Background— Lymphatic network and chemokine-mediated signals are essential for leukocyte traffic during the proximal steps of alloimmune response. We aimed to determine the role of lymphatic vessels and their principal growth signaling pathway, vascular endothelial growth factor (VEGF)-C/D/VEGFR-3, during acute and chronic rejection in cardiac allografts. Methods and Results— Analysis of heterotopically transplanted rat cardiac allografts showed that chronic rejection increased VEGF-C ⁺ inflammatory cell and hyaluronan receptor-1 (LYVE-1) ⁺ lymphatic vessel density. Allograft lymphatic vessels were VEGFR-3 ⁺ , contained antigen-presenting cells, and produced dendritic cell chemokine CCL21. Experiments with VEGFR-3/LacZ mice or mice with green fluorescent protein–positive bone marrow cells as cardiac allograft recipients showed that allograft lymphatic vessels originated almost exclusively from donor cells. Intraportal adenoviral VEGFR-3-Ig (Ad.VEGFR-3-Ig/VEGF-C/D-Trap) perfusion was used to inhibit VEGF-C/D/VEGFR-3 signaling. Recipient treatment with Ad.VEGFR-3-Ig prolonged rat cardiac allograft survival. Ad.VEGFR-3-Ig did not affect allograft lymphangiogenesis but was linked to reduced CCL21 production and CD8 ⁺ effector cell entry in the allograft. Concomitantly, Ad.VEGFR-3-Ig reduced OX62 ⁺ dendritic cell recruitment and increased transcription factor Foxp3 expression in the spleen. In separate experiments, treatment with a neutralizing monoclonal VEGFR-3 antibody reduced arteriosclerosis, the number of activated lymphatic vessels expressing VEGFR-3 and CCL21, and graft-infiltrating CD4 ⁺ T cells in chronically rejecting mouse cardiac allografts. Conclusions— These results show that VEGFR-3 participates in immune cell traffic from peripheral tissues to secondary lymphoid organs by regulating allograft lymphatic vessel CCL21 production and suggest VEGFR-3 inhibition as a novel lymphatic vessel–targeted immunomodulatory therapy for cardiac allograft rejection and arteriosclerosis.

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.

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. 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. 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. 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.

Ex vivo lung perfusion (EVLP) is an excellent platform to apply novel therapeutics, such as gene and cell therapies, before lung transplantation. We investigated the concept of human donor lung engineering during EVLP by combining gene and cell therapies. Premodified cryopreserved mesenchymal stromal cells with augmented anti-inflammatory interleukin-10 production (MSC^IL-10) were administered during EVLP to human lungs that had various degrees of underlying lung injury. Cryopreserved MSC^IL-10 had excellent viability, and they immediately and efficiently elevated perfusate and lung tissue IL-10 levels during EVLP. However, MSC^IL-10 function was compromised by the poor metabolic conditions present in the most damaged lungs. Similarly, exposing cultured MSC^IL-10 to poor metabolic, and especially acidic, conditions decreased their IL-10 production. In conclusion, we found that “off-the-shelf” MSC^IL-10 therapy of human lungs during EVLP is safe and feasible, and results in rapid IL-10 elevation, and that the acidic target-tissue microenvironment may compromise the efficacy of cell-based therapies.

Angiopoietin-1 (Ang1) and Ang2 regulate the maintenance of normal vasculature by direct endothelial and indirect smooth muscle cell (SMC) effects. Dysfunction of vascular wall cells is considered central in cardiac allograft vasculopathy (CAV), where inflammation and arterial injury initiate subsequent intimal SMC proliferation. In this study, we investigated the effect of exogenous Ang1 and Ang2 in chronically rejecting rat cardiac allografts by intracoronary adeno-associated virus (AAV)-mediated gene transfer. Bioluminescent imaging of AAV-transfected syngeneic grafts revealed gradual and stable transgene expression in graft cardiomyocytes. In cardiac allografts, both AAV-Ang1 and AAV-Ang2 decreased inflammation and increased antiapoptotic Bcl-2 mRNA and Bcl-2/Bax ratio at 8 weeks. Only AAV-Ang2 decreased the development of CAV, whereas AAV-Ang1 activated arterial SMC and increased PDGF-A mRNA in the allograft. Collectively, our results show that exogenous Ang1 and Ang2 have similar antiinflammatory and antiapoptotic effects in cardiac allografts. Prolonged AAV-mediated Ang1 transgene expression also induced SMC activation, whereas AAV-Ang2 lacked the SMC activating effects and decreased CAV. Our results thus highlight the common protective and diverse SMC effects of Ang1 and Ang2 in cardiac allograft microenvironment and the importance of timing of angiopoietins to achieve therapeutic effects.

Objectives. Lung transplantation remains the only available treatment option for many end-stage lung diseases. We evaluated our long-term lung transplantation results and the impact of chronic lung allograft dysfunction (CLAD). Design. Adult de novo lung transplants (2003-2015, n=175) in a nationwide single transplant center were retrospectively analyzed. Kaplan–Meier survival and Cox regression analysis were used to evaluate the effect of CLAD. Results. Recipient and graft 1-, 5- and 10-year survival estimates were 94%, 79% and 64%, and 93%, 75% and 59%, respectively. CLAD affected 43% of patients at a median of 2.3 years after transplantation, and impaired recipient (p = .03) and graft survival (p = .001) with the most advanced CLAD stage, and restrictive CLAD phenotype, resulting in worst graft survival. CLAD was the primary cause of death in 54% of all patients, and in 80% of patients with an established CLAD diagnosis. CLAD, high-risk cytomegalovirus serostatus, and recipient preoperative sensitization increased graft loss hazard ratio. CLAD was the only significant investigated risk factor for graft loss in multivariate regression analysis. Conclusions. Although very favourable lung transplant patient long-term survival was achieved, CLAD significantly impaired recipient and graft survival. Identification of risk factors and therapeutic options for CLAD may further improve lung transplantation results.