- Email: [email protected]
Spontaneous plaque disruptions, dissection, erosion and intraplaque hemorrhage in murine vein grafts can be attenuated by TIMP-1 overexpression
Abstracts
patients with coronary atherothrombosis (13). Finally, we have recently shown a significant correlation between TRX, PRX-1 and NADPH activity in asymptomatic subjects, suggesting a coordinated antioxidant response to increased oxidative stress observed in atherothrombosis (14). In conclusion, oxidative stress is a main determinant of pathological remodelling of the vascular wall, linked in part to the presence of hemoglobin within disease tissue and to the imbalance between pro and anti-oxydant molecules. Understanding the complex mechanisms underlying redox balance could help to define novel potential targets to decrease atherothrombostic risk. References 1. Michel, J.B., et al., 2011. Novel aspects of the pathogenesis of aneurysms of the abdominal aorta in humans. Cardiovasc Res. 90, 18–27. 2. Michel, J.B., 2011 Mar 12. Intraplaque haemorrhages as the trigger of plaque vulnerability. Eur Heart J. 3. Nagy, E., et al., 2010. Red cells, hemoglobin, heme, iron, and atherogenesis. Arterioscler Thromb Vasc Biol 30, 1347–1353. 4. Balla, J., et al., 2007. Heme, heme oxygenase, and ferritin: how the vascular endothelium survives (and dies) in an iron-rich environment. Antioxid Redox Signal. 9, 2119–2137. 5. Levy, A.P., et al., 2010. Haptoglobin: basic and clinical aspects. Antioxid Redox Signal 12, 293–304. 6. Kalet-Litman, S., et al., 2010. The haptoglobin 2–2 genotype is associated with increased redox active hemoglobin derived iron in the atherosclerotic plaque. Atherosclerosis 209, 28–31. 7. Moreno, P.R., et al., 2008. Haptoglobin genotype is a major determinant of the amount of iron in the human atherosclerotic plaque. J Am Coll Cardiol 52, 1049–1051. 8. Martinez-Pinna et al. Decreased circulating transferrin and iron levels in patients with abdominal aortic aneurysm (Arterioscler Thromb Vasc Biol, submitted). 9. Altamura, C., et al., 2009. Ceruloplasmin/Transferrin system is related to clinical status in acute stroke. Stroke. 40, 1282–8. 10. Nishihira, K., et al., 2008. Thioredoxin in coronary culprit lesions: possible relationship to oxidative stress and intraplaque hemorrhage. Atherosclerosis. 201, 360–7. 11. Martinez-Pinna, R., et al., 2010. Increased levels of thioredoxin in patients with abdominal aortic aneurysms (AAAs). A potential link of oxidative stress with AAA evolution Atherosclerosis. 212, 333–338. 12. Martinez-Pinna, R., et al., 2011. Identification of peroxiredoxin-1 as a novel biomarker of abdominal aortic aneurysm. Arterioscler Thromb Vasc Biol. 31, 935–43. 13. Hokamaki, J., et al., 2005. Plasma thioredoxin levels in patients with unstable angina. Int J Cardiol. 99, 225–31. 14. Madrigal-Matute et al. Peroxiredoxin-1/Thioredoxin levels are associated to intima media thickness and NADPH oxidase activity in subjects with subclinical atherosclerosis (Arterioscler Thromb Vasc Biol, in revision). doi:10.1016/j.vph.2011.08.100
L.14.4 Prognostic value of intraplaque hemorrhage Gerard Pasterkamp UMC Utrecht, The Netherlands E-mail address: [email protected] Identification of patients at risk for secondary manifestations of atherosclerotic disease progression is based mainly on established risk factors. The AtheroExpress study, is a biobank of atherosclerotic carotid lesions that were collected from patients who underwent carotid
341
endarterectomy and underwent clinical follow-up yearly, up to 3 years after carotid endarterectomy. Using this biobank, we have identified several plaque protein markers which level was associated with secondary cardiovascular events during follow-up (vascular death, nonfatal stroke, nonfatal myocardial infarction, vascular intervention). Local atherosclerotic plaque composition is also thought to be an important determinant of acute cardiovascular events, but no prospective studies have been performed. Using the same AtheroExpress biobank with follow-up, we investigated whether carotid atherosclerotic plaque composition as determined by histology is associated with the occurrence of future vascular events. Macrophage infiltration, large lipid core, calcifications, collagen, and smooth muscle cell infiltration were not associated with clinical outcome. Carotid plaque hemorrhage or marked intraplaque vessel formation, however, was associated with an increased risk of secondary cardiovascular events independent of clinical risk factors and medication use. This demonstrates that not only plaque protein levels but also atherosclerotic plaque composition contains information that can be used as an independent predictor of future cardiovascular events.
doi:10.1016/j.vph.2011.08.101
O.14.1 Spontaneous plaque disruptions, dissection, erosion and intraplaque hemorrhage in murine vein grafts can be attenuated by TIMP-1 overexpression Margreet R. de Vriesa,b, Hans W.M. Niessene, LowikClemens Löwikc, J. Wouter Jukemab,d, Paul H.A. Quaxa,b a Einthoven Laboratory for Experimental Vascular Medicine, Leiden University Medical Center, Leiden, The Netherlands b Department of Surgery, Leiden University Medical Center, Leiden, The Netherlands c Department of Endocrinology, Leiden University Medical Center, Leiden, The Netherlands d Department of Cardiology, Leiden University Medical Center, Leiden, The Netherlands e Department of Pathology and Cardiac Surgery, ICaR-VU, VU University Medical Center, Amsterdam, The Netherlands E-mail address: [email protected] (M. R. de Vries) Plaque rupture and erosion are the most common underlying causes of cardiovascular death. Matrixmetalloproteinases (MMP) play a key role in these processes. We hypothesize that overexpression of the endogenous inhibitor of MMPs TIMP-1 results in decreased plaque disruptions in vein grafts in hypercholesterolemic ApoE3Leiden mice. Vein graft lesions in ApoE3Leiden mice consist of foamcells, smooth muscle cells (SMC) and extracellular matrix with necrotic cores, calcifications and inflammatory cells. Lesion neovessels consist of endothelial cells and basement membranes, supporting mural cells were infrequently seen. 79% of 47 vein grafts studied showed spontaneous disruption, 1/3 of these disruptions were dissections, 1/3 were leaky microvessels with extravasated erythrocytes and 1/3 were erosions or intramural thrombosis. Also vein grafts with combined leaky vessels and dissections or erosions were seen. Systemic overexpression of TIMP-1 (and Luciferase as control) was obtained by plasmid electroporation. Near InfraRed Fluorescense imaging was used to detect in vivo (inhibition of) MMP activity and thus TIMP-1 plasmid functionality. TIMP-1 overexpression resulted in a decrease of 73% in MMP activity in the vein graft-region after 28 d and a 40% reduction in vein graft thickening after 28 d. The lesions showed significant less disruptions (90%) than the control Luciferase group resulting in a more stable phenotype with significant more SMC, collagen and significant less macrophages and fibrinogen.
342
Abstracts
Here we show that murine ApoE3L vein grafts are a relevant model to study plaque stability and subsequent disruptions. Furthermore we demonstrate that MMP inhibition reduced plaque disruptions resulting in a more stable plaque phenotype. doi:10.1016/j.vph.2011.08.102
O.14.2 Mature endothelial cell therapy stabilizes abdominal aortic aneurysm through paracrine mechanisms and recruitment of local vascular cells Grégory Franck, Jianping Dai, Stéphanie Michineau, Saravath Ngo, Anne-Marie Guinault, Eric Allaire, Marianne Gervais CNRS EAC 7054, Creteil, France E-mail address: [email protected] (G. Franck) Background: Abdominal aortic aneurysms (AAAs) expand as a consequence of extracellular matrix (ECM) destruction and vascular smooth muscle cell (VSMC) depletion. Loss of the endothelium and its replacement by a thick mural thrombus is although a common structural feature of human AAA. Since the endothelium exerts anti-thrombotic properties and since the thrombus participates in AAA expansion, we hypothesize that restoration of a functional endothelium by cell therapy would stabilize AAA. Our goal was thus to study the impact of local seeding of mature endothelial cells on AAA evolution in rats. Method and results: Rat aortic endothelial cells (RAEC) were established from 8 week-old male Fischer rat aortas. Preventive or stabilizing effect of RAEC on AAA formation was evaluated in the xenograft model in rats. 5.106 RAEC (n=16) or 200 μl of serum-free medium (control, n=14) were seeded endovascularly either immediately (D0, prevention) or 14 days (D14, stabilization) after surgery, i.e. on alreadyformed AAA. Animals were sacrificed 7 (prevention: D0+7; stabilization: D14+7) or 28 (prevention: D0+28; stabilization: D14+28) days after cell therapy. In vivo, local RAEC seeding prevented AAA formation (diameter increase at D0+7: 22±2 vs 35±3% and D0+28: 49±6 vs 81±6% in RAEC and control rats, respectively, p<0.05) and stabilized already-formed AAA in rats (diameter increase at D14+7: 3.7±2.8 vs 13.1±3.5% and D14+28: –0.9±0.8% vs 26.1±13.5% in RAEC and control rats, p<0.01). This stabilizing effect was associated with a decrease in MMP-dependent proteolysis and macrophage infiltration in the injured aorta, the re-establishment of the luminal endothelial monolayer, the reconstitution of new aortic wall rich in VSMC and ECM components, and the resorption of the intraluminal thrombus. We further showed that transplanted RAEC did not participate directly to the aortic cellular reconstruction but exerted their healing proprieties through paracrine mechanisms, involving the upregulation of endothelial-derived stabilizing factors, such as endothelial NO synthase, basic Fibroblast Growth Factor and VE-cadherin mRNAs and the recruitment of resident vascular cells but not circulating progenitor cells into the new aortic wall. Conclusion: Endovascular cell therapy using mature endothelial cells prevents AAA development and stabilizes already-formed AAA in rats, by re-establishing the endothelial lining and reconstructing a new ECM and VSMC-rich aortic wall through a paracrine mechanism. doi:10.1016/j.vph.2011.08.103
SESSION 15 Systems biology applied to cardiovascular disease L.15.1 Network biology in the post-genomic era Ignat Drozdov
BHF Centre of Excellence, King's College London, London, UK E-mail address: [email protected] Cardiovascular disease (CVD) remains the most prevalent cause of death in developed nations and it is increasing in prevalence in developing countries. Thus, there is substantial interest in identification of pharmacologically targeted pathways and novel biomarkers that recognize persons who are at risk for the development of CVD and who could be targeted for preventive measures. Advancements in our understanding of the molecular basis of CVD suggest that a cardiovascular phenotype is rarely a consequence of an aberration in a single gene. Instead, it reflects a variety of biological and environmental changes that are transmitted across an array of complex reactions. We combine in silico modeling with high-throughput data collection techniques, such as mass spectrometry and gene expression profiling, and interrogate molecular interactomes that form the basis of CVD pathogenesis. I will discuss how we study the underlying regulation in diseases including left ventricular cardiac hypertrophy (LVH), atherosclerosis, and diabetes by combining genomes, proteomes, and phenotypes using the concepts of network biology. Additionally, I will introduce our findings that emphasize temporal nature of CVDs and offer a model of signal transduction in LVH. Finally, I will present how information within cellular interactomes may be used for effective biomarker selection and pharmacotherapy. Taken together, the emerging tools of network biology offer an unbiased platform to explore molecular complexity of CVD, potentially leading to the identification of novel disease modules and pathways.
doi:10.1016/j.vph.2011.08.104
L.15.2 Surfing the data tsunami — How bioinformatics can help to mine transcriptomic data Anton J.G. Horrevoets MCBI, VU University Medical Center, Amsterdam, The Netherlands E-mail address: [email protected] The past decade has shown an ever increasing amount of genomic data, including full-genome DNA-sequences for multiple organisms, transcriptome data for mRNA and microRNA expression, and proteomic profiling data. As an example, currently the number of micro-array transcriptomes deposited at GEO database at NCBI approaches 600,000 profiles. Whereas technology to produce data is ever increasing in quality and speed, the computational approaches to make optimal use of these data are severely lagging behind. More disturbing even is the increasing communication gap between computational bioinformaticians and clinical research or basic research scientists. We have assembled a suite of bioinformatics tools, both commercial and from freely available websources that enable experimental scientists to mine transcriptomics data both from their isolated experiments, and how to combine these with the many datasets that are available from other labs in public databases. Mining these public datasets based on specific research questions proves highly efficient and supplies great added value to limited private datasets. Approaches to combine such data with genomic sequences and/or proteomic data prove especially fruitful in discovering the signaling pathways, gene networks and pivotal control molecules that underlie apparently complex expression data. Finding such pivotal molecules based on unbiased genome-wide data from experimental model systems or patient materials has proven a very efficient key to steer further experimentation in experimental models and to classify individual patient groups. Based on a number of real life examples, these approaches will be presented for practical application by experimental scientists. Organizing and visualizing these multilevel and multiscale datasets into distinct pathways prove a key concept in translating bioinformatics and
patients with coronary atherothrombosis (13). Finally, we have recently shown a significant correlation between TRX, PRX-1 and NADPH activity in asymptomatic subjects, suggesting a coordinated antioxidant response to increased oxidative stress observed in atherothrombosis (14). In conclusion, oxidative stress is a main determinant of pathological remodelling of the vascular wall, linked in part to the presence of hemoglobin within disease tissue and to the imbalance between pro and anti-oxydant molecules. Understanding the complex mechanisms underlying redox balance could help to define novel potential targets to decrease atherothrombostic risk. References 1. Michel, J.B., et al., 2011. Novel aspects of the pathogenesis of aneurysms of the abdominal aorta in humans. Cardiovasc Res. 90, 18–27. 2. Michel, J.B., 2011 Mar 12. Intraplaque haemorrhages as the trigger of plaque vulnerability. Eur Heart J. 3. Nagy, E., et al., 2010. Red cells, hemoglobin, heme, iron, and atherogenesis. Arterioscler Thromb Vasc Biol 30, 1347–1353. 4. Balla, J., et al., 2007. Heme, heme oxygenase, and ferritin: how the vascular endothelium survives (and dies) in an iron-rich environment. Antioxid Redox Signal. 9, 2119–2137. 5. Levy, A.P., et al., 2010. Haptoglobin: basic and clinical aspects. Antioxid Redox Signal 12, 293–304. 6. Kalet-Litman, S., et al., 2010. The haptoglobin 2–2 genotype is associated with increased redox active hemoglobin derived iron in the atherosclerotic plaque. Atherosclerosis 209, 28–31. 7. Moreno, P.R., et al., 2008. Haptoglobin genotype is a major determinant of the amount of iron in the human atherosclerotic plaque. J Am Coll Cardiol 52, 1049–1051. 8. Martinez-Pinna et al. Decreased circulating transferrin and iron levels in patients with abdominal aortic aneurysm (Arterioscler Thromb Vasc Biol, submitted). 9. Altamura, C., et al., 2009. Ceruloplasmin/Transferrin system is related to clinical status in acute stroke. Stroke. 40, 1282–8. 10. Nishihira, K., et al., 2008. Thioredoxin in coronary culprit lesions: possible relationship to oxidative stress and intraplaque hemorrhage. Atherosclerosis. 201, 360–7. 11. Martinez-Pinna, R., et al., 2010. Increased levels of thioredoxin in patients with abdominal aortic aneurysms (AAAs). A potential link of oxidative stress with AAA evolution Atherosclerosis. 212, 333–338. 12. Martinez-Pinna, R., et al., 2011. Identification of peroxiredoxin-1 as a novel biomarker of abdominal aortic aneurysm. Arterioscler Thromb Vasc Biol. 31, 935–43. 13. Hokamaki, J., et al., 2005. Plasma thioredoxin levels in patients with unstable angina. Int J Cardiol. 99, 225–31. 14. Madrigal-Matute et al. Peroxiredoxin-1/Thioredoxin levels are associated to intima media thickness and NADPH oxidase activity in subjects with subclinical atherosclerosis (Arterioscler Thromb Vasc Biol, in revision). doi:10.1016/j.vph.2011.08.100
L.14.4 Prognostic value of intraplaque hemorrhage Gerard Pasterkamp UMC Utrecht, The Netherlands E-mail address: [email protected] Identification of patients at risk for secondary manifestations of atherosclerotic disease progression is based mainly on established risk factors. The AtheroExpress study, is a biobank of atherosclerotic carotid lesions that were collected from patients who underwent carotid
341
endarterectomy and underwent clinical follow-up yearly, up to 3 years after carotid endarterectomy. Using this biobank, we have identified several plaque protein markers which level was associated with secondary cardiovascular events during follow-up (vascular death, nonfatal stroke, nonfatal myocardial infarction, vascular intervention). Local atherosclerotic plaque composition is also thought to be an important determinant of acute cardiovascular events, but no prospective studies have been performed. Using the same AtheroExpress biobank with follow-up, we investigated whether carotid atherosclerotic plaque composition as determined by histology is associated with the occurrence of future vascular events. Macrophage infiltration, large lipid core, calcifications, collagen, and smooth muscle cell infiltration were not associated with clinical outcome. Carotid plaque hemorrhage or marked intraplaque vessel formation, however, was associated with an increased risk of secondary cardiovascular events independent of clinical risk factors and medication use. This demonstrates that not only plaque protein levels but also atherosclerotic plaque composition contains information that can be used as an independent predictor of future cardiovascular events.
doi:10.1016/j.vph.2011.08.101
O.14.1 Spontaneous plaque disruptions, dissection, erosion and intraplaque hemorrhage in murine vein grafts can be attenuated by TIMP-1 overexpression Margreet R. de Vriesa,b, Hans W.M. Niessene, LowikClemens Löwikc, J. Wouter Jukemab,d, Paul H.A. Quaxa,b a Einthoven Laboratory for Experimental Vascular Medicine, Leiden University Medical Center, Leiden, The Netherlands b Department of Surgery, Leiden University Medical Center, Leiden, The Netherlands c Department of Endocrinology, Leiden University Medical Center, Leiden, The Netherlands d Department of Cardiology, Leiden University Medical Center, Leiden, The Netherlands e Department of Pathology and Cardiac Surgery, ICaR-VU, VU University Medical Center, Amsterdam, The Netherlands E-mail address: [email protected] (M. R. de Vries) Plaque rupture and erosion are the most common underlying causes of cardiovascular death. Matrixmetalloproteinases (MMP) play a key role in these processes. We hypothesize that overexpression of the endogenous inhibitor of MMPs TIMP-1 results in decreased plaque disruptions in vein grafts in hypercholesterolemic ApoE3Leiden mice. Vein graft lesions in ApoE3Leiden mice consist of foamcells, smooth muscle cells (SMC) and extracellular matrix with necrotic cores, calcifications and inflammatory cells. Lesion neovessels consist of endothelial cells and basement membranes, supporting mural cells were infrequently seen. 79% of 47 vein grafts studied showed spontaneous disruption, 1/3 of these disruptions were dissections, 1/3 were leaky microvessels with extravasated erythrocytes and 1/3 were erosions or intramural thrombosis. Also vein grafts with combined leaky vessels and dissections or erosions were seen. Systemic overexpression of TIMP-1 (and Luciferase as control) was obtained by plasmid electroporation. Near InfraRed Fluorescense imaging was used to detect in vivo (inhibition of) MMP activity and thus TIMP-1 plasmid functionality. TIMP-1 overexpression resulted in a decrease of 73% in MMP activity in the vein graft-region after 28 d and a 40% reduction in vein graft thickening after 28 d. The lesions showed significant less disruptions (90%) than the control Luciferase group resulting in a more stable phenotype with significant more SMC, collagen and significant less macrophages and fibrinogen.
342
Abstracts
Here we show that murine ApoE3L vein grafts are a relevant model to study plaque stability and subsequent disruptions. Furthermore we demonstrate that MMP inhibition reduced plaque disruptions resulting in a more stable plaque phenotype. doi:10.1016/j.vph.2011.08.102
O.14.2 Mature endothelial cell therapy stabilizes abdominal aortic aneurysm through paracrine mechanisms and recruitment of local vascular cells Grégory Franck, Jianping Dai, Stéphanie Michineau, Saravath Ngo, Anne-Marie Guinault, Eric Allaire, Marianne Gervais CNRS EAC 7054, Creteil, France E-mail address: [email protected] (G. Franck) Background: Abdominal aortic aneurysms (AAAs) expand as a consequence of extracellular matrix (ECM) destruction and vascular smooth muscle cell (VSMC) depletion. Loss of the endothelium and its replacement by a thick mural thrombus is although a common structural feature of human AAA. Since the endothelium exerts anti-thrombotic properties and since the thrombus participates in AAA expansion, we hypothesize that restoration of a functional endothelium by cell therapy would stabilize AAA. Our goal was thus to study the impact of local seeding of mature endothelial cells on AAA evolution in rats. Method and results: Rat aortic endothelial cells (RAEC) were established from 8 week-old male Fischer rat aortas. Preventive or stabilizing effect of RAEC on AAA formation was evaluated in the xenograft model in rats. 5.106 RAEC (n=16) or 200 μl of serum-free medium (control, n=14) were seeded endovascularly either immediately (D0, prevention) or 14 days (D14, stabilization) after surgery, i.e. on alreadyformed AAA. Animals were sacrificed 7 (prevention: D0+7; stabilization: D14+7) or 28 (prevention: D0+28; stabilization: D14+28) days after cell therapy. In vivo, local RAEC seeding prevented AAA formation (diameter increase at D0+7: 22±2 vs 35±3% and D0+28: 49±6 vs 81±6% in RAEC and control rats, respectively, p<0.05) and stabilized already-formed AAA in rats (diameter increase at D14+7: 3.7±2.8 vs 13.1±3.5% and D14+28: –0.9±0.8% vs 26.1±13.5% in RAEC and control rats, p<0.01). This stabilizing effect was associated with a decrease in MMP-dependent proteolysis and macrophage infiltration in the injured aorta, the re-establishment of the luminal endothelial monolayer, the reconstitution of new aortic wall rich in VSMC and ECM components, and the resorption of the intraluminal thrombus. We further showed that transplanted RAEC did not participate directly to the aortic cellular reconstruction but exerted their healing proprieties through paracrine mechanisms, involving the upregulation of endothelial-derived stabilizing factors, such as endothelial NO synthase, basic Fibroblast Growth Factor and VE-cadherin mRNAs and the recruitment of resident vascular cells but not circulating progenitor cells into the new aortic wall. Conclusion: Endovascular cell therapy using mature endothelial cells prevents AAA development and stabilizes already-formed AAA in rats, by re-establishing the endothelial lining and reconstructing a new ECM and VSMC-rich aortic wall through a paracrine mechanism. doi:10.1016/j.vph.2011.08.103
SESSION 15 Systems biology applied to cardiovascular disease L.15.1 Network biology in the post-genomic era Ignat Drozdov
BHF Centre of Excellence, King's College London, London, UK E-mail address: [email protected] Cardiovascular disease (CVD) remains the most prevalent cause of death in developed nations and it is increasing in prevalence in developing countries. Thus, there is substantial interest in identification of pharmacologically targeted pathways and novel biomarkers that recognize persons who are at risk for the development of CVD and who could be targeted for preventive measures. Advancements in our understanding of the molecular basis of CVD suggest that a cardiovascular phenotype is rarely a consequence of an aberration in a single gene. Instead, it reflects a variety of biological and environmental changes that are transmitted across an array of complex reactions. We combine in silico modeling with high-throughput data collection techniques, such as mass spectrometry and gene expression profiling, and interrogate molecular interactomes that form the basis of CVD pathogenesis. I will discuss how we study the underlying regulation in diseases including left ventricular cardiac hypertrophy (LVH), atherosclerosis, and diabetes by combining genomes, proteomes, and phenotypes using the concepts of network biology. Additionally, I will introduce our findings that emphasize temporal nature of CVDs and offer a model of signal transduction in LVH. Finally, I will present how information within cellular interactomes may be used for effective biomarker selection and pharmacotherapy. Taken together, the emerging tools of network biology offer an unbiased platform to explore molecular complexity of CVD, potentially leading to the identification of novel disease modules and pathways.
doi:10.1016/j.vph.2011.08.104
L.15.2 Surfing the data tsunami — How bioinformatics can help to mine transcriptomic data Anton J.G. Horrevoets MCBI, VU University Medical Center, Amsterdam, The Netherlands E-mail address: [email protected] The past decade has shown an ever increasing amount of genomic data, including full-genome DNA-sequences for multiple organisms, transcriptome data for mRNA and microRNA expression, and proteomic profiling data. As an example, currently the number of micro-array transcriptomes deposited at GEO database at NCBI approaches 600,000 profiles. Whereas technology to produce data is ever increasing in quality and speed, the computational approaches to make optimal use of these data are severely lagging behind. More disturbing even is the increasing communication gap between computational bioinformaticians and clinical research or basic research scientists. We have assembled a suite of bioinformatics tools, both commercial and from freely available websources that enable experimental scientists to mine transcriptomics data both from their isolated experiments, and how to combine these with the many datasets that are available from other labs in public databases. Mining these public datasets based on specific research questions proves highly efficient and supplies great added value to limited private datasets. Approaches to combine such data with genomic sequences and/or proteomic data prove especially fruitful in discovering the signaling pathways, gene networks and pivotal control molecules that underlie apparently complex expression data. Finding such pivotal molecules based on unbiased genome-wide data from experimental model systems or patient materials has proven a very efficient key to steer further experimentation in experimental models and to classify individual patient groups. Based on a number of real life examples, these approaches will be presented for practical application by experimental scientists. Organizing and visualizing these multilevel and multiscale datasets into distinct pathways prove a key concept in translating bioinformatics and