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I should like to comment on a recent paper in the Annals of the Rheumatic Diseases, which demonstrates the expression of angiopoietin-1 in the synovium of patients with rheumatoid arthritis (RA).1 Angiopoietin-1 was demonstrated in most of the RA synovial membranes examined by immunohistochemistry, but there was a marked discrepancy in the amount and distribution of angiopoietin-1 at the mRNA level (as demonstrated by in situ hybridisation) compared with that seen at the protein level (as demonstrated by immunohistochemistry). This was not commented on by the authors of the paper but is rather surprising, particularly as such a discrepancy is not seen even with cytokine expression, which has a labile mRNA due to AUUUA-rich areas of the 3′ untranslated region. It is not stated whether angiopoietin-1 mRNA has similar such regions and whether its mRNA is labile, but even this is unlikely to explain the discrepancy between the results of in situ hybridisation and immunohistochemistry for angiopoietin-1.
Using serial sections of RA synovial membranes, the authors stated that both CD68 positive macrophages and CD68 negative fibroblasts in the lining layer of the synovium contain angiopoietin-1, yet this is not very evident in the images displayed in fig 1. It surely would have been preferable to perform dual immunohistochemistry for CD68 and angiopoietin-1, or even to combine in situ hybridisation for angiopoietin-1 mRNA with immunohistochemistry for CD68 to demonstrate more definitively which cells in the RA synovial membrane are producing angiopoietin-1.
We thank Dr Smith for his interest in our study and for his comments about the differences between protein levels of angiopoietin-1 in RA synovium detected by immunohistochemistry and mRNA levels for angiopoietin-1 detected by in situ hybridisation. Immunohistochemical analysis frequently showed angiopoietin-1 protein in the synovial lining layer, as well as in cells within the sublining tissues, both in perivascular areas and in areas remote from vessels. Analysis of angiopoietin-1 mRNA expression by in situ hybridisation showed mRNA in these sites, but at low levels and with significantly less frequent detection of mRNA within the synovial lining layer. All the tissue samples evaluated by in situ hybridisation in this study, however, were paraffin embedded samples. It is known that during the processing of tissues into paraffin blocks, mRNA can be lost, even when care is taken to avoid RNase contamination. Owing to the limitations of this technique, we went on to examine both mRNA and protein expression in cultured synovial fibroblasts in vitro. We demonstrated angiopoietin-1 mRNA expression by northern blot analysis in unstimulated, as well as in tumour necrosis factor α stimulated, synovial fibroblasts, and confirmed the production of angiopoietin-1 protein by these cells using an enzyme linked immunosorbent assay (ELISA).
We believe that the serial sections in fig 1 of our previous paper1 show that angiopoietin-1 protein is present in both CD68 expressing and non-expressing cells. Neither serial sections comparing angiopoietin-1 expression by in situ hybridisation and immunohistochemistry nor dual immunohistochemistry for CD68 and angiopoietin-1 is likely to yield new information.
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