Wound repair [45,53], suggesting age contributes to dysregulated tissue repair. Our data of reduced FPR2/ALX together with the elevated PGE2 levels with age suggests an inflamm-aging process is present in injured tendons. In support of this concept, IL-1b stimulated tendon explants derived from uninjured horses aged 10 years and above showed a diminished capacity to express FPR2/ ALX compared to individuals less than 10 years of age. Interestingly, tendon derived cells from older horses also had a reduced response to IL-1b induced PGE2 production compared toProstaglandins and Lipoxins in Tendinopathyyoung horses (Fig. S1). Taken together these data suggest aged individuals are less capable of mounting a protective response to tendon inflammation by this mechanism compared to younger individuals. Immunosenescence may therefore represent a potential mechanism for tendon re-injury via this pathway and in the development of Vitamin D2 site chronic injury and explain, at least in part, the high frequency of tendon injuries sustained by aged individuals [17,18]. We hypothesise that `inflamm-aging’ is an important component in the complex aetiology of tendinopathy, to which additional contributing factors such as load-induced proteolytic matrix disruption [9] and lower tendon metabolic activity with age [54] are superimposed. Furthermore, as we did not observe any relationship between LXA4 levels with age in samples of natural disease or in vitro, it appears that it is the ability to generate the FPR2/ALX receptor that is impeded by age and not synthesis of the LXA4 ligand, diminishing the ability to mediate downstream processes culminating in a deficit to resolve tendon inflammation. From the findings in the present study, we conclude that PGE2 is implicated in the development of tendon inflammation and its ensuing resolution. Temporal analysis suggests class switching of lipid mediators occurs in early stage injury, driving synthesis of LXA4. However, ageing individuals exhibit a reduced capacity to resolve tendon inflammation via the FPR2/ALX receptor, which may present a mechanism for the development of chronic tendinopathy. Improved understanding of injury pathogenesis throughout the phases of tendon repair will facilitate identification of novel therapeutic targets to modulate or prevent disease progression, such as the use of FPR2/ALX agonists.added and the sample vortexed 15857111 and allowed to stand at room temperature for 5 minutes. Tissue was pelleted by centrifugation at 16,0006g and the supernatant harvested. Acetone was removed by evaporation at room temperature for 18 hours and the residual supernatant used for determination of PGE2 and PGF2a measurement via radioimmunoassay as described by Cheng et al. [56].Measurement of Lipoxin A4 in Tendon Extracts and Tissue Culture MediaLXA4 levels were determined as an indicator of resolution of inflammation in the supernatant prepared earlier 1317923 to determine prostaglandin levels in extracts of normal (n = 8), sub-acute (n = 7) and chronic injured SDFTs (n = 6) and in samples of tissue culture media from cytokine stimulated tendon explants as described later. LXA4 was separated from the supernatant used to determine prostaglandin levels by JSI-124 passage through Bond Elut C18 columns (Agilent Technologies, USA) followed by elution with methyl formate. Samples were evaporated to dryness in a stream of nitrogen and the resulting residues used to determine LXA4 levels by ELISA (Neogen Corp, USA) according to the manufacture.Wound repair [45,53], suggesting age contributes to dysregulated tissue repair. Our data of reduced FPR2/ALX together with the elevated PGE2 levels with age suggests an inflamm-aging process is present in injured tendons. In support of this concept, IL-1b stimulated tendon explants derived from uninjured horses aged 10 years and above showed a diminished capacity to express FPR2/ ALX compared to individuals less than 10 years of age. Interestingly, tendon derived cells from older horses also had a reduced response to IL-1b induced PGE2 production compared toProstaglandins and Lipoxins in Tendinopathyyoung horses (Fig. S1). Taken together these data suggest aged individuals are less capable of mounting a protective response to tendon inflammation by this mechanism compared to younger individuals. Immunosenescence may therefore represent a potential mechanism for tendon re-injury via this pathway and in the development of chronic injury and explain, at least in part, the high frequency of tendon injuries sustained by aged individuals [17,18]. We hypothesise that `inflamm-aging’ is an important component in the complex aetiology of tendinopathy, to which additional contributing factors such as load-induced proteolytic matrix disruption [9] and lower tendon metabolic activity with age [54] are superimposed. Furthermore, as we did not observe any relationship between LXA4 levels with age in samples of natural disease or in vitro, it appears that it is the ability to generate the FPR2/ALX receptor that is impeded by age and not synthesis of the LXA4 ligand, diminishing the ability to mediate downstream processes culminating in a deficit to resolve tendon inflammation. From the findings in the present study, we conclude that PGE2 is implicated in the development of tendon inflammation and its ensuing resolution. Temporal analysis suggests class switching of lipid mediators occurs in early stage injury, driving synthesis of LXA4. However, ageing individuals exhibit a reduced capacity to resolve tendon inflammation via the FPR2/ALX receptor, which may present a mechanism for the development of chronic tendinopathy. Improved understanding of injury pathogenesis throughout the phases of tendon repair will facilitate identification of novel therapeutic targets to modulate or prevent disease progression, such as the use of FPR2/ALX agonists.added and the sample vortexed 15857111 and allowed to stand at room temperature for 5 minutes. Tissue was pelleted by centrifugation at 16,0006g and the supernatant harvested. Acetone was removed by evaporation at room temperature for 18 hours and the residual supernatant used for determination of PGE2 and PGF2a measurement via radioimmunoassay as described by Cheng et al. [56].Measurement of Lipoxin A4 in Tendon Extracts and Tissue Culture MediaLXA4 levels were determined as an indicator of resolution of inflammation in the supernatant prepared earlier 1317923 to determine prostaglandin levels in extracts of normal (n = 8), sub-acute (n = 7) and chronic injured SDFTs (n = 6) and in samples of tissue culture media from cytokine stimulated tendon explants as described later. LXA4 was separated from the supernatant used to determine prostaglandin levels by passage through Bond Elut C18 columns (Agilent Technologies, USA) followed by elution with methyl formate. Samples were evaporated to dryness in a stream of nitrogen and the resulting residues used to determine LXA4 levels by ELISA (Neogen Corp, USA) according to the manufacture.
