Creating superior lungs for transplantation with next-generation gene therapy during ex vivo lung perfusion.

Engineering donor organs to better tolerate the harmful non-immunological and immunological responses inherently related to solid organ transplantation would improve transplant outcomes. Our enhanced knowledge of ischemia-reperfusion injury, alloimmune responses and pathological fibroproliferation after organ transplantation, and the advanced toolkit available for gene therapies, have brought this goal closer to clinical reality. Ex vivo organ perfusion has evolved rapidly especially in the field of lung transplantation, where clinicians routinely use ex vivo lung perfusion (EVLP) to confirm the quality of marginal donor lungs before transplantation, enabling safe transplantation of organs originally considered unusable. EVLP would also be an attractive platform to deliver gene therapies, as treatments could be administered to an isolated organ before transplantation, thereby providing a window for sophisticated organ engineering while minimizing off-target effects to the recipient. Here, we review the status of lung transplant first-generation gene therapies that focus on inducing transgene expression in the target cells. We also highlight recent advances in next-generation gene therapies, that enable gene editing and epigenetic engineering, that could be used to permanently change the donor organ genome and to induce widespread transcriptional gene expression modulation in the donor lung. In a future vision, dedicated organ repair and engineering centers will use gene editing and epigenetic engineering, to not only increase the donor organ pool, but to create superior organs that will function better and longer in the recipient.

Access site complications in thoracic endovascular aortic repair.

BACKGROUND: Percutaneous access and use of vascular closure devices facilitate thoracic endovascular aortic repair (TEVAR) procedures during local anesthesia and allow immediate detection of signs of spinal ischemia. However, the very large bore access (usually ≥22F sheath) associated with TEVAR increases the risk of vascular complications. In this study, we sought to define the safety and feasibility of two percutaneous femoral artery closure devices during TEVAR, in terms of access site vascular complications and major, life-threatening, or fatal bleeding (≥major) within 48 hours. Access site vascular complications were defined as technical failure of vascular closure or later formation of pseudoaneurysm. METHODS: From March 2010 to December 2022, 199 transfemoral TEVAR were performed at Helsinki University Central Hospital, Finland. We retrospectively categorized these into three groups, based on surgeon preference for the access technique and femoral artery closure method: (1) surgical cut-down and vessel closure, n = 85 (42.7%), (2) percutaneous access and vascular closure with suture-based ProGlide, n = 56 (28.1%), or (3) percutaneous access and vascular closure with ultrasound-guided plug-based MANTA, n = 58 (29.1%). The primary outcome measure was technical success of vascular closure and access site vascular complications during index hospitalization. Secondary outcome measures were ≥major bleeding, early mortality, and hospital stay. RESULTS: The technical success rate was 97.6% vs 91.1% vs 93.1% for surgical cut-down, ProGlide, and MANTA, respectively (P = .213). The rate of access site vascular complication was 3.5% vs 8.9% vs 10.3%, respectively (P = .290), with two pseudoaneurysms detected postoperatively and conservatively managed in the MANTA group. The vascular closure method was not associated with increased risk of ≥major bleeding, early mortality, or hospital stay on univariate analysis. Predictors for ≥major bleeding after TEVAR in multivariable analysis were urgent procedure (odds ratio: 2.8, 95% confidence interval: 1.4-5.5; P = .003) and simultaneous aortic branch revascularization (odds ratio: 2.7, 95% confidence interval: 1.3-5.4; P = .008). CONCLUSIONS: In this study, the technical success rates of the percutaneous techniques demonstrated their feasibility during TEVAR. However, the number of access site complications for percutaneous techniques was higher compared with open approach, although the difference was not statistically significant. In the lack of evidence, the safety of the new MANTA plug-based vascular closure for TEVAR warrants further investigation.

Transcriptomic Landscape of Circulating Extracellular Vesicles in Heart Transplant Ischemia-Reperfusion.

Ischemia-reperfusion injury (IRI) is an inevitable event during heart transplantation, which is known to exacerbate damage to the allograft. However, the precise mechanisms underlying IRI remain incompletely understood. Here, we profiled the whole transcriptome of plasma extracellular vesicles (EVs) by RNA sequencing from 41 heart transplant recipients immediately before and at 12 h after transplant reperfusion. We found that the expression of 1317 protein-coding genes in plasma EVs was changed at 12 h after reperfusion. Upregulated genes of plasma EVs were related to metabolism and immune activation, while downregulated genes were related to cell survival and extracellular matrix organization. In addition, we performed correlation analyses between EV transcriptome and intensity of graft IRI (i.e., cardiomyocyte injury), as well as EV transcriptome and primary graft dysfunction, as well as any biopsy-proven acute rejection after heart transplantation. We ultimately revealed that at 12 h after reperfusion, 4 plasma EV genes (ITPKA, DDIT4L, CD19, and CYP4A11) correlated with both cardiomyocyte injury and primary graft dysfunction, suggesting that EVs are sensitive indicators of reperfusion injury reflecting lipid metabolism-induced stress and imbalance in calcium homeostasis. In conclusion, we show that profiling plasma EV gene expression may enlighten the mechanisms of heart transplant IRI.

Mesenchymal Stromal Cell Therapy in Lung Transplantation.

Lung transplantation is often the only viable treatment option for a patient with end-stage lung disease. Lung transplant results have improved substantially over time, but ischemia-reperfusion injury, primary graft dysfunction, acute rejection, and chronic lung allograft dysfunction (CLAD) continue to be significant problems. Mesenchymal stromal cells (MSC) are pluripotent cells that have anti-inflammatory and protective paracrine effects and may be beneficial in solid organ transplantation. Here, we review the experimental studies where MSCs have been used to protect the donor lung against ischemia-reperfusion injury and alloimmune responses, as well as the experimental and clinical studies using MSCs to prevent or treat CLAD. In addition, we outline ex vivo lung perfusion (EVLP) as an optimal platform for donor lung MSC delivery, as well as how the therapeutic potential of MSCs could be further leveraged with genetic engineering.

Upregulation of Coagulation Factor VIII and Fibrinogen After Pulmonary Endarterectomy in Patients with Chronic Thromboembolic Pulmonary Hypertension.

OBJECTIVES: Chronic thromboembolic pulmonary hypertension (CTEPH) is associated with thrombotic states including elevated coagulation factor VIII (FVIII). Pulmonary endarterectomy (PEA) is the main treatment for CTEPH, and efficient anticoagulation is essential to prevent thromboembolism recurrence after surgery. We aimed to characterize longitudinal changes in FVIII and other coagulation biomarkers after PEA. METHODS: Coagulation biomarker levels were measured at baseline and up to 12 months after operation in 17 consecutive patients with PEA. Temporal patterns of coagulation biomarkers, and correlation of FVIII with other coagulation biomarkers, were analyzed. RESULTS: Baseline FVIII levels were elevated in 71% of the patients (mean 216 ± 67 IU/dl). FVIII doubled 7 days after PEA, peaking at 471 ± 87 IU/dl, and gradually returned to respective baseline levels within 3 months. Postoperative fibrinogen levels were also elevated. Antithrombin decreased at 1 to 3 days, D-dimer increased at 1 to 4 weeks, and thrombocytosis was observed at 2 weeks. CONCLUSIONS: FVIII is elevated in most patients with CTEPH. After PEA, early but transient elevation of FVIII and fibrinogen, and delayed reactive thrombocytosis, occurs, and warrants careful postoperative anticoagulation to prevent thromboembolism recurrence.

Successful 3-day lung preservation using a cyclic normothermic ex vivo lung perfusion strategy.

BACKGROUND: Cold static preservation (CSP) at higher temperatures (10°C) has been recently shown as an optimal strategy up to 24-36h of preservation. Here, we hypothesized that alternating 10°C static storage with cycles of normothermic ex vivo lung perfusion (EVLP) would provide conditions for cellular "recharge", allowing for multi-day lung preservation. METHODS: Donor lungs from male Yorkshire pigs were preserved using 10°C CSP with two cycles of 4h EVLP. After a total of 3 days of preservation, a left lung transplant was performed followed by 4h of graft evaluation. As controls, 2 lungs were preserved solely with continuous 10°C preservation for 3 days and transplanted. FINDINGS: For animals receiving lungs preserved using a cyclic EVLP protocol, lung function and histological structures were stable and the recipient systemic partial pressure of oxygen/fraction of inspired oxygen (P/F Ratio) after excluding the contralateral lung was 422 ± 61 mmHg. In contrast, lungs preserved solely in continuous cold static storage at 10°C for 72h developed massive lung failure, resulting in recipient death. Metabolomic analysis revealed that EVLP plays a critical role in the re-vitalization of key central carbon energy metabolites (Glucose, Succinate, N-Acetyl Aspartate) and reducing the expression of the inflammasome activation marker CASP1. INTERPRETATION: In conclusion, we demonstrate for the first time the feasibility of 3-day lung preservation leading to excellent early post-transplant outcomes. The thoughtful combination of cold storage (10°C) and intermittent EVLP can open new opportunities in organ transplantation. FUNDING: This work was supported by the UHN Foundation (Grant#1013612).

Near-infrared fluorescence imaging during ex vivo lung perfusion: Noninvasive real-time evaluation of regional lung perfusion and edema.

OBJECTIVE: Ex vivo lung perfusion (EVLP) is an excellent platform to evaluate donor lung function before transplantation, but novel methods are needed to accurately confirm transplant quality. Near-infrared fluorescence (NIRF) imaging with indocyanine green (ICG) has been used in various clinical perioperative applications to evaluate tissue perfusion. We used NIRF imaging during pig and human EVLP to evaluate donor lung perfusion and edema. METHODS: Pig lungs with various degrees of lung injury (n = 10) and human lungs rejected from clinical transplantation (n = 3) were imaged during EVLP using intravascular ICG and a SPY Elite (Stryker) NIRF imaging unit. Optimal ICG and imaging conditions, and perfusion and edema quantification methods, were established. Pig lung transplants with extended graft preservation (n = 5) and control native lungs (n = 6) were also imaged. RESULTS: A single ICG dose resulted in sustained donor lung NIRF throughout the EVLP. Even and homogenous ICG signal was demonstrated in areas of normal lung. Low NIRF was present in regions with poor tissue perfusion, and rapid, intense ICG accumulation occurred in damaged and edematous areas. Segmental perfusion defects were common in the peripheral and elevated regions of the lungs, and serial imaging showed gradual perfusion recovery during EVLP. Impaired microvascular reperfusion, indicated by a decreased NIRF ingress rate, was detected in transplanted pig lungs early after reperfusion. CONCLUSIONS: NIRF imaging enables noninvasive real-time evaluation of lung perfusion and edema during EVLP. Prospective clinical studies are needed to determine the role of NIRF imaging in donor lung assessment and selection, and prediction of posttransplant outcomes.

Chronic lung allograft dysfunction subtype analysis by computed tomography volumetry.

BACKGROUND: Chronic lung allograft dysfunction (CLAD) limits long-term survival after lung transplantation. Of the two subtypes, restrictive allograft syndrome (RAS) is characterized by a larger lung volume decrease and worse prognosis than bronchiolitis obliterans syndrome (BOS). We used computed tomography (CT) volumetry to classify CLAD subtypes and determined their clinical impact. METHODS: Adult primary lung transplants performed 2003-2015 (n = 167) were retrospectively evaluated for CLAD and subclassified with CT volumetry. Lung volume decrease of < 15% from baseline resulted in BOSCT-vol and ≥15% resulted in RASCT-vol diagnosis. Clinical impact of CLAD subtypes was defined, and the prognostic value of different lung function, radiological, and lung volume parameters present at the time of CLAD diagnosis were compared. RESULTS: CLAD affected 43% of patients and was classified with CT volumetry as BOSCT-vol in 89% and RASCT-vol in 11%. Median graft survival estimate in RASCT-vol was significantly decreased compared to BOSCT-vol (1.6 vs. 9.7 years, P = .038). At CLAD onset, RASCT-vol diagnosis (P = .05), increased lung density (P = .007), and more severe FEV1 (P = .004) decline from baseline, increased graft loss risk in multivariate analysis. CONCLUSIONS: CT volumetry serves to identify lung transplant patients with a poor clinical outcome but should be validated in prospective trials.

Pseudo Heparin Resistance After Pulmonary Endarterectomy: Role of Thrombus Production of Factor VIII.

Pulmonary endarterectomy (PEA) is the main treatment for chronic thromboembolic pulmonary hypertension (CTEPH). Postoperative unfractionated heparin dosing can be monitored by activated partial thromboplastin time (APTT) or by anti-factor Xa activity (anti-Xa). In pseudo heparin resistance, APTT response to heparin is blunted due to elevated Factor VIII (FVIII) which can underestimate anticoagulation. We examined possible pseudo heparin resistance after PEA and assessed the impact of FVIII. APTT response to heparin before and after operation was determined in 13 PEA patients anticoagulated with unfractionated heparin. APTT and anti-Xa concordance was analyzed from paired postoperative samples, and antithrombin, fibrinogen and FVIII levels were measured. Single-cell RNA sequencing was used to characterize FVIII gene expression in PEA specimens of 5 patients. APTT response to heparin was blunted after PEA. APTT and anti-Xa were discordant in 36% of postoperative samples and most common discordant patterns were subtherapeutic APTT with therapeutic (16%) or supratherapeutic (11%) anti-Xa. Overall, APTT underestimated anticoagulation relative to anti-Xa in one-third of the samples. FVIII levels were elevated before surgery, increased substantially 1 and 3 days (median 4.32 IU/mL) after PEA, and were higher in discordant than concordant samples. Single-cell RNA sequencing showed FVIII gene expression in PEA specimen endothelial cells. Pseudo heparin resistance is common after PEA likely due to highly elevated postoperative FVIII levels indicating that anti-Xa reflects postoperative heparinization better than APTT in these patients. FVIII production by the pulmonary artery endothelium may participate in local prothrombotic processes important for CTEPH pathogenesis.

Plasma proteome of brain-dead organ donors predicts heart transplant outcome.

BACKGROUND: The pathophysiological changes related to brain death may affect the quality of the transplanted organs and expose the recipients to risks. We probed systemic changes reflected in donor plasma proteome and investigated their relationship to heart transplant outcomes. METHODS: Plasma samples from brain-dead multi-organ donors were analyzed by label-free protein quantification using high-definition mass spectrometry. Unsupervised and supervised statistical models were used to determine proteome differences between brain-dead donors and healthy controls. Proteome variation and the corresponding biological pathways were analyzed and correlated with transplant outcomes. RESULTS: Statistical models revealed that donors had a unique but heterogeneous plasma proteome with 237 of 463 proteins being changed compared to controls. Pathway analysis showed that coagulation, gluconeogenesis, and glycolysis pathways were upregulated in donors, while complement, LXR/RXR activation, and production of nitric oxide and reactive oxygen species in macrophages pathways were downregulated. In point-biserial correlation analysis, lysine-specific demethylase 3A was moderately correlated with any grade and severe PGD. In univariate and multivariate Cox regression analyses myosin Va and proteasome activator complex subunit 2 were significantly associated with the development of acute rejections with hemodynamic compromise within 30 days. Finally, we found that elevated levels of lysine-specific demethylase 3A and moesin were identified as predictors for graft-related 1-year mortality in univariate analysis. CONCLUSIONS: We show that brain death significantly changed plasma proteome signature Donor plasma protein changes related to endothelial cell and cardiomyocyte function, inflammation, and vascular growth and arteriogenesis could predict transplant outcome suggesting a role in donor evaluation.

Reconstruction of Donor Anomalous Pulmonary Vein During Lung Transplantation.

Pulmonary vein anomalies are rare but can complicate lung transplantation, especially if unrecognized during the lung procurement operation. We describe a case of a partial anomalous pulmonary venous return of a donor lung detected at the time of transplant reperfusion. The anomalous donor right upper pulmonary vein was successfully connected to the recipient atrial cuff using a bovine pericardium conduit constructed with a vascular stapler. This reconstruction has been durable and has remained patent for over 5 years.

Engineered mesenchymal stromal cell therapy during human lung ex vivo lung perfusion is compromised by acidic lung microenvironment.

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 (MSCIL-10) were administered during EVLP to human lungs that had various degrees of underlying lung injury. Cryopreserved MSCIL-10 had excellent viability, and they immediately and efficiently elevated perfusate and lung tissue IL-10 levels during EVLP. However, MSCIL-10 function was compromised by the poor metabolic conditions present in the most damaged lungs. Similarly, exposing cultured MSCIL-10 to poor metabolic, and especially acidic, conditions decreased their IL-10 production. In conclusion, we found that "off-the-shelf" MSCIL-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.

Donor Simvastatin Treatment in Heart Transplantation.

BACKGROUND: Ischemia-reperfusion injury may compromise the short-term and long-term prognosis after heart transplantation. Experimental studies show that simvastatin administered to the organ donor is vasculoprotective and inhibits cardiac allograft ischemia-reperfusion injury. METHODS: Eighty-four multiorgan donors were randomly assigned to receive 80 mg of simvastatin (42 donors) via nasogastric tube after declaration of brain death and upon acceptance as a cardiac donor, or to receive no simvastatin (42 donors). The primary efficacy end point was postoperative plasma troponin T and I levels during the first 24 hours after heart transplantation. Secondary end points included postoperative hemodynamics, inflammation, allograft function, rejections and rejection treatments, and mortality. Results: Organ donor simvastatin treatment significantly reduced the heart recipient plasma levels of troponin T by 34% (14 900 ± 12 100 ng/L to 9800 ± 7900 ng/L, P=0.047), and troponin I by 40% (171 000 ± 151 000 ng/L to 103 000 ± 109 000 ng/L, P=0.023) at 6 hours after reperfusion, the levels of NT-proBNP (N-terminal pro-B-type natriuretic peptide) by 36% (32 800 ± 24 300 ng/L to 20 900 ± 15 900 ng/L; P=0.011) at 1 week, and the number of rejection treatments with hemodynamic compromise by 53% within the first 30 days (P=0.046). Donor simvastatin treatment did not affect donor lipid levels but was associated with a specific transplant myocardial biopsy gene expression profile, and a decrease in recipient postoperative plasma levels of CXCL10 (C-X-C motif chemokine 10), interleukin-1α, placental growth factor, and platelet-derived growth factor-BB. Postoperative hemodynamics, biopsy-proven acute rejections, and mortality were similar. No adverse effects were seen in recipients receiving noncardiac solid organ transplants from simvastatin-treated donors. CONCLUSIONS: Donor simvastatin treatment reduces biomarkers of myocardial injury after heart transplantation, and-also considering its documented general safety profile-may be used as a novel, safe, and inexpensive adjunct therapy in multiorgan donation. CLINICAL TRIAL REGISTRATION: URL: https://www.clinicaltrials.gov. Unique identifier: NCT01160978.

Hypoxia-inducible factor controls immunoregulatory properties of myeloid cells in mouse cardiac allografts - an experimental study.

Hypoxia-inducible factors (HIFs) play a critical role in inflammatory properties of myeloid-derived cells. The effect of HIFs on myeloid-derived cell functions in organ transplantation remains unknown, however. We transplanted hearts into transgenic mice with myeloid cell-targeted deletions of HIF-1α or its negative regulator von Hippel-Lindau (VHL) to investigate the effects of HIF-1α inactivation or HIF pathway activation, respectively, on ischemia-reperfusion injury (IRI) and acute rejection. Deletion of VHL in myeloid cells enhanced mRNA expression of anti-inflammatory genes IDO, Arg-1, and HO-1 in vitro. In vivo, VHL-/- myeloid-derived cells of allograft recipients alleviated IRI and acute rejection, evidenced by reduced cardiomyocyte damage, decreased proinflammatory cytokine mRNA levels, and absence of inflammatory infiltrate at 5 days after transplantation. Ultimately, allograft survival was significantly prolonged. In vitro, VHL-/- myeloid-derived cells dose-dependently inhibited T-cell proliferation. Myeloid cells with HIF-1α-deletion retained proinflammatory qualities in vitro and in vivo. Deletion of VHL in myeloid cells of nonimmunosuppressed cardiac allograft recipients reduced myocardial injury and acute rejection. We suggest that HIF transcription factors induce a regulatory phenotype in myeloid-derived cells, which may be harnessed as a novel therapeutic strategy to regulate immune responses after heart transplantation.

Vascular Endothelial Growth Factor-B Overexpressing Hearts Are Not Protected From Transplant-Associated Ischemia-Reperfusion Injury.

OBJECTIVES: Cardiac vascular endothelial growth factor-B transgene limits myocardial damage in rat infarction models. We investigated whether heart transplant vascular endothelial growth factor-B overexpression protected against ischemia-reperfusion injury. MATERIALS AND METHODS: We transplanted hearts heterotopically from Dark Agouti to Wistar Furth rats. To characterize the role of vascular endothelial growth factor-B in ischemia-reperfusion injury, we transplanted either long-term human vascular endothelial growth factor-B transgene overexpressing hearts from Wistar Furth rats or short-term adeno-associated virus 9-human vascular endothelial growth factor-B-transduced hearts from Dark Agouti rats into Wistar Furth rats. Heart transplants were subjected to 2 hours of cold and 1 hour of warm ex vivo ischemia. Samples were collected 6 hours after reperfusion. RESULTS: Two hours of cold and 1 hour of warm ischemia increased vascular endothelial growth factor-B mRNA levels 2-fold before transplant and 6 hours after reperfusion. Transgenic vascular endothelial growth factor-B overexpression caused mild cardiac hypertrophy and elevated cardiac troponin T levels 6 hours after reperfusion. Laser Doppler measurements indicated impaired epicardial tissue perfusion in these transgenic transplants. Recombinant human vascular endothelial growth factor-B increased mRNA levels of cytochrome c oxidase and extracellular ATPase CD39, suggesting active oxidative phosphorylation and high ATP production. Adeno-associated virus 9-mediated vascular endothelial growth factor-B overexpression in transplanted hearts increased intragraft macrophages 1.5-fold and proinflammatory cytokine interleukin 12 p35 mRNA 1.6-fold, without affecting recipient serum cardiac troponin T concentration. CONCLUSIONS: Vascular endothelial growth factor-B expression in transplanted hearts is linked to ischemia and ischemia-reperfusion injury. Cardiac transgenic vascular endothelial growth factor-B overexpression failed to protect heart transplants from ischemia-reperfusion injury.

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.

VEGF Pathways in the Lymphatics of Healthy and Diseased Heart.

Cardiac lymphatic system is a rare focus of the modern cardiovascular research. Nevertheless, the growing body of evidence is depicting lymphatic endothelium as an important functional unit in healthy and diseased myocardium. Since the discovery of angiogenic VEGF-A in 1983 and lymphangiogenic VEGF-C in 1997, an increasing amount of knowledge has accumulated on the essential roles of VEGF ligands and receptors in physiological and pathological angiogenesis and lymphangiogenesis. Tissue adaptation to several stimuli such as hypoxia, pathogen invasion, degenerative process and inflammation often involves coordinated changes in both blood and lymphatic vessels. As lymphatic vessels are involved in the initiation and resolution of inflammation and regulation of tissue edema, VEGF family members may have important roles in myocardial lymphatics in healthy and in cardiac disease. We will review the properties of VEGF ligands and receptors concentrating on their lymphatic vessel effects first in normal myocardium and then in cardiac disease.

Platelet-derived Growth Factor-B Protects Rat Cardiac Allografts From Ischemia-reperfusion Injury.

BACKGROUND: Microvascular dysfunction and cardiomyocyte injury are hallmarks of ischemia-reperfusion injury (IRI) after heart transplantation. Platelet-derived growth factors (PDGF) have an ambiguous role in this deleterious cascade. On one hand, PDGF may exert vascular stabilizing and antiapoptotic actions through endothelial-pericyte and endothelial-cardiomyocyte crosstalk in the heart; and on the other hand, PDGF signaling mediates neointimal formation and exacerbates chronic rejection in cardiac allografts. The balance between these potentially harmful and beneficial actions determines the final outcome of cardiac allografts. METHODS AND RESULTS: We transplanted cardiac allografts from Dark Agouti rat and Balb mouse donors to fully major histocompatibility complex-mismatched Wistar Furth rat or C57 mouse recipients with a clinically relevant 2-hour cold ischemia and 1-hour warm ischemia. Ex vivo intracoronary delivery of adenovirus-mediated gene transfer of recombinant human PDGF-BB upregulated messenger RNA expression of anti-mesenchymal transition and survival factors BMP-7 and Bcl-2 and preserved capillary density in rat cardiac allografts at day 10. In mouse cardiac allografts PDGF receptor-ß, but not -α intragraft messenger RNA levels were reduced and capillary protein localization was lost during IRI. The PDGF receptor tyrosine kinase inhibitor imatinib mesylate and a monoclonal antibody against PDGF receptor-α enhanced myocardial damage evidenced by serum cardiac troponin T release in the rat and mouse cardiac allografts 6 hours after reperfusion, respectively. Moreover, imatinib mesylate enhanced rat cardiac allograft vasculopathy, cardiac fibrosis, and late allograft loss at day 56. CONCLUSIONS: Our results suggest that PDGF-B signaling may play a role in endothelial and cardiomyocyte recovery from IRI after heart transplantation.

Systemic overexpression of matricellular protein CCN1 exacerbates obliterative bronchiolitis in mouse tracheal allografts.

Obliterative bronchiolitis (OB) involves airway epithelial detachment, fibroproliferation, and inflammation, resulting in chronic rejection and transplant failure. Cysteine-rich 61 (CCN1) is an integrin receptor antagonist with a context-dependent role in inflammatory and fibroproliferative processes. We used a mouse tracheal OB model to investigate the role of CCN1 in the development of lung allograft OB. C57Bl/6 mice received a systemic injection of CCN1-expressing adenoviral vectors 2 days prior to subcutaneous implantation of tracheal allografts from major MHC-mismatched BALB/c mice. We treated another group of tracheal allograft recipients with cyclic arginine-glycine-aspartic acid peptide to dissect the role of αvß3-integrin signaling in mediating CCN1 effects in tracheal allografts. Allografts were removed 4 weeks after transplantation and analyzed for luminal occlusion, inflammation, and vasculogenesis. CCN1 overexpression induced luminal occlusion (P < 0.05), fibroproliferation, and smooth muscle cell proliferation (P < 0.05). Selective activation of αvß3-integrin receptor failed to mimic the actions of CCN1, and blocking failed to inhibit the effects of CCN1 in tracheal allografts. In conclusion, CCN1 exacerbates tracheal OB by enhancing fibroproliferation via an αvß3-integrin-independent pathway. Further experiments are required to uncover its potentially harmful role in the development of OB after lung transplantation.