EUROPEAN  TISSUE  REPAIR  SOCIETY

HEALING OF VENOUS ULCERS IS ASSOCIATED WITH A REDUCTION IN WOUND TUMOUR NECROSIS FACTOR ALPHA
Michael Murphy, Beaumont Hospital, Ireland

Impaired angiogenesis in tissues of patients with Chronic venous disease (CVD) may be of primary importance in the pathogenesis of venous ulcers. The aim of this study was to measure the effect of venous ulcer wound fluid (WF) on vascular endothelial cell apoptosis and to determine the levels of the pro- and anti-angiogenic cytokines Vascular Endothelial Growth Factor (VEGF) and Tumour Necrosis Factor alpha (TNFa) in WF from non-healing chronic venous ulcers compared to healing ulcers. Patients with nonhealing chronic venous ulcers were selected from a specialised clinic. Wound fluid was aspirated from the ulcers after application of an occlusive adherent dressing. Further samples were obtained from ulcers undergoing compression treatment, all of which demonstrated healing as determined by a measured reduction in ulcer size. The effect of WF on vascular endothelial cell apoptosis was measured after application to cell cultures in concentrations of 1% in standard media. The percentage of apoptotic cells were measured using annexin V antibody staining and FACS analyses. Levels of VEGF and TNFa proteins in WF were measured using sandwich ELISA assays.
Data from 16 patients, 8 with nonhealing and 8 with healing Ulcers in presented. WF from nonhealing ulcers caused a significant increase in the apoptosis of vascular endothelium compared to WF from healing ulcers (mean 38% Vs 7%, p<0.05). Concentrations of both VEGF and TNFa were significantly higher in nonhealing (mean 7000pg/ml and 5000pg/ml) compared to healing ulcer WF (2000pg/ml and 500pg/ml).
These data demonstrate a significant alteration in the cytokine content of healing compared to nonhealing ulcers and suggests a causative role for the antiangiogeneic factor TNFa in the resistance to healing of chronic venous ulcers.

EFFECTS OF IRON ON SOD, LDH AND FERRITIN LEVELS IN DERMAL FIBROBLASTS
Sim Yeoh, Fremantle Hospital, Australia

Wound fluid from chronic venous ulcers has been shown in our department to induce lipid peroxidation in dermal fibroblasts. It is possible that this damage could result from induction of oxygen free radicals due to the presence of iron. Iron can participate in superoxide-driven Fenton reaction and insufficient superoxide dismutase (SOD) activity can lead to lipid peroxidation and cellular damage.
The aim of this study was to determine whether SOD and lactate dehydrogenase (LDH) activities, and ferritin levels in dermal fibroblasts are influenced by iron in culture.
Dermal fibroblasts were derived from neonatal foreskin, normal elderly skin and non-healing chronic venous leg ulcers. Fibroblasts were exposed to 1.5mM ferrous sulphate and cell lysates were measured for SOD (RANSOD colorimetric assay), LDH (UV test) and ferritin (immuno-turbidimetric assay). CLONTECH Atlas™ cDNA array technology was used to assess SOD gene expression.
The study showed no significant effect of iron on SOD and LDH activities. Iron significantly increased intracellular ferritin synthesis (p<0.0001). Leg ulcer fibroblasts had higher levels of SOD and LDH activities (p<0.0001) compared to neonatal and elderly fibroblasts. In response to iron, leg ulcer fibroblasts increased their ferritin synthesis less than the other fibroblasts (p<0.0001). Atlas™ array study showed that SOD was not differentially expressed in response to iron treatment by the dermal fibro-blasts.
Lower ferritin levels in leg ulcer fibroblasts suggest they may be susceptible to damage as indirectly demonstrated by the SOD and LDH activities. While ferritin may be a source of iron in pathological wounds it was not possible to clearly demonstrate from this study that iron causes free radical induced damage in fibroblasts.

GENE ACTIVATION AND CONTRACTILE FORCE REGULATION IN 3D FIBROBLAST POPULATED COLLAGEN LATTICES
Frank Hirche, University of Cologne, Germany

Fibroblasts mediated connective tissue contraction and remodeling is essential in normal and pathological wound healing including granulation tissue formation and fibrosis. The regulation of contractile force generation and associated modulation of gene expression in human fibro-blasts is still poorly understood.
Therefore we investigated the effect of mechanical tension on gene expression and cellular morphology using primary human dermal fibroblasts cultured in three dimensional collagen lattices either under low (relaxed lattice) or high tension (stressed lattice). In addition we measured the isometric contractile force generated by stressed lattices to investigate the signal mechanism regulating tension maintenance.
Analysis of differential gene expression employing cDNA microarrays, subtractive and Northern hybridization demonstrated that application of tension led to differential regulation of genes and cellular morphology. Tension induced a myofibroblastic phenotype, and production of extracellular matrix constituents and protease inhibitors, while repressing production of proteases and inflammatory mediators.
In stressed lattices the specific Rho-kinase inhibitor Y-27632 induced a dose-dependent, complete and reversible force decrease, associated with disappearance of stress fibres and development of numerous thin branched cellular extensions. In contrast to Rho-kinase inhibition increasing intracellular cAMP by db-cAMP and IBMX or Forsko-lin resulted only in an incomplete relaxation and slight reduction in stress fibres. It is therefore concluded that the Rho-kinase signaling cascade predominantly regulates force maintenance in fibroblast populated collagen lattices.

CYTO-MECHANICS AND HEALING

CYTOMECHANICS AND CELL-GENERATED FORCES
Robert Brown, Tissue Repair Unit, University College London, UK

Cytomechanics is the application of mechanical engineering principles at the cell level. It is characterised by a complete interdependence of extracellular matrix (ECM) material properties, mechanical loading and adaptive cell responses, making the study of any one element problematic. In many forms of tissue repair the major need is to understand how adaptive cell responses give rise to the material properties of new ECM.
Since ECM architecture is influenced by local mechanical loads our aim has been to identify how key mechanical cues elicit cell responses. Models have included the use of uniaxial loading to determine alignment, collagen and MMP production (reflecting remodelling). These demonstrated that fibroblasts, which are subject to, and are able to align with, predictable strains in their ECM increase collagen deposition. However, where local cues conflict and prevent adaptive responses, MMP production increases, suggesting cell migration or ECM removal. The general conclusion is that resident cell behaviour to mechanical cues, is one of stress shielding, using adaptive responses including shape/alignment changes and local production/removal of collagen. In turn, this dictates that (i) fibre composition and architecture (i.e., material properties) and (ii) the pattern of cell-matrix attachment to pericellular ECM are central determinants. In effect, the existing pericellular matrix (e.g., soft, random granulation tissue or stiff, collagenous scar) has a major influence on downstream, cell-mediated remodelling of that tissue.

INTRACELLULAR DELIVERY OF THE N-TERMINAL PEPTIDE OF SMOOTH MUSCLE ACTIN INHIBITS MYOFIBROBLAST CONTRACTION
Boris Hinz, Christine Chaponnier and Giulio Gabbiani Department of Pathology, C.M.U., University of Geneva, Switzerland

Myofibroblasts are generally considered to be responsible for granulation tissue contraction and for the soft tissue retractions taking place during fibro-contractive diseases. No efficient medical therapy exists at present for these lesions. The main marker of fibroblast-myofibroblast modulation is the expression of a smooth muscle actin (aSMA), the actin isoform typical of vascular smooth muscle cells. Moreover, there is a correlation between aSMA expression and contractile activity of fibroblastic cells in vitro and in vivo. Actin isoforms differ essentially in their N-terminus; thus this domain represents a likely candidate for specialized functions mediated through specific binding. The N-terminal sequence AcEEED appears to be important for aSMA polymerization and incorporation into stress fibers. When microinjected into cultured fibroblasts it produces the disappearance of aSMA specific immuno-detection. We have constructed a fusion peptide (FP) including Ac-EEED and the antennapoedia third helix sequence that allows cell penetration. We show that the FP localizes in stress fibers rapidly after administration; it then inhibits: 1) the spontaneous retractile activity of fibroblasts placed on a silicone substrate or within a collagen gel, 2) the contraction of granulation tissue strips stimulated by SM agonists and 3) the in vivo contraction of a rat wound spontaneously healing or of a wound splinted for 10 days by a rigid plastic frame. Our results support the assumption that aSMA plays an important role in wound contraction and furnish the basis for a new cytoskeleton-based therapeutic approach for pathological scarring and/or fibro-contractive diseases.

REGULATION OF MYOFIBROBLAST DIFFERENTIATION AND CONTRACTION
J.J. Tomasek, and C.J. Haaksma, Dept of Cell Biology, University of Oklahoma Health Sciences Ctr, Oklahoma City, USA

Myofibroblasts are specialised fibroblasts that have acquired a contractile phenotype including stress fibres, fibronexus adhesion complexes, extracellular fibronectin fibrils, and may express a-smooth-muscle actin. Myofibro-blasts have been proposed to play a key role in generating the force responsible for granulation tissue contraction during wound healing. Using three dimensional collagen lattices, we have demonstrated that mechanical stress will promote the modulation of fibroblasts into myofibroblasts. Once formed, myofibroblasts are capable of generating sustained isometric tension and transmitting this force to the surrounding extracellular matrix. We have also examined the intracellular signals regulating myofibroblasts force generation in both cultured myofibroblasts, as well as myofibroblasts present in granulation tissue. The Rho kinase inhibitor Y27632 inhibited myofibroblast contraction and this inhibition was reversed by the phosphatase inhibitor calyculin. In addition, increasing intracellular Ca2+ was insufficient to promote contraction. Together these results support a model whereby Rho/Rho kinase-mediated inhibition of myosin light chain phosphatase is necessary for myofibroblast contraction, in contrast to smooth muscle cells where Ca2+ -activation of myosin light chain kinase alone is sufficient to promote contraction. The question that arises is why myofibroblasts and smooth muscle cells would have different intracellular regulation. The answer may lie in the difference in function. Activation of the Rho/Rho kinase pathway may allow myofibro-blasts to continuously generate force as needed during tissue contraction, while responsiveness to intracellular calcium would allow smooth muscle cells to rapidly alter force generation in response to external stimuli. (This work was funded by NIH grant 1 RO1 GM60661-01).