RA and OA, two common conditions with different clinical features [1
], have in common an increased tendency for new blood vessel formation [3
]. The importance of VEGF in the pathogenesis of RA and OA has been emphasized in several recent reports [7
]. Following chronic inflammation, an upregulation of VEGF increases vascular permeability [20
], resulting in edema, protein leakage, and probably granulation tissue formation (pannus) with erosion of the articular cartilage and progressive destruction of the joint. In a previous study [8
], we showed that VEGF is overexpressed in RA and OA, and such pathologic synovia are highly vascularized as compared with normal controls. Similarly, the expression of PD-ECGF, another potent factor for angiogenesis, was considerably enhanced in the arthritic synovial membranes. In the present study, we found a varying degree of expression of HIF-αs in the synovial lining and stromal cells of RA and OA, whereas normal synovium was persistently negative. The lack of HIF-1α expression by the normal synovium was also reported by Hollander and coworkers [21
]. In the latter study, however, HIF-1α was more prominent in RA than in OA, which was not confirmed in our study, which included a larger number of specimens. As HIF-αs are directly involved in the upregulation of VEGF, it might be suggested that VEGF overexpression in arthritides is probably a result of HIF pathway activation.
There were differences in the expression of HIF-αs between RA and OA. Thus, although the extent of detection of HIF-αs was more or less similar in both conditions, HIF-1α expression in OA only was significantly associated with standard MVD, VEGF/KDR activated MVD, PD-ECGF expression and with the antiapoptotic protein bcl-2. In the case of RA, the high standard MVD was independent of the extent of HIF-1α expression, whereas the VEGF/KDR activated MVD was persistently higher than that in OA, irrespective of the extent of HIF-1α staining. Similarly, the extent of PD-ECGF expression in the synovial rheumatoid stroma was significantly higher than that in the osteoarthritic, regardless of the magnitude of HIF-1α reactivity.
Hypoxic stimulation is the primary cause for intracellular HIF-1α accumulation, not because of increased mRNA transcription or translation but rather as a result of a redox-sensitive stabilization [22
]. Following HIF-α heterodimerization with the HIF-1β unit, the complex enters into the nucleus, binds to DNA at the hypoxia response elements of target genes (i.e. VEGF), and induces transcription. Although upregulation of the HIF pathway may also occur as a result of a genetic alteration [23
], the nonmalignant nature of RA and OA suggests that hypoxic signaling may be implicated in the pathogenesis of these diseases. However, a range of growth factor signalling pathways and cytokines can also upregulate HIF-αs (e.g. tumor necrosis factor-α, epidermal growth factor, insulin growth factor-II and thrombin) [25
The direct association of the extent of HIF-1α with MVD and the VEGF/KDR activated MVD in OA is consistent with the notion that VEGF is induced by hypoxia after activation of the HIF-α pathway. This is further reinforced by the direct association between HIF-1α expression and the expression of proteins PD-ECGF and bcl-2. Oxidative stress is probably a major stimulus for the expression of PD-ECGF [29
], whereas hypoxic induction of bcl-2 was shown to prevent apoptotic cell death induced by hypoxia [30
]. It is possible then that within the degenerative context of the osteoarthritic disease, impaired vascular homeostasis results in focal, still progressively expanding, hypoxic regions in the synovium. In these areas, upregulation of HIF-αs leads to overexpression of VEGF and PD-ECGF by the synovial lining and stromal cells, and to the genesis of a defective vascular network with poor survival ability. As previously shown, the activation of the OA vasculature is low, despite the over-production of VEGF [8
]. Although a relationship between HIF-1α and VEGF/KDR activated MVD was observed in the present study, the magnitude of the increase was limited. Given that the importance of the VEGF/KDR pathway in mediating endothelial cell survival has repeatedly been confirmed [34
], the survival ability of the OA vasculature may be hindered despite an upregulated HIF/VEGF system.
In RA, high MVD, high activated VEGF/KDR pathway, and upregulated PD-ECGF expression were constant features, independent of the extent of the activated HIF-α pathway. This may mean that angiogenesis is not exclusively dependent on the extent of HIF reactivity and that hypoxia is not the only factor that upregulates the HIF/VEGF pathway. It is well known that a variety of cytokines are produced by lymphocytes in the context of the rheumatoid pathology (i.e. interleukin-1 and tumor necrosis factor [37
]) and that blocking such cytokines induces clinical remission of the disease [39
]. Cytokines released by the immune response system may directly stimulate both HIF-α dependent mRNA transcription [40
] and HIF-independent VEGF or PD-ECGF overexpression [41
]. The latter mechanism is less likely to be engaged in OA, where the synovial tissues bear reduced cytokine expression as compared with RA [45
]. By contrast, a dense vasculature, characterized by a VEGF/KDR-activated status [8
] and pannus formation, is probably a primary event in RA.
Our finding that bcl-2 is predominantly expressed in OA whereas rheumatoid synovium lacks expression of this anti-apoptotic protein is in direct contrast to a previously reported study by Perlman and coworkers [46
]. Although this discrepancy is difficult to explain, forced bcl-2 down-regulation failed to induce cell death in rheumatoid fibroblasts, suggesting that bcl-2 is probably of minor importance in the pathology of the synovium [46
]. An experimental study from the same group concluded that the expression of bcl-2 is temporally expressed, so that its role in RA may be confined to just a step in the development of rheumatic pathology [47
]. In accordance with the diminished role of bcl-2 in RA is a study conducted by Chu and coworkers [48
], which showed lack of bcl-2 involvement in apoptosis in RA.
It is concluded that activation of the HIF-α pathway occurs in both RA and OA, although for unrelated reasons. Hypoxia, consistent with an impaired vascular homeostasis, may hinder the angiogenic effect of the upregulated HIF/VEGF pathway in OA. Deranged vascular homeostasis should not be attributed to a defective HIF pathway, but rather to a defective communication between VEGF and vascular receptors. Furthermore, the intact VEGF-dependent angiogenic and vascular survival pathway of RA appears to be cytokine, rather than hypoxia, stimulated. This premise is schematically represented in Fig. .
Figure 5 Schematic representation of the suggested pathogenetic model in OA and RA. HIF, hypoxia inducible factor; TP, thymidine phosphorylase (referred to in the text as platelet-derived endothelial cell growth factor [PD-ECGF]); VEGF, vascular endothelial growth (more ...)