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Comparative Study
. 2009;11(3):R64.
doi: 10.1186/ar2689. Epub 2009 May 8.

Hypoxia upregulates angiogenesis and synovial cell migration in rheumatoid arthritis

Mohammed A Akhavani  1 , Leigh Madden, Ian Buysschaert, Branavan Sivakumar, Norbert Kang, Ewa M Paleolog
Affiliations
Comparative Study

Hypoxia upregulates angiogenesis and synovial cell migration in rheumatoid arthritis

Mohammed A Akhavani et al. Arthritis Res Ther. 2009.

Abstract

Introduction: Rheumatoid arthritis (RA) is characterised by invasion of cartilage, bone and tendon by inflamed synovium. Previous studies in our laboratory have shown that hypoxia is a feature of RA synovitis. In the present study, we investigated the consequences of hypoxia on angiogenesis and synovial fibroblast migration in RA.

Methods: Synovial tissue was harvested from RA patients, and synovial membrane cells were cultured under conditions either of hypoxia (1% oxygen) or normoxia (21% oxygen). Protein levels of matrix metalloproteinases (MMPs) and angiogenic factors were measured, while RNA was extracted for PCR quantification of MMPs/tissue inhibitors of MMP (TIMPs) and angiogenic factors. Migration of RA synovial fibroblasts through collagen, and the effect of RA synovial cell supernatants in an in vitro angiogenesis assay, were utilised to determine the functional relevance of changes in mRNA/protein.

Results: We observed upregulation under hypoxic conditions of MMPs responsible for collagen breakdown, specifically collagenase MMP-8, and the gelatinases MMP-2 and MMP-9, at both mRNA and protein levels. Increased MT1-MMP mRNA was also observed, but no effect on TIMP-1 or TIMP-2 was detected. RA fibroblast migration across collagen was significantly increased under hypoxic conditions, and was dependent on MMP activity. Furthermore, expression of angiogenic stimuli, such as vascular endothelial growth factor (VEGF), and VEGF/placental growth factor heterodimer, was also increased. Crucially, we show for the first time that hypoxia increased the angiogenic drive of RA cells, as demonstrated by enhanced blood vessel formation in an in vitro angiogenesis assay.

Conclusions: Hypoxia may be responsible for rendering RA synovial lining proangiogenic and proinvasive, thus leading to the debilitating features characteristic of RA.

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Figures

Figure 1
Figure 1
Hypoxia differentially modulates the angiogenic balance. Rheumatoid arthritis synovial cells were exposed to either 21% oxygen (normoxia) or 1% oxygen (hypoxia) for 24 hours. mRNA levels of (a) vascular endothelial growth factor (VEGF) and (b) placental growth factor (PlGF) were measured by quantitative PCR (n = 19). The change in threshold cycle (ΔCt) values was calculated for each mRNA using the 2-ΔΔCt method against the housekeeping gene acidic ribosomal protein. The fold changes in mRNA levels were related to a reference sample (human cDNA). In parallel, release of (c) PlGF and (d) VEGF/PlGF heterodimer was measured by ELISA of cell culture supernatants (n = 13 to 15). Data were analysed versus normoxia by Wilcoxon signed rank test: *P < 0.05, ***P < 0.001.
Figure 2
Figure 2
Rheumatoid arthritis synovial cells exposed to hypoxia express more proangiogenic activity. Rheumatoid arthritis (RA) synovial cells were exposed to either 21% oxygen (normoxia) or 1% oxygen (hypoxia) for 24 hours. Cell supernatants were filtered, and the protein fraction was resuspended in fresh medium. Angiogenesis in response to RA synovial cell supernatants was assessed after 11 days, using CD31 expression quantified by colorimetric assay. (a) Representative data, with cells exposed to either vascular endothelial growth factor (VEGF) (2 ng/ml), RA synovial cell supernatants or no stimulus. Data are means of triplicate determinations, and were analysed by one-way analysis of variance: ***P < 0.001. (b) Comparison of angiogenesis in response to normoxic and hypoxic RA synovial cell supernatants. Data are means of paired triplicate determinations for six separate patients, and were analysed by paired t-test: **P < 0.01. (c) to (f) Representative images showing morphology of the formed tubes stained for CD31 at day 11 (objective magnification, ×40): (c) untreated, (d) VEGF treated (2 ng/ml), (e) normoxic RA synovial cell supernatants and (f) hypoxic RA synovial cell supernatants from the same patient.
Figure 3
Figure 3
Hypoxia modulates gelatinase expression by rheumatoid arthritis synovial cells. Rheumatoid arthritis synovial cells were exposed to either 21% oxygen (normoxia) or 1% oxygen (hypoxia) for 24 hours. mRNA levels of (a) matrix metalloproteinase (MMP)-2 and (c) MMP-9 were measured by quantitative PCR (n = 16 to 18). The change in threshold cycle (ΔCt) values was calculated for each mRNA using the 2-ΔΔCt method against the housekeeping gene acidic ribosomal protein. The fold changes in mRNA levels were related to a reference sample (human cDNA). In parallel, release of (b) MMP-2 and (d) MMP-9 protein was measured by ELISA of cell culture supernatants (n = 12). Data were analysed versus normoxia by Wilcoxon signed rank test (b, c) or paired t test (a, b) as appropriate: *P < 0.05, **P < 0.01.
Figure 4
Figure 4
Hypoxia modulates collagenase expression by rheumatoid arthritis synovial cells. Rheumatoid arthritis synovial cells were exposed to either 21% oxygen (normoxia) or 1% oxygen (hypoxia) for 24 hours. mRNA levels of (a) matrix metalloproteinase (MMP)-8 and (c) MMP-13 were measured by quantitative PCR (n = 10 to 14). The change in threshold cycle (ΔCt) values was calculated for each mRNA using the 2-ΔΔCt method against the housekeeping gene acidic ribosomal protein. The fold changes in mRNA levels were related to a reference sample (human cDNA). In parallel, release of (b) MMP-8 and (d) MMP-13 protein was measured by ELISA of cell culture supernatants (n = 12 to 13). Data were analysed versus normoxia by Wilcoxon signed rank test: *P < 0.05, **P < 0.01.
Figure 5
Figure 5
Cell migration through type-I collagen is enhanced under hypoxic conditions: effect of matrix metalloproteinase inhibition. Rheumatoid arthritis fibroblast migration was investigated under normoxic (21% oxygen) and hypoxic (1% oxygen) conditions over a 48-hour culture period. The top chamber was coated with porcine type-I collagen, followed by the addition of 20,000 fibroblasts per well. The chambers were subsequently placed in a 24-well culture plate, with the lower chambers containing DMEM plus 10% FCS. A universal MMP inhibitor (GM6001; 10 μM) was added to both the bottom and top chambers of selected inserts, before exposure to either normoxia or hypoxia. Migration from six different patients, assayed in triplicate, is shown. Data presented as the mean ± standard error of the mean and were analysed by repeated-measures one-way analysis of variance, with Bonferroni's post hoc test for multiple comparisons: **P < 0.01, ***P < 0.001.
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