Vincent Sarrazy

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.

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.

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.

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.

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.

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.

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.

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, β-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.

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.

Mechanisms of pathological scarring: Role of myofibroblasts and current developments: Pathological scarring mechanisms

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.