PLoS One. 2014 Oct 7;9(10):e109524. doi: 10.1371/journal.pone.0109524.

Angiopoietin-like 4 is over-expressed in rheumatoid arthritis patients: association with pathological bone resorption.


Swales C1, Athanasou NA2, Knowles HJ1.
  • 1Botnar Research Centre, Nuffield Department of Orthopaedics Rheumatology and Musculoskeletal Sciences, University of Oxford, Oxford, United Kingdom.
  • 2Pathology Department, Nuffield Orthopaedic Centre, University of Oxford, Oxford, United Kingdom.



INTRODUCTION: Osteoclasts are responsible for the bone loss associated with rheumatoid arthritis (RA). The secreted adipokine angiopoietin-like 4 (ANGPTL4) specifically increases osteoclast-mediated bone resorption. We have investigated expression of ANGPTL4 and its regulatory transcription factor, hypoxia-inducible factor-1 alpha (HIF-1α), in osteoclasts and other cells within rheumatoid synovium. We have also examined whether circulating levels of ANGPTL4 differ in RA patients compared with that in normal controls or patients with osteoarthritis (OA).

RESULTS: Immunohistochemical analysis revealed that bone-apposing osteoclasts within the rheumatoid synovium express both ANGPTL4 and HIF-1α. ANGPTL4 was also strongly expressed in synovial lining cells, endothelial cells, stromal cells, CD68+ macrophages and plasma cells within RA synovium. Little ANGPTL4 was evident in normal synovial tissue. This reflected the over-expression of HIF-1α in rheumatoid versus normal synovial tissue. The concentration of ANGPTL4 was higher in both the serum and the synovial fluid of RA patients than in patients with OA or normal controls. High serum ANGPTL4 associated with elevated levels of the serum marker of bone resorption, receptor activator for nuclear factor κB ligand (RANKL).

CONCLUSIONS: Over-expression of ANGPTL4 in multiple cell types within the rheumatoid synovium potentially provides a local pool of ANGPTL4 to stimulate osteoclast-mediated bone resorption in RA. Additionally, correlation of high serum ANGPTL4 with circulating RANKL suggests that ANGPTL4 may represent a novel marker for bone destruction in RA.

PMID: 25289668



Osteoclasts are large, multi-nucleated cells that digest (resorb) bone. Although a vital regulator of bone remodelling during development, in the mature skeleton they are mostly present in pathological bone loss conditions such as rheumatoid arthritis (RA), osteoporosis and cancer (primary bone tumours or cancer metastasis to bone).

Our laboratory has been studying the effect of hypoxia on bone resorption by mature osteoclasts, and has found that resorption activity is increased 3- to 4-fold at 2-5% O2 [1]. This enhanced activity is regulated by the hypoxia-inducible transcription factor HIF-1a, which both drives production of ATP under hypoxia to provide energy for accelerated resorption and increases production of resorption-promoting factors such as the adipokine angiopoietin-like 4 (ANGPTL4) [2, 3].

Pathological bone loss in RA is caused by osteoclasts. Both bone loss and micro-environmental hypoxia are characteristic of RA and are poor prognostic indicators. In the current paper, we therefore asked the question whether ANGPTL4 is present in RA and whether its expression levels correlate with markers of bone loss in this condition [4].

Given our specific interest in osteoclasts, we first analysed expression of ANGPTL4 and its regulatory transcription factor, HIF-1a, in these cells. Within the rheumatoid synovium, cytoplasmic ANGPTL4 was observed in over 95% of osteoclasts while HIF-1a was expressed by ≤50% of osteoclasts. Expression of HIF-1a and ANGPTL4 was seen to co-localise in resorbing osteoclasts at the synovium-bone interface. This is the first description of expression of either HIF-1a or ANGPTL4 in RA osteoclasts.

ANGPTL4 (and HIF-1a) was also strongly expressed by the synovial lining cells, endothelial cells and stromal fibroblasts within RA synovial tissue, as well as in CD68+ macrophages and plasma cells in the immediate vicinity of lymphoid aggregates. Expression of ANGPTL4 in multiple cell types within the rheumatoid synovium could generate a micro-environment that supports and promotes bone erosion.

We therefore investigated whether levels of secreted ANGPTL4 are also elevated in the synovial fluid of RA patients. ANGPTL4 was 3-fold higher in synovial fluid from RA patients than in non-inflammatory osteoarthritis (OA) patients.

As obtaining synovial fluid is very invasive, we next analysed serum levels of ANGPTL4 in RA patients. Serum ANGPTL4 showed a 10-fold increase in RA patients over either OA patients or normal controls. This was largely driven by a ‘high ANGPTL4’ sub-population of RA patients, with serum levels well outside the normal range. In accordance with our hypothesis that ANGPTL4 drives bone erosion in RA, ‘high ANGPTL4’ patients had significantly more sRANKL (a circulating marker of bone erosion) in their serum than either ‘low ANGPTL4’ RA patients or controls.

Although a larger study cohort is required, with matched serum, synovial tissue/fluid and radiological data, this study indicates that ANGPTL4 might represent not only a serum marker of either progression of joint damage or response to treatment, but also a potential therapeutic target for prevention of bone erosion in RA.



  1. Knowles HJ, Athanasou NA: Acute hypoxia and osteoclast activity: a balance between enhanced resorption and increased apoptosis. J Pathol 2009, (218):256-264.
  2. Knowles HJ, Cleton-Jansen AM, Korsching E, Athanasou NA: Hypoxia-inducible factor regulates osteoclast-mediated bone resorption: role of angiopoietin-like 4. FASEB J 2010, 24(12):4648-4659.
  3. Morten KJ, Badder L, Knowles HJ: Differential regulation of HIF-mediated pathways increases mitochondrial metabolism and ATP production in hypoxic osteoclasts. J Pathol 2013, 229(5):755-764.
  4. Swales C, Athanasou NA, Knowles HJ: Angiopoietin-like 4 is over-expressed in rheumatoid arthritis patients: association with pathological bone resorption. PloS One 2014, 9(10):e109524.




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