Introduction Cysteine-rich angiogenic inducer 61 (Cyr-61) and connective tissue growth factor (CTGF) – also known as CCN1 and CCN2 respectively – are involved in healing and wound repair. CCN2 has been found to be over-expressed in fibroblasts in conditions of exaggerated healing, fibrosis, and scar formation, whereas CCN1 has been shown to limit fibrosis through cellular senescence of myofibroblasts [Jun 2011].

This is of interest as the histological changes seen in tendinopathy can be likened to an exaggerated wound healing and display characteristics of fibrosis.

It is known that tendons need a certain degree of strain to keep healthy, and this pilot study set out to find whether CCN1 and CCN2 are expressed by tendon cells, and how they are affected by cyclical strain. We also wanted to study how these genes are affected by TGF-β; a known agent in fibrosis and scar formation.

Methods Rat Achilles tendon was used to establish primary cell cultures. Cells in passage 3 and were seeded with a density of 1.75×10⁵cells/well in 6-well plates with a collagen I pre-coated elastic membrane (BioFlex BF-3001C). After serum-starvation in 1% FBS for 24 h, the plates were positioned in a FlexCell strain unit FX-4000. Using a frequency of 1 Hz, a total of 120 min cyclic tension of 10% strain was performed. Another group was exposed to TGF-β (10 ng/ml). Control samples were kept in the same conditions (with and without TGF-β), but not strained.

RNA was extracted using the RNeasy kit from Qiagen and RT-qPCR was performed to analyse the expression of the CCN1 and CCN2 genes.

Immunohistochemistry was used to verify that tendon cells in culture express CCN1 and CCN2.

Results Cultured rat Achilles tendon cells expressed both CCN1 and CCN2 as shown by immuno-histochemistry.

RT-qPCR, showed a slight decrease in both CCN1 and CCN2 following 120 min of 10% strain in the tendon cell cultures.

TGF-β increased CCN1- and CCN2-expression, but in the strained cells this effect on CCN2 was abolished (Figure 1).

• Download figure
• Open in new tab
• Download powerpoint

Abstract 13 Figure 1

RT-qPCR analysis of CCN1 and CCN2 following strain and/or TGF-β administration

Discussion The downregulation of CCN1 and CCN2 following cyclic strain, was unexpected, as skin fibroblasts – cells similar to tenocytes – are known to react to strain with an upregulation of CCN2 [Chaqour 2006]. However, endothelial cells have been shown to downregulate CCN2 when strained, and this is explained by the notion that physiological shear stress protects vessels from fibrosis and atherosclerotic disease [Chaqour 2006].

It may very well be that tendons are protected in a similar manner, with tensile strain promoting an anti-fibrotic environment by downregulation of CCN2. It would seem that CCN1, with anti-fibrotic abilities, is upregulated in response to the profibrotic TGF-β.

Further studies are required, but regulation of CCN1 and CCN2 may play a role in preventing fibrotic disease – such as tendinopathy – in tendons.

References Jun et al. Nat Rev Drug Discov. 2011;10:945–63

Chaqour et al. FEBS J. 2006;273:3639–3649