The association of endotypes with carotid ultrasonographic measurements at baseline, after 30 months, and with the 3-year atherosclerotic cardiovascular disease (ASCVD) risk was estimated by linear (β , SE) and Cox [hazard ratio (HR), 95% confidence interval (CI)] regression models. Crude estimates were adjusted by common cardiovascular risk factors, and baseline ultrasonographic measures. Improvement in ASCVD risk prediction was evaluated by C -statistic and by net reclassification improvement with reference to SCORE2, c-IMTmean-max, and presence of carotid plaques. An ensemble stacking model was used to predict endotypes in an independent validation cohort, the PIVUS (n = 1061). We identified four endotypes able to differentiate carotid atherosclerosis risk profiles from mild (endotype 1) to severe (endotype 4). SHAP identified endotype-shared variables (age, biological sex, and systolic blood pressure) and endotype-specific biomarkers. In the IMPROVE, as compared to endotype 1, endotype 4 associated with the thickest c-IMT at baseline (β , SE) 0.36 (0.014), the highest number of plaques 1.65 (0.075), the fastest c-IMT progression 0.06 (0.013), and the highest ASCVD risk (HR, 95% CI) (1.95, 1.18–3.23). Baseline and progression measures of carotid subclinical atherosclerosis and ASCVD risk were associated with the predicted endotypes in the PIVUS. Endotypes consistently improved measures of ASCVD risk discrimination and reclassification in both study populations. Conclusions We report four replicable subclinical carotid atherosclerosis—endotypes associated with progression of atherosclerosis and ASCVD risk in two independent populations. Our approach based on endotypes can be applied for precision medicine in ASCVD prevention. [ABSTRACT FROM AUTHOR]

Abstract: Objective: To examine the role of platelets and platelet-derived products on cyclooxygenase-2 (Cox-2) induction in adherent monocytes and to address the signaling pathways involved. Methods: Platelets and monocytes were obtained from peripheral blood of healthy donors. Adherent monocytes were co-cultured with autologous platelets or platelet releasates or exposed to mediators contained in platelet α-granules (either from platelet source or recombinant) for 4–24 h. Cox-2 protein and mRNA were determined by Western and RT-PCR analysis, respectively. Thromboxane B2 (TxB2) and prostaglandin E2 (PGE2) synthesis as index of Cox-2 activity, and levels of transforming growth factor-β1 (TGF-β1) in platelet releasates were measured by enzyme immunoassay (EIA). Results: Activated platelets induce rapid and transient Cox-2 de novo synthesis in adherent monocytes. The effect is dependent upon the platelet number but not upon cell–cell contact. Platelet-induced Cox-2 was not affected by prevention of platelet TxA2 synthesis or microparticle formation but was blunted by inhibition of platelet α-granule secretion. TGF-β1, either platelet-derived or recombinant (rTGF-β1), induced Cox-2 expression and activity in adherent monocytes at concentrations within the range of those detected in releasates from activated platelets; this effect was not shared by recombinant platelet-derived growth factor (rPDGFBB). The time course of Cox-2 induction by TGF-β1 in monocytes was identical to that observed with platelet releasates. Moreover, TGF-β1 receptor blockade completely abolished platelet-induced Cox-2 expression. p38 MAPK activation represents a common transduction pathway through which activated platelets and rTGF-β1 induce Cox-2 in monocytes. Conclusion: These findings suggest that TGF-β1 released by activated platelets has a pivotal role in Cox-2 induction in monocytes and further supports the key role of platelets in the inflammatory and reparative responses. [Copyright &y& Elsevier]

Abstract: Objective: To assess whether indobufen, a reversible inhibitor of platelet cyclooxygenase (Cox) activity, affects tissue factor (TF) in human monocytes and to investigate the relationship between Cox-derived products and TF. Methods: TF was evaluated in isolated adherent monocytes, both resting and lipopolysaccharide (LPS)-stimulated, in terms of procoagulant activity, protein, and mRNA levels. The expression of TF surface antigen was determined in LPS-stimulated whole blood monocytes by flow cytometry. The levels of the stable thromboxane A2 (TxA2) metabolite, TxB2, and of prostaglandin E2 (PGE2) were measured in monocyte supernatant by immunoenzymatic techniques. Cox-1 and Cox-2 protein level, tyrosine phosphorylation, and mitogen-activated protein kinase (MAP-kinase) activation were determined by Western blot analysis. Results: Indobufen prevents TF expression and activity both in isolated and in whole blood monocytes. Reduction of TxA2 synthesis, coupled with a lack of effect on PGE2 levels and prevention of ERK1/2 phosphorylation are highlighted as the mechanisms through which indobufen negatively affects TF. Conclusions: Data show that indobufen down-regulates TF in monocytes. This novel activity, coupled with the antiplatelet effect of the drug, may add benefit for its use in the management of atherothrombosis. [Copyright &y& Elsevier]

Abstract: Objective: This study examines whether cyclooxygenase 2 (Cox-2) synthesis in human endothelial cells involves different signaling pathways when induced by the proinflammatory cytokine tumor necrosis factor-α (TNFα) or by the tumor and angiogenic promoter phorbol ester (PMA). Moreover, the hypothesis that reactive oxygen species (ROS) and an altered redox status within the cell are fundamental steps for Cox-2 synthesis is verified. Methods: Human endothelial cells isolated from umbilical vein (HUVEC) were exposed to PMA and TNFα and Cox-2 protein and mRNA levels were evaluated by Western blot and Real-Time Quantitative Reverse Transcription–PCR analysis. Prostaglandin E2 (PGE2) and 6-keto prostaglandin F1α (6-keto-PGF1α) levels were measured in cell medium as an index of Cox-2 activity. Intracellular ROS formation was detected by flow cytometry in HUVEC loaded with the oxidant-sensitive 2′,7′-dichlorofluorescein diacetate (DCFH-DA) and by nitroblue tetrazolium (NBT) reduction. Reduced and oxidized glutathione (GSH and GSSG) were measured by HPLC. Results: Data show that TNFα and PMA signal for early Cox-2 induction through distinct pathways. PMA-induced Cox-2 expression involves a small GTPase-dependent pathway acting via tyrosine kinase, activation of protein kinase C (PKC) and of the mitogen-activated protein kinase (MAPK) ERK1/2. Conversely, MAPK p38 is critical for Cox-2 induction by TNFα. Of interest, intracellular ROS generation and consequent GSH/GSSG ratio reduction represents a common step through which PMA and TNFα signal for early Cox-2 induction. In addition, we provide evidence that phosphatidylinositol 3 (PI3)-kinase activation plays a regulatory role for Cox-2 synthesis in HUVEC. Conclusion: Cox-2 represents a critical link among vascular homeostasis, inflammatory response, angiogenesis and tumor growth. The finding that two independent pathways and an overlapping upstream event signal for Cox-2 induction in HUVEC may be of relevance to develop strategies aimed at selectively interfering with Cox-2 regulating pathways. [Copyright &y& Elsevier]

Objective: The objective of this study was to explore the relationship between monocyte differentiation into macrophages and cyclooxygenase-2 (Cox-2) expression, based upon the observation that high amounts of this enzyme, colocalizing mainly with macrophages, have been found in human atherosclerotic lesions. Moreover, the hypothesis that reactive oxygen species (ROS) could be important as mediators of Cox-2 expression during monocyte differentiation was verified. Although ROS are known as modulators of gene expression profile, their involvement in monocyte differentiation has not been explored previously. Methods: Human adherent monocytes and the promonocytic cell line U937 were differentiated into macrophages by phorbol ester (PMA). Cox-2 was evaluated in terms of protein, mRNA and activity. Intracellular ROS formation was measured by the oxidant sensitive dye 2′,7′-dichlorofluorescein diacetate. NADPH oxidase subunit p47phox was evaluated by Western blot analysis. Results: Functionally active Cox-2 is expressed during PMA-induced monocyte transition into macrophages and ROS driven by the NADPH oxidase play a critical role in this event. Conclusion: Monocyte differentiation into macrophages, possibly triggered by unquenched ROS, may contribute to the increased inflammatory response within atheromata. [Copyright &y& Elsevier]

Objective: Oxidized low-density lipoproteins (ox-LDL) or their components suppress macrophage inflammatory response by down-regulating cytokine synthesis, nitric oxide synthase and inducible cyclooxygenase (Cox-2). This event is crucial for the pathophysiological process leading to the formation of atherosclerotic plaque. Our present study focused on the mechanisms through which oxidized phospholipids inhibit LPS-induced Cox-2 expression in human macrophages. Methods: Macrophages were incubated with a mixture of oxidized fragmented phospholipids (ox-PAPC), present in modified LDL, and then exposed to LPS. Cox-2 was evaluated in terms of protein levels, mRNA and activity. Results: Ox-PAPC dose-dependently inhibited Cox-2 protein, mRNA and activity by preventing NF-κB binding to DNA. This effect was consequent to alterations of the degradation pattern of IκBα. Moreover, ox-PAPC markedly prevented extracellular signal-regulated kinase (ERK2) activation, leading to Cox-2 expression, whereas activation of the transcription factor peroxisome proliferator-activated receptors (PPARs) was not influenced. Conclusion: ox-PAPC down-regulates LPS-induced Cox-2 expression in human macrophages by targeting both NF-κB/IκB and ERK2 pathways. An altered inflammatory response by macrophages within atheromata may contribute to the progression of atherosclerosis. [Copyright &y& Elsevier]

Objective: To examine the role of platelets and platelet-derived products on cyclooxygenase-2 (Cox-2) induction in adherent monocytes and to address the signaling pathways involved., Methods: Platelets and monocytes were obtained from peripheral blood of healthy donors. Adherent monocytes were co-cultured with autologous platelets or platelet releasates or exposed to mediators contained in platelet alpha-granules (either from platelet source or recombinant) for 4-24 h. Cox-2 protein and mRNA were determined by Western and RT-PCR analysis, respectively. Thromboxane B2 (TxB2) and prostaglandin E2 (PGE2) synthesis as index of Cox-2 activity, and levels of transforming growth factor-beta1 (TGF-beta1) in platelet releasates were measured by enzyme immunoassay (EIA)., Results: Activated platelets induce rapid and transient Cox-2 de novo synthesis in adherent monocytes. The effect is dependent upon the platelet number but not upon cell-cell contact. Platelet-induced Cox-2 was not affected by prevention of platelet TxA2 synthesis or microparticle formation but was blunted by inhibition of platelet alpha-granule secretion. TGF-beta1, either platelet-derived or recombinant (rTGF-beta1), induced Cox-2 expression and activity in adherent monocytes at concentrations within the range of those detected in releasates from activated platelets; this effect was not shared by recombinant platelet-derived growth factor (rPDGFBB). The time course of Cox-2 induction by TGF-beta1 in monocytes was identical to that observed with platelet releasates. Moreover, TGF-beta1 receptor blockade completely abolished platelet-induced Cox-2 expression. p38 MAPK activation represents a common transduction pathway through which activated platelets and rTGF-beta1 induce Cox-2 in monocytes., Conclusion: These findings suggest that TGF-beta1 released by activated platelets has a pivotal role in Cox-2 induction in monocytes and further supports the key role of platelets in the inflammatory and reparative responses.

Objective: This study examines whether cyclooxygenase 2 (Cox-2) synthesis in human endothelial cells involves different signaling pathways when induced by the proinflammatory cytokine tumor necrosis factor-alpha (TNF alpha) or by the tumor and angiogenic promoter phorbol ester (PMA). Moreover, the hypothesis that reactive oxygen species (ROS) and an altered redox status within the cell are fundamental steps for Cox-2 synthesis is verified., Methods: Human endothelial cells isolated from umbilical vein (HUVEC) were exposed to PMA and TNF alpha and Cox-2 protein and mRNA levels were evaluated by Western blot and Real-Time Quantitative Reverse Transcription-PCR analysis. Prostaglandin E(2) (PGE(2)) and 6-keto prostaglandin F(1 alpha) (6-keto-PGF(1 alpha)) levels were measured in cell medium as an index of Cox-2 activity. Intracellular ROS formation was detected by flow cytometry in HUVEC loaded with the oxidant-sensitive 2',7'-dichlorofluorescein diacetate (DCFH-DA) and by nitroblue tetrazolium (NBT) reduction. Reduced and oxidized glutathione (GSH and GSSG) were measured by HPLC., Results: Data show that TNF alpha and PMA signal for early Cox-2 induction through distinct pathways. PMA-induced Cox-2 expression involves a small GTPase-dependent pathway acting via tyrosine kinase, activation of protein kinase C (PKC) and of the mitogen-activated protein kinase (MAPK) ERK1/2. Conversely, MAPK p38 is critical for Cox-2 induction by TNF alpha. Of interest, intracellular ROS generation and consequent GSH/GSSG ratio reduction represents a common step through which PMA and TNF alpha signal for early Cox-2 induction. In addition, we provide evidence that phosphatidylinositol 3 (PI3)-kinase activation plays a regulatory role for Cox-2 synthesis in HUVEC., Conclusion: Cox-2 represents a critical link among vascular homeostasis, inflammatory response, angiogenesis and tumor growth. The finding that two independent pathways and an overlapping upstream event signal for Cox-2 induction in HUVEC may be of relevance to develop strategies aimed at selectively interfering with Cox-2 regulating pathways.

Objective: Oxidized low-density lipoproteins (ox-LDL) or their components suppress macrophage inflammatory response by down-regulating cytokine synthesis, nitric oxide synthase and inducible cyclooxygenase (Cox-2). This event is crucial for the pathophysiological process leading to the formation of atherosclerotic plaque. Our present study focused on the mechanisms through which oxidized phospholipids inhibit LPS-induced Cox-2 expression in human macrophages., Methods: Macrophages were incubated with a mixture of oxidized fragmented phospholipids (ox-PAPC), present in modified LDL, and then exposed to LPS. Cox-2 was evaluated in terms of protein levels, mRNA and activity., Results: Ox-PAPC dose-dependently inhibited Cox-2 protein, mRNA and activity by preventing NF-kappaB binding to DNA. This effect was consequent to alterations of the degradation pattern of IkappaBalpha. Moreover, ox-PAPC markedly prevented extracellular signal-regulated kinase (ERK2) activation, leading to Cox-2 expression, whereas activation of the transcription factor peroxisome proliferator-activated receptors (PPARs) was not influenced., Conclusion: ox-PAPC down-regulates LPS-induced Cox-2 expression in human macrophages by targeting both NF-kappaB/IkappaB and ERK2 pathways. An altered inflammatory response by macrophages within atheromata may contribute to the progression of atherosclerosis.