Meeting of the British Microcirculation Society

Bristol, April 7-8, 2003

Symposium on Recent Advances in Lymphatic Research

The Second Lymphatic Valve System

G. W. Schmid-Schonbein, J. Trzewik, E. Mendoza, F.A. DeLano

Department of Bioengineering, The Whitaker Institute for Biomedical Engineering, University of California San Diego, La Jolla, Calif., USA

The transport mechanism for fluid uptake from the tissue into lymphatics remains uncertain. Intra-luminal bi-leaflet valves prevent fluid reflux along lymphatic channels, but these are insufficient to provide unidirectional flow into initial lymphatics. Thus we present evidence that initial lymphatics have a separate (primary) valve system that permits fluid to enter from the interstitium but prevents escape back into the tissue. The transport of fluorescent microspheres across endothelium of initial lymphatics in cremaster muscle under general anaesthesia (40 mg/kg nembutal) was investigated. The results show that microspheres can readily pass from the interstitium across the endothelium into the lumen of the initial lymphatics, but do not leave the lumen even after elevation of the pressure. We propose a model for the primary valves at the level of lymphatic endothelial cells. The model consists of two overlapping endothelial extensions at junctions in the initial lymphatics but without interendothelial adhesion molecules. One cell extension is attached to the adjacent connective tissue while the other is free to bend into the lymphatic lumen when the lymphatic pressure falls below the adjacent interstitial fluid pressure. Thereby the cell junction opens a gap permitting entry of interstitial fluid into the lymphatic lumen. When the lymphatic fluid pressure rises above the adjacent interstitial fluid pressure, the endothelial extensions contact each other to close the junction. These results suggest that initial lymphatics have a primary valve system at the level of the endothelium. The primary valves in conjunction with the secondary (intralymphatic) valves provide the mechanism that facilitates the unidirectional flow in lymphatics.

Supported by NSF IBN 98 76379.

Critical Role of VEGF-C in Embryonic and Postnatal Lymphangiogenesis

M.J. Karkkainen(a), P. Haiko(a), J. Partanen(b), J. Taipale(a), C. Betsholtz(c), K. Alitalo(a)

a Molecular/Cancer Biology Laboratory and Ludwig Institute for Cancer Research, Haartman Institute and Helsinki University Hospital, Biomedicum Helsinki, University of Helsinki, Helsinki, Finland; b Biotechnology Institute, University of Helsinki, Helsinki, Finland; c Department of Medical Biochemistry, University of Goteborg, Goteborg, Sweden

Lymphatic vessels have an essential role in transporting tissue fluid, extravasated plasma proteins and cells back to the blood circulation. The lymphatic system also contributes to immunological defence mechanisms and to fat adsorption from the digestive tract. Abnormal function or growth of the lymphatic vessels is involved in various diseases such as tumour metastasis and lymphoedema. Vascular endothelial growth factor C (VEGF-C) mediates proliferation, migration and survival signals to the lymphatic endothelial cells by binding to two tyrosine kinase receptors, VEGFR-2 and VEGFR-3. However, the mechanisms regulating lymphangiogenesis are not fully understood. The targeted inactivation of the mouse VEGF-C gene appears to reveal some of the mechanisms involved.

Mechanisms of Lymphangiogenesis: Interplay between Biophysical and Biomolecular Regulators

M.A. Swartz

Chemical Engineering Department, McCormick School of Engineering and Applied Sciences, Northwestern University, Sheridan, Evanston, Ill., USA

Unlike angiogenesis, the driving forces and physiological processes that initiate and control lymphangiogenesis are mostly unknown. We hypothesize that the mechanical environment of the interstitium, which dictates the convective transport of proteins (e.g. proteases and growth factors), is a physiological regulator of lymphatic development just as hypoxia is a regulator of blood angiogenesis. We have shown in a new lymphangiogenesis model that interstitial flow mediates the formation of fluid channels along which lymphatic endothelial cells (LECs) migrate, proliferate, and then reorganise into a functionally optimal network of lymphatic capillaries. We present this original mechanism based on experiments using a novel in vivo model of lymphangiogenesis. In our model, a portion of tail skin is excised and replaced with a collagen gel (‘collagen dermal equivalent’, or CDE) to monitor lymphatic development over time. Lymph flow within the tail is purely unidirectional (distal to proximal) and a comparison of lymphatic growth between the distal and proximal ends thus reflects differences only in interstitial flow direction, since epithelial cell migration and angiogenesis occur from both ends equally. We observed lymphatic development within this collagen ‘window’ and saw that fluid channel formation occurs prior to lymphatic endothelial cell (LEC) migration and organisation, and that lymphatic migration, protease streaming and VEGF-C upregulation occurred primarily in the direction of fluid flow. This supports the hypothesis that interstitial fluid flow is an important regulator of lymphangiogenesis.

Proteomic Technologies to Study Lymphangiogenesis

L.V. Leak(a,b,c), E.F. Petricoin(b), L.A. Liotta(c), M. Jones(d)

a Department of Anatomy, Howard University, College of Medicine, Washington, D.C., USA; b Division of Therapeutic Products, FDA, Bethesda, Md., USA; c Laboratory of Pathology, NCI, NIH, Bethesda, Md., USA; d ardiovascular Branch, NHLBI, NIH, Bethesda, Md., USA

Little is known about protein expression changes in lymphatic endothelial cells (LEC) undergoing phenotypic changes during formation of new lymphatic vessels (lymphangiogenesis). The aim of this study was to determine differential protein expression profiles in LEC from quiescence through differentiation (lymphangiogenesis). LEC isolated from ovine mesenteric lymphatics were grown to confluence, stimulated to form tubular structures and processed for 2-D PAGE, immunoblotting and probed with antibodies to proteins of interest. Based on the results of image analysis of 2-D PAGE gels of LEC lysate, over 1,500 protein spots were seen in silver stained gels, and major changes in protein numbers viewed during proliferation and tube formation over that of the confluent quiescent monolayer cultures. Similarly, analysis of gels of conditioned medium revealed an increase in proteins secreted by proliferating LEC. Analysis of major protein spots of 40 kDa and pI of 5.3 with mass spectrometry (MS) and immunoblotting indicated that actin is downregulated as LEC change from quiescence to proliferation. However, as LECs undergo phenotypic modulations to form tubular structures, actin is upregulated. The analysis of protein gel spots by MS provides a means for the identification and characterisation of protein alterations taking place in association with morphological correlates. The application of this technology to identify and characterise the LEC proteome during various physiological modulations will provide valuable information on alterations of protein constituents during physiological and pathological states.

Tumour Lymphangiogenesis

M. Skobe

Derald H. Ruttenberg Cancer Center, Mount Sinai School of Medicine, New York, N.Y., USA

Lymphatic microvasculature is an important entry point for leukocytes and tumour cells. The earliest steps of tumour metastases involve tumour-cell interactions with the lymphatic capillary endothelium, but the nature of these interactions is unclear. Using an orthotopic breast cancer model in immunosuppressed mice, we showed lymphatics surrounding and within experimental tumours and that overexpression of the lymphangiogenesis factor VEGF-C resulted in enhanced infiltration of tumours by lymphatics. Moreover, VEGF-C- induced increase of tumour lymphangiogenesis resulted in enhanced breast cancer metastasis to regional lymph nodes and lungs. In human melanoma xenotransplants, moreover, VEGF-C also induced angiogenesis and recruitment of macrophages. This could be explained by different proteolytic processing of VEGF-C by these tumours, indicating that in vivo processing plays a key role in determining biological function of VEGF-C. To explore mechanisms of tumour cell interactions with lymphatic endothelium in vitro, we isolated primary lymphatic and microvascular endothelial cells from human skin. The two cell lineages express distinct sets of vascular markers, respond differently to growth factors and extracellular matrix and interact in a specific way with tumour cells. Comparative microarray analysis revealed unique gene expression profiles that distinguish lymphatic and blood vascular endothelium. The molecular profile of lymphatic endothelium reflects characteristic functional and structural features of lymphatic capillaries and suggests that lymphatic endothelium plays an active role in trafficking of macromolecules and cells.

Biology, Structure and Regulation of the Lymphatic Endothelial Hyaluronan Receptor LYVE-1

D.G. Jackson

MRC Human Immunology Unit, Weatherall Institute of Molecular Medicine, John Radcliffe Hospital, Headington, Oxford, UK

It is currently unclear how leukocytes and tumour cells gain access or traffic through the lymphatic network to regional lymph nodes. Clearly, an understanding of these p\rocesses will be fundamental to the development of new therapies for inflammation and metastatic cancer. One of the main impediments to such research has been the lack of specific markers to distinguish lymphatic vessels or to isolate lymphatic endothelial cells for experimental manipulation. In recent years however, we and other groups have identified new markers of lymphatic endothelium, and one of these, the hyaluronan receptor LYVE-1, has proved useful in studies of tumour lymphangiogenesis and tumour metastasis. Here we outline the characteristics of LYVE-1, including studies into its 3D structure, regulation and likely physiological function. In addition, we describe the application of LYVE-1 antibodies to study lymphangiogenesis in human cancers and to isolate primary lymphatic endothelial cells for detailed molecular analyses.

Lymphatic Pathophysiology – New Insights

P.S. Mortimer

Division of Physiological Medicine (Dermatology), St George’s Hospital Medical School, London, UK

Lymphoedema is swelling due to excess accumulation of fluid in the tissues caused by inadequate lymph drainage. Lymph drainage may be reduced because of (i) congenital or acquired reduction in the number of lymphatic vessels, (ii) increased resistance to lymph flow, (iii) lymph reflux, or (iv) vessel contractile dysfunction. Recent discoveries in the genetics of hereditary lymphoedema have improved our knowledge of the phenotypic abnormalities. Vascular endothelial growth factor receptor 3 (VEGFR-3) signalling is essential for formation and maintenance of initial lymphatic vessels. In Milroy’s disease, missense mutations in VEGFR-3 manifest at birth with below knee swelling due to hypoplasia of peripheral lymphatics (Karkkainen et al.: Nature Genet 2000;25:153-159). In lymphoedemadistichiasis (LD), for which the gene is also known, peripheral leg lymphatics are normal or even increased in number. Lymph reflux is likely and the frequent co-existence of venous reflux in LD may relate to the common origins of veins and lymphatics (Brice et al.: J Med Genet 2002;39:478-483). Arm lymphoedema is acquired following breast cancer treatment involving axillary lymph node removal and targeting of lymph drainage routes by radiotherapy. Recent work indicates that the traditional view of lymphatic obstruction to the whole arm is unlikely and that regional differences in lymph flow contribute to variable distribution of swelling e.g. sparing of the hand (Stanton et al.: Clin Sci 2001;100:131-140). Recent observations of cutaneous angiogenesis and possibly lymphangiogenesis in the forearm (Mellor et al.: J Vasc Res 2000;37:501-512 and Microcirc 2002;9:207-219) will stimulate further research.

Oral Communications

In situ Measurements of Ca^sup 2+^ Signals in Arteriolar Smooth Muscle Cells and Endothelial Cells

T. Burdyga(a), A. Shmygol(a), D.A. Eisner(b), S. Wray(a)

a The Physiological Laboratory, University of Liverpool, UK; b Unit of Cardiac Physiology, University of Manchester, Manchester, UK

In the present study we report the first local and global Ca^sup 2+^ measurements made from in situ terminal arterioles (diameter ~8- 40 [mu]m) from the rat ureter muscle strips using Ca^sup 2+^ indicator Fluo-4 and wide-field fast speed confocal imaging. The smooth muscle cells were arranged circumferentially and could make up to two full turns of the vessel. We find the terminal arterioles to be extremely active, both spontaneously and in response to noradrenaline stimulation. Under resting conditions, endothelial cells produced a wide range of Ca^sup 2+^ signals – from local events (Ca^sup 2+^ puffs and Ca^sup 2+^ spikes) to global events (abortive and full size Ca^sup 2+^ waves, which could pass from cell to cell), whereas the vascular myocytes showed the presence of Ca^sup 2+^ sparks discharged by at least one discharging site. Global events in vascular myocytes could be induced by either caffeine (10 mM) or noradrenaline ( 10 [mu]M). Caffeine-induced Ca^sup 2+^ waves were preceded with an increase in the frequency of Ca^sup 2+^ sparks and only one Ca^sup 2+^ wave per cell could be generated. In contrast, agonist-induced Ca^sup 2+^ waves were preceded by a slow rise of intracellular Ca^sup 2+^, which, after reaching a threshold, triggered a spreading Ca^sup 2+^ wave. Several Ca^sup 2+^ waves per cell could be generated by vascular myocytes in the presence of an agonist. Ca^sup 2+^ waves appeared asynchronously and produced contraction of individual smooth muscle cells. We conclude that under resting conditions endothelial cells in the terminal arterioles show much higher levels of spontaneous calcium activity with large variation in spatial and temporal characteristics in comparison with smooth muscle cells.

Localisation of Intermediate-Conductance Calcium-Activated K+ (IK) Channels: Relevance to EDHF

J.M. Hinton, P.D. Langton

Department of Physiology, University of Bristol, Bristol, UK

We sought to identify the K+ channel underlying the charybdotoxin (ChTX) sensitive component of endothelium-dependent hyperpolarisation (EDHF). Endothelial cells release a factor that hyperpolarizes the underlying smooth muscle, producing vasodilatation. The identity of this EDHF. which is neither nitric oxide nor prostacyclin, remains controversial. However, it is well established that EDHF-mediated responses can be abolished by a combination of the toxins apamin and ChTX, but not by either toxin alone. Male Wistar rats were sacrificed by stunning and cervical dislocation. Relaxation of third order mesenteric arteries elicited by acetylcholine (0.0110 [mu]M) was abolished by the toxin apamin (50 nM) in combination with each of the potent, selective IK inhibitors TRAM-39 (50 nM; p < 0.05; n = 6) and TRAM-34 (50 nM; p < 0.05; n = 6). Using an anti-IK antibody we performed immunoconfocal microscopy of transverse sections of arteries. Immunoreactivity was observed in the endothelial cell layer only. Western blotting of arterial homogenates confirmed the presence of a band at the predicted molecular weight for the IK channel, ~63 kDa. Herein, we demonstrate that in mesenteric resistance arteries, EDHF mediated relaxation and hyperpolarisation is, in part, mediated by an IK channel located on endothelium, but not smooth muscle. As the contribution of EDHF to endothelium-dependent relaxation increases as the vessel size decreases, these data suggest that endothelial cell IK channels play a pivotal role in the regulation of local blood flow within the resistance vasculature and in systemic blood pressure.

Supported by the BHF.

Impact of Six Months Hormone Replacement Therapy on Microvascular Function in Healthy and Type 2 Diabetic Postmenopausal Women

K.M. Gooding, G. Spyer, K.M. MacLeod, A.C. Shore

Institute of Biomedical and Clinical Science, Peninsula Medical School, Exeter, UK

The effect of hormone replacement therapy (HRT) on cardiovascular risk and the vascular system in women is currently very contentious. Its impact on the macrocirculation has previously been explored, however little data is available on its impact on the microcirculation. Thus this study aimed to determine the impact of HRT on microvascular function in healthy and type 2 diabetic postmenopausal women (n = 20 and 17, respectively). Microvascular function was assessed by skin maximum hyperaemia, skin hyperaemic response to iontophoretically applied acetylcholine (ACh) (endothelial-dependent vasodilator) and sodium nitroprusside (SNP) (endothelial-independent vasodilator), finger nailfold capillary pressure and the microvascular filtration coefficient. Microvascular assessments were repeated following 6 months oral HRT (combined HRT or unopposed oestradiol for hysterectomised women). Following 6 months HRT there were no significant changes in microvascular assessments in healthy women. In diabetic women there was a reduction in the skin hyperaemic response to ACh (median peak ACH response: 1.95 (25th, 75th centiles: 1.54, 2.30)V vs. post 1.53 (1.30. 1.91)V) (p = 0.011, Wilcoxon signed rank test) and SNP (median peak SNP response: 1.59 (1.37, 1.99) versus 1.35 (0.92, 1.63)V) (p = 0.011) with HRT, but no other changes. Thus suggesting that HRT does not affect microvascular function in healthy women, and adversely affects it in diabetic women. These findings may help to explain why HRT does not confer the predicted cardiovascular protection.

Store-Operated Calcium Influx Is Attenuated in Rat Retinal Arterioles with Persistent Diabetes

T.M. Curtis(a), C.N. Scholfield(b)

a Department of Ophthalmology and Vision Science, Queen’s University of Belfast, Royal Victoria Hospital, Belfast, UK; b Smooth Muscle Group, Belfast, UK

We have previously shown that diabetes inhibits store-operated Ca^sup 2+^ influx in rat retinal microvascular smooth muscle cells through the increased activation of protein kinase-C (PKC) (Curtis TM et al.: Pflugers Arch 2002;443S:S196). As adjunct to this work, in the present study we have examined the time-course over which this pathology develops. Cytosolic Ca^sup 2+^ was estimated in freshly isolated rat retinal arterioles from streptozotocin-induced diabetic animals of 3- and 1-month disease duration and non- diabetic animals using fura-2 microfluorimetry. Store-operated Ca^sup 2+^ influx was assessed by depleting [Ca^sup 2+^]^sub i^ stores with Ca^sup 2+^-free medium/5 [mu]M cyclopiazonic acid over 10 min and subsequently measuring the rate of rise in Ca^sup 2+^ on adding 2 mM or 10 mM Ca^sup 2+^. Store-operated Ca^sup 2+^ influx was attenuated in microvessels after 3 months of diabetes. The reduced Ca^sup 2+^ entry was reversed by 2-hour exposure to staurosporine and mimicked in microvessels from non-diabetic animals by 10-min exposure to the PKC activator phorbol myristate acetate. The effect was absent in retinal microvessels from non-diabetic animals incubated for 6 h in 25 mM D-glucose and in microvessels from animals after only 1 month o\f diabetes. These results demonstrate that the inhibition of store-operated Ca^sup 2+^ influx in retinal microvessels from diabetic animals is slow in onset. They also contradict the dogma that hyperglycaemia can account for the pathological activation of PKCs in diabetes by rapid de novo synthesis of diacylglycerols.

Identification of a Blood-Brain Barrier Cell-Cell Adhesion Molecule

P. Clark, M.J. Williams, J.A. Firth

Division of Biomedical Sciences, Imperial College London, London, UK

Cadherins are a family of transmembrane proteins known to mediate homotypic cell-cell adhesion at adherens junctions of epithelial and endothelial cells, and are believed to be responsible for organising other intercellular junctions, including tight junctions. Although the endothelia of blood-brain barrier (BBB) microvessels form more elaborate, tighter junctions than in non-brain vessels, few qualitative differences in their composition have been described. We cloned a fragment of cadherin-10 in a screen for cadherins in human placental endothelia. Previous studies have shown that, in humans, cadherin-10 mRNA is predominantly expressed in brain, but its localisation and function have not been described (Kools et al.: FEBS Lett 1999;452:328-334). By PCR, we find that cadherin-10 is strongly expressed in adult mouse brain, a panel of other tissues giving a weak or negative signal. We raised antisera to cadherin-10 and have immunostained both human and mouse brain. Cadherin-10 staining is restricted to cerebral microvessels, pial vessels and to the ependyma and tanycytes of some ventricular linings, but not in larger brain arteries or the capillaries of the choroid plexus. In brain capillaries, cadherin-10 co-localised with endothelial markers, PECAM-1 Ulex lectin, and the BBB marker, GLUT-1, suggesting that this cadherin is an endothelial junction molecule specific to BBB, that could confer brain endothelial junctions with their unique properties.

This work is supported by The Wellcome Trust.

Histamine Induces Changes in Cardiac Capillary Endothelial Cells in situ, Which Are Similar to Morphometric Changes Observed following Ischaemia

M.C. Glyn(a), J.G. Lawrenson(b), B.J. Ward(a)

a William Harvey Research Institute, Biomedical Sciences, Queen Mary and Westfield College, University of London, UK; a Department of Optometry and Visual Science, City University, London, UK

Vasoactive agents such as histamine and thrombin have been shown to increase vascular permeability in vivo. Increased vascular permeability, in vitro, correlates with changes in the endothelial cell cytoskeleton, for example, an increase in stress fibre formation, and cell contraction mediated by the actomyosin contractile system. Previously, we demonstrated that myocardial ischaemia alters capillary dimensions and endothelial cell shape which are likely to be effected by the actomyosin contractile system. We observed in morphometric studies, that phalloidin and, separately, inhibition of a regulator of stress fibre assembly. Rho kinase, prevent changes in capillary dimensions on reperfusion, demonstrating that reperfusion-induced changes similarly engage the actomyosin contractile system. Here, in Langendorff perfused rat hearts, we demonstrate that histamine (10^sup -4^ M for 45 min) induces a reduction in capillary calibre simultaneously with changes in other endothelial cell dimensions, in situ. While the cross- sectional areas of endothelial cells were not altered by histamine, whole capillary and luminal cross-sectional areas and abluminal and luminal perimeters were significantly reduced compared with those in myocardium perfused without histamine. These changes were similar to those observed following myocardial ischaemia and provide further evidence supporting active endothelial cell contraction in capillaries mediated by the actomyosin contractile system.

Combretastatin A-4-Phosphate (CA-4-P) Activates NF[kappa]B in Human Endothelial Cells

A.C. Brooks, C.S. Parkins, C. Kanthou, G.M. Tozer

Tumour Microcirculation Group, Gray Cancer Institute, Northwood, Middlesex, UK

The tumour vascular targeting agent CA-4-P induces endothelial cellular adhesion molecule (CAM) expression, and subsequent neutrophil adhesion. CAM expression is dependent upon the activation of transcription factors, including NF[kappa]B, activated by Rho GTPases. We recently demonstrated Rho GTPase activation by CA-4-P in human endothelial cells (Kanthou and Tozer: Blood 2002;99:2060- 2069). The aim of this study was to investigate CA-4-P-induced NF[kappa]B activation. Nuclear fractions were obtained from HUVEC incubated with TNF-[alpha] (100 U/ml), or CA-4-P (0.001-0.1 [mu]M). To determine the role of Rho in CA-4-P-induced NF[kappa]B activation, cells were pre-treated with the RhoA inhibitor, C3 exoenzyme, or the Rho kinase inhibitors Y27632 and HA1077. NF[kappa]B-DNA interactions were examined using an EMSA. and NF[kappa]B components identified using supershift assays. Following treatment with TNF-[alpha] or CA-4-P, for 1 h, two densely labelled bands were observed on the EMSA blot corresponding to NF[kappa]B- DNA complexes. These were inhibited by excess unlabeled NF[kappa]B probe, whilst supershift assays revealed NF[kappa]B in the form of a p50[middot]p65 heterodimer. Pre-incubation of cells with C3 exoenzyme prior to CA-4-P exposure, prevented formation of NF[kappa]B-DNA complex bands, while Y27632 or HA1077 were ineffective. These data suggest that CA-4-P activates NF[kappa]B via mechanisms involving Rho, but not Rho kinase. This pathway is likely to be important for expression of CAM following vascular-targeted therapy.

The authors would like to thank Cancer Research UK for funding and OXiGENE Europe AB for supply of CA-4-P.

The Rate of Increase, Rather than the Concentration ([Ca^sup 2+^]^sub c^), of Cytosolic Ca^sup 2+^ Regulates Hydraulic Conductivity (Lp) in vivo

C.A. Glass, T.M. Pocock, D.O. Bates

Microvascular Research Laboratories, Department of Physiology, University of Bristol, UK

L^sub P^ increases when [Ca^sup 2+^]^sub c^ increases (He et al.: AJP 1993;265: H1019) and it is assumed that the [Ca^sup 2+^]^sub c^ itself is responsible for the L^sub p^ increase. L^sub P^ often reaches its maximum value before [Ca^sup 2+^]^sub c^ does, however, so to tease out the rate from the concentration increase we have compared time courses for L^sub P^ and Ca^sup 2+^ responses in vessels where Ca^sup 2+^ enters by store-mediated Ca^sup 2+^ entry or is released from stores. Vessels were treated with a Ca^sup 2+^ influx blocker, SK&F96365 (SKF, 100 [mu]M), a sarco-endoplasmic reticulum Ca^sup 2+^ ATPase inhibitor, thapsigargin (TG, 100 nM) and a Ca^sup 2+^ ionophore, ionomycin (IM, 5 [mu]M). Single mesenteric microvessels of MS222-anaesthetised frogs were perfused with SKF/TG/ IM for 20 min and L^sub P^ measured using the Landis-Michel method (Michel et al.: QJEPCMS 1974;59: 283) or [Ca^sup 2+^]^sub c^ as described by He et al. The mean + or – SE time (min) taken for the L^sub p^ to peak (3.6 + or – 0.9. n = 10) was significantly less than the time for the [Ca^sup 2+^]^sub c^ to peak (9.4 + or – 2.8, n = 8, p < 0.05, unpaired t-test) but not significantly different to the time to the maximum rate of increase of [Ca^sup 2+^]^sub c^ (4.4 + or - 2.0, n = 8, p > 0.05). Similarly when 30 [mu]M ATP was perfused, the time to the maximum L^sub p^ was 1.2 + or – 0.7 (n = 12) compared with a time to maximum [Ca^sup 2+^]^sub c^ of 2.1 + or – 0.7 (n = 6, p < 0.05) and rate of increase of [Ca^sup 2+^]^sub c^ of 1.1 + or - 0.4 (n = 6, p > 0.05). These data suggest that the rate of rise of [Ca^sup 2+^]^sub c^ causes the increase in L^sub P^ in vivo and not the absolute [Ca^sup 2+^]^sub c^.

Supported by BHF 2000 BB03, PG30, SS57.

The Effect of Type 1 Diabetes on the Endothelial Barrier of the Human Placenta

C. Gray, Y. Littler, L. Leach

School of Biomedical Sciences, University of Nottingham, UK

The aim of this study was to investigate whether the junctional integrity of human placental vessels was compromised in pregnancies complicated by type 1 diabetes. Molecular organisation of adherens junctional molecules, leakage of 76-kDa TRITC-Dextran, and vascular endothelial growth factor (VEGF) levels were analysed in perfused human term placenta from normal pregnancies (n = 4) and pregnancies complicated with type 1 diabetes (n = 4). Using systematic random sampling and confocal microscopy, placental vessels from the diabetic group were found to be highly permeable to 76-kDa TRITC- Dextran; there was a significant increase in number of hot-spots in the abluminal side of blood vessels. There were statistically significant decreases in the percentage of vascular profiles showing immunolocalisation of vascular endothelial cadherin (from 92 to 44%) and [beta]-catenin (from 88 to 39%) in type 1 placentae compared to normal. The former also showed a significant increase (from 8 to 28%) in the percentage of vessels with junctional phospho-[beta]- catenin. Increased levels of VEGF were seen in these placentae, VEGF being present in trophoblast and vascular layers. The results suggest that the endothelial barrier of placentae from pregnancies complicated by type 1 diabetes mellitus is compromised. The observed leakage may be due to phosphorylation and loss of junctional molecules influenced by increased levels of placental VEGF.

Vascular Endothelial Growth Factor (VEGF) Induces Vacuoles in Vessel Walls when Microvascular Pressure (Pc) Is Low

C.R. Neal, D.O. Bates

Microvascular Research Laboratories, Department of Physiology, University of Bristol, Bristol, UK

Serial section reconstruction of the walls of microvessels perfused with VEGF solutions has shown that the ultrastructure changes differ in frog and rat, although the increase in hydraulic conductivity (L^sub p^) is similar (Neal et al.: J Physiol 1998;506P:24). In the frog, transcellular openings through the endothelial cell\s occurred with the transient increase in L^sub p^, however, in rats the route through the endothelial cells was vacuolar. In a recent set of experiments on the mesenteric capillaries of pithed frogs we showed that VEGF increased L^sub p^ 3- fold when Pc was 30 cm H2O but 10-fold when Pc was 15 cm H2O (Neal and Bates: J Physiol 2001;536P:114). We now describe the morphology associated with these permeability increases. We have used a combined light and electron microscopy technique where large numbers (> 1,000) of ultrathin (100 nm) serial sections were rapidly surveyed in the light microscope prior to transmission electron microscopy (TEM) of selected sections. When Pc was 15 cm H2O, abundant vacuoles (46) were apparent in clusters along the length of 4/5 vessels (80 [mu]m) (vacuolar size range 0.2-3.3 [mu]m diameter). No endothelial gaps were found along 138 [mu]m of vessel walls. In the fourth vessel, few vacuoles were found along with 2-4 small endothelial gaps in a 28-[mu]m section of vessel wall. When Pc was 30 cm H2O, 2 vessels showed only few vacuoles and no endothelial gaps in 132 [mu]m of vessel length. We have yet to establish the percentage of these vacuoles that act as channels.

Funded by The Wellcome Trust (58083) and BHF (BB2000003).

Effects of Nitric Oxide on in vivo Models of Angiogenesis in Mouse Skeletal Muscle

J. Williams, O. Baum, A. Zakrzewicz, S. Egginton

Angiogenesis Research Group, Department of Physiology, The Medical School, University of Birmingham, Birmingham, UK

The effects of inhibiting nitric oxide production were studied in two different models of angiogenesis in mouse skeletal muscle: (1) increased shear stress caused by oral administration of prazosin (50 mg[middot]l^sup -1^ in drinking water) for up to 14 days; (2) muscle overload for up to 14 days (extirpation of a synergistic muscle under hypnorm/hypnovel anaesthesia). Inhibition of nitric oxide was achieved either pharmacologically, through co-administration of N(omega)-Nitro-L-Arginine (L-NNA; 100 mg[middot]l^sup -1^ in drinking water), or by using genetic knockouts of nitric oxide synthase (NOS) isoforms, eNOS or nNOS (on a C57/B16 background). Increases in capillary density and capillary:fibre ratio (determined from GSL-1 stained frozen sections) of 25-30% in m. extensor digitorum longus seen in response to prazosin administration were not seen with co-administration of L-NNA or in the eNOS knockout, but were seen in the nNOS knockout mice. Capillarity was increased in overloaded muscles in all situations tested, regardless of nitric oxide inhibition. This shows that angiogenesis in these two models can proceed via two distinct pathways, one of which is dependent on nitric oxide production, specifically from eNOS, the other of which is nitric oxide independent.

Supported by the British Heart Foundation.

Retinal Angiogenesis in vitro by Serum from Patients with Pre- Proliferative Diabetic Retinopathy Is Mediated by Advanced Glycation Endproducts (AGEs)

A.W. Stitt, C. McGoldrick, D. McCance, T.A. Gardiner

Ophthalmology and Vision Science, Queen’s University of Belfast, Royal Victoria Hospital, Belfast, UK

The current study has determined the angiogenic potential of retinal microvascular endothelial cells grown in serum from diabetic patients at either the vasogenerative or vasoproliferative stage of diabetic retinopathy (DR). A modulatory role for serum-derived proteins that had been modified by advanced glycation was also studied. A novel in vitro model of retinal angiogenesis was developed which incorporates key phenomena of cell proliferation, migration, tube formation and invasion. Invasion of pre-formed retinal vascular tubes into a secondary matrix was quantified after exposure to non-diabetic serum or serum from diabetic patients. Using this system the angiogenic potential of serum from patients classified as well-controlled (WC), as assessed by the HbA1c test, was compared with that from a poorly-controlled (PC) group. Serum from non-diabetic patients showed significantly greater angiogenic potential than diabetic serum (p < 0.0001). while that from the WC diabetic group was significantly pro-angiogenic relative to the PC group. AGE-modified albumin caused a dose-dependent inhibition of angiogenesis (p < 0.001) when compared to native albumin controls. The data suggest a significant suppression of angiogenesis by retinal microvascular endothelial cells during diabetes. DR may have a significant anti-angiogenic aetiology, an effect mediated, at least in part, by serum-derived AGEs. This may have implications for retinal microvascular repair during vaso-degeneration.

Evidence for Prostaglandin-Mediated Coupling of Endothelial Cell and Osteoblast Behaviour

C.E. Clarkin, A.A. Pitsillides, C.P.D. Wheeler-Jones

Department of Veterinary Basic Sciences, Royal Veterinary College, London, UK

Osteoblasts (bone-forming cells) produce angiogenic factors in response to pro-osteogenic stimuli, including prostaglandin E^sub 2^ (PGE^sub 2^). We have evaluated the potential roles of vascular endothelial growth factor (VEGF) and PGE^sub 2^ as autocrine/ paracrine signalling molecules in bone by investigating their effects on primary osteoblasts (OBs) and human umbilical vein endothelial cells (HUVECs). In ECs VEGF^sub 165^ induced early and robust ERK1/2 activation, late COX-2 protein induction and the release of prostanoids (PGI^sub 2^ and PGE^sub 2^). In contrast, VEGF-treated human and rat-derived OBs showed increased prostanoid release in the absence of ERK1/2 activation or COX-2 induction. While VEGF is strongly mitogenic for ECs, exogenous VEGF^sub 165^ had little effect on OB proliferation, a finding consistent with an apparent lack of VEGFR2 (Flk-1) protein expression by immunoblotting. This differential sensitivity was not apparent with PGE^sub 2^; thus, PGE^sub 2^-treated HUVEC and OBs both exhibited enhanced ERK1/2 phosphorylation, COX-2 induction, and PGI^sub 2^ synthesis. Preliminary studies using a VEGFR tyrosine kinase inhibitor showed complete blockade of VEGF-mediated responses and partial inhibition of PGE^sub 2^-induced ERK1/2 activation, COX-2 induction and PGI^sub 2^ release. These results suggest that: (i) in bone, VEGF preferentially targets endothelial cells; (ii) since PGE^sub 2^ stimulates OB VEGF production, OB-derived VEGF exerts a selective paracrine action on ECs, ensuring continued PGE^sub 2^ production and coupling of OB and EC behaviour, and (iii) PGE^sub 2^ may signal through VEGF receptors in endothelial cells.

Exogenous Migration Stimulating Factor Stimulates Angiogenesis and Fibroblast Migration in vivo

S.P. Bennett(a), R. Baillie(a), G.D. Griffiths(a), G.P. Leese(a), S.L. Schor(a), C. Green(b), A.M. Schor(a)

a Unit of Cell and Molecular Biology, The Dental School, University of Dundee, Dundee, UK; b Northwick Park Institute for Medical Research, Northwick, Middlesex, UK

Migration Stimulating Factor (MSF), an isoform of fibronectin produced by fetal fibroblasts and also found in adult surgical wound fluid, has been shown to stimulate fibroblast migration in vitro and angiogenesis in the chick chorioallantoic membrane model. This study examines the effect of exogenous MSF on cell migration and angiogenesis in vivo. Implants of human collagen (Alloderm) soaked in either a concentration of MSF (1 g/ml, 100 ng/ml and 10 ng/ml) or PBS (control) were implanted subcutaneously in rats. Implants and surrounding tissue were removed after 3 or 7 days. Fibroblast counts were determined semi-quantitatively with light microscopy by two independent observers and graded (0-3). Vascularity was assessed by estimating microvascular volume using stereological point counting on sections stained for von Willebrand Factor. The number of fibroblasts in the implant and in the surrounding tissue was greater in all the MSF groups than the control group at day 3. There was an increase in vascularity in all the MSF-soaked implants compared with controls at day 3 (p < 0.05) with no evidence of a dose response. By day 7, cell and vessel numbers were similar. MSF stimulates early fibroblast migration and angiogenesis in this rat wound healing model. MSF may have a therapeutic role early in wound healing before endogenous regulatory processes stimulate fibroblast migration and angiogenesis.

VEGF^sub 165^b, a Novel Splice Variant of VEGF, Is Not Angiogenic in vivo in the Rabbit Cornea

J. Woolard(a), L. Morbidelli(b), M. Ziche(b), S.J. Harper(a), D.O. Bates(a)

a Microvascular Research Laboratories, Department of Physiology, University of Bristol, Bristol, UK; b Department of Molecular Biology, University of Siena, Siena, Italy

Vascular endothelial growth factor (VEGF) is a potent angiogenic factor in vivo. VEGF^sub 165^b, a splice variant of VEGF, dose dependently inhibits VEGF^sub 165^-mediated endothelial proliferation and migration, and vasodilatation (Bates et al.: Cancer Res 2002;62:4123-4131). To determine if VEGF^sub 165^b stimulates blood vessel growth in vivo, we measured the effects of VEGF^sub 165^b and VEGF^sub 165^ on angiogenesis in the rabbit cornea. Stable MCF-7 cell lines were generated expressing VEGF^sub 165^, VEGF^sub 165^b, or empty expression vector (pcDNA^sub 3^) that expressed 17.8, 4.9 and 0.08 fg VEGF/cell/hour, respectively. For each cell line 2.5 x 10^sup 5^ cells were implanted into a micro- pocket incised into the cornea of female New Zealand albino rabbits (n = 4) under local anaesthesia. Animals were observed daily and angiogenic responses measured according to the product of vessel density and distance from the limbus, as previously described (Gimbrone et al.: JNCI 1974;52:413-427). Five days after implantation, angiogenic scores for VEGF^sub 165^, VEGF^sub 165^b or pcDNA^sub 3^ were 1.0 + or – 0.3, 0.12 + or – 0.06 and 0.3 + or – 0.1. respectively. At day 10 post-implantation, this increased to 2.18 + or – 0.2, 0.25 + or – 0.2 and 0.43 + or – 0.1, respectively\. Responses to VEGF^sub 165^ were significantly greater than VEGF^sub 165^b or pcDNA^sub 3^ (p < 0.01, two-way ANOVA). VEGF^sub 165^b- transfected cells caused no significant angiogenesis, despite producing 4 times more VEGF- i.e. VEGF^sub 165^b is not angiogenic in vivo. These findings suggest a role for VEGF^sub 165^b as a potential anti-angiogenic factor.

The Role of Advanced Glycation End-Products (AGEs) in Pericyte Dysfunction during Diabetic Retinopathy

S. Hughes, T. Curtis, T.A. Gardiner, A. W. Stitt

Ophthalmology and Vision Science, Queen’s University of Belfast, Royal Victoria Hospital, Belfast, UK

Advanced glycation end-products (AGEs) have been implicated in the pathogenesis of diabetic retinopathy (DR) and it is evident that there maybe important links between AGE-crosslinking of basement membrane (BM) and premature pericyte dysfunction in the retinal microvasculature. The objective of this study was to determine if AGE-crosslinking of BM components could alter retinal pericyte function. Bovine retinal pericytes (BRPs) were exposed to control or AGE-modificd BM (Matrigel) over time. Cell attachment, spreading and death to these substrates were quantified. BRP attachment was also assessed in the presence of peptides (5 mM) such as the RGD and YIGSR which sought to replace AGE-modified cell-recognition motifs in the underlying BM. Thin filaments was localised with fluorescently-tagged phalloidin and visualised using confocal microscopy, while endothelin-1 (ET-1, 10 mM) induced rises in intracellular calcium were assessed microflurometrically with the dye Fura-2AM. BRPs exposed to AGE-BM showed reduced attachment and spreading and increased levels of apoptosis. There was a concomitant alteration in microfilament configuration with the cells. The cells also demonstrated attenuations in intracellular Ca^sup 2+^ following ET-1 stimulation. Supplementing AGE-BM with YIGSR peptide enhanced cell attachment, whereas RDG was slightly inhibitory. In conclusion, AGE-modified BM can induce a range of pathophysiological responses in BRPs in vitro. These findings may have implications for the pathogenic role of AGEs in retinal microvascular dysfunction and death during DR.

The Impaired Response of Endothelial Cells to an Ischaemic Injury in Habitual Smoking Adults

M.A. Moloney, J.J. Kelly, C. Condron, G. Roche-Nagle, F.M. Fennessy, D. Bouchier-Hayes

Department of Surgery, R.C.S.I., Beaumont Hospital, Dublin, Ireland

We previously reported that a brief ischaemia reperfusion insult results in shedding of endothelial cells from the vascular bed and that this provokes a mobilisation of progenitor endothelial cells (EPC) for repair. We hypothesise that in cigarette smokers the magnitude of resulting injury is increased and the reparative response impaired. We tested this hypothesis in a clinical model of short-term ischaemia by characterising the response of circulating endothelial cells (CECs) to 10 min of upper limb ischaemia in matched groups of non-smoking and healthy smoking young males. A blood pressure cuff induced an ischaemia-reperfusion (I-R) injury’ to the upper limb, blood samples taken pre and post insult (ipsi- and contralateral upper limbs). Endothelial cell populations were determined by flow cytometry using CD45, CD133 and CD145.

A greater number of CECs were shed into the circulation following insult in the smokers compared to the controls, but a decreased number of EPC were observed. In conclusion, the magnitude of injury and efficacy of repair was significantly different in the smoking group. This has implications for cardiovascular function, disease and therapeutic interventions.

Induction of Heme Oxygenase-1 (HO-1) by Moderately Oxidised LDL in Vascular Smooth Muscle Cells

F.Y.L. Li(a), A. Anwar(a), A. Rupani(a), D.S. Leake(b), R.C.M. Siow(a)

a Centre for Cardiovascular Biology and Medicine, King’s College, Guy’s Campus, London, UK; b School of Animal and Microbial Sciences, University of Reading, Whiteknights, Reading, UK

Oxidatively modified low-density lipoproteins (LDL) play a central role in atherogenesis (Steinberg: Nat Med 2002;8:1211- 1217). Vitamin C (vit-C) attenuates oxidised LDL induced apoptosis in vascular smooth muscle cells (SMC; Siow et al.: Arter Thromb Vase Biol 1999;19:2387-2394). The antioxidant stress protein HO-1 catabolises heme to generate the vasodilator carbon monoxide and antioxidants biliverdin and bilirubin, and is induced by highly oxidised LDL (oxLDL) in SMC (Siow et al.: Cardiovasc Res 1999;41:385- 394). We have investigated whether vit-C modulates HO-1 induction in SMC treated with moderately oxidized LDL (mLDL), a species containing higher levels of lipid hydroperoxides. Human aortic SMC were pretreated with vit-C (24 h, 100 [mu]M) before treatment in the absence of vit-C with diethylmaleate (DEM, 100 [mu]M an electrophilic agent), human native LDL (nLDL), mLDL or oxLDL (0-300 [mu]g/ml) for 24 h. Western analysis revealed that mLDL, oxLDL and DEM induced HO-1 expression in a dose-dependent manner. HO-1 was not detected in control or nLDL treated SMC, but mLDL enhanced HO-1 expression to a significantly greater extent than oxLDL. vit-C significantly attenuated DEM, mLDL and oxLDL mediated HO-1 induction. These findings show that HO-1 expression can be differentially enhanced by mLDL and oxLDL and that vit-C affords protection against the more atherogenic mLDL species.

Support from Wellcome Trust and Guy’s and St. Thomas’ Charitable Foundation.

Combretastatin A-4-Phosphate Causes a Rapid Increase in Tumour Vascular Permeability: As Detected by Intravital Multiphoton Fluorescence Microscopy

I. Wilson, S. Ameer-Beg, R. Hodgkiss, P. Barber, B. Vojnovic, G. Tozer

Gray Cancer Institute, Mount Vernon Hospital, Northwood, UK

Combretastatin A-4-Phosphate (CA-4-P) is a tubulin binding agent, which depolymerises the tubulin cytoskeleton, increases endothelial cell permeability in vitro and selectively shuts down tumour blood flow in vivo. The aim of this study was to develop a method of measuring vascular permeability in vivo and determine the effect of CA-4-P. P22 rat sarcomas were grown in dorsal skin-flap window chambers implanted into BD9 rats. Animals were anaesthetised (Hypnorm/midazolam), positioned on the microscope and body temperature maintained. Following i.v. injection of FITC-dextran/ albumin a time series was taken of the fluorescence intensity and lifetime. The plasma clearance constant (k^sub i^) of the fluorophore was calculated. For high-molecular-weight compounds, which show little backflux into the vasculature, k^sub i^ is closely related to the permeability-surface area product. Fluorescence lifetime imaging revealed that FITC-albumin had significantly different lifetimes between intra- and extravascular compartments indicating unquenching due to molecular changes or breakdown. In contrast, FITC-dextran lifetime measurements did not alter. k^sub i^ for FITC-dextran was increased from ~4.2 x 10^sup -3^ to ~8.8 x 10^sup -3^ ml plasma/ml tissue/min^sup -1^ following an i.p. injection of 30 mg/kg CA-4-P. Intravital multiphoton fluorescence microscopy is a valuable tool for functional imaging of tumour vasculature. These results support the hypothesis that CA-4-P causes a rapid increase in vascular permeability, which sets off a cascade of events leading to vascular shutdown.

This work was supported by Cancer Research UK.

Distribution Volumes of Native and Charge-Modified Albumin in DMBA-Induced Rat Mammary Tumours

H. Wiig, O. Tenstad, C. Gyenge

Department of Physiology and Locus on Circulatory Research, University of Bergen, Bergen, Norway

Interstitial collagen and glycosaminoglycans limit the space available for plasma proteins and other macromolecules, a phenomenon called volume exclusion. In tumours, studies of exclusion phenomena are of special interest since, in this tissue, the interstitium is a major barrier to drug delivery. Our objectives were to decide whether the extracellular matrix excludes albumin from a substantial part of the tumour interstitial fluid in vivo, and whether fixed charges in the matrix influences the distribution volume of macromolecules. Mammary tumours were induced in rats by gavage of di- methyl-benzanthracene (DMBA). Steady-state concentrations of radiolabelled native (pI 5.0) and cationized (pI 7.3) albumin were established in plasma and interstitial fluid by continuous infusion of tracer for up to 7 days using an osmotic minipump. Before termination of the experiment, the rats were anaesthetized (pentobarbital. 50 mg/kg i.p.) and nephrectomized, and ^sup 51^Cr- EDTA allowed 2 h circulation time to be used as an extracellular fluid volume marker. Final tracer distribution volumes were calculated from their respective concentrations in tumour fluid isolated by centrifugation (Wiig et al.: Am J Physiol 2003;284:H416- H424). Native albumin was excluded from on average 39% (+ or – 8.8, n = 8) of the tumour interstitial fluid phase. Charge neutralization resulted in an increased probe distribution volume and thus a reduction in excluded volume fraction, averaging 19% (+ or – 8.0. n = 8) for cationized albumin. Our experiments suggest that fixed negative charges in the tumour interstitium strongly influence the macromolecular distribution volume.

Inhibition of VEGF-Stimulated Angiogenesis by SU5416 Attached to Three Potential Prodrug Systems

L. Maskell(a), J. Newsome(b), E. Blanche(b), C.J. Moody(b), J.L. Whatmore(b)

a Institute for Biomedical and Clinical Sciences, Peninsula Medical School, Exeter, UK; b School of Chemistry, University of Exeter, Exeter, UK

Angiogenesis is essential to tumour growth and is critically regulated by vascular endothelial growth factor (VEGF). Thus, inhibition of VEGF-induced angiogenesis is a potential anti-tumour therapy. Previously, we confirmed that SU5416 (a Flk-1 inhibitor) fully inhibited VEGF-induced angiogenesis. This study examines the inhibition of VEGF-s\timulated angiogenesis by SU5416 attached to three potentially tumour-selective prodrug systems designed to release the active drug only within the target endothelial cells. Human umbilical vein endothelial cells (HUVECs) were seeded onto fibrin matrices. After 24 h. SU5416 or its prodrugs i.e. drug plus delivery system (in DMSO) and VEGF (100 ng/ml) were added. Controls received DMSO and VEGF. Three prodrugs were tested-EBL106 and EBL115 that chemically dissociate and EBL107 that does not. Formation of tubular structures was analysed. Intracellular release of the active free drug from the delivery system was examined by fluorescence microscopy utilising fluorescently labelled delivery molecules. SU5416 inhibited angiogenesis by 99.46% at 10 [mu]M (p < 0.001, n = 15). Angiogenesis was significantly inhibited by EBL106 (7% at 10 [mu]M, p < 0.005. n = 9) and EBL115 (47% at 10 [mu]M, p < 0.005, n = 9). EBL107 did not significantly inhibit angiogenesis. Fluorescence microscopy confirmed the dissociation of the prodrug for EBL106 within HUVECs. We conclude that VEGF-stimulated angiogenesis is significantly inhibited by SU5416 attached to delivery systems that release the free drug within endothelial cells.

Ex vivo Tumour Imaging Using a Monoclonal Antibody to CD 105 in Kidneys from Patients with Renal Cell Carcinoma

C.B. Costello, C. Li, S. Duff, D. Butterworth, A. Khan, M. Perkins, S. Ovens, A. Al-Mowallad, S. Kumar

Department of Pathology, Medical School, Manchester University, Manchester, UK

There is strong evidence that CD 105 Mab E9. which is highly reactive with angiogenic endothelial cells, could be a useful reagent to target vasculature of solid tumours in man. We evaluated its localisation using human kidney as an ex vivo model. Perfusion through a renal artery of ^sup 99^Tc^sup m^-labelled purified Mab E9 in freshly excised kidney from patients with renal carcinoma was performed. In all cases immunoscintigraphs showed the presence of well-defined radioactive hotspots which matched the position of the tumour as identified by pre-surgery MRI scan and subsequently by histological examination. That the labelling of tumour was specific was confirmed when prior perfusion of unlabelled Mab E9 in a kidney completely blocked the localisation of ^sup 99^Tc^sup m^-conjugated Mab E9. Radioactivity in aliquots of tissue blocks from 5 kidneys representing tumour and normal kidney showed that the mean values, adjusted per gram wet weight, for radioactive counts were significantly greater in tumours compared with the normal looking kidney. Cryostat sections of tumour tissue using FITC-labelled rabbit anti-mouse secondary antibody showed a strong and uniform localisation of Mab E9 in tumour microvessels. Interestingly, in an occasional microvessel segment, chimeric staining of endothelial cells (EC) was seen. Whether anti-CD105 Mab would be of use for tumour imaging/vascular targeting in cancer patients requires further studies on its localisation and pharmacokinetics in vivo.

Annexin 1 Deficiency Alters Leukocyte Extravasation in the Mouse as Determined by Flow Cytometry

B.E. Chatterjee, S. Yona, R.J. Flower, M. Perretti

Department of Biochemical Pharmacology, The William Harvey Research Institute, London, UK

No studies have yet addressed the role of the endogenous annexin 1 (ANX-A1) on the process of cell recruitment. Here we have used ANX- A1 KO mice (Hannon R et al.: FASEB J 2003; DOI 10.1096/fj.02- 0239fje) and a FACS protocol to address this point. Zymosan or IL- 1[beta] was injected intraperitoneally, and cavities washed at selected time points. Cells were stained with FITC-labelled Gr-1 mAb to stain preferentially murine neutrophils: PE-labelled F4/80 mAb to monitor monocytes and macrophages; and biotin-labelled anti-CD11b mAb (clone 5C6) used to determine the extent of white blood cell activation. Zymosan injection increased 4-hour Gr-1+ (polymorphonuclear cell (PMN)) accumulation into the peritoneal cavity of WT mice. Cell influx then declined up to 24 h post- injection, though remaining still higher than at time 0. IL-1[beta] showed a similar profile of Gr-1+ cell accumulation in WT mice. In these mice, the presence of F4/80+ cells (monocytes and macrophages) was decreased following zymosan, but not IL-1[beta], injection. In ANX-A1 KO mice significantly higher 4-hour PMN (Gr-1+) influx and lower F4/80+ cells were measured after zymosan, but not IL-1[beta], injection. CD11b expression was equal between ANX-A1 KO and WT mice. These data indicated that gene deficiency for the glucocorticoid- regulated protein ANX-A1 is associated with a higher degree of PMN extravasation in a stimulus-dependent manner.

This work was supported by the MRC (G78/7211) and William Harvey Research Foundation (London UK).

Macrophages Stimulate Angiogenesis in the Dorsal Skinfold Chamber Model

L. Bingle, C.E. Lewis, K. Corke, M. W.H. Reed, N.J. Brown

Clinical Sciences and Genomic Medicine, University of Sheffield, UK

Tumour-associated macrophages (TAM) are widespread in breast cancer and high levels of TAM infiltration correlate with increased tumour angiogenesis. TAM are thought to promote tumour angiogenesis by accumulating specifically in avascular, hypoxic areas of breast tumours, where their expression of vascular endothelial growth factor (VEGF) is upregulated. Here, we provide evidence that macrophages exhibit potent pro-angiogenic activity in vivo. Breast tumour spheroids (small, avascular tumour masses with a central hypoxic/necrotic core, grown from non-adherent cultures of the breast cancer cell line, T47D) were infiltrated with human macrophages and VEGF levels released from the spheroids measured by ELISA. VEGF release by TAM-infiltrated spheroids was up to 5-fold higher than that by non-infiltrated spheroids in vitro. TAM- infiltrated and non-infiltrated spheroids, coated with alginate (to prevent cell migration into or from the spheroid in vivo), were implanted into dorsal skinfold chambers in mice. In vivo microscopy and image analysis were used to quantify changes in the number and length of blood vessels in areas adjacent to spheroids. After 3 days, significantly (p = 0.01) more blood vessels formed around TAM- infiltrated spheroids than around non-infiltrated spheroids, vessel length was shorter (p = 0.008) and the number of junctions was greater (p = 0.025). By day 7 the only significant difference was seen in the vessel length (p = 0.021). From these studies we conclude that in the initial period after spheroid implantation TAM markedly stimulate VEGF release by spheroids, which may result in the observed early increase in angiogenesis.

Secretion, Cell-Surface Binding, and Endocytosis of Xanthine Oxidoreductase by the Vascular Endothelium

J. Hewinson(a), C.R. Stevens(a), D.R. Blake(a,b), T.M. Millar(a)

a Department of Medical Sciences, University of Bath, Bath, UK; b RNHRD NHS Trust, Bath, UK

This study aims to determine the mechanism of secretion and endocytosis for xanthine Oxidoreductase (XOR) from the vascular endothelium. The molybdoflavoenzyme xanthine Oxidoreductase catalyses the production of reactive oxygen and nitrogen species (RONS) and has been associated with many pathological processes, including ischaemia-reperfusion, breast cancer and adult respiratory distress syndrome (ARDS). XOR is found in many organs of the body including the liver, mammary gland, pancreas, and the blood. Circulating XOR has been shown in patients with liver disease and linked to ARDS in patients suffering from pancreatitis. We are interested in discovering the mechanisms and control of exocytosis and endocytosis of XOR by vascular endothelial cells and ultimately associate these processes with the disease states mentioned. In studies using human umbilical vein endothelial cells (HUVECs) in culture we have shown, using immunofluorescence, flow cytometry and enzyme assays, that HUVEC can bind purified exogenous enzyme to the cell surface. Endogenous XOR was expressed in the cytoplasm with a granular appearance. Following stimulation with histamine or thrombin, cells released endogenous XOR in a time- and dose- dependent manner which mirrored stimulated von Willebrand Factor release. Therefore, using inflammatory mediators, we have shown a mechanism for inducing localised endothelial XOR release and capture that could contribute to free radical damage and pathology both systemically and at localised sites distant from release.

Mechanosensitive Modulation of Hyaluronan (HA) Secretion Involves Protein Kinase C, Ca^sup 2+^ and MAPK

T. Momberger(a), J.R. Levick(b), R.M. Mason(a)

a Cell and Molecular Biology, Faculty of Medicine, Imperial College, London, UK; b Department of Physiology, St George’s Hospital Medical School, London, UK

HA secreted into joints in vivo influences fluid kinetics and is stimulated by joint distension. We investigated the transduction pathways in vitro. Primary rabbit synoviocyte cultures on 6 well plates received 10% static stretch (FX-2000 Flexercell). HA secretion into the media over 3 h was analysed by ELISA-type assay. Protein kinase C (PKC) isoform activation was assessed by a shift to the particulate fraction. HA synthase (HAS) mRNA was assessed by RTPCR. 10 min stretch and 170 min rest, or 180 min continuous stretch, stimulated HA secretion by 57 + or – 18% and 57 + or – 7%, respectively (mean + or – SEM; n = 10 and 17 plates; p < 0.01, Wilcoxon). Mainly HAS-2 isoform mRNA (and some HAS-3) was expressed. Stretch did not significantly increase HAS mRNA, but cycloheximide and actinomysin D reduced basal and stimulated HA secretion, which thus depends on new protein synthesis. Stretch stimulation was blocked by the PKC inhibitor bisindolylmaleimide (BIM III), specific cPKC inhibitor Go 6976, and by phorbol-induced PKC downregulation. PKC[alpha] and [delta] were activated (membrane-bound) by stretch. HA synthesis was reduced by Ca^sup 2+^ chelation. BayK (Ca^sup 2+^ channelactivator) activated PKC[alpha] and increased HA synthesis. Stretch phosphorylated MAPK (ERK1/2). Inhibition of MAPK phosphorylation by PD98059 reduced HA secretion. Stretch-induced MAPK activation was inhibited by BIM III or intracellular Ca^sup 2+^ chelator BAPTA AM. We infer that mechanotransducers may activate Ca^sup 2+^ influx and a PKC[alpha]-MAPK cascade that increases the transcription, translation and synthesis of HAS or an associated regulatory protein.

Solute Transport in Extracellular Matrix under Dynamic Load

Z.Y. Li, Y. Lu, D.L. Bader, W. Wang

Medical Engineering Division, Queen Mary, University of London, London, UK

To gain insight into the effects of dynamic loading on solute transport in extracellular matrices, we have applied a binary mixture theory and modelled the extracellular matrix as a deformable porous medium. Based on the theory, every single point of the matrix is simultaneously occupied by liquid as well as solid phases. When there is relative movement between the two phases, interaction in the form of a drag force is introduced (Wang and Parker: J Fluid Mech 1995:283:287-305). The simplified two-dimensional model consists of a matrix filled with cells and immersed in a solution with known solute concentration at the bottom of a rigid impermeable rectangular well. Only the top surface of the matrix is in direct contact with the solution, where the solute diffuses into the matrix and is consumed by the cells distributed within the matrix. Mechanical load is applied on the top surface of the matrix, causing its deformation and extracellular fluid movement. Resulting cell density is related to tissue deformation. Solute diffusion is coupled to the extracellular fluid flow contributing to solute transport within the matrix. The simplified model was solved numerically for the solid matrix deformation, fluid velocity and solute concentration distribution under dynamic loading conditions. Comparisons on solute transport were made between different loading frequencies and amplitudes. The model can be applied to interpret experiments on small solute, e.g. glucose and oxygen, transport in soft tissues, such as articular cartilage, and has the potential to relate cell metabolic rate at different locations in the tissue to external mechanical stimuli.

Effect of Chain Length on Hyaluronan Permeation across the Joint- to-Lymph Barrier

S. Sabaratnam, J.R. Levick

Department of Physiology, St George’s Hospital Medical School, London, UK

Hyaluronan (HA) chain length is reduced in arthritic effusion. This attenuates the buffering of trans-synovial fluid escape by HA. Reduced buffering by short-chain HA may be due to easier HA escape through the synovial lining. This study examines the effect of reducing the HA chain length on HA transmission from joint cavity into the periarticular lymphatic system. Rooster HA of ~2,000 kDa (HA2000, 0.2 mg[middot]ml^sup -1^) or sonicated HA of ~140 kDa (HA140, 4 mg[middot]ml^sup -1^) was infused along with fluorescein- dextran20 (FD) under constant pressure into knee joint cavities of anaesthetised rabbits. After trans-synovial filtration had established a steady state, femoral lymph was sampled every 15 min for 3 h. Samples were analysed for HA and FD by high pressure liquid chromatography. The HA140 concentration in lymph as a % of infusate HA140 concentration, namely 53 + or – 5% (mean + or – SEM, 30 samples), was higher than HA2000 % concentration (31 + or – 3%, 85 samples: p = 0.0001. t test). Dilution of joint lymph by skin/ muscle lymph was estimated from FD % concentration in femoral lymph. Reflection fraction was calculated as lymph HA % concentration divided by lymph FD % concentration. For HA140 the reflection fraction was 0.16 + or – 0.03 (5 rabbits, 21 lymph samples). For HA2000 the reflection fraction was >3 x bigger, 0.54 + or – 0.03 (17 rabbits, 82 samples: p < 0.0001, unpaired t test). It is concluded that short-chain HA experiences less molecular sieving than long- chain HA during fluid drainage from joint cavity to lymphatic. This is presumably because the small radius of gyration (solvated domain radius) of short-chain HA reduces its reflection by the pores of the synovial interstitial matrix, which is the main barrier to HA permeation.

Poster Communications

Evaluation of the Time Course of the Veni-Arteriolar Response to Venous Congestion in the Human Calf

M.D. Brown, M.A. Oldfield

School of Sport and Exercise Sciences, University of Birmingham, Birmingham, UK

Elevation of venous pressure by a congestion cuff or limb dependency invokes the veni-arteriolar response leading to pre- capillary constriction in muscle, skin and subcutaneous tissues. The triggering of this response depends on the size of pressure increments. Limb blood flow measured several minutes after a single large (>25 mm Hg) pressure increase is reduced, but during cumulative small (<10 mm Hg) increases, limb blood flow measured after 5 min at each pressure was sustained even when cuff pressure approached mean arterial pressure (Gamble et al.: J Physiol 1993:464:407-422). To determine the time course of activation of the veni-arteriolar response, calf blood flow was measured by strain gauge plethysmography at intervals between 3 s and 5 min after venous congestion by a thigh cuff in 9