Hypoxia augments cytokine (transforming growth factor-beta (TGF-beta) and IL-1)-induced vascular endothelial growth factor secretion by human synovial fibroblasts

Abstract

Vascular endothelial growth factor (VEGF) is abundant in synovium and synovial fluids, where it probably contributes to vascular permeability and angiogenesis in arthritic joints. To investigate the probable sources of VEGF in synovium, we compared the ability of several cytokines (TGF-beta, platelet-derived growth factor (PDGF), IL-1, tumour necrosis factor (TNF), basic fibroblast growth factor (bFGF) that are associated with arthritis and angiogenesis, to stimulate secretion of VEGF protein by human synovial fibroblasts. TGF-beta was the strongest inducer of VEGF secretion; six times more VEGF was secreted when cells were stimulated by TGF-beta than when stimulated by PDGF or IL-1 for 24 h. TNF-alpha and bFGF did not stimulate any secretion of VEGF. The stimulatory effects of TGF-beta and IL-1 on VEGF secretion were additive. Hypoxic culture alone also stimulated VEGF secretion, but more importantly, hypoxic culture conditions doubled the rate of VEGF secretion stimulated by the cytokines TGF-beta and IL-1. When dermal and synovial fibroblasts were stimulated identically by hypoxia and cytokines (TGF-beta and IL-1), synovial fibroblasts secreted four times more VEGF than did dermal fibroblasts. Thus in rheumatoid arthritis, the capacity of synovial fibroblasts in the hypoxic environment to secrete large amounts of VEGF in response to cytokines such as TGF-beta probably contributes significantly to angiogenesis in the synovium.

Fig. 1

Secretion of vascular endothelial growth factor (VEGF) by synovial fibroblasts stimulated with cyokines. The cytokines indicated were added to replicate wells containing confluent monolayers of human synovial fibroblasts (approx. 20 000 cells/well in 12-well plates) in 0.5 ml of serum-free medium. All cytokines were added at 10 ng/ml into triplicate wells to ensure saturating concentrations. After 2, 6, 24 and 48 h of culture, medium was removed and the concentration of VEGF attained in each well determined by ELISA analysis. In all cases VEGF was undetectable in medium after 2 h of culture (data not shown). The mean of triplicate wells and the standard error are shown. These data are representative of six experiments performed with cell lines derived from tissue from four different patients. TNF, Tumour necrosis factor; PDGF, platelet-derived growth factor.

Fig. 2

Kinetics and dose–response of induction of mRNA for vascular endothelial growth factor (VEGF) isoforms by TGF-β. Confluent cultures of synovial fibroblasts were incubated for 48 h in serum-free medium. TGF-β was then added to fresh medium at various concentrations for the times indicated. Total RNA was isolated and 5 μg per sample were analysed by an RNase protection assay designed to detect individual VEGF isoforms. Two micrograms of total RNA were used for actin transcript protection to assess whether equal RNA was present in each sample. (a) Autoradiograph of a polyacrylamide gel. Lane P, radioactive probe alone; lane Y, control with 5 μg yeast RNA. TGF-β was added to cell cultures at 4 ng/ml for 2, 6, 12 and 24 h, at 0.1, 2 and 4 ng/ml for 6 h, or was not added as indicated above. VEGF isoforms of 189, 165 and 121 amino acids are indicated. (b) Densitometric analysis of VEGF₁₆₅ bands induced by TGF-β after 6 h and expressed relative to VEGF₁₆₅ transcripts of cells incubated 24 h without TGF-β.

Fig. 3

Kinetics and dose–response of induction of mRNA for vascular endothelial growth factor (VEGF) isoforms by IL-1. (a) Confluent cultures of synovial fibroblasts were incubated for 48 h in serum-free medium. IL-1 was then added to fresh medium at various concentrations for the times indicated. Total RNA was isolated and 5 μg per sample were analysed by an RNase protection assay designed to detect individual VEGF isoforms. Two micrograms of total RNA was used for actin transcript protection to assess whether equal RNA was present in each sample. Autoradiograph of a polyacrylamide gel. Lane P, radioactive probe alone; lane Y, control with 5 μg yeast RNA. IL-1 was added to cell cultures at 0, 1, 4 and 16 ng/ml for 12 h, or 4 ng/ml for 2, 6 and 12 h as indicated above. VEGF isoforms of 189, 165 and 121 amino acids are indicated. (b) Densitometric analysis of VEGF₁₆₅ bands induced by various doses of IL-1 after 12 h and expressed relative to VEGF₁₆₅ transcripts of cells incubated for 24 h without IL-1.

Fig. 4

Induction of vascular endothelial growth factor (VEGF) mRNA expression by TGF-β and IL-1. Synovial fibroblasts were grown to confluence in T-75 flasks, incubated for another 48 h in serum-free medium and then fresh medium was added with or without TGF-β, IL-1 or both combined (10 ng/ml). After 6 h at 37°C total RNA was isolated. (a) Northern hybridization. Total RNA (10 μg/lane) and RNA size standards were electrophoresed and blotted onto nylon membranes and hybridized with a VEGF-specific ³²P-labelled cDNA. The VEGF transcript is seen at 4.2 kb. The blot was stripped and rehybridized with a 28S RNA-specific labelled probe (28S). (b) Densitometric analysis. The bands on exposed films were imaged and digitized and the number of pixels associated with each band quantified. Induction of VEGF mRNA transcripts in cells treated with TGF-β and IL-1 combined for 6 h was increased approx. 25-fold above control cells.

Fig. 5

Combined effect of hypoxic culture and cytokines on vascular endothelial growth factor (VEGF) mRNA levels. After 48 h of culture in serum-free medium, confluent fibroblast cultures were changed to fresh medium with or without addition of TGF-β (10 ng/ml) and IL-1 (10 ng/ml) added together. Replicate flasks were cultured under either normoxic (21% oxygen) or hypoxic (3% oxygen) conditions for 6 h or 24 h. Total RNA was isolated from cells in six flasks in each case, and Northern hybridization for VEGF performed with 5 μg total RNA. The blot was rehybridized for 28S RNA to assess equality of loading The absence or presence of cytokines, the incubation time period and normoxic or hypoxic culture are indicated in the figure.

Fig. 6

Effect of hypoxia on cytokine-induced vascular endothelial growth factor (VEGF) secretion. Synovial fibroblasts and dermal fibroblasts were grown to confluence in 48-well plates, cultured in the absence of serum for 24 h and cytokines (TGF-β and IL-1) were added at 10 ng/ml to fresh serum-free medium. Cells were then incubated in either normoxic (21% oxygen) or hypoxic conditions (3% oxygen). At the times indicated, the medium was collected from triplicate wells and assayed by ELISA for secreted VEGF. The data were normalized to constant cell number (5 × 10³) and the mean concentration of VEGF and the s.e.m. are shown.