Kyoko Hijiya

Protective effect of plasmin in marginal donor lungs in an ex vivo lung perfusion model

BACKGROUND Donor lung thrombi are considered an important etiology for primary graft dysfunction in lung transplantation. We hypothesized that thrombolysis before lung transplantation could alleviate ischemia-reperfusion injury. This study was designed to evaluate the effect of the fibrinolytic agent plasmin on lungs damaged by thrombi in an ex vivo lung perfusion (EVLP) system. METHODS Rats were divided into control, non-plasmin, and plasmin groups (n = 7 each). In the control and plasmin groups, cardiac arrest was induced by withdrawal of mechanical ventilation without heparinization. Ventilation was restarted 150 minutes after cardiac arrest. The lungs were flushed, and the heart and lungs were excised en bloc. The lungs were perfused in the EVLP system for 60 minutes, and plasmin or placebo was administered upon EVLP initiation. RESULTS Fibrin/fibrinogen degradation products in the perfusate were significantly higher in the plasmin group than in the control and non-control groups (p < 0.001 for both). Plasmin administration significantly decreased pulmonary vascular resistance (plasmin vs non-plasmin, p = 0.011) and inhibited the exacerbation of dynamic compliance (plasmin vs non-plasmin, p = 0.003). Lung weight gain was less in the plasmin group than in the non-plasmin group (p = 0.04). CONCLUSIONS Our results confirmed that plasmin administration in an EVLP model dissolved thrombi in the lungs, resulting in reconditioning of the lungs as assessed by various physiologic parameters.

Plasmin administration during ex vivo lung perfusion ameliorates lung ischemia–reperfusion injury

BACKGROUND Donor lung thrombus is considered a significant etiology for primary graft dysfunction (PGD). We hypothesized that thrombolysis in ex vivo lung perfusion (EVLP) before lung transplantation could alleviate ischemia-reperfusion injury (IRI), resulting in a decreased incidence of PGD. METHODS Rats were divided into control (n = 5), non-plasmin (n = 7) and plasmin (n = 7) groups. In the non-plasmin and plasmin groups, cardiac arrest was induced by withdrawal of ventilation without heparinization. After 120 minutes of warm ischemia, the lungs were ventilated and flushed. Hearts and both lungs were excised en bloc. The lungs were perfused and ventilated in the EVLP for 30 minutes, and plasmin or placebo was administered on EVLP initiation. The lungs were then stored at 4°C for 90 minutes and finally perfused with rat blood for 80 minutes. We assessed physiologic and histologic findings during reperfusion and the correlation between physiologic data during EVLP and after reperfusion. RESULTS Physiologic results were better in the plasmin group than in the non-plasmin group. The plasmin group lungs had fewer signs of histologic injury. Caspase-3 and -7 activity in the plasmin group was lower in the non-plasmin group. Pulmonary vascular resistance (PVR) during EVLP correlated with that at the end of reperfusion. CONCLUSIONS Plasmin administration during EVLP protected the donor lungs after reperfusion. We also found that several physiologic values in EVLP may be predictive markers of lung function after reperfusion.

Protective effect of pre-recovery surfactant inhalation on lungs donated after cardiac death in a canine lung transplantation model

BACKGROUND Warm ischemia-reperfusion injury related to donation after cardiac death is a crucial issue in transplantation. Because surfactant function deteriorates in lungs during warm ischemia, we hypothesized pre-recovery surfactant inhalation would mitigate warm ischemia-reperfusion injury. METHODS We rendered donor dogs cardiac dead and left them at room temperature. All animals received ventilation for 60 minutes starting at 240 minutes after cardiac arrest. The animals were divided into 2 groups: NS (normal saline, n = 7) group, which received aerosolized normal saline, and SF (surfactant; n = 5), which received aerosolized surfactant. The lungs were flushed and procured, and the left lung was transplanted into recipient dogs. At 45 minutes of reperfusion, the right pulmonary artery was ligated, and the left transplanted lung function was evaluated. RESULTS In the NS group, 2 of 7 dogs died at 75 minutes after reperfusion, whereas all 5 animals in the SF group survived for 240 minutes after reperfusion. The SF group showed significantly better dynamic compliance, oxygenation, and wet-to-dry weight ratio. Furthermore, the SF group had higher levels of high-energy phosphates in the lung tissues and lower levels of interleukin-8, tumor necrosis factor-α, and protein in the bronchoalveolar lavage fluid. Histologically, the lungs in the SF group showed fewer signs of interstitial edema and hemorrhage and significantly less neutrophilic sequestration than those of the NS group. CONCLUSIONS Our results indicated pre-recovery surfactant inhalation improved graft function, maintained adenine nucleotide levels, and prevented alveolar-capillary barrier leakage, resulting in the attenuation of warm ischemia-reperfusion injury.

Outcomes of various transplant procedures (single, sparing, inverted) in living-donor lobar lung transplantation

Objectives: In standard living‐donor lobar lung transplantation (LDLLT), the right and left lower lobes from 2 healthy donors are implanted. Because of the difficulty encountered in finding 2 donors with ideal size matching, various transplant procedures have been developed in our institution. The purpose of this retrospective study was to compare outcomes of nonstandard LDLLT with standard LDLLT. Methods: Between June 2008 and January 2016, we performed 65 LDLLTs for critically ill patients. Functional size matching was performed by estimating graft forced vital capacity based on the donors measured forced vital capacity and the number of pulmonary segments implanted. For anatomical size matching, 3‐dimensional computed tomography volumetry was performed. In cases of oversize mismatch, single‐lobe transplant or downsizing transplant was performed. In cases of undersize mismatch, native upper lobe sparing transplant or right‐left inverted transplant was performed. In right‐left inverted transplants, the donors right lower lobe was inverted and implanted into the recipients left chest cavity. Results: Twenty‐nine patients (44.6%) received nonstandard LDLLT, including 12 single‐lobe transplants, 7 native upper lobe sparing transplants, 6 right‐left inverted transplants, 2 sparing + inverted transplants, and 2 others. Thirty‐six patients (57.4%) received standard LDLLT. Three‐ and five‐year survival rates were similar between the 2 groups (89.1% and 76.6% after nonstandard LDLLT vs 78.0% and 71.1% after standard LDLLT, P = .712). Conclusions: Various transplant procedures such as single, sparing and inverted transplants are valuable options when 2 donors with ideal size matching are not available for LDLLT.

Hypoxia-inducible factor 1 promotes chemoresistance of lung cancer by inducing carbonic anhydrase IX expression

Lung cancer treatment is difficult owing to chemoresistance. Hypoxia‐inducible factor 1 (HIF‐1) and HIF‐1‐induced glycolysis are correlated with chemoresistance; however, this is not evident in lung cancer. We investigated the effect of HIF‐1α and carbonic anhydrase IX (CAIX), a transmembrane protein neutralizing intracellular acidosis, on chemoresistance and prognosis of lung cancer patients after induction chemoradiotherapy. Associations of HIF‐1α, glucose transporter 1 (GLUT1), and CAIX with chemoresistance of lung cancer were investigated using A549 lung cancer cells under normoxia or hypoxia in vitro. HIF‐1α‐induced reprogramming of glucose metabolic pathway in A549 cells and the effects of HIF‐1 and CAIX on the cytotoxicity of vinorelbine were investigated. Immunohistochemical analyses were performed to determine HIF‐1α, GLUT1, and CAIX expression levels in cancer specimens from lung cancer patients after induction chemoradiotherapy. Hypoxia induced HIF‐1α expression in A549 cells. Moreover, hypoxia induced GLUT1 and CAIX expression in A549 cells in a HIF‐1‐dependent manner. Glucose metabolic pathway was shifted from oxidative phosphorylation to glycolysis by inducing HIF‐1α in A549 cells. HIF‐1 and CAIX induced chemoresistance under hypoxia, and their inhibition restored the chemosensitivity of A549 cells. The expression levels of HIF‐1α, GLUT1, and CAIX were associated with poor overall survival of lung cancer patients after induction chemoradiotherapy. HIF‐1 and CAIX affected the chemosensitivity of A549 cells and prognosis of lung cancer patients. Therefore, inhibition of HIF‐1 and CAIX might improve prognosis of lung cancer patients after induction chemoradiotherapy. Further analysis might be helpful in developing therapies for lung cancer.

Inhibition of Toll-like receptor 4 signaling ameliorates lung ischemia-reperfusion injury in acute hyperglycemic conditions.

BACKGROUND Recent lung transplantation studies have shown that peri-operative hyperglycemia is an important factor affecting recipient survival; however, its underlying mechanisms are not well understood. We hypothesized that acute hyperglycemia exacerbates lung ischemia-reperfusion injury (IRI) through up-regulation of Toll-like receptor 4 (TLR4) signaling pathways. METHODS C57BL/6Ncr mice were divided into 3 treatment groups: sham; IRI; and IRI under acute hyperglycemic conditions (IRI+HG). Mice in the IRI and IRI+HG groups were exposed to IRI via clamping the left hilum for 1 hour, followed by reperfusion for 2 hours. Acute hyperglycemia was established by glucose injection. The severity of lung injury and TLR4 signaling pathway activity were assessed. Further, we performed a pharmacologic blockade of TLR4 signaling to determine the effect of TLR4 signaling inhibition on lung injury. RESULTS Compared with normoglycemic mice, hyperglycemic mice had 2-fold higher blood glucose levels (p < 0.001). Pulmonary compliance was significantly lower, and airway resistance was significantly higher, in the IRI+HG group than in the IRI group (p < 0.05). Levels of inflammatory cytokines in bronchoalveolar lavage fluid were significantly higher in the IRI+HG group than in the IRI group. Correspondingly, TLR4 signaling pathways were up-regulated in the IRI+HG group. Moreover, pharmacologic inhibition of TLR4 signaling significantly decreased lung injury markers under hyperglycemic conditions. CONCLUSIONS Acute hyperglycemia exacerbated lung IRI and was associated with up-regulation of TLR4 signaling pathways. Pharmacologic inhibition of TLR4 signaling ameliorated lung IRI with acute hyperglycemia. Targeting TLR4 appears to be a promising approach to managing coexisting pathologies in lung transplant recipients.

Association of Local Intrapulmonary Production of Antibodies Specific to Donor Major Histocompatibility Complex Class I With the Progression of Chronic Rejection of Lung Allografts

Background Antibody-mediated rejection may lead to chronic lung allograft dysfunction, but antibody-mediated rejection may develop in the absence of detectable donor-specific antibody (DSA) in recipient serum. This study investigated whether humoral immune responses develop not only systemically but locally within rejected lung allografts, resulting in local production of DSA. Methods Lewis rats received orthotopic left lung transplantation from Lewis (syngeneic control) or Brown-Norway (major histocompatibility complex-mismatched allogeneic) donor rats. Rats that underwent allogeneic lung transplantation were subsequently administered cyclosporine until day 14 (short immunosuppression) or day 35 (long immunosuppression). The lung grafts and spleens of recipient animals were tissue cultured for 4 days, and the titer of antibody against donor major histocompatibility complex molecules was assayed by flow cytometry. Explanted lung grafts were also evaluated pathologically. Results By day 98, DSA titers in supernatants of lung graft (P = 0.0074) and spleen (P = 0.0167) cultures, but not serum, from the short immunosuppression group were significantly higher than titers in syngeneic controls. Cultures and sera from the long immunosuppression group showed no production of DSA. Microscopically, the lung grafts from the short immunosuppression group showed severe bronchiole obliteration and parenchymal fibrosis, along with lymphoid aggregates containing T and B cells, accompanying plasma cells. These findings suggestive of local humoral immune response were not observed by days 28 and 63. Conclusions DSA can be locally produced in chronically rejected lung allografts, along with intragraft immunocompetent cells. Clinical testing of DSA in serum samples alone may underestimate lung allograft dysfunction.

Novel thermographic detection of regional malperfusion caused by a thrombosis during ex vivo lung perfusion

OBJECTIVES Although ex vivo lung perfusion (EVLP) has been clinically applied as a novel rig to evaluate marginal donor lungs, no parameters have been reported to objectively detect regional lung damage during EVLP. The aim of this study was to investigate whether regional donor lung malperfusion-related damage caused by a thrombus could be detected by thermography during EVLP. METHODS Lewis rats were divided into two groups: the Thrombosis group and the Control group (n = 6 in each group). All rats were heparinized and the lungs were flushed with 20 ml of Steen solution. In the Thrombosis group, a 30-mg artificial thrombus was inserted into the left main pulmonary artery. All the lungs were perfused and ventilated using the EVLP system. Perfusion flow was increased every 2 min up to 10 ml/min. The lungs were evaluated by collecting thermographical and physiological data during EVLP. RESULTS Pulmonary artery pressure was higher and lung compliance was lower in the Thrombosis group compared with those in the Control group (P = 0.0005 and <0.0001, respectively). Macroscopically, no differences were seen between the perfused area and the malperfused area, whereas significant differences were detected between them by thermography. The surface temperature of both lungs in the Control group and the right lungs in the Thrombosis group rose with increasing perfusion flow, whereas the surface temperature of the left lungs in the Thrombosis group did not rise (P < 0.0001). CONCLUSIONS Although physiological data could possibly imply the existence of thrombi in the Thrombosis group, it could not reveal which area was obstructed by thrombi; however, thermography could detect a malperfused region. Thermographical evaluation may become a promising strategy to detect regional damage in donor lungs.

Excellent outcome of donor lobectomy with various surgical techniques for the interlobar artery

OBJECTIVES: Donor lobectomies in living-donor lobar lung transplantation sometimes require additional surgical procedures on the interlobar pulmonary artery. We retrospectively reviewed the surgical techniques and outcomes of living-donor lobectomy. METHODS: Between 2008 and 2015, 116 living-donor lobectomies were performed in 64 consecutive living-donor lobar lung transplantations (52 bilateral, 12 single). The surgical techniques and outcomes were reviewed retrospectively. RESULTS: Right lower lobectomies were performed in 69 donors, left lower lobectomies in 45 and middle lobectomies in 2. In 61 living donors (52.6%), small branches of the pulmonary artery were sacrificed for anatomical reasons. Pulmonary arterioplasties to preserve the pulmonary artery branches were performed in 13 donors (11.2%). Thirteen left lower lobectomies with pulmonary arterioplasty were performed to preserve lingular branches, as the lingular branch originated far distal to the superior segmental branch of the left lower lobe. Pulmonary arterioplasty with an autologous pericardial patch in 10 donors and with an end-to-end anastomosis in three donors was performed. All surgical procedures had no complications. Three months after lobectomy, the ratio of the pulmonary function test results to the preoperative values and the complication rate showed no significant differences between donors with and without pulmonary arterioplasties, and donors with and without sacrifice of small branches. All living donors resumed their previous lifestyles without restriction. CONCLUSIONS: Although additional surgical procedures were often required in living-donor lobectomies, the donor outcomes were satisfactory. Arterioplasty to close the interlobar artery in the donor was needed in left-sided cases only.

Surgical management of bronchial stumps in lobar lung transplantation

Background: The validity of lobar lung transplantation (LT) has been established in both living‐donor lobar lung transplantation (LDLLT) and cadaveric‐donor lung transplantation (CLT). However, bronchial stump management in lobar LT has not been precisely documented. Thus, we retrospectively analyzed our strategies for bronchial stump management in lobar LT. Methods: Between June 2008 and August 2016, 145 LTs (72 LDLLTs and 73 CLTs) were performed at our institution. Bronchial stumps were left in 14 LDLLTs. Eight patients underwent bilateral CLTs with downsizing lobectomy. We avoided leaving donor bronchial stumps by lobar‐to‐lobar bronchial anastomosis, and left recipient bronchial stumps if necessary. We retrospectively reviewed the bronchial stump management methods and outcomes in these 22 patients. Results: Among the 14 LDLLTs, right‐to‐left inverted lobar LT and right single‐lobe LT with left pneumonectomy were performed in 12 and 2 patients, respectively. Among the 8 CLTs, 11 lobectomies were performed because of oversized grafts and/or localized pneumonia. Twenty‐three lobar‐to‐lobar bronchial anastomoses were performed, and there were 21 recipient bronchial stumps in total. Three bronchial stumps were left in the donor graft, the middle bronchus in all cases. No complications related to lobar‐to‐lobar bronchial anastomoses were observed. All bronchial stumps healed well without developing a bronchopleural fistula. The 3‐year overall survival rate was 88.1% (95% confidence interval, 58.8%‐97.0%). Conclusions: We successfully avoided leaving bronchial stumps in the donor graft, except in the middle bronchus, by performing lobar‐to‐lobar bronchial anastomoses in lobar LTs. Excellent healing of lobar‐to‐lobar bronchial anastomoses and bronchial stumps was observed.