Calafiore cardioplegia offers better myocardial protection in adult and senescent rat hearts than Del Nido cardioplegia.

OBJECTIVES: We compared the cardioprotective capacity of Del Nido cardioplegia and warm Calafiore blood cardioplegia in an experimental setting during 90 min of ischaemia. METHODS: 20 adult and 20 senescent rat hearts were isolated and mounted on a blood-perfused, pressure-controlled Langendorff apparatus. After a stabilization period, cardiac arrest (90 min) was induced by the administration of either Calafiore (Cala) or Del Nido solution (DNS). While Cala was given warm and intermittently, DNS was given as a cold single shot. During 90 min of reperfusion, cardiac function and metabolism were evaluated and biomarker levels were measured. After the end of the experiment, hearts were prepared for electronmicroscopic investigation. RESULTS: Hearts exposed to Cala recovered faster during reperfusion compared with hearts administered DNS (Cala vs DNS at 30 min reperfusion: left ventricular developed pressure 72, SD: 22% of baseline (BL) versus 40, SD: 32% of BL, p < .001, and positive derived left ventricular pressure over time was better in both adult and senescent Cala groups (96, SD: 31% of BL) than in the DNS groups (39, SD: 27% of BL, p < .001). Ischaemic contractures were seen in the DNS groups starting after 30 min of ischaemia, whereas no rise in diastolic pressure was observed for the Cala groups. Accordingly, lactate production was higher after DNS (1.23 mg/dl (SD 0.87) than after Cala (0.33 mg/dl (SD 0.68), p = .015) at the beginning of reperfusion. Troponin I levels at the end of reperfusion were higher after DNS treatment in adult hearts (DNS: 287.9 SD: 147.7 ng/mL vs Cala 91.2: SD: 94.7 ng/mL, p = .02) and in senescent hearts (DNS: 376.5 (SD: 162.8) ng/ml versus Cala 104.7 (SD: 150.2) ng/ml, p = .025). Electron microscopy showed that the cellular oedema index was higher in adult DNS hearts (1.2 ± 0.2) than in adult Cala hearts (0.8 ± 0.1, p = .012), whereas the VS ratio was similar (0.18 ± 0.01 vs 0.17 ± 0.03). CONCLUSION: Calafiore cardioplegia offers better myocardial protection from ischaemia/reperfusion-related damage in isolated perfused adult and senescent rat hearts than Del Nido cardioplegia.

Maggot excretion products from the blowfly Lucilia sericata contain contact phase/intrinsic pathway-like proteases with procoagulant functions.

For centuries, maggots have been used for the treatment of wounds by a variety of ancient cultures, as part of their traditional medicine. With increasing appearance of antimicrobial resistance and in association with diabetic ulcers, maggot therapy was revisited in the 1980s. Three mechanisms by which sterile maggots of the green bottle fly Lucilia sericata may improve healing of chronic wounds have been proposed: Biosurgical debridement, disinfecting properties, and stimulation of the wound healing process. However, the influence of maggot excretion products (MEP) on blood coagulation as part of the wound healing process has not been studied in detail. Here, we demonstrate that specific MEP-derived serine proteases from Lucilia sericata induce clotting of human plasma and whole blood, particularly by activating contact phase proteins factor XII and kininogen as well as factor IX, thereby providing kallikrein-bypassing and factor XIa-like activities, both in plasma and in isolated systems. In plasma samples deficient in contact phase proteins, MEP restored full clotting activity, whereas in plasma deficient in either factor VII, IX, X or II no effect was seen. The observed procoagulant/intrinsic pathway-like activity was mediated by (chymo-) trypsin-like proteases in total MEP, which were significantly blocked by C1-esterase inhibitor or other contact phase-specific protease inhibitors. No significant influence of MEP on platelet activation or fibrinolysis was noted. Together, MEP provides contact phase bypassing procoagulant activity and thereby induces blood clotting in the context of wound healing. Further characterisation of the active serine protease(s) may offer new perspectives for biosurgical treatment of chronic wounds.

RNase1 prevents the damaging interplay between extracellular RNA and tumour necrosis factor-α in cardiac ischaemia/reperfusion injury.

Despite optimal therapy, the morbidity and mortality of patients presenting with an acute myocardial infarction (MI) remain significant, and the initial mechanistic trigger of myocardial "ischaemia/reperfusion (I/R) injury" remains greatly unexplained. Here we show that factors released from the damaged cardiac tissue itself, in particular extracellular RNA (eRNA) and tumour-necrosis-factor α (TNF-α), may dictate I/R injury. In an experimental in vivo mouse model of myocardial I/R as well as in the isolated I/R Langendorff-perfused rat heart, cardiomyocyte death was induced by eRNA and TNF-α. Moreover, TNF-α promoted further eRNA release especially under hypoxia, feeding a vicious cell damaging cycle during I/R with the massive production of oxygen radicals, mitochondrial obstruction, decrease in antioxidant enzymes and decline of cardiomyocyte functions. The administration of RNase1 significantly decreased myocardial infarction in both experimental models. This regimen allowed the reduction in cytokine release, normalisation of antioxidant enzymes as well as preservation of cardiac tissue. Thus, RNase1 administration provides a novel therapeutic regimen to interfere with the adverse eRNA-TNF-α interplay and significantly reduces or prevents the pathological outcome of ischaemic heart disease.

Pneumococcal phosphoglycerate kinase interacts with plasminogen and its tissue activator.

Streptococcus pneumoniae is not only a commensal of the nasopharyngeal epithelium, but may also cause life-threatening diseases. Immune-electron microscopy studies revealed that the bacterial glycolytic enzyme, phosphoglycerate kinase (PGK), is localised on the pneumococcal surface of both capsulated and non-capsulated strains and colocalises with plasminogen. Since pneumococci may concentrate host plasminogen (PLG) together with its activators on the bacterial cell surface to facilitate the formation of plasmin, the involvement of PGK in this process was studied. Specific binding of human or murine PLG to strain-independent PGK was documented, and surface plasmon resonance analyses indicated a high affinity interaction with the kringle domains 1-4 of PLG. Crystal structure determination of pneumococcal PGK together with peptide array analysis revealed localisation of PLG-binding site in the N-terminal region and provided structural motifs for the interaction with PLG. Based on structural analysis data, a potential interaction of PGK with tissue plasminogen activator (tPA) was proposed and experimentally confirmed by binding studies, plasmin activity assays and thrombus degradation analyses.

Extracellular nucleic acids as novel alarm signals in the vascular system. Mediators of defence and disease.

Upon vascular injury or tissue damage, the exposed intracellular material such as nucleic acids, histones and other macromolecules may come into contact with vessel wall cells and circulating blood cells and may thus, have an enduring influence on wound healing and body defence processes. This short review summarizes recent work related to extracellular DNA and RNA and their role as prominent alarm signals and inducers of different defence reactions related to innate immunity and thrombus formation. Of particular importance are DNA-histone complexes (nucleosome material) that, having been expelled during stimulation of the neutrophils, not only trap and eliminate bacteria but also promote thrombus formation in the arterial and venous system. Consequently therefore, the administration of DNase exhibits strong antithrombotic functions. Similarly, extracellular RNA provokes activation of the contact phase system of blood coagulation and, by interacting with specific proteins and cytokines, it promotes vascular permeability and oedema formation. The development of RNA-mediated thrombosis, vasogenic oedema or proinflammatory responses are counteracted by the administration of RNase1 in several pathogenetic animal models. Thus, extracellular nucleic acids appear not only to function as host alarm signals that serve to amplify the defence response, but they also provide important links to thrombus formation as part of the innate immune system.

Elevated B-cell activating factor BAFF, but not APRIL, correlates with CSF cerebellar autoantibodies in pediatric opsoclonus-myoclonus syndrome.

Childhood opsoclonus-myoclonus syndrome (OMS) occurs idiopathic or, in association with a neuroblastoma, as a paraneoplastic syndrome. Since autoantibodies were identified in some patients, an autoimmune pathogenesis has been suspected. While the newly discovered B-cell activating factors BAFF and APRIL are involved in systemic autoimmune diseases, their association with neuroimmunological diseases is hardly understood. We here investigated the BAFF and APRIL levels in serum and cerebrospinal fluid (CSF) of OMS patients and their correlation with surface-binding autoantibodies. BAFF and APRIL were both determined by ELISA, and autoantibodies to cerebellar granular neurons (CGN) have been investigated by flow cytometry in 17 OMS patients, 16 neuroblastoma (NB) patients, 13 controls and 11 children with inflammatory neurological diseases (IND). BAFF, but no APRIL, was elevated in the CSF of OMS children and IND children. However, in contrast to IND patients, OMS patients did not have a blood-brain-barrier disturbance, indicating that BAFF was produced intrathecally in OMS patients, but not in IND patients. CSF BAFF levels showed a correlation with CSF CGN autoantibodies (r(2)=0.58, p<0.05). These data indicate that an activated B-cell system in the cerebrospinal fluid is involved in the pathogenesis of OMS, and BAFF may be a candidate parameter for the activation of B-cell immune system.

Role of coagulation and fibrinolysis in lung and renal fibrosis.

Elevated procoagulant and suppressed fibrinolytic activities are regularly encountered in different forms of clinical and experimental fibrosis of the lungs and the kidneys. Although primarily serving to provide a provisional matrix of repair largely consisting of fibrin and fibronectin, the involved procoagulant serine proteases and protease inhibitors may also exert distinct cellular downstream signaling events modifying the fibrotic response. In this review, evidence for an impaired regulation of coagulation and fibrinolysis factors in clinical and experimental lung and renal fibrosis is provided and the role of PAR (protease activated receptor) induced profibrotic and HGF (hepatocyte growth factor) elicited antifibrotic cellular events is worked out. In view of experiments obtained in animal models of lung and renal fibrosis, the potential therapeutic usefulness of anticoagulant or profibrinolytic strategies is discussed.

[Physiology of blood coagulation and fibrinolysis: biochemistry]. / Physiologie der Blutgerinnung und Fibrinolyse: Biochemie.

The principles of initiator and amplifications reactions of blood coagulation and fibrinolysis will be presented and discussed in relation to various regulatory pathways of haemostasis. In particular, cell surface-dependent activation and inhibition reactions are characteristics of multicomponent enzyme complexes that also allow the endogenous control of the haemostasis system. The understanding of these relationships in complications of haemostasis has lead to different strategies for the therapeutic intervention with pro- and anticoagulant substances.

Identification of CML-modified proteins in hemofiltrate of diabetic patients by proteome analysis.

The posttranslational modification of extra- and intracellular proteins by non-enzymatic glycation results in the formation of advanced glycation end products (AGEs) in physiological systems and is associated with the loss of protein structure and function. Modification by N (epsilon)-carboxymethyl lysine (CML) correlates with the risk for retinopathy in diabetes mellitus and has been discussed as a marker for the prediction of mortality in hemodialysis patients. AGEing of proteins is particularly increased under hyperglycemia associated with different late complications of diabetes mellitus. Modification of proteins to form AGE residues is significantly more enhanced in patients suffering from chronic renal disease than in hyperglycemia and is associated with increased risk for cardiovascular complications and inflammation in patients with chronic renal insuffiency. In order to identify and define the protein "substrates" for non-enzymatic glycation we used a proteome approach combining two-dimensional gel electrophoresis and immunoblotting with Edman protein sequencing to identify specific CML-modified proteins in human hemofiltrate, which essentially resembles plasma with respect to protein composition. Albumin, Ig kappa chain, prostaglandin D2 synthase, lysozyme C, plasma retinol binding protein and beta-2-microglobulin were identified as the major CML-modified proteins. CML-modified fragments of these proteins were also found in hemofiltrate. All identified proteins have in common that they appeared in hemofiltrate predominantly in their CML-modified form(s). Further studies of the functional roles of proteins identified by this new experimental approach could lead to the development of diagnostic tools to follow the progression of diabetes and contribute to the understanding of the pathogenesis of AGE-related diseases.

Extracellular RNA. A new player in blood coagulation and vascular permeability.

Upon vascular injury, locally controlled haemostasis prevents life threatening blood loss and ensures wound healing. Intracellular material derived from damaged cells at these sites will become exposed to cells and plasma proteins and could thereby influence vascular homeostasis, blood coagulation and defense mechanisms. Recently, this concept was documented by several studies indicating that extracellular nucleic acids, and RNA in particular, serve as promoter of blood coagulation in vivo and significantly increase the permeability across brain endothelial cells in vitro and in vivo. As procoagulant cofactor and "natural foreign material", RNA triggers the contactphase pathway of blood coagulation and thereby contributes to pathological thrombus formation. Administration of RNase significantly delayed occlusive thrombus formation and prevented edema formation in different animal models. Thus, extracellular RNA derived from damaged and necrotic cells may serve as a natural danger signal that contributes to initiation of host defense mechanisms, while antagonizing RNase provides new regimens for antithrombotic and vessel-protective therapies.

New autoantibodies in pediatric opsoclonus myoclonus syndrome.

Opsoclonus-myoclonus syndrome (OMS) is a rare neurologic disorder comprising the main symptoms of eye-movement disturbances, muscle jerks, and severe ataxia. In children and adults, some cases are associated with a tumor as a paraneoplastic syndrome, whereas in children the paraneoplastic form is almost exclusively associated with neuroblastoma. The detection of autoantibodies in some OMS sera led to the hypothesis that the syndrome is of autoimmune origin. Beside autoantibodies against intracellular proteins, such as anti-Hu, alpha-enolase, and KHSRP, specific binding of autoantibodies to the surface of neuroblastoma cells and cerebellar granular neurons have been found. Antiproliferative and proapoptotic effects of these autoantibodies on neuroblastoma cell lines were noted as well. These results support the concept of a humoral autoimmune process in the pathogenesis of OMS.

Cellular origin of pro-coagulant and (anti)-fibrinolytic factors in bleomycin-injured lungs.

Excessive pro-coagulant and decreased fibrinolytic activities in the alveolar compartment have been repeatedly documented for inflammatory and fibrotic lung diseases. The current authors determined the contribution of different resident lung cells to the altered local production of coagulation- and fibrinolysis-system components in bleomycin-injured mouse lungs via cell-specific and quantitative assessment of mRNA levels of various pro-coagulant and (anti)-fibrinolytic factors. Laser-assisted microdissection technology was used to sample specific cell populations in combination with subsequent mRNA analysis by real-time quantitative reverse transcriptase-PCR. Additionally, western blot analysis, immunohistochemistry and activity assays were performed. Following bleomycin challenge, the strongest induction of tissue factor and plasminogen activator inhibitor (PAI)-1 mRNA expression was observed in alveolar macrophages (approximately 250- and 60-fold induction, respectively). These factors were also upregulated in alveolar type II cells, but to an approximately six-fold lesser extent. In contrast, PAI-2 expression was induced exclusively in alveolar macrophages. A slight increase of urokinase-type plasminogen activator (uPA) expression was also observed in alveolar macrophages (two-fold induction), but uPA activity was reduced due to a disproportionate increase of PAI production. Alveolar macrophages and, to a lesser extent, alveolar type II cells are the main sources of locally produced pro-coagulant and anti-fibrinolytic factors in bleomycin-injured lungs.

HCG in the regulation of placental angiogenesis. Results of an in vitro study.

Placental vascular development is essential for fetal growth and development. Inadequate placental vascular development is associated with early pregnancy losses and other pregnancy related pathologies. In addition to the ubiquitous, well-characterized angiogenic factors like vascular endothelial growth factor (VEGF) or basic fibroblast growth factor (bFGF), some pregnancy-specific factors (e.g. human chorionic gonadotropin (hCG), insulin-like growth factor-II (IGF-II) or alpha fetoprotein (AFP) were recently described to play a possible regulatory role in this process. In the present study we described an improved separation method for human placental microvascular endothelial cells (HPMVEC) and their functional characterization. Using the combination of enzymatic digestion and multistep immunomagnetic sorting with CD31 antibodies a model for villous vascularization was established. Isolated cells took up ac-dil-LDL, spontaneously formed capillary-like structures, and expressed common endothelial markers such as vascular endothelial growth factor receptor-2 (VEGFR-2), angiopoetin-1 and -2, Tie-2, CD144, thrombomodulin, and von Willebrand factor (vWF) as shown by RT-PCR, flow cytometry and Western blot analysis. The expression of the hCG/LH receptor in the placental vascular tree was verified both in vitro and in vivo. hCG stimulated proliferation of HPMVEC in a dose specific manner. While hCG alone had no significant effect on endothelial cell apoptosis, the combination of VEGF-A and hCG protected HPMVEC from staurosporine-induced apoptosis. hCG significantly stimulated sprout formation when compared to controls in a spheroid angiogenesis assay. Our results demonstrate a modified and reproducible method allowing studies of placental vascular development and provide new insights into the possible role of trophoblastic factors in this process.

IgG subclass distribution of autoantibodies in pediatric opsoclonus-myoclonus syndrome.

Opsoclonus-myoclonus syndrome (OMS) in children is a rare disorder including a severe eye movement disturbance, myoclonia, ataxia and often developmental retardation. Both OMS forms, idiopathic or neuroblastoma-associated (paraneoplastic), have been suspected to be autoimmune. Recently, autoantibodies have been found in OMS sera. We here show that autoantibodies in OMS, both intracellular and surface binding, belong mainly to the IgG3 subclass, although the total serum IgG3 level is normal. These results support the autoimmune hypothesis and point to a protein autoantigen as antigenic target.

Endothelial survival factors and spatial completion, but not pericyte coverage of retinal capillaries determine vessel plasticity.

Pericyte loss and capillary regression are characteristic for incipient diabetic retinopathy. Pericyte recruitment is involved in vessel maturation, and ligand-receptor systems contributing to pericyte recruitment are survival factors for endothelial cells in pericyte-free in vitro systems. We studied pericyte recruitment in relation to the susceptibility toward hyperoxia-induced vascular remodeling using the pericyte reporter X-LacZ mouse and the mouse model of retinopathy of prematurity (ROP). Pericytes were found in close proximity to vessels, both during formation of the superficial and the deep capillary layers. When exposure of mice to the ROP was delayed by 24 h, i.e., after the deep retinal layer had formed [at postnatal (p) day 8], preretinal neovascularizations were substantially diminished at p18. Mice with a delayed ROP exposure had 50% reduced avascular zones. Formation of the deep capillary layers at p8 was associated with a combined up-regulation of angiopoietin-1 and PDGF-B, while VEGF was almost unchanged during the transition from a susceptible to a resistant capillary network. Inhibition of Tie-2 function either by soluble Tie-2 or by a sulindac analog, an inhibitor of Tie-2 phosphorylation, resensitized retinal vessels to neovascularizations due to a reduction of the deep capillary network. Inhibition of Tie-2 function had no effect on pericyte recruitment. Our data indicate that the final maturation of the retinal vasculature and its resistance to regressive signals such as hyperoxia depend on the completion of the multilayer structure, in particular the deep capillary layers, and are independent of the coverage by pericytes.

Integrin-mediated leukocyte adhesive interactions: regulation by haemostatic factors.

Leukocyte recruitment to sites of inflammation, infection or vascular injury is a complex event that is orchestrated by a tightly coordinated sequence of interactions between leukocytes and cells of the vessel wall, especially endothelial cells. These interactions are controlled by the expression and activation of various adhesion receptors and protease systems. This review will focus on novel aspects of the regulation of integrin-dependent leukocyte adhesion by haemostatic factors. Here, so-called non-haemostatic properties of endogenous proteins such as high molecular weight kininogen, urokinase receptor, urokinase, as well as plasminogen and its cleavage product angiostatin in leukocyte adhesion and transmigration will be summarized. The crosstalk between haemostatic factors and inflammatory reactions may contribute to a better understanding of inflammatory vascular disorders and to the development of novel therapeutical concepts.