Introduction Rotator cuff healing after injury and/or repair is often compromised by excessive fibrosis both at the lesion site and in the surrounding tendon, particularly in patients with diabetes. Diabetic and hyperglycaemic patients have a higher incidence of tendinopathy than healthy populations. Proposed explanations, including a direct effect of glucose on tenocytes, are speculative. Metformin, a safe and widely-prescribed anti-hyperglycaemic agent, has direct effects on heart, nasal and kidney fibroblasts, preventing differentiation into myofibroblasts and reducing fibrosis.1,2 Its effects on tendon are unknown.
This study thus aimed to determine the molecular changes that occur in tendon explants with or without metformin in both a normoglycaemic and a hyperglycaemic environment.
Methods Ovine infraspinatus tendon explants were cultured for 5 or 17 days in normal (5.5 mM) or high (25 mM) glucose in isotonic media (+10% FBS). After 8 days in the 17-day cultures, cells had grown out from the explants onto the plastic, so both outgrowth cells and explants were continued separately in varied glucose media. In a second experiment, explants were similarly cultured for 15 days with the addition of 0.5 mM or 1 mM metformin. Total RNA was isolated from tendon and cells and realtime RT-PCR performed using primers for matrix proteins, enzymes involved in matrix metabolism and other cellular factors.
Results Stress depriving tendon in culture significantly affected 14 of 16 matrix protein genes and all 13 matrix-modifying enzymes tested compared to ex vivo tendon. COL2A1, COMP, DCN, ELN, FMOD, VCAN and LOXL4 expression decreased; the other 19 changed genes increased. Higher glucose concentrations in the media significantly increased tendon explant expression of 8 of 16 matrix protein genes (COL1A1, COL3A1, COL5A1, COMP, DCN, ELN, FBN1 and LUM), five of 13 enzymes (ADAMTS4, ADAMTS5, MMP13, LOXL3 and LOXL4) and CTGF. Increased glucose had no significant effect on any gene expression by outgrowth cells, however these cells had significantly elevated expression of smooth muscle actin (ACTA2; p < 0.001) compared to explants.
Under normoglycaemic conditions, metformin halved tendon explant CTGF (p = 0.016) and COL1A1 (p = 0.017) expression. Increased MMP13 expression was partially abrogated by metformin (p < 0.025). Gene expression of TGFB (p < 0.034), scleraxis (SCX; p ≤ 0.007) and VCAN (p ≤ 0.003) were restored to ex vivo levels by metformin. In hyperglycaemic media, increased CTGF expression was prevented (p = 0.039).
Discussion Hyperglycaemic conditions significantly affected tenocyte matrix metabolism only when the cells were within their extracellular matrix (ECM) microenvironment. This suggests that the effects of excess glucose on tenocytes, though substantial, are indirect and via modifications to the ECM, perhaps by the action of advanced glycation endproducts. Fibrosis has not been widely recognised in association with tendon injury, however, a recent study reports induction of genes associated with epithelial (epitenon) to mesenchymal transition following acute Achilles tenotomy and repair in rats.3 Peripheral fibrosis of the epitenon and joint capsule are common in conditions such as frozen shoulder. This evidence, and the appearance of myofibroblasts from tendon stress-deprived in culture, suggests that targeting these cells and associated excessive fibrosis with metformin may be a potential therapy for limiting tendinopathy and enhancing tendon repair after injury.
The changes seen in these tendon explants by the addition of metformin would be expected to result in a reversal of collagen accumulation in tendon. The prevention of versican loss may also decrease interfasicular adhesion. Ongoing studies will determine whether metformin has beneficial effects in reducing tendon lesion fibrosis, and normalising tenocyte phenotype, in an animal model.
References 1 Bai J, et al. PLoS One. 2013;8:e72120
2 Park IH, et al. Otolaryngol Head Neck Surg. 2014;150:148–153
3 Sugg KB, et al. J Orthop Res. 2014;32:944–951
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