SR-B1-/-ApoE-R61h/h Mice Mimic Human Coronary Heart Disease.

Cardiovascular diseases are the leading cause of death in the modern world. Atherosclerosis underlies the majority of these pathologies and may result in sudden life-threatening events such as myocardial infarction or stroke. Current concepts consider a rupture (resp. erosion) of "unstable/vulnerable" atherosclerotic plaques as a primary cause leading to thrombus formation and subsequent occlusion of the artery lumen finally triggering an acute clinical event. We and others described SR-B1-/-ApoE-R61h/h mice mimicking clinical coronary heart disease in all major aspects: from coronary atherosclerosis through vulnerable plaque ruptures leading to thrombus formation/coronary artery occlusion, finally resulting in myocardial infarction/ischemia. SR-B1-/-ApoE-R61h/h mouse provides a valuable model to study vulnerable/occlusive plaques, to evaluate bioactive compounds as well as new anti-inflammatory and "anti-rupture" drugs, and to test new technologies in experimental cardiovascular medicine. This review summarizes and discuss our knowledge about SR-B1-/-ApoE-R61h/h mouse model based on recent publications and experimental observations from the lab.

Non-FDG imaging of atherosclerosis: will imaging of MMPs assess plaque vulnerability?

Acute ruptures of atherosclerotic plaques with subsequent occlusion account for the vast majority of clinical events such as myocardial infarction or stroke. New imaging approaches focusing on the visualization of inflammation in the vessel wall could emerge as tools for individualized risk assessment and prevention of events. To this end, PET employing (18)F-fluorodeoxyglucose (FDG) has recently been introduced for the first clinical trials. Although this approach nicely visualizes plaques inflammation questions remain with respect to if and how this inflammatory signal can be employed for predicting individual plaque rupture. Molecular imaging of proteases such as matrix-metalloproteinases (MMPs) involved in several steps in plaque progression driving plaques into vulnerable, rupture-prone states seems a promising alternative approach. This review introduces and discusses the vulnerable plaque concept, animal models with human-like plaque ruptures and the potential of a FDG versus a non-FDG MMP-targeted strategy to image rupture-prone plaques.

TGF-ß1-induced EMT of non-transformed prostate hyperplasia cells is characterized by early induction of SNAI2/Slug.

BACKGROUND: Epithelial-mesenchymal transition (EMT) underlying cancer cell invasion and metastasis has been thoroughly studied in prostate cancer. Although EMT markers have been clinically observed in benign prostate hyperplasia, molecular events underlying the onset and progression of EMT in benign prostate cells have not been described. METHODS: EMT in BPH-1 cells was induced by TGF-ß1 treatment and the kinetics of expression of EMT markers, regulators, and selected miRNAs was assessed by western blotting and quantitative RT-PCR. RESULTS: EMT in BPH-1 cells was accompanied by rapid up-regulation of SNAI2/Slug and ZEB1 transcription factors, while changes in expression levels of ZEB2 and miR-200 family members were observed after extended time intervals. Invasive phenotype with EMT hallmarks, characterizing tumorigenic clones derived from BPH-1 cells, was associated with increased mRNA levels of SNAI2, ZEB1, and ZEB2, but was not associated with significant changes in basal levels of miR-200 family members. RNA interference revealed that SNAI2/Slug is crucial for TGF-ß1-induced vimentin up-regulation and migration of BPH-1 cells. CONCLUSIONS: This study suggests that in BPH-1 cells the transcription factor SNAI2/Slug is important for EMT initiation, while the ZEB family of transcription factors in cooperation with the miR-200 family may oppose the reversal of the EMT phenotype.

Dynamic Monitoring of Cellular Remodeling Induced by the Transforming Growth Factor-ß1.

The plasticity of differentiated adult cells could have a great therapeutic potential, but at the same time, it is characteristic of progression of serious pathological states such as cancer and fibrosis. In this study, we report on the application of a real-time noninvasive system for dynamic monitoring of cellular plasticity. Analysis of the cell impedance profile recorded as cell index using a real-time cell analyzer revealed its significant increase after the treatment of prostate epithelial cells with the transforming growth factor-ß1. Changes in the cell index profile were paralleled with cytoskeleton rebuilding and induction of epithelial-mesenchymal transition and negatively correlated with cell proliferation. This novel application of such approach demonstrated a great potential of the impedance-based system for noninvasive and real-time monitoring of cellular fate.

Imaging Reveals the Connection Between Spontaneous Coronary Plaque Ruptures, Atherothrombosis, and Myocardial Infarctions in HypoE/SRBI-/- Mice.

UNLABELLED: The hyperlipidemic mouse model HypoE/SRBI(-/-) has been shown to develop occlusive coronary atherosclerosis followed by myocardial infarctions and premature deaths in response to high-fat, high-cholesterol diet (HFC). However, the causal connection between myocardial infarctions and atherosclerotic plaque rupture events in the coronary arteries has not been investigated so far. The objective of this study was to assess whether diet-induced coronary plaque ruptures trigger atherothrombotic occlusions, resulting in myocardial infarctions in HFC-fed HypoE/SRBI(-/-) mice. METHODS: HypoE/SRBI(-/-) mice were characterized with respect to the individual dynamics of myocardial infarctions and features of infarct-related coronary atherosclerosis by serial noninvasive molecular and functional imaging, histopathology, and a pharmaceutical intervention. Detailed histologic analysis of whole mouse hearts was performed when spontaneously occurring acute myocardial infarctions were diagnosed by imaging. RESULTS: Using the imaging-triggered approach, we discovered thrombi in 32 (10.8%) of all 296 atherosclerotic coronary plaques in 14 HFC-fed HypoE/SRBI(-/-) mice. These thrombi typically were found in arteries presenting with inflammatory plaque phenotypes. Acetylsalicylic acid treatment did not attenuate the development of atherosclerotic coronary plaques but profoundly reduced the incidence of premature deaths, the number of thrombi (7 in 249 plaques), and also the degree of inflammation in the culprit lesions. CONCLUSION: HFC-induced ruptures of coronary plaques trigger atherothrombosis, vessel occlusions, myocardial infarctions, and sudden death in these mice. Thus, the HypoE/SRBI(-/-) mouse model mimics major features of human coronary heart disease and might therefore be a valuable model for the investigation of molecular and cellular parameters driving plaque rupture-related events and the development of new interventional approaches.

Sample preparation for high-resolution 3D confocal imaging of mouse skeletal tissue.

High-resolution confocal imaging is a vital tool for analyzing the 3D architecture and detailed spatial distribution of cells in situ. However, imaging of skeletal tissue has remained technically challenging because of its calcified nature. Here we describe a protocol that allows high-resolution imaging of skeletal tissue with preservation of cellular morphology and tissue architecture. The procedure involves tissue fixation, decalcification and cryosectioning of the mouse skeletal tissue to generate thick sections. The thick sections generated by this procedure are not only compatible with the analysis of genetically expressed fluorescent proteins but they also preserve antigenicity, thus enabling diverse combinations of antibody labeling. Further, this procedure also permits other fluorescence techniques such as TUNEL and ethynyl deoxyuridine (EdU) incorporation assays. Images resulting from the confocal imaging can be assessed qualitatively and quantitatively to analyze various parameters such as distribution and interrelationships of cell types. The technique is straightforward and robust, highly reproducible and can be completed in ∼11 d.

TGF-ß1 signaling plays a dominant role in the crosstalk between TGF-ß1 and the aryl hydrocarbon receptor ligand in prostate epithelial cells.

Crosstalk between the aryl hydrocarbon receptor (AhR) and transforming growth factor-ß1 (TGF-ß1) signaling has been observed in various experimental models. However, both molecular mechanism underlying this crosstalk and tissue-specific context of this interaction are still only partially understood. In a model of human non-tumorigenic prostate epithelial cells BPH-1, derived from the benign prostatic hyperplasia, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) persistently activates the AhR signaling pathway and induces expression of xenobiotic metabolizing enzymes, such as CYP1A1 or CYP1B1. Here we demonstrate that TGF-ß1 suppresses the AhR-mediated gene expression through multiple mechanisms, involving inhibition of AhR expression and down-regulation of nuclear AhR, via a SMAD4-dependent pathway. In contrast, TCDD-induced AhR signaling does not affect either TGF-ß1-regulated gene expression or epithelial-to-mesenchymal transition. These observations suggest that, in the context of prostate epithelium, TGF-ß1 signaling plays a dominant role in the crosstalk with AhR signaling pathway. Given the importance of TGF-ß1 signaling in regulation of prostate epithelial tissue homeostasis, as well as the recently revealed role of AhR in prostate development and tumorigenesis, the above findings contribute to our understanding of the mechanisms underlying the crosstalk between the two signaling pathways in the prostate-specific context.

TGF-beta1 suppresses IL-6-induced STAT3 activation through regulation of Jak2 expression in prostate epithelial cells.

Chronic inflammation plays an important role in the initiation and progression of various human diseases including benign prostatic hyperplasia or prostate cancer. Here we show that the proinflammatory cytokine interleukin-6 (IL-6) has prosurvival effects and chronically activates the Jak2/STAT3 signalling pathway in a model of benign prostatic hyperplasia (BPH-1). We demonstrate that the antiinflammatory cytokine transforming growth factor-beta1 (TGF-beta1), which also permanently activates its canonical signalling pathway through SMAD proteins in BPH-1 cells, modifies the effects of IL-6 on cell proliferation. Importantly, TGF-beta1 inhibits IL-6 signal transduction by decreasing the phosphorylation levels of STAT3. This effect is associated with decreased expression of Jak2 at both mRNA and protein levels. Moreover, we showed that TGF-beta1 inhibits IL-6-induced expression of the cancer-associated gene MUC1. These observations demonstrated a novel interaction between TGF-beta1 and IL-6 signalling and suggested another mechanism of how defects in TGF-beta signalling, frequently associated with prostate pathologies, can contribute to the disruption of tissue homeostasis.

Genotoxic polycyclic aromatic hydrocarbons fail to induce the p53-dependent DNA damage response, apoptosis or cell-cycle arrest in human prostate carcinoma LNCaP cells.

Exposure to polycyclic aromatic hydrocarbons (PAHs) has been positively associated with prostate cancer, but knowledge of the formation of PAH-DNA adducts and related genotoxic events in prostatic cells is limited. In the present study, benzo[a]pyrene (BaP), a potent mutagenic PAH, formed significant levels of DNA adducts in cell lines derived from human prostate carcinoma. When analyzing the effect of BaP on the induction of CYP1 enzymes participating in the metabolic activation of PAHs in LNCaP cells, we found that BaP induced expression of CYP1A1 and CYP1A2, but not CYP1B1 enzyme. Despite a significant amount of DNA adducts being formed by BaP and, to a lesser extent also by another strong genotoxin, dibenzo[a,l]pyrene, neither apoptosis nor cell-cycle arrest were induced in LNCaP cells. LNCaP cells were not sensitized to the induction of apoptosis by PAHs even through inhibition of the phosphoinositide-3-kinase/Akt pro-survival pathway. The lack of apoptosis was not due a disruption of expression of pro-apoptotic and pro-survival members of the Bcl-2 family of apoptosis regulators. In contrast to other genotoxic stimuli, genotoxic PAHs failed to induce DNA double-strand breaks, as illustrated by the lack of phosphorylation of histone H2AX or checkpoint kinase-2. BaP did not activate p53, as evidenced by the lack of p53 accumulation, phosphorylation at Ser15, or induction of p53 transcriptional targets. Taken together, although genotoxic PAHs produced significant levels of DNA adducts in a model of human prostate carcinoma cells, they did not activate the mechanisms leading to elimination of cells with significant damage to DNA, presumably due to their failure to activate the p53-dependent DNA damage response.

Multiple defects in negative regulation of the PKB/Akt pathway sensitise human cancer cells to the antiproliferative effect of non-steroidal anti-inflammatory drugs.

Antitumorigenic effects of non-steroidal anti-inflammatory drugs (NSAIDs) are well established in several types of cancer disease. However, the mechanisms driving these processes are not understood in all details. In our study, we observed significant differences in sensitivity of cancer epithelial cell lines to COX-independent antiproliferative effects of NSAIDs. The prostate cancer cell line LNCaP, lacking both critical enzymes in the negative control of PKB/Akt activation, PTEN and SHIP2, was the most sensitive to these effects, as assessed by analysing the cell cycle profile and expression of cell cycle regulating proteins. We found that p53 protein and its signalling pathway is not involved in early antiproliferative action of the selected NSAID-indomethacin. RNAi provided evidence for the involvement of p21(Cip1/Waf1), but not GDF-15, in antiproliferative effects of indomethacin in LNCaP cells. Interestingly, we also found that indomethacin activated PKB/Akt and induced nuclear localisation of p21(Cip1/Waf1) and Akt2 isoform. Our results are in agreement with other studies and suggest that maintaining of the p21(Cip1/Waf1) level and its intracellular localisation might be influenced by Akt2. Knock-down of SHIP2 by RNAi in PTEN negative prostate and colon cancer cell lines resulted in higher sensitivity to antiproliferative effects of indomethacin. Our data suggest novel mechanisms of NSAIDs antiproliferative action in cancer epithelial cells, which depends on the status of negative regulation of the PKB/Akt pathway and the isoform-specific action of Akt2. Thus, unexpectedly, multiple defects in negative regulation of the PKB/Akt pathway may contribute to increased sensitivity to chemopreventive effects of these widely used drugs.