Inhibition of Vascular Endothelial Growth Factor Receptors 1 and 2 Attenuates Natural Killer Cell and Innate Immune Responses in an Experimental Model for Obliterative Bronchiolitis.

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+ cells and Cd4+ 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.

Eosinophils in transbronchial biopsies: a predictor of chronic lung allograft dysfunction and reduced survival after lung transplantation - a retrospective single-center cohort study.

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.

Extending cytomegalovirus prophylaxis in high-risk (D+/R-) lung transplant recipients from 6 to 9 months reduces cytomegalovirus disease: A retrospective study.

RATIONALE: Cytomegalovirus (CMV)-seronegative recipients receiving a seropositive allograft (D+/R-) are at a high risk of developing CMV disease. Our program increased the duration of CMV prophylaxis from 6 to 9 months in May 2013. Here, we present the impact on the incidence of CMV infection, disease, side effects, rejection, and other factors. METHODS: Retrospective cohort of 241 CMV (D+/R-) patients transplanted between January 1, 2008, and December 31, 2017. Blood CMV testing was done according to protocol. All patients received ganciclovir/valganciclovir as prophylaxis. We compared the incidence and timing of CMV infection and disease up to 6 months after cessation of prophylaxis between patients who received 9 months (May 2013 onwards) and a historical control group who received 6 months of prophylaxis (prior to May 2013). CMV infection was defined as detectable CMV viremia in the absence of symptoms. CMV disease was defined as CMV syndrome or tissue-invasive disease. Side effects of prophylaxis and CMV resistance were recorded. RESULTS: A total of 116 patients were included in the 6-month group and 125 in the 9-month group. The extended 9-month CMV prophylaxis delayed the onset of CMV infection (median time to CMV infection after lung transplantation 295 vs 353 days, P < .01) but did not significantly reduce the incidence of CMV infection (65% vs 64%, P = .06, log-rank). The 9-month prophylaxis delayed the onset and decreased the incidence of CMV disease from 50% in the 6-month group to 42% (P = .02 log-rank). There was no difference in the rate of adverse effects (leukopenia in 32% in both groups, P = .53) or development of CMV resistance between the two groups (4 cases in both groups, P = .92). There were no significant differences in overall survival or the rate of chronic lung allograft dysfunction between the groups. CONCLUSIONS: Extending duration of CMV prophylaxis from 6 to 9 months resulted in a delayed and decreased incidence of CMV disease in our lung transplant population. The absolute risk reduction achieved by extended CMV prophylaxis was 8%. The incidence of CMV infection, and ganciclovir resistance and side effects were similar between the two groups. Our results suggest that extending CMV prophylaxis in the highest risk CMV D+/R- group is effective in reducing CMV disease.

Epithelial cell death markers in bronchoalveolar lavage correlate with chronic lung allograft dysfunction subtypes and survival in lung transplant recipients-a single-center retrospective cohort study.

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.

De Novo DQ Donor-Specific Antibodies Are Associated with Chronic Lung Allograft Dysfunction after Lung Transplantation.

RATIONALE: 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. OBJECTIVES: To describe the incidence, characteristics, and impact of dnDSA after lung transplantation. METHODS: 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. MEASUREMENTS AND MAIN RESULTS: 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. CONCLUSIONS: 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.

Bone morphogenetic protein-inducer tilorone identified by high-throughput screening is antifibrotic in vivo.

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-ß 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-ß 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.

Critical role of VEGF-C/VEGFR-3 signaling in innate and adaptive immune responses in experimental obliterative bronchiolitis.

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.

Donor simvastatin treatment abolishes rat cardiac allograft ischemia/reperfusion injury and chronic rejection through microvascular protection.

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-ß1 signaling, and myocardial fibrosis. In vitro, simvastatin inhibited transforming growth factor-ß1-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.

Long-term Outcomes of Lung Transplant With Ex Vivo Lung Perfusion.

Importance: 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. Objective: To assess the long-term outcomes of transplant recipients of donor lungs treated with EVLP. Design, Setting, and Participants: 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. Exposure: Donor lungs undergoing EVLP. Main Outcomes and Measures: 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. Results: 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. Conclusions and Relevance: 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.

VEGFR-1 and -2 regulate inflammation, myocardial angiogenesis, and arteriosclerosis in chronically rejecting cardiac allografts.

OBJECTIVE: Interplay between inflammation and angiogenesis is important in pathological reparative processes such as arteriosclerosis. We investigated how the two vascular endothelial growth factor receptors VEGFR-1 and -2 regulate these events in chronically rejecting cardiac allografts. METHODS AND RESULTS: Chronic rejection in mouse cardiac allografts induced primitive myocardial, adventitial, and intimal angiogenesis with endothelial expression of CD31, stem cell marker c-kit, and VEGFR-2. Experiments using marker gene mice or rats as cardiac allograft recipients revealed that replacement of cardiac allograft endothelial cells with recipient bone marrow- or non-bone marrow-derived cells was rare and restricted only to sites with severe injury. Targeting VEGFR-1 with neutralizing antibodies in mice reduced allograft CD11b+ myelomonocyte infiltration and allograft arteriosclerosis. VEGFR-2 inhibition prevented myocardial c-kit+ and CD31+ angiogenesis in the allograft, and decreased allograft inflammation and arteriosclerosis. CONCLUSIONS: These results suggest interplay of inflammation, primitive donor-derived myocardial angiogenesis, and arteriosclerosis in transplanted hearts, and that targeting VEGFR-1 and -2 differentially regulate these pathological reparative processes.

Angiogenic growth factors in cardiac allograft rejection.

Normal adult vasculature is in a quiescent state. In transplanted hearts, peri- and postoperative ischemic and alloimmune stimuli may be interpreted as inadequate tissue perfusion leading to activation of angiogenic signaling. Although this may have protective functions, improper activation of cardiac allograft endothelial cells and smooth muscle cells may actually result in impaired survival of cardiac allografts. In this paper, we review the current knowledge on angiogenic growth factors, vascular endothelial growth factor, angiopoietins, and platelet-derived growth factor in cardiac allografts. We also discuss the potential for therapies aimed at angiogenic growth factors in preventing and treating cardiac allograft rejection and transplant coronary artery disease.

Increased myeloid cell hypoxia-inducible factor-1 delays obliterative airway disease in the mouse.

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-1α that controls the activity of HIF-1. We investigated the effect of myeloid cell-targeted gene deletion of HIF-1α 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-1α 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-1α activity in myeloid cells of the recipient by HIF-1α 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.

Vascular endothelial growth factor enhances cardiac allograft arteriosclerosis.

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.

Combined vascular endothelial growth factor and platelet-derived growth factor inhibition in rat cardiac allografts: beneficial effects on inflammation and smooth muscle cell proliferation.

BACKGROUND: Perivascular inflammation and subsequent smooth muscle cell (SMC) proliferation are central in the development of cardiac allograft arteriosclerosis. We examined the effect of combined inhibition of proinflammatory vascular endothelial growth factor (VEGF) and SMC mitogen platelet-derived growth factor (PDGF) in rat cardiac allografts. METHODS: Heterotopic cardiac transplantations were performed between fully major histocompatibility mismatched rat strains receiving cyclosporine A immunosuppression. In situ hybridization and immunohistochemistry were performed to examine VEGF and PDGF ligand and receptor (R) expression. Protein tyrosine kinase inhibitors PTK787 and imatinib were used to inhibit VEGFR and PDGFR activity, respectively. Rat coronary artery SMC migration and proliferation assays were used to examine the effect of VEGF and PDGF and tyrosine kinase inhibitors in vitro. RESULTS: Both ligand and receptor expression of VEGF and PDGF were detected in chronically rejecting allografts. In vitro, PDGF-BB mediated rat coronary artery SMC migration and proliferation was completely inhibited with imatinib and partially with PTK787. In vivo, combined treatment with PTK787 and imatinib significantly reduced the formation of neointimal lesions in arteries of cardiac allografts at 8 weeks, producing a greater effect than either drug alone. PTK787, in contrast with imatinib, reduced the number of ED1 macrophages and PDGF-B immunoreactivity in the allografts at 4 weeks. CONCLUSIONS: Blocking VEGF and PDGF receptor signaling in cardiac allografts has distinctive effects on inflammation and SMC proliferation, suggesting that targeting both inflammation and pathologic vascular remodeling may be needed to inhibit cardiac allograft arteriosclerosis.

Functional outcomes and quality of life after normothermic ex vivo lung perfusion lung transplantation.

BACKGROUND: Ex vivo lung perfusion (EVLP) is an effective method to assess and improve the function of otherwise unacceptable lungs, alleviating the shortage of donor lungs. The early results with EVLP have been encouraging, but longer-term results, including functional and patient-reported outcomes, are not well characterized. METHODS: This retrospective single-center study included all lung transplants performed between September 2008 and December 2012. We investigated whether survival or rate of chronic lung allograft dysfunction (CLAD) differed in recipients of EVLP-treated lungs compared with contemporaneous recipients of conventional donor lungs. We also studied functional (highest forced expiratory volume in 1 second predicted, change in 6-minute walk distance, number of acute rejection episodes) and quality of life outcomes. RESULTS: Of 403 lung transplants that were performed, 63 patients (15.6%) received EVLP-treated allografts. Allograft survival for EVLP and conventional donor lung recipients was 79% vs 85%, 71% vs 73%, and 58% vs 57% at 1, 3, and 5 years after transplant, respectively (log-rank p = not significant). Freedom from CLAD was also similar (log-rank p = 0.53). There were no significant differences in functional outcomes such as highest forced expiratory volume in 1 second predicted (76.5% ± 23.8% vs 75.8% ± 22.8%, p = 0.85), change in 6-minute walk distance (194 ± 108 meters vs 183 ± 126 meters, p = 0.57), or the number of acute rejection episodes (1.5 ± 1.4 vs 1.3 ± 1.3, p = 0.36). The EVLP and conventional donor groups both reported a significantly improved quality of life after transplantation, but there was no intergroup difference. CONCLUSION: EVLP is a safe and effective method of assessing and using high-risk donor lungs before transplantation and leads to acceptable long-term survival, graft function, and improvements of quality of life that are comparable with conventionally selected donor lungs.

Platelet-derived growth factor regulates cytomegalovirus infection-enhanced obliterative bronchiolitis in rat tracheal allografts.

BACKGROUND: Cytomegalovirus (CMV) infection is a risk factor for the development of obliterative bronchiolitis (OB) after lung transplantation. METHODS: In the rat tracheal allograft model, rat CMV (RCMV) infection is associated with accelerated OB through enhanced alloimmune activation and increased smooth muscle cell (SMC) proliferation. Using this model, we investigated the role of platelet-derived growth factor (PDGF) in RCMV infection-enhanced OB. RESULTS: Immunohistochemistry and in situ hybridization revealed that RCMV infection significantly up-regulates PDGF ligand and receptor expression in inflammatory and SMC-like cells in tracheal allografts. Selective inhibition of PDGF receptor tyrosine kinase activity by CGP 53716 prevents the development of OB in RCMV-infected allograft recipients. CONCLUSION: The results of this study emphasize the key regulatory role of PDGF in the pathogenesis of RCMV infection-enhanced OB, suggesting a novel strategy for the prevention of this fibroproliferative disorder.

Platelet-derived growth factor receptor inhibition reduces allograft arteriosclerosis of heart and aorta in cholesterol-fed rabbits.

BACKGROUND: Crosstalk between pro-inflammatory cytokines and platelet-derived growth factor (PDGF) regulates smooth-muscle-cell proliferation in cardiac-allograft arteriosclerosis. In this study, we tested the effect of STI 571, a novel orally active protein tyrosine kinase (PTK) inhibitor selective for PDGF receptor (PDGF-R) on transplant and accelerated arteriosclerosis in hypercholesterolemic rabbits. METHODS: Cardiac allografts were transplanted heterotopically from Dutch Belted to New Zealand White rabbits. A 0.5% cholesterol diet was begun 4 days before transplantation. Recipients received STI 571 5 mg/kg per day or vehicle intraperitoneally throughout the study period of 6 weeks. Cyclosporine A was given as background immunosuppression. RESULTS: In cardiac allografts of vehicle-treated rabbits, 76.2+/-2.1% of medium-sized arteries were affected by intimal thickening, and the percentage of arterial occlusion was 45.0+/-5.0%. Treatment with STI 571 reduced the incidence of affected medium-sized arteries to 41.2+/-8.1% (P <0.05) and the arterial occlusion to 27.6+/-5.0% ( P<0.05). In addition, we observed that STI 571 treatment reduced intimal lesion formation in proximal ascending aorta of transplanted hearts from 72.3+/-19.9 to 12.7+/-1.9 microm ( P<0.05). Our results also show that STI 571 significantly inhibited accelerated arteriosclerosis in medium-sized arteries of recipients' own hearts. CONCLUSIONS: The results of the present study suggest that PDGF-R activation may regulate the development of transplant and accelerated arteriosclerosis in hypercholesterolemic rabbits. Thus, PTK inhibitors may provide new strategies for prevention of these fibroproliferative vascular disorders.

Effect of simvastatin on development of obliterative airway disease: an experimental study.

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-1ß, 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.