Assessment of the predictive value of plasma calprotectin in the evolution of SARS-Cov-2 primo-infection.

BACKGROUND: The COVID-19 epidemic still calls for anticipation aimed at preventing the overloading of critical care services. With this in mind, the predictive value of easily accessible biomarkers is to be assessed. OBJECTIVE: Secretion of calprotectin is stimulated during an inflammatory process, especially in the cytokine storm. We tried to determine whether early plasma concentration of calprotectin in patients with primary SARS-CoV-2 infection could predict an adverse outcome in cases of COVID-19. METHODS: We included 308 patients with a primary diagnosis of SARS-CoV-2 confirmed by PCR. Heparinized tube samples, collected within the first 24 h of hospitalization, were used for biomarker assays, in which plasma calprotectin was included. Data from the patients' medical records and severity groups established subsequent to diagnosis at the end of hospitalization were collected. RESULTS: Early plasma calprotectin concentration is significantly associated with progression to a severe form of COVID-19 in patients with primary infection (Relative Risk: 2.2 [1.6-2.7]). In multivariate analysis, however, it does not appear to provide additional information compared to other parameters (age, GFR, CRP…). CONCLUSION: Our study shows that while an early single blood test for calprotectin could help to predict the progression of a primary SARS-CoV-2 infection, it is not superior to the other parameters currently used in emergency medicine. However, it paves the way for future considerations, such as the interest of this biomarker for high-risk infected patients (immunocompromised individuals…). Finally, the usefulness of early serial measurements of plasma calprotectin to assess progression towards severity of COVID-19 requires further assessment.

Physical activity and odds of coronary heart disease among Lebanese women.

BACKGROUND: It is known that physical activity (PA) is protective against cardiovascular morbidity and mortality. However, few studies have examined the association between PA, sedentary lifestyle and coronary heart disease (CHD) in women. This case-control study investigates the relationship between PA and sedentary behavior on CHD odds in Lebanese women over forty. METHODS: One thousand five hundred selected Lebanese women (300 cases and 1200 controls) were included between 2018-2019. Cases were hospitalized women newly diagnosed with CHD, whereas the control groups were free of any heart diseases. Data on socio-demographic, lifestyle, cardiovascular factors, PA and sedentary lifestyle were collected. Multivariate logistic regressions, adjusted for covariates, were performed to investigate the association of PA domains and sedentary behavior with CHD. RESULTS: A sedentary lifestyle combined with low activity levels increased the odds of CHD. Among cases, 46.7% participated in moderate or vigorous PA against almost 60.3% of controls. 36.3% of coronary patients had more than 10 h/day of sedentary time, with a positive correlation with CHD (adjusted OR: 1.533, 95%CI: 1.046-2.247). Conversely, moderate and high levels (respectively 600-3000 and ≥ 3000 metabolic equivalents [MET]-minutes/week) of domestic/garden PA revealed lower CHD odds (OR: 0.566, 95%CI: 0.396-0.808 and 0.193, 0.065-0.578 respectively). The detrimental influence of sedentary lifestyle appeared to be significantly reversed by weekly moderate PA, especially as weekly sedentary time was less (OR: 0.616, 95%CI: 0.427-0.888/ 6 to10h of sedentary time and OR: 0.537, 95% CI: 0.37-0.779/ ≤ 6 h), and except sedentary time exceeding 10 h daily. Two PA patterns revealed lower CHD odds: transport-related and domestic/garden PA, as early as low amount, even after adjustment for possible confounders. CONCLUSION: The current study highlights the importance of combating sedentary behaviors and engaging in regular, easily accessible PA to reduce the odds of coronary disease among aging women. Therefore, better information regarding the benefits of physical activities such as transportation-related activities or gardening would be helpful in enhancing the prevention of CHD in aging women.

Risk Factors for Coronary Heart Disease Among Lebanese Women: A Case-Control Study.

Purpose: Women are increasingly concerned by coronary heart disease (CHD), with peculiarities of their own, particularly concerning risk factors. The aim of the study was to assess the risk factors for CHD in Lebanese women over forty. Patients and Methods: A case-control study was carried out in 6 hospitals in Beirut and Mount-Lebanon, from December 2018 to December 2019 including 1500 patients (1200 controls and 300 cases). Women were stratified into pre- and post-menopausal groups. Personal and medical data were collected from hospital records and during an interview where validated questionnaires were used. Binary logistic regressions were performed to investigate potential predictors of CHD in the 2 groups. Results: In post-menopausal women, dyslipidemia (adjusted odds ratio [aOR], 3.018; 95% confidence interval, 2.102-4.332), hypertension (aOR: 2.449, [1.386-4.327]), a family history of CHD (aOR: 2.724, [1.949-3.808]), cigarette smoking (aOR: 2.317, [1.574-3.410]) and common non-rheumatic joint pain (aOR: 1.457, [1.053-2.016]) were strongly associated with CHD. Conversely, living in Mount Lebanon seemed protective, compared to Beirut (aOR: 0.589, [0.406-0.854]), as well as having a moderate monthly income (aOR: 0.450, [0.220-0.923]), adhering to a Mediterranean diet (aOR: 0.965, [0.936-0.994]), and practicing physical activity [PA] (aOR: 0.396, [0.206-0.759] and 0.725, [0.529-0.992], respectively for high and moderate vs low PA). In pre-menopausal women, dyslipidemia (aOR: 6.938, [1.835-26.224]), hypertension (aOR: 6.195, [1.318-29.119]), family histories of dyslipidemia (aOR: 6.143, [1.560-24.191]) and CHD (aOR: 4.739, [1.336-16.805]) reached statistical significance. Conclusion: The identification of factors associated with CHD in women, some of which are frequent and trivialized in post-menopause, underlines the need to put in place specific and dedicated CHD prevention strategies in women.

The effects of periodontal treatment on diabetic patients: The DIAPERIO randomized controlled trial.

AIM: To assess whether periodontal treatment can lead to clinical, glycaemic control and quality of life improvements in metabolically unbalanced diabetic patients (type 1 or type 2) diagnosed with periodontitis. METHODS: In this open-labelled randomized controlled trial, diabetic subjects (n = 91) were given "immediate" or "delayed" periodontal treatment (full-mouth non-surgical scaling and root planing, systemic antibiotics, and oral health instructions). The main outcome was the effect on glycated haemoglobin (HbA1C ) and fructosamine levels. The General Oral Health Assessment Index and the SF-36 index were used to assess quality of life (QoL). RESULTS: Periodontal health significantly improved after periodontal treatment (p < 0.001). Periodontal treatment seemed to be safe but had no significant effects on glycaemic control based on HbA1C (adjusted mean difference with a 95% confidence interval (aMD) of 0.04 [-0.16;0.24]) and fructosamine levels (aMD 5.0 [-10.2;20.2]). There was no obvious evidence of improvement in general QoL after periodontal treatment. However, there was significant improvement in oral health-related QoL (aMD 7.0 [2.4;11.6], p = 0.003). CONCLUSION: Although periodontal treatment showed no clinical effect on glycaemic control in this trial, important data were provided to support periodontal care among diabetic patients. Periodontal treatment is safe and improves oral health-related QoL in patients living with diabetes. ISRCTN15334496.

Head-to-head comparison of 10 natriuretic peptide assays.

BACKGROUND: The aim of the study was to compare NT-proBNP and BNP levels in fresh samples from heart failure (HF) patients measured using 10 immunoassays and to assess their agreement. METHODS: NT-proBNP (CobasH232(®), Elecsys(®), Vidas(®), Vista(®), XPand(®), Vitros(®)) and BNP (Triage(®), Access(®), CentaurXP(®), Architect(®)) levels were measured in 39 heparin and 19 EDTA samples, respectively. RESULTS: The Pearson correlation coefficient ranged between 0.929 (Triage(®-)Centaur(®)) and 0.994 (Access(®)-Architect(®)) for BNP assays and between 0.972 (Vidas(®)-Cobas H232(®)) and 0.999 (Vitros(®)-Vidas(®)) for NT-proBNP assays. Passing Bablok regression analyses showed a significant difference in the slopes [0.80 (Centaur(®)-Triage(®)) to 1.84 (Architect(®)-Centaur(®))] and intercepts [-55 ng/L (Architect(®)-Centaur(®)) to 48 ng/L (Access(®)-Triage®)] for BNP assays, and a lower heterogeneity between NT-proBNP assays [0.83 (Vidas(®)-Elecsys(®)) to 1.20 (Vitros(®)-Vidas(®)) and -97 ng/L (XPand(®)-CobasH232(®)) to 51 ng/L (CobasH232(®)-Elecsys(®)) for slopes and intercepts, respectively]. The concordance correlation coefficient revealed a poor (ρc<0.90) to moderate (ρc=0.90-0.95) agreement in 4/6 pairs of BNP assays and an almost perfect (ρc>0.99) agreement in 5/15 pairs of NT-proBNP assays. The acceptable difference limit reflecting the number of individual discrepant results between two assays, ranged between 15.1% (Access(®)-CentaurXP(®)) and 34.5% (Architect(®)-Triage(®)) for BNP assays, and between 10.9% (Vidas(®)-Vitros(®)) and 55% (CobasH232(®)-Xpand(®)) for NT-proBNP assays. CONCLUSIONS: This study stresses the lack of transferability of the results obtained using different techniques to measure BNP and NT-proBNP levels in fresh samples. Individual reference ranges and HF diagnostic cut-offs should be assessed for each commercial NP immunoassay. We recommend to systematically monitoring HF patients using the same assay (BNP or NT-proBNP) over the time.

Predictive value of the heart-type fatty acid-binding protein and the Pulmonary Embolism Severity Index in patients with acute pulmonary embolism in the emergency department.

OBJECTIVES: Heart-type fatty acid-binding protein (h-FABP), sensitive troponins, natriuretic peptides, and clinical scores such as the Pulmonary Embolism Severity Index (PESI) are candidates for risk stratification of patients with acute pulmonary embolism (PE). The aim was to compare their respective prognostic values to predict an adverse outcome at 1 month. METHODS: The authors prospectively included 132 consecutive patients with confirmed acute PE. On admission to the emergency department (ED), plasma concentrations of h-FABP, sensitive cardiac troponin I-Ultra (cTnI-Ultra), and brain natriuretic peptide (BNP) were measured and the PESI calculated in all patients. The combined 30-day outcomes of interest were death, cardiac arrest, mechanical ventilation, use of catecholamines, and recurrence of acute PE. RESULTS: During the first 30 days, 14 (10.6%) patients suffered complications. Among the biomarkers, h-FABP above 6 µg/L was a stronger predictor of an unfavorable outcome (odds ratio [OR] = 17.5, 95% confidence interval [CI] = 4.2 to 73.3) than BNP > 100 pg/mL (OR = 5.7, 95% CI = 1.6 to 20.4) or cTnI-Ultra > 0.05 µg/L (OR = 3.4, 95% CI = 1.1 to 10.9). The PESI classified 83 of 118 patients (70.3%) with favorable outcomes and only one of 14 (7%) with adverse outcomes in low class I or II (OR = 30.8, 95% CI = 3.2 to 299.7). The areas under the receiver operating characteristic (ROC) curves (AUCs) were 0.90 (95% CI = 0.81 to 0.98) for h-FABP, 0.89 (95% CI = 0.82 to 0.96) for PESI, 0.79 (95% CI = 0.67 to 0.90) for BNP, and 0.76 (95% CI = 0.64 to 0.87) for cTnI-Ultra. The combination of h-FABP with PESI was a particularly useful prognostic indicator because none of the 79 patients (59.8%) with h-FABP < 6 ng/mL and PESI class < III had an adverse outcome. CONCLUSIONS: h-FABP and the PESI are superior to BNP and cTnI-Ultra as markers for risk stratification of patients with acute PE. The high sensitivity of their combination identified a large number of low-risk patients in the ED.

Copeptin improves the diagnostic performance of sensitive troponin I-Ultra but cannot rapidly rule out non-ST-elevation myocardial infarction at presentation to an emergency department.

STUDY OBJECTIVE: We assess the performance of a single multimarker strategy, using a combination of sensitive troponin I-Ultra and copeptin assays to rule out non-ST-elevation myocardial infarction (NSTEMI) at presentation to an emergency department (ED). METHODS: A secondary analysis was carried out on 587 consecutive patients with chest pain who presented to the ED without ST elevation on ECG and were included in a single-site, prospective observational study. Samples for copeptin and combination of sensitive troponin I-Ultra assays were collected at presentation. The performance of the combination of copeptin and combination of sensitive troponin I-Ultra for NSTEMI was calculated in the whole cohort and after stratification by thrombosis in myocardial infarction (TIMI) risk score. RESULTS: NSTEMI was diagnosed in 87 patients (14.8%). The sensitivity and the negative predictive value of the combination of copeptin and combination of sensitive troponin I-Ultra were 96.6% (95% confidence interval [CI] 90.3% to 99.3%) and 99.1% (95% CI 97.4% to 99.8%), respectively, for a cutoff level of copeptin less than 12 pmol/L. Among the 243 patients with a low TIMI score, all 8 who had an NSTEMI were detected with the combination (sensitivity 100%; 95% CI 63.1% to 100%), and 158 were a combination of sensitive troponin I-Ultra and copeptin negative and had no NSTEMI (negative predictive value 100%; 95% CI 97.7% to 100%). CONCLUSION: In this study, the combination of sensitive troponin and copeptin measurements had a high sensitivity and negative predictive value for NSTEMI diagnosis, especially among subjects with a low TIMI risk score. However, the sensitivity was too low to rule out NSTEMI with a single-draw strategy at ED presentation. Future studies are needed on the low-risk TIMI group to further investigate this preliminary finding.

Combination of copeptin and troponin assays to rapidly rule out non-ST elevation myocardial infarction in the emergency department.

OBJECTIVES: The aim of this study was to analyze the diagnostic accuracy and the clinical usefulness of the combination of troponin I (cTnI) and copeptin measured at presentation with an automated assay to rapidly rule out non-ST elevation myocardial infarction (NSTEMI) in patients with suspected cardiac chest pain presenting to an emergency department (ED). METHODS: This study was an ancillary analysis of a prospective observational study. Copeptin and cTnI levels were sampled at presentation in 641 consecutive patients admitted to the ED for chest pain with onset within the last 12 hours and without ST elevation on a 12-lead electrocardiogram (ECG). Copeptin was measured with an automated assay and troponin with conventional assay. The performance of a combination of cTnI and copeptin for NSTEMI diagnosis was studied, the clinical utility was assessed by multivariate analysis, and an area under the curve (AUC) calculation was used to determine accuracy. RESULTS: NSTEMI was diagnosed in 95 patients (15%). The sensitivity and negative predictive value (NPV) of the combination of copeptin and cTnI measures were 90.4% (95% confidence interval [CI] = 88.2% to 92.7%) and 97.6% (95% CI = 96.4% to 98.7%) versus 55.3% (95% CI = 51.5% to 59.2%) and 92.8% (95% CI = 90.8% to 94.8%) with cTnI alone. The AUC of the combination of copeptin and cTnI was 0.89 (95% CI = 0.85% to 0.92%) and was significantly higher than the AUC of cTnI alone (0.77, 95% CI = 0.72% to 0.82%, p < 0.05). The patient classification was slightly improved when copeptin was added to the usual diagnostic tools used for NSTEMI management. CONCLUSIONS: In this study, determination of copeptin, in addition to cTnI, improves early diagnostic accuracy of NSTEMI. However, the sensitivity of this combination even using a conventional troponin assay remains insufficient to safely rule out NSTEMI at the time of presentation.

[Inventory of the use of natriuretic peptides in France]. / État des lieux de l'utilisation des peptides natriurétiques en France.

Since the introduction of routine assay for natriuretic peptides (NP), there is an increasing number of clinical applications for these assays. Due to the continuously increasing number of prescription of those tests, a reappraisal of the use of natriuretic peptide assays, namely BNP and NT-proBNP in France was necessary. This was achieved through a national survey to obtain a detailed description of NP prescription and realization by French laboratories. A questionnaire was sent in April 2010 to hospital and private clinical chemists. Statistical analysis of results concerned 584 answers. This survey demonstrated an equivalent use of BNP and NT-proBNP both in public or private laboratories together with a huge heterogeneity of tests used within labs. Medical prescription heterogeneity both in public or private sectors confirms the large implication of those tests in clinical diagnosis. These assays are not yet standardized, so clinicians and biologists should be very careful when interpreting the results for diagnostic or therapeutic monitoring.

Diagnostic accuracy of quantitative heart-fatty acid binding protein assays compared with Cardiodetect(®) in the early detection of acute coronary syndrome.

BACKGROUND: Heart-fatty acid binding protein (h-FABP) has been proposed as a cardiac marker for the early detection of acute coronary syndrome (ACS). In a study of 677 patients admitted to the emergency department (ED) for chest pain, we found that a semiquantitative point-of-care test that detects h-FABP (Cardiodetect(®)) had low sensitivity for the prediction of ACS. OBJECTIVE: The aim of this ancillary study was to analyze and compare the performance of h-FABP for early ACS diagnosis in this large cohort of unselected patients, using a quantitative immunoassay and Cardiodetect(®). METHODS: h-FABP was measured with a ready-to-use, solid-phase, enzyme-linked immunosorbent assay (ELISA) in 677 patients admitted to the ED with chest pain and suspected non-ST-segment elevation ACS. Two physicians, blinded to the results of the marker, categorized patients as having or not having non-ST-segment elevation ACS. RESULTS: Non-ST-segment elevation ACS was diagnosed in 185 patients (27.3%). The median h-FABP level was higher in patients with ACS (1.36µg/L, interquartile range [IQR] 0.59-3.55) than in those without ACS (0.58µg/L, IQR 0.24-1.34; P<0.01). The area under the curve was 0.68 (95% confidence interval [CI] 0.63-0.73). h-FABP did not improve the performance of a model that included the usual diagnostic tools for ACS management (odds ratio 0.92, 95% CI 0.32-2.70). The classification agreement between the ELISA and Cardiodetect(®) was 92.1% (kappa 0.39). CONCLUSION: In this study, we confirmed that measurement of h-FABP was insufficient to be used as a marker of ACS and NSTEMI in ED, whatever the analytical technique used.

Clinical assessment of ischemia-modified albumin and heart fatty acid-binding protein in the early diagnosis of non-ST-elevation acute coronary syndrome in the emergency department.

OBJECTIVES: Heart fatty acid-binding protein (h-FABP) and ischemia-modified albumin (IMA) have recently been evaluated, but to the best of our knowledge, no study has reported an analysis of these two markers for the detection of early myocardial infarction and myocardial ischemia in a large cohort of consecutive patients presenting to an emergency department (ED). This study evaluates the diagnostic accuracy and the clinical utility of h-FABP and IMA for non-ST-segment elevation acute coronary syndrome (ACS) diagnosis in the first hour of management in an ED. METHODS: In a prospective 11-month study, 677 patients admitted to the ED with chest pain and suspected non-ST-segment elevation ACS were enrolled. On presentation, blood samples were obtained for the measurement of the biomarkers h-FABP (immunodetection with CardioDetect) and IMA (albumin cobalt-binding test). Two physicians, blinded to the results of the markers, independently categorized patients as having or not having non-ST-segment elevation ACS. RESULTS: Of the 677 patients who were prospectively recruited, non-ST-segment elevation ACS was diagnosed in 185 (27.3%). While IMA was not predictive of the ACS diagnosis (odds ratio [OR] = 1.23; 95% CI = 0.87 to 1.81), h-FABP was predictive of ACS diagnosis (OR = 4.65; 95% CI = 2.39 to 9.04) with specificity at 96.8% (95% CI = 95.4% to 98.1%) and sensitivity at 13.5% (95% CI = 10.9% to 16.1%). However, h-FABP did not add significant additional information to a predictive model that included the usual diagnostic tools for non-ST-elevation ACS management (p = 0.40). CONCLUSIONS: In this study on a large cohort of patients admitted to an ED for chest pain, IMA and h-FABP did not provide valuable information for ACS diagnosis.

Integrin alpha(v)beta(3), metalloproteinases, and sphingomyelinase-2 mediate urokinase mitogenic effect.

Plasminogen activators are implicated in the pathogenesis of several diseases such as inflammatory diseases and cancer. Beside their serine-protease activity, these agents trigger signaling pathways involved in cell migration, adhesion and proliferation. We previously reported a role for the sphingolipid pathway in the mitogenic effect of plasminogen activators, but the signaling mechanisms involved in neutral sphingomyelinase-2 (NSMase-2) activation (the first step of the sphingolipid pathway) are poorly known. This study was carried out to investigate how urokinase plasminogen activator (uPA) activates NSMase-2. We report that uPA, as well as its catalytically inactive N-amino fragment ATF, triggers the sequential activation of MMP-2, NSMase-2 and ERK1/2 in ECV304 cells that are required for uPA-induced ECV304 proliferation, as assessed by the inhibitory effect of Marimastat (a MMP inhibitor), MMP-2-specific siRNA, MMP-2 defect, and NSMase-specific siRNA. Moreover, upon uPA stimulation, uPAR, MT1-MMP, MMP-2 and NSMase-2 interacted with integrin alpha(v)beta(3), evidenced by co-immunoprecipitation and immunocytochemistry experiments. Moreover, the alpha(v)beta(3) blocking antibody inhibited the uPA-triggered MMPs/uPAR/integrin alpha(v)beta(3) interaction, NSMase-2 activation, Ki67 expression and DNA synthesis in ECV304. In conclusion, uPA triggers interaction between integrin alpha(v)beta(3), uPAR and MMPs that leads to NSMase-2 and ERK1/2 activation and cell proliferation. These findings highlight a new signaling mechanism for uPA, and suggest that, upon uPA stimulation, uPAR, MMPs, integrin alpha(v)beta(3) and NSMase-2 form a signaling complex that take part in mitogenic signaling in ECV304 cells.

E-cadherin/beta-catenin/T-cell factor pathway is involved in smooth muscle cell proliferation elicited by oxidized low-density lipoprotein.

The E-cadherin/beta-catenin/T-cell factor (Tcf) signaling pathway plays a crucial role in embryogenesis and carcinogenesis and has recently emerged in atherosclerosis. The aim of this work was to investigate whether this signaling pathway is involved in smooth muscle cell proliferation induced by oxidized low-density lipoprotein (LDL). In human aortic smooth muscle cells, mitogenic concentration of mildly oxidized LDL induced the activation of beta-catenin, as assessed by the dissociation of the beta-catenin/cadherin complex, and the concomitant rise of active beta-catenin in the cytosol. The oxidized LDL-induced rise of active beta-catenin required metalloproteinase activation, as well as epidermal growth factor receptor and Src signaling, as assessed by the use of pharmacological inhibitors and cells overexpressing a SrcK-inactive form. The concomitant phosphatidylinositol 3-kinase/Akt activation and glycogen synthase kinase 3-beta phosphorylation induced the inhibition of the proteasomal degradation of beta-catenin. Then active beta-catenin associated with Tcf4 and translocated into the nucleus. This enhanced the expression of the cell cycle activator cyclin D1. This crucial role of beta-catenin in the mitogenic effect of oxidized LDL was confirmed by silencing beta-catenin by specific small interfering RNA that blocked DNA synthesis. Immunohistochemistry staining of stable and disrupted plaques from carotid endarterectomy sections showed a correlation between active beta-catenin and Ki67, a proliferation marker, and a more intense staining in the smooth muscle cell layer surrounding the lipid core of disrupted plaques. In conclusion, the beta-catenin pathway is required for the mitogenic effect of oxidized LDL on human aortic smooth muscle cells. This study highlights the putative important role of the E-cadherin/beta-catenin/Tcf signaling pathway in atherosclerosis.

Intestinal cholesterol transport proteins: an update and beyond.

PURPOSE OF REVIEW: Various studies have delineated the causal role of dietary cholesterol in atherogenesis. Strategies have thus been developed to minimize cholesterol absorption, and cholesterol transport proteins found at the apical membrane of enterocytes have been extensively investigated. This review focuses on recent progress related to various brush-border proteins that are potentially involved in alimentary cholesterol transport. RECENT FINDINGS: Molecular mechanisms responsible for dietary cholesterol and plant sterol uptake have not been completely defined. Growing evidence, however, supports the concept that several proteins are involved in mediating intestinal cholesterol transport, including SR-BI, NPC1L1, CD36, aminopeptidase N, P-glycoprotein, and the caveolin-1/annexin-2 heterocomplex. Other ABC family members (ABCA1 and ABCG5/ABCG8) act as efflux pumps favoring cholesterol export out of absorptive cells into the lumen or basolateral compartment. Several of these cholesterol carriers influence intracellular cholesterol homeostasis and are controlled by transcription factors, including RXR, LXR, SREBP-2 and PPARalpha. The lack of responsiveness of NPC1L1-deficient mice to ezetimibe suggests that NPC1L1 is likely to be the principal target of this cholesterol-lowering drug. SUMMARY: The understanding of the role, genetic regulation and coordinated function of proteins mediating intestinal cholesterol transport may lead to novel ways of treating cardiovascular disease.

Activation of the {beta}-catenin/T-cell-specific transcription factor/lymphoid enhancer factor-1 pathway by plasminogen activators in ECV304 carcinoma cells.

Besides its involvement in clot lysis, the plasminogen activator (PA) system elicits various cellular responses involved in cell migration, adhesion, and proliferation and plays a key role in the progression of cancers. beta-Catenin interacts with E-cadherins and functions as transcriptional coactivator of the Wnt-signaling pathway, which is implicated in tumor formation when aberrantly activated. We report that tissue-type plasminogen activator (tPA) elicited tyrosine phosphorylation and cytosolic accumulation of an active (non-serine-threonin phosphorylated, nonubiquitinated) form of beta-catenin in ECV304 carcinoma cells. tPA-dependent beta-catenin activation is mediated through epidermal growth factor receptor (EGFR) transactivation (via Src), suggested by the inhibitory effects of AG1478 and PP2 (specific inhibitors of EGFR and Src, respectively) and by the lack of beta-catenin activation in EGFR-negative B82 fibroblasts. EGFR phosphorylation and beta-catenin activation were inhibited by plasminogen activator inhibitor 1 and pertussis toxin, two inhibitors of the urokinase-type plasminogen activator (uPA)/uPA receptor system. beta-Catenin activation was correlated with the phosphorylation of glycogen synthase kinase-3beta through a phosphatidylinositol 3-kinase/Akt-dependent mechanism. Gel shift experiments revealed the activation of beta-catenin/T-cell-specific transcription factor (Tcf)/lymphoid enhancer factor-1 (Lef) transcriptional complex, evidenced by an increased binding of nuclear extracts to oligonucleotides containing the cyclin D1 Lef/Tcf site. beta-Catenin silencing through small interfering RNA and antisense oligonucleotides inhibited both the tPA-mediated cyclin D1 expression and cell proliferation. A similar activation of the beta-catenin pathway was triggered by amino-terminal fragment, the NH(2)-terminal catalytically inactive fragment of tPA, thus suggesting that this effect was independent of the proteolytic activity of plasminogen activators. In conclusion, the beta-catenin/Lef/Tcf pathway is activated by tPA and is involved in cell cycle progression and proliferation.

Role for matrix metalloproteinase-2 in oxidized low-density lipoprotein-induced activation of the sphingomyelin/ceramide pathway and smooth muscle cell proliferation.

BACKGROUND: Oxidized LDLs (oxLDLs) and matrix metalloproteinases (MMPs) are present in atherosclerotic lesions. OxLDLs activate various signaling pathways potentially involved in atherogenesis. OxLDLs induce smooth muscle cell (SMC) proliferation mediated by the activation of the sphingomyelin/ceramide pathway and tyrosine kinase receptors. MMPs are also able to induce SMC migration and proliferation in addition to extracellular matrix degradation. The present study was designed to investigate whether MMPs play a role in the mitogenic effect of oxLDLs. METHODS AND RESULTS: OxLDLs induce the release of activated MMP-2 in SMC culture medium. MMP-2 was identified by its 65-kDa gelatinase activity on zymography and by using specific blocking antibodies and MMP-2-/- cells. MMP inhibitors (batimastat and Ro28-2653) and the blocking antibodies anti-MMP-2 and anti-membrane type 1-MMP inhibited the oxLDL-induced sphingomyelin/ceramide pathway activation and subsequent activation of ERK1/2 and DNA synthesis but did not inhibit the oxLDL-induced epidermal growth factor receptor and platelet-derived growth factor receptor activation. Exogenously added activated MMP-2 or membrane type 1-MMP-1 triggered the activation of both sphingomyelin/ceramide and ERK1/2 pathways and DNA synthesis. Conversely, suppression of MMP-2 expression in MMP-2-/- cells or in SMCs treated by small-interference RNA also blocked both sphingomyelin/ceramide signaling and DNA synthesis. CONCLUSIONS: Together, these data demonstrate that MMP-2 plays a pivotal role in oxLDL-induced activation of the sphingomyelin/ceramide signaling pathway and subsequent SMC proliferation. These pathways may constitute a potential therapeutic target for modulating the oxLDL-induced proliferation of SMCs in atherosclerosis or restenosis.

The sphingomyelin/ceramide pathway is involved in ERK1/2 phosphorylation, cell proliferation, and uPAR overexpression induced by tissue-type plasminogen activator.

Plasminogen activators (tPA and uPA) are serine proteases that convert the circulating zymogen plasminogen to active plasmin and mediate fibrin degradation. These multifunctional proteins trigger various biological events such as extracellular matrix degradation, cell adhesion, migration, and proliferation, through not yet fully characterized mechanisms. We report that, in smooth muscle cells and ECV-304 carcinoma cells, tPA and ATF (the N-terminal catalytically inactive fragment of tPA) elicited DNA synthesis that requires activation of the sphingomyelin/ceramide/sphingosine-1-phosphate (Spm/Cer/S1P), signaling pathway and was blocked by D-erythro-2-(N-myristoylamino)-1-phenyl-propanol (D-MAPP) and N-N'-dimethyl sphingosine (DMS), two classical inhibitors of sphingosine-1-phosphate biosynthesis. Binding of tPA to its receptor uPAR triggered the coordinated activation of two key enzymes of the Spm/Cer/S1P pathway, the neutral sphingomyelinase and the sphingosine kinase-1 that was mediated by a common pertussis toxin (PTX)-sensitive mechanism. The tPA-induced sphingosine kinase-1 activation was mediated by Src, since it was inhibited by herbimycin A and in SrcK- cells (overexpressing a dominant negative kinase defective form of Src) and by ERK1/2 (early phase peaking at 15 min). Sphingosine kinase-1 activation was followed by a second phase of ERK1/2 phosphorylation (peaking at 120 min) and subsequent DNA synthesis, which were inhibited by D-MAPP and DMS, by anti-EGD-1 antibodies and in SrcK- cells (in which the mitogenic signaling was rescued by sphingosine-1-phosphate). Altogether, these data underline a pivotal role for the Spm/Cer/S1P pathway in the tPA-induced mitogenic signaling.

Oxidized LDL-induced smooth muscle cell proliferation involves the EGF receptor/PI-3 kinase/Akt and the sphingolipid signaling pathways.

OBJECTIVE: Oxidized low-density lipoprotein (oxLDL)-induced smooth muscle cell (SMC) proliferation requires the coactivation of various signaling pathways, namely sphingomyelin/ceramide/sphingosine-1-phosphate, epithelial growth factor receptor (EGFR), and phosphoinositide 3-kinase (PI-3K) pathways. This study aimed to clarify the respective role and the hypothetical cross-talk between sphingomyelin/ceramide/sphingosine-1-phosphate, EGFR, and PI-3K/Akt pathways in the balance between mitogenic and cytotoxic effects elicited by oxLDL. METHODS AND RESULTS: Coimmunoprecipitation experiments and the use of inhibitors and dominant-negative mutant showed that oxLDL-induced PI-3K activation is dependent on EGFR. PI-3K activation is independent of the sphingomyelin/ceramide/sphingosine-1-phosphate pathway, because PI-3K inhibition by LY294002 or dominant-negative Deltap85 mutant does not abrogate sphingomyelin hydrolysis, and, conversely, the use of permeant C2-ceramide and of N,N-dimethyl-sphingosine, a sphingosine kinase inhibitor, does not alter PI-3K activity. Activation of Akt/PKB by oxLDL requires PI-3K, as shown by the inhibition by LY204002 and in Deltap85 SMC. The inhibition of Akt/PKB by PI-3K inhibitor LY204002 or by overexpression of kinase-dead Akt shifted the mitogenic effect of oxLDL toward apoptosis, thus suggesting that the PI-3K/Akt pathway acts as a survival pathway. CONCLUSIONS: SMC proliferation elicited by moderate concentrations of oxLDL involves the sphingomyelin/ceramide/sphingosine-1-phosphate pathway, which leads to extracellular regulated kinase 1/2 activation and DNA synthesis, and the EGFR/PI-3K/Akt pathway, which prevents the apoptotic effect of oxLDL.