R. Arnaudova

Donor Simvastatin Treatment Prevents Ischemia‐Reperfusion and Acute Kidney Injury by Preserving Microvascular Barrier Function

Ischemia‐reperfusion injury (IRI) after kidney transplantation may result in delayed graft function. We used rat renal artery clamping and transplantation models to investigate cholesterol‐independent effects of clinically relevant single‐dose peroral simvastatin treatment 2 h before renal ischemia on microvascular injury. The expression of HMG‐CoA reductase was abundant in glomerular and peritubular microvasculature of normal kidneys. In renal artery clamping model with 30‐min warm ischemia, simvastatin treatment prevented peritubular microvascular permeability and perfusion disturbances, glomerular barrier disruption, tubular dysfunction and acute kidney injury. In fully MHC‐mismatched kidney allografts with 16‐h cold and 1‐h warm ischemia, donor simvastatin treatment increased the expression of flow‐regulated transcription factor KLF2 and vasculoprotective eNOS and HO‐1, and preserved glomerular and peritubular capillary barrier integrity during preservation. In vitro EC Weibel–Palade body exocytosis assays showed that simvastatin inhibited ischemia‐induced release of vasoactive angiopoietin‐2 and endothelin‐1. After reperfusion, donor simvastatin treatment prevented microvascular permeability, danger‐associated ligand hyaluronan induction, tubulointerstitial injury marker Kim‐1 immunoreactivity and serum creatinine and NGAL levels, and activation of innate and adaptive immune responses. In conclusion, donor simvastatin treatment prevented renal microvascular dysfunction and IRI with beneficial effects on adaptive immune and early fibroproliferative responses. Further studies may determine potential benefits in clinical cadaveric kidney transplantation.

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.

Tricyclic Benzo[cd]azulenes Selectively Inhibit Activities of Pim Kinases and Restrict Growth of Epstein-Barr Virus-Transformed Cells

Oncogenic Pim family kinases are often overexpressed in human hematopoietic malignancies as well as in solid tumours. These kinases contribute to tumorigenesis by promoting cell survival and by enhancing resistance against chemotherapy and radiation therapy. Furthermore, we have recently shown that they increase the metastatic potential of adherent cancer cells. Here we describe identification of tricyclic benzo[cd]azulenes and their derivatives as effective and selective inhibitors of Pim kinases. These compounds inhibit Pim autophosphorylation and abrogate the anti-apoptotic effects of Pim kinases. They also reduce cancer cell motility and suppress proliferation of lymphoblastoid cell lines infected and immortalized by the Epstein-Barr virus. Thus, these novel Pim-selective inhibitors provide promising compounds for both research and therapeutic purposes.

Combined donor simvastatin and methylprednisolone treatment prevents ischemia-reperfusion injury in rat cardiac allografts through vasculoprotection and immunomodulation.

Background Ischemia-reperfusion injury (IRI) and allograft dysfunction remain as two of the major clinical challenges after heart transplantation. Here, we investigated the effect of donor treatment with simvastatin and methylprednisolone on microvascular dysfunction and immunomodulation during IRI in rat cardiac allografts subjected to prolonged ischemia time. Methods The DA rats received simvastatin, methylprednisolone, or both 2 hr before heart donation. The allografts were subjected to 4-hr hypothermic preservation and transplanted to the fully major histocompatibility complex–mismatched WF rat recipients. Results Six hours after reperfusion, donor treatment either with simvastatin alone or with high dose of methylprednisolone alone or in combination with simvastatin and methylprednisolone significantly reduced cardiac troponin T release and the number of allograft infiltrating ED1+ macrophages MPO+ neutrophils. However, the combination donor treatment was superior in the prevention of IRI and significantly prolonged allograft survival. Donor simvastatin treatment inhibited allograft microvascular RhoA GTPase pathway activation, whereas methylprednisolone prevented activation of innate immune response and mRNA expression of hypoxia-inducible factor-1&agr; and its multiple target genes. Conclusions Our results show that donor treatment in combination with simvastatin and methylprednisolone prevents IRI and has beneficial effect on allograft survival in rat cardiac allografts. Minimizing microvascular injury and the activation of innate immunity may offer a novel therapeutic strategy to expand the donor pool and furthermore improve the function of the marginal donor organs.

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.

Nucleophilic Substitution of Hydrogen Facilitated by Quinone Methide Moieties in Benzo[cd]azulen-3-ones

The built-in o- and p-QM (QM = quinone methide) moieties in benzo[cd]azulen-3-ones account for an easy switch between the bridged 10π- and 6π-aromatic systems in organic synthesis. We report conjugate additions, oxidative nucleophilic substitutions of hydrogen, and reversible Michael additions under very mild conditions. In the presence of thiol nucleophiles, the protonated σH-adducts could be isolated and characterized. The typical preference for either the o- or p-QM moiety led to high regioselectivity. Furthermore, the inhibitory potency of the novel benzo[cd]azulenes against the human Pim-1 kinase was evaluated.

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.