Factors affecting the respiratory system cell proliferation

Chronic airway inflammatory diseases appear with a different frequency and severity in people with different genders. In these diseases increased cholinergic transmission is observed, as well as release of inflammatory and growth factors in the airway. Chronic airway diseases are often characterized by remodeling of the airway wall, in which an increase in SMC mass is observed. In the treatment of some of these diseases macrolides are used, due to not only their antmicrobial and anti-inflammatory effect, but also to their direct effect on the airway cells. The aim of the present study was to examine the effect of gender hormones, muscarinic agonists, inflammatory and growth factors as well as the antibiotic azithromycin, on the airway SMCs. Rabbit tracheal and human bronchial SMCs were used. The proliferation of SMCs was estimated using the radioactive thymidine incorporation and the Cell Titer 96® AQueous One Solution Assay method, which estimates cell number. The cell viability and the number of lysosomes were estimated with the biological dyes Trypan blue and Lysotracker red respectively. The signal pathways that are activated, were studied using pathway inhibitors as well as Western blot analysis, while intracellular proteins were tracked by indirect immunofluorescence. Gender hormones increase the male and female adult rabbit tracheal SMC proliferation, within the first 24h and their effect is mediated by classic androgen receptors and activation of the PI3K and MAPK signal pathways. Muscarinic agonists increase only rabbit SMC proliferation and their effect seems to be mediated by M3 muscarinic receptors and the PI3K and MAPK signal pathways. TNFα, TGF, bFGF and PDGF act as mitogens in human bronchial SMCs and increase the radioactive thymidine incorporation in rabbit tracheal SMCs, through activation of the above mentioned pathways, but have no synergetic effect with muscarinic agonists. Azithromycin inhibits the rabbit and human airway SMC proliferation and causes autophagy. Its effect on rabbit tracheal SMCs was dose-dependent and time-dependent and did not lead the cells to apoptosis, while in human bronchial SMCs it decrease the cell number. The results of the present study show that airway SMCs react towards a variety of factors that are present in the airways in normal and pathological conditions. The nature, the intensity and the duration of the stimuli determines the cellular response which includes activation of signal pathways leading to proliferation or activation of procedures necessary for cell survival. The effect of these stimuli is mediated by binding to cellular receptors and activating the PI3K and MAPK signal pathways. The way the cells react is depended on their source as well as the culture conditions.Στις χρόνιες φλεγμονώδεις παθήσεις του αναπνευστικού εμφανίζεται διαφορά στη συχνότητα και τη βαρύτητα της νόσου σε άτομα διαφορετικού φύλου, αλλά και αυξημένη χολινεργική διαβίβαση, και απελευθέρωση φλεγμονωδών και αυξητικών παραγόντων. Οι χρόνιες νόσοι του αναπνευστικού συχνά χαρακτηρίζονται από αναδιαμόρφωση του τοιχώματος των αεραγωγών, στην οποία συμμετέχει και η αύξηση της μάζας των ΛΜΚ. Στην θεραπεία μερικών από αυτές τις παθήσεις χορηγούνται μακρολίδες, εξαιτίας όχι μόνο της αντιμικροβιακής και αντιφλεγμονώδους δράσης τους αλλά και λόγω της απευθείας επίδρασης τους στα κύτταρα των αεραγωγών. Σκοπός της παρούσας μελέτης ήταν να διερευνηθεί η επίδραση των γεννητικών ορμονών, των μουσκαρινικών αγωνιστών, φλεγμονωδών και αυξητικών παραγόντων και της αζιθρομυκίνης, στα ΛΜΚ των αεραγωγών. Χρησιμοποιήθηκαν ΛΜΚ τραχείας κουνελιού και βρόγχων ανθρώπου. Ο πολλαπλασιασμός εκτιμήθηκε με τη μέθοδο της ενσωμάτωσης ραδιενεργού θυμιδίνης σε νεοσυντιθέμενα μόρια DNA, και με τη μέθοδο Cell Titer 96® AQueous One Solution Assay, με την οποία εκτιμάται ο αριθμός των κυττάρων. Η βιωσιμότητα των κυττάρων και ο αριθμός των λυσοσωμάτων εκτιμήθηκαν με χρώσεις με Trypan blue και Lysotracker red, αντίστοιχα. Τα σηματοδοτικά μονοπάτια που ενεργοποιούνται, μελετήθηκαν με τη χρήση αναστολέων και με ανοσοαποτύπωση κατά Western, ενώ ενδοκυττάριες πρωτεΐνες εντοπίστηκαν με έμμεσο ανοσοφθορισμό. Οι γεννητικές ορμόνες επάγουν τον πολλαπλασιασμό ΛΜΚ τραχείας αρσενικού και θηλυκού κουνελιού τις πρώτες 24 ώρες της επώασης και η δράση τους μεσολαβείται μέσω των κλασικών υποδοχέων ανδρογόνων και των μονοπατιών της ΡΙ3Κ και των ΜΑΡΚ. Οι μουσκαρινικοί αγωνιστές επάγουν μόνο τον πολλαπλασιασμό των ΛΜΚ κουνελιού και η δράση τους φαίνεται να μεσολαβείται μέσω Μ3 μουσκαρινικών υποδοχέων και των μονοπατιών της ΡΙ3Κ και των ΜΑΡΚ. Οι παράγοντες TNFα, TGF, bFGF και PDGF έχουν μιτογόνο δράση σε ΛΜΚ βρόγχων ανθρώπου και επάγουν την ενσωμάτωση ραδιενέργειας από ΛΜΚ τραχείας κουνελιού, μέσω ενεργοποίησης των παραπάνω μονοπατιών, αλλά δεν εμφανίζουν συνεργική δράση με τους μουσκαρινικούς αγωνιστές. Η αζιθρομυκίνη ελάττωσε τον πολλαπλασιασμό και προκάλεσε αυτοφαγία σε ΛΜΚ αεραγωγών κουνελιού και ανθρώπου. Η δράση της στα ΛΜΚ τραχείας κουνελιού ήταν δοσοεξαρτώμενη, χρονοεξαρτώμενη και δεν οδήγησε σε απόπτωση, ενώ στα ΛΜΚ βρόγχων ανθρώπου ελάττωσε τον αριθμό των κυττάρων. Τα αποτελέσματα της παρούσας μελέτης δείχνουν ότι τα ΛΜΚ των αεραγωγών αντιδρούν απέναντι σε μια πληθώρα παραγόντων, οι οποίοι βρίσκονται στους αεραγωγούς σε φυσιολογικές αλλά και παθολογικές καταστάσεις. Η φύση, η ένταση και η διάρκεια του ερεθίσματος καθορίζει την κυτταρική απόκριση που περιλαμβάνει ενεργοποίηση σηματοδοτικών μονοπατιών που οδηγούν το κύτταρο σε πολλαπλασιασμό ή σε ενεργοποίηση διαδικασιών απαραίτητων για την επιβίωσή του. Ο τρόπος με τον οποίο αντιδρούν τα κύτταρα εξαρτάται τόσο από την προέλευση τους αλλά και από τις συνθήκες της καλλιέργειας

Camphene as a Protective Agent in Myocardial Ischemia/Reperfusion Injury

Myocardial ischemia/reperfusion injury (I/R) and the resulting heart failure is one of the main causes of mortality and morbidity worldwide. Camphene has been shown to have anti-inflammatory and hypolipidemic properties; however, its role in the protection of the heart from ischemia and reperfusion has not been investigated. The cardioprotective role of camphene and the mechanism that mediates its action against I/R injury was evaluated in the present study. A single dose of camphene was administered in adult rats prior to ex vivo I/R induction. Infarct size was measured using 2,3,5-triphenyltetrazolium chloride (TTC) staining and cardiomyocyte injury was assessed by determining the release of the enzyme lactate dehydrogenase (LDH). Camphene pretreatment provided significant protection reducing myocardial infarct size and cell death after I/R. The effect was correlated with the reduction in oxidative stress as evidenced by the determination of protein carbonylation, GSH/GSSG ratio, the increase in mitochondrial content as determined by CS activity, and the modulation of antioxidant defense mechanisms (expression of Nrf2 and target genes and activities of CAT, MnSOD, and GR). Furthermore, ferroptosis was decreased, as demonstrated by downregulation of GPx4 expression and reduction in lipid peroxidation. The results suggest that camphene can protect the heart against I/R injury by maintaining redox homeostasis and can hold therapeutic potential for mitigating the detrimental effects of I/R in the heart

Critical-Illness: Combined Effects of Colistin and Vasoactive Drugs: A Pilot Study

Colistin is often used as a last resort for treating multidrug-resistant infections, particularly in critically ill patients in intensive care units. Nonetheless, its side effects, including myopathy, require careful monitoring. Vasoconstrictive drugs are also used in intensive care to increase blood pressure and improve blood flow to vital organs, which can be compromised in critically ill patients. The exact mechanism of colistin-induced muscle toxicity is of significant interest due to its potential intensive-care clinical implications. Colistin alone or in combination with vasoconstrictive agents was administrated in non-septic and LPS-induced septic animals for 10 days. Histopathological evaluation of the gastrocnemius muscle and dot-blot protein tissue analysis were performed. Increased intramuscular area, de-organization of the muscle fibers and signs of myopathy were observed in colistin-treated animals. This effect was ameliorated in the presence of vasoconstrictive drugs. Administration of colistin to septic animals resulted in a decrease of AMPK and cyclin-D1 levels, while it had no effect on caspase 3 levels. Vasoconstrictive drugs’ administration reversed the effects of colistin on AMPK and cyclin D1 levels. Colistin’s effects on muscle depend on septic state and vasoconstriction presence, highlighting the need to consider these factors when administering it in critically ill patients

Long-term exposure to muscarinic agonists decreases expression of contractile proteins and responsiveness of rabbit tracheal smooth muscle cells

Background: Chronic airway diseases, like asthma or COPD, are characterized by excessive acetylcholine release and airway remodeling. The aim of this study was to investigate the long-term effect of muscarinic agonists on the phenotype and proliferation of rabbit tracheal airway smooth muscle cells (ASMCs). Methods: ASMCs were serum starved before treatment with muscarinic agonists. Cell phenotype was studied by optical microscopy and indirect immunofluorescence, using smooth muscle a-actin, desmin and SM-Myosin Heavy Chain (SM-MHC) antibodies. [N-methyl-H-3] scopolamine binding studies were performed in order to assess M-3 muscarinic receptor expression on isolated cell membranes. Contractility studies were performed on isolated ASMCs treated with muscarinic agonists. Proliferation was estimated using methyl-[3H] thymidine incorporation, MTT or cell counting methods. Involvement of PI3K and MAPK signalling pathways was studied by cell incubation with the pathway inhibitors LY294002 and PD98059 respectively. Results: Prolonged culture of ASMCs with acetylcholine, carbachol or FBS, reduced the expression of a-actin, desmin and SM-MHC compared to cells cultured in serum free medium. Treatment of ASMCs with muscarinic agonists for 3-15 days decreased muscarinic receptor expression and their responsiveness to muscarinic stimulation. Acetylcholine and carbachol induced DNA synthesis and increased cell number, of ASMCs that had acquired a contractile phenotype by 7 day serum starvation. This effect was mediated via a PI3K and MAPK dependent mechanism. Conclusions: Prolonged exposure of rabbit ASMCs to muscarinic agonists decreases the expression of smooth muscle specific marker proteins, down-regulates muscarinic receptors and decreases ASMC contractile responsiveness. Muscarinic agonists are mitogenic, via the PI3K and MAPK signalling pathways