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1. Dysregulated vascular endothelial growth factor (VEGF) expression has been reported in several pathological states based upon evidence of elevated serum VEGF levels. Using two immunoassays for VEGF, this study determines normal plasma and serum VEGF ranges, determines which are more likely to reflect circulating VEGF levels and investigates a potential contribution of VEGF from platelets to VEGF levels detected in serum. 2. The presence of soluble VEGF receptor, sflt-1, at a molar excess of 7:1 significantly reduced measured VEGF levels in both assays. Serum VEGF levels were higher than plasma levels in children [(mean +/- S.E.M.) 306.1 +/- 39.4 versus 107.4 +/- 24.9 pg/ml, P < 0.0001] and adults (249.4 +/- 46.4 versus 76.1 +/- 10.7 pg/ml, P < 0.0001). Serum VEGF increased with clotting time (P = 0.0005 t0 compared with 2 h samples); plasma VEGF levels were not affected by time between sampling and centrifugation. 3. Calcium-induced clotting of platelet-rich but not platelet-poor plasma induced VEGF release with a proportional response between platelet count and VEGF level and isolated platelets released significant quantities of VEGF upon incubation with thrombin. Reverse transcriptase-PCR studies confirmed that platelets express VEGF121 and VEGF165 mRNA. 4. These data suggest that plasma is the preferred medium to measure VEGF levels; a significant and highly variable platelet-mediated secretion of VEGF during the clotting process invalidates the use of serum as an indicator of circulating VEGF levels in disease states.

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The aims of this study were (i) to determine whether PlGF, VEGF and PlGF/VEGF heterodimers are detected in synovial fluid (SF) and plasma samples from patients with a range of arthropathies; (ii) to describe whether any correlation exists between SF PlGF, VEGF and PlGF/VEGF heterodimer levels and the total and differential SF leucocyte counts; and (iii) to investigate the regulation of peripheral blood mononuclear cell (PBMC) VEGF secretion by stimuli relevant to inflammatory joints. PlGF, VEGF and PlGF/VEGF heterodimer levels were measured in the SF and plasma of patients with a range of arthropathies and normal controls by ELISA. Western blotting for PlGF was performed on SF from three patients with rheumatoid arthritis (RA) and primary inflammatory arthropathies. VEGF was quantified in cell culture supernatants after stimulation with lipopolysaccharide (LPS), PlGF or cobalt ions of PBMC isolated from RA patients and controls. PlGF and VEGF were detected in all SF samples. PlGF/VEGF heterodimers were detected in 10.2% of SF samples, most frequently in RA samples. Western blotting confirmed the presence of PlGF in RA SF. PlGF was detected in 52% of RA and 31% of control plasma samples, and VEGF was detected in 38% of RA and 38% of control plasma samples. PlGF/VEGF heterodimers were detected in 21% of RA samples and none of the control samples. In primary inflammatory arthropathy patients, SF PlGF and VEGF levels correlated significantly with the SF total leucocyte count and the neutrophil count. PlGF was the most potent inducer of PBMC VEGF production in both RA and control subjects. This is the first report of the detection of PlGF and PlGF/VEGF heterodimers in the SF of patients with inflammatory arthropathies, and we have shown for the first time that PlGF up‐regulates PBMC VEGF production. PlGF may therefore play a key role in the production of VEGF in the inflammatory joint.

Aim-To investigate vascular endothelial growth factor (VEGF) mRNA expression in glomerular disease in the context of heavy proteinuria. Methods-Non-radioisotopic in situ hybridisation was performed using a cocktail of 12 deoxyoligonucleotides complementary to VEGF mRNA labelled during solid phase synthesis with 2,4-dinitrophenyl. Archival renal biopsies were studied from cases of minimal change nephropathy, membranous nephropathy, diabetic nephropathy, and controls, matched for age, sex, race, and storage time. Hybrid detection used NBT/ BCIP colorimetric development. Conclusions-Using non-radioisotopic in situ hybridisation, VEGF mRNA is almost exclusively expressed by visceral glomerular epithelial cells. Abnormal numbers of cells are seen in both minimal change and diabetic nephropathy. As VEGF exists in a number of functionally distinct isoforms, further study of qualitative VEGF isoform expression in diagnostic groups is indicated. (J Clin Pathol 1999;52:735-738)

This study was designed to investigate VEGF production from peripheral blood mononuclear cells (PBMC) from patients with rheumatoid arthritis (RA) compared with healthy controls and to identify the predominant cellular source in PBMC isolated from RA patients. The regulation of PBMC VEGF production by cytokines and synovial fluid (SF) was studied. PBMC were isolated from RA patients and healthy controls and stimulated with lipopolysaccharide (LPS), IL-1beta, IL-4, IL-6, IL-8, IL-10, TNF-alpha and transforming growth factor-beta (TGF-beta) isoforms for varying time points up to 72 h at 37 degrees C/5% CO2. The effect of SF on VEGF secretion by PBMC was also studied. Supernatant VEGF levels were measured using a flt-1 receptor capture ELISA. RA patients had significantly higher spontaneous production of VEGF compared with controls, and monocytes were identified as the predominant cellular source. RA PBMC VEGF production was up-regulated by TGF-beta isoforms and TNF-alpha and down-regulated by IL-4 and IL-10, with no effect observed with IL-1beta, IL-6 and IL-8. Antibody blocking experiments confirmed that TNF-alpha and not TGF-beta isoforms in SF increased VEGF secretion by RA PBMC. These results emphasize the importance of monocytes as a source of VEGF in the pathophysiology of RA. Several cytokines known to be present in SF can modulate the level of VEGF secretion, but the predominant effect of SF in VEGF up-regulation is shown to be dependent on TNF-alpha.

Increased circulating VEGF levels are not responsible for the proteinuria observed during relapses of SSNS. Further studies are warranted to investigate intrarenal VEGF expression.

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