The hypoxic synovial environment regulates expression of vascular endothelial growth factor and osteopontin in juvenile idiopathic arthritis.
OBJECTIVE
Synovial angiogenesis, a critical determinant of juvenile idiopathic arthritis (JIA) pathogenesis, is sustained by various mediators, including vascular endothelial growth factor (VEGF) and osteopontin (OPN). We characterized the contribution of the local hypoxic environment to VEGF and OPN production by monocytic cells recruited to the synovium in JIA.


METHODS
Paired synovial fluid (SF) and peripheral blood (PB) samples were collected from 20 patients with JIA. Mononuclear cells (MC) were isolated, and monocytic cells were purified by adherence, maintained in a hypoxic environment, or subjected to reoxygenation. VEGF and OPN protein concentrations were tested in SF, plasma, and culture supernatants by ELISA, and mRNA expression was assessed in freshly purified and cultured cells by reverse transcriptase-polymerase chain reaction. Synovial tissue was obtained at synovectomy from 4 patients with JIA, and analyzed by immunohistochemistry for VEGF, OPN, CD68, and hypoxia-inducible factor-1alpha (HIF-1alpha).


RESULTS
VEGF mRNA expression was increased in SFMC and SF monocytic cells compared to matched PBMC and PB monocytic cells or SF lymphocytes, correlating with significantly higher protein levels in SF relative to plasma samples. Accordingly, OPN mRNA expression in SF monocytic cells was associated with significant increase of SF protein. Immunohistochemistry revealed the presence of both factors in synovial tissues at the level of the lining and sublining layers, which colocalized with intense CD68 and HIF-1alpha staining, suggesting production by hypoxic synovial monocytic cells. VEGF and OPN expression was abrogated upon SF monocytic cell reoxygenation and maintained by exposure to prolonged hypoxia.


CONCLUSIONS
Hypoxic synovial monocytic cells are a likely source of VEGF and OPN in JIA. These data point to a role for hypoxia in the perpetuation of synovitis in JIA.
Bosco
Maria Carla
MC
Laboratory of Molecular Biology, Department of Pediatrics, G. Gaslini Institute, Genova, Italy. mariacarlabosco@ospedale-gaslini.ge.it
Delfino
Silvana
S
Ferlito
Francesca
F
Puppo
Maura
M
Gregorio
Andrea
A
Gambini
Claudio
C
Gattorno
Marco
M
Martini
Alberto
A
Varesio
Luigi
L
D000293:Adolescent; D015703:Antigens, CD / Q000378:metabolism; D015214:Antigens, Differentiation, Myelomonocytic / Q000378:metabolism; D001171:Arthritis, Juvenile / Q000378:metabolism* / Q000473:pathology / Q000601:surgery; D015415:Biomarkers / Q000378:metabolism; D015687:Cell Hypoxia / Q000502:physiology*; D002478:Cells, Cultured; D002648:Child; D002675:Child, Preschool; D005260:Female; D015870:Gene Expression; D006801:Humans; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000378:metabolism; D008297:Male; D009000:Monocytes / Q000378:metabolism* / Q000473:pathology; D053495:Osteopontin / Q000235:genetics / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism; D013582:Synovial Fluid / Q000166:cytology / Q000378:metabolism; D013583:Synovial Membrane / Q000378:metabolism* / Q000473:pathology; D042461:Vascular Endothelial Growth Factor A / Q000235:genetics / Q000378:metabolism*; D055815:Young Adult; D000097382:CD68 Molecule
D015703:Antigens, CD; D015214:Antigens, Differentiation, Myelomonocytic; D015415:Biomarkers; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D053495:Osteopontin; D012333:RNA, Messenger; D042461:Vascular Endothelial Growth Factor A; C067980:CD68 antigen, human; D000097382:CD68 Molecule; C497442:HIF1A protein, human; C467484:VEGFA protein, human