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Sains Malaysiana 46(10)(2017):
1895–1902 http://dx.doi.org/10.17576/jsm-2017-4610-28
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) YANG-GYU
PARK1,
JAWUN
CHOI1,
INKYU
SONG1,
CHRISTOPHER
J.
JACKSON2,
SANG-YOUEL
PARK1
& JAE-WON SEOL1* 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 Australia Received: 4 March 2016/Accepted:
13 March 2017 ABSTRACT 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 ABSTRAK 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 REFERENCES Akhavani, M.A., Madden, L., Buysschaert, I., Sivakumar, B.,
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