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. 1998 Oct;43(4):525–536. doi: 10.1136/gut.43.4.525

Expression of basic fibroblast growth factor in intact and ulcerated human gastric mucosa

M Hull 1, J Brough 1, D Powe 1, G Carter 1, D Jenkins 1, C Hawkey 1
PMCID: PMC1727263  PMID: 9824581

Abstract

Background—Basic fibroblast growth factor (bFGF) promotes angiogenesis and healing of gastric ulcers in rats, and bFGF expression is up regulated in such ulcers. However, little is known about expression of bFGF in human gastric mucosa. 
Aims—To investigate bFGF expression in intact human gastric mucosa and gastric ulcers and to determine whether low bFGF content or altered binding by mucosa is associated with ulceration. 
Subjects—Endoscopy outpatients, gastrectomy patients, and organ donors. 
Methods—bFGF was isolated by heparin affinity chromatography and characterised by western blotting and endothelial cell bioassay. bFGF was measured by immunoassay and its distribution defined by immunohistochemistry and in situ hybridisation. Binding of bFGF by heparan sulphate proteoglycans was investigated by sodium chloride and heparin extraction. 
Results—Bioactive bFGF (19 kDa) was detected in normal mucosa but bFGF mRNA was not found. bFGF expression was up regulated in granulation tissue endothelial cells, mononuclear cells, and epithelial cells at the ulcer rim. Gastric ulcer patients had constitutively low bFGF concentrations in intact antral mucosa which were not explained by changes in binding to heparan sulphate proteoglycans. 
Conclusions—bFGF expression is up regulated in human gastric ulcers. Low intact mucosal bFGF content is associated with gastric ulceration. 



Keywords: basic fibroblast growth factor; gastric mucosa; heparan sulphate proteoglycan; peptic ulceration

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Figure 1 .

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Elution of bFGF from human gastric mucosa using heparin affinity chromatography and a 0-3 mol/l NaCl gradient. bFGF content of each fraction was assessed using a bFGF ELISA. A peak of bFGF was detected in fractions 30-40 corresponding to elution by 1.8-2.8 mol/l NaCl. 


Figure 2 .

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Western blot analysis of gastr ic mucosal bFGF. Fractions 30-40 from heparin affinity chromatography were pooled, freeze dried, and reconstituted in 100 µl H2O. Gastric mucosa contained a 19 kDa isoform of bFGF compared with 16.4 kDa N-terminal truncated recombinant bFGF. 


Figure 3 .

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Mitogenic activity of gastric mucosal bFGF. HuGE cell number was assayed using a formazan colorimetric assay (Promega, Madison, Wisconsin). We have previously shown a good correlation (r2=0.99) between absorbance at 492 nm and HuGE cell number using this assay.12 Briefly, 103 HuGE cells were added to individual 1% gelatin coated wells of a 96 well plate and incubated for 48 hours in 5% CO2 at 37°C in the absence of endothelial cell growth supplement. Gastric mucosal bFGF or control buffer with or without Mab3H3 (working concentration 50 µg/ml) was added to HuGE cell monolayers and incubated for a further 24 hours before assay. Data are expressed as the mean (SEM) multiple of the absorbance at 492 nm of the negative control (n=4 wells). Gastric mucosal bFGF promoted proliferation of HuGE cells which was almost completely inhibited by Mab3H3. Cell number was not affected by the presence of Mab3H3 per se (data not shown). 


Figure 4 .

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Immunohistochemistry for bFGF in normal tissue. (A) Normal antral mucosa. Localisation of bFGF to surface and glandular epithelium, lamina propria extracellular matrix (ECM) (asterisk), muscularis, and blood vessels (arrow). Bar=80 µm. (B) Normal antral mucosa. Patchy distribution of bFGF in glandular epithelium. Bar=20 µm. (C) Dupytren's contracture. Staining of sweat glands, blood vessels, and ECM (asterisk). Bar=40 µm. (D) Dupytren's contracture. Preabsorption of Mab52 by recombinant human bFGF (×9 molar excess). No staining. Scale as for (C). 


Figure 5 .

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Intact mucosal bFGF concentrations in gastric ulcer patients and matched non-ulcer controls. *p<0.001. 


Figure 6 .

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Immunohistochemistry for bFGF in antral gastric mucosa. (A) H pylori associated gastritis. Strongly stained mononuclear cells present in a chronic inflammatory infiltrate (arrow). Bar=50 µm. (B) Reactive (type C) gastritis. Prominent lamina propria smooth muscle fibres positive for bFGF (asterisk). Bar=100 µm. 


Figure 7 .

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(A) bFGF extraction from antral mucosa of control (non-GU) and gastric ulcer (GU) patients by NaCl. *p<0.05. (B) bFGF extraction from antral mucosa of non-GU and GU patients by 100 µg/ml heparin and 3.0 mol/l NaCl. +p=0.03. 


Figure 8 .

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Increased bFGF concentrations at the rim of gastric ulcers compared with intact gastric mucosa. The thick line joins the mean values for the two groups. The thin lines join individual pairs of data. 


Figure 9 .

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Immunohistochemistry for bFGF in gastric ulcers. (A) Granulation tissue. Staining of capillary endothelium (arrow) and extracellular matrix (ECM) (asterisk). Bar=20 µm. (B) Epithelium at the ulcer rim. Increased staining of surface (arrow head) and glandular epithelium compared with intact mucosa. Bar=20 µm. (C) Granulation tissue. Staining of fibroblasts. Scale as for (B). (D) Smooth muscle breached by ulceration. Intense staining of smooth muscle cells. Bar=30 µm. 


Figure 10 .

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In situ hybridisation of bFGF mRNA with a 450 bp 35S labelled cDNA probe. (A) Gastric ulcer granulation tissue. Signal over mononuclear cells and endothelial cells of a capillary (asterisk). Bar=40 µm. (B) Gastric ulcer. Signal over smooth muscle cells breached by ulcer granulation tissue. 


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