Raimo Tuuminen

Targeting Lymphatic Vessel Activation and CCL21 Production by Vascular Endothelial Growth Factor Receptor-3 Inhibition Has Novel Immunomodulatory and Antiarteriosclerotic Effects in Cardiac Allografts

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.

Donor Simvastatin Treatment Abolishes Rat Cardiac Allograft Ischemia/Reperfusion Injury and Chronic Rejection Through Microvascular Protection

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-1&agr;, 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-&bgr;1 signaling, and myocardial fibrosis. In vitro, simvastatin inhibited transforming growth factor-&bgr;1–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.

PDGF-A, -C, and -D but not PDGF-B Increase TGF-β1 and Chronic Rejection in Rat Cardiac Allografts

Objective—Chronic rejection is the main reason for the poor long-term survival of heart transplant recipients and is characterized by cardiac allograft inflammation, fibrosis, and arteriosclerosis. We examined the specific roles of different platelet-derived growth factor (PDGF) ligands (A–D)—potent mesenchymal cell mitogens—in rat cardiac allografts. Methods and Results—PDGFR-&agr; mRNA was upregulated in acutely-rejecting, and PDGF-A and PDGF-C mRNA in chronically-rejecting cardiac¢hatn allografts. In acute rejection, PDGFR-&agr; immunoreactivity increased in the media of arteries. In chronically-rejecting allografts, immunoreactivity of all PDGF ligands and receptors—except that of PDGF-B ligand—was found in the intima of arteries, and the expression of PDGF-A and PDGF-C was seen in cardiomyocytes. Intracoronary adeno-associated virus-2 (AAV2)-mediated PDGF-A and -D gene transfer enhanced cardiac allograft inflammation. AAV2-PDGF-A, AAV2-PDGF-C, and AAV2-PDGF-D significantly upregulated profibrotic TGF-&bgr;1 mRNA and accelerated cardiac fibrosis and arteriosclerosis. In contrast, AAV2-PDGF-B did not aggravate chronic rejection. Conclusions—We found that alloimmune response induces PDGF-A, PDGF-C, and PDGF-D expression in the graft vasculature. PDGF-A, PDGF-C, and PDGF-D mediated profibrotic and proarteriosclerotic effects in transplanted hearts involving the TGF-&bgr;1 pathway. Inhibition of signaling of all PDGF-ligands except that of PDGF-B may thus be needed to inhibit chronic rejection in cardiac allografts.

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.

Increased Th17 rather than Th1 alloimmune response is associated with cardiac allograft vasculopathy after hypothermic preservation in the rat

BACKGROUND Preservation injury decreases patient survival and promotes the development of cardiac allograft vasculopathy. We investigated the sequential effects of hypothermic preservation on ischemia-reperfusion injury (IRI), subsequent innate immune activation, and adaptive immune response in rat cardiac allografts. METHODS Allografts were transplanted from fully major histocompatibility complex-mismatched Dark Agouti to Wistar Furth rats without pre-operative hypothermia or after 4 hours of hypothermic preservation. Recipients received cyclosporine A immunosuppression. The allografts were recovered at 6 hours (n = 6, 7), 24 hours (n = 6), 10 days (n = 5), and 8 weeks (n = 5). Immunohistochemical, histologic, and reverse-transcription polymerase chain reaction analysis was performed. RESULTS In IRI, significantly increased messenger RNA (mRNA) levels for Toll-like receptor 4, hyaluronan synthases (HAS)1-2 (p = 0.03), high-mobility group box 1 (p = 0.05), CD80/83 (p = 0.01, p = 0.048), and the cytokines tumor necrosis factor-alpha (p = 0.004), interferon-gamma (p = 0.012), and interleukin (IL)-6 (p = 0.019) were seen in allografts subjected to hypothermic preservation. During established alloimmune response, allografts subjected to hypothermic preservation expressed prominent infiltration of CD4+ T cells (p = 0.043) and dendritic cells (p = 0.029) and significantly up-regulated mRNA levels of CD80 (p = 0.036), chemokine (C-C motif) ligand 21 (p = 0.008), C-C chemokine receptor type 7 (p = 0.003), vascular endothelial growth factor-C (p = 0.016), and vascular endothelial growth factor receptor-3 (p = 0.02). These allografts also showed prominent mRNA upregulation of Foxp3 (p = 0.014), IL-17 (p = 0.038), and IL-23 (p = 0.043). Preservation significantly increased the incidence and intensity of allograft arteriosclerosis (p < 0.05) and cardiac fibrosis (p = 0.003) at 8 weeks. CONCLUSION Our results demonstrate that preservation injury induced a cascade leading to an innate immune response that modulated the adaptive immune response towards Th17 rather than Th1 T-cell response in rat cardiac allografts and ultimately enhanced cardiac fibrosis and arterial occlusion. Our results also suggest that this immune response was not regulated by the calcineurin inhibitor cyclosporine A.

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.

Differential Effects of Pharmacological HIF Preconditioning of Donors Versus Recipients in Rat Cardiac Allografts

Ischemia‐reperfusion injury (IRI) induces hypoxia‐inducible factor‐1 (HIF‐1) in the myocardium, but the consequences remain elusive. We investigated HIF‐1 activation during cold and warm ischemia and IRI in rat hearts and cardiac syngrafts. We also tested the effect of HIF‐α stabilizing prolyl hydroxylase inhibitor (FG‐4497) on IRI or allograft survival. Ex vivo ischemia of the heart increased HIF‐1α expression in a time‐ and temperature‐dependent fashion. Immunohistochemistry localized HIF‐1α to all cardiac cell types. After reperfusion, HIF‐1α immunoreactivity persisted in smooth muscle cells and cardiomyocytes in the areas with IRI. This was accompanied with a transient induction of protective HIF‐1 downstream genes. Donor FG‐4497 pretreatment for 4 h enhanced IRI in cardiac allografts as evidenced by an increase in cardiac troponin T release, cardiomyocyte apoptosis, and activation of innate immunity. Recipient FG‐4497 pretreatment for 4 h decreased infiltration of ED1+ macrophages, and mildly improved the long‐term allograft survival. In syngrafts donor FG‐4497 pretreatment increased activation of innate immunity, but did not induce myocardial damage. We conclude that the HIF‐1 pathway is activated in heart transplants. We suggest that pharmacological HIF‐α preconditioning of cardiac allografts donors would not lead to clinical benefit, while in recipients it may result in antiinflammatory effects and prolonged allograft survival.

Low intravitreal angiopoietin‐2 and VEGF levels in vitrectomized diabetic patients with simvastatin treatment

To investigate the intravitreal levels of potent vasoactive, angiogenic and extracellular matrix remodelling factors in the diabetic patients with simvastatin treatment.

Cardiomyocyte-targeted HIF-1α gene therapy inhibits cardiomyocyte apoptosis and cardiac allograft vasculopathy in the rat

BACKGROUND Hypoxia-inducible factor-1 (HIF-1), a key transcription factor in hypoxia, affects a wide range of adaptive cell functions. We examined the kinetics of endogenous HIF-1alpha during acute and chronic rejection, and the effect of exogenous HIF-1alpha in chronically rejecting rat cardiac allografts. METHODS Heterotopic cardiac transplantations were performed between major MHC-mismatched Dark Agouti and Wistar-Furth rats. Cyclosporine A (CsA) was used to prevent acute rejection in the chronic rejection model. The effect of HIF-1alpha overexpression was investigated by adeno-assocated virus 2 (AAV2)-mediated gene transfer of a constitutively stabilized form of mouse HIF-1alpha (AAV-HIF-1alpha). The analysis of allografts was based on histology, immunohistochemistry and quantitative reverse transcript-polymerase chain reaction (RT-PCR). RESULTS Acute and chronic rejection significantly induced HIF-1alpha mRNA in rat cardiac allografts when compared with syngeneic controls. Immunohistochemistry localized significantly increased HIF-1alpha immunoreactivity to vascular smooth muscle cells, vascular endothelial cells, post-capillary venules and graft-infiltrating mononuclear inflammatory cells of the allograft, whereas expression in cardiomyocytes remained unchanged. Regression analysis revealed a linear correlation between the progression of cardiac allograft vasculopathy (CAV) and HIF-1alpha immunoreactivity in post-capillary venules and graft-infiltrating mononuclear inflammatory cells in chronically rejecting rat cardiac allografts. AAV-HIF-1alpha enhanced cardiomyocyte HIF-1alpha production and significantly reduced cardiomyocyte apoptosis and the development of CAV in chronically rejecting rat cardiac allografts. CONCLUSIONS We found that acute and chronic rejection increased HIF-1alpha mRNA and protein levels in rat cardiac allografts. On the other hand, cardiomyocyte-targeted HIF-1alpha gene transfer inhibited cardiomyocyte apoptosis and the development of CAV, suggesting a novel therapeutic strategy for HIF-1alpha in cardiac allografts.

The Finnish national guideline for diagnosis, treatment and follow‐up of patients with wet age related macular degeneration

Age‐related macular degeneration (AMD) is the main cause of visual impairment in developed countries. Several improvements in the visualization of posterior segment of the eye together with the introduction of intravitreal anti‐VEGF treatment have revolutionized the prognosis of the wet form of AMD (wAMD). Increasing incidence of wAMD together with the limited resources of society and of the healthcare system poses challenges for the provision and development of care. In context of these current aspects, we aimed to set evidence‐based medical guidelines for diagnosis, treatment and follow‐up of patients with wAMD.