A Novel Artificial Coronary Plaque to Model Coronary Heart Disease.

BACKGROUND: Experimental coronary artery interventions are currently being performed on non-diseased blood vessels in healthy animals. To provide a more realistic pathoanatomical scenario for investigations on novel interventional and surgical therapies, we aimed to fabricate a stenotic lesion, mimicking the morphology and structure of a human atherosclerotic plaque. METHODS: In an interdisciplinary setting, we engineered a casting mold to create an atherosclerotic plaque with the dimensions to fit in a porcine coronary artery. Oscillatory rheology experiments took place along with long-term stability tests assessed by microscopic examination and weight monitoring. For the implantability in future in vivo setups, we performed a cytotoxicity assessment, inserted the plaque in resected pig hearts, and performed diagnostic imaging to visualize the plaque in its final position. RESULTS: The most promising composition consists of gelatin, cholesterol, phospholipids, hydroxyapatite, and fine-grained calcium carbonate. It can be inserted in the coronary artery of human-sized pig hearts, producing a local partial stenosis and interacting like the atherosclerotic plaque by stretching and shrinking with the vessel wall and surrounding tissue. CONCLUSION: This artificial atherosclerotic plaque model works as a simulating tool for future medical testing and could be crucial for further specified research on coronary artery disease and is going to help to provide information about the optimal interventional and surgical care of the disease.

Immature human engineered heart tissues engraft in a guinea pig chronic injury model.

Engineered heart tissue (EHT) transplantation represents an innovative, regenerative approach for heart failure patients. Late preclinical trials are underway, and a first clinical trial started recently. Preceding studies revealed functional recovery after implantation of in vitro-matured EHT in the subacute stage, whereas transplantation in a chronic injury setting was less efficient. When transplanting matured EHTs, we noticed that cardiomyocytes undergo a dedifferentiation step before eventually forming structured grafts. Therefore, we wanted to evaluate whether immature EHT (EHTIm) patches can be used for transplantation. Chronic myocardial injury was induced in a guinea pig model. EHTIm (15×106 cells) were transplanted within hours after casting. Cryo-injury led to large transmural scars amounting to 26% of the left ventricle. Grafts remuscularized 9% of the scar area on average. Echocardiographic analysis showed some evidence of improvement of left-ventricular function after EHTIm transplantation. In a small translational proof-of-concept study, human scale EHTIm patches (4.5×108 cells) were epicardially implanted on healthy pig hearts (n=2). In summary, we provide evidence that transplantation of EHTIm patches, i.e. without precultivation, is feasible, with similar engraftment results to those obtained using matured EHT.

Culture of care in animal research - Expanding the 3Rs to include people.

Research on animals is essential for science and medical progress. While it is still necessary to conduct this research, it is essential to apply the highest standards in animal welfare, including animal husbandry and care. Furthermore, it is important to recognize the special relationship between research animals and the people who care for them. Caring for research animals can be extremely fulfilling and meaningful, but it also comes with challenges, particularly when caring for animals experiencing pain or distress. These challenges can lead to work-related mental stress. To get more insight into the challenges of working in animal research, we organized a panel discussion at the GV-SOLAS (German Society for Laboratory Animal Science) and IGTP (Interest Group Animal Caretakers) conference 2021 about work wellbeing. This discussion was the first of its kind in Germany. The active panel contributions included the view of an ethical philosopher, a scientist, a lecturer for laboratory animal science, an animal facility manager and an animal caretaker. They gave insights from their perspective into key factors that can affect human wellbeing in animal research. Keys ideas included stigmatization of work, tension between research aims and animal wellbeing, and the importance of supportive culture to overcome work-related strains, as well as lack of education and supportive environments to cope with emotional stress in the workplace. Overall, the discussion has shown that we must also promote human wellbeing when promoting culture of care in animal research, because there is strong relationship between culture of care and individual performance.

Human engineered heart tissue transplantation in a guinea pig chronic injury model.

Myocardial injury leads to an irreversible loss of cardiomyocytes (CM). The implantation of human engineered heart tissue (EHT) has become a promising regenerative approach. Previous studies exhibited beneficial, dose-dependent effects of human induced pluripotent stem cell (hiPSC)-derived EHT patch transplantation in a guinea pig model in the subacute phase of myocardial injury. Yet, advanced heart failure often results from a chronic remodeling process. Therefore, from a clinical standpoint it is worthwhile to explore the ability to repair the chronically injured heart. In this study human EHT patches were generated from hiPSC-derived CMs (15 × 106 cells) and implanted epicardially four weeks after injury in a guinea pig cryo-injury model. Cardiac function was evaluated by echocardiography after a follow-up period of four weeks. Hearts revealed large transmural myocardial injuries amounting to 27% of the left ventricle. EHT recipient hearts demonstrated compact muscle islands of human origin in the scar region, as indicated by a positive staining for human Ku80 and dystrophin, remuscularizing 5% of the scar area. Echocardiographic analysis demonstrated no significant functional difference between animals that received EHT patches and animals in the cell-free control group (fractional area change 36% vs. 34%). Thus, EHT patches engrafted in the chronically injured heart but in contrast to the subacute model, grafts were smaller and EHT patch transplantation did not improve left ventricular function, highlighting the difficulties for a regenerative approach.

Histological and SEM Assessment of Blood Stasis in Kidney Blood Vessels after Repeated Intra-Arterial Application of Radiographic Contrast Media.

BACKGROUND: After application of iodinated contrast media (CM), a pronounced deterioration of the microcirculation in skin and myocardium was reported. Clinically, the repeated application of CM, especially, led to an increase of the renal resistance index (RRI). With respect to the transiency of the RRI increase, it is reasonable to assume that the deterioration of blood flow could be due to transient blood stasis caused by reversible morphologic cell alterations due to osmotic discrepancies between CM and human blood. Therefore, the hypothesis was investigated whether CM are able to induce in vivo such blood stasis and cell deformations in the renal vasculature of well-hydrated pigs. METHODS: The in vivo study was performed as a prospective randomized examination to compare the effects of two different CM in 16 pigs (German Landrace). Pigs were randomized to receive either Iodixanol (n = 8), or Iopromide (n = 8). Each animal received 10 injections separated by 5-min intervals via the suprarenal aorta at a rate of 10 mL/s according to the usual procedure during a cardiac catheter examination. Finally, the kidneys were explanted and processed for histology (H & E staining and fibrin staining according to Weigert) as well as for scanning electron microscopy (SEM) with regards to morphologic correlates explaining the changes in the microcirculation. RESULTS: In each of the predefined four categories of vascular diameters, blood stasis were found, but clearly more often after application of Iopromide than after application of Iodixanol (p < 0.001). In addition, Iopromide induced more blood stasis in all of the examined kidney regions compared to Iodixanol (p = 0.0001). There were no obstructive events in the middle cortex following the application of Iodixanol. Except for the region around a puncture channel of a placed-in catheter probe, no fibrin was detected in Weigert's fibrin-stained samples, neither around the histologically assessed thrombi nor in vessels with blood stasis. Complementary SEM analyses revealed in a few cases only a slight generation of fibrin and thrombi and deformations, such as echinocyte and "box-like" deformations. CONCLUSIONS: According to previous in vitro studies, pathological erythrocyte deformations, such as echinocyte and box-like formation of erythrocytes, were observed also in vivo. In addition, blood stasis and/or thrombi could be detected in histological samples from explanted kidneys from young pigs after repeated in vivo administration of CM. In only a few cases, mural platelet aggregates within minimal fibrin meshes occurred only after the application of Iopromide.

Growth-related micromorphological characteristics of the porcine common carotid artery.

The purpose of this study was to gain knowledge about the micromorphology of the porcine common carotid artery (CCA) during the period of growth over the bodyweight range of 10-40 kg. CCA samples from German landrace pigs (DL) aged either 2 or 3 months (DL-2 and DL-3) were compared with samples from Göttingen minipigs (GM) aged either 18 or 40 months (GM-18 and GM-40) using transmitted light (phase-contrast mode) and transmission electron microscopy. The GM-18, GM-40 and the DL-3 groups had typical muscular artery histological characteristics. Contrasting to this, the 2-month-old DL pigs had a transitional artery type being characterized by a significantly higher proportion of elastic fibres and a significantly lower number of smooth muscle cells than did the 1 month older DL-3. During the period of maturation, the tunica media of the CCA in GM animals thickened by 1.3× and in DL animals by 2.5× resulting in an overall increased vessel wall thickness. The cumulated thickness of the tunica interna (endothelium, stratum subendotheliale and internal elastic lamina) and the tunica media (including the external elastic lamina) of DL-3 and GM-40 pigs were similar to each other and comparable to that of humans. With an increasing vessel wall thickness, the luminal diameter decreased in GM by 19% and in DL by 11%. Additionally, in the older age groups, GM-40 and DL-3, the internal elastic lamina principally was continuous, but there were also interrupted large segments of elastic lamina separated by gaps. In addition, the principal internal elastic lamina was duplicated in several places.

Increased compensatory kidney workload results in cellular damage in a short time porcine model of mixed acidemia - Is acidemia a 'first hit' in acute kidney injury?

Acute kidney injury (AKI) corrupts the outcome of about 50% of all critically ill patients. We investigated the possible contribution of the pathology acidemia on the development of AKI. Pigs were exposed to acidemia, acidemia plus hypoxemia or a normal acid-base balance in an experimental setup, which included mechanical ventilation and renal replacement therapy to facilitate biotrauma caused by extracorporeal therapies. Interestingly, extensive histomorphological changes like a tubular loss of cell barriers occurred in the kidneys after just 5 hours exposure to acidemia. The additional exposure to hypoxemia aggravated these findings. These 'early' microscopic pathologies opposed intra vitam data of kidney function. They did not mirror cellular or systemic patterns of proinflammatory molecules (like TNF-α or IL 18) nor were they detectable by new, sensitive markers of AKI like Neutrophil gelatinase-associated lipocalin. Instead, the data suggest that the increased renal proton excretion during acidemia could be an 'early' first hit in the multifactorial pathogenesis of AKI.

Effect of three different forms of handling on the variation of aggression-associated parameters in individually and group-housed male C57BL/6NCrl mice.

Mice are social animals hence group-housing of mice is preferred over individual housing. However, aggression in group-housed male mice under laboratory housing conditions is a well-known problem leading to serious health issues, including injury or death. Therefore, group-housed mice are frequently separated for welfare reasons. In this study, we investigated the effect of 3 different handling methods (tail, forceps, tube) in 2 different housing conditions (single vs. group) on the variance of aggression-associated parameters in male C57BL/6NCrl mice over 8 weeks. Blood glucose concentration, body weight, body temperature, plus number and severity of bite wounds and barbering intensity in group-housed mice were recorded. An assessment of nest complexity was also performed weekly. Feces were collected in week 3 and 7 for analysis of corticosterone metabolites. We also monitored the level of aggression by recording the behavior of group-housed animals after weekly cage cleaning. An open field test followed by a social novel object test, a light/dark box test, a hotplate and a resident-intruder test were performed at the end of the 8-week handling period. Post-mortem, we assessed organ weights. We found that forceps-handled mice, independent of the housing condition, had significantly higher levels of stress-induced-hyperthermia and enhanced aggression after cage cleaning, and they performed worse in the nest complexity test. In addition, handling male mice by the tail seems to be most effective to reduce aggressiveness after transferring animals into new cages, thereby representing an appropriate refinement.

Instant Outcome Evaluation of Microwave Ablation With Subtraction CT in an In Vivo Porcine Model.

OBJECTIVES: The aim of this study was to investigate whether the accuracy of multislice contrast-enhanced computed tomography (MS-CECT) may be improved by performing additional subtraction CT. MATERIALS AND METHODS: Thirty-five microwave ablations were performed under CT guidance in 12 healthy and anesthetized pigs. Preablation and postablation MS-CECT scans were obtained in arterial and venous contrast phases. These scans were reconstructed and subtracted from each other. Lesion size was measured in a region of interest drawn around the ablation area. Computed tomography measurements were compared with standardized macroscopic images of explanted liver tissue, obtained immediately after ablation. Paired correlation and Bland-Altman analyses were performed for assessing agreement between modalities and ratings. RESULTS: The correlation between lesion size measured in CT and histology was very strong for subtracted images (r = 0.91; 95% confidence interval [CI], 0.8-0.96) and strong for standard MS-CECT images (r = 0.85; 95% CI, 0.68-0.93). Interrater agreement for all measurements was excellent (intraclass correlation coefficient, 0.99; 95% CI, 0.98-0.99 for subtraction and intraclass correlation coefficient, 0.99; 95% CI, 0.98-1.00 for MS-CECT). All differences were statistically significant (P < 0.05). CONCLUSIONS: Subtraction CT was superior to nonsubtracted MS-CECT in measurement of liver lesion size after microwave ablation in a porcine model, achieving a very strong correlation with pathologic measurement and a significantly lower overestimation of lesion size compared with MS-CECT.

Multipolar RFA of the liver: Influence of intrahepatic vessels on ablation zones and appropriateness of CECT in detecting ablation dimensions - Results of an in-vivo porcine liver model.

BACKGROUND: Radiofrequency ablation (RFA) is an important treatment option for hepatic tumors and metastases. Post-ablation recurrence rates are reported up to 36.5 percent and seem to depend on tumor size, intrahepatic localization of tumors and adjacent hepatic vessels. Multipolar RFA has the potential to overcome/reduce these limitations. Experimental and standardized data on achievable lesion sizes, influence of hepatic vessels and non-invasive evaluation of complete ablation is still insufficient. OBJECTIVES: The aim of this study was to evaluate the influence of intrahepatic vessels on shape and size of multipolar RF-ablation zones in healthy porcine liver and to evaluate the appropriateness of immediate post-ablation contrast-enhanced computed tomography (CECT) in detecting RF-ablation dimensions. MATERIAL AND METHODS: We conducted multipolar RFAs in each of the livers of 10 healthy, narcotized and laparotomized domestic pigs by inserting three parallel probes with a constant probe distance and a constant energy supply. In 4 ablations we interrupted hepatic blood flow using Pringle's maneuver. Immediate post-ablation CECT scans were acquired. After euthanasia the livers were sliced perpendicularly to the probes at the probes' active centers. CECT scans were reconstructed equivalently in order to compare RF-lesion size and shape to the macroscopic sections. RESULTS: In total, 19 RF-lesions were analyzed. Every RF-lesion that was ablated during physiological liver perfusion showed an irregular and cloverleaf-like shape (n = 15). Interrupting the hepatic blood flow during RFA led to well-defined, round and homogeneous ablation zones which were 3.8 times larger compared to RF-lesions ablated during continuous hepatic perfusion (n = 4). We found an excellent correlation between immediate post-ablation CECT slices and macroscopic sections when comparing RF-lesion diameters and area, although CECT tended to overestimate ablation dimensions. CONCLUSIONS: The interruption of hepatic blood flow using Pringle's maneuver during multipolar RFA with three applicators significantly reduces heat sink effects of hepatic vessels and generates large and coherent ablation zones. This approach should be considered in each case of ablation planning adjacent to larger hepatic vessels or when ablating larger tumor volumes. Immediate post-ablation CECT has limited value in detecting incomplete RFA periprocedurally.

Sex-related differences in the wheel-running activity of mice decline with increasing age.

Laboratory mice of both sexes having free access to running wheels are commonly used to study mechanisms underlying the beneficial effects of physical exercise on health and aging in human. However, comparative wheel-running activity profiles of male and female mice for a long period of time in which increasing age plays an additional role are unknown. Therefore, we permanently recorded the wheel-running activity (i.e., total distance, median velocity, time of breaks) of female and male mice until 9months of age. Our records indicated higher wheel-running distances for females than males which were highest in 2-month-old mice. This was mainly reached by higher running velocities of the females and not by longer running times. However, the sex-related differences declined in parallel to the age-associated reduction in wheel-running activities. Female mice also showed more variances between the weekly running distances than males, which were recorded most often for females being 4-6months old but not older. Additional records of 24-month-old mice of both sexes indicated highly reduced wheel-running activities at old age. Surprisingly, this reduction at old age resulted mainly from lower running velocities and not from shorter running times. Old mice also differed in their course of night activity which peaked later compared to younger mice. In summary, we demonstrated the influence of sex on the age-dependent activity profile of mice which is somewhat contrasting to humans, and this has to be considered when transferring exercise-mediated mechanism from mouse to human.

Comparison of bipolar radiofrequency ablation zones in an in vivo porcine model: Correlation of histology and gross pathological findings.

BACKGROUND: Continuing research ex vivo and in vivo with animal models is performed to advance the oncological safety of radiofrequency ablation (RFA) of liver tumors. In these experiments, frequently imaging modalities (e.g. MRI or CT) or macro-morphological measurements are used to determine the full extent of the different ablation zones inside of RFA lesions. However, no systematic study has been performed so far, which verified the accuracy of the macro-morphological findings. Therefore, the present study aimed to correlate histological and gross pathological findings of bipolar radiofrequency ablation zones of porcine livers with regard to cell viability in vivo. METHODS: Bipolar RFA was performed in the liver of anaesthetized female domestic pigs under CT-guidance using an internally cooled 20 mm RFA applicator. Afterwards RFA cross sections of the liver were made in a perpendicular orientation to the applicator. Ablation zones were initially documented by photography and thereafter prepared for histological analysis. Latter was based on HE-staining and NADH-diaphorase cell viability staining. Micro- and macro-morphological sections were digitally analyzed along the cross-section area for statistical correlation. RESULTS: Three different RF ablation zones could be differentiated. A central zone showing no cell viability (white zone) was surrounded by a red zone. The red zone could be divided into an inner zone of viable and non-viable cells (red zone 1), followed by a zone of edema with mostly viable cells (red zone 2).Micro- and macro-morphological data showed a strong correlation for the white zone (r = 0.95, p < 0.01), the red zone 1 (r = 0.85, p < 0.01), and the red zone 2 (r = 0.89, p < 0.01). CONCLUSION: White zone and red zone could clearly be distinguished in gross pathology and histology after bipolar RFA of porcine liver tissue in vivo. The red zone could be differentiated into an inner zone of viable and non-viable cells and an outer zone with high cell viability and intercellular edema. A strong correlation of micro- and macro-morphology could be shown for all three ablation zones. With this knowledge, gross pathological examination can be used as a reliable indicator of lethally damaged tissue in bipolar RFA of in vivo porcine liver.

Hypoxia-mediated alterations and their role in the HER-2/neuregulated CREB status and localization.

The cAMP-responsive element-binding protein (CREB) is involved in the tumorigenicity of HER-2/neu-overexpressing murine and human tumor cells, but a link between the HER-2/neu-mediated CREB activation, its posttranslational modification and localization and changes in the cellular metabolism, due to an altered (tumor) microenvironment remains to be established. The present study demonstrated that shRNA-mediated silencing of CREB in HER-2/neu-transformed cells resulted in decreased tumor formation, which was associated with reduced angiogenesis, but increased necrotic and hypoxic areas in the tumor. Hypoxia induced pCREBSer133, but not pCREBSer121 expression in HER-2/neu-transformed cells. This was accompanied by upregulation of the hypoxia-inducible genes GLUT1 and VEGF, increased cell migration and matrix metalloproteinase-mediated invasion. Treatment of HER-2/neu+ cells with signal transduction inhibitors targeting in particular HER-2/neu was able to revert hypoxia-controlled CREB activation. In addition to changes in the phosphorylation, hypoxic response of HER-2/neu+ cells caused a transient ubiquitination and SUMOylation as well as a co-localization of nuclear CREB to the mitochondrial matrix. A mitochondrial localization of CREB was also demonstrated in hypoxic areas of HER-2/neu+ mammary carcinoma lesions. This was accompanied by an altered gene expression pattern, activity and metabolism of mitochondria leading to an increased respiratory rate, oxidative phosphorylation and mitochondrial membrane potential and consequently to an enhanced apoptosis and reduced cell viability. These data suggest that the HER-2/neu-mediated CREB activation caused by a hypoxic tumor microenvironment contributes to the neoplastic phenotype of HER-2/neu+ cells at various levels.

Perioperative support reduces mortality of obese BALB/c mice after ovariectomy.

The incidence of obesity is on the rise in most western countries and represents major risks to health. Obesity causes complex metabolic dysfunctions and can be associated with a large number of secondary diseases. To investigate causal mechanisms of obesity and develop better options for treatment, researchers study the condition in animal models. In addition to genetically engineered animal models, diet-induced obesity is often used because it occurs similarly in animals as it does in humans. For several types of investigations that use obesity models, investigators must carry out surgical interventions and they frequently encounter severe perioperative complications induced by anesthesia. In an example of this problem, we observed 100% mortality in obese BALB/c mice after ovariectomy, despite no obvious surgical complications. We supposed that a failure to recover from surgery was the primary cause of this increased mortality. Therefore, to support their recovery from surgery we administered atropine to obese mice in order to facilitate blood circulation, and we also increased the oxygen content of the ambient air. With this specific support before and after surgery, we increased the survival rate of obese ovariectomized mice up to 83%. These results confirm the assumption that obesity is a risk factor for the recovery of obese animal models after ovariectomy, and they highlight the need to provide additional interventions for such experimental animals.

Biocompatibility of a novel zinc stent with a closed-cell-design.

Biomaterials made of zinc have been widely described to be antioxidative, hypothrombogenic, antiinflammatory and antiproliferative. Additionally in vivo zinc is toxic only in high concentrations and can completely be metabolized in vivo. Due to these properties zinc based vascular stents might be able to reduce the rate of restenosis in comparison to bare metal stents and zinc stents might be also able to limit the foreign body reaction. In the presented study we tested the biocompatibility and degradability of a stent made of zinc and characterized by a closed-cell-design to achieve high opening force and to increase stent stiffness. After 100 days of enzymatic and hydrolytic degradation in 15 ml blood serum (fetal calf serum) a significant loss of weight (1.72 wt% ) was measured. Zinc was compared to other metals in terms of degradation rates. After six weeks of incubation in physiologic sodium chloride solution zinc showed the slowest degradation time, 6 times less than stainless steel and 4 times less than magnesium. In the tests for cytotoxic effects the degraded zinc stent caused no changes in the LDH-release and cell membrane integrity (3T3 cells, mouse fibroblasts) respectively, in the cell activity/proliferation (MTS assay) and in the morphological characteristics of the cells and cell layers in comparison to the control material (polystyrene). Based on these results the tested zinc stent proved to be non-cytotoxic and to be characterized by degradation characteristics which might be advantageous in comparison to magnesium and stainless steel.

Human microvascular endothelial cells displaying reduced angiogenesis and increased uptake of lipids during in vitro culture.

Human microvascular ECs from the neonatal foreskin of two donors purchased from one distributor were used in an angiogenesis assay under the same culture conditions. Different angiogenic potency was apparent in these two batches (ECang and ECnon-ang). During the cultivation period of three weeks, ECang ran through all stages of angiogenesis starting from proliferation to migration up to the formation of three-dimensional capillary-like structures. Despite of expression of endothelial markers, ECnon-ang showed excessive intracellular storage of lipids in form of multilamellar bodies and decreased angiogenic potency in contrast to its counterpart, ECang. Results indicate that lipid metabolism differs in ECang versus ECnon-ang. This study points up that these differences are based on the different donors and presents a novel and valuable model for the study of mechanisms of atherosclerosis in endothelial cells in vitro.

Value or waste: Perfusion imaging following radiofrequency ablation - early experience.

BACKGROUND: Radiofrequency ablation (RFA) is an evolving technique in treatment of hepatic malignant tumors. By heating local tissue it leads to coagulative necrotic areas around the ablation probe. Temperature falls with increasing distance to the probe, risking incomplete necrosis at the margins of the RFA-induced lesion. Therefore, immediate non-invasive and precise detection of incomplete ablation is necessary for early enlargement of the ablation if needed. OBJECTIVES: This in vivo pig study compares early experiences of immediate post-interventional computed tomography (CT) perfusion volume analysis to macroscopic and CT image evaluation in healthy pig liver. MATERIAL AND METHODS: RFA was performed in vivo in healthy pig livers. Different CT perfusion algorithms (Maximum slope analysis and Patlak plot) were used to quantify three different perfusion parameters. Data points were acquired from rectangular grids. These grids were semiautomatically overlayed to macroscopic images documented after liver explantation. Each data point was visually assigned to zones defined as "inner" and "outer necrotic zone", "margin" or "vital tissue". RESULTS: Significant differences between necrotic zones and vital tissue are shown for equivalent blood volume (p <  0.0001), arterial flow (p <  0.01) and flow extraction product (p <  0.001). Looking at equivalent blood volume and flow extraction product, there were also significant differences (EquivBV: p <  0.0001, FE: p <  0.001) between margins, necrotic and vital areas. CONCLUSIONS: In a porcine model these early results could show that all of the used CT perfusion parameters allowed discrimination of necrosis from vital tissue after RFA at high levels of significance. In addition, the parameters EquivBV and FE that give an estimate of the tissue blood volume and the permeability, were able to precisely discern different zones also seen macroscopically. From this data CT perfusion analysis could be precise tool for measurement and visualization of ablated liver lesions and for immediate detection of incomplete ablation areas.

Colorectal Carcinogenesis: Connecting K-RAS-Induced Transformation and CREB Activity In Vitro and In Vivo.

UNLABELLED: Oncogenic transformation is often associated with an increased expression of the cAMP response element binding (CREB) transcription factor controlling the expression of genes involved in cell proliferation, cell cycle, apoptosis, and tumor development, but a link between K-RAS(V12)-induced transformation and CREB has not yet been determined. Therefore, the constitutive and/or inhibitor-regulated mRNA and protein expression of CREB and signal transduction components and growth properties of parental fibroblasts, K-RAS(V12)-transformed counterparts, shCREB K-RAS(V12) transfectants and human colon carcinoma cells were determined. Increased CREB transcript and protein levels accompanied by an enhanced CREB activity was detected in K-RAS(V12)-transformed murine fibroblasts and K-RAS(V12)-mutated human tumor cells, which is dependent on the MAPK/MEK, PI3K, and/or PKC signal transduction. Immunohistochemical (IHC) staining of colorectal carcinoma lesions and murine tumors, with known KRAS gene mutation status, using antibodies specific for CREB and phospho-CREB, revealed a mechanistic link between CREB expression and K-RAS(V12)-mutated colorectal carcinoma lesions when compared with control tissues. Silencing of CREB by shRNA and/or treatment with a CREB inhibitor (KG-501) reverted the neoplastic phenotype of K-RAS(V12) transformants as demonstrated by a more fibroblast-like morphology, enhanced apoptosis sensitivity, increased doubling time, decreased migration, invasion and anchorage-independent growth, reduced tumorigenesis, and enhanced immunogenicity in vivo. The impaired shCREB-mediated invasion of K-RAS(V12) transformants was accompanied by a transcriptional downregulation of different matrix metalloproteinases (MMP) coupled with their reduced enzymatic activity. IMPLICATIONS: CREB plays a key role in the K-RAS(V12)-mediated neoplastic phenotype and represents a suitable therapeutic target for murine and human K-RAS(V12)-induced tumors.

Interleukin-1 potently contributes to 25-hydroxycholesterol-induced synergistic cytokine production in smooth muscle cell-monocyte interactions.

OBJECTIVES: Inflammation is essential for atherogenesis. Cholesterol, a cardiovascular risk factor, may activate inflammation in the vessel wall during this process. Cytokine-mediated interactions of human monocytes with vascular smooth muscle cells (SMCs) may perpetuate this process. METHODS: We investigated the capacity of the cholesterol metabolite 25-hydroxycholesterol to induce inflammatory mediators in cocultures of freshly isolated monocytes with SMCs. We determined the role of interleukin-(IL)-1 in this interaction using qPCR, bioassays, ELISA and western blot. Cocultures with SMC to monocyte ratios from 1:4 to 1:20 were tested. RESULTS: In separate SMC and monocyte cultures (monocultures) 25-hydroxycholesterol only poorly activated IL-1, IL-6 and MCP-1 production, whereas LPS stimulated much higher cytokine levels than unstimulated cultures. In contrast, cocultures of SMCs and monocytes stimulated with 25-hydroxycholesterol produced hundredfold higher cytokine levels than the corresponding monocultures. Blocking experiments with IL-1-receptor antagonist showed that IL-1 decisively contributed to the 25-hydroxycholesterol-induced synergistic IL-6 and MCP-1 production. The presence of intracellular IL-1ß precursor, released mature IL-1ß, and caspase-1 p10 indicated that the inflammasome was involved in this process. Determination of IL-1-mRNA in Transwell experiments indicated that the monocytes are the major source of IL-1, which subsequently activates the SMCs, the primary source of IL-6 in the coculture. CONCLUSION: Taken together, these interactions between local vessel wall cells and invading monocytes may multiply cholesterol-triggered inflammation in the vessel wall, and IL-1 may play a key role in this process. The data also indicate that lower cholesterol levels than expected from monocultures may suffice to initiate inflammation in the tissue.