Deletion of EP4 in S100a4-lineage cells reduces scar tissue formation during early but not later stages of tendon healing

AbstractTendon injuries heal via scar tissue rather than regeneration. This healing response forms adhesions between the flexor tendons in the hand and surrounding tissues, resulting in impaired range of motion and hand function. Mechanistically, inflammation has been strongly linked to adhesion formation, and Prostaglandin E2 (PGE2) is associated with both adhesion formation and tendinopathy. In the present study we tested the hypothesis that deletion of the PGE2 receptor EP4 in S100a4-lineage cells would decrease adhesion formation. S100a4-Cre; EP4flox/flox (EP4cKOS100a4) repairs healed with improved gliding function at day 14, followed by impaired gliding at day 28, relative to wild type. Interestingly, EP4cKOS100a4 resulted in only transient deletion of EP4, suggesting up-regulation of EP4 in an alternative cell population in these mice. Loss of EP4 in Scleraxis-lineage cells did not alter gliding function, suggesting that Scx-lineage cells are not the predominant EP4 expressing population. In contrast, a dramatic increase in α-SMA+, EP4+ double-positive cells were observed in EP4cKOS100a4 suggesting that EP4cKOS100a4 repairs heal with increased infiltration of EP4 expressing α-SMA myofibroblasts, identifying a potential mechanism of late up-regulation of EP4 and impaired gliding function in EP4cKOS100a4 tendon repairs.</jats:p

Obesity/Type II diabetes alters macrophage polarization resulting in a fibrotic tendon healing response

<div><p>Type II Diabetes (T2DM) dramatically impairs the tendon healing response, resulting in decreased collagen organization and mechanics relative to non-diabetic tendons. Despite this burden, there remains a paucity of information regarding the mechanisms that govern impaired healing of diabetic tendons. Mice were placed on either a high fat diet (T2DM) or low fat diet (lean) and underwent flexor tendon transection and repair surgery. Healing was assessed via mechanical testing, histology and changes in gene expression associated with collagen synthesis, matrix remodeling, and macrophage polarization. Obese/diabetic tendons healed with increased scar formation and impaired mechanical properties. Consistent with this, prolonged and excess expression of extracellular matrix (ECM) components were observed in obese/T2DM tendons. Macrophages are involved in both inflammatory and matrix deposition processes during healing. Obese/T2DM tendons healed with increased expression of markers of pro-inflammatory M1 macrophages, and elevated and prolonged expression of M2 macrophages markers that are involved in ECM deposition. Here we demonstrate that tendons from obese/diabetic mice heal with increased scar formation and increased M2 polarization, identifying excess M2 macrophage activity and matrix synthesis as a potential mechanism of the fibrotic healing phenotype observed in T2DM tendons, and as such a potential target to improve tendon healing in T2DM.</p></div

Relative expression of collagen and matrix metalloproteinase genes are altered in HFD.

<p>Relative mRNA expression of (A) type III (<i>Col3a1</i>) and (B) type I collagen (<i>Col1a1</i>), (C) <i>Mmp9</i>, and (D) <i>Mmp2</i> were determined by RT-PCR between 3 and 28 days post-repair. Data were normalized to the internal control <i>β-actin</i>. Fold changes are reported relative to LFD day 3 expression. White bars = LFD; black bars = HFD, (*) indicates p<0.05, (**) indicates p<0.001, (***) indicates p<0.0001 between HFD and LFD.</p

Primer sequences for quantitative PCR experiments.

<p>Primer sequences for quantitative PCR experiments.</p

HFD leads to increased body weight and impairs glucose tolerance.

<p>A—Body weights from mice in both HFD (black bars) and LFD groups (white bars) were compared at the time of surgery and the time of harvest. At both points, the HFD group weighed significantly more than the LFD group. B—Elevated fasting blood glucose levels were observed in HFD (black bars), relative to LFD mice (white bars). C—Mice from the HFD group (black line) demonstrated an impaired ability to respond to glucose loading relative to LFD (red line), as shown by significantly elevated blood glucose levels at 30, 60, and 120 minutes after delivering a glucose bolus. (*) Indicates p<0.05.</p

HFD impairs return to normal MTP flexion angle and gliding resistance.

<p>Incremental loading of the proximal end of the FDL was performed to measure the gliding resistance and the metatarsophalangeal (MTP) flexion angle in the hindpaw. A—LFD-fed mice (white bars) show substantial improvement in flexion angle 28 days post-repair, while the MTP flexion angle is significantly lower in the HFD group (black bars) at the latest time-point. B—Gliding resistance is significantly higher in the HFD group (black bars) 28 days post-repair relative to LFD (white bars), suggesting that T2DM interferes with the restoration of normal gliding and flexion after repair. (*) Indicates p<0.05.</p