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Definitions for chronic lung allograft dysfunction (CLAD) phenotypes were recently revised (2019 ISHLT consensus). Post-CLAD onset phenotype transition may occur as a result of change in obstruction, restriction, or RAS-like opacities (RLO). We aimed to assess the prevalence and prognostic implications of these transitions. This was a single-center, retrospective cohort study of bilateral lung transplants performed in 2009-2015. CLAD phenotypes were determined per ISHLT guidelines. CLAD phenotype transition was defined as a sustained change in obstruction, restriction or RLO. We specifically focused on phenotype changes based on RLO emergence. Association of RLO development with time to death or retransplant were assessed using Kaplan-Meier and Cox proportional hazards models. Among 211 patients with CLAD, 47 (22.2%) experienced a phenotype transition. Nineteen patients developed RLO. Development of RLO phenotype after CLAD onset was associated with a shorter time to death/retransplant when considering the entire CLAD patient cohort (HR = 4.00, CI 2.74-5.83, P

BackgroundThe published experience of lung transplantation in acute respiratory distress syndrome (ARDS) is limited. The aim of this study was to investigate the contemporary results of lung transplantation attempts in ARDS in major European centres.MethodsWe conducted a retrospective multicentre cohort study of all patients listed for lung transplantation between 2011 and 2019. We surveyed 68 centres in 22 European countries. All patients admitted to the waitlist for lung transplantation with a diagnosis of “ARDS/pneumonia” were included. Patients without extracorporeal membrane oxygenation (ECMO) or mechanical ventilation were excluded. Patients were followed until 1 October 2020 or death. Multivariable analysis for 1-year survival after listing and lung transplantation was performed.Results55 centres (81%) with a total transplant activity of 12 438 lung transplants during the 9-year period gave feedback. 40 patients with a median age of 35 years were identified. Patients were listed for lung transplantation in 18 different centres in 10 countries. 31 patients underwent lung transplantation (0.25% of all indications) and nine patients died on the waitlist. 90% of transplanted patients were on ECMO in combination with mechanical ventilation before lung transplantation. On multivariable analysis, transplantation during 2015–2019 was independently associated with better 1-year survival after lung transplantation (OR 10.493, 95% CI 1.977–55.705; p=0.006). 16 survivors out of 23 patients with known status (70%) returned to work after lung transplantation.ConclusionsLung transplantation in highly selected ARDS patients is feasible and outcome has improved in the modern era. The selection process remains ethically and technically challenging.

CASE PRESENTATION: A 63-year-old man with a left single lung transplant for end-stage combined restrictive and obstructive lung disease developed persistent pulmonary infiltrates and recurrent gram-negative bacteremia post-transplant. Bronchoalveolar lavage fluid revealed a nematode on Papanicolau staining compatible with Strongyloides stercoralis larvae on day 50 post-transplant. Although Strongyloides serology performed post-transplant was negative, a retrospective review of the medical record revealed marked peripheral blood eosinophilia on several occasions before transplantation. Despite reduction in immunosuppression and treatment with albendazole and ivermectin, the patient developed another episode of Escherichia coli bacteremia. He died 3 months post-transplant from pulmonary and neurological complications. DIAGNOSIS: Strongyloides hyper-infection. DISCUSSION: Strongyloides hyper-infection syndrome is known to occur in immunocompromised patients, but it has only been reported once in a lung transplant recipient. This case illustrates the importance of screening for parasitic infections before transplantation in patients with marked eosinophilia, especially among immigrants from countries in which Strongyloides is endemic. Hyper-infection syndrome may appear years after infection in the context of immunosuppression or immunodeficiency. This case also highlights the association between Strongyloides hyper-infection and bacteremia with enteric organisms.

Funding Information: Supported by the Helsinki University Hospital , the Sigrid Juselius Foundation , the Academy of Finland , Finska Läkaresällskapet , the Research and Science Foundation of Farmos , The Paulo Foundation , Jalmari and Rauha Ahokas Foundation , Aarne Koskelo Foundation , Päivi and Sakari Sohlberg Foundation , Finnish Pulmonary Association , Biomedicum Helsinki Foundation , Jane and Aatos Erkko Foundation , and the University of Helsinki . Funding Information: Supported by the Helsinki University Hospital, the Sigrid Juselius Foundation, the Academy of Finland, Finska L?kares?llskapet, the Research and Science Foundation of Farmos, The Paulo Foundation, Jalmari and Rauha Ahokas Foundation, Aarne Koskelo Foundation, P?ivi and Sakari Sohlberg Foundation, Finnish Pulmonary Association, Biomedicum Helsinki Foundation, Jane and Aatos Erkko Foundation, and the University of Helsinki. Publisher Copyright: © 2022 American Society for Investigative Pathology Obliterative bronchiolitis (OB) after lung transplantation is a nonreversible, life-threatening complication. Herein, the role of vascular endothelial growth factor receptor (Vegfr)-1 and -2 was investigated in the development of obliterative airway disease (OAD), an experimental model for OB. The nonimmunosuppressed recipients underwent transplantation with fully major histocompatibility complex mismatched heterotopic tracheal allografts and received Vegfr1 and -2-specific monoclonal antibodies either alone or in combination, or rat IgG as a control. The treatment with Vegfr1- or -2-blocking antibody significantly decreased intragraft mRNA expression of natural killer cell activation markers early after transplantation. This was followed by reduced infiltration of Cd11b thorn cells and Cd4 thorn T cells as well as down-regulated mRNA expression of proinflammatory chemokines and profibrotic growth factors. However, blocking of both Vegfr1 and -2 was necessary to reduce luminal occlusion. Furthermore, concomitant inhibition of the calcineurin activation pathway almost totally abolished the development of OAD. This study proposes that blocking of Vegf receptors blunted natural killer cell and innate immune responses early after transplantation and attenuated the development of OAD. The results of this study suggest that further studies on the role of Vegfr1 and -2 blocking in development of obliterative airway lesions might be rewarding. (Am J Pathol 2022, 192: 254-269; https://doi.org/10.1016/ j.ajpath.2021.10.018)

Long-term outcomes after lung transplantation remain inferior to those of other solid organ groups. The significance of eosinophils detected on transbronchial biopsies (TBBx) after lung transplantation and their relationship to long-term outcomes remain unknown. A retrospective single-center cohort study was performed of patients transplanted between January 01, 2001, and July 31, 2018, who had at least 1 TBBx with evaluable parenchymal tissue. Multivariable Cox proportional hazard models were used to assess the associations between eosinophil detection and: all-cause mortality and Chronic Lung Allograft Dysfunction (CLAD). 8887 TBBx reports from 1440 patients were reviewed for the mention of eosinophils in the pathology report. 112 (7.8%) patients were identified with eosinophils on at least one TBBx. The median (95% CI) survival time for all patients was 8.28 (7.32-9.31) years. Multivariable analysis, adjusted for clinical variables known to affect post-transplant outcomes, showed that the detection of eosinophils was independently associated with an increased risk of death (HR 1.51, 95% CI 1.24-1.85, p < 0.01) and CLAD (HR 1.35, 95% CI 1.07-1.70, P = 0.01). Eosinophils detected in TBBx are associated with an increased risk of CLAD and death. There may be benefit in specifically reporting the presence of eosinophils in TBBx reports and incorporating their presence in clinical decision-making.

Background Letermovir, a new viral terminase complex inhibitor, has been approved for the prevention of cytomegalovirus (CMV) infection in hematopoietic stem cell transplant patients. However, data on the efficacy and safety of letermovir for the treatment of CMV infection in transplant recipients remain scarce. Methods We performed a single-center retrospective study of stem cell and organ transplant recipients who received letermovir for the treatment of CMV infection from November 2017 to October 2018. Results Six patients were included and five were evaluable. All received letermovir in the context of a refractory or resistant CMV infection including asymptomatic CMV viremia (n=3), CMV syndrome (n=1) and CMV pneumonitis and colitis (n=1). The three asymptomatic patients experienced a decrease of the viral load to less than 200 IU/mL after letermovir therapy. One patient displayed a partial viral load response (2 log of viral load reduction) but a good clinical response, and one who received a suboptimal dose of letermovir experienced an increase of viremia. There were no treatment-related adverse effects. Conclusion We demonstrate mixed efficacy in patients with refractory CMV infection suggesting that letermovir may be a useful therapeutic adjunct, potentially in combination with other antivirals.

BackgroundChronic lung allograft dysfunction (CLAD) is the principal cause of graft failure in lung transplant recipients and prognosis depends on CLAD phenotype. We used a machine learning computed tomography (CT) lung texture analysis tool at CLAD diagnosis for phenotyping and prognostication compared with radiologist scoring.MethodsThis retrospective study included all adult first double lung transplant patients (January 2010–December 2015) with CLAD (censored December 2019) and inspiratory CT near CLAD diagnosis. The machine learning tool quantified ground-glass opacity, reticulation, hyperlucent lung and pulmonary vessel volume (PVV). Two radiologists scored for ground-glass opacity, reticulation, consolidation, pleural effusion, air trapping and bronchiectasis. Receiver operating characteristic curve analysis was used to evaluate the diagnostic performance of machine learning and radiologist for CLAD phenotype. Multivariable Cox proportional hazards regression analysis for allograft survival controlled for age, sex, native lung disease, cytomegalovirus serostatus and CLAD phenotype.Results88 patients were included (57 bronchiolitis obliterans syndrome (BOS), 20 restrictive allograft syndrome (RAS)/mixed and 11 unclassified/undefined) with CT a median 9.5 days from CLAD onset. Radiologist and machine learning parameters phenotyped RAS/mixed with PVV as the strongest indicator (area under the curve (AUC) 0.85). Machine learning hyperlucent lung phenotyped BOS using only inspiratory CT (AUC 0.76). Radiologist and machine learning parameters predicted graft failure in the multivariable analysis, best with PVV (hazard ratio 1.23, 95% CI 1.05–1.44; p=0.01).ConclusionsMachine learning discriminated between CLAD phenotypes on CT. Both radiologist and machine learning scoring were associated with graft failure, independent of CLAD phenotype. PVV, unique to machine learning, was the strongest in phenotyping and prognostication.

Lung transplantation has become an invaluable approach for the treatment of end-stage respiratory diseases. However, survival after lung transplant remains limited due to chronic lung allograft dysfunction (CLAD), a fibrotic process affecting the airway and/or parenchymal compartments of the lung graft leading to a significant and persistent deterioration in lung function [1, 2]. With our accelerating understanding of CLAD, it has become evident that CLAD is a heterogeneous process comprised of multiple patterns of physiological and radiological features, which have strong implications for post-CLAD survival [3]. Footnotes This manuscript has recently been accepted for publication in the European Respiratory Journal . It is published here in its accepted form prior to copyediting and typesetting by our production team. After these production processes are complete and the authors have approved the resulting proofs, the article will move to the latest issue of the ERJ online. Please open or download the PDF to view this article. Conflict of interest: Dr. Levy has nothing to disclose. Conflict of interest: Dr. Huszti has nothing to disclose. Conflict of interest: Dr. Berra has nothing to disclose. Conflict of interest: Dr. Renaud-Picard has nothing to disclose. Conflict of interest: Dr. Kawashima has nothing to disclose. Conflict of interest: Dr. Takahagi has nothing to disclose. Conflict of interest: Dr. Moshkelgosha has nothing to disclose. Conflict of interest: Dr. Ghany has nothing to disclose. Conflict of interest: Dr. Fuchs has nothing to disclose. Conflict of interest: Dr. Chow has nothing to disclose. Conflict of interest: Dr. Keshavjee has nothing to disclose. Conflict of interest: Dr. Singer has nothing to disclose. Conflict of interest: Dr. Tikkanen has nothing to disclose. Conflict of interest: Dr. Martinu has nothing to disclose.

RationaleThe diffusing capacity of the lung for carbon monoxide corrected for haemoglobin (DLCOcor) measures gas movement across the alveolar–capillary interface. We hypothesised that DLCOcor is a sensitive measure of injurious allograft processes disrupting this interface.ObjectivesTo determine the prognostic significance of the DLCOcor trajectory on chronic lung allograft dysfunction (CLAD) and survival.MethodsA retrospective analysis was conducted of all bilateral lung transplant recipients at a single centre, between January 1998 and January 2018, with one or more DLCOcor measurements. Low baseline DLCOcor was defined as the failure to achieve a DLCOcor >75% predicted. Drops in DLCOcor were defined as >15% below recent baseline.Results1259 out of 1492 lung transplant recipients were included. The median (range) time to peak DLCOcor was 354 (181–737) days and the mean±sdDLCOcor was 80.2±21.2% pred. Multivariable analysis demonstrated that low baseline DLCOcor was significantly associated with death (hazrd ratio (HR) 1.68, 95% CI 1.27–2.20; pDLCOcor was not independently associated with CLAD after adjustment for low baseline forced expiratory volume in 1 s or forced vital capacity. Any DLCOcor declines ≥15% were significantly associated with death, independent of concurrent spirometric decline. Lower percentage predicted DLCOcor values at CLAD onset were associated with shorter post-CLAD survival (HR 0.75 per 10%-unit change, pConclusionLow baseline DLCOcor and post-transplant declines in DLCOcor were significantly associated with survival, independent of spirometric measurements. We propose that DLCOcor testing may allow identification of a subphenotype of baseline and chronic allograft dysfunction not captured by spirometry. There may be benefit in routine monitoring of DLCOcor after lung transplantation to identify patients at risk of poor outcomes.

The mortality rate for individuals on the wait list for lung transplant is 15% to 25%, and still only 20% of lungs from multiorgan donors are used for lung transplant. The lung donor pool may be increased by assessing and reconditioning high-risk extended criteria donor lungs with ex vivo lung perfusion (EVLP), with similar short-term outcomes.To assess the long-term outcomes of transplant recipients of donor lungs treated with EVLP.This retrospective cohort single-center study was conducted from August 1, 2008, to February 28, 2017, among 706 recipients of donor lungs not undergoing EVLP and 230 recipients of donor lungs undergoing EVLP.Donor lungs undergoing EVLP.The incidence of chronic lung allograft dysfunction and allograft survival during the 10-year EVLP era were the primary outcome measures. Secondary outcomes included donor characteristics, maximum predicted percentage of forced expiratory volume in 1 second, acute cellular rejection, and de novo donor-specific antibody development.This study included 706 patients (311 women and 395 men; median age, 50 years [interquartile range, 34-61 years]) in the non-EVLP group and 230 patients (85 women and 145 men; median age, 46 years [interquartile range, 32-55 years]) in the EVLP group. The EVLP group donors had a significantly lower mean (SD) Pao2:fraction of inspired oxygen ratio than the non-EVLP group donors (348 [108] vs 422 [88] mm Hg; P .001), higher prevalence of abnormal chest radiography results (135 of 230 [58.7%] vs 349 of 706 [49.4%]; P = .02), and higher proportion of smoking history (125 of 204 [61.3%] vs 322 of 650 [49.5%]; P = .007). More recipients in the EVLP group received single-lung transplants (62 of 230 [27.0%] vs 100 of 706 [14.2%]; P .001). There was no significant difference in time to chronic lung allograft dysfunction between the EVLP and non-EVLP group (70% vs 72% at 3 years; 56% vs 56% at 5 years; and 53% vs 36% at 9 years; log-rank P = .68) or allograft survival between the EVLP and non-EVLP groups (73% vs 72% at 3 years; 62% vs 58% at 5 years; and 50% vs 44% at 9 years; log-rank P = .97) between the 2 groups. All secondary outcomes were similar between the 2 groups.Since 2008, 230 of 936 lung transplants (24.6%) in the Toronto Lung Transplant Program were performed after EVLP assessment and treatment. Use of EVLP-treated lungs led to an increase in the number of patients undergoing transplantation, with comparable long-term outcomes.

BACKGROUND Chronic lung allograft dysfunction (CLAD) is a heterogeneous condition. Characterization of CLAD phenotypes is essential to enhance the understanding of pathogenesis and guide new therapies. The study objective was to validate the new International Society for Heart and Lung Transplantation (ISHLT) CLAD classification system and further explore patients who do not fall into the defined CLAD sub-categories. METHODS We performed a single-center, retrospective cohort study of adult, first, bilateral lung transplants performed from 2010 to 2015. Patients with CLAD were classified on the basis of the 2019 ISHLT consensus document. CLAD phenotypes and other potential predictors of survival after CLAD onset were assessed using Kaplan–Meier and Cox proportional hazards models. RESULTS Among the 174 subjects with CLAD, 104 (59.8%) had bronchiolitis obliterans syndrome (BOS), 16 (9.2%) restrictive allograft syndrome (RAS), 9 (5.2%) mixed, and 19 (10.9%) undefined phenotype. A total of 26 patients (14.9%) did not match any of these 4 categories and remained unclassified. Allograft survival post-CLAD onset was longer for patients with BOS (median, 500 days) than patients with RAS (median, 372 days) or mixed (median, 328 days). The 45 patients (26.8%) with undefined/unclassified phenotype were combined and recategorized on the basis of the presence or absence of characteristic RAS-like opacities on chest imaging; those with RAS-like opacities had significantly worse allograft survival than patients with BOS (hazard ratio, 2.14; 95% confidence interval, 1.17–3.93; p = 0.014) and similar survival to RAS or mixed phenotype. CONCLUSIONS The new ISHLT CLAD phenotype classification is informative with regards to post-CLAD outcomes. Chest imaging demonstrating persistent parenchymal or pleural fibrosis may be used for risk-stratification of patients who do not match the major CLAD phenotypes.

Chronic lung allograft dysfunction (CLAD) remains the leading cause of late death after lung transplantation. Epithelial injury is thought to be a key event in the pathogenesis of CLAD. M30 and M65 are fragments of cytokeratin-18 released specifically during epithelial cell apoptosis and total cell death, respectively. We investigated whether M30 and M65 levels in bronchoalveolar lavage (BAL) correlate with CLAD subtypes: restrictive allograft syndrome (RAS) versus bronchiolitis obliterans syndrome (BOS). BALs were obtained from 26 patients with established CLAD (10 RAS, 16 BOS) and 19 long-term CLAD-free controls. Samples with concurrent infection or acute rejection were excluded. Protein levels were measured by ELISA. Variables were compared using Kruskal-Wallis, Mann-Whitney U test and Chi-squared tests. Association of M30 and M65 levels with post-CLAD survival was assessed using a Cox PH models. M65 levels were significantly higher in RAS compared to BOS and long-term CLAD-free controls and correlated with worse post-CLAD survival. Lung epithelial cell death is enhanced in patients with RAS. Detection of BAL M65 may be used to differentiate CLAD subtypes and as a prognostic marker in patients with established CLAD. Understanding the role of epithelial cell death in CLAD pathogenesis may help identify new therapeutic targets to improve outcome.

Acute cellular rejection (ACR) is a significant risk factor for chronic lung allograft dysfunction (CLAD). Although clinically manifest and higher grade (≥A2) ACR is generally treated with augmented immunosuppression, management of minimal (grade A1) ACR remains controversial. In our program, patients with subclinical and spirometrically stable A1 rejection (StA1R) are routinely not treated with augmented immunosuppression. We hypothesized that an untreated first StA1R does not increase the risk of CLAD or death compared to episodes of spirometrically stable no ACR (StNAR). The cohort was drawn from all consecutive adult, first, bilateral lung transplantations performed between 1999 and 2017. Biopsies obtained in the first-year posttransplant were paired with (forced expiratory volume in 1 second FEV1 ). The first occurrence of StA1R was compared to a time-matched StNAR. The risk of CLAD or death was assessed using univariable and multivariable Cox proportional hazards models. The analyses demonstrated no significant difference in risk of CLAD or death in patients with a first StA1R compared to StNAR. This largest study to date shows that, in clinically stable patients, an untreated first A1 ACR in the first-year posttransplant is not significantly associated with an increased risk for CLAD or death. Watchful-waiting approach may be an acceptable tactic for stable A1 episodes in lung transplant recipients.

Purpose Chronic Lung Allograft Dysfunction (CLAD) is a heterogeneous disease. Three major phenotypes: bronchiolitis obliterans syndrome (BOS), restrictive allograft syndrome (RAS) and mixed phenotype, are defined based on combinations of lung physiology and chest imaging. Total lung capacity (TLC)≤90% of baseline is used to define RAS/Mixed; however, many centers do not routinely obtain TLC and use instead forced vital capacity (FVC)≤80%. The objective of the current study was to assess the diagnostic accuracy of FVC≤80% for determining RAS/Mixed in a large center that routinely monitors both TLC and FVC. Methods This was a retrospective cohort study of all consecutive adult, first, bilateral lung transplants performed 2010-2015. All patients with CLAD were included. Spirometry, lung volumes, and chest imaging were available. RAS/mixed were determined based on 2019 ISHLT definitions using TLC≤90% and the presence of RAS-like opacities. Sensitivity, specificity, positive likelihood ratio (PLR), negative likelihood ratio (NLR), and diagnostic accuracy of FVC≤80% combined with RAS-like opacities for diagnosis of RAS/Mixed were computed. Results A total of 174 patients developed CLAD: 25(14.4%) met criteria for RAS/mixed by TLC. The sensitivity, specificity, PLR, NLR and diagnostic accuracy for FVC to determine RAS/Mixed was 84.0% (95%CI 63.9-95.5), 95.3% (95%CI 90.6-98.1), 17.9 (95%CI 8.5-37.6), 0.17 (95%CI 0.07-0.41) and 93.7% (95%CI 89.0-96.8). Conclusion Low FVC has an overall good diagnostic value for estimating RAS/mixed phenotype. Given the high specificity, it is possible to rule in RAS/mixed phenotype with spirometry. However, it cannot be ruled out confidently based on spirometry alone. In CLAD patients with a clinical suspicion of RAS/mixed phenotype and a preserved FVC, measurement of TLC is important for a more accurate diagnosis.

Purpose Bronchiolitis obliterans syndrome (BOS), restrictive allograft syndrome (RAS), and mixed are major phenotypes of CLAD. In previous, older studies, CMV viremia has been associated with reduced survival. We hypothesized that, in a more modern era of CMV prophylaxis, CMV viremia would remain associated with death, but also represent a risk factor for CLAD and specifically RAS/mixed phenotype. Methods This is a retrospective cohort study of all consecutive adult, first, bilateral-/single-lung transplants performed 2010-2016 who had ≥4 pulmonary function tests and ≥1 CMV-PCR measurement. CMV-PCR levels were divided into 3 categories: undetectable, Results Of 645 patients, 257 developed CLAD (143 BOS, 44 RAS/mixed, 66 undefined/unclassified) and 388 were CLAD-free at last follow-up. In univariate analysis, CMV-PCR≥1000 IU/mL was a significant risk factor for CLAD (HR 1.33, p=0.05). In multivariate analysis, CMV-PCR was not a significant risk factor for CLAD, whereas CMV serostatus mismatch was (Table). CMV-PCR was not a significant risk factor for RAS/mixed (CMV-PCR Conclusion CMV viremia was significantly associated with death/re-transplantation. Conversely, CMV viremia was not strongly associated with CLAD or RAS/mixed, while CMV serostatus mismatch was. CMV serostatus mismatch may have an impact on CLAD through mechanisms independent of viremia. In light of these paradoxical results, we are further assessing the relationship between immunosuppression, immune function, and CMV.

Purpose Lung and heart-lung transplant candidates and recipients are susceptible to mycobacterial infections that can cause morbidity and mortality. We characterized the incidence, clinical characteristics and treatment of mycobacterial findings and assessed their impact on patient outcomes. Methods We conducted a retrospective review of patients who underwent lung or heart-lung transplantation in our center between June 1988 and May 2020 and identified patients with mycobacterial findings, in most cases positive sputum or bronchoscopy sample cultures. Pre-transplant patients are screened for mycobacterial infections with bronchoscopy and sputum samples. Post-transplant patients undergo surveillance bronchoscopies several times during the first post-transplant year, annually thereafter, and when clinically warranted. The decision to initiate anti-mycobacterial treatment is based on clinical, radiologic and mycobacterial sample results, with the aim to treat mycobacterial disease and monitor colonization. Results In a cohort of 355 patients, 15/355 (4.2%) patients had a pre-transplant mycobacterial finding and 9/15 received anti-mycobacterial treatment. In one patient, mycobacterium tuberculosis infection recurred post-transplant. No deaths were related to mycobacterial infections in patients with a pre-transplant mycobacterial finding. In post-transplant patients, 27/355 (7.6%) patients had a mycobacterial finding and 5/27 received anti-mycobacterial treatment. One patient died due to disseminated mycobacterium abscessus infection. Mycobacterial findings pre- or post-transplant did not affect overall patient survival compared to non-infected patients (Log Rank p = 0.312). Conclusion Mycobacterial findings are rare in patients undergoing lung or heart-lung transplantation, and do not seem to affect overall survival of patients. Most post-transplant mycobacterial findings represent colonization, but disseminated mycobacterial infection may be fatal.

Purpose Airway epithelial injury is thought to be a key event in the pathogenesis of Chronic lung allograft dysfunction (CLAD). We investigated whether markers of epithelial activity and injury in bronchoalveolar lavage (BAL) correlate with CLAD diagnosis and major CLAD phenotypes: bronchiolitis obliterans syndrome (BOS) vs. restrictive allograft syndrome (RAS)-related phenotypes (including RAS, mixed phenotype, and all other patients with RAS-like opacities). Methods CLAD status and phenotypes were retrospectively determined in a cohort of all consecutive adult, first, bilateral lung transplants performed 2010-2015. All patients with RAS-related phenotypes were included and 1:1 matched with BOS patients based on time from transplant to CLAD-onset. CLAD-free subjects were used as controls. Proteins that maintain the barrier function of the airway epithelial mucosa (club cell secretory protein (CCSP), surfactant protein-D (SP-D) and epithelial mucins: MUC1, MUC5AC, MUC5B, MUC16), as well as epithelial cell death markers (M30&M65 representing apoptosis and overall death, respectively), were measured in BAL obtained within 6-months from CLAD onset using a double-sandwich ELISA or a multiplex bead assay. Protein levels were compared using Kruskal-Wallis and Mann-Whitney-U-test. Association between protein levels and graft survival was assessed using Cox proportional hazards models, adjusted for CMV serology mismatch status and phenotype. Results Fifty-four CLAD (27 BOS, 11 RAS, 7 mixed, 9 others with RAS-like opacities) patients and 26 CLAD-free controls were included. Median BAL levels were significantly higher in patients with CLAD compared to CLAD-free controls for M30 (124.5 vs. 88.9), MUC1 (6.8 vs. 3.28) and MUC16 (121.0 vs. 31.1). When comparing CLAD phenotypes, M30 was significantly higher in patients with RAS-related phenotypes compared to BOS (160.9 vs. 114.6). In multivariable models, levels of M30 and MUC5B were associated with allograft survival after CLAD onset independent of phenotype (P Conclusion Airway epithelial mucin and cell death markers are enhanced in the BAL fluid of patients with CLAD and can assist in differentiating between CLAD phenotypes. Abnormal airway mucin expression and epithelial cell death may be involved in the airway inflammatory response of CLAD and thus aid in future selection of targeted therapies.

Purpose Allograft infection after lung transplant (LTx) has a significant impact on outcome and has been associated with chronic lung allograft dysfunction (CLAD). Commensal bacteria (CB) are commonly isolated from bronchoalveolar lavages (BAL) in LTx recipients but the clinical significance is unknown. Our objective was to evaluate the association of high growth of CB (≥106 CFU/L) in BAL cultures with subsequent infection or CLAD. Methods We conducted a single-center, retrospective, cohort study of consecutive adult, first bilateral LTx performed 2010-2017. Patients who had no evidence of BAL infection and were alive and CLAD-free throughout the first 6-months post-transplant were included. Infection was defined as the presence of treatment-requiring pathogens in BAL. Outcomes of patients with ≥1CB within the first 6-months post-transplant were compared to patients with no CB isolation episodes within the same time interval. Association of CB with subsequent clinically significant BAL infections using logistic regression models and with time to CLAD was assessed using Cox Proportional Hazards models. Results The study cohort consisted of 202 patients with a median of 7 (IQR 7, 8) bronchoscopies per patient. Seventy of the 202 (34.7%) had subsequent BAL infection at some point post-transplant. Median time to CLAD was 497 days (IQR 131, 1043). Fifty-four (26.7%) patients had at least one episode of CB within the first 6-months post-transplant. In a multivariable analysis accounting for age, sex, primary lung disease, acute rejection score and CMV-mismatch, there was no difference in risk for subsequent infections (OR=0.83, 95% CI 0.40-1.69, P=0.60) or CLAD (HR=0.97, 95% CI 0.50-1.87, P=0.92) between patients with ≥1CB episode versus none. Conclusion We found that in the absence of specific infectious pathogens, isolation of commensal bacteria from BAL of LTx recipients during the first 6-months is not associated with an increased risk of subsequent infections or development of CLAD.

Purpose Surveillance bronchoscopies with bronchoalveolar lavage (BAL) and transbronchial biopsies (TBB) are used in the evaluation of patients after lung transplant to detect acute cellular rejection (ACR) or infection. We have previously shown that a focused BAL protein signature early post-transplant may predict CLAD or death in a subset of clinically-stable patients with A1-grade (minimal) ACR. The aim of the current study was to determine whether similar BAL markers would have similar predictive characteristics irrespective of ACR grade. Methods The cohort consisted of all adult, first, bilateral lung transplants performed 2010-2017. Clinical status was categorized as unstable or stable based on presence or absence of ≥10% concurrent drop in FEV1. Clinically stable patients with grade AX TBB (inadequate biopsies, which are not routinely treated at our center, thus avoiding the confounding effect of ACR treatment) during the first-year post-transplant, not preceded by ACR (grade A≥1 or grade B≥1) were included. IL6, S100A8, IL10, TNF-receptor1, IL-1α, pentraxin3 and CXCL10, previously shown to be associated with worse outcomes, were measured in the BAL using a multiplex bead assay. Association with subsequent CLAD or death was assessed using Cox proportional hazards models adjusted for age, sex, native lung disease, and CMV-mismatch. Results 107 patients matched the inclusion criteria with a stable AX occurring at a median time of 188 days (IQR 96-279) post-transplant. Median time from AX TBB to CLAD or death was 462 (IQR 330, 928) and 582 days (IQR 251-1410), respectively. In multivariable models, levels of CXCL10 and IL10 were associated with both CLAD development and death while IL6, S100A8 and pentraxin3 were only associated with death (P Conclusion A focused BAL protein signature in clinically stable patients with ungradable TBB, early post-transplant, may be informative in determining increased risk for worse outcome and may benefit from a more aggressive management strategy.

The development of de novo donor-specific HLA antibodies (dnDSA) after transplantation is associated with graft failure, mortality, and cost. There is no effective therapeutic intervention to prevent dnDSA or ameliorate associated injury. The aims of this study were to identify specific HLA factors associated with dnDSA development and to propose primary prevention strategies that could reduce the incidence of dnDSA without prohibitively limiting access to transplant. The investigation cohort included heart transplant recipients from 2008 to 2015 (n = 265). HLA typing was performed and HLA antibody testing was undertaken before and after transplantation. HLAMatchmaker analysis was performed for persistent dnDSA to identify potentially more immunogenic eplet differences. Validation was performed in recipients of lung transplants from 2008 to 2013 (n = 433). The majority of recipients with dnDSA had antibodies to identical eplet positions on DQ2 and DQ7. A high-risk epitope mismatch (found in DQA1*05 + DQB1*02/DQB1*03:01(7)) was associated with a 4.2- and 4.9-fold increased risk of dnDSA in heart and lung recipients respectively. HLA electrostatic potential modeling provided a plausible explanation for this observed immunogenicity. A theoretical allocation algorithm avoiding high-risk epitope mismatches was generated and predicted to reduce dnDSA by up to 72% without additional testing, eplet analysis, or cost.

The growing demand for suitable lungs for transplantation drives the quest for alternative strategies to expand the donor pool. The aim of this study is to evaluate the outcomes of lung transplantation (LTx) with donation after circulatory determination of death (DCDD) and the impact of selective ex vivo lung perfusion (EVLP). From 2007 to 2013, 673 LTx were performed, with 62 (9.2%) of them using DCDDs (seven bridged cases). Cases bridged with mechanical ventilation/extracorporeal life support were excluded. From 55 DCDDs, 28 (51%) underwent EVLP. Outcomes for LTx using DCDDs and donation after neurological determination of death (DNDD) donors were similar, with 1 and 5-year survivals of 85% and 54% versus 86% and 62%, respectively (p = 0.43). Although comparison of survival curves between DCDD + EVLP versus DCDD-no EVLP showed no significant difference, DCDD + EVLP cases presented shorter hospital stay (median 18 vs. 23 days, p = 0.047) and a trend toward shorter length of mechanical ventilation (2 vs. 3 days, p = 0.059). DCDDs represent a valuable source of lungs for transplantation, providing similar results to DNDDs. EVLP seems an important technique in the armamentarium to safely increase lung utilization from DCDDs; however, further studies are necessary to better define the role of EVLP in this context.

Intragraft DSA can be found in explanted lungs of patients with chronic lung allograft dysfunction in the absence of circulating DSA. New diagnostic approaches to assess for intragraft DSA may add to our diagnostic arsenal in treating AMR.http://bit.ly/31kS00b

Idiopathic pulmonary fibrosis (IPF) is a progressive lung disease with a poor prognosis and very few therapeutic options. On the molecular level, patients with IPF have increased amounts of the bone morphogenetic protein (BMP) inhibitor gremlin in their lungs, which results in decreased BMP signaling, and an increase in transforming growth factor-β signaling. Based on these findings, we hypothesized that restoration of the impaired BMP signaling would offer a novel strategy for the prevention of fibrosis progression or for the treatment of pulmonary fibrosis. We used reporter cell lines and high-throughput screening of a chemical compound library as an approach to finding molecules that increase BMP signaling in lung epithelial cells, without increasing transforming growth factor-β signaling. The most promising candidate drug was analyzed further by studying its effects on BMP target gene expression, Smad protein phosphorylation, and a mouse model of silica-induced pulmonary fibrosis. The most promising drug candidate, tilorone, induced BMP signaling in the reporter cells and increased the expression of BMP-7 and a BMP target gene, Id3, in lung epithelial A549 cells. In a mouse model of pulmonary fibrosis, tilorone decreased lung hydroxyproline content and the expression of collagen genes Col1A1 and Col3A1. Mice treated with tilorone showed markedly decreased histological changes, compared with untreated mice. These findings indicate that tilorone has biologically significant antifibrotic properties.

BACKGROUND: Obliterative bronchiolitis after lung transplantation is characterized by chronic airway inflammation leading to the obliteration of small airways. Hypoxia-inducible factor-1 (HIF-1) is a master regulator of cellular responses to hypoxia and inflammation. The Von Hippel-Lindau protein (pVHL) drives the degradation of oxygen-sensitive subunit HIF-1 alpha that controls the activity of HIF-1. We investigated the effect of myeloid cell targeted gene deletion of HIF-1 alpha or its negative regulator pVHL on the development of obliterative airway disease (OAD) in the recipients of tracheal allografts, a mouse model for obliterative bronchiolitis after lung transplantation. METHODS: Tracheal allografts were heterotopically transplanted from BALB/c donor mice to fully major histocompatibility complex mismatched recipient mice with HIF-1 alpha or VHL gene deletion in myeloid cells. The recipients were left non-immunosuppressed or received tacrolimus daily. Histologic, immunohistochemical, and real-time reverse transcription polymerase chain reaction analyses were performed at 3, 10, and 30 days. RESULTS: In the absence of immunosuppression, myeloid cell-specific VHL deficiency of the recipient mice improved epithelial recovery, decreased inflammatory cell infiltration and expression of pro-inflammatory cytokines, increased regulatory forkhead box P3 messenger RNA expression, and reduced OAD development in tracheal allografts. In the presence of tacrolimus immunosuppression, loss of HIF-1 alpha activity in myeloid cells of the recipient by HIF-1 alpha gene deletion accelerated OAD development in mouse tracheal allografts. CONCLUSIONS: Activity of the HIF-pathway affects the development of allograft rejection, and our results suggest that myeloid cell-specific VHL-deficiency that potentially increases HIF-activity decreases allograft inflammation and the subsequent development of OAD in mouse tracheal allografts. (C) 2016 International Society for Heart and Lung Transplantation. All rights reserved.

Despite increasing evidence about the role of donor-specific human leukocyte antigen (HLA) antibodies in transplant outcomes, the incidence and impact of de novo donor-specific antibodies (dnDSA) after lung transplantation remains unclear.To describe the incidence, characteristics, and impact of dnDSA after lung transplantation.We investigated a single-center cohort of 340 lung transplant recipients undergoing transplant during 2008 to 2011. All patients underwent HLA-antibody testing quarterly pretransplant and at regular intervals over the first 24 months after transplant. The patients received modified immunosuppression depending on their pretransplant sensitization status. Risk factors for dnDSA development, as well as the associations of dnDSA with patient survival and chronic lung allograft dysfunction (CLAD), were determined using multivariable analysis.The cumulative incidence of dnDSA was 47% at a median of 86 days (range, 44-185 d) after lung transplantation. Seventy-six percent of recipients with dnDSA had DQ-DSA. Male sex and the use of ex vivo lung perfusion were associated with an increased risk of dnDSA, whereas increased HLA-DQB1 matching was protective. DQ-dnDSA preceded or coincided with the diagnosis of CLAD in all cases. Developing dnDSA (vs. no dnDSA) was associated with a twofold increased risk of CLAD (hazard ratio, 2.04; 95% confidence interval, 1.13-3.69). This association appeared to be driven by the development of DQ-dnDSA.dnDSA are common after lung transplantation, with the majority being DQ DSA. DQ-dnDSA are associated with an increased risk of CLAD. Strategies to prevent or treat DQ-dnDSA may improve outcomes for lung transplant recipients.

Chronic inflammation, a hallmark of obliterative bronchiolitis, is known to induce lymphangiogenesis. We therefore studied the role of lymphangiogenic vascular endothelial growth factor C (VEGF-C), its receptor VEGFR-3, and lymphangiogenesis during development of experimental obliterative bronchiolitis [ie, obliterative airway disease (OAD)] in rat tracheal allografts. The functional importance of VEGF-C was investigated by adenovirus-mediated overexpression of VEGF-C (AdVEGF-C), and by inhibition of VEGF-C activity with VEGFR-3-Ig (AdVEGFR-3-Ig). Analyses included histology, immunohistochemistry, and real-time RT-PCR 10 and 30 days after transplantation. In the course of OAD development, lymphangiogenesis was induced in the airway wall during the alloimmune response, which was reversed by cyclosporine A in a dose-dependent fashion. VEGF-C overexpression in tracheal allografts induced epithelial activation, neutrophil chemotaxis, and a shift toward a Th17 adaptive immune response, followed by enhanced lymphangiogenesis and the development of OAD. In contrast, inhibition of VEGF-C activity with VEGFR-3-Ig inhibited lymphangiogenesis and angiogenesis and reduced infiltration of CD4(+) T cells and the development of OAD. Lymphangiogenesis was linked to T-cell responses during the development of OAD, and VEGF-C/VEGFR-3 signaling modulated innate and adaptive immune responses in the development of OAD in rat tracheal allografts. Our results thus suggest VEGFR-3-signaling as a novel strategy to regulate T-cell responses in the development of obliterative bronchiolitis after lung transplantation.

Background Obliterative bronchiolitis after lung transplantation is characterized by airway inflammation leading to obliteration of small airways. Statins are known to have lipid-independent immunomodulatory properties. We investigated the effect of simvastatin treatment on innate and adaptive immune responses and the development of obliterative airway disease (OAD). Methods In fully MHC-mismatched rat tracheal allograft recipients, we used simvastatin at different doses (0.1 to 20 mg/kg/day orally) to assess its effect on OAD development. No immunosuppressive treatment was administered. Histologic, immunohistochemical and real-time RT-PCR analyses were performed 3, 10 and 30 days after transplantation. Results Simvastatin treatment with doses ranging from 0.5 to 20 mg/kg/day significantly enhanced early epithelial recovery and reduced the development of OAD. No dose response was observed. Simvastatin treatment markedly reduced IL-23 mRNA and lymphocyte chemokine CCL20 production, and the infiltration of CD4 + and CD8 + T cells into allografts already at 3 days. At 10 days, simvastatin significantly attenuated the production of pro-inflammatory cytokines, IL-1β, TNF-α, MCP-1 and IP-10, and Th17-polarizing cytokines, IL-6 and IL-17e, and inhibited allograft infiltration by inflammatory cells. The protective effects of simvastatin on inflammation and OAD were partially mediated through nitric oxide synthase. Conclusions Simvastatin treatment inhibited adaptive T-cell alloimmune activation as depicted by reduced expression of lymphocyte chemokine and pro-inflammatory cytokine mRNA and reduced allograft infiltration by inflammatory cells. Importantly, simvastatin inhibits the development of OAD and this effect is partially mediated by increased nitric oxide activity. These results suggest a role for simvastatin in the prevention of obliterative bronchiolitis.

Endothelin-1 (ET-1) expression is increased after lung transplantation in association with ischemia reperfusion injury and acute rejection. However, little is known of the role of ET-1 during the development of obliterative bronchiolitis. In this study, we investigated the biological significance of ET-1 in obliterative airway disease development using a rat tracheal allograft model. Immunoreactivity of ET-1 and its receptors ET-RA and ET-RB was increased four-fold in allografts compared with syngrafts and localized to mononuclear cells and smooth muscle cells of the myofibroproliferative lesion and airway wall, indicating that ET-1 may mediate its effects in both a paracrine and autocrine manner in smooth muscle cells. Inhibition of ET-1 action by a nonselective ET-1 receptor antagonist, bosentan, significantly decreased tracheal occlusion, which was linked to delayed epithelial necrosis, suppressed smooth muscle cell proliferation, and a marked reduction in the number of interleukin-1beta and interleukin-2 immunoreactive cells. Our findings show that endogenous ET-1 activation is associated with obliteration of the airway wall, and blocking signaling downstream of ET-1 receptors leads to attenuation of obliterative airway disease. The results suggest that ET-1 has a proproliferative and proinflammatory role in the development of obliterative bronchiolitis.

Background — Cardiac allograft arteriosclerosis is a complex process of alloimmune response, chronic inflammation, and smooth muscle cell proliferation that includes cross talk between cytokines and growth factors. Methods and Results — Our results in rat cardiac allografts established alloimmune response as an alternative stimulus capable of inducing vascular endothelial growth factor (VEGF) mRNA and protein expression in cardiomyocytes and graft-infiltrating mononuclear inflammatory cells, which suggests that these cells may function as a source of VEGF to the cells of coronary arteries. Linear regression analysis of these allografts with different stages of arteriosclerotic lesions revealed a strong correlation between intragraft VEGF protein expression and the development of intimal thickening, whereas blockade of signaling downstream of VEGF receptor significantly reduced arteriosclerotic lesions. In addition, in cholesterol-fed rabbits, intracoronary perfusion of cardiac allografts with a clinical-grade adenoviral vector that encoded mouse VEGF 164 enhanced the formation of arteriosclerotic lesions, possibly secondary to increased intragraft influx of macrophages and neovascularization in the intimal lesions. Conclusions — Our findings suggest a positive regulatory role between VEGF and coronary arteriosclerotic lesion formation in the allograft cytokine microenvironment.

T cell activation is a proximal event in the initiation of chronic rejection that may ultimately lead to obliterative bronchiolitis (OB) after lung transplantation. In addition to primary signals generated by the T cell receptor, T cell activation relies on costimulatory signals, of which the most important are mediated via interaction between CD28 and its ligands B7-1 and B7-2. In nontreated rat tracheal allografts, B7-2, but not B7-1, expression peaked 10 d after transplantation, unlike in syngeneic grafts, where no B7-2 upregulation was observed. Selective blockade of the CD28/B7-1 T cell costimulatory pathway by a mutant form of CTLA4Ig (cytotoxic T lymphocyte antigen 4 immunoglobulin), CTLA4IgY100F, did not affect epithelial injury or degree of luminal occlusion in rat tracheal allografts. Treatment with CTLA4Ig fusion protein, which blocks both CD28/B7-1 and CD28/B7-2 interaction, significantly delayed the development of epithelial injury and airway occlusion. Immunohistochemical analyses of allografts showed that selective inhibition of the CD28/B7-1 pathway did not affect cytokine expression. In contrast, treatment with CTLA4Ig was associated with a significant decrease in the intragraft production of tumor necrosis factor alpha, interleukin 2, and interferon gamma, as well as slightly increased intragraft expression of interleukin 10. In conclusion, CTLA4Ig-mediated costimulatory blockade delays epithelial injury and attenuates obliterative changes and is associated with marked suppression of helper T cell type 1 (Th1)-dominated cytokine response. These observations emphasize the role of the CD28/B7-2 costimulatory pathway in regulating proinflammatory and Th1 cytokine responses and thereby in the development of epithelial and graft injury gradually leading to obliteration of the airway lumen.

Background: Activins are members of the TGF-ß superfamily of growth factors. First, we identified by expression array screening that activin-B and follistatin are upregulated in human idiopathic pulmonary fibrosis (IPF). Next, we wanted to clarify their specific role in lung fibrosis formation. Methods: We used specific antibodies for activin-A and -B subunits and follistatin to measure and localize their levels in idiopathic pulmonary fibrosis and control lung biopsies. To inhibit activin signaling, we used soluble activin type IIB receptor fused to the Fc portion of human IgG1 (sActRIIB-Fc) in two different mouse models of pulmonary fibrosis. Results: Activin-B and follistatin mRNA levels were elevated in the human IPF lung. Immunoreactivity to activin-A, -B and follistatin localized predominantly to the hyperplastic, activated alveolar epithelium, but was also seen in inflammatory cells. Mice treated with sActRIIB-Fc showed increased skeletal muscle mass and a clear reduction in alveolar cell counts in bronchoalveolar lavage fluid, but no significant antifibrotic effect in the lung was observed. Conclusions: The upregulation of activin-B and follistatin in IPF is a novel finding. Our results indicate that activin inhibition is not an efficient tool for antifibrotic therapy, but could be useful in reducing alveolar cellular response to injury. Activin-B and follistatin levels may be useful as biomarkers of IPF. peerReviewed

In this study, the prevention of rat cytomegalovirus (RCMV) infection-enhanced experimental obliterative bronchiolitis in rat tracheal allografts was investigated. RCMV infection markedly enhanced cell proliferation and histological changes of obliterative bronchiolitis, a form of chronic rejection after lung transplantation. These alterations were linked to increased interleukin (IL)-2 and tumor necrosis factor-alpha (TNF-alpha) immunoreactivity, and reduction of IL-10 expression. In recipient rats with acute RCMV infection, prophylaxis with either ganciclovir (DHPG) or hyperimmune serum (HIS) totally prevented RCMV infection-enhanced tracheal occlusion. DHPG treatment initiated during acute RCMV infection also reduced lesion development but markedly less than DHPG prophylaxis. Treatment of acute RCMV infection with HIS alone or in combination with DHPG had no significant effect on tracheal occlusion. Inhibition of the transcription of cytokines by high doses of cyclosporine A significantly reduced RCMV infection-enhanced tracheal obliteration. In rats with chronic RCMV infection, obliterative alterations were prevented by DHPG prophylaxis initiated at the time of transplantation. Prophylaxis either with DHPG or HIS did not affect the amount of infectious RCMV recovered from host salivary glands, nor were there differences seen in RCMV major immediate early DNA expression in tracheal allografts between different antiviral drug regimens. Immunohistochemical analysis of allografts revealed that inhibition of tracheal occlusion by antiviral prophylaxis was associated with a reduction in the number of ED1(+) macrophages and cells staining for Th1 cytokines and TNF-alpha, while immune modulation by cyclosporine A up-regulated IL-10 production. In conclusion, the results of the present study suggest that the CMV infection-enhanced chronic rejection develops independently of viral load but requires both immune activation and simultaneous CMV gene expression beyond immediate early genes.

Background— Ischemia/reperfusion injury may have deleterious short- and long-term consequences for cardiac allografts. The underlying mechanisms involve microvascular dysfunction that may culminate in primary graft failure or untreatable chronic rejection. Methods and Results— Here, we report that rat cardiac allograft ischemia/reperfusion injury resulted in profound microvascular dysfunction that was prevented by donor treatment with peroral single-dose simvastatin, a 3-hydroxy-3-methylglutaryl coenzyme A reductase and Rho GTPase inhibitor, 2 hours before graft procurement. During allograft preservation, donor simvastatin treatment inhibited microvascular endothelial cell and pericyte RhoA/Rho-associated protein kinase activation and endothelial cell–endothelial cell gap formation; decreased intragraft mRNA levels of hypoxia-inducible factor-1α, inducible nitric oxide synthase, and endothelin-1; and increased heme oxygenase-1. Donor, but not recipient, simvastatin treatment prevented ischemia/reperfusion injury–induced vascular leakage, leukocyte infiltration, the no-reflow phenomenon, and myocardial injury. The beneficial effects of simvastatin on vascular stability and the no-reflow phenomenon were abolished by concomitant nitric oxide synthase inhibition with N -nitro- l -arginine methyl ester and RhoA activation by geranylgeranyl pyrophosphate supplementation, respectively. In the chronic rejection model, donor simvastatin treatment inhibited cardiac allograft inflammation, transforming growth factor-β1 signaling, and myocardial fibrosis. In vitro, simvastatin inhibited transforming growth factor-β1–induced microvascular endothelial-to-mesenchymal transition. Conclusions— Our results demonstrate that donor simvastatin treatment prevents microvascular endothelial cell and pericyte dysfunction, ischemia/reperfusion injury, and chronic rejection and suggest a novel, clinically feasible strategy to protect cardiac allografts.

Platelet-derived growth factor (PDGF) is an important smooth muscle cell mitogen, and vascular endothelial growth factor (VEGF) is a known angiogenic and proinflammatory growth factor. We hypothesized that specific therapy aimed at these growth factors might inhibit the development of experimental obliterative airway disease (OAD).In fully mismatched rat tracheal allografts, we used imatinib and PTK/ZK, either alone or in combination, to block PDGF and VEGF receptor protein tyrosine kinase (RTK) action, respectively. Prophylaxis was initiated at the time of transplantation. Early treatment was commenced on Day 7 during the inflammatory phase and late treatment on Day 14 during the fibroproliferative phase of OAD. No immunosuppression was administered.Prophylaxis with either PTK/ZK or imatinib alone significantly reduced OAD, and combined prophylaxis completely prevented its development. Early treatment with PTK/ZK and imatinib also effectively reduced the development of OAD. Late treatment failed to show significant efficacy. Blocking VEGF RTK action with PTK/ZK reduced the activation of allograft blood vessels and the number of lymph vessels in the allograft airway wall, and significantly diminished allograft inflammation, whereas PDGF blockade with imatinib inhibited the growth of smooth muscle cells in the proliferating lesion.Combined prophylactic PDGF and VEGF RTK blockade completely prevents the development of OAD. Also, when early treatment with PTK/ZK and imatinib is commenced during the inflammatory phase of OAD development, it significantly attenuates the development of tracheal occlusion, suggesting that these drugs could potentially be used to treat bronchiolitis obliterans syndrome in its early phase.

Obliterative bronchiolitis (OB) is the major limitation for long-term survival of lung allograft recipients. We investigated the role of vascular endothelial growth factor (VEGF) in the development of OB in rat tracheal allografts. In nonimmunosuppressed allografts, VEGF mRNA and protein expression vanished in the epithelium and increased in smooth muscle cells and mononuclear inflammatory cells with progressive loss of epithelium and airway occlusion compared with syngeneic grafts. Intragraft VEGF overexpression by adenoviral transfer of a mouse VEGF(164) gene increased early epithelial cell proliferation and regeneration but increased microvascular remodeling and lymphangiogenesis and luminal occlusion by more than 50% compared with AdlacZ-treated allografts. Although VEGF receptor inhibition decreased early epithelial regeneration in noninfected allografts, it reduced microvascular remodeling, lymphangiogenesis, intragraft traffic of CD4(+) and CD8(+) T cells, and the degree of luminal occlusion. Simultaneous VEGF gene transfer and platelet-derived growth factor receptor inhibition with imatinib preserved respiratory epithelium and totally prevented luminal occlusion. In conclusion, our findings indicate that VEGF has a dual role in transplant OB. Our results suggest that VEGF may protect epithelial integrity. On the other hand, VEGF may enhance luminal occlusion by increasing the recruitment of mononuclear inflammatory cells with platelet-derived growth factor acting as a final effector molecule in this process.

Transplant coronary artery disease (TxCAD) as a manifestation of chronic rejection is a major limitation to long-term survival of heart transplant recipients. Although the exact molecular and cellular mechanisms contributing to neointimal formation are unknown, it has been generally believed that smooth muscle cells (SMC) of donor origin migrate from the media into the subendothelial layer of the vascular wall, where SMC proliferate and synthesize extracellular matrix resulting in intimal thickening. However, recent observations indicate that hematopoietic and vascular progenitor cells derived from recipient bone marrow may contribute to the arteriosclerotic lesion formation in the coronary arteries of the transplant. On the other hand, studies on postnatal hematopoiesis indicate that angiogenic growth factors such as vascular endothelial growth factor (VEGF) and angiopoietin-1 (Ang1) may regulate the recruitment of these cells into distant organs. Furthermore, embryonic VEGFR-2 /CD34+ stem cells may serve as vascular progenitor cells and their differentiation into endothelial cells and SMC may be regulated by VEGF and platelet-derived growth factor (PDGF), respectively. In this review, we discuss the role of angiogenic growth factors such as VEGF, Ang, and PDGF in the recruitment of hematopoietic and vascular progenitor cells in TxCAD and suggest novel therapies targeted at homing, differentiation and proliferation of these cells in the allograft.