Αξιολόγηση μοριακής δράσης ανασυσταμένης HDL, που περιέχει ανθρώπινες απολιποπρωτεΐνες apoA-I ή apoE3 αγρίου τύπου, στα ανθρώπινα ενδοθηλιακά κύτταρα αορτής και ειδικότερα στην έκφραση miRNAs

Η αθηροσκλήρωση είναι η κύρια αιτία καρδιαγγειακής νόσου παγκοσμίως. Οι διαθέσιμες όμως θεραπείες παρουσιάζουν σημαντικούς περιορισμούς ως προς την αποτελεσματικότητα ή/και την ασφάλειά τους. Οι διαταραχές της ακεραιότητας και της λειτουργίας του ενδοθηλίου διαδραματίζουν κεντρικό ρόλο σε όλα τα στάδια της αθηροσκλήρωσης. Ο πολλαπλασιασμός και η μετανάστευση των ενδοθηλιακών κυττάρων (ECs) αποκαθιστούν την ακεραιότητα του ενδοθηλίου, ενώ η διατήρηση της ομοιόστασης της χοληστερόλης είναι απαραίτητη για την ομαλή λειτουργία του. Αν και έχει δειχθεί in vivo πως η χορήγηση ανασυσταμένης HDL (rHDL) λειτουργεί αθηροπροστατευτικά αυξάνοντας την ανάστροφη ροή χοληστερόλης και η επίδρασή των rHDL apoA-I και apoE3 στη γονιδιακή έκφραση των ECs έχει αποτελέσει αντικείμενο προηγούμενης μελέτης της ομάδας μας, η πιθανή επίδραση τους στην έκφραση miRNAs των ECs δεν έχει ακόμα μελετηθεί. Σκοπός της παρούσας εργασίας ήταν ο εντοπισμός πιθανών αθηροπροστατευτικών επιδράσεων των συγκεκριμένων τύπων rHDL στα ECs και ο χαρακτηρισμός των ενδεχόμενων ρόλων των miRNAs στους μηχανισμούς μέσω των οποίων ασκούνται. Χρησιμοποιώντας μικροσυστοιχίες oλιγονουκλεοτιδίων (GeneChip™ miRNA 2.0 Array, Affymetrix) που αξιολογούν αλλαγές έκφρασης όλων των γνωστών miRNAs, μελετήθηκαν φυσιολογικά ανθρώπινα ECs αορτής μετά από έκθεση στους δύο τύπους rHDL. Μετά από λεπτομερή βιοπληροφορική ανάλυση των αποτελεσμάτων των μικροσυστοιχιών με κριτήρια ανάλυσης την αλλαγή έκφρασης με λόγο (fold change, FC) ≥ ±1,5 και πιθανότητα λάθους (false discovery rate, FDR) ≤ 0,05 παρατηρήθηκαν αλλαγές σε 8 miRNAs. Τα miR-3188, miR-3185, miR-1231, miR-3195, miR-2861, miR-1915 και miR-638 υποεκφράζονταν σε στατιστικά σημαντικό βαθμό μετά τη χορήγηση rHDL-apoΑ-Ι και το miR-503 μετά τη χορήγηση rHDL-apoE3. Δεν βρέθηκαν miRNAs με στατιστικά σημαντική υπερέκφραση. Ακολούθησε η αναζήτηση πειραματικά επιβεβαιωμένων και η πρόβλεψη πιθανών στόχων για το καθένα. Ανάλυση με το πρόγραμμα Ingenuity Pathway Analysis έδειξε πως τόσο τα miRNAs όσο και οι στόχοι τους, εμπλέκονται σε εύρος κυτταρικών λειτουργιών, κάποιες από τις οποίες σχετίζονται άμεσα με τη λειτουργία των ECs και ενδεχομένως με την αθηροσκλήρωση. Ο λειτουργικός χαρακτηρισμός των στόχων των miRNAs έδειξε ότι στους στόχους των mir-3185 και miR-2861 περιλαμβάνονται σημαντικά γονίδια του μεταβολισμού χοληστερόλης, ενώ οι στόχοι του miR-503 περιλαμβάνουν σημαντικούς ρυθμιστές της αγγειογένεσης. Βιβλιογραφικά βρέθηκε ότι το miR-503 είναι ρυθμιστής της αγγειογένεσης. Η υπερέκφρασή του την εμποδίζει, μειώνοντας τον πολλαπλασιασμό και τη μετανάστευση των ECs, ενώ η αναστολή του έχει τα αντίθετα αποτελέσματα. Ανάμεσα στους στόχους του βρίσκονται γονίδια με σημαντική επίδραση στον πολλαπλασιασμό (EFNB2, FLT1, NR4A1, ADAMTS4) και τη μετανάστευση (EFNB2, FLT1, PTGIR, COL4A1) των ECs. Ο συνδυασμός με τα δεδομένα της προηγούμενης μελέτης γονιδιακής έκφρασης υποδεικνύει ότι η rHDL-apoE3 θα μπορούσε να επάγει τον πολλαπλασιασμό των ECs μέσω ενός άξονα rHDL-apoE3 – miR-503↓ – mRNA στόχων↑. Αν και μέχρι στιγμής δεν έχει αναφερθεί κάποιος ρόλος για τα miR-3185 και miR-2861 στη ρύθμιση του μεταβολισμού της χοληστερόλης, ανάμεσα στους πιθανούς στόχους τους βρίσκονται γονίδια σημαντικά για όλες σχεδόν τις λειτουργίες του. Από αυτά, τα HMGCR, MVK και CYP51A1 κωδικοποιούν για ένζυμα που συμμετέχουν άμεσα στο μονοπάτι βιοσύνθεσης χοληστερόλης. Ο συνδυασμός με τα δεδομένα της προηγούμενης μελέτης υποδεικνύει την πιθανή ύπαρξη ενός άξονα της μορφής rHDL-apoA-I (ή η εκροή χοληστερόλης που προκαλεί) – miR-3185 και miR-2861↓ – mRNA των HMGCR, MVK και CYP51A1↑ – επαγωγή της βιοσύνθεσης χοληστερόλης. Συμπερασματικά, η παρούσα μελέτη είναι η πρώτη που εξετάζει την επίδραση των rHDL-apoA-I και rHDL-apoE3 στην έκφραση miRNAs από φυσιολογικά ΗΑΕCs καθώς και το ρόλο των miRNAs στις πιθανές αθηροπροστατευτικές δράσεις της rHDL. Το miR-503 έχει τη δυνατότητα να επάγει τον πολλαπλασιασμό και τη μετανάστευση των ECs, συμβάλλοντας στην αποκατάσταση της ενδοθηλιακής συνέχειας, ενώ τα miR-3185 και miR-2861, μπορούν να επάγουν την ελεγχόμενη βιοσύνθεση χοληστερόλης, συμβάλλοντας στη διατήρηση της ομοιόστασής της στα ECs. Μέσω αυτών των δράσεων τα miR-3185, miR-2861 και miR-503 έχουν τη δυνατότητα να διαμεσολαβούν αθηροπροστατευτικές επιδράσεις των rHDL-apoA-I και rHDL-apoE3 στα ECs, αποτελώντας πολλά υποσχόμενους θεραπευτικούς στόχους, που αξίζει να μελετηθούν σε μεγαλύτερο βάθος και περισσότερα επίπεδα, ώστε να τεθούν οι βάσεις για την περαιτέρω αξιοποίησή τους στο χώρο της πρόληψης και αντιμετώπισης της αθηροσκλήρωσης.Despite atherosclerosis being the leading cause of cardiovascular disease worldwide, there is no available treatment without important effectivity and/or safety issues. Damage of the endothelial monolayer integrity is an important initiating event in the pathogenesis of atherosclerosis; however increased endothelial cell (EC) proliferation and migration can restore its integrity following vascular injury. Furthermore, the maintenance of cholesterol homeostasis is essential for EC function and its perturbations can contribute to the emergence of several pathologies. Although it has been shown that reconstituted HDL (rHDL) administration is atheroprotective, partly due to the increased reverse cholesterol transport it mediates and a previous study of our group has studied the effects of rHDL particles containing either apoA-I or apoE3 on EC gene expression, their effect on miRNA expression are still unknown. The aim of this study was to identify potential atheroprotective effects of discoid rHDL particles containing human wild type apoA-I or apoE3 and phospholipids and characterize the potential roles of miRNAs in the mechanisms by which these could be exercised. We studied normal human aortic ECs after exposure to the two types of rHDL, using oligonucleotide miRNA expression microarrays (GeneChip™ miRNA 2.0 Array Affymetrix). Multilevel bioinformatical analysis using 1.5-fold and ≤ 0.05 false discovery rate thresholds identified 8 significantly changed miRNAs: miR-3188, miR-3185, miR-1231, miR-3195, miR-2861, miR-1915 and miR-638 were under-expressed after rHDL-apoA-I treatment and so was miR-503 after the rHDL-apoE3 one. There were no significantly changed miRNAs over-expressed. Next we identified the experimentally validated and predicted targets for each one of the 8 miRNAs. Ingenuity Pathway Analysis indicated that both the miRNAs and their targets affect several cellular functions, some of which could affect EC function and atherosclerosis. The in depth functional characterization of the predicted target genes of miR-3185 and miR-2861, revealed that some of them are key cholesterol biosynthesis genes, while the targets of miR-503 include key genes regulating angiogenesis. PubMed analysis showed that miR-503 is an important regulator of angiogenesis; its over-expression inhibits it by reducing EC proliferation and migration, while its inhibition has the opposite effects. Furthermore, genes with significant effects on EC proliferation (EFNB2, FLT1, NR4A1, and ADAMTS4) and migration (EFNB2, FLT1, PTGIR and COL4A1) can be found among its validated and predicted target genes. Combination of the above with data from the preceding gene expression study, allows us to make the assumption that rHDL-apoE3 can theoretically induce EC proliferation through an rHDL-apoE3 – miR-503 expression reduction – target mRNA increase, axis. Although, there are no reports indicating that miR-3185 and miR-2861 have a part in cholesterol metabolism regulation so far, several genes participating in almost every part of it, could be found among their predicted targets. Of these, HMGCR, MVK and CYP51A1 code for enzymes that directly participate in the cholesterol biosynthesis pathway. Again, combination of the above with data from the preceding gene expression study suggest the existence of an axis were rHDL-apoA-I itself or the cholesterol efflux it causes, down-regulates miR- viii 3185 and miR -2861, up-regulating HMGCR, MVK and CYP51A1 expression leading to an increase in cholesterol biosynthesis. In conclusion the present study is the first to examine the effects of rHDL-apoA-I and rHDL-apoE3 on EC miRNA expression, as well as the role of the affected miRNAs in mediating the probable atheroprotective effects of rHDL on ECs. Through this study we found that miR-503 has the potential to induce the proliferation and migration of ECs, contributing to the restoration of endothelial integrity, whereas miR-3185 and miR-2861 could potentially induce cholesterol biosynthesis in a controlled manner, contributing to the maintenance of EC cholesterol homeostasis. Through these effects, miR-3185, miR-2861 and miR-503 could mediate potentially atheroprotective rHDL-apoA-I and rHDL-apoE3 functions. This makes rHDL in general and these miRNAs in particular possible therapeutic targets worth exploring more deeply, since the deeper understanding of their effects on the endothelium at the cellular and molecular level promises to facilitate the development of new therapies to prevent from or treat atherosclerosis

BioFire (R) FilmArray (R) Pneumonia Panel for Severe Lower Respiratory Tract Infections: Subgroup Analysis of a Randomized Clinical Trial

Introduction: The epidemiology of severe lower respiratory tract infections (LRTI) is constantly changing. We aimed to describe it using the BioFire (R) FilmArray (R) Pneumonia plus (PNplus) Panel. Methods: In a sub-study of the PROGRESS trial, sputum samples of 90 patients with sepsis and LRTI were retrospectively studied. The primary endpoint was the comparative detection rate of pathogens between conventional microbiology and PNplus Panel; secondary endpoints were microbiology and the association with the inflammatory host response. Results: Fifty-six patients with community-acquired pneumonia without risk factors for multidrug-resistant (MDR) pathogens and another 34 patients with risk factors for MDR were studied; median pneumonia severity index (PSI) was 113 (88-135). PNplus detection rate was 72.2% compared to 10% by conventional microbiology (p < 0.001); Streptococcus pneumoniae was the most common pathogen. PSI and procalcitonin were greater among patients with bacterial pathogens than viral pathogens. Median procalcitonin was 0.49 ng/ml and 0.18 ng/ml among patients with >= 10(5) and < 10(5) copies/ml of detected bacteria, respectively (p = 0.004). Resistance reached 14.4%. Conclusion: PNplus detects severe pneumonia pathogens at a greater rate than conventional microbiology. High levels of inflammation accompany bacterial detection

Early Start of Oral Clarithromycin Is Associated with Better Outcome in COVID-19 of Moderate Severity: The ACHIEVE Open-Label Single-Arm Trial

Introduction The anti-inflammatory effect of macrolides prompted the study of oral clarithromycin in moderate COVID-19. Methods An open-label non-randomized trial in 90 patients with COVID-19 of moderate severity was conducted between May and October 2020. The primary endpoint was defined at the end of treatment (EOT) as no need for hospital re-admission and no progression into lower respiratory tract infection (LRTI) for patients with upper respiratory tract infection and as at least 50% decrease of the respiratory symptoms score without progression into severe respiratory failure (SRF) for patients with LRTI. Viral load, biomarkers, the function of mononuclear cells and safety were assessed. Results The primary endpoint was attained in 86.7% of patients treated with clarithromycin (95% CIs 78.1-92.2%); this was 91.7% and 81.4% among patients starting clarithromycin the first 5 days from symptoms onset or later (odds ratio after multivariate analysis 6.62; p 0.030). The responses were better for patients infected by non-B1.1 variants. Clarithromycin use was associated with decreases in circulating C-reactive protein, tumour necrosis factor-alpha and interleukin (IL)-6; by increase of production of interferon-gamma and decrease of production of interleukin-6 by mononuclear cells; and by suppression of SARS-CoV-2 viral load. No safety concerns were reported. Conclusions Early clarithromycin treatment provides most of the clinical improvement in moderate COVID-19

Early treatment of COVID-19 with anakinra guided by soluble urokinase plasminogen receptor plasma levels: a double-blind, randomized controlled phase 3 trial

Early increase of soluble urokinase plasminogen activator receptor (suPAR) serum levels is indicative of increased risk of progression of coronavirus disease 2019 (COVID-19) to respiratory failure. The SAVE-MORE double-blind, randomized controlled trial evaluated the efficacy and safety of anakinra, an IL-1 alpha/beta inhibitor, in 594 patients with COVID-19 at risk of progressing to respiratory failure as identified by plasma suPAR >= 6 ng ml(-1), 85.9% (n = 510) of whom were receiving dexamethasone. At day 28, the adjusted proportional odds of having a worse clinical status (assessed by the 11-point World Health Organization Clinical Progression Scale (WHO-CPS)) with anakinra, as compared to placebo, was 0.36 (95% confidence interval 0.26-0.50). The median WHO-CPS decrease on day 28 from baseline in the placebo and anakinra groups was 3 and 4 points, respectively (odds ratio (OR) = 0.40, P < 0.0001); the respective median decrease of Sequential Organ Failure Assessment (SOFA) score on day 7 from baseline was 0 and 1 points (OR = 0.63, P = 0.004). Twenty-eight-day mortality decreased (hazard ratio = 0.45, P = 0.045), and hospital stay was shorter.The SAVE-MORE phase 3 study demonstrates the efficacy of anakinra, an IL-1 alpha/beta inhibitor, in patients with COVID-19 and high serum levels of soluble plasminogen activator receptor

Early treatment of COVID-19 with anakinra guided by soluble urokinase plasminogen receptor plasma levels: a double-blind, randomized controlled phase 3 trial

Early increase of soluble urokinase plasminogen activator receptor (suPAR) serum levels is indicative of increased risk of progression of coronavirus disease 2019 (COVID-19) to respiratory failure. The SAVE-MORE double-blind, randomized controlled trial evaluated the efficacy and safety of anakinra, an IL-1 alpha/beta inhibitor, in 594 patients with COVID-19 at risk of progressing to respiratory failure as identified by plasma suPAR >= 6 ng ml(-1), 85.9% (n = 510) of whom were receiving dexamethasone. At day 28, the adjusted proportional odds of having a worse clinical status (assessed by the 11-point World Health Organization Clinical Progression Scale (WHO-CPS)) with anakinra, as compared to placebo, was 0.36 (95% confidence interval 0.26-0.50). The median WHO-CPS decrease on day 28 from baseline in the placebo and anakinra groups was 3 and 4 points, respectively (odds ratio (OR) = 0.40, P < 0.0001); the respective median decrease of Sequential Organ Failure Assessment (SOFA) score on day 7 from baseline was 0 and 1 points (OR = 0.63, P = 0.004). Twenty-eight-day mortality decreased (hazard ratio = 0.45, P = 0.045), and hospital stay was shorter. The SAVE-MORE phase 3 study demonstrates the efficacy of anakinra, an IL-1 alpha/beta inhibitor, in patients with COVID-19 and high serum levels of soluble plasminogen activator receptor

Effect of anakinra on mortality in patients with COVID-19: a systematic review and patient-level meta-analysis

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