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.

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.

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.

Alanine-rich amphiphilic peptide containing the RGD cell adhesion motif: a coating material for human fibroblast attachment and culture.

We studied the self-assembly of peptide A6RGD (A: alanine, R: arginine, G: glycine, D: aspartic acid) in water, and the use of A6RGD substrates as coatings to promote the attachment of human cornea stromal fibroblasts (hCSFs). The self-assembled motif of A6RGD was shown to depend on the peptide concentration in water, where both vesicle and fibril formation were observed. Oligomers were detected for 0.7 wt% A6RGD, which evolved into short peptide fibres at 1.0 wt% A6RGD, while a co-existence of vesicles and long peptide fibres was revealed for 2-15 wt% A6RGD. A6RGD vesicle walls were shown to have a multilayer structure built out of highly interdigitated A6 units, while A6RGD fibres were based on ß-sheet assemblies. Changes in the self-assembly motif with concentration were reflected in the cell culture assay results. Films dried from 0.1-1.0 wt% A6RGD solutions allowed hCSFs to attach and significantly enhanced cell proliferation relative to the control. In contrast, films dried from 2.5 wt% A6RGD solutions were toxic to hCSFs.

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.

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.

Thermoreversible lysozyme hydrogels: properties and an insight into the gelation pathway.

The gelation behaviour of aqueous solutions of hen egg white lysozyme (HEWL) in the presence of 20 mM DTT in the concentration range 0.7 to 4.0 mM has been investigated using microDSC, FTIR, cryoTEM, SANS and oscillatory rheology. The macroscopic critical gelation concentration, Cgel, was found to be ∼ 3.0 mM. The disruption of the disulfide bonds by the DTT and the destabilisation of the protein were found to be a prerequisite for the formation of ß-sheet rich fibrils under the mild conditions used in this work. Using our methodology the hydrogels obtained have a pH of 7, hence are suitable for cell culture, and are also thermoreversible. The hydrogel melting temperature was found to increase with increasing concentration and a similar structure was observed across the concentration range investigated. Our results suggest these systems are composed of a well defined regular network where single ß-sheet rich fibrils (∼ 3 nm diameter) form initially, then two of these fibrils associate two-by-two to form junctions (∼ 6 nm diameter) and then on cooling further aggregate to form larger bundles of fibres. The network mesh size was found to decrease with increasing concentration. Our results suggest that below Cgel small unconnected gel-like aggregates exist that have a similar structure to the hydrogels obtained above Cgel. Using our data we propose a model for the denaturation and gelation behaviour of our system. During the first heating an α-helix to ß-sheet molecular transition for the protein conformation occurs resulting in ß-sheet rich fibrils forming through the self-assembly of ß-sheet rich denaturated proteins. At high temperature the solution contains ß-sheet rich fibrils with dissolved protein. On cooling an increase in the amount of ß-sheet was observed via FTIR suggesting that as the temperature is decreased more and more protein forms ß-sheet rich fibrils. At the gelation temperature these fibrils associate two-by-two to form the network junctions resulting in the macroscopic gelation of the sample. Our results suggest the network junctions are formed via specific hydrophobic interactions. The hydrogels elastic modulus was found to scale as C2.45 for C > Cgel.

Enzymatic hydrolysis combined with mechanical shearing and high-pressure homogenization for nanoscale cellulose fibrils and strong gels.

Toward exploiting the attractive mechanical properties of cellulose I nanoelements, a novel route is demonstrated, which combines enzymatic hydrolysis and mechanical shearing. Previously, an aggressive acid hydrolysis and sonication of cellulose I containing fibers was shown to lead to a network of weakly hydrogen-bonded rodlike cellulose elements typically with a low aspect ratio. On the other hand, high mechanical shearing resulted in longer and entangled nanoscale cellulose elements leading to stronger networks and gels. Nevertheless, a widespread use of the latter concept has been hindered because of lack of feasible methods of preparation, suggesting a combination of mild hydrolysis and shearing to disintegrate cellulose I containing fibers into high aspect ratio cellulose I nanoscale elements. In this work, mild enzymatic hydrolysis has been introduced and combined with mechanical shearing and a high-pressure homogenization, leading to a controlled fibrillation down to nanoscale and a network of long and highly entangled cellulose I elements. The resulting strong aqueous gels exhibit more than 5 orders of magnitude tunable storage modulus G' upon changing the concentration. Cryotransmission electron microscopy, atomic force microscopy, and cross-polarization/magic-angle spinning (CP/MAS) 13C NMR suggest that the cellulose I structural elements obtained are dominated by two fractions, one with lateral dimension of 5-6 nm and one with lateral dimensions of about 10-20 nm. The thicker diameter regions may act as the junction zones for the networks. The resulting material will herein be referred to as MFC (microfibrillated cellulose). Dynamical rheology showed that the aqueous suspensions behaved as gels in the whole investigated concentration range 0.125-5.9% w/w, G' ranging from 1.5 Pa to 105 Pa. The maximum G' was high, about 2 orders of magnitude larger than typically observed for the corresponding nonentangled low aspect ratio cellulose I gels, and G' scales with concentration with the power of approximately three. The described preparation method of MFC allows control over the final properties that opens novel applications in materials science, for example, as reinforcement in composites and as templates for surface modification.

The effect of platelet-derived growth factor ligands in rat cardiac allograft vasculopathy and fibrosis.

BACKGROUND: In chronic rejection, parenchymal fibrosis and cardiac allograft vasculopathy (CAV) characterized by neointimal growth are the leading causes of graft loss for heart transplant recipients. During alloimmune responses a variety of cytokines, adhesion proteins, and growth factors, such as platelet-derived growth factor (PDGF), are up-regulated. The PDGF family (AA, AB, BB, CC, DD), which acts mainly on connective tissue cells, is considered to be a potent mitogenic and chemotactic factor for fibroblasts and vascular smooth muscle cells. In this study, we evaluated the effects of PDGF ligands in chronic rejection. METHODS: Heterotopic heart transplantations were performed between fully major histocompatability complex-mismatched Dark Agouti to Wistar Furth rats receiving cyclosporine immunosuppression. Allograft coronary arteries were perfused with a recombinant adeno-associated virus (AAV) encoding enhanced green fluorescence protein (EGFP) as a control gene or PDGF-A, -B, -C, -D. Allografts were harvested at 100 days for morphometric analysis of CAV and fibrosis. RESULTS: AAV-mediated transgene expression was detected by EGFP immunoreactivity across the graft section (at 100 days) in AAV EGFP-perfused allografts. AAV PDGF-A, -C, and -D perfusion resulted in accelerated CAV and fibrosis. In contrast, AAV PDGF-B perfusion did not induce arteriosclerotic changes or fibrosis in cardiac allografts. CONCLUSIONS: AAV PDGF-A, -C, and -D overexpression accelerated the development of chronic rejection, whereas PDGF-B did not. Our results suggested that more targeted therapy with monoclonal antibodies blocking the active sites of PDGF-A, -C, and -D may produce beneficial effects on heart transplant survival.

Effect of graft preservation and acute rejection on hypoxia-inducible factor-1 in rat cardiac allografts.

Hypoxia plays an integral part in cardiac transplantation as prolonged graft preservation is an individual risk factor for the development of cardiac allograft vasculopathy (CAV). In this study we characterized the role of hypoxia-inducible factor-1 (HIF-1) during prolonged graft preservation, ischemia-reperfusion (I/R), acute rejection, and chronic rejection. Heart transplantations were performed from Dark Agouti (DA) to Wister-Furth (allo) or DA to DA (syn) rats, without immunosuppression (acute rejection model, harvested at day 5) or with cyclosporine (chronic rejection model, harvested at day 60). To study the effect of preservation on HIF-1 regulation, normal DA hearts were subjected to different cold ischemia times with or without 45 minutes of additional warm ischemia. The role of I/R was studied by harvesting syngrafts at different time points after reperfusion. Real-time reverse-transcriptase polymerase chain reaction quantified total HIF-1 mRNA, while enzyme-linked immunosorbent assay and immunohistochemistry quantified and localized HIF-1 protein. Our results show that HIF-1 nuclear immunoreactivity is increased during graft preservation and I/R leads to loss of nuclear HIF-1 immunoreactivity. Acute rejection induced HIF-1 in mRNA level. Our findings thus indicated that HIF-1 is activated during transplantation and suggested that manipulation of the HIF-1 pathway might reveal new therapeutic options to manage CAV.

ATP requirements and small interfering RNA structure in the RNA interference pathway.

We examined the role of ATP in the RNA interference (RNAi) pathway. Our data reveal two ATP-dependent steps and suggest that the RNAi reaction comprises at least four sequential steps: ATP-dependent processing of double-stranded RNA into small interfering RNAs (siRNAs), incorporation of siRNAs into an inactive approximately 360 kDa protein/RNA complex, ATP-dependent unwinding of the siRNA duplex to generate an active complex, and ATP-independent recognition and cleavage of the RNA target. Furthermore, ATP is used to maintain 5' phosphates on siRNAs. A 5' phosphate on the target-complementary strand of the siRNA duplex is required for siRNA function, suggesting that cells check the authenticity of siRNAs and license only bona fide siRNAs to direct target RNA destruction.

Synergistic inhibition of Listeria monocytogenes on cold-smoked rainbow trout by nisin and sodium lactate.

The inhibition of Listeria monocytogenes and mesophilic aerobic bacteria in cold-smoked rainbow trout by nisin, sodium lactate or their combination was studied. Nisin (4000-6000 IU/ml), sodium lactate (60%) or their combination (1:1) were injected into rainbow trout at an industrial scale before the smoking process, or injected into the finished smoked product. Both types of fish samples were smoked, sliced and vacuum-packed according to normal practice in the plant. Packages were opened and L. monocytogenes was inoculated (10(3)-10(4) log colony forming units (cfu)/g) onto the fish samples, which were then vacuum packed again. Samples were stored at 8 degrees C for 17 days or at 3 degrees C for 29 days. Listeria and mesophilic aerobic bacteria counts were measured once a week. The effects of treatments on sensory characteristics and storage stability were also analyzed. Both nisin and lactate inhibited the growth of L. monocytogenes in smoked fish, but the combination of the two compounds was even more effective. The combination of nisin and sodium lactate injected into smoked fish decreased the count of L. monocytogenes from 3.26 to 1.8 log cfu/g over 16 days of storage at 8 degrees C. The level of L. monocytogenes remained almost constant (4.66-4.92 log cfu/g) for 29 days at 3 degrees C in the samples injected before smoking and which contained both nisin and sodium lactate. The treatments did not affect the sensory characteristics of cold-smoked rainbow trout. Based on a triangle test, the sensory quality of all test samples remained unchanged for 23 days of storage at 3 degrees C, whereas the control fish prepared without additives or additional salt remained unchanged only for 16 days.

Adenoids provide a microenvironment for the generation of CD4(+), CD45RO(+), L-selectin(-), CXCR4(+), CCR5(+) T lymphocytes, a lymphocyte phenotype found in the middle ear effusion.

Adenoidectomy in children with otitis media with effusion reduces inflammation in the middle ear by an unknown mechanism. Potentially, the adenoids of these children may serve as a site for the differentiation of lymphocytes, which after entering blood circulation eventually extravasate in the middle ear mucosa and thereby contribute to excessive inflammation. During lymphocyte extravasation various adhesion molecules and chemokines play a crucial role. To evaluate possible connections between the adenoids and middle ear inflammation, the expression of the chemokine receptors CXCR4 and CCR5 and the lymphocyte homing receptor L-selectin were analyzed in adenoidal and middle ear lymphocytes. It was found that most CD4(+) T lymphocytes in the middle ear effusion express the memory phenotype marker CD45RO and the chemokine receptors CXCR4 and CCR5, but are negative for the lymphocyte homing receptor L-selectin. This cell phenotype was rare in peripheral blood but was found much more frequently in the adenoids. The results suggest that the adenoids provide a microenvironment for the generation for CD4(+), CD45RO(+), L-selectin(-), CXCR4(+) and CCR5(+) T lymphocytes. Further, these cells may include cells that have the capacity to home to the middle ear mucosa. As the adenoidal CD4(+) memory phenotype CD45RO(+) T cells expressed the activation antigen CD69 and included cells expressing the HIV co-receptors CXCR4 and CCR5 at a high level, they may be permissive for HIV infection.

Adapting atomic force microscopy for cell biology.

We present details of our AFM modifications to produce an adaptable imaging system for the cell biologist. We have designed and validated a new inverted microscope interface and a scan head with increased Z-range, based upon the TopoMetrix Explorer AFM. We have utilised these changes, together with home-made glass ball cantilevers, to obtain topographical information over cells with large Z-dimension (over 15 microm high), and mapped calcitonin-calcitonin receptor binding forces in living bone cells. We conclude that modified AFM can be used to evaluate intermolecular events in living cells and that this approach will ensure general application to the study of receptor-ligand interactions under truly physiological conditions.

Synergistic antimicrobial effect of nisin whey permeate and lactic acid on microbes isolated from fish.

The antimicrobial activity of a combination of lactic acid and whey permeate fermented by a nisin-producing Lactococcus lactis strain was tested by the agar diffusion method using bacteria isolated from fish as test organisms. Lactic acid inhibited all bacterial strains studied, but nisin whey permeate inhibited Gram-positive bacteria only. The combination was more effective than lactic acid alone against Pseudomonas fluorescens and Staphylococcus hominis isolated from fish, and Pseudomonas aeruginosa ATCC9721 and Micrococcus luteus ATCC9341.

Molecular epidemiology of HIV-1 based on phylogenetic analysis of in vivo gag p7/p9 direct sequences.

Viruses of different geographical origin are circulating in Finland. We wanted to test whether phylogenetic analysis of patient lymphocyte proviral quasispecies sequences could be used to group different strains into genetic lineages. The gag p7/p9 coding region was analyzed using solid-phase direct sequencing from 30 patients in Finland and Estonia. Proviral sequences were found to represent at least four, possibly even five, different, highly diverged major lineages. Different methods of phylogenetic analysis resulted in the same conclusion. Serial samples from the same patients, taken over a period of several years showed limited variation over time. Cases of potential patient-to-patient transmission or common source of infection were identified based on the sequence analysis. Compared to similar analyses of longer genome segments, the gag p7/p9 nucleic acid binding protein coding region produces analogous results in phylogenetic analysis. The method can be used as a rapid way of determining the genetic subtype of HIV-1 strains circulating in populations.