Inhaled nitric oxide does not prevent postpneumonectomy pulmonary edema in pigs.

OBJECTIVE: Increase in lung permeability is an inevitable consequence of pneumonectomy in relation to inflammatory injury and increased perfusion flow. We tested whether inhaled nitric oxide, a potent vasodilatator and anti-inflammatory agent, prevents postpneumonectomy edema in the first 24 hours after pneumonectomy in pigs. METHODS: We assessed hemodynamics, gas exchange, extravascular lung water estimated with the double-indicator dilution method, and lung neutrophil sequestration measured on the basis of lung myeloperoxidase activity at 1 and 24 hours after left pneumonectomy in 14 pigs randomly assigned to inhaled nitric oxide (10 ppm) or control groups. RESULTS: Extravascular lung water content markedly increased at 1 and 24 hours after pneumonectomy, with no difference between the 2 groups. Hemodynamics did not differ between the 2 groups. Myeloperoxidase activity was higher and PaO2 values were lower in the nitric oxide group compared with in the control group. CONCLUSIONS: Over the 24 hours after pneumonectomy, intraoperative inhaled nitric oxide levels neither improved gas exchange nor attenuated accumulation of lung water. On the contrary, they were associated with an increase in lung neutrophil sequestration and deterioration of arterial oxygenation, suggesting the occurrence of an early and toxic effect of nitric oxide.

Role of E-selectin in cell apoptosis induced by allogeneic blood perfusion in isolated mouse lung.

BACKGROUND: In a model of mouse isolated lung, we have recently demonstrated that E-selectin is involved in the activation of endothelial cells induced by allogeneic blood perfusion. In the present study, we explored the signaling pathway of apoptosis induced by E-selectin triggering. METHODS: Lungs were perfused for 3 hours with fresh blood in the absence or presence of an anti-E-selectin monoclonal antibody, or a protein kinase C (PKC), protein tyrosine phosphatase (PTP), or protein tyrosine kinase (PTK) inhibitor. The number of apoptotic cells in lung sections was determined by a TUNEL method. mRNAs for Fas, FasL and caspase-8, and for Bad, Bax, Bcl-w, Bcl-xL and caspase-9, for the FasL and the mitochondrial cytochrome-c pathways of apoptosis, respectively, and mRNA for the effector caspase-3 were quantified in lung tissues by RT-PCR. PTP and Src-PTK activities were also measured. RESULTS: After 3 hours of allogeneic perfusion, we observed a significant increase in: 1) the number of apoptotic cells in lung sections, 2) mRNA levels of FasL, Bcl-xL, caspase-8 and caspase-3, and 3) PTP activity (P < 0.05 compared with isogeneic perfusion). Surprisingly, mRNA levels of the proapoptotic genes Bad and Bax were significantly decreased (P < 0.05). PTK activity and caspase-9 mRNA level were not affected. Blocking anti-E-selectin mAbs and inhibitors for PKC, PTP, and PTK resulted in a significant reduction of apoptosis. CONCLUSIONS: In our model, the engagement of E-selectin induced by endothelial cell allogeneic activation appeared to be a prerequisite for lung apoptosis, which involved FasL and increase of PTP activity. Blockade of apoptosis with selective inhibitors may be a promising approach to the treatment/prevention of lung graft injury.

E-selectin early overexpression induced by allogeneic activation in isolated mouse lung.

BACKGROUND: The interaction between host lymphocytes and graft endothelial cells plays an important role in graft rejection. METHODS: Using our model of isolated ventilated lung from female mouse perfused with fresh blood from either isogeneic or allogeneic male mouse for 3 hours without noticeable ischemia, we have investigated the kinetics of the early events after endothelial cell triggering by E-selectin engagement. RESULTS: Isogeneic perfusion induced nonspecific endothelial cell activation, which was characterized by up-regulation of E-selectin, intercellular adhesion molecule (ICAM)-1, and of the pro-inflammatory cytokines tumor necrosis factor (TNF)-alpha, interleukin (IL)-2, and lymphotoxin-alpha (mRNAs by real-time polymerase chain reaction). Allogeneic perfusion was characterized after 3 hours by an additional loose adhesion of lymphocytes mediated by the E-selectin and related to the allogeneic activation of endothelial cells. These in turn expressed the I-A molecule (immunostaining). ICAM-1 and lymphocyte function-associated antigen (LFA)-3 mRNA levels were significantly increased in lung extracts after 2 hours, then vascular cell adhesion molecule (VCAM)-1 and TNF-alpha mRNAs after 3 hours without evidence of TNF-alpha production (enzyme-linked immunoadsorbent assay). The major participation of the E-selectin in early allogeneic activation by way of the protein kinase (PK)C pathway was confirmed by using a neutralizing anti-CD62E monoclonal antibody or the inhibitory PKC 19-31 fragment. CONCLUSIONS: Altogether, our results demonstrate that E-selectin expression (1) is not a consequence of TNF-alpha triggering, (2) up-regulates its own expression and expression of I-A, VCAM-1, TNF-alpha, and lymphotoxin-alpha mRNAs, and (3) down-regulates expression of LFA-3 and ICAM-1 mRNAs. In conclusion, in our physiologic model, the E-selectin highly participates in the loose adhesion of allogeneic lymphocytes and in the early activation of endothelial cell and therefore in structural and functional lung alterations.

Heterotopic en bloc tracheobronchial transplantation with direct revascularization in pigs.

OBJECTIVE: This article describes the application of a novel aortic tube technique for directly revascularized tracheobronchial transplantation with dual blood supply in pigs. METHODS: Eleven adult Large White pigs underwent heterotopic tracheal transplantation with a dual revascularization technique (inferior thyroid artery and bronchial artery). Seven tracheobronchial grafts were perfused ex vivo, and hemodynamic data were collected. RESULTS: At the last evaluation, 6 pigs had normally epithelialized mucus-producing allografts with correct morphologic conformation and cartilage viability. The histopathologic examination revealed homogeneous tissue regardless of biopsy site (trachea, carina, or bronchi), demonstrating the efficacy of the revascularization procedure. Four animals had early ischemic necrosis develop, 2 from acute rejection and 2 from technical mishap. One additional pig had acute rejection starting on the 14th postoperative day. The CD4(+)/CD8(+) ratio was maintained close to or above 0.8 in the subgroup with rejection and below 0.6 in the animals that were correctly immunosuppressed. Pressure-flow curves in 7 ex vivo tracheobronchial grafts showed a nonsignificant difference (P <.12) in vascular resistance between the bronchial artery territory (lower resistance) and the inferior thyroid artery territory. CONCLUSIONS: For the first time, a transplantation technique encompassing the entire trachea, carina, and stem bronchi has been made possible. By means of the dual inferior thyroid and bronchial artery axis, we were able to obtain a structurally healthy and functional graft to replace the main airway.

Regression of postobstructive vasculopathy after revascularization of chronically obstructed pulmonary artery.

OBJECTIVES: Pulmonary vascular resistance decreases dramatically after pulmonary thromboendarterectomy and further improves in time. This may reflect the slow regression of postobstructive pulmonary vasculopathy. We hypothesized that postobstructive pulmonary vasculopathy may regress after reperfusion in a piglet model of chronic (5 weeks) left pulmonary artery obstruction. METHODS: The ligated left pulmonary artery was reimplanted into the pulmonary arterial trunk. Pulmonary artery blood flow and pressure were measured 2 days and 5 weeks after reperfusion. Pulmonary artery smooth muscle thickness, endothelium-dependent relaxation, and left lung endothelial nitric oxide synthase activity and expression were assessed 5 weeks after ligation (n = 10) and 5 weeks after reperfusion (n = 10), and compared with a sham group (n = 10). Patency of the anastomoses and systemic blood supply to the lung were assessed by pulmonary angiography and nonselective thoracic aortography, respectively. RESULTS: Angiography showed that pulmonary artery anastomoses were patent in all animals. Five weeks after reperfusion, left pulmonary blood flows were similar to those in the sham animals, and systemic blood supply to the left lung decreased. Left pulmonary vascular resistance decreased by 50% at 5 weeks after reperfusion compared with 2 days after reperfusion (P =.0009). Medial muscle thickness of the left pulmonary artery greater than 600 microm increased 5 weeks after ligation and regressed to sham values 5 weeks after reperfusion (P =.001). Endothelium-dependent relaxation was only partially restored 5 weeks after reperfusion, whereas left lung endothelial nitric oxide synthase expressions and activities returned to sham values. CONCLUSIONS: This study shows that postobstructive pulmonary vasculopathy induced by ligation of the pulmonary artery for 5 weeks regresses after reperfusion, accounting for the progressive improvement in hemodynamics after thromboendarterectomy.

Induction of pulmonary angiogenesis by adenoviral-mediated gene transfer of vascular endothelial growth factor.

BACKGROUND: We hypothesized that gene transfer of vascular endothelial growth factor (VEGF) mediated by an adenovirus vector might induce pulmonary artery angiogenesis in a lamb model of pulmonary artery hypoplasia. METHODS: Thirteen fetal lambs had left pulmonary artery banding at 106 days of gestation. Following birth, 3 groups were divided: VEGF group (n = 5) and beta-GAL group (n = 4) received an adenoviral vector encoding respectively for human VEGF165 and for galactosidase A. A control group (n = 4) had neither gene nor virus. Viral suspensions were selectively instilled in the left bronchus 6.5 days after birth. Five nonoperated lambs constituted the normal group. Euthanasia was performed at 30 days of age. Gene transfer was confirmed by blue coloration of left lung obtained with Xgal solution in an additional experiment. Histomorphometric evaluation was performed. All groups were compared with ANOVA test and paired test was used to compare right and left lung in each animal. RESULTS: Left lung was similarly hypoplastic in all operated lambs. Left pulmonary artery hypoplasia present in all operated groups was significantly less pronounced in VEGF group. The number of pleural arteries was similarly increased in left lung of all operated lambs. Left lung arterial density was higher in VEGF group than in all other groups. The percentage of parenchyma of left lung was lower in beta-GAL group than in all others, partially returned to normal in VEGF group. CONCLUSIONS: In this model, transbronchial VEGF gene transfer induces pulmonary angiogenesis, proximal pulmonary artery growth and contributes to lung parenchyma recovery.

Fibrinogen Poissy II (gammaN361K): a novel dysfibrinogenemia associated with defective polymerization and peptide B release.

A fibrinogen variant was identified in a pregnant patient with disseminated intravascular coagulation and abruptio placentae. This dysfibrinogen was also found in four asymptomatic members of the patient's family. Coagulation studies showed prolongation of both the thrombin and reptilase times, and discrepancy was noted between the levels of plasma fibrinogen as determined by a kinetic versus an immunological determination or light-scattering assay. Studies on purified fibrinogen revealed an impaired release of fibrinopeptide B by thrombin related to a delayed thrombin-induced fibrin polymerization. DNA sequencing revealed a heterozygous T <-- A point mutation in position 9373 of the gamma-chain gene (exon 9), which substituted a K for an N at position 361.

Adenovirus-mediated gene transfer to the transplanted piglet heart after intracoronary injection.

BACKGROUND: The advent of cardiac gene therapy in clinical practice requires a more efficient and safer myocardial gene delivery in large animals. A new approach to adenovirus-mediated intracoronary gene transfer in the piglet, using a heterotopic heart transplantation model, was designed to maximize the duration of contact between the vector and the heart in noncoronary flow conditions. METHODS: Recombinant adenoviruses harboring a nucleus-localized beta-galactosidase gene under the control of a viral promoter were injected into the coronary vessels of the harvested hearts at a dose ranging from 10(10) to 2 x 10(11) pfu. The graft was maintained for 75 min in saline solution and then implanted in the abdomen of recipients. Gene transfer to allografts was evaluated 4 days after grafting by immunohistochemical and enzymatic analysis of beta-galactosidase expression. RESULTS: Transgene expression was detected in all cardiac areas and up to 64, 44, 32, and 15% of positive nuclei were estimated in the left ventricle wall in four animals out of eleven. In the remaining animals, transgene expression was focally distributed, mainly in the left ventricle wall. PCR analysis revealed the presence of adenoviral sequences, albeit minimal, in exposed organs such as the liver and lung. CONCLUSIONS: This procedure demonstrated that direct intracoronary gene transfer can be achieved using an ex vivo gene transfer strategy.

Endothelial cell early activation induced by allogeneic lymphocytes in isolated perfused mouse lung.

BACKGROUND: The early inflammatory response induced following vascularized organ allotransplantation is mediated by adhesin-ligand interactions between blood leukocytes and allogeneic endothelial cells. In the present study, we focused on the role of allogeneic blood lymphocytes in early immune alterations following lung reperfusion. METHODS: We developed an experimental model of isolated and ventilated lung in female C57BL/6 mouse perfused with either isogeneic (C57BL/6) or allogeneic (C3H/He) male mouse fresh blood for 3 hours. SRY DNA quantification by polymerase chain reaction (PCR) was used to assess the presence of perfusate cells in lung extract. Using quantitative reverse transcriptase (RT)-PCR, we measured mRNA expression both of the adhesion molecules--intercellular adhesion molecule (ICAM)-1, lymphocyte function-associated antigen (LFA)-3, vascular cell adhesion molecule (VCAM)-1, and endothelial leukocyte adhesion molecule (ELAM)-1--and of tumor necrosis factor (TNF)-alpha in lung tissue. RESULTS: The number of blood lymphocytes is significantly decreased in allogeneic versus isogeneic condition at 3 hours of perfusion, without evidence of increased apoptosis or necrosis. In parallel, SRY DNA quantity recovered in the lung was 2.5 times higher in allogeneic condition, which was related to cells loosely adherent to endothelial cells. Lastly, the levels of mRNAs of all adhesion molecules and of TNF-alpha were significantly increased in allogeneic versus isogeneic conditions. CONCLUSION: These results demonstrate that an early interaction between allogeneic blood lymphocytes and vascular endothelial cells is correlated with a high mRNA expression both of adhesion molecules and of TNF-alpha in the perfused lung. Our model of a mouse lung perfused with fresh blood appears to be a useful clinical assessment system for in-depth investigation of early cell activation and the resulting intragraft immune alterations.