Abstract

Introduction

Rheumatoid arthritis (RA), a polyarticular disease of autoimmune nature [1], and osteoarthritis (OA) a noninflammatory degenerative disease of the articular cartilage [2], have in common an increased tendency for new blood vessel formation [3-5]. This phenomenon, however, may not necessarily proceed in a similar manner in the two conditions. Neoangiogenesis is important in the development of new cartilage and mineralization in OA [6], whereas the same process contributes to synovitis, pannus formation and articular cartilage destruction in RA [7]. In a previous study [8], we showed increased levels of expression of the angiogenic factors vascular endothelial growth factor (VEGF) and platelet-derived endothelial cell growth factor (PD-ECGF; also known as thymidine phosphorylase) and, as a consequence, an increased microvessel density (MVD) of the entire synovial vasculature in both RA and OA, relative to normal. However, the presence of an activated synovial vasculature ('VEGF/kinase insert domain protein receptor [KDR] complex' expression) was high only in the case of RA. This failure of OA to activate the VEGF/KDR pathway, in the presence of increased VEGF expression, is consistent with the degenerative nature of the disease, whereas the profoundly upregulated VEGF/KDR pathway in RA pursues a destructive angiogenesis-related course.

Hypoxia inducible factor (HIF)-1α and HIF-2α are important transcription factors, regulating VEGF gene responses to hypoxic stimuli. Reduction in the degradation rate of HIF-αs, as occurs under hypoxic stress, results in accumulation of HIF-1α and HIF-2α proteins and upregulation of the angiogenic process [9,10]. The direct link between accumulation of HIF-αs and overexpression of VEGF [11,12], and the important role of the VEGF angiogenic pathway in arthritides suggest a central role for HIF-αs in the pathogenesis of RA and OA.

In the present study, we investigated the immunohistochemical expression of HIF-1α and HIF-2α in synovial tissues in RA and OA. The results were related to the angiogenic process in the synovial membrane and to the antiapoptotic protein bcl-2. More specifically, the results were analyzed with reference to MVD, VEGF, and the activation of the angiogenic pathways VEGF/KDR and PD-ECGF.

Material and methods

Formalin-fixed paraffin-embedded synovial tissues were retrieved from the files of the Departments of Pathology, Democritus University of Thrace, Alexandroupolis, Greece, and Nuffield Orthopaedic Centre, Oxford, UK. The material was from 22 cases of active RA, 34 cases of OA and 22 nonarthritic cases derived from hip joint replacement following fracture. Table ​Table11 shows the patient characteristics. Histological confirmation of the arthritic pathology was performed on haematoxylin and eosin stained sections.

Table 1

Patient characteristics

	Fracture	Rheumatoid arthritis	Osteoarthritis
Number of patients	22	22	34
Age range (median; years)	55–64 (58)	28–72 (62)	69–78 (72)
Sex
Male	8	12	14
Female	14	10	20
Location
Hip	22	8	25
Knee	0	10	9
Wrist	0	4	0

Open in a separate window

Results

HIF-α expression in synovial lining cells

HIF-1α and HIF-2α expression was both cytoplasmic and nuclear in arthritic synovial tissues (Fig. 1a,1b). The median percentage of synovial lining cells exhibiting cytoplasmic and/or nuclear HIF-1α expression was 50% (range 10–80%; 95% confidence interval [CI] 40–61%) in RA and 60% (range 20–100%; 95% CI 53–73%) in OA. For HIF-2α, the median percentage of positive synovial cells was 60% (range 10–80%; 95% CI 40–63%) in RA and 50% (range 0–100%; 95% CI 43–66%) in OA (Table ​(Table2).2). Normal synovia were, in most cases, unreactive, and only in a small percentage of cases (6/22 and 4/22 for HIF-1α and HIF-2α, respectively) exhibited a weak and focal cytoplasmic reactivity that in no case exceeded 20% of the cells present. This difference in the frequency of HIF-1α and HIF-2α between the arthritides and the normal tissues was statistically significant (Fig. ​(Fig.2;2; P < 0.0001).

Open in a separate window

Figure 1

(a)Mixed nuclear and cytoplasmic expression of synovial lining and stromal cells in a case of RA. Note a similar reactivity in the lymphoid and plasma cell component. (b) Mixed nuclear and cytoplasmic expression of synovial lining cells, stromal cells, and endothelial cells in a case of osteoarthritis (OA). (c) Mixed nuclear and cytoplasmic expression of endothelial cells in a case of RA.

Open in a separate window

Figure 2

Overexpression of HIF-1α and HIF-2α in OA and RA synovium, relative to normal.

Table 2

Percentage of synovial cells expressing HIF-1α and HIF-2α proteins in normal, rheumatoid, and osteoarthritic synovium

	Normal (n= 22)		OA (n = 34)		RA (n = 22)
	HIF-1α	HIF-2α	HIF-1α	HIF-2α	HIF-1α	HIF-2α
Minimum	0.00	0.00	20.00	0.00	10.00	10.00
25th percentile	0.00	0.00	40.00	30.00	35.00	25.00
Median	0.00	0.00	60.00	50.00	50.00	60.00
75th percentile	10.00	0.00	90.00	90.00	75.00	80.00
Maximum	20.00	10.00	100.0	100.0	80.00	80.00
Mean	5.45	1.81	63.53	55.29	50.91	50.91
Standard deviation	9.11	3.94	29.12	32.50	24.08	28.10
Standard error	1.94	0.84	4.99	5.57	5.13	5.99
Lower 95% CI	1.41	0.06	53.37	43.96	40.23	39.55
Upper 95% CI	9.49	3.56	73.69	66.63	61.59	63.37

Open in a separate window

CI, confidence interval; HIF, hypoxia inducible factor; OA, osteoarthritis; RA, rheumatoid arthritis.

The pathologic synovial tissues were grouped into categories of high and low HIF-α reactivity (Table ​(Table3),3), using a 40% cytoplasmic/nuclear reactivity as a cut-off point. This represents the percentage of HIF positive synovial cells corresponding to the lowest 95% CI value of HIF reactivity noted in the arthritic material.

Table 3

Expression of HIF-1α and HIF-2α in normal, rheumatoid, and osteoarthritic synovium

	Fractures	RA	OA	P
HIF-1α
Negative	16	0	0
Low	6	10	13	<0.0001
High	0	12	21
HIF-2α
Negative	18	0	0
Low	4	8	14	<0.0001
High	0	14	20

Open in a separate window

HIF, hypoxia inducible factor; OA, osteoarthritis; RA, rheumatoid arthritis.

Analysis of the extent of HIF-α expression

Table ​Table44 shows the association between the extent of HIF-αs expression in RA and OA and the various parameters investigated. The most important findings are as follows. First, In OA a high HIF-1α, but not HIF-2α, synovial lining/stromal cell expression was significantly associated with increased standard MVD, VEGF/KDR activated MVD, and PD-ECGF expression. Second, the activated MVD and synovial stromal cell PD-ECGF reactivity were significantly higher in RA than in OA (P < 0.001), and this was independent of HIF-α expression (Fig. 3a,3b). Finally, extensive bcl-2 expression by synovial membrane cells was noted only in OA, and this was directly associated with HIF-1α expression (Fig. ​(Fig.44).

Open in a separate window

Figure 3

(a) Relationship of HIF-1α expression in OA and RA with VEGF/KDR vascular activation pathway. Note that the degree of VEGF/KDR microvessel density is directly correlated with the degree of HIF-1α expression only in the case of OA; VEGF/KDR is consistently high in RA, and higher than in OA. (b) Relationship of HIF-1α expression in osteoarthritis (OA) and rheumatoid arthritis (RA) with stromal cell thymidine phosphorylase (TP; referred to in the text as platelet-derived endothelial cell growth factor [PD-ECGF]) reactivity. Note that the degree of TP expression is directly correlated with the degree of HIF-1α expression only in the case of OA; TP expression is consistently high in RA, and higher than in OA.

Open in a separate window

Figure 4

Relationship of HIF-1α expression in OA and RA with bcl-2 reactivity. Bcl-2 protein is almost exclusively expressed in OA and is significantly related to the extent of HIF-1α expression.

Table 4

Correlation of HIF-1α and HIF-2α expression with MVD, VEGF/KDR activated MVD, and PD-ECGF expression by synovial lining and stromal cells, and with bcl-2 expression, in the rheumatoid and osteoarthritic synovium

	HIF-1α			HIF-2α
	Low	High	P	Low	High	P
Rheumatoid arthritis
Standard MVD	57 ± 10	63 ± 7	0.16	61 ± 6	59 ± 10	0.63
Activated MVD	27 ± 12	34 ± 10	0.14	31 ± 10	31 ± 12	0.97
% PD-ECGF lin. cells	50 ± 13	55 ± 24	0.57	50 ± 30	54 ± 12	0.65
% PD-ECGF str. cells	82 ± 10	81 ± 9	0.95	80 ± 10	82 ± 9	0.51
% bcl-2	4 ± 8	2 ± 4	0.54	0 ± 0	5 ± 7	-
Osteoarthritis
Standard MVD	58 ± 10	73 ± 18	0.01	68 ± 23	66 ± 11	0.71
Activated MVD	15 ± 3	18 ± 4	0.01	16 ± 3	17 ± 4	0.46
% PD-ECGF lin. cells	51 ± 14	68 ± 21	0.01	55 ± 18	66 ± 21	0.14
% PD-ECGF str. cells	30 ± 17	49 ± 26	0.01	34 ± 18	47 ± 21	0.13
% bcl-2	5 ± 8	32 ± 25	0.0007	18 ± 25	25 ± 23	0.46

Open in a separate window

HIF, hypoxia inducible factor; lin., synovial lining; MVD, microvessel density; OA, osteoarthritis; PD-ECGF, platelet-derived endothelial cell growth factor; RA, rheumatoid arthritis; str., stromal.

Discussion

RA and OA, two common conditions with different clinical features [1,2], have in common an increased tendency for new blood vessel formation [3-5]. The importance of VEGF in the pathogenesis of RA and OA has been emphasized in several recent reports [7,19]. 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 [23,24], 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) [25-28].

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 [30-33]. 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 [34-36], 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 [37,38]) 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 [41-44]. 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. ​Fig.55.

Open in a separate window

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 factor.

Competing interests

None declared.

Abbreviations

APAAP = alkaline phosphatase/antialkaline phosphatase; CI = confidence interval; HIF = hypoxia inducible factor; KDR = kinase insert domain protein receptor; mAb = monoclonal antibody; MVD = microvessel density; OA = osteoarthritis; PD-ECGF = platelet-derived endothelial cell growth factor; RA = rheumatoid arthritis; TBS = tris-buffered saline; VEGF = vascular endothelial growth factor.

References