SummaryCo-stimulation via CD154 binding to CD40, pivotal for both innate and adaptive immunity, may also link haemostasis to vascular remodelling. Here we demonstrate that human platelet-bound or recombinant soluble CD154 (sCD154) elicit the release from and tethering of ultra-large (UL) von Willebrand factor (vWF) multimers to the surface of human cultured endothelial cells (ECs) exposed to shear stress. This CD40-mediated ULVWF multimer release from the Weibel-Palade bodies was triggered by consecutive activation of TRAF6, the tyrosine kinase c-Src and phospholipase Cγ1 followed by inositol-1,4,5 tris-phosphate-mediated calcium mobilisation. Subsequent exposure to human washed platelets caused ULVWF multimer-platelet string formation on the EC surface in a shear stress-dependent manner. Platelets tethered to these ULVWF multimers exhibited P-selectin on their surface and captured labelled monocytes from the superfusate. When exposed to shear stress and sCD154, native ECs from wild-type but not CD40 or vWF-deficient mice revealed a comparable release of ULVWF multimers to which murine washed platelets rapidly adhered, turning P-selectin-positive and subsequently capturing monocytes from the perfusate. This novel CD154-provoked ULVWF multimerplatelet string formation at normal to fast flow may contribute to vascular remodelling processes requiring the perivascular or intravascular accumulation of pro-inflammatory macrophages such as arteriogenesis or atherosclerosis.

A pivotal autocrine mechanism following endothelial cell activation is the release of angiopoietin‐2 (Ang‐2) which subsequently antagonizes the binding of Ang‐1 to the Tie‐2 receptor. In this context, we noted that hypertension in mice reduces the abundance of Ang‐2 stored in arterial endothelial cells and hypothesized that this Ang‐2 release depends on an increase in circumferential wall tension or stretch – a hallmark of hypertension. In fact, stretching isolated perfused mouse arteries or human cultured endothelial cells triggered rapid Ang‐2 release in a calcium‐dependent manner. Interestingly, Ang‐1 abolished stretch‐induced Ang‐2 release both in the isolated perfused mouse arteries and in the cultured endothelial cells. Subsequent analyses of the latter indicated that pre‐treatment with Ang‐1 robustly inhibits their stretch‐induced calcium response as well as the stretch‐mediated expression of pro‐inflammatory adhesion molecules. Collectively, our results suggest that stretch induces the release of Ang‐2 from human cultured endothelial cells and that Ang‐1 counteracts these Ang‐2 functions by attenuating the stretch‐dependent intracellular calcium release which may at least in part explain its anti‐inflammatory effect in stretch‐stimulated endothelial cells.

AIMSAdequate endothelial cell stimulation is a prerequisite for the adaptive remodelling of macro- and microvessels. A pivotal autocrine mechanism following endothelial cell activation is the release of angiopoietin-2 (Ang-2), which subsequently antagonizes the binding of Ang-1 to the Tie-2 receptor, thus sensitizing the endothelial cells to pro-angiogenic and/or pro-inflammatory stimuli. Based on the observation that hypertension in mice reduces the abundance of Ang-2 stored in arterial endothelial cells, this study was aimed at testing the hypothesis that an increase in wall stress (WS) or stretch-a hallmark of hypertension-is sufficient to release Ang-2 from endothelial cells.METHODS AND RESULTSIn fact, stretching of isolated perfused mouse arteries or human cultured endothelial cells rapidly elicited an increased release of Ang-2. In the cultured endothelial cells, this was preceded by a transient rise in intracellular free calcium, abrogated through calcium chelation and accompanied by a decrease in Tie-2 phosphorylation. Interestingly, Ang-1 abolished the stretch-induced release of Ang-2 from both cultured and native endothelial cells through inhibiting the stretch-dependent mobilization of intracellular calcium.CONCLUSIONCollectively, these results indicate that increased WS or stretch facilitates the release of Ang-2 from endothelial cell Weibel-Palade bodies, and that Ang-1 can block this by attenuating the stretch-mediated rise in intracellular calcium.

Objective. Ischemia-reperfusion injury is associated with a high rate of primary organ dysfunction and thereby contributes substantially to morbidity and mortality in the course of liver transplantation. In the present study, the impact of metabolic preconditioning with fructose on ischemia-reperfusion injury in the isolated perfused rat liver model is evaluated. Methods. Fasted rats received a single intravenous fructose injection to induce metabolic preconditioning (fructose group) or a volume equivalent of normal saline (control group) 10 min before liver explantation. After 26 h of cold storage, livers were reperfused for 90 min at 37°C with Krebs-Henseleit buffer. The parameters used to quantify ischemia-reperfusion injury included hepatic oxygen consumption, enzyme release, and cell viability. Results. During reperfusion, livers in the fructose group consumed more oxygen than livers in the control group (p < 0.005), indicating ATP synthesis as a result of glycolytic fructose degradation. Moreover, cell injury was reduced by fructose administration, as reflected by a lower enzyme release during both cold ischemia and reperfusion (p < 0.05). Finally, hepatocyte viability at the end of reperfusion was significantly higher in the fructose group (p < 0.01). However, there was no significant difference between the two experimental groups in reference to the viability of endothelial cells. Conclusion. In clinical use, metabolic preconditioning with fructose prior to organ recovery might contribute to a reduction in the incidence of primary organ dysfunction after liver transplantation.

BACKGROUND:Nonsteroidal anti-inflammatory drugs (NSAID) represent a one of the most widely used anti-inflammatory substances. Their anti-inflammatory effects are mainly based on inhibition of cyclooxygenase. The potential direct effect of NSAID on leukocyte migration was poorly investigated. Using time-lapse microscopy and 96-well fluorescence-based assay, we studied the effect of three different NSAID, ketoprofen, diclofenac and SC-560, on leukocyte haptokinesis and haptotaxis in vivo and in vitro.RESULTS:NSAID induced an immediate inhibiting effect on leukocyte migration both in vitro and in vivo. This effect was dose-dependent and was not restricted to a specific type of leukocytes. The inhibition of leukocyte migration by NSAID was partially re-stored after removal of inhibiting agent. Only complete blockade of leukocyte migration was accompanied by a strong reduction of [Ca(2+)]i.CONCLUSIONS:NSAID strongly supress leukocyte migration. The results of the present study may have important clinical implications since blockade of leukocyte migration can be achieved after topical application of NSAID.

In 2007, 2.7 million vertebrates were used for animal experiments and other scientific purposes in Germany alone. Since 1998 there has been an increase in the number of animals used for research purposes, which is partly attributable to the growing use of transgenic animals. These animals are, for instance, used as in vivo models to mimic human diseases like diabetes, cancer or Alzheimer's disease. Here, transgenic model organisms serve as valuable tools, being instrumental in facilitating the analysis of the molecular mechanisms underlying human diseases, and might contribute to the development of novel therapeutic approaches. Due to variable and, sometimes low, efficiency (depending on the species used), however, the generation of such animals often requires a large number of embryo donors and recipients. The experts evaluated methods that could possibly be utilised to reduce, refine or even replace experiments with transgenic vertebrates in the mid-term future. Among the promising alternative model organisms available at the moment are the fruit fly Drosophila melanogaster and the roundworm Caenorhabditis elegans. Specific cell culture experiments or three-dimensional (3D) tissue models also offer valuable opportunities to replace experiments with transgenic animals or reduce the number of laboratory animals required by assisting in decision-making processes. Furthermore, at the workshop an in vitro technique was presented which permits the production of complete human antibodies without using genetically modified ("humanised") animals. Up to now, genetically modified mice are widely used for this purpose. Improved breeding protocols, enhanced efficiency of mutagenesis as well as training of laboratory personnel and animal keepers can also help to reduce the numbers of laboratory animals. Well-trained staff in particular can help to minimise the pain, suffering and discomfort of animals and, at the same time, improve the quality of data obtained from animal experiments. This, in turn, can lead to a reduction in the numbers of animals needed for each experiment. The experts also came to the conclusion that the numbers of laboratory animals can be reduced by open access to a central database that provides detailed documentation of completed experiments involving transgenic animals. This documentation should not be restricted to experiments with substantial scientific results that warrant publication, but should also include those with "negative" outcome, which are usually not published. Capturing all kinds of results within such a database provides added value to the respective scientists and the scientific community as a whole; it could also help to stimulate collaborations and to ensure funding for future research. An important aspect to be considered in the generation of this kind of database is the quality and standardisation of the information provided on existing in vitro models and the respective opportunities for their use. The experts felt that the greatest potential for reducing the numbers of laboratory animals in the near future realistically might not be offered by the complete replacement of transgenic animal models but by opportunities to examine specific questions to a greater degree using in vitro models, such as cell and tissue cultures including organotypic models. The use of these models would considerably reduce the number of in vivo experiments using transgenic animals. However, the overall number of experimental animals may still be increasing or remain unaffected, e.g. when transgenic animals continue to serve as the source of primary cells and organs/tissues for in vitro experiments.

BACKGROUND:Pancreatic cancer (PaCa) is a fatal human cancer due to its exceptional resistance to all current anticancer therapies. The cytoprotective enzyme heme oxygenase-1 (HO-1) is significantly overexpressed in PaCa and seems to play an important role in cancer resistance to anticancer treatment. The inhibition of HO-1 sensitized PaCa cells to chemo- and radiotherapy in vitro. Therefore, we investigated the effects of HO-1 and its metabolites biliverdin, carbon monoxide and iron on PaCa cells. PaCa cell lines with divergent HO-1 expression patterns were used in a murine orthotopic cancer model. HO-1 expression and activity was regulated by zinc (inhibition) and cobalt (induction) protoporphyrin. Furthermore, the influence of cellular HO-1 levels and its metabolites on effects of standard chemotherapy with gemcitabine was tested in vivo and in vitro.RESULTS:High HO-1 expression in PaCa cell lines was associated with increased chemoresistance in vitro. Chemoresistance to gemcitabine was increased during HO-1 induction in PaCa cells expressing low levels of HO-1. The inhibition of HO-1 activity in pancreatic tumors with high HO-1 boosted chemotherapeutic effects in vivo significantly. Furthermore, biliverdin and iron promoted PaCa resistance to chemotherapy. Consequently, specific iron chelation by desferrioxamine revealed profound anticancerous effects.CONCLUSION:In summary, the inhibition of HO-1 and the chelation of iron in PaCa cells were associated with increased sensitivity and susceptibility of pancreatic tumors to chemotherapy in vivo. The metabolites biliverdin and iron seem to be involved in HO-1-mediated resistance to anticancer treatment. Therefore, HO-1 inhibition or direct interference with its metabolites may evolve new PaCa treatment strategies.

Extravascular neutrophil migration is poorly characterized in vivo. To test the hypothesis that this migration is a non-random process, we used videomicroscopy to monitor neutrophils in irises of living mice with endotoxin-induced uveitis (EIU). Paths of individual cells were analyzed. Nearly all of these cells were moving in divergent directions, and mean displacement plots indicated that the predominant movement was random. The paths of some cells were fit to bivariate autoregressive integrated moving average models that revealed at least two modes of movement: random search and linear trend. Cell speed was significantly reduced by the actin inhibitor, cytochalasin D. The pattern of migration for neutrophils is in marked contrast to what we previously described for antigen-presenting cells in the iris, but somewhat resembles recent descriptions for T cells within a lymph node. Characterization of extravascular migration of neutrophils has important implications for understanding infection and immunity.

BACKGROUND:Phosphodiesterase-3 inhibition has been shown to attenuate hepatic warm ischemia-reperfusion injury. The aim of this study was to investigate the effect of milrinone, phosphodiesterase-3 inhibitor, on post-ischemic microcirculation of rat livers by intravital microscopy. MATERIALS AND METHODS:Male Wistar rats were randomly assigned to three groups; group A, milrinone pre-treatment; group B, ischemic pre-conditioning; and group C, no pre-treatment. All animals underwent a 60-min warm ischemia of the left lateral liver lobe. Microvascular perfusion and leukocyte-endothelial interaction were observed by intravital videomicroscopy. Hepatocellular viability and cellular damage were quantified by adenosine triphosphate tissue concentration as well as alanine aminotransferase and lactate dehydrogenase blood levels, respectively. RESULTS:In groups A and B, cyclic AMP hepatic tissue concentration was elevated significantly. After reperfusion, microvascular perfusion in hepatic sinusoids was significantly better maintained, and the number of adherent leukocytes was reduced in sinusoids and in post-sinusoidal venules in these rats. Serum transaminase blood levels were suppressed significantly in these groups compared with controls. CONCLUSION:The demonstrated improvement of hepatic microcirculation is certainly derived from milrinone induced cell protection in ischemia reperfusion of the liver. This effect is outlined by improved energy status and reduced liver enzyme liberation and mimics the effect of ischemic pre-conditioning.

L-selectin belongs to the C-type lectin family of glycoproteins and is constitutively expressed on most leukocytes. L-selectin mediates leukocyte rolling in inflamed microvessels and high endothelial venules (HEV) via binding to specific carbohydrate structures on selectin ligands. Previous studies using sialidase treatment suggested a role of sialic acid residues in L-selectin-dependent rolling. To investigate the role of the alpha2,3-sialyltransferase (ST3Gal)-IV on L-selectin ligand activity in vivo, we studied leukocyte rolling in inflamed venules of the cremaster muscle and in Peyer's patch HEV of ST3Gal-IV-deficient mice and littermate control mice. In cremaster muscle venules with or without TNF-alpha treatment, L-selectin-dependent rolling was almost completely abolished in ST3Gal-IV(-/-) mice. In both models, L-selectin interacts with P-selectin glycoprotein ligand-1 (PSGL-1) presented by adherent leukocytes and leukocyte fragments, but not with endothelial L-selectin ligands. In contrast, L-selectin-dependent rolling in Peyer's patch HEV, which is mediated by unknown endothelial L-selectin ligands, was not impaired in the absence of ST3Gal-IV. Our in vivo data show that PSGL-1, the molecule responsible for L-selectin-mediated leukocyte interactions in inflammation, is dependent on ST3Gal-IV, while alpha2,3-sialylation by ST3Gal-IV is not necessary for L-selectin ligand activity on high endothelial cells of Peyer's patch HEV.

A previous study showed, in a rat adipocutaneous flap model, that acute ischemic preconditioning (IP) can be achieved not only by preclamping of the flap pedicle, but also by a brief extremity ischemia prior to flap ischemia. The purpose of this study was to determine whether remote IP is also effective in other tissues such as muscle flaps. Twenty male Wistar rats were divided into three experimental groups. The rat cremaster flap in vivo microscopy model was used for assessment of ischemia/reperfusion injury. In the control group (CG, n = 8), a 2‐hr flap ischemia was induced after preparation of the cremaster muscle. In the “classic” IP group (cIP, n = 6), a brief flap ischemia of 10 min was induced by preclamping the pedicle, followed by 30 min of reperfusion. A 10‐min ischemia of the contralateral hindlimb was induced in the remote IP group (rIP, n = 6). The limb was then reperfused for 30 min. Flap ischemia and the further experiment were performed as in the CG. In vivo microscopy was performed after 1 hr of flap reperfusion in each animal. A significantly higher red blood cell velocity in the first‐order arterioles and capillaries, a higher capillary flow, and a decreased number of leukocytes adhering to the endothelium of the postcapillary venules were observed in both preconditioned groups by comparison to the control group ( P < 0.05). The differences within the preconditioned groups were not significant for these parameters. Our data show that ischemic preconditioning and improvement of flap microcirculation can be achieved not only by preclamping of the flap pedicle, but also by induction of an ischemia/reperfusion event in a body area distant from the flap prior to elevation. These findings indicate that remote IP is a systemic phenomenon, leading to an enhancement of flap survival. Our data suggest that remote IP could be performed simultaneously with flap elevation in the clinical setting without prolongation of the operation and without invasive means. © 2002 Wiley‐Liss, Inc. MICROSURGERY 22:221–226 2002

Not only is the renin angiotensin system or its components found morphologically in many organs, it also exerts many different regulatory functions such as contributing to systemic homeostasis as well as to organ-specific regulation. The presence of the components of the renin angiotensin system in the pancreas was discovered only a few years ago. Physiological and pathophysiological stimuli were able to modify, in part, the gene expression and the occurrence of some of these components. Because of the important clinical significance of pancreatic diseases such as pancreatitis, research should follow every traces of the renin angiotensin system in the pancreas: impairment of microcirculation via hypoxia mediated up-regulation with the subsequent further deterioration of the oxygen supply seems to be the most obvious mechanism. There are many possible approaches to a better understanding of problems that are associated with diseases such as different kinds of pancreatitis; basic studies in animal models are oriented toward microcirculation, cellular function and the time course of modified gene expression after stimuli such as hypoxia; a clinical approach must reevaluate different correlations between clinical parameters of hypertension and those of pancreatic diseases.

PURPOSE:Numerous investigations have stressed the significance of leukocytes in early angiogenesis. Leukocytes invade the cornea, and the location of their extravasation corresponds to the site of vessel ingrowth. The interactions between leukocytes and vascular endothelium are mediated by various proteins, including adhesion molecules such as intercellular adhesion molecule-1 (ICAM-1). In this study, the role of ICAM-1 during early corneal angiogenesis was evaluated in vivo.METHODS:Corneal neovascularization was induced in New Zealand White rabbits by use of intrastromal pellets containing 750 ng vascular endothelial growth factor (VEGF). The fluorescent dye rhodamine 6G was used to stain leukocytes in vivo. Leukocyte adhesion and vessel growth were quantified in vivo by high-resolution fluorescence angiography. To inhibit ICAM-1 interactions a microemulsion containing anti-ICAM-1 antibody was applied topically.RESULTS:Limbal vessels showed increased leukocyte adhesion 24 hours after pellet implantation: The number of rolling and sticking leukocytes was significantly increased compared with the number in control animals (P < 0.01). Treatment with anti-ICAM-1 antibody resulted in reduced leukocyte sticking and increased leukocyte rolling. The area covered by new blood vessels was significantly diminished in eyes treated with anti-ICAM-1 (P < 0.05).CONCLUSIONS:The results support the hypothesis that ICAM-1-mediated leukocyte adhesion is a key event in early angiogenesis. This model may serve for investigation of the significance of adhesion molecules by in vivo observation and quantification.

· Purpose: The purpose of our study was to establish an animal model to study the microcirculation in corneal neovascularization in the living animal atraumatically. · Methods: Corneal neovascularization was induced in New Zealand white rabbits by a standard micropocket assay utilizing pellets with 250 ng basic fibroblast growth factor. Anesthesia consisted of intramuscular injections of ketamine and xylazine. Intravital microscopy was performed without preparation of the cornea. Rhodamine 6G was used as fluorescent dye to stain leukocytes. Fluorescein-isothiocyanate-dextran served as plasma marker. Microcirculation analysis was done off-line by digital video imaging with special analysis software and included the following parameters: vessel diameters, blood velocity, and differentiation of leukocytes according to their interaction with endothelium into free-floating, rolling and sticking leukocytes. · Results: Vessel diameters in venular trunk vessels showed diameters of 54.0 ± 13.3 μm with 1.1 ± 0.5 mm/s flow; 29.4 ± 16.3% of all leukocytes were attached to the vascular wall. The number of sticking leukocytes was found to be 17.8 ± 36.0 cells/mm 2 endothelial surface. Values are given for arteriolar trunk and branch as well as venular branch vessels. · Conclusions: This method for in vivo microscopic observation and quantification of the vasculature of the ocular surface seems to be suitable for evaluation of microhemodynamic and leukocyte measurements in mature neovascular vessels. It allows atraumatic experiments without corneal preparation procedures which disturb the microcirculation. The results concerning microhemodynamics and adherence of leukocytes are in a range comparable to other microcirculation studies. This new model could provide insight into the pathophysiology of microcirculatory disorders of the anterior eye segment, e.g. during angiogenesis.

Rats were given a magnesium- (Mg) depleted (Mgd), or a Mg-standard (Mgst) or a Mg-enriched (Mge) diet, with 20 mg/kg/day cyclosporine (Cy) or olive oil per os for 90 days (6 groups). Anti-renin antibody was applied and the percent of renin-positive glomeruli (RI) was taken. Sodium excretion (NaU), relative heart weight (HW), as a measure of hypertension, and total femur Mg were measured. Compared to dietary controls, femur Mg was reduced under Cy and Mgd or Mgst indicating Mg deficiency. RI was higher in all Cy groups (p < 0.01), and NaU was lower in Mgd + Cy and in Mgst + Cy (p < 0.01). Correspondingly, HW was found to be significantly higher in Mgd + Cy and Mgst + Cy. In animals under Mge + Cy, there were no differences in NaU and HW compared to controls. The results indicate a relation between Cy-related hypertension and Mg status: Mg deficiency seems to enhance the hypertensive effect of Cy via sodium retention.

In rapidly growing male Sprague-Dawley rats with an initial body weight of 100 +/- 10 g, we investigated how alimentary magnesium (Mg) supply, Mg metabolism and ciclosporine (Ci)-associated nephrotoxicity are interrelated. Food with 100 ppm Mg (1Mg) or 1,000 ppm Mg (stMg) or 10,000 ppm Mg (rMg), Ci 20 mg/kg body weight daily or olive oil were applied for 3 months (n = 10/group). Mg concentrations in various compartments were measured by atomic absorption spectrophotometry. Creatinine clearance (Jaffe), urinary N-acetyl-beta-D-glucosaminidase (NAG) activity (fluorometrically), urinary sodium excretion (flame photometry) and osmolality were measured. Histomorphological examination was done and renal renin expression was studied by monoclonal antibodies. Ci reduced the Mg concentration of the femur under 1Mg (72.6 +/- 9.7 vs. 112.6 +/- 14.3 mmol/kg dry substance, p < 0.05) and under stMg (150.6 +/- 16.6 vs. 194.1 +/- 10.2 mmol/kg dry substance, p < 0.05), thus indicating Ci-related Mg deficiency. This was due to a significant increase in Mg excretion in Ci treatment compared to dietary controls. Under rMg, there was no difference between Ci-treated and control animals. Ci treatment lowered creatinine clearance in 1Mg (1.42 +/- 0.05 vs. 3.02 +/- 0.58 ml/min) and in stMg (1.04 +/- 0.45 vs. 2.18 +/- 0.51 ml/min), NAG/creatinine and urinary sodium excretion were negatively affected by Ci under 1Mg and stMg. Histomorphology showed macrocalcifications due to Mg deficiency and Ci-specific findings, which were markedly enhanced in 1Mg and stMg. Animals with plentiful Mg supply had no functional alterations due to Ci and no or weakly expressed histomorphological lesions. Renin-positive stained cells were higher in Ci-treated animals. This seems to be functionally relevant under 1Mg and stMg, since it was associated with sodium retention and elevated relative heart weight, indicating hypertension. Alimentary or drug-induced Mg deficiency plays a relevant role in the pathophysiology of chronic Ci nephrotoxicity. Our data suggest that Mg supplementation is helpful to reduce Ci toxicity, even if there is 'normal' alimentary Mg intake.