Establishment of a matrix-associated transepithelial resistance invasion assay to precisely measure the invasive potential of synovial fibroblasts.
OBJECTIVE
Synovial fibroblasts (SFs) contribute to several aspects of the pathogenesis of rheumatoid arthritis (RA) and have been implicated most prominently in the progressive destruction of articular cartilage. Targeting the invasive phenotype of RASFs has therefore gained increasing attention, but the precise measurement of their invasive capacity and the evaluation of potential treatment effects constitute a challenge that needs to be addressed. This study used a novel in vitro invasion assay based on the breakdown of transepithelial electrical resistance to determine the course of fibroblast invasion into extracellular matrix.


METHODS
A matrix-associated transepithelial resistance invasion (MATRIN) assay was used to assess SFs from patients with RA in comparison with SFs from patients with osteoarthritis (OA). The SFs were grown on a commercially available collagen mix that was placed onto the upper side of a Transwell polycarbonate membrane. In addition, freshly isolated cartilage extracts were studied to assess the conditions in vivo. Under this membrane, a monolayer of MDCK-C7 cells was seeded to create a high electrical resistance.


RESULTS
Invasion of fibroblasts into the matrix affected the integrity of the MDCK-C7 monolayer and led to a measurable decrease and subsequent breakdown of electrical resistance. Unlike in the assay with OASFs, which did not achieve a breakdown of resistance up to 72 hours, RASFs exhibited a pronounced invasiveness in this assay, with a 50% breakdown after 42 hours. Treatment of fibroblasts with either a matrix metalloproteinase inhibitor or antibodies against beta1 integrin significantly reduced the invasiveness of RASFs.


CONCLUSIONS
The MATRIN assay is a valuable and sensitive biologic assay system that can be used to determine precisely the invasive potential of RASFs in vitro, and thus would be suitable for screening anti-invasion compounds.
Wunrau
Christina
C
Institute of Experimental Musculoskeletal Medicine, University Hospital Muenster, Domagkstrasse 3, Muenster, Germany.
Schnaeker
Eva-Maria
EM
Freyth
Katharina
K
Pundt
Noreen
N
Wendholt
Doreen
D
Neugebauer
Katja
K
Hansen
Uwe
U
Pap
Thomas
T
Dankbar
Berno
B
D000818:Animals; D000906:Antibodies / Q000276:immunology / Q000494:pharmacology; D001172:Arthritis, Rheumatoid / Q000473:pathology*; D001681:Biological Assay / Q000379:methods*; D002450:Cell Communication / Q000502:physiology; D002460:Cell Line; D045744:Cell Line, Tumor; D002465:Cell Movement / Q000187:drug effects / Q000502:physiology*; D002478:Cells, Cultured; D004285:Dogs; D017097:Electric Impedance; D005109:Extracellular Matrix / Q000473:pathology*; D005347:Fibroblasts / Q000473:pathology*; D006801:Humans; D019012:Integrin beta1 / Q000276:immunology; D007668:Kidney / Q000166:cytology; D008545:Melanoma / Q000473:pathology; D045726:Metalloproteases / Q000037:antagonists & inhibitors; D010003:Osteoarthritis / Q000473:pathology*; D012878:Skin Neoplasms / Q000473:pathology; D013583:Synovial Membrane / Q000473:pathology*
D000906:Antibodies; D019012:Integrin beta1; D045726:Metalloproteases