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INCREASED ANGIOGENESIS AND TUMOUR SIZE IN 3-INTEGRIN DEFICIENT MICE IS ASSOCIATED WITH INCREASED VEGF-RECEPTOR LEVELS
Louise Reynolds¹, Lorenza Wyder¹, Julie Lively² XiaoshuHuang³, Dean Sheppard³, Ian Hart¹, Richard Hynes² and Kairbaan Hodivala-Dilke¹. Imperial Cancer Research Fund, St Thomas' Hospital, London, UK; CCR, Howard Hughes Medical Institute, MIT, Cambridge, MA 02139, USA; Lung Biology Center, University of California, San Fransisco, CA 94143, USA

v expression has been reported to be upregulated on endothelial cells during neovascularisation. Here we investigated the effect of -integrin deficiency on angiogenesis in vivo and in vitro. We report that in both retinopathy of prematurity models and tumour assays that angiogenesis is supported in -integrin knockout and /5-double knockout mice. Immunohistochemical staining of B16F0 subcutaneous tumour sections revealed that the -deficient angiogenic vessels express various endothelial markers including PECAM-1, von Willebrands factor (vWF), VE-cadher-in, vascular endothelial growth factor receptor-2 (VEGFR2/Flk-1) and the angiopoietin receptor (Tie2/Tek). In addition, analysis of tumours grown in -null mice established that vessel density (number of vessels per unit area) and tumour size was significantly increased when compared to wild types. In order to measure vessel growth in vitro ex vivo aortic vessel culture assays were performed and indicated that -deficient vessels respond to exogenous VEGF with increased microvessel sprouting when compared to wild types. Since v3 and v5-integrins are receptors for vitronectin and there is evidence that v5 is a receptor for VEGF and it could be imagined that in -knockout mice v5-integrins may play a compensatory role. Endothelial cells isolated from the mice were deficient in v-integrin expression, but had normal expression levels of other integrins. In adhesion and migration assays -null cells had baseline responses to vitronectin and normal responses to fibronectin, collagen and laminin, suggesting that v5 is not playing a compensatory role in -null endothelial cells. However, examination of Flk-1 by western blotting and FACs analysis established a two-fold increase in Flk-1 levels in -null endothelial cells. Taken together, these data demonstrate that -integrin deficiency enhances angiogenesis and that this is associated with an increase VEGF and its receptor Flk-1.

WOUND HEALING IN THE ATHEROSCLEROTIC PLAQUE
Andrew C Newby, Bristol Heart Institute, Bristol Royal Infirmary, Bristol, UK

Wounding to the intimal surface of the atherosclerotic plaque is the main cause of myocardial infarction and hence sudden cardiac death. It is therefore by far the most common acutely fatal wound in pathobiology. The wounding agent is pulsatile blood flow acting on a chronically inflamed intima, in particular new or episodic inflammation on an established lesion. Plaque healing post-myocardial infarction (or coronary angioplasty) is efficient, which demonstrates the adequate capacity for repair of the vessel wall. The atherosclerosis plaque is itself, arguably, a non-healing wound. It occurs initially in response to injury caused by oxidised low-density lipoproteins. The response involves macrophage and T-cell invasion, angiogenesis and fibrosis, all components of wound healing. Why therefore do plaques not stabilise but instead rupture? Causes might include excessive inflammation leading to matrix degradation by metalloproteinases, senescence or apoptosis of smooth muscle cells or inadequate angiogenesis. Statins decrease serum LDL concentration, reduce plaque inflammation, promote fibrosis and reduce the risk of myocardial infarction by approximately 30%. There is clinical potential for other treatments that more directly promote healing of the atherosclerotic plaque.

RESEARCH STUDIES

THE ROLE OF IRON IN LIPODERMATOSCLEROSIS
Sarah Herrick, Linda Treharne, Philip Coleridge Smith, Geoffrey J Laurent, Department of Medicine, University College London, London, UK

Many patients with chronic venous insufficiency develop skin changes in the lower leg prior to venous ulceration. This condition, known as lipodermatosclerosis ( LDS), is characterised by skin thickening and hyperpigmentation caused by deposition iron in the form of haemosiderin. Although iron is essential for many metabolic processes, it acts as a catalyst in the formation of oxygen free radicals and if unregulated can disrupt cell membranes and cause tissue damage. We hyposthesise that iron accumulates in dermal cells in LDS and chronic iron overload causes cell damage and subsequent ulceration. The cellular distribution of iron in lower leg skin from patients with varying stages of venous disease was assessed by histological and ultrastructural analysis. In LDS skin, a variety of cell types, including inflammatory cells and fibroblasts contained iron. However, skin from patients with venous insufficiency but without skin changes and normal control skin showed no evidence of iron deposition. The in vitro effect of iron on the viability of dermal fibroblasts was assessed using colorimetric assays. Dermal fibroblasts subjected to iron with a carrier resulted in a decrease in cell viability whereas iron or carrier alone had no effect. This decrease was attenuated in the presence of the iron chelator, deferoxamine. These results suggest that excess iron accumulates in dermal fibroblasts in LDS and this may lead to a decrease in cell viability this study provides important information on mechanisms involved in iron induced tissue damage in venous disease.
This study was funded by Johnson and Johnson Medical

DISTINCT EXPRESSION OF THE NITRIC OXIDE SYNTHASE (NOS) AND ARGINASE PATHWAY DURING CUTANEOUS WOUND HEALING IN RATS
M.B. Witte, S. Wagner, H.D. Becker, Department of Surgery, University of Tübingen, Tübingen, Germany

Introduction: Previous work has demonstrated the importance of the nitric oxide synthase (NOS) pathway for wound healing. Arginine, a semi-essential amino acid, is the substrate for this pathway. Arginine can also be metabolised via arginase. Little is known about the function of this alternate pathway that is acting during healing. The present work investigates the temporal expression of these two pathways.
Methods: PVA sponges were implanted subcutaneously into male Lewis rats and harvested at different time points after wounding. The wound cell population was isolated and wound fluid harvested. Wound cells were analysed for arginase activity (nmol urea/min/mg cellular protein) and NOS activity (NO2 in cell culture supernatant/mg/protein). Arginase activity was also measured in wound fluid.
Results: Nitrite synthesis and arginase activity are maximal directly after wounding. Whereas nitrite synthesis fades rapidly thereafter until day 10, arginase activity remains elevated more sustained until day 21. Wound fluid arginase activity peaks around day 10, suggesting that inflammatory cells are the main source of extracellular arginase activity which is probably derived from cell death. Induction of the arginase pathway is solely due to upregulation of the arginase I isoform (AI) and not the AII isoform.
Conclusion: Distinct metabolism of L-arginine via arginase and NOS indicates a crucial role of both enzymes. The functional role of the arginase pathway for wound healing is currently investigated.
Supported by a grant from fortune of the University of Tübingen.