Cholesterol efflux pathways hinder KRAS-driven lung tumor progenitor cell expansion.

Cholesterol efflux pathways could be exploited in tumor biology to unravel cancer vulnerabilities. A mouse model of lung-tumor-bearing KRASG12D mutation with specific disruption of cholesterol efflux pathways in epithelial progenitor cells promoted tumor growth. Defective cholesterol efflux in epithelial progenitor cells governed their transcriptional landscape to support their expansion and create a pro-tolerogenic tumor microenvironment (TME). Overexpression of the apolipoprotein A-I, to raise HDL levels, protected these mice from tumor development and dire pathologic consequences. Mechanistically, HDL blunted a positive feedback loop between growth factor signaling pathways and cholesterol efflux pathways that cancer cells hijack to expand. Cholesterol removal therapy with cyclodextrin reduced tumor burden in progressing tumor by suppressing the proliferation and expansion of epithelial progenitor cells of tumor origin. Local and systemic perturbations of cholesterol efflux pathways were confirmed in human lung adenocarcinoma (LUAD). Our results position cholesterol removal therapy as a putative metabolic target in lung cancer progenitor cells.

Kindlin-2 Mediates Mechanical Activation of Cardiac Myofibroblasts.

We identify the focal adhesion protein kindlin-2 as player in a novel mechanotransduction pathway that controls profibrotic cardiac fibroblast to myofibroblast activation. Kindlin-2 is co-upregulated with the myofibroblast marker α-smooth muscle actin (α-SMA) in fibrotic rat hearts and in human cardiac fibroblasts exposed to fibrosis-stiff culture substrates and pro-fibrotic TGF-ß1. Stressing fibroblasts using ferromagnetic microbeads, stretchable silicone membranes, and cell contraction agonists all result in kindlin-2 translocation to the nucleus. Overexpression of full-length kindlin-2 but not of kindlin-2 missing a putative nuclear localization sequence (∆NLS kindlin-2) results in increased α-SMA promoter activity. Downregulating kindlin-2 with siRNA leads to decreased myofibroblast contraction and reduced α-SMA expression, which is dependent on CC(A/T)-rich GG(CArG) box elements in the α-SMA promoter. Lost myofibroblast features under kindlin-2 knockdown are rescued with wild-type but not ∆NLS kindlin-2, indicating that myofibroblast control by kindlin-2 requires its nuclear translocation. Because kindlin-2 can act as a mechanotransducer regulating the transcription of α-SMA, it is a potential target to interfere with myofibroblast activation in tissue fibrosis.

Early postnatal soluble FGFR3 therapy prevents the atypical development of obesity in achondroplasia.

BACKGROUND: Achondroplasia is a rare genetic disease is characterized by abnormal bone development and early obesity. While the bone aspect of the disease has been thoroughly studied, early obesity affecting approximately 50% of them during childhood has been somewhat neglected. It nevertheless represents a major health problem in these patients, and is associated to life-threatening complications including increasing risk of cardiovascular pathologies. We have thus decided to study obesity in patients and to use the mouse model to evaluate if soluble FGFR3 therapy, an innovative treatment approach for achondroplasia, could also impact the development of this significant complication. METHODS AND FINDINGS: To achieve this, we have first fully characterized the metabolic deregulations in these patients by conducting a longitudinal retrospective study, in children with achondroplasia Anthropometric, densitometric measures as well as several blood parameters were recorded and compared between three age groups ranging from [0-3], [4-8] and [9-18] years old. Our results show unexpected results with the development of an atypical obesity with preferential fat deposition in the abdomen that is remarkably not associated with classical complications of obesity such as diabetes or hypercholosterolemia. Because it is not associated with diabetes, the atypical obesity has not been studied in the past even though it is recognized as a real problem in these patients. These results were validated in a murine model of achondroplasia (Fgfr3ach/+) where similar visceral adiposity was observed. Unexpected alterations in glucose metabolism were highlighted during high-fat diet. Glucose, insulin or lipid levels remained low, without the development of diabetes. Very interestingly, in achondroplasia mice treated with soluble FGFR3 during the growth period (from D3 to D22), the development of these metabolic deregulations was prevented in adult animals (between 4 and 14 weeks of age). The lean-over-fat tissues ratio was restored and glucose metabolism showed normal levels. Treating Fgfr3ach/+ mice with soluble FGFR3 during the growth period, prevented the development of these metabolic deregulations in adult animals and restored lean-over-fat tissues ratio as well as glucose metabolism in adult animals. CONCLUSION: This study demonstrate that achondroplasia patients develop an atypical obesity with preferential abdominal obesity not associated with classical complications. These results suggest that achondroplasia induces an uncommon metabolism of energy, directly linked to the FGFR3 mutation. These data strongly suggest that this common complication of achondroplasia should be included in the clinical management of patients. In this context, sFGFR3 proved to be a promising treatment for achondroplasia by normalizing the biology at different levels, not only restoring bone growth but also preventing the atypical visceral obesity and some metabolic deregulations.

Isolation of Astrocytes Displaying Myofibroblast Properties and Present in Multiple Sclerosis Lesions.

A wide heterogeneity of lesions can affect the central nervous system (CNS). In all situations where neurons are damaged, including multiple sclerosis (MS), a common reactive astrocytosis is present. Sedimentation field-flow fractionation (SdFFF) was used to sort astrocyte subpopulations. After SdFFF elution, cells, prepared from rat newborn cortex, were cultured and analyzed by immunocytofluorescence for glial fibrillary acidic protein (GFAP) and α-smooth muscle (SM) actin (a specific marker for myofibroblasts) expression. Cell contractile capacity was studied. Samples from patients with MS were also analyzed. Three main fractions (F1, F2, and F3) were isolated and compared with the total eluted population (TP). TP, F1, F2, and F3, contained respectively 74, 96, 12, and 98% of GFAP expressing astrocytes. In F3, astrocytes only expressed GFAP while in F1, astrocytes expressed both GFAP and α-SM actin. In F2 and TP, α-SM actin expression was barely detected. F3-derived cells showed higher contractile capacities compared with F1-derived cells. In one specific case of MS known as Baló's concentric MS, astrocytes expressing both GFAP and α-SM actin were detected. Using SdFFF, a population of astrocytes presenting myofibroblast properties was isolated. This subpopulation of astrocytes was also observed in a MS sample suggesting that it could be involved in lesion formation and remodeling during CNS pathologies.

Disruption of Glut1 in Hematopoietic Stem Cells Prevents Myelopoiesis and Enhanced Glucose Flux in Atheromatous Plaques of ApoE(-/-) Mice.

RATIONALE: Inflamed atherosclerotic plaques can be visualized by noninvasive positron emission and computed tomographic imaging with (18)F-fluorodeoxyglucose, a glucose analog, but the underlying mechanisms are poorly understood. OBJECTIVE: Here, we directly investigated the role of Glut1-mediated glucose uptake in apolipoprotein E-deficient (ApoE(-/-)) mouse model of atherosclerosis. METHODS AND RESULTS: We first showed that the enhanced glycolytic flux in atheromatous plaques of ApoE(-/-) mice was associated with the enhanced metabolic activity of hematopoietic stem and multipotential progenitor cells and higher Glut1 expression in these cells. Mechanistically, the regulation of Glut1 in ApoE(-/-) hematopoietic stem and multipotential progenitor cells was not because of alterations in hypoxia-inducible factor 1α signaling or the oxygenation status of the bone marrow but was the consequence of the activation of the common ß subunit of the granulocyte-macrophage colony-stimulating factor/interleukin-3 receptor driving glycolytic substrate utilization by mitochondria. By transplanting bone marrow from WT, Glut1(+/-), ApoE(-/-), and ApoE(-/-)Glut1(+/-) mice into hypercholesterolemic ApoE-deficient mice, we found that Glut1 deficiency reversed ApoE(-/-) hematopoietic stem and multipotential progenitor cell proliferation and expansion, which prevented the myelopoiesis and accelerated atherosclerosis of ApoE(-/-) mice transplanted with ApoE(-/-) bone marrow and resulted in reduced glucose uptake in the spleen and aortic arch of these mice. CONCLUSIONS: We identified that Glut1 connects the enhanced glucose uptake in atheromatous plaques of ApoE(-/-) mice with their myelopoiesis through regulation of hematopoietic stem and multipotential progenitor cell maintenance and myelomonocytic fate and suggests Glut1 as potential drug target for atherosclerosis.

Neural Stem Cell Properties of an Astrocyte Subpopulation Sorted by Sedimentation Field-Flow Fractionation.

Astrocytes encompass a heterogeneous cell population. Using sedimentation field-flow fractionation (SdFFF) method, different, almost pure, astrocyte subpopulations were isolated. Cells were collected from cortex of newborn rats and sorted by SdFFF to obtain different fractions, which were subjected to protein analysis and characterized by immunocytofluorescence. The behavior of the cells was analyzed in vitro, under culture conditions used for neural stem cells. These culture conditions were also applied to cells derived from an adult cortical tissue after traumatic brain injury (TBI). Finally, the astrocytic neural stem-like cells were transplanted in damaged sciatic nerve. Protein analysis indicated a high expression of glial fibrillary acidic protein (GFAP) and vimentin in fraction F3-derived cells. These cells formed neurospheres when cultured with epidermal growth factor and large colonies in a collagen-containing semi-solid matrix. Neurospheres expressed GFAP and nestin and were able in addition to generate neurons expressing MAP2 and oligodendrocytes expressing Olig2. When transplanted in a damaged nerve, cells of F3-derived neurospheres colonized the damaged area. Finally, after TBI in adult rats, cells able to form neurospheres containing a subpopulation of astrocytes expressing vimentin were obtained. Using the SdFFF method, an astrocyte subpopulation presenting stem cell properties was isolated from a newborn rat cortex and from an injured adult rat cortex. The specific activation of this astrocyte subpopulation may provide a potential therapeutic approach to restore lost neuronal function in injured or diseased brain.

Maintenance of Macrophage Redox Status by ChREBP Limits Inflammation and Apoptosis and Protects against Advanced Atherosclerotic Lesion Formation.

Enhanced glucose utilization can be visualized in atherosclerotic lesions and may reflect a high glycolytic rate in lesional macrophages, but its causative role in plaque progression remains unclear. We observe that the activity of the carbohydrate-responsive element binding protein ChREBP is rapidly downregulated upon TLR4 activation in macrophages. ChREBP inactivation refocuses cellular metabolism to a high redox state favoring enhanced inflammatory responses after TLR4 activation and increased cell death after TLR4 activation or oxidized LDL loading. Targeted deletion of ChREBP in bone marrow cells resulted in accelerated atherosclerosis progression in Ldlr(-/-) mice with increased monocytosis, lesional macrophage accumulation, and plaque necrosis. Thus, ChREBP-dependent macrophage metabolic reprogramming hinders plaque progression and establishes a causative role for leukocyte glucose metabolism in atherosclerosis.

Integrins αvß5 and αvß3 promote latent TGF-ß1 activation by human cardiac fibroblast contraction.

AIMS: Pathological tissue remodelling by myofibroblast contraction is a hallmark of cardiac fibrosis. Myofibroblasts differentiate from cardiac fibroblasts under the action of transforming growth factor-ß1 (TGF-ß1), which is secreted into the extracellular matrix as a large latent complex. Integrin-mediated traction forces activate TGF-ß1 by inducing a conformational change in the latent complex. The mesenchymal integrins αvß5 and αvß3 are expressed in the heart, but their role in the activation of TGF-ß1 remains elusive. Here, we test whether targeting αvß5 and αvß3 integrins reduces latent TGF-ß1 activation by cardiac fibroblasts with the goal to prevent the formation of α-smooth muscle actin (α-SMA)-expressing cardiac myofibroblasts and their contribution to fibrosis. METHODS AND RESULTS: Using a porcine model of induced right ventricular fibrosis and pro-fibrotic culture conditions, we show that integrins αvß5 and αvß3 are up-regulated in myofibroblast-enriched fibrotic lesions and differentiated cultured human cardiac myofibroblasts. Both integrins autonomously contribute to latent TGF-ß1 activation and myofibroblast differentiation, as demonstrated by function-blocking peptides and antibodies. Acute blocking of both integrins leads to significantly reduced TGF-ß1 activation by cardiac fibroblast contraction and loss of α-SMA expression, which is restored by adding active TGF-ß1. Manipulating integrin protein levels in overexpression and shRNA experiments reveals that both integrins can compensate for each other with respect to TGF-ß1 activation and induction of α-SMA expression. CONCLUSIONS: Integrins αvß5 and αvß3 both control myofibroblast differentiation by activating latent TGF-ß1. Pharmacological targeting of mesenchymal integrins is a possible strategy to selectively block TGF-ß1 activation by cardiac myofibroblasts and progression of fibrosis in the heart.

Deficiency of ATP-binding cassette transporter B6 in megakaryocyte progenitors accelerates atherosclerosis in mice.

OBJECTIVE: The ATP-binding cassette (ABC) transporter B6 (ABCB6) is highly expressed in megakaryocyte progenitors, but its role in platelet production and disease has not been elucidated. APPROACH AND RESULTS: Among various ABC transporters, ABCB6 was highly expressed in megakaryocyte progenitors, exhibiting the same pattern of expression of genes involved in heme synthesis pathway. Transplantation of Abcb6 deficient (Abcb6(-/-)) bone marrow into low density lipoprotein receptor deficient recipient mice resulted in expansion and proliferation of megakaryocyte progenitors, attributable to increased reactive oxygen species production in response to porphyrin loading. The enhanced megakaryopoiesis in Abcb6(-/-) bone marrow-transplanted mice was further illustrated by increased platelet counts, mean platelet volume, and platelet activity. Platelets from Abcb6(-/-) bone marrow-transplanted mice had higher levels of chemokine (C-C motif) ligand 5, which was associated with increased plasma chemokine (C-C motif) ligand 5 levels. There were also increased platelet-leukocyte aggregates, which resulted in leukocyte activation. Abcb6(-/-) bone marrow-transplanted mice had accelerated atherosclerosis which was associated with deposition of the chemotactic agent, chemokine (C-C motif) ligand 5 in atherosclerotic plaques, resulting in increased macrophage accumulation. CONCLUSIONS: Our findings identify a new role of ABCB6 in preventing atherosclerosis development by dampening platelet production, reactivity, and chemokine (C-C motif) ligand 5 deposition in atherosclerotic lesions.

Fast astrocyte isolation by sedimentation field flow fractionation.

Astrocytes play a key role during central nervous system (CNS) repair and glial scar formation. After CNS damage, an extensive deposition of the extracellular matrix produced by the activated astrocytes limits the extension of the lesion but impairs axon outgrowth and functional recovery. Until now, methods to obtain astrocytes need long culture period and laborious cell culture conditions and do not allow the isolation of pure astrocyte preparation. In this study, we used sedimentation field flow fractionation (SdFFF) to rapidly sort well preserved astrocyte population. Four main cell fractions, the total eluted population (TP), and fractions F1, F2, and F3, were isolated by SdFFF from rat newborn cortex. After elution, cells were cultured for one week, and analyzed by immunocytofluorescence using antibodies against specific epitopes: glial fibrillary acidic protein (GFAP), O4, ß-III tubulin, and CD 68, labelling respectively astrocytes, oligodendrocytes, neurons, and microglial cells. SdFFF eluted cells were compared with the cells obtained with the classical method. Results showed that SdFFF appeared to be a rapid (one week) and effective method to sort enriched populations of viable and functional astrocytes. In particular, F1 and F3 fractions contained high percentage of GFAP expressing cells (95.6% and 98.0%, respectively). Results also showed that F1 derived cell cultures contained large astrocytes that spread in the culture dish while in fraction F3 derived cell cultures, astrocytes were small, showing a tendency to aggregate and displaying higher migratory capacities than those of fraction F1. Thanks to SdFFF, isolation of almost pure astrocyte populations was rapidly obtained. In addition, the isolation of different astrocyte subpopulations showing different behaviors offers a new perspective to better understand the glial scar formation and remodeling after CNS damage.

The role of the myofibroblast in tumor stroma remodeling.

Since its first description in wound granulation tissue, the myofibroblast has been recognized to be a key actor in the epithelial-mesenchymal cross-talk that plays a crucial role in many physiological and pathological situations, such as regulation of prostate development, ventilation-perfusion in lung alveoli or organ fibrosis. The presence of myofibroblasts in the stroma reaction to epithelial tumors is well established and many data are accumulating which suggest that the stroma compartment is an active participant in tumor onset and/or evolution. In this review we summarize the evidence in favor of this concept, the main mechanisms that regulate myofibroblast differentiation and function, as well as the biophysical and biochemical factors possibly involved in epithelial-stroma interactions, using liver carcinoma as main model, in view of achieving a better understanding of tumor progression mechanisms and of tools directed toward stroma as eventual therapeutic target.

The mechanical memory of lung myofibroblasts.

Fibroblasts differentiate into the highly synthetic and contractile myofibroblast phenotype when exposed to substrates with an elastic modulus corresponding to pathologically stiff fibrotic tissue. Cellular responses to changes in substrate stiffness are typically analyzed after hours or days, which does not enable the monitoring of myofibroblast persistence, a hallmark of fibrosis. To determine long-lasting effects on the fibrotic behavior of lung fibroblasts, we followed a novel approach of explanting and repeatedly passaging fibroblasts on silicone substrates with stiffness representing various states of lung health. Fibrotic activity was determined by assaying for myofibroblast proliferation, cell contractility, expression of α-smooth muscle actin, extracellular matrix and active TGFß1. As predicted, myofibroblast activity was low on healthy soft substrates and increased with increasing substrate stiffness. However, explanting and mechanically priming lung fibroblasts for 3 weeks on pathologically stiff substrates resulted in sustained myofibroblast activity even after the cells were returned to healthy soft cultures for 2 weeks. Such primed cells retained higher fibrotic activity than cells that had been exclusively cultured on soft substrates, and were not statistically different from cells continuously passaged on stiff surfaces. Inversely, priming lung fibroblasts for 3 weeks on soft substrates partially protected from myofibroblast activation after the shift to stiff substrates. Hence, mechano-sensed information relating to physical conditions of the local cellular environment could permanently induce fibrotic behavior of lung fibroblasts. This priming effect has important implications for the progression and persistence of aggressive fibrotic diseases such as idiopathic pulmonary fibrosis.

Erythropoietin restores C-fiber function and prevents pressure ulcer formation in diabetic mice.

Pressure-induced vasodilatation (PIV), a cutaneous physiological neurovascular (C-fiber/endothelium) mechanism, is altered in diabetes and could possibly contribute to pressure ulcer development. We wanted to determine whether recombinant human erythropoietin (rhEPO), which has protective neurovascular effects, could prevent PIV alteration and pressure ulcer formation. We developed a skin pressure ulcer model in mice by applying two magnetic plates to the dorsal skin. This induced significant stage 2 ulcers (assessed visually and histologically) in streptozotocin-treated mice with 8 weeks of diabetes compared with very few in controls. Control and streptozotocin mice received either no treatment or systematic rhEPO (3,000 UI kg(-1) intraperitoneally, twice a week) during the last 2 weeks of diabetes. After 8 weeks of diabetes, we assessed ulcer development, PIV, endothelium-dependent vasodilation, C-fiber-mediated nociception threshold, and skin innervation density. Pretreatment with rhEPO fully prevented ulcer development in streptozotocin mice and also fully restored C-fiber nociception, skin innervation density, and significantly improved PIV, but had no effect on endothelium-dependent vasodilation. Our finding that rhEPO treatment protects the skin against pressure-induced ulcers in diabetic mice encourages evaluation of the therapeutic potential for non-hematopoietic analogs of EPO in preventing neuropathic diabetic ulcers.

TLR4 signal transduction pathways neutralize the effect of Fas signals on glioblastoma cell proliferation and migration.

The Fas pathway is described as an activator of the glioblastoma proliferation by increasing the pathogenicity of this tumour. The lipopolysaccharide (LPS) pathway depending on Toll-like receptor 4 (TLR4) could limit the glioblastoma spreading. Here, Fas and TLR4 pathways were activated in glioblastoma cell lines by an agonist antibody and/or LPS treatment. Activation of the Fas pathway or of the TLR4 pathway induced cell proliferation. However, simultaneous treatment with agonist antibody and LPS decreased proliferation. This anti-proliferative effect was caspase dependent, and a decreased cell migration and matrix metalloproteinase (MMP)-9 expression were also observed. Both TLR4 and MMP-9 were highly expressed in human glioblastoma tissues. These data suggest that TLR4 signal transduction pathways neutralize proliferation and migration induced by Fas pathway activation in glioblastoma cell lines.

Mechanisms of pathological scarring: role of myofibroblasts and current developments.

Myofibroblasts play a key role in the wound-healing process, promoting wound closure and matrix deposition. These cells normally disappear from granulation tissue by apoptosis after wound closure, but under some circumstances, they persist and may contribute to pathological scar formation. Myofibroblast differentiation and apoptosis are both modulated by cytokines, mechanical stress, and, more generally, cell-cell and cell-matrix interactions. Tissue repair allows tissues and organs to recover, at least partially, functional properties that have been lost through trauma or disease. Embryonic skin wounds are repaired without scarring or fibrosis, whereas skin wound repair in adults always leads to scar formation, which may have functional or esthetic consequences, as in the case of hypertrophic scars, for example. Skin wound repair involves a precise remodeling process, particularly in the dermal compartment, during which fibroblasts/myofibroblasts play a central role. This article reviews the origins of myofibroblasts and their role in normal and pathological skin wound healing. This article focuses on traumatic skin wound healing, but largely, the same mechanisms apply in other physiological and pathological settings. Tissue healing in other organs is examined by comparison, as well as the stromal reaction associated with cancer. New approaches to wound/scar therapy are discussed.

Photodynamic effects of porphyrin-polyamine conjugates in human breast cancer and keratinocyte cell lines.

Porphyrin-polyamine conjugates bearing two (cis or trans position) or four spermidine or spermine units were synthesized. We studied the photostability, the hydrophilic/lipophilic balance of porphyrin-polyamine derivatives and the production of singlet oxygen. All these compounds possess physicochemical features required for their use in PDT. Then, we investigated the photocytotoxic efficacy of these porphyrin-polyamine derivatives and the cell death pathway implicated. All compounds appear to be more efficient than Photofrin® to induce HaCat and MCF7 cell death, essentially by apoptosis. Collectively, these data show that porphyrin-polyamine conjugates could be promising phototherapeutic agents.

Fibrogenic cell phenotype modifications during remodelling of normal and pathological human liver in cultured slices.

BACKGROUND: The debate concerning the potential remodelling and/or reversibility of cirrhotic lesions and biliary fibrosis is still open. AIMS/METHODS: In this work, we have used the precision-cut liver slice (PCLS) model, which maintains cell-cell and cell-matrix interactions to study, by immunohistochemistry, the behaviour of the different fibrogenic cells, i.e. hepatic stellate cells (HSC) and portal fibroblasts, in cultured (for 1 week) PCLS derived from normal and fibrotic human livers. RESULTS: In normal liver, before and after culture, α-smooth muscle (SM) actin was present only in the vessel walls. Platelet-derived growth factor (PDGF) receptor-ß was expressed before and after culture by portal fibroblasts, and appeared after culture in HSC. Before culture, CD 34 was not expressed in parenchyma, but appeared after culture in sinusoidal endothelial cells. In cirrhotic lesions, before culture, α-SM actin, PDGF receptor-ß and Thy-1 were expressed in septa; after culture, α-SM actin expression disappeared but the expression of the PDGF receptor-ß and Thy-1 was maintained. In cholestatic liver specimens, α-SM actin, PDGF receptor-ß and Thy-1 expression, which was present before culture in enlarged portal areas, disappeared after culture, and apoptosis was detected. In the parenchyma of both cirrhotic and cholestatic livers, the expression of the PDGF receptor-ß and of CD 34, which was not observed before culture, was present in HSC and sinusoidal endothelial cells, respectively, after culture. CONCLUSIONS: These results indicate that during remodelling of pathological tissues in cultured liver slices, the myofibroblastic cells derived from HSC or from portal fibroblasts show different behaviours, suggesting different mechanisms of activation/deactivation.

Double immunohistochemistry with horseradish peroxidase and alkaline phosphatase detection systems.

We describe here a protocol optimized for formalin-fixed paraffin-embedded tissue sections that enables the detection of two antigens. This technique allows immunohistochemistry to be performed with detection systems allowing observation by light microscopy. This chapter discusses the choice of appropriate protocols as well as the choice of visualization systems.In doing so, we provide examples of representative results obtained with this protocol and describe necessary controls; additionally, we discuss common problems associated with this methodology, and detail troubleshooting recommendations. Although this method has been optimized for liver sections, it may be applicable for performing double immunostaining in a variety of tissue samples.

Proteomic analysis of differentially expressed proteins in peripheral cholangiocarcinoma.

Cholangiocarcinoma is an adenocarcinoma of the liver which has increased in incidence over the last thirty years to reach similar levels to other liver cancers. Diagnosis of this disease is usually late and prognosis is poor, therefore it is of great importance to identify novel candidate markers and potential early indicators of this disease as well as molecules that may be potential therapeutic targets. We have used a proteomic approach to identify differentially expressed proteins in peripheral cholangiocarcinoma cases and compared expression with paired non-tumoral liver tissue from the same patients. Two-dimensional fluorescence difference gel electrophoresis after labeling of the proteins with cyanines 3 and 5 was used to identify differentially expressed proteins. Overall, of the approximately 2,400 protein spots visualised in each gel, 172 protein spots showed significant differences in expression level between tumoral and non-tumoral tissue with p < 0.01. Of these, 100 spots corresponding to 138 different proteins were identified by mass spectrometry: 70 proteins were over-expressed whereas 68 proteins were under-expressed in tumoral samples compared to non-tumoral samples. Among the over-expressed proteins, immunohistochemistry studies confirmed an increased expression of 14-3-3 protein in tumoral cells while α-smooth muscle actin and periostin were shown to be overexpressed in the stromal myofibroblasts surrounding tumoral cells. α-Smooth muscle actin is a marker of myofibroblast differentiation and has been found to be a prognostic indicator in colon cancer while periostin may also have a role in cell adhesion, proliferation and migration and has been identified in other cancers. This underlines the role of stromal components in cancer progression and their interest for developing new diagnostic or therapeutic tools.

DNA photocleavage by porphyrin-polyamine conjugates.

A series of polyamine-porphyrin conjugates bearing two (cis or trans position) or four units of spermidine or spermine was synthesized. We studied the binding of these cationic porphyrins to calf thymus DNA by the means of UV-vis spectroscopy and we investigated their ability to cleave plasmid DNA in the presence of light. DNA binding and DNA photocleavage abilities were found to depend on structural characteristics as (a) the relative positions of the side chains on the porphyrin ring and (b) the nature of the attached side chains (spermidine or spermine). DNA cleavage was also studied in the presence of a singlet oxygen quencher (NaN(3)) and in the presence of a hydroxyl radical scavenger (mannitol). Singlet oxygen was the major species responsible for the cleavage of DNA previously observed. Collectively, these data show that polyamine-porphyrin conjugates could be promising phototherapeutic agents.