Serum levels of vascular endothelial growth factor (VEGF) are markedly elevated in patients with Wegener's granulomatosis.

OBJECTIVES: Necrotizing vasculitis and granuloma formation are the predominant features of Wegener's granulomatosis (WG). We have investigated the importance of vascular endothelial growth factor (VEGF) in monitoring disease activity in WG. METHODS: Serum VEGF levels were determined in 23 patients with active WG, 21 healthy controls and 25 patients with urinary infection, by ELISA using commercially available antibodies to VEGF. RESULTS: VEGF levels were enormously elevated in patients with WG compared to both controls and patients with urinary infection (P < 0.0001). Of the 23 patients, 21 (91.3%) had VEGF levels above the cut-off value (3.3 ng/ml, calculated as the mean of the controls + 2 S.D.). Further analysis of the data showed that VEGF levels did not correlate with age, sex, incidence of classic antineutrophil cytoplasmic antibodies (c-ANCA) or duration of the disease (P > 0.05), but there was correlation with disease activity (r = 0.51, P < 0.01). VEGF levels were higher in patients with major compared to those with minor disease activity (P < 0.01). However, there was no significant correlation between VEGF levels and the Birmingham scores for vascular activity and damage. CONCLUSION: VEGF levels are raised in WG patients compared to normal controls and may be a marker of disease activity. Further studies on serial blood samples from a large cohort of patients with WG and other systemic vasculitides are needed to evaluate the specificity and usefulness of VEGF levels in monitoring disease activity.

Glucosaminylmuramyl dipeptide (GMDP) modulates endothelial cell activities in vitro but has no effect on angiogenesis in vivo.

OBJECTIVE AND DESIGN: The aim of the study was to evaluate the effects of GMDP on angiogenesis in vivo and as a modulator of human umbilical vein endothelial cell proliferation, cell surface antigen expression and cell adhesion in vitro. MATERIALS: Human umbilical vein endothelial cells (HUVEC), fertilized white leghorn chicken eggs, antibodies against adhesion molecules and glucosaminylmuramyl dipeptide (GMDP). TREATMENT: GMDP [0.01-100 micrograms/ml] applied to cell cultures for 6-72 h and to the chick chorioallantoic membrane (CAM) for four days. METHODS: Angiogenic activity of GMDP in vivo was assessed using the CAM assay; HUVEC proliferation was measured by tritiated thymidine incorporation and cell cycle studies; cell surface antigen expression by indirect immunofluorescence and flow cytometry; cell adhesion by quantification of [3H]-thymidine labeled leukocyte adherence to HUVEC monolayers. Statistical analysis was performed using one-way ANOVA and if necessary was followed by Duncan's multiple range test for variables. RESULTS: GMDP induced [3H]-thymidine incorporation in a concentration- and time-dependent manner (p < 0.003) and significantly increased the porportion of cells in the S phase of the cell cycle (p < 0.03). It weakly augmented the expression of ICAM-1 and CD31 but not adhesion of leukocytes to HUVEC monolayers GMDP was not angiogenic in the CAM assay. CONCLUSIONS: GMDP can modulate endothelial cell activity without the induction of angiogenesis in vivo which may have implications for its use as a therapeutic agent.

Localisation and cellular origin of hyaluronectin.

Hyaluronectin is an extracellular matrix glycoprotein which specifically binds to hyaluronan. Isoforms of hyaluronectin are present in nervous and mesenchymal tissues but, while the nervous tissue isoform has been characterised in some detail, less is known about the mesenchymal isoform. Although its tissue localisation suggests a role in tumour development, neither its cellular origin nor its exact function are known. In this study we demonstrate hyaluronectin synthesis in fibroblasts and smooth muscle cells in vitro. The pattern of immunolocalisation of hyaluronectin in fibroblasts depended on the cell type, length of time spent by the cells in culture and cell density. Immunoreactivity in sparsely plated migratory cells was seen mainly in a patchy distribution at the attached cell surface and in the migration tracks left by the cells on the subtratum. In stationary cells a more uniform distribution associated with the attached cell surface was observed, while in confluent cultures hyaluronectin immunoreactivity was mainly seen as a network of fibrillar material above the cell. The pattern of staining was distinct from that of other hyaluronan-binding proteins. Immunoprecipitation, using antihyaluronectin antibodies, of the substratum-attached material deposited by human fetal fibroblasts revealed a family of proteins ranging from 22 to 90 kDa, the major protein being of approximately 60 kDa. These results lead us to propose that hyaluronectin plays an important role in cell migration, probably by regulation of hyaluronan distribution and binding.

Isolation and characterisation of a hyaluronan binding protein, hyaluronectin, from human placenta and its colocalisation with hyaluronan.

Hyaluronan (HA) is a major component of the extracellular matrix and is known to influence cell behaviour and to play a role in angiogenesis, morphogenesis and tissue remodelling, although little is known concerning the regulation of these effects. Until now its detection in the placenta has been by indirect methods, which has led to conflicting conclusions as to its distribution and hence its role. Hyaluronectin (HN) is one of a group of proteins with HA binding ability which may regulate the effects of HA. Although nervous tissue HN has been partly characterised with regard to its distribution, structure and biochemistry, little is known about the mesenchymal isoform and its distribution in placenta has not previously been reported. Using specific probes we have characterised the distribution of HA and HN in human placental tissue. At all stages of development studied (8, 10, 12, 30 and 38 wk gestation) HA and HN were unequivocally colocalised, being distributed in the extracellular matrix of stromal tissue of placental villi, chorioallantoic membranes and umbilical cord. Particularly strong immunoreactivity was observed in the villous stroma immediately adjacent to fibrinoid depositions at sites of denudation of the trophoblast layer. Extraction and characterisation of the HN from placental villi have revealed 4 major glycoproteins of 47, 52, 57 and 67 kDa, this being a different pattern and smaller molecular range than observed for the nervous tissue form. This is the first direct demonstration of the presence of HA and HN in the placenta and identifies an abundant new source of mesenchymal HN. The functions of mesenchymal HN are unknown but may include ion exchange, immunosuppression and regulation of the effects of HA in such roles as maintenance of tissue architecture, cell migration and angiogenesis.

Sera of children with renal tumours contain low-molecular-mass hyaluronic acid.

The molecular mass of hyaluronic acid (HA) rather than its serum concentration alone may be a hallmark of certain types of malignancy. A radiometric assay was used to measure HA levels in 35 children with renal tumours [33 Wilms' tumours and 2 bone metastasizing renal tumours of childhood (BMRTC)] and 20 normal siblings of children with cancer. The HA level in the sera of normal children was barely detectable and had a molecular mass of 1-5 x 10(5). In both Wilms' and BMRTC patients, very high levels of HA were found in preoperative serum samples; these fell dramatically following surgical excision of the tumours. A novel finding of our study was the presence of low-molecular-mass HA (similar to the angiogenic fragments of HA) in the sera of BMRTC patients. In contrast, high-molecular-mass HA (which is not angiogenic) was found in the sera of Wilms' patients (2 x 10(6) kDa). Following surgery in BMRTC patients, not only did serum HA levels fall to a value within normal ranges, but also the HA which remained was of high molecular mass.