Diabetes, Advanced Glycation, and Tendinopathy

Introduction: Diabetes mellitus is a major risk factor for tendon pain, injury, and pathology. Surprisingly, tendon problems persist in diabetic patients with superior blood glucose control (HbA1c<6.5), suggesting that alternative mechanisms contribute to this problem. Advanced glycation end products (AGEs) have been implicated in several diabetes-related complications, but their role in diabetic tendon pathology has not fully been explored. To expand our understanding of AGE-mediated tendon pathology, the following five studies were completed.

Study 1: Streptozotocin-induced diabetes alters transcription of multiple genes necessary for extracellular matrix remodeling in rat patellar tendon. RNA was isolated from the patellar tendon of non-diabetic (control, n=9), 1-week diabetic (acute, n=8), 10-weeks diabetic (chronic, n=7), and insulin treated 10-weeks diabetic (insulin, n=8) rats. Determination of mRNA transcripts was completed using droplet digital PCR (ddPCR). Our findings indicated that STZ-induced diabetes results in rapid and large changes in the expression of several genes that are key to extracellular matrix (ECM) remodeling, maintenance, and maturation.

Study 2: Advanced glycation end products suppress mitochondrial function and proliferative capacity of Achilles tendon-derived fibroblasts. Using an in vitro cell culture system, rat Achilles tendon fibroblasts were treated with glycolaldehyde-derived AGEs (0, 50, 100, and 200μg/ml) for 48 hours in normal glucose (5.5mM) and high glucose (25mM) conditions. Our findings demonstrate that tendon fibroblasts treated with AGEs display reduced ATP production, electron transport efficiency, and proliferative capacity. These impairments were coupled with alterations in mitochondrial DNA content and expression of genes associated with ECM remodeling, mitochondrial energy metabolism, and apoptosis.

Study 3: Descriptive transcriptome analysis of tendon derived fibroblasts following in vitro exposure to advanced glycation end products. Rat Achilles tendon fibroblasts were treated with glycolaldehyde-derived AGEs (200μg/ml) for 48 hours in normal glucose (5.5mM) conditions. Total RNA was isolated and the PolyA⁺ library was sequenced. We demonstrate that tendon fibroblasts treated with 200μg/ml of AGEs differentially express 2,159 gene targets compared to fibroblasts treated with an equal amount of bovine serum albumin (BSA)-Control. Our findings suggest that AGEs disrupt the tendon fibroblast transcriptome on a large scale and that these pathways may contribute to the development and progression of diabetic tendinopathy.

Study 4: Evaluation of tendon healing in a mouse model of elevated serum advanced glycation end products following tendon injury. Mice received daily BSA-Control or AGE-BSA injections (200μg/ml) for two weeks prior to creation of a tendon injury in the central third of both patellar tendons. Animals assigned to an exercise group began a moderate treadmill protocol (13 meters/min, five days/week, five weeks) one week following injury and all animals continued to receive injections until termination. We demonstrated that based on our injection dose and schedule, that serum AGEs are significantly elevated to ~200μg/ml, levels that are typically seen in type II diabetic patients. Additionally, a main effect for AGEs was observed in genes related to cell proliferation (Mybl2), mitochondrial function (Bcs1l), and growth factors (Fgf2). However, moderate treadmill exercise did not alter gene markers, such as Ctgf and Fgf2, which are makers of a tendon healing response. Our findings suggest that AGEs modulate tendon gene expression following patellar tendon injury, with no effect of moderate treadmill exercise.

Study 5: Serum levels of advanced glycation end products and their relationship to patellar tendon properties in diabetes. Subjects (n=32) from a full spectrum of diabetes status, including no history of diabetes were recruited for a cross-sectional study. A fasted blood sample was collected and magnetic resonance imaging (MRI) of the knee was completed. Both current HbA1c and previous diagnosis was used to stratify collected data. Additionally, a full correlation matrix of all measured variables was created to establish relationships that could be used to predict tendon pathology in diabetes. Our findings demonstrate that diabetes is associated with smaller patellar tendon dimensions, which is in disagreement with the literature. Further, we show that changes to body weight normalized (BWN) tendon cross-sectional area (CSA) occur independent of circulating N^ε-(carboxymethyl)lysine (CML) levels. These new data suggest that alternative mechanisms contributing to tendon pathology in diabetes deserve attention.