The α5β1 integrin provides matrix survival signals for normal and osteoarthritic human articular chondrocytes in vitro

Authors


Abstract

Objective

Chondrocyte cell death may play an important role in the development of arthritis. The goal of the present study was to evaluate the role of the extracellular matrix (ECM) in promoting chondrocyte survival via signals through the integrin family of ECM receptors.

Methods

Chondrocytes were isolated by sequential enzymatic digestion from normal ankle cartilage of organ donors and from osteoarthritic (OA) knee tissue obtained from patients undergoing total knee replacement. Cell survival in monolayer and in suspension culture was measured using fluorescent labels after treatment with specific integrin-blocking antibodies and echistatin, a disintegrin peptide. A quantitative enzyme-linked immunosorbent assay for histone-associated DNA fragments and morphologic evaluation by electron microscopy were used to evaluate apoptosis.

Results

Freshly isolated chondrocytes died when plated in serum-free media at low density on poly-L-lysine, but showed >95% survival on fibronectin (FN). A monoclonal blocking antibody to the α5-integrin subunit (FN receptor) significantly inhibited survival on FN, whereas control antibodies had no effect. Likewise, treatment of freshly isolated chondrocytes in serum-free alginate-suspension culture with the α5-blocking antibody resulted in cell death in a dose-dependent manner, with 20 μg/ml of the antibody reducing normal chondrocyte survival to 20% of that in controls, and OA chondrocyte survival to 23% of that in controls. Antibody inhibition of αv and α1 integrins or treatment with echistatin did not cause cell death. Addition of insulin-like growth factor 1 (IGF-1; 100 ng/ ml) was not able to improve survival of α5-antibody–treated cells. However, treatment with 10% fetal bovine serum improved normal chondrocyte survival to 98% (a 5.1-fold increase) and OA chondrocyte survival to 64% (a 2.8-fold increase). Cell death due to α5 inhibition was associated with apoptosis.

Conclusion

These results demonstrate that chondrocyte survival signals are transmitted via the α5β1 FN receptor. Inhibition of matrix survival signals mediated by α5β1 also inhibits the ability of IGF-1 to promote survival, suggesting that IGF-1–mediated survival signaling may require a cosignal from α5β1.

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