Epigenetic Status of The Human MMP11 Gene Promoter is Altered in Patellar Tendinopathy.

Author:Rickaby, Rebecca
Position:Research article - Report
 
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Introduction

Patellar tendinopathy (PT), is an exercise-associated injury that results in significant disability and can compromise the career of an athlete (Peers and Lysens, 2005). The prevalence of PT is high in sports, such as volleyball (44.6% [+ or -] 6.6%) and basketball (31.9% [+ or -] 6.8%) (Lian et al., 2005) and it accounts for up to 15% of the soft tissue injuries reported in military cohorts (Rutland et al., 2010). Recently, a study by Bode et al. (2017) found prevalence rates as high as 13.4% in young elite level soccer players (Bode et al., 2017). Furthermore, in addition to the type of sports participation, the duration of training, female gender and hamstring flexibility have all been shown to affect the risk of PT (Morton et al., 2017). There is also some evidence to suggest that both back pain and a family history of tendon problems might be related to the risk of PT (Morton et al., 2017).

From a histological perspective, the development of PT is associated with neovascularisation, an increase in collagen type III fibers and an accumulation of glycosaminoglycans (Rosso et al., 2015). Such changes may, in part, be due to the altered expression of matrix metalloproteinase (MMP) enzymes that have been shown to have an important role in regulating tendon extracellular matrix (ECM) homeostasis (Del Buono et al., 2013). Interestingly, genetic variation within genes (like the MMPs), that code for enzymes that regulate ECM homeostasis, are known to impact on Achilles tendinopathy risk (Raleigh and Collins, 2012; Collins and Raleigh, 2009). However, genetic association studies conducted to identify risk variants have sometimes yielded conflicting data. For example, Raleigh et al. (2009) demonstrated that the G allele of the rs679620 variant within the MMP3 gene increased the risk of Achilles tendinopathy in a South African study, whereas the same variant in a British cohort did not increase risk (El Khoury et al., 2016). Likewise, the COL5A1 rs12722 T variant has been shown to increase risk of Achilles tendinopathy in both South African and Australian cohorts (September et al., 2009) but in British Caucasians this variant does not modify risk (Brown et al., 2017).

We recently suggested that epigenetic modifications, such as DNA methylation, might explain discrepant association signals sometimes obtained in studies on human patellar tendinopathy (El Khoury et al., 2018). Indeed, we demonstrated that methylation changes to a CpG site upstream of the ADAMTS4 promoter were associated with patellar tendinopathy (El Khoury et al., 2018). However, to date, and as far as we are aware, no other research has been published to address the impact of DNA methylation on the risk of human tendinopathy.

The MMP11 enzyme catalytically degrades several components of the extracellular matrix (ECM) including aggrecan, fibronectin and laminin (Araki and Mimura, 2017). Although the detailed role that MMP11 plays in ECM homeostasis is poorly understood, the expression of MMP11 in Achilles tendinopathy is substantially affected. For example, it has been shown that MMP11 RNA levels are both five and six-fold higher in painful and ruptured Achilles tendon respectively, compared to control tissue (Jones et al., 2006). Furthermore, MMP11 expression decreases nearly twofold in human Achilles tenocytes that have been subjected to mechanical strain (Jones et al., 2013). Although these observations may infer a role for MMP11 in tendinopathy, the mechanisms underlying the observations are unknown.

The MMP11 gene was originally mapped to chromosome 22 (Anglard et al., 1995). Multiple transcripts are encoded within the region 24,110,413-24,126,503 (GRch37-hp19 at: https://grch37.ensembl.org/index.html) and expression is known to be altered by DNA methylation (Navarro et al., 2012). Specifically, hypomethylation is associated with transcriptional upregulation of MMP11 (Navarro et al., 2012). With this in mind, we hypothesized that the methylation status of the MMP11 promoter might differ in DNA isolated from patellar tendinopathy compared to normal tendon. If this were the case, such differences in methylation might explain why MMP11 expression is substantially altered in tendinopathy and lead the way to novel strategies that could reduce the incidence of these injuries.

Methods

Patellar tendon tissue was obtained from 10 males with healthy patellar tendons (CON) and 10 males with patellar tendinopathy (PT). The CON samples were from patients undergoing ACL reconstruction surgeries using a patellar tendon graft and had no history of tendinopathy. Controls also had magnetic resonance imaging (MRI) to confirm their injury. The PT samples were from patients undergoing surgical debridement for recalcitrant overuse patellar tendinopathy (Parkinson et al., 2010). The PT specimens all had ultrasound scans (US) or MRI_confirmation of the diagnosis by a medical doctor. PT samples were taken from the proximal tendon. Normal (CON) tendons were also taken from the proximal end, at the margins of the graft. Participants were Caucasian, aged from 19 to 41 years and they were all otherwise healthy. All participants provided written informed consent and approval for this study was obtained from the University of Northampton's School of...

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