published_or_final_version / Medical Sciences / Master / Master of Medical Sciences

Leukocyte-specific protein 1 (LSP1), an F-actin binding protein, is involved in neutrophil recruitment into peritoneum. Because mechanisms of excessive migration of activated neutrophils into inflamed lungs, credited with tissue damage, are not fully understood, we explored the hitherto unknown expression and role of LSP1 in neutrophil migration in a mouse model of acute lung inflammation. We induced acute lung inflammation through intranasal E. coli lipopolysacharide (LPS) (80μg) in wild-type 129/SvJ (WT) and LSP1 deficient (LSP1-/-) mice. WT (n=10) and LSP1-/- (n=11) mice showed significant neutrophilia and more neutrophils in bronchoalveolar lavage (BAL) at 9 hour post-LPS challenge compared to respective saline-treated controls (WT=7; LSP1-/-=10). LPS treatment induced more BAL neutrophils (P<0.001), myeloperoxidase concentrations and Gr-1+ neutrophils in lung tissues in WT mice compared to LSP1-/- mice. Lung myeloperoxidase and Gr-1+ (P<0.05) were higher in LPS-treated WT compared to the LSP1-/- mice. Lung tissue and BAL fluid KC, MCP-1, MIP-1α and MIP-1β concentration and vascular permeability were not different between LPS-treated WT and LSP1-/- mice but TNF-α concentration was higher in LPS-treated WT mice. Hematoxylin and eosin staining showed more septal congestion in LPS-treated WT mice compared to LSP1-/- mice. LSP1 expression was increased in lungs from LPS-treated mice compared to saline control. The autopsied lungs from septic humans, compared to their respective controls, showed increased expression of LSP1. These data show that LSP1 expression is modulated in acute lung inflammation and that LSP1 deficiency reduces neutrophil migration into acute lung inflammation.

Είναι γνωστό ότι οι μεταγγίσεις ομόλογου αίματος ασκούν ανοσοκατασταλτική δράση, ευνοόντας την υποτροπή των καρκινικών όγκων και των μεταστάσεων. Επιπλέον η περιεγχειρητική αναιμία θεωρείται ανεξάρτητος προγνωστικός παράγων σε ασθενείς που υποβάλλονται σε ογκολογικές επεμβάσεις. Οι μείζονες θωρακικές επεμβάσεις, όπως αυτές για καρκίνο του πνεύμονα συνοδεύονται από αυξημένες απώλειες αίματος και ανάγκες για μετάγγιση οδηγώντας σε επακόλουθη αύξηση της νοσηρότητας και θνησιμότητας. Παράλληλα η ευεργετική επίδραση της απροτινίνης είναι ευρέως γνωστή όσον αφορά τις Καρδιοχειρουργικές επεμβάσεις. Σκοπό των μελετών μας αποτέλεσε, από τη μία η πιθανή επίδραση των μεταγγίσεων ομολόγου αίματος και της προεγχειρητικής αναιμίας στην απώτερη επιβίωση των ασθενών που υπεβλήθησαν σε εκτομή μη μικροκυτταρικού καρκίνου του πνεύμονα και από την άλλη η επίδραση της απροτινίνης στις ανάγκες για μετάγγιση και την αιμορραγική διάθεση, χορηγούμενη σε πολύ χαμηλή δόση σε μείζωνες πνευμονικές εκτομές. Κατά την πρώτη μας μελέτη, 331 ασθενείς (άνδρες/γυναίκες=295/36, μέσης ηλικίας 64 ± 9 έτη), οι οποίοι υπεβλήθησαν σε ριζική εκτομή μη μικροκυτταρικού καρκίνου πνεύμονα, εξετάσθηκαν προοπτικά. Ο μέσος χρόνος μετεγχειρητικής παρακολούθησης ήταν 27,2 μήνες. Η συνολική επιβίωση των ασθενών εξετάσθηκε συγκριτικά με την χορήγηση μεταγγίσεων ομολόγου αίματος και την περιεγχερητική αναιμία. Οι παράμετροι αυτοί εξετάσθηκαν αρχικά για τον συνολικό πληθυσμό ασθενών και εν συνεχεία ξεχωριστά για τους ασθενείς σταδίου I. Οι ασθενείς ταξινομήθηκαν ανάλογα με την χορήγηση περιεχειρητικής μετάγγισης αίματος στην Ομάδα Α (μεταγγιζόμενοι) και Ομάδα Β (μη μεταγγιζόμενοι) και ανάλογα με το επίπεδο της προεγχειρητικής αιμοσφαιρίνης (Hb) στην Ομάδα 1 (Hb<12 g/dl) και Ομάδα 2 (Hb≥12 g/dl) αντίστοιχα. Κατά τη δέυτερη μας μελέτη, μια υποομάδα 59 ασθενών (μέσης ηλικίας 58 ± 13.25 έτη) οι οποίοι υπεβλήθησαν σε μείζονα θωρακοχειρουργική επέμβαση ταξινομήθηκαν στην ομάδα ελέγχου (Ομάδα Α) και στην ομάδα απροτινίνης (Ομάδα Β). Η δεύτερη ομάδα (n=29) έλαβε κατά την εισαγωγή της αναισθησίας διεγχειρητικά 500.000 I.U απροτινίνης, ακολουθούμενη από μία δεύτερη ισόποση δόση αμέσως μετά το κλείσιμο της θωρακοτομής. Τα αποτελέσματά μας για την πρώτη μελέτη κατέγραψαν ποσοστό μετάγγισης 25,7%. Η μονοπαραγοντική ανάλυση για το σύνολο του πλυθησμού ανέδειξε συνολική επιβίωση μικρότερη στην Ομάδα Α (μεταγγιζόμενοι) (μέση επιβίωση 33.6 μήνες, 5-ετής επιβίωση 25.1%) σε σύγκριση με την Ομάδα B (μέση επιβίωση 48.0 μήνες, 5-ετής επιβίωση 37.3%) (p=0.001). Παρατηρήσαμε επίσης ότι οι ασθενείς με προεγχειρητική τιμή Hb<12 g/dl (Ομάδα 1) (μέση επιβίωση 33.0 μήνες, 5-ετής επιβίωση 21.3%), παρουσίασαν μικρότερη επβίωση συγκρινόμενοι με την Ομάδα 2 (μέση επιβίωση 49.3 μήνες, 5-ετής επιβίωση 40%) (p=0.002). Η πολυπαραγοντική ανάλυση του συνόλου των ασθενών ανέδειξε ότι η προεγχειρητική αναιμία αποτελεί ανεξάρτητο προγνωστικό παράγοντα για την επιβίωση, ενώ η μετάγγιση με μονάδες ομόλογου αίματος όχι. Παρατηρήσαμε επίσης κατά την πολυπαραγοντική ανάλυση ότι στους ασθενείς του σταδίου I, οι μεταγγίσεις ομόλογου αίματος αποτέλεσαν ανεξάρτητο προγνωστικό παράγοντα για την απώτερη επιβίωση, γεγονός που δεν επιβεβαιώθηκε για την προεγχειρητική αναιμία. Στην δεύτερη μελέτη μας, η μέση παροχή αιματηρού περιεχομένου που κατεγράφη από τους σωλήνες θώρακος κατά την 1η και 2η μετεγχειρητική ημέρα στην Ομάδα Β (απροτινίνης) ήταν σημαντικά ελαττωμένη (412.6±199.2 έναντι 764.3±213.9 ml, p<0.000 και 248.3±178.5 έναντι 455.0±274.6, p<0.001 αντίστοιχα). Παρομοίως, οι ανάγκες μετάγγισης με φρέσκο κατεψυγμένο πλάσμα ήταν λιγότερες στην Ομάδα της απροτινίνης. Επιπλέον, τόσο ο διεγχειρητικός χρόνος, όσο και η διάρκεια παραμονής στο νοσοκομείο ήταν χαμηλότερα υπέρ της Ομάδας Β, χωρίς όμως να αγγίζουν την στατιστική σημαντικότητα (84.6±35.2 έναντι 101.2±52.45 λεπτών και 5.8±1.6 έναντι 7.2±3.6 ημερών αντίστοιχα) (p<0.064). το συνολικό ποσοστό μετάγγισης δεν δέφερε σημαντικά ανάμεσα στις δύο ομάδες. Δεν παρατηρήθηκαν ανεπιθύμητες ενέργειες κατά τη χορήγηση της ουσίας. Τα συμπεράσματά μας συνιστούν ότι οι μεταγγίσεις ομολόγου αίματος επηρεάζουν αρνητικά την απώτερη επιβίωση των ασθενών με πρώιμο μη μικροκυτταρικό καρκίνο (σταδίου I) του πνεύμονα, χωρίς να παρατηρείται όμως η ίδια επίδραση στην επιβίωση αυτών με χειρουργικά εξαιρέσιμη πιο προχωρημένη νόσο. Τα ευρήματα αυτά καταδεικνύουν ότι οι μεταγγίσεις ομολόγου αίματος πιθανώς να ασκούν ανοσοτροποποιητική δράση στα πρώιμα στάδια της νόσου, ενώ για τα πιο προχωρημένα το φιανόμενο αυτό δεν είναι προφανές. Επιπλέον, η χορήγηση της απροτινίνης σε πολύ χαμηλή δόση, συνοδεύεται από ελάττωση των μετεγχειρητικών απωλειών αίματος και αναγκών για μετάγγιση των παραγώγων του αίματος. Με βάση τα δεδομένα αυτά θεωρούμε την χορήγηση της απροτινίνης ως ασφαλή και ευεργετική στις μείζονες θωρακοχειρουργικές επεμβάσεις. / It has been postulated that transfusions have immunosuppressive effects that promote tumor growth and metastasis. Moreover perioperative anaemia is considered an independent prognostic factor on outcome in patients operated for malignancy. Major thoracic operations (such as lung cancer resections) are associated with increased blood losses and transfusion requirements leading to increased mortality and morbidity. In addition, the blood saving effect of aprotinin has been well documented in cardiac surgery. The aims of our studies were to evaluate, on the one hand the possible influence of red blood cell (RBC) transfusions and perioperative anaemia on survival in patients operated for non-small cell lung carcinoma (NSCLC); and on the other hand, we have tested the influence of aprotinin using an ultra-low dose drug regime on major pulmonary operations concerning bleeding diathesis and need for transfusion. In our first study, 331 consecutive patients, male/female=295/36, (mean age 64±9 years), who underwent radical surgery for NSCLC were prospectively enrolled in this cohort and followed up for a mean of 27.2 months. The overall survival of patients was analyzed in relation to RBC transfusions and perioperative anemia. These parameters were analyzed in the whole cohort of patients and separately for stage I patients. Patients were divided according to perioperative transfusion, into Group A (transfused) and Group B (non-transfused) and according to the preoperative haemoglobin (Hb) level into Group 1 (Hb<12g/dl) and Group 2 (Hb≥12gr/dl) respectively. Furthermore in our second study, a subgroup of 59 patients, of mean age 58±13.25 years (mean±SD) undergoing general major thoracic procedures were randomized into placebo (Group A) and treatment–aprotinin group (Group B). The group B (n=29) received 500.000 IU of aprotinin after induction to anesthesia and a repeat dose immediately after chest closure. In our fisrt study, the overall transfusion rate was 25.7%. Univariate analysis showed that in the whole cohort of patients overall survival was significantly shorter in Group A (mean 33.6 months, 5-year survival 25.1%) compared to Group B (mean 48.0 months, 5-year survival 37.3%) (p=0.001). It also showed that patients with preoperative Hb level<12g/dl (Group 1), (mean of 33.0 months, 5-year survival 21.3%) had shorter survival compared to Group 2 patients (mean 49.3 months and 5-year survival 40.0%) respectively (p=0.002). Multivariate analysis in the whole cohort of patients showed that preoperative anemia was an independent risk factor for survival while RBC transfusion was not. In particular for stage I patients, it was shown that RBC transfusion was an independent prognostic factor for long-term survival as detected by multivariate analysis (p=0.043), while anemia was not. In our second study, the two groups were similar in terms of age, gender, diagnosis, pathology, comorbidity and operations performed. The mean drainage of the first and second postoperative day in group B was significantly reduced (412.6±199.2 vs. 764.3±213.9 ml, p<0.000, and 248.3±178.5 vs. 455.0±274.6, p<0.001). Similarly, the need for fresh frozen plasma transfusion was lower in group B, p<0.035. Both the operation time and the hospital stay were also less for group B but without reaching statistical significance (84.6±35.2 vs 101.2±52.45 min. and 5.8±1.6 vs 7.2±3.6 days respectively, p<0.064). The overall transfusion rate did not differ significantly. No side effects of aprotinin were noted Our conclusions suggested that RBC transfusions affect adversely the survival of stage I NSCLC patients, while do not exert any effect on survival of patients with surgically resectable more advanced disease, where preoperative anemia is an independent negative prognostic factor. These findings indicate that RBC transfusion might exert an immunomodulatory effect on patients with early disease while in more advanced stages this effect is not apparent. Additionally perioperative ultra-low dose aprotinin administration was associated with a reduction of total blood losses and blood product requirements. We therefore consider the use of aprotinin safe and effective in major thoracic surgery.

It has been suggested that some adult bone marrow cells (BMC) can localize to the injured tissues and develop tissue-specific characteristics including those of the pulmonary epithelium. In Chapter 2 we show that the combination of mild airway injury as a conditioning regimen to direct the site of BMC localization and transtracheal delivery of short-term cultured BMC enhances airway localization and adoption of an epithelial-like phenotype expressing Clara cell secretory protein (CCSP) and pro-surfactant protein-C. Bone marrow cells from transgenic mice expressing green fluorescent protein driven by the epithelial-specific cytokeratin-18 promoter were injected transtracheally into airway-injured wild-type recipients. BMC retention in the lung was observed to be at least 120 days following cell delivery with increasing transgene expression over time. The results indicate that targeted delivery of BMC can promote airway regeneration. Although bone marrow stem/progenitor cells can develop into lung epithelial cells, the specific subpopulation remains unknown. In Chapter 3 we identify a newly discovered population of murine and human BMC that express CCSP. These CCSP+ cells increase in the bone marrow and blood after airway injury and can be expanded in culture. CCSP+ cells are unique in that they express both hematopoietic and mesenchymal stromal cell markers and can give rise to various lung epithelial lineages in vitro. Importantly, bone marrow transplant of CCSP+ cells to CCSP knockout recipients confirms that bone marrow CCSP+ cells contribute to airway epithelium after airway injury. In Chapter 4 we enrich for a stem/progenitor cell population within the CCSP+ using the stem cell antigen (Sca)-1 as a marker. Here we identified a putative epithelial stem/progenitor cell that can be induced to differentiate into various lung epithelial cell lineages expressing markers exclusive to airway or alveolar epithelial cells when cultured under an air liquid interface. These cells also have self-renewal potential in vitro that can proliferate in vivo and repopulate the injured airway epithelium. This newly discovered epithelial-like cells may play a central role in the bone marrow contribution to lung repair and are exciting candidates for cell-based targeted therapy for treatment of lung diseases.

Rationale: Activation of the platelet-derived growth factor receptors-α and -β (PDGF-Rα and -Rβ) is critical in the formation of secondary crests/septa during alveologenesis, and its regulation has been found to be disrupted in rat lung injury models. Objective: To determine whether the process of secondary septation can occur after transient pharmacologic inhibition of PDGF-R action during postnatal days (P)1 – 7 in rats. Hypothesis: The initial process of secondary crest formation is time-limited and, if missed, will result in a permanent loss of alveoli. Methods: Imatinib mesylate, a PDGF-R inhibitor, was injected intraperitoneally from P1 – 7. Pups were sacrificed on P2, 4, 8, 14, 28 and 65 for studies of alveolar development. Main results: The injection of imatinib inhibited PDGF-R action, resulting in a permanent decrease in alveolar number in treated rats. Conclusions: Inhibition of secondary septation during the first 7 days of life resulted in a decrease in alveolar number lasting into early adult life. This is consistent with a critical time window for secondary septation, which, if disrupted, results in long-term adverse effects on lung development.

It has been suggested that some adult bone marrow cells (BMC) can localize to the injured tissues and develop tissue-specific characteristics including those of the pulmonary epithelium. In Chapter 2 we show that the combination of mild airway injury as a conditioning regimen to direct the site of BMC localization and transtracheal delivery of short-term cultured BMC enhances airway localization and adoption of an epithelial-like phenotype expressing Clara cell secretory protein (CCSP) and pro-surfactant protein-C. Bone marrow cells from transgenic mice expressing green fluorescent protein driven by the epithelial-specific cytokeratin-18 promoter were injected transtracheally into airway-injured wild-type recipients. BMC retention in the lung was observed to be at least 120 days following cell delivery with increasing transgene expression over time. The results indicate that targeted delivery of BMC can promote airway regeneration. Although bone marrow stem/progenitor cells can develop into lung epithelial cells, the specific subpopulation remains unknown. In Chapter 3 we identify a newly discovered population of murine and human BMC that express CCSP. These CCSP+ cells increase in the bone marrow and blood after airway injury and can be expanded in culture. CCSP+ cells are unique in that they express both hematopoietic and mesenchymal stromal cell markers and can give rise to various lung epithelial lineages in vitro. Importantly, bone marrow transplant of CCSP+ cells to CCSP knockout recipients confirms that bone marrow CCSP+ cells contribute to airway epithelium after airway injury. In Chapter 4 we enrich for a stem/progenitor cell population within the CCSP+ using the stem cell antigen (Sca)-1 as a marker. Here we identified a putative epithelial stem/progenitor cell that can be induced to differentiate into various lung epithelial cell lineages expressing markers exclusive to airway or alveolar epithelial cells when cultured under an air liquid interface. These cells also have self-renewal potential in vitro that can proliferate in vivo and repopulate the injured airway epithelium. This newly discovered epithelial-like cells may play a central role in the bone marrow contribution to lung repair and are exciting candidates for cell-based targeted therapy for treatment of lung diseases.

Rationale: Activation of the platelet-derived growth factor receptors-α and -β (PDGF-Rα and -Rβ) is critical in the formation of secondary crests/septa during alveologenesis, and its regulation has been found to be disrupted in rat lung injury models. Objective: To determine whether the process of secondary septation can occur after transient pharmacologic inhibition of PDGF-R action during postnatal days (P)1 – 7 in rats. Hypothesis: The initial process of secondary crest formation is time-limited and, if missed, will result in a permanent loss of alveoli. Methods: Imatinib mesylate, a PDGF-R inhibitor, was injected intraperitoneally from P1 – 7. Pups were sacrificed on P2, 4, 8, 14, 28 and 65 for studies of alveolar development. Main results: The injection of imatinib inhibited PDGF-R action, resulting in a permanent decrease in alveolar number in treated rats. Conclusions: Inhibition of secondary septation during the first 7 days of life resulted in a decrease in alveolar number lasting into early adult life. This is consistent with a critical time window for secondary septation, which, if disrupted, results in long-term adverse effects on lung development.

Mechanically ventilated patients are at risk of developing Ventilator-Associated Lung Injury (VALI). Improved ventilation strategies by lung-protective settings may cause hypercapnia. This study investigated whether attenuation of VALI is attributed to protective ventilation with low tidal volume (VT) or hypercapnia. Lung injury was induced in rats by instillation of 1.25M HCl. Ten rats each were ventilated for 4 hours with: Conventional Normocapnia (highVT), Lung-Protective Ventilation (VT¬ 8mL/Kg), Injurious Normocapnia (highVT, added dead space), Conventional Hypercapnia (highVT, inhaled CO2), Protective Hypercapnia (VT 8mL/Kg, inhaled CO2) and Permissive Hypercapnia (VT 8mL/Kg, hypoventilation). Lung-Protective Ventilation reduced pulmonary edema compared to Conventional and Injurious Normocapnia. Therapeutic hypercapnia reduced alveolar damage and inflammation by reducing IL-6 and MCP-1 in the lung, and IL-1? and TNF-? systemically. Therapeutic hypercapnia may be more effective in attenuating some of the biomarkers of VALI and protecting the lung than protective ventilation alone.

Cystic Fibrosis (CF) is caused by mutations in CFTR, a protein for chloride efflux in epithelial cells. VIP is a peptide that activates CFTR and improves membrane stability; VIP has 3 receptors VPAC1, VPAC2 and PAC1 that can cause CFTR phosphorylation. VIP-knockout (VIPKO) mice experience inflammation and reduced CFTR membrane localization comparable to CF phenotypes, that’s reversible after 3 weeks of VIP treatment (VIPKOT). In this thesis western blotting showed VPAC1 and VPAC2 expression increased in VIPKO and VIPKOT lung and duodenum tissues. The expression and maturation of CFTR was unchanged in both VIPKO and VIPKOT tissues. The results showed absence of VIP caused increased receptor expression in VIPKO mice, after VIP treatment VIPKO mice maintained increased receptor expression. VIP treatment reduces inflammation and restores existing CFTR membrane localization in VIPKO mice. VIP receptor expression may be important for future treatment of CF for CFTR localization and reducing tissue inflammation.

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