Sains Malaysiana 46(10)(2017): 1895–1902


Differential Effects of Activated Protein C on Synovial Fibroblasts in Response to Hypoxia and Normoxia

(Perbezaan Kesan Protein C yang Diaktifkan pada Sinovium Fibroblas dalam Tindak Balas kepada Hipoksia dan Normosia)




1Bio-Safety Research Institute, College of Veterinary Medicine, Chonbuk National University, Iksan, Jeonbuk 54596, Republic of Korea


2Sutton Arthritis Research Laboratories, Institute of Bone and Joint Research, Kolling Institute of Medical Research, University of Sydney at Royal North Shore Hospital, St. Leonards, NSW2065



Received: 4 March 2016/Accepted: 13 March 2017




Rheumatoid arthritis (RA) is a chronic disease characterized by inflammation of the joints and their lining or synovium. Previous studies showed that the synovium in RA patients is more hypoxic than normal synovium. Activated protein C (APC) has anticoagulant and anti-inflammatory effects and is highly expressed in the joints of RA patients. We examined the effect of APC on RA and normal synovial fibroblasts under hypoxic conditions. Human synovial fibroblasts were isolated from the synovial tissues of RA patients and normal controls and cells were exposed to recombinant APC under normoxic (21% oxygen) or hypoxic (1% oxygen) conditions. Cell proliferation was measured using MTT assays. Cell lysates and conditioned media were collected and assayed for matrix metalloproteinase (MMP)-2, MMP-9 and p38 using zymography and western blots. Proliferation of both normal and RA synovial fibroblasts dose-dependently increased after APC treatment in normoxic conditions. Under hypoxia, APC enhanced RA cell proliferation but had no effect on normal fibroblasts. MMP-2 production and activation were significantly augmented by APC in both cell types under normoxia and hypoxia conditions. However, activated MMP-2 was more reduced in cells under hypoxia than normoxia. APC substantially reduced the phosphorylation of p38 in normal and RA synovial fibroblasts under hypoxia. No difference in p38 phosphorylation was observed under normoxia. The receptor for APC, endothelial protein C receptor (EPCR), was elevated in normal fibroblasts under hypoxic conditions whereas in RA cells, EPCR was highly expressed under both normoxic and hypoxic conditions. We found that hypoxia enhanced the effect of APC on RA synovial fibroblasts through activation of MMP2 and inhibition of p38 phosphorylation. Our results suggested that APC may suppress joint destruction and progression of inflammation in a hypoxic RA environment.


Keywords: Activated protein C; hypoxia; MMP-2; rheumatoid arthritis; synovial fibroblast




Reumatoid artritis (RA) adalah penyakit kronik yang dicirikan oleh keradangan sendi serta lapisan atau sinovium. Kajian terdahulu menunjukkan bahawa sinovium dalam pesakit RA lebih hipoksik daripada sinovium biasa. Protein yang diaktifkan C (APC) mempunyai kesan antikoagulan dan anti-radang yang sering terjadi dalam sendi pesakit RA. Kami mengkaji kesan APC pada RA dan sinovium fibroblas normal dalam keadaan hipoksia. Sinovium fibroblas manusia telah diasingkan daripada tisu sinovium pesakit RA dan kawalan normal, dan sel terdedah kepada APC rekombinan dalam keadaan oksigen (21% oksigen) atau hipoksik (1% oksigen). Proliferasi sel diukur menggunakan ujian MTT. Sel lisat dan media bersyarat dikumpulkan dan diuji pada matriks metaloproteinase (MMP) -2, MMP-9 dan p38 menggunakan zimografi dan western blots. Proliferasi pada kedua-dua dos biasa sinovium fibroblas dan RA meningkat bergantung pada rawatan APC dalam keadaan normosik. Di bawah hipoksia, APC meningkatkan percambahan sel RA tetapi tidak mempunyai kesan pada fibroblas biasa. Pengeluaran dan pengaktifan MMP-2 secara signifikan ditambah oleh APC dalam kedua-dua jenis sel di bawah keadaan normoksia dan hipoksia. Walau bagaimanapun, MMP-2 diaktifkan berkurangan dalam sel di bawah hipoksia daripada normoksia. APC secara substansial mengurangkan fosforilasi p38 dalam sinovium fibroblas normal dan RA di bawah hipoksia. Tiada perbezaan dalam p38 fosforilasi diperhatikan di bawah normoksia. Reseptor untuk APC dan reseptor protein endotelium C (EPCR) dinaikkan pada fibroblas biasa di bawah keadaan hipoksik manakala dalam sel RA, EPCR dinyatakan di bawah kedua-dua keadaan normoksik dan hipoksik. Kami mendapati bahawa hipoksia meningkatkan kesan APC terhadap sinovium fibroblas RA melalui pengaktifan MMP2 dan perencatan p38 fosforilasi. Keputusan kami mencadangkan supaya APC dapat menghentikan kemusnahan sendi dan perkembangan keradangan dalam persekitaran RA hipoksik.

Kata kunci: Hipoksia; MMP-2; protein C diaktifkan; reumatoid artritis; sinovium fibroblas


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