A. Raissadati

Progress in Late Results Among Pediatric Cardiac Surgery Patients A Population-Based 6-Decade Study With 98% Follow-Up

Background— Surgical treatment of congenital cardiac defects in Finland started >60 years ago. We analyzed the survival of all the pediatric cardiac surgery patients operated on before 2010. Methods and Results— Data were obtained retrospectively from a pediatric cardiac surgery database. Patient status was received from the Finnish Population Registry. Survival was determined with the Kaplan–Meier method, and the survival rate was compared with a sex- and age-matched general population. Between 1953 and 2009, 13 876 cardiac operations were performed on 10 964 pediatric patients in Finland. Follow-up coverage was 98%. The 60-year survival for the entire study was 70% versus 86% for the general population. The number and proportion of severe cardiac defects increased in the 2000s. The long-term survival of patients with severe defects improved significantly across decades. For instance, the 22-year survival rate of patients with transposition of the great arteries operated on in 1953 to 1989 and in 1990 to 2009 improved from 71% to 93% (hazard ratio for death, 0.29; 95% confidence interval, 0.17–0.49; P<0.0001), respectively. The mean patient age at operation decreased from 8.9 to 2.2 years (95% confidence interval, 6.2–7.1; P<0.0001). The early mortality of patients decreased from a maximum of 7% in the 1970s to 3% in the 2000s (95% confidence interval, 0.05–0.08; P<0.0001). Conclusions— Patients are diagnosed and treated at an increasingly younger age. Advanced diagnostics, surgical methods, and postoperative intensive care have led to substantial improvements in both early and late results among pediatric cardiac surgery patients.

Angiopoietin-2 Inhibition Prevents Transplant Ischemia-Reperfusion Injury and Chronic Rejection in Rat Cardiac Allografts

Transplant ischemia‐reperfusion injury (Tx‐IRI) and allograft dysfunction remain as two of the major clinical challenges after heart transplantation. We investigated the role of angiopoietin‐2 (Ang2) in Tx‐IRI and rejection using fully MHC‐mismatched rat cardiac allografts. We report that plasma levels of Ang2 were significantly enhanced in the human and rat recipients of cardiac allografts, but not in the rat recipients of syngrafts, during IRI. Ex vivo intracoronary treatment of rat cardiac allografts with anti‐Ang2 antibody before 4‐h cold preservation prevented microvascular dysfunction, endothelial cell (EC) adhesion molecule expression and leukocyte infiltration, myocardial injury and the development of cardiac fibrosis and allograft vasculopathy. Recipient preoperative and postoperative treatment with anti‐Ang2 antibody produced otherwise similar effects without effecting microvascular dysfunction, and in additional experiments prolonged allograft survival. Recipient preoperative treatment alone failed to produce these effects. Moreover, ex vivo intracoronary treatment of allografts with recombinant Ang2 enhanced Tx‐IRI and, in an add‐back experiment, abolished the beneficial effect of the antibody. We demonstrate that neutralization of Ang2 prevents EC activation, leukocyte infiltration, Tx‐IRI and the development of chronic rejection in rat cardiac allografts. Our results suggest that blocking Ang2 pathway is a novel, clinically feasible, T cell–independent strategy to protect cardiac allografts.

Donor Heart Treatment With COMP‐Ang1 Limits Ischemia‐Reperfusion Injury and Rejection of Cardiac Allografts

The major cause of death during the first year after heart transplantation is primary graft dysfunction due to preservation and ischemia‐reperfusion injury (IRI). Angiopoietin‐1 is a Tie2 receptor‐binding paracrine growth factor with anti‐inflammatory properties and indispensable roles in vascular development and stability. We used a stable variant of angiopoietin‐1 (COMP‐Ang1) to test whether ex vivo intracoronary treatment with a single dose of COMP‐Ang1 in donor Dark Agouti rat heart subjected to 4‐h cold ischemia would prevent microvascular dysfunction and inflammatory responses in the fully allogeneic recipient Wistar Furth rat. COMP‐Ang1 reduced endothelial cell–cell junction disruption of the donor heart in transmission electron microscopy during 4‐h cold ischemia, improved myocardial reflow, and reduced microvascular leakage and cardiomyocyte injury of transplanted allografts during IRI. Concurrently, the treatment reduced expression of danger signals, dendritic cell maturation markers, endothelial cell adhesion molecule VCAM‐1 and RhoA/Rho‐associated protein kinase activation and the influx of macrophages and neutrophils. Furthermore, COMP‐Ang1 treatment provided sustained anti‐inflammatory effects during acute rejection and prevented the development of cardiac fibrosis and allograft vasculopathy. These results suggest donor heart treatment with COMP‐Ang1 having important clinical implications in the prevention of primary and subsequent long‐term injury and dysfunction in cardiac allografts.

Ischemia-Reperfusion Injury Enhances Lymphatic Endothelial VEGFR3 and Rejection in Cardiac Allografts.

Organ damage and innate immunity during heart transplantation may evoke adaptive immunity with serious consequences. Because lymphatic vessels bridge innate and adaptive immunity, they are critical in immune surveillance; however, their role in ischemia–reperfusion injury (IRI) in allotransplantation remains unknown. We investigated whether the lymphangiogenic VEGF‐C/VEGFR3 pathway during cardiac allograft IRI regulates organ damage and subsequent interplay between innate and adaptive immunity. We found that cardiac allograft IRI, within hours, increased graft VEGF‐C expression and lymphatic vessel activation in the form of increased lymphatic VEGFR3 and adhesion protein expression. Pharmacological VEGF‐C/VEGFR3 stimulation resulted in early lymphatic activation and later increase in allograft inflammation. In contrast, pharmacological VEGF‐C/VEGFR3 inhibition during cardiac allograft IRI decreased early lymphatic vessel activation with subsequent dampening of acute and chronic rejection. Genetic deletion of VEGFR3 specifically in the lymphatics of the transplanted heart recapitulated the survival effect achieved by pharmacological VEGF‐C/VEGFR3 inhibition. Our results suggest that tissue damage rapidly changes lymphatic vessel phenotype, which, in turn, may shape the interplay of innate and adaptive immunity. Importantly, VEGF‐C/VEGFR3 inhibition during solid organ transplant IRI could be used as lymphatic‐targeted immunomodulatory therapy to prevent acute and chronic rejection.

Late outcome after paediatric heart transplantation in Finland

OBJECTIVES We studied the long-term survival and rejection episodes of paediatric heart transplant recipients. METHODS We included all paediatric patients (≤18 years) who underwent heart transplantation during 1991-2014 in Finland. Data were obtained retrospectively from a paediatric cardiac surgery database. Patient status was received from the Finnish population registry. All patients underwent yearly routine postoperative endomyocardial biopsies and coronary angiographies. RESULTS Between 1991 and 2014, 68 heart transplantations were performed. The early mortality (<30 days after surgery) rate was 10% and follow-up coverage was 100%. The 10- and 15-year survival rates for all patients were 68% (95% confidence internal, CI, 56-80%) and 65% (95% CI 53-78%), respectively, including early mortality. The 1-year survival rate was 100% when excluding early operative mortality. Indications for heart transplantation were cardiomyopathy in 57% and cardiac malformations in 43% of patients, with similar long-term survival between the groups. During 23 years of follow-up, 43 patients (70%) had at least one rejection episode and 17 patients (29%) at least a grade 1 coronary artery vasculopathy finding. Patients with early rejection episodes (<3 months) had a higher incidence of late rejection episodes (P = 0.025). Older age at operation was a significant risk factor for the development of coronary artery vasculopathy (hazard ratio 1.1, 95% CI 1.0-1.3, P = 0.012). CONCLUSIONS First-year survival was excellent. Asymptomatic rejection episodes were common among patients. Early rejection episodes are a risk factor for late rejection episodes and show a trend towards an increased risk of late death. Coronary artery vasculopathy remains a major challenge for late graft survival.

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 αvβ3‐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 αvβ3‐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 αvβ3‐integrin‐independent pathway. Further experiments are required to uncover its potentially harmful role in the development of OB after lung transplantation.

Ex vivo intracoronary gene transfer of adeno-associated virus 2 leads to superior transduction over serotypes 8 and 9 in rat heart transplants

Heart transplant gene therapy requires vectors with long‐lasting gene expression, high cardiotropism, and minimal pathological effects. Here, we examined transduction properties of ex vivo intracoronary delivery of adeno‐associated virus (AAV) serotype 2, 8, and 9 in rat syngenic and allogenic heart transplants. Adult Dark Agouti (DA) rat hearts were intracoronarily perfused ex vivo with AAV2, AAV8, or AAV9 encoding firefly luciferase and transplanted heterotopically into the abdomen of syngenic DA or allogenic Wistar–Furth (WF) recipients. Serial in vivo bioluminescent imaging of syngraft and allograft recipients was performed for 6 months and 4 weeks, respectively. Grafts were removed for PCR‐, RT‐PCR, and luminometer analysis. In vivo bioluminescent imaging of recipients showed that AAV9 induced a prominent and stable luciferase activity in the abdomen, when compared with AAV2 and AAV8. However, ex vivo analyses revealed that intracoronary perfusion with AAV2 resulted in the highest heart transplant transduction levels in syngrafts and allografts. Ex vivo intracoronary delivery of AAV2 resulted in efficient transgene expression in heart transplants, whereas intracoronary AAV9 escapes into adjacent tissues. In terms of cardiac transduction, these results suggest AAV2 as a potential vector for gene therapy in preclinical heart transplants studies, and highlight the importance of delivery route in gene transfer studies.

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-&bgr;, but not -&agr; 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-&agr; 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.

Simvastatin pretreatment reduces caspase-9 and RIPK1 protein activity in rat cardiac allograft ischemia-reperfusion.

BACKGROUND In transplantation-associated ischemia/reperfusion injury (Tx-IRI), tumor necrosis factor alpha and damage-associated molecular patterns promote caspase-8 and -9 apoptotic and receptor-interacting protein kinase-1 and -3 (RIPK1/3) necroptotic pathway activation. The extent of cell death and the counterbalance between apoptosis and regulated necrosis eventually determine the immune response of the allograft. Although simvastatin prevents Tx-IRI, its role in apoptotic and necroptotic activity remains unsolved. METHODS Rat allograft donors and recipients were treated with a single-dose of simvastatin 2h prior to allograft procurement and reperfusion, respectively. Intragraft caspase-3, -8, and -9 and RIPK1 and -3 mRNA expression was analysed by quantitative RT-PCR and protein activity measured by immunohistochemistry and luminescent assays 6h after reperfusion. Lactate and lactate dehydrogenase (LDH) levels were analysed from allograft recipient and from hypoxic endothelial cell cultures having treated with activated simvastatin. RESULTS When compared to without cold ischemia, prolonged 4-hour cold ischemia significantly enhanced intragraft mRNA expression of caspase-3 and -9, and RIPK1 and -3, and elevated protein activity of caspase-9 and RIPK1 in the allografts. Simvastatin pretreatment decreased mRNA expression of caspase-3 and -9, and RIPK1 and -3 and protein activity of caspase-9 and RIPK1 in the allografts. Intragraft caspase-8 mRNA expression remained constant regardless of cold ischemia or simvastatin pretreatment. Simvastatin pretreatment attenuated lactate and LDH levels, both in the allograft recipients and in hypoxic endothelial cell cultures. CONCLUSIONS The beneficial effects of simvastatin pretreatment in cardiac allograft IRI may involve prevention of apoptosis and necroptosis.

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.