PDGF-BB regulates splitting angiogenesis in skeletal muscle by limiting VEGF-induced endothelial proliferation.

VEGF induces normal or aberrant angiogenesis depending on its dose in the microenvironment around each producing cell in vivo. This transition depends on the balance between VEGF-induced endothelial stimulation and PDGF-BB-mediated pericyte recruitment, and co-expression of PDGF-BB normalizes aberrant angiogenesis despite high VEGF doses. We recently found that VEGF over-expression induces angiogenesis in skeletal muscle through an initial circumferential vascular enlargement followed by longitudinal splitting, rather than sprouting. Here we investigated the cellular mechanism by which PDGF-BB co-expression normalizes VEGF-induced aberrant angiogenesis. Monoclonal populations of transduced myoblasts, expressing similarly high levels of VEGF alone or with PDGF-BB, were implanted in mouse skeletal muscles. PDGF-BB co-expression did not promote sprouting and angiogenesis that occurred through vascular enlargement and splitting. However, enlargements were significantly smaller in diameter, due to a significant reduction in endothelial proliferation, and retained pericytes, which were otherwise lost with high VEGF alone. A time-course of histological analyses and repetitive intravital imaging showed that PDGF-BB co-expression anticipated the initiation of vascular enlargement and markedly accelerated the splitting process. Interestingly, quantification during in vivo imaging suggested that a global reduction in shear stress favored the initiation of transluminal pillar formation during VEGF-induced splitting angiogenesis. Quantification of target gene expression showed that VEGF-R2 signaling output was significantly reduced by PDGF-BB co-expression compared to VEGF alone. In conclusion, PDGF-BB co-expression prevents VEGF-induced aberrant angiogenesis by modulating VEGF-R2 signaling and endothelial proliferation, thereby limiting the degree of circumferential enlargement and enabling efficient completion of vascular splitting into normal capillary networks despite high VEGF doses.

External inosculation as a feature of revascularization occurs after free transplantation of murine liver grafts.

The induction of angiogenesis is essential for successful engraftment of freely transplanted cells or cellular composites. How to augment angiogenesis to ensure an appropriate viability of the grafts is still under investigation. This study evaluated the proangiogenic capability of different syngeneic free liver transplants and elucidated the origin of the newly formed vascular network via use of an eGFP(+) /eGFP(-) (enhanced green fluorescent protein) cross-over design. Using intravital fluorescence microscopy, we found that neonatal and resected murine liver transplants implanted into dorsal skinfold chambers display a significantly enhanced vascularization compared to regular adult transplants. Immunohistochemically, less tissue hypoxia, apoptosis and macrophage infiltration was observed in the neonatal and resected transplants, which is in line with improved vascularization of those grafts. Additionally, electron microscopy revealed morphological hallmarks of liver cells. eGFP(+) liver transplants implanted on eGFP(-) recipients displayed vascular sprouting from the grafts themselves and connection to the recipients` microvasculature, which also undergoes transient proangiogenic response. This process is described as external inosculation, with microvessels exhibiting a chimeric nature of the endothelial lining. These data collectively show that proliferative stimulation is taking effect on angiogenic properties of free transplants and might provide a novel tool for modulating the revascularization of free grafts.

RAGE blockade and hepatic microcirculation in experimental endotoxaemic liver failure.

BACKGROUND: Activation of the receptor for advanced glycation endproducts (RAGE) causes sustained activation of multiple inflammatory pathways. Therefore, RAGE has potential as a new therapeutic target in sepsis. The aim of this study was to analyse whether RAGE blockade in vivo prevents microcirculatory dysfunction and subsequent tissue injury in endotoxaemic liver failure. METHODS: The hepatic microcirculation was analysed using intravital fluorescence microscopy in murine livers exposed to galactosamine/lipopolysaccharide (G/L) and treated with an anti-RAGE antibody (abRAGE) either 12 h before or h after exposure to G/L. Blood and liver tissue samples were harvested for analysis of leucocyte tissue infiltration, apoptotic and necrotic damage as well as RAGE downstream pathway signalling. RESULTS: Sinusoidal perfusion failure in livers exposed to G/L was reduced significantly by both pretreatment and post-treatment with abRAGE. Hepatic inflammation induced by exposure to G/L was also attenuated by abRAGE administration, as shown by a 55 per cent reduction in sinusoidal leucocyte stasis, a 65 per cent decrease in venular leucocyte rolling and adhesion, and an 85 per cent reduction in leucocyte tissue infiltration. Treatment with abRAGE markedly reduced hepatocellular apoptosis and necrosis in livers exposed to G/L, and blunted the rise in plasma high-mobility group protein B1 levels. Finally, G/L-induced activation of the mitogen-activated protein kinase cascade was also reduced significantly by blockade of RAGE. CONCLUSION: RAGE plays an important role in mediating endotoxaemic liver damage. RAGE blockade may have potential therapeutic value. SURGICAL RELEVANCE: The innate immune response to endoxaemia is initiated by a group of pattern recognition receptors, including the receptor for advanced glycation endproducts (RAGE). As RAGE is well known for perpetuation of inflammatory processes, blockade of this receptor might be of particular value in reducing or even halting endoxaemia-related organ disorders. Using intravital fluorescence microscopy this study demonstrated in vivo that pretreatment, but also post-treatment, with a RAGE-blocking antibody attenuated hepatic microcirculatory deterioration and leucocyte recruitment, and thus diminished liver injury in a murine model of endotoxaemic organ failure. These data underline the important role of RAGE in the innate immune response and support the potential therapeutic value of blocking this pattern recognition receptor.

Diabetes aggravates acute pancreatitis and inhibits pancreas regeneration in mice.

AIMS/HYPOTHESIS: It is well established that acute pancreatitis often causes diabetes and that a high blood glucose level associated with pancreatitis is a marker of poor prognosis. The aim of this study was to evaluate if diabetes merely reflects the severity of pancreatitis or whether it can also aggravate the progression of this disease in a vicious circle. METHODS: Reversible acute oedematous pancreatitis was induced in untreated and streptozotocin-treated diabetic mice by injection of cerulein. Progression of pancreatitis was studied by immunohistochemistry, ELISA and various other enzyme assays. The production of regenerating islet-derived 3ß (REG3ß) was determined by western blot and immunohistochemistry. RESULTS: While cerulein treatment in non-diabetic mice resulted in acute pancreatitis followed by regeneration of the pancreas within 7 days, diabetes aggravated pancreatitis, inhibited the regeneration of the exocrine tissue and led to strong atrophy of the pancreas. The aggravation of pancreatitis by diabetes was characterised by decreased production of the anti-inflammatory protein REG3ß, increased inflammation, augmented oedema formation and increased cell death during the acute phase of pancreatitis (p < 0.05). During the regenerative phase, diabetes augmented inflammation, increased cell death, reduced acinar cell expansion and increased the expansion of duct as well as interstitial cells, resulting in the formation of tubular complexes (p < 0.05). Administration of insulin reversed the observed phenotype in diabetic mice. CONCLUSIONS/INTERPRETATION: Diabetes aggravates acute pancreatitis and suppresses regeneration of the exocrine tissue. Thus, diabetes is not just a concomitant phenomenon of pancreatitis, but can have a fundamental influence on the progression of acute pancreatitis.

Ghrelin protects musculocutaneous tissue from ischemic necrosis by improving microvascular perfusion.

Persistent ischemia in musculocutaneous tissue may lead to wound breakdown and necrosis. The objective of this experimental study was to analyze, whether the gastric peptide ghrelin prevents musculocutaneous tissue from necrosis and to elucidate underlying mechanisms. Thirty-two C57BL/6 mice equipped with a dorsal skinfold chamber containing ischemic musculocutaneous tissue were allocated to four groups: 1) ghrelin; 2) N(ω)-nitro-l-arginine methyl ester (l-NAME); 3) ghrelin and l-NAME; and 4) control. Microcirculation, inflammation, angiogenesis, and tissue survival were assessed by fluorescence microscopy. Inducible and endothelial nitric oxide synthase (iNOS I and eNOS), vascular endothelial growth factor (VEGF), as well as nuclear factor κB (NF-κB) were assessed by Western blot analysis. Ghrelin-treated animals showed an increased expression of iNOS and eNOS in critically perfused tissue compared with controls. This was associated with arteriolar dilation, increased arteriolar perfusion, and a sustained functional capillary density. Ghrelin further upregulated NF-κB and VEGF and induced angiogenesis. Finally, ghrelin reduced microvascular leukocyte-endothelial cell interactions, apoptosis, and overall tissue necrosis (P < 0.05 vs. control). Inhibition of nitric oxide by l-NAME did not affect the anti-inflammatory and angiogenic action of ghrelin but completely blunted the ghrelin-induced tissue protection by abrogating the arteriolar dilation, the improved capillary perfusion, and the increased tissue survival. Ghrelin prevents critically perfused tissue from ischemic necrosis. Tissue protection is the result of a nitric oxide synthase-mediated improvement of the microcirculation but not due to induction of angiogenesis or attenuation of inflammation. This might represent a promising, noninvasive, and clinically applicable approach to protect musculocutaneous tissue from ischemia.

The dorsal skinfold chamber: window into the dynamic interaction of biomaterials with their surrounding host tissue.

The implantation of biomaterials into the human body has become an indispensable part of almost all fields of modern medicine. Accordingly, there is an increasing need for appropriate approaches, which can be used to evaluate the suitability of different biomaterials for distinct clinical indications. The dorsal skinfold chamber is a sophisticated experimental model, which has been proven to be extremely valuable for the systematic in vivo analysis of the dynamic interaction of small biomaterial implants with the surrounding host tissue in rats, hamsters and mice. By means of intravital fluorescence microscopy, this chronic model allows for repeated analyses of various cellular, molecular and microvascular mechanisms, which are involved in the early inflammatory and angiogenic host tissue response to biomaterials during the initial 2-3 weeks after implantation. Therefore, the dorsal skinfold chamber has been broadly used during the last two decades to assess the in vivo performance of prosthetic vascular grafts, metallic implants, surgical meshes, bone substitutes, scaffolds for tissue engineering, as well as for locally or systemically applied drug delivery systems. These studies have contributed to identify basic material properties determining the biocompatibility of the implants and vascular ingrowth into their surface or internal structures. Thus, the dorsal skinfold chamber model does not only provide deep insights into the complex interactions of biomaterials with the surrounding soft tissues of the host but also represents an important tool for the future development of novel biomaterials aiming at an optimisation of their biofunctionality in clinical practice.

In vivo imaging of bile accumulation and biliary infarction after common bile duct ligation in rats.

Obstructive cholestasis is caused by mechanical constriction or occlusion leading to reduced bile flow. Serious complications such as jaundice and even death may follow. Little is known about the initial phase of cholestasis and its consequences for the hepatic microarchitecture. This in vivo study aimed to characterize the nature and kinetics of developing obstructive cholestasis and focused on areas with biliary stasis and infarction by visualizing the autofluorescence of bile acids using intravital microscopy of the liver over a period of 30 h after bile duct ligation in rats. The innovation resided in performing fluorescence microscopy without applying fluorescent dyes. In animals subjected to obstructive cholestasis, the most significant changes observed in vivo were the concomitant appearance of (1) areas with bile accumulation increasing in size (6 h: 0.163 ± 0.043, 18 h: 0.180 ± 0.086, 30 h: 0.483 ± 0.176 mm(2)/field) and (2) areas with biliary infarction (6 h: 0.011 ± 0.006, 18 h: 0.010 ± 0.004, 30 h: 0.010 ± 0.050 mm(2)/field) as well as (3) a relation between the formation of hepatic lesions and enzyme activity in serum. The sequential in vivo analysis presented herein is a new method for the in vivo visualization of the very early changes in the hepatic parenchyma caused by obstructive cholestasis.

Tumour growth following portal branch ligation in an experimental model of liver metastases.

BACKGROUND: Portal branch ligation (PBL) is being used increasingly before hepatectomy for colorectal metastases. This study evaluated the effect of PBL on angiogenesis, growth factor expression and tumour growth in a mouse model of hepatic colorectal metastases. METHODS: CT26.WT cells were implanted into the left liver lobe of BALB/c mice. Animals underwent PBL of the left liver lobe or sham treatment. Angiogenesis, microcirculation, growth factor expression, cell proliferation and tumour growth were studied over 14 and 21 days by intravital multifluorescence microscopy, laser Doppler flowmetry, immunohistochemistry and western blotting. RESULTS: Left hilar blood flow and tumour microcirculation were significantly diminished during the first 7 days after PBL. This resulted in tumour volume being 20 per cent less than in sham controls by day 14. Subsequently, PBL-treated animals demonstrated recovery of left hilar blood flow and increased expression of hepatocyte growth factor and transforming growth factor alpha, associated with increased cell proliferation and acceleration of growth by day 21. CONCLUSION: PBL initially reduced vascular perfusion and tumour growth, but this was followed by increased growth factor expression and cell proliferation. This resulted in delayed acceleration of tumour growth, which might explain the stimulated tumour growth observed occasionally after PBL.

Toll-like receptor 2-deficiency on bone marrow-derived cells augments vascular healing of murine arterial lesions.

AIMS: Neointimal hyperplasia contributes to arterial restenosis after percutaneous transluminal coronary angioplasty or vascular surgery. Neointimal thickening after arterial injury is determined by inflammatory processes. We investigated the role of the innate immune receptor toll-like receptor 2 (TLR2) in neointima formation after arterial injury in mice. MATERIALS AND METHODS: Carotid artery injury was induced by 10% ferric chloride in C57Bl/6J wild type (WT), TLR2 deficient (B6.129-Tlr2tm1Kir/J, TLR2-/-) and WT mice treated with a TLR2 blocking antibody. 21 days after injury, carotid arteries were assessed histomorphometrically and for smooth muscle cell (SMC) content. To identify the contribution of circulating cells in mediating the effects of TLR2-deficiency, arterial injury was induced in WT/TLR2-/--chimeric mice and the paracrine modulation of bone marrow-derived cells from WT and TLR2-/- on SMC migration compared in vitro. KEY FINDINGS: TLR2-/- mice and WT mice treated with TLR2 blocking antibodies exhibited reduced neointimal thickening (23.7 ± 4.2 and 6.5 ± 3.0 vs. 43.1 ± 5.9 µm, P < 0.05 and P < 0.01), neointimal area (5491 ± 1152 and 315 ± 76.7 vs. 13,756 ± 2627 µm2, P < 0.05 and P < 0.01) and less luminal stenosis compared to WT mice (8.5 ± 1.6 and 5.0 ± 1.3 vs. 22.4 ± 2.2%, both P < 0.001n = 4-8 mice/group). The phenotypes of TLR2-/- vs. WT mice were completely reverted in WT/TLR2-/- bone marrow chimeric mice (5.9 ± 1.5 µm neointimal thickness, 874.2 ± 290.2 µm2 neointima area and 2.7 ± 0.6% luminal stenoses in WT mice transplanted with TLR2-/- bone marrow vs. 23.6 ± 5.1 µm, 3555 ± 511 µm2 and 12.0 ± 1.3% in WT mice receiving WT bone marrow, all P < 0.05, n = 6/group). Neointimal lesions of WT and WT mice transplanted with TLR2-/- bone marrow chimeric mice showed increased numbers of SMC (10.8 ± 1.4 and 12.6 ± 1.4 vs. 3.8 ± 0.9 in TLR2-/- and 3.5 ± 1.1 cells in WT mice transplanted with TLR2-/- bone marrow, all P < 0.05, n = 6). WT bone marrow cells stimulated SMC migration more than TLR2-deficient bone marrow cells (1.7 ± 0.05 vs. 1.3 ± 0.06-fold, P < 0.05, n = 7) and this effect was aggravated by TLR2 stimulation and diminished by TLR2 blockade (1.1 ± 0.03-fold after stimulation with TLR2 agonists and 0.8 ± 0.02-fold after TLR2 blockade vs. control treated cells defined as 1.0, P < 0.05, n = 7). SIGNIFICANCE: TLR2-deficiency on hematopoietic but not vessel wall resident cells augments vascular healing after arterial injury. Pharmacological blockade of TLR2 may thus be a promising therapeutic option to improve vessel patency after iatrogenic arterial injury.

Paclitaxel-coated stents to prevent hyperplastic proliferation of ureteral tissue: from in vitro to in vivo.

Ureteric stents have become an indispensable tool in the armamentarium of every urologist. However, they carry their own morbidity resulting mostly from infectious or abacterial fouling and biofilm formation, and/or urothelial hyperplastic reaction. All of these may interact and lead to clinical complications. Many different stent designs and coatings have been proposed. In this study, we focused on the effect of paclitaxel-coated stents on hyperplastic proliferation of ureteral tissue, using as example anastomotic strictures after ureteroureterostomy in a rat model. Human urothelial cells (SV-HUC-1) were used to determine paclitaxel dosages in vitro. Polyurethane stents were coated with a paclitaxel containing biodegradable polymer and studied in a ureteroureterostomy rat model. 48 male 9-week-old Sprague-Dawley rats underwent either sham surgery (n = 16) or ureteroureterostomy with sutured anastomosis, and consecutive stenting with either a paclitaxel-coated or an uncoated stent (16 per group), respectively. The animals received daily intraperitoneal injections of 5-bromo-2-deoxyuridine (20 mg/ml, 100 mg/kg body weight) during the first eight postoperative days, and were sacrificed on day 28. Healing of the ureteral anastomosis and proliferation of urothelial cells was examined histologically and immunohistochemically. In vitro, a concentration of 10 ng/mm2 paclitaxel can be considered as non-toxic, while still exerting an anti-proliferative effect on urothelial cells. Histologically, typical wound healing processes were seen at the site of the ureteral anastomosis in vivo. Proliferation of urothelial cells was significantly lower in animals with paclitaxel-coated stents compared to those with uncoated stents (LI 41.27 vs. 51.58, p < 0.001). Our results indicate that stenting of ureteral anastomoses with paclitaxel-coated stents can reduce hyperplastic proliferation of ureteral tissue. Paclitaxel-coated stents thus might be able to prevent not only scar-induced postoperative stenosis after reconstructive surgery, but also hyperplastic urothelial reaction in non-anastomotic stent patients as part of their inflammatory response to the foreign material.

[Microcirculatory failure of sublingual perfusion in septic-shock patients. Examination by OPS imaging and PiCCO monitoring]. / Sublinguale Mikrozirkulationsstörungen bei Patienten im septischen Schock: Untersuchungen mittels OPS-Imaging-System und PiCCO-Monitoring.

BACKGROUND: Haemodynamic monitoring of septic patients is impeded by the discrepancy between the macrohaemodynamics and the microcirculation of internal organs. Pulse contour analysis (PiCCO) provides new parameters for an improved assessment of the volume status of critically ill patients. However, changes in regional circulation, in particular those affecting the splanchnic perfusion, have proven to be especially important. The aim of our study was to compare macrohaemodynamic parameters (PiCCO) with microcirculation (OPS imaging) in severely septic patients with multiple organ failure. PATIENTS AND METHODS: In seven patients suffering from septic shock and multiple organ failure (APACHE II score > 25) repeated examinations at a twenty-four hour interval were carried out by PiCCO monitoring and OPS imaging. OPS data were recorded for twenty seconds at 6 different buccal and sublingual localisations, adequately reflecting microvascular perfusion of the liver and the small intestine. Data were videotaped for off-line analysis, calculating current velocity in small and large venules (< 25 and > 25 microm), as well as functional capillary density. RESULTS: Significant correlations were found for current velocity in small venules with systemic vascular resistance (r(2) = 0.252, p < 0.05), mean arterial blood pressure (r(2) = 0.259, p < 0.05), and pH value (r(2) = 0.265, p < 0.05). In addition, a significant correlation was found between the oxygen transport index and the density of small vessels (r(2) = 0.355; p < 0.05). CONCLUSION: According to our findings, data acquired through PiCCO monitoring may be used for a rough estimation of the microcirculation during severe sepsis and multiple organ failure. For an assessment of the local conditions of perfusion, however, there are limits in the use of the parameters that were the object of our research. For the measurement at localisations which are accessible non-invasively and representative of the splanchnic perfusion, OPS is the more accurate method for characterisation of the microcirculation, although a more extensive and time-consuming analysis is needed.

Intraluminal three-dimensional optical coherence tomography - a tool for imaging of the Eustachian tube?

OBJECTIVES: The cause of Eustachian tube dysfunction often remains unclear. Therefore, this study aimed to examine the feasibility and possible diagnostic use of optical coherence tomography in the Eustachian tube ex vivo. METHODS: Two female blackface sheep cadaver heads were examined bilaterally. Three conditions of the Eustachian tube were investigated: closed (resting position), actively opened and stented. The findings were compared (and correlated) with segmented histological cross-sections. RESULTS: Intraluminal placement of the Eustachian tube with the optical coherence tomography catheter was performed without difficulty. Regarding the limited infiltration depth of optical coherence tomography, tissues can be differentiated. The localisation of the stent was accurate as was the lumen. CONCLUSION: The application of optical coherence tomography in the Eustachian tube under these experimental conditions is considered to be a feasible, rapid and non-invasive diagnostic method, with possible diagnostic value for determining the luminal shape and superficial lining tissue of the Eustachian tube.

[18F]-florbetaben PET/CT Imaging in the Alzheimer's Disease Mouse Model APPswe/PS1dE9.

BACKGROUND: Positron-emission-tomography (PET) using 18F labeled florbetaben allows noninvasive in vivo-assessment of amyloid-beta (Aß), a pathological hallmark of Alzheimer's disease (AD). In preclinical research, [18F]-florbetaben-PET has already been used to test the amyloid-lowering potential of new drugs, both in humans and in transgenic models of cerebral amyloidosis. The aim of this study was to characterize the spatial pattern of cerebral uptake of [18F]-florbetaben in the APPswe/ PS1dE9 mouse model of AD in comparison to histologically determined number and size of cerebral Aß plaques. METHODS: Both, APPswe/PS1dE9 and wild type mice at an age of 12 months were investigated by smallanimal PET/CT after intravenous injection of [18F]-florbetaben. High-resolution magnetic resonance imaging data were used for quantification of the PET data by volume of interest analysis. The standardized uptake values (SUVs) of [18F]-florbetaben in vivo as well as post mortem cerebral Aß plaque load in cortex, hippocampus and cerebellum were analyzed. RESULTS: Visual inspection and SUVs revealed an increased cerebral uptake of [18F]-florbetaben in APPswe/ PS1dE9 mice compared with wild type mice especially in the cortex, the hippocampus and the cerebellum. However, SUV ratios (SUVRs) relative to cerebellum revealed only significant differences in the hippocampus between the APPswe/PS1dE9 and wild type mice but not in cortex; this differential effect may reflect the lower plaque area in the cortex than in the hippocampus as found in the histological analysis. CONCLUSION: The findings suggest that histopathological characteristics of Aß plaque size and spatial distribution can be depicted in vivo using [18F]-florbetaben in the APPswe/PS1dE9 mouse model.

Nitric oxide reduces organ injury and enhances regeneration of reduced-size livers by increasing hepatic arterial flow.

BACKGROUND: Reduced-size livers suffer from portal hyperperfusion, diminished arterial blood flow and the risk of postoperative liver injury. The aim of this experimental study was to unravel the role of nitric oxide in this setting. METHODS: Rats underwent 85 per cent partial hepatectomy and either substitution of nitric oxide with molsidomine or inhibition of nitric oxide synthase (NOS) with N(G)-nitro-L-arginine methyl ester. Untreated hepatectomized animals served as controls and unresected animals as the sham group. RESULTS: Ultrasonic flowmetry following partial hepatectomy revealed a marked increase in portal venous inflow with a concomitant decrease in hepatic arterial inflow. Nitric oxide substitution counteracted the decline in hepatic arterial inflow and caused a significantly greater increase in cell proliferation after partial hepatectomy compared with control or NOS-inhibited animals. Hepatectomized animals further profited from nitric oxide substitution, as indicated by reduced aminotransferase release and improved liver function. CONCLUSION: Nitric oxide improves the postoperative course of rats with reduced-size livers by modulating hepatic macrohaemodynamics and mediating regeneration and cytoprotection, but not by reducing hepatic hyperperfusion and the accompanying sinusoidal shear stress.

Decitabine demonstrates antileukemic activity in B cell precursor acute lymphoblastic leukemia with MLL rearrangements.

BACKGROUND: Promotor hypermethylation of CpG islands is common in B cell precursor acute lymphoblastic leukemia (BCP-ALL) with mixed lineage leukemia (MLL) gene rearrangements. Hypomethylating agents (HMA) such as azacitidine (AZA) and decitabine (DEC) reduce DNA hypermethylation by incorporation into DNA and were successfully introduced into the clinic for the treatment of myeloid neoplasias. METHODS: Here, we investigated whether HMA induce comparable biological effects in MLL-positive BCP-ALL. Further, efficacy of HMA and concomitant application of cytostatic drugs (cytarabine and doxorubicin) were evaluated on established SEM and RS4;11 cell lines. In addition, promising approaches were studied on BCP-ALL cell line- and patient-derived xenograft models. RESULTS: In general, DEC effects were stronger compared to AZA on MLL-positive BCP-ALL cells. DEC significantly reduced proliferation by induction of cell cycle arrest in G0/G1 phase and apoptosis. Most sensitive to HMA were SEM cells which are characterized by a fast cell doubling time. The combination of low-dose HMA and conventional cytostatic agents revealed a heterogeneous response pattern. The strongest antiproliferative effects were observed when ALL cells were simultaneously exposed to HMA and cytostatic drugs. Most potent synergistic effects of HMA were induced with cytarabine. Finally, the therapeutic potential of DEC was evaluated on BCP-ALL xenograft models. DEC significantly delayed leukemic proliferation in xenograft models as demonstrated longitudinally by non-invasive bioluminescence as well as 18F-FDG-PET/CT imaging. Unexpectedly, in vivo concomitant application of DEC and cytarabine did not enhance the antiproliferative effect compared to DEC monotherapy. CONCLUSIONS: Our data reveal that DEC is active in MLL-positive BCP-ALL and warrant clinical evaluation.

Continuous intra-arterial application of substance P induces signs and symptoms of experimental complex regional pain syndrome (CRPS) such as edema, inflammation and mechanical pain but no thermal pain.

Substance P is involved in nociception in both the peripheral nervous system and the CNS and has been documented to play a crucial role in the complex regional pain syndrome (CRPS). So far, however, most experimental animal models are restricted to the effect of neurokinin-1 receptor blockers to inhibit substance P and do not directly evaluate its action. Thus, this study was conducted to test the hypothesis that local application of substance P causes signs and symptoms of CRPS. For this purpose rats received a continuous infusion of either substance P or saline over 24 h delivered by a mini-osmotic pump connected to an intrafemoral catheter. Animals were analyzed at either day 1 (n=6, each group) or day 4 (n=5, each group) after start of infusion. Substance P application caused a significant and long-lasting decrease in paw withdrawal thresholds upon mechanical stimulation, while animals did not present with thermal allodynia at days 1 and 4 after onset of infusion. In addition, severe s.c. edema was observed in all animals receiving substance P. In vivo fluorescence microscopy of the extensor digitorum longus muscle of the affected hind paw revealed enhanced leukocyte-endothelial cell interaction with a significant rise in the number of leukocytes both rolling along and firmly adhering to the wall of postcapillary venules, while saline-exposed animals were free of this local inflammatory response. Muscle cell apoptosis, as assessed by in vivo bisbenzimide staining, terminal deoxynucleotidyl transferase nick end labeling analysis and caspase 3-cleavage, could not be observed in either of the animals. In summary, the present study indicates that substance P is responsible for neurogenic inflammation, including local cell response, edema formation and mechanical pain, while it seems not to contribute to the generation of thermal allodynia.

Antileukoproteinase protects against hepatic inflammation, but not apoptosis in the response of D-galactosamine-sensitized mice to lipopolysaccharide.

BACKGROUND AND PURPOSE: There is major evidence for the strong bi-directional interrelation of parenchymal cell apoptosis and leukocyte accumulation and inflammation in acute liver injury. Therefore, the aim of this in vivo study was to investigate the anti-apoptotic and anti-inflammatory potential of antileukoproteinase (ALP) in a murine model of acute liver failure. EXPERIMENTAL APPROACH: C57BL/6J mice were given galactosamine (D-GalN) and E. coli lipopolysaccharide (LPS) followed by administration of saline or ALP. Besides survival rate, hepatic tissue damage and inflammatory response were analyzed by intravital fluorescence microscopy 6 hours after treatment. In addition, immunohistochemical analysis of NFkappaB-p65 and hepatocellular apoptosis, plasma levels of AST/ALT, TNF-alpha and IL-10 were determined. KEY RESULTS: Administration of D-GalN/LPS provoked hepatic damage, including marked leukocyte recruitment and microvascular perfusion failure, as well as hepatocellular apoptosis and enzyme release. NFkappaB-p65 became increasingly detectable in hepatocellular nuclei, accompanied by a rise of TNF-alpha and IL-10 plasma levels. ALP markedly reduced intrahepatic leukocyte accumulation, nuclear translocation of NFkappaB and plasma levels of TNF-alpha and IL-10. Moreover, liver enzyme levels indicated the absence of necrotic parenchymal cell death. In contrast, ALP failed to block both apoptosis and caspase-3 levels and the mortality rate of ALP-treated animals was comparable to that of saline-treated mice. CONCLUSIONS AND IMPLICATIONS: ALP could effectively prevent D-GalN/LPS-associated intrahepatic inflammatory responses by inhibition of NFkappaB activity, but not apoptosis-driven mortality. Thus, a protease-inactivating approach such as application of ALP seems to be inadequate in damaged liver where apoptosis represents the predominant mode of cell death.

Pathomechanisms of ischemia-reperfusion injury as the basis for novel preventive strategies: is it time for the introduction of pleiotropic compounds?

Ischemia-reperfusion-associated tissue dysfunction and organ failure still represent major complications in transplantation surgery. The pathomechanisms involve microvascular perfusion failure, ie, no-reflow and tissue hypoxia despite reperfusion and reoxygenation. However, postischemic reperfusion also provokes an inflammatory response, ie, reflow paradox, with activation of macrophages, recruitment of leukocytes, and accumulation of platelets, involving surface adhesion molecules such as P-selectin, P-selectin glycoprotein ligand (PSGL)-1, Mac-1, and intercellular adhesion molecule (ICAM)-1. These inflammatory cells produce cytokines, chemokines, lipid mediators, and oxygen radicals, which all may contribute to the manifestation of injury, including apoptosis, necrosis, and necrapoptosis. Although specific inhibition of single mediators, such as tumor necrosis factor (TNF)-alpha, interleukin (IL)-1, and oxygen radicals, or distinct molecules, such as P-selectin and ICAM-1, has been shown to be protective in the experimental setting, these single-agent antimediator and antimolecule approaches did not find their way into clinical practice. Clinically, University of Wisconsin (UW) solution for organ preservation is still the major milestone for prevention of ischemia- and reperfusion-associated injury. Characteristically, this treatment strategy does not represent an anti-single mediator approach, but exerts protection by influencing multiple pathways involved in hypoxic and inflammatory injury, potentially restoring the overall homeostasis. This type of pleiotropic action may also be achieved by single pharmacological compounds, such as statins, erythropoietin, hemoxygenase-1, and L-glycine. In recent experimental studies, these compounds have been shown to be effective to reduce post-ischemic-reperfusion injury, and, additionally, to be associated with less side effects. Accordingly, these pleiotropic substances may represent ideal candidates for pharmacological preconditioning in patient treatment, and, thus, should be further evaluated in clinical trials.

Rapamycin induces regression of endometriotic lesions by inhibiting neovascularization and cell proliferation.

BACKGROUND AND PURPOSE: Rapamycin is a widely used drug with antifungal, immunosuppressant and antiangiogenic effects. Herein, we studied whether immunosuppressive doses of rapamycin are capable of influencing endometriotic lesions. EXPERIMENTAL APPROACH: We tested in vitro the potential of rapamycin to inhibit endothelial cell sprouting using the aortic ring assay and we further studied its effect on the expression of proliferating cell nuclear antigen (PCNA), apoptotic cell death-associated activated caspase-3 and vascular endothelial growth factor (VEGF) in cultured endometrial tissue fragments. In addition, we analyzed the drug in vivo after induction of endometriotic lesions by transplanting isolated endometrial fragments into the dorsal skinfold chamber of Syrian golden hamsters. Using intravital fluorescence microscopy, we repetitively analyzed angiogenesis, neovascularization and microcirculatory parameters over a time period of 14 days in rapamycin-treated animals and DMSO-treated controls. KEY RESULTS: Administration of rapamycin significantly reduced the size of the endometriotic lesions. This was associated by inhibition of VEGF-mediated angiogenesis as indicated by a suppression of endothelial cell sprouting in vitro and a reduction of microvessel density in endometriotic lesions in vivo. Moreover, rapamycin directly inhibited cell proliferation within endometrial tissue, while manifestation of apoptotic cell death remained unaffected. CONCLUSIONS AND IMPLICATIONS: Our data indicate that administration of rapamycin may represent a novel therapeutic approach for an antiangiogenic treatment of endometriosis.

Protective effects of PARP inhibition on liver microcirculation and function after haemorrhagic shock and resuscitation in male rats.

OBJECTIVE: The aim of this study was to investigate the impact of the water-soluble poly-(ADP)-ribose-polymerase (PARP) inhibitor 5-aminoisoquinolinone (5-AIQ) on liver microcirculation and function after haemorrhagic shock and resuscitation. DESIGN: Controlled, randomized animal study. SETTING: University animal care facility and research laboratory. SUBJECT: Male Sprague-Dawley rats were subjected to haemorrhagic shock for 1 h, followed by resuscitation with shed blood and crystalloid solution for a total of 5 h. INTERVENTIONS: The PARP inhibitor 5-AIQ (3 mg/kg; n=7) or vehicle (n=7) was administered 5 min prior to resuscitation. Sham-operated animals without induction of shock served as controls (n=7). MEASUREMENTS AND RESULTS: Using intravital fluorescence microscopy hepatic microcirculation was assessed at baseline, end of shock phase as well as 1 h and 5 h after resuscitation. Systemic arterial blood pressure and bile flow were continuously monitored. 5-AIQ treatment attenuated shock/resuscitation-induced increase of intrahepatic leukocyte-endothelial cell interaction with a marked reduction of both sinusoidal leukostasis and venular leukocyte adherence. Moreover, nutritive perfusion was found improved, guaranteeing sufficient oxygen supply to tissue, as indicated by low NADH autofluorescence, which was not different to that in controls. Most notably, excretory liver function reached baseline level over 5 h of reperfusion in 5-AIQ-treated animals. CONCLUSIONS: In the present setting of shock/resuscitation in male rats the PARP inhibitor 5-AIQ proved to be very effective in ameliorating compromised liver microcirculation and function. Further research has to confirm that PARP inhibition is a suitable tool in the acute treatment of patients suffering from reduced circulating blood volume and thus microcirculatory organ dysfunction.