A gene-based approach to experimental heart transplant rejection

Heart transplantation is often the last resort for end-stage heart disease. Despite increasing success in this medical field, transplant recipients remain at significant risk for both early and late allograft failure. The cascade of events leading to heart transplant rejection are initiated by donor brain death, progress throughout ex vivo preservation of the organ, are exacerbated during reperfusion, and culminate in cardiac allograft vasculopathy (CAV). The aim of this thesis was to evaluate the efficiency of adeno-associated virus (AAV) as a vector for gene therapy of the heart transplant, elaborate on the role of vascular endothelial growth factor B (VEGF-B) in heart transplant ischemia-reperfusion injury (TX-IRI) and hypoxia-inducible factor (HIF) in the inflammatory properties of allograft-infiltrating myeloid-derived cells. The long-term kinetics and safety of AAV serotypes 2, 8, and 9 were evaluated by perfusing the coronary tree of rat heart transplants with each serotype and comparing the reporter gene expression at set time-points and inflammatory response at the end-point of the study. We studied the role of VEGF-B in ischemia-reperfusion injury of cardiac allografts by transgene- and AAV-mediated overexpression of VEGF-B in rat cardiac allografts. The significance of HIF as an immunoregulatory switch in myeloid-derived cells was determined by using transgenic mice with myeloid cell-targeted activation or knock-out (KO) of HIF-1α and -2α as heart transplant recipients. We found that AAV2 was most effective in transducing heart transplants after intracoronary injection, whereas AAV9 was most effective when injected systemically into the transplant donor. Adeno-associated virus serotype 9 caused a mild inflammatory response in cardiac allografts, whereas AAV2 and 8 did not. Chronic, but not short-term, VEGF-B overexpression in rat cardiac transplants resulted in cardiomyocyte hypertrophy and higher energy demands, with subsequent higher susceptibility towards TX-IRI. HIF-1α and -2α activation in recipient myeloid cells established an immunoregulatory phenotype that significantly suppressed both TX-IRI and acute rejection and prolonged allograft survival. Our results highlight the importance of the route of administration on heart transplant AAV gene therapy and suggest AAV2 as the preferred vector for intracoronary perfusion and AAV9 for systemic delivery in experimental rat heart transplant models. Vascular endothelial growth factor B may regulate heart energy demand and thus might play an important role in transplant ischemic tolerance. Hypoxia-inducible factor-1α and -2α act as important switches for the immunoregulatory phenotype of myeloid cells, and may offer a viable therapeutic target to alleviate allograft rejection.Sydämensiirto on usein viimeinen vaikean sydäntaudin hoitomuoto. Alan kehityksistä huolimatta sydänsiirteen akuutti ja krooninen hyljintä ovat edelleen merkittäviä sydämensiirron pitkäaikaistuloksia heikentäviä tekijöitä. Hyljintään johtava patofysiologinen tapahtumaketju saa alkunsa siirteen luovuttajan aivokuolemasta, etenee sydänsiirteen ex vivo- kylmäsäilytyksen ja reperfuusion aikana, ja kulminoituu siirteeseen kohdistuvaan vastaanottajan immuunijärjestelmän aktivaatioon sekä siirteen vaskulopatiaan. Tässä väitöskirjatutkimuksessa arvioidaan Adeno-associated viruksen (AAV) tehoa ja turvallisuutta sydänsiirteen geeniterapiavektorina, selvitetään verisuonikasvutekijä vascular endothelial growth factor B:n (VEGF-B) osuutta sydänsiirteen iskemia-reperfuusiovauriossa (IRI), sekä hypoksiassa aktivoituvan transkriptiotekijä hypoxia-inducible factor:in (HIF) vaikutuksia vastaanottajan sydänsiirrettä vahingoittavien myeloidilinjan tulehdussolujen proinflammatorisissa mekanismeissa. AAV-serotyyppien 2, 8 ja 9 pitkäaikaiskinetiikkaa ja turvallisuutta arvioitiin perfusoimalla rotan sydänsiirteen sepelvaltimopuusto kullakin serotyypillä ja vertailemalla reportterigeenin ilmentymistä ja sydänsiirteen tulehdusvastetta keskenään. VEGF-B:n roolia sydänsiirteen IRI:ssa tutkittiin yli-ilmentämällä kasvutekijää sydänsiirteessä sekä transgeenisesti, että AAV- virusvektoreiden välityksellä. HIF:n osuutta sydänsiirteen hyljintäreaktion synnyssä tutkittiin käyttämällä myeloidilinjan soluihin keskitettyä HIF-1α ja -2α yli- tai ali-ilmentäviä transgeenisiä hiiriä sydänsiirteen vastaanottajina. AAV2 oli tehokkain virusvektori sydänsiirteessä sepelvaltimoon injisoitaessa, kun taas AAV9 oli tehokkain sydänsiirteen luovuttajalle suonensisäisesti annettuna. AAV9 aiheutti lievän tulehduksellisen vasteen sydänsiirteissä. Pitkäaikainen, mutta ei lyhytaikainen VEGF-B yli- ilmentäminen sydänsiirteessä aiheutti sydänlihassolujen hypertrofian ja korkeamman metabolisen tarpeen, altistaen siten sydänsiirrettä IRI:n haittavaikutuksille. HIF-1α sekä HIF-2α aktivaatio sydänsiirteen vastaanottajan myeloidilinjan soluissa aiheutti näissä immunoregulatorisen fenotyypin, joka vähensi merkittävästi sydänsiirteen IRI:ä ja akuuttia hyljintäreaktiota, ja pidensi merkittävästi sydänsiirteen selviytymistä. Tulokset korostavat virusvektorien antoreittien tärkeyttä sydänsiirteen geeniterapiassa. VEGF- B säätelee sydämen metaboliaa ja voi mahdollisesti vaikuttaa sydänsiirteen iskemiatoleranssiin. HIF-1α ja HIF-2α ovat tärkeitä myeloidilinjan valkosolujen tulehduksellisten toimintojen säätelijöitä, joita voisi hyödyntää allogeenisen sydänsiirteen hyljinnän ehkäisemisessä

Chronic Disease Burden After Congenital Heart Surgery : A 47-Year Population-Based Study With 99% Follow-Up

Background Postoperative morbidity is an increasingly important outcome measure of patients who have undergone congenital heart surgery (CHS). We examined late postoperative morbidity after CHS on the basis of patients' government-issued medical special reimbursement rights. Methods and Results Between 1953 and 2009, 10 635 patients underwent CHS at Conclusions Chronic cardiac and noncardiac sequelae are common after CHS regardless of the severity of the defect, underscoring the importance of long-term follow-up of all patients after CHS.Peer reviewe

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

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 PathologyObliterative 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)Peer reviewe

Lapsena sydänleikkauksella hoidettujen ennuste on parantunut

Lievissä sydänvioissa elinajanodote vastaa normaaliväestön tasoa. Sydämen vajaatoiminta ja äkkikuolemat ovat vähentyneet merkittävästi vuoden 1990 jälkeen leikatuilla potilailla. Aortan koarktaation leikkauksen jälkeisiä pitkäaikaisongelmia ovat kohonnut verenpaine sekä rekoarktaatio. Fallot’n tetralogian ja yksikammioisen sydämen vuoksi leikatuilla potilailla yleisiä ovat takyarytmiat ja sydämen vajaatoiminta. Valtasuonten transpositiossa eteistunnelointiin liittyvät rytmihäiriöt ja sydämen vajaatoiminta ovat vähentyneet merkittävästi uuden valtasuonten vaihtoleikkauksen myötä. Potilaat tarvitsevat sydämen seurantaa sekä tehokasta liitännäissairauksien primaarista ja sekundaarista ehkäisyä.Peer reviewe

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-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.Peer reviewe

The effect of sildenafil on pleural and peritoneal effusions after the TCPC operation

Background We evaluated whether the administration of sildenafil in children undergoing the TCPC operation shortened the interval from the operation to the removal of the pleural and peritoneal drains. Methods We retrospectively reviewed the data of 122 patients who had undergone the TCPC operation between 2004 and 2014. Patients were divided into two groups on the basis of their treatments. Sildenafil was orally administered pre-operatively in the morning of the procedure or within 24 hours after the TCPC operation to the sildenafil group (n = 48), which was compared to a control group (n = 60). Fourteen patients were excluded from the study. Results The primary outcome measure was the time from the operation to the removal of the drains. The study groups had similar demographics. The median [interquartile range] time for the removal of drains (sildenafil group 11 [8-19] vs control group 11 [7-16] d, P = .532) was comparable between the groups. The median [interquartile range] fluid balance on the first post-operative day was significantly higher (P = .001) in the sildenafil group compared with controls (47 [12-103] vs 7 [-6-67] mL kg(-1)). The first post-operative day fluid balance was a significant predictor for a prolonged need for drains in the multivariate analysis. Conclusions Sildenafil administration, pre-operatively or within 24 hours after the TCPC operation, did not reduce the required time for pleural and peritoneal drains but was associated with a significantly higher positive fluid balance.Peer reviewe

Development of Human Leukocyte Antigen (HLA) Antibodies Against Vascular Homograft Donor in Pediatric Heart Transplant Recipients

Background: The appearance of human leukocyte antigen (HLA) antibodies after solid organ transplantation predisposes recipients to graft dysfunction. In theory, vascular homografts, which are widely used in children with congenital heart defects, may cause allosensitization. Material/Methods: In this single-center retrospective study, the presence of pre-existing HLA antibodies in pediatric heart trans- plant (HTx) recipients with a vascular homograft was evaluated in a cohort of 12 patients. HLA antibodies were screened before and after HTx and positive screening results were confirmed and identified using the Luminex (R) single antigen bead method. Endomyocardial biopsies (EMB) and coronary angiography studies were re-evaluated to assess the prevalence of acute rejections and coronary artery change in these patients. Results: At the time of HTx, 8 patients (67%) had HLA antibodies detected by the Luminex assay, none of which were heart donor specific (DSA). All patients had negative leukocyte crossmatch. One patient developed DSAs against homograft donor prior to HTx. After the HTx, 5 patients (42%) developed DSAs against the heart donor and 4 patients (40%) against the homograft donor. In 2 patients (17%), the antibodies were against both heart and homograft donors. The rejection rate or prevalence of coronary artery vasculopathy did not differ significantly between the homograft cohort and our historical controls. Conclusions: Our results suggest that the prevalence of DSAs against homograft donor prior to HTx is relatively rare. However, almost half of the patients developed DSAs against homograft post-HTx. The clinical importance of these antibodies warrants further studies.Peer reviewe

Outcome of pediatric cardiac surgery

Pediatric heart surgery aims to correct or palliate cardiac malformations, and to maximize the quality of life of the patients. This thesis investigates the late outcome after surgery for congenital cardiac defects to assess the quality and progress of treatment during the last decades. It also studies outcomes after the arterial switch operation (ASO) for treatment of transposition of the great arteries (TGA) and pediatric heart transplantation. Data was obtained from a national database containing all pediatric heart operations performed since 1953 in one of five university- and one regional hospital in Finland. Causes of death were obtained from Statistics Finland and categorized into congenital heart defect (CHD)- related and non-CHD-related causes of death. CHD-related deaths were further divided into heart failure-, post-reoperative early-, cardiovascular-, and sudden deaths. Between 1953 and 2009, 13,786 cardiac operations were performed on 10,964 pediatric patients in Finland. Follow-up coverage was 98%. Early operative mortality was 5.6% for all operations. 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, whereas surgery for simple defects decreased. Operative age and early operative mortality decreased significantly among all defect groups. Long-term survival of all patients improved significantly during recent decades. The most common cause of death was heart failure, which decreased among the majority of defect groups. Sudden death was reduced to zero among patients with simple defects and TGA, but remained a challenge among patients with univentricular heart configurations. ASO resulted in markedly improved early operative mortality and late outcome after correction of TGA when compared to the Mustard and Senning operations, with zero sudden deaths. Early mortality after pediatric heart transplantation was 10%, with a late outcome of 68% at 10 years after the transplantation. In conclusion, both early and long-term outcome after surgery for CHD s has improved. The reason is multifaceted, including advances in diagnostic tools, perioperative care, intensive care, and surgical techniques, allowing earlier treatment of increasingly severe defects. Sudden deaths and heart failure have markedly diminished, but remain a risk factor among patients with severe defects. These results underscore the importance of long-term follow-up after surgery for severe defects.Monet synnynnäiset sydänviat johtavat hoitamattomana lapsen kuolemaan varhaisessa iässä. Useimmat näistä ovat korjattavissa sydänleikkauksella tai katetrisaatiotoimenpiteellä. Hoitojen tavoittena on potilaan oireiden vähentäminen ja elämänlaadun parantaminen. Tämä väitöskirja tarkastelee lapsena sydänleikattujen potilaiden pitkäaikaistuloksia hoidon laadun ja kehityksen arvioimiseksi. Tutkimuksessa tarkastellaan erikseen myös valtasuonten transposition (TGA) hoidossa käytetyn arterial switch menetelmän (ASO) ja lasten sydänsiirtojen pitkäaikaistuloksia. Tutkimuksessa käytetään lasten sydänleikkausten tietokantaa, ProCardio:ta. Tietokanta perustettiin vuonna 1995, ja se sisältää tiedot kaikista Suomessa tehdyistä lasten sydänleikkauksista vuodesta 1953 lähtien. Kuolinsyyt on jaettu kardiogeenisiin ja ei- kardiogeenisiin, ja kardiogeeniset kuolinsyyt edelleen sydämen vajaatoimintaan, kardiovaskulaariseen, perioperatiiviseen ja äkilliseen kuolemaan. Vuosien 1953 ja 2009 välillä Suomessa tehtiin 13 786 sydänleikkausta 10 694:lle lapsipotilaalle. Seurannan kattavuus oli 98%. Leikkauskuolleisuus oli yhteensä 5.6%. 60 vuoden pitkäaikaisselviytyminen oli 70% kaikilla potilailla, ja 86% sukupuolen-, iän-, synnyinajan-, ja sairaanhoitopiirin suhteen samankaltaistetulla kontrolliväestöllä. Vaikeiden sydänvikojen osuus kaikista leikatuista sydänvioista nousi tasaisesti 2000-luvulle asti, samalla kun yksinkertaisten sydänvikojen leikkaukset vähenivät. Leikkausikä pieneni ja leikkauskuolemat vähenivät merkittävästi kaikissa sydänvikaryhmissä, ja pitkäaikaisselviytyminen parani viime vuosikymmenten aikana leikattujen keskuudessa. Tavallisin kuolinsyy oli sydämen vajaatoiminta, jonka esiintyvyys väheni merkittävästi suurimmassa osassa sydänvikaryhmiä. Äkkikuolemien esiintyvyys laski nollaan yksinkertaisten sydänvikojen ja TGA-potilaiden keskuudessa, mutta muodosti huomattavan pitkäaikaisriskin yksikammioisen sydänvian vuoksi leikatuilla potilailla. Mustard- ja Senning-menetelmiin verrattuna ASO-menetelmä paransi merkittävästi leikkaustuloksia ja pitkäaikaisselviytymistä TGA-potilailla, ja laski äkkikuolemien esiintyvyyden nollaan. Leikkauskuolleisuus lasten sydänsiirron jälkeen oli 10%, ja pitkäaikaisselviytyminen oli 68% 10 vuotta leikkauksesta. Tutkimus osoitti, että leikkauskuolemien määrä on laskenut ja pitkäaikaistulokset parantuneet sydänleikatuilla lapsilla. Syitä tulosten paranemiseen on useita, kuten diagnostisten menetelmien, pre- ja post-operatiivisen hoidon, tehohoidon, sekä leikkaustekniikoiden kehittyminen, jotka mahdollistavat aikaisemman hoidon yhä vaikeampia sydänvikoja omaaville potilaille. Äkkikuolemat ja kuolemat sydämen vajaatoiminnan seurauksena ovat vähentyneet merkittävästi, mutta ovat edelleen riski vaikeimpien sydänvikojen omaavien potilaiden keskuudessa. Tulokset korostavat vaikean sydänvian omaavien lasten pitkäaikaisseurannan tärkeyttä

Late Causes of Death After Pediatric Cardiac Surgery A 60-Year Population-Based Study

BACKGROUND Comprehensive information regarding causes of late post-operative death following pediatric congenital cardiac surgery is lacking. OBJECTIVES The study sought to analyze late causes of death after congenital cardiac surgery by era and defect severity. METHODS We obtained data from a nationwide pediatric cardiac surgery database and Finnish population registry regarding patients who underwent cardiac surgery at RESULTS Overall, 10,964 patients underwent 14,079 operations, with 98% follow-up. Early mortality ( CONCLUSIONS CHD-related deaths have decreased markedly but remain a challenge after surgery for severe cardiac defects. Premature deaths are generally more common among patients than the control population, warranting long-term follow-up after congenital cardiac surgery. (C) 2016 by the American College of Cardiology Foundation.Peer reviewe

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 2 h 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 6 h 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. (C) 2016 Elsevier B.V. All rights reserved.Peer reviewe