Serpine1 Knockdown Enhances MMP Activity after Flexor Tendon Injury in Mice: Implications for Adhesions Therapy

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Injuries to flexor tendons can be complicated by fibrotic adhesions, which severely impair the function of the hand. Plasminogen activator inhibitor 1 (PAI‐1/SERPINE1), a master suppressor of fibrinolysis and protease activity, is associated with adhesions. Here, we used next‐generation RNA sequencing (RNA‐Seq) to assess genome‐wide differences in messenger RNA expression due to PAI‐1 deficiency after zone II flexor tendon injury. We used the ingenuity pathway analysis to characterize molecular pathways and biological drivers associated with differentially expressed genes (DEG). Analysis of hundreds of overlapping and DEG in PAI‐1 knockout (KO) and wild‐type mice (C57Bl/6J) during tendon healing revealed common and distinct biological processes. Pathway analysis identified cell proliferation, survival, and senescence, as well as chronic inflammation as potential drivers of fibrotic healing and adhesions in injured tendons. Importantly, we identified the activation of PTEN signaling and the inhibition of FOXO1‐associated biological processes as unique transcriptional signatures of the healing tendon in the PAI‐1/Serpine1 KO mice. Further, transcriptomic differences due to the genetic deletion of PAI‐1 were mechanistically linked to PI3K/Akt/mTOR, PKC, and MAPK signaling cascades. These transcriptional observations provide novel insights into the biological roles of PAI‐1 in tendon healing and could identify therapeutic targets to achieve scar‐free regenerative healing of tendons. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 38:43–58, 2020

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

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Transcriptomic Analysis of Cellular Pathways in Healing Flexor Tendons of Plasminogen Activator Inhibitor 1 (PAI‐1/Serpine1) Null Mice

Freeberg

Easa

Lillis

et al. 2019

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PAI‐1 mediates TGF‐β1‐induced myofibroblast activation in tenocytes via mTOR signaling

Alenchery

Ajalik

Jerreld

et al. 2023

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Transforming growth factor‐beta (TGF‐β1) induces plasminogen activator inhibitor 1 (PAI‐1) to effect fibrotic pathologies in several organs including tendon. Recent data implicated PAI‐1 with inhibition of phosphatase and tensin homolog (PTEN) suggesting that PAI‐1‐induced adhesions involves phosphoinositide 3‐kinase/protein kinase B/mammalian target of rapamycin (mTOR) signaling. Ergo, we investigated effects of TGF‐β1, PAI‐1, and mTOR signaling crosstalk on myofibroblast activation, senescence, and proliferation in primary flexor tenocytes from wild‐type (WT) and PAI‐1 knockout (KO) mice. PAI‐1 deletion blunted TGF‐β1‐induced myofibroblast activation in murine flexor tenocytes and increased the gene expression of Mmp‐2 to confer protective effects against fibrosis. While TGF‐β1 significantly reduced phosphorylation of PTEN in WT cells, PAI‐1 deletion rescued the activation of PTEN. Despite that, there were no differences in TGF‐β1‐induced activation of mTOR signaling (AKT, 4EBP1, and P70S6K) in WT or KO tenocytes. Phenotypic changes in distinct populations of WT or KO tenocytes exhibiting high or low mTOR activity were then examined. TGF‐β1 increased alpha‐smooth muscle actin abundance in WT cells exhibiting high mTOR activity, but this increase was blunted in KO cells exhibiting high 4EBP1 activity but not in cells exhibiting high S6 activity. DNA damage (γH2AX) was increased with TGF‐β1 treatment in WT tenocytes but was blunted in KO cells exhibiting high mTOR activity. Increased mTOR activity enhanced proliferation (Ki67) in both WT and KO tenocytes. These findings point to a complex nexus of TGF‐β1, PAI‐1, and mTOR signaling in regulating proliferation, myofibroblast differentiation, and senescence in tenocytes, which could define therapeutic targets for chronic tendon adhesions and other fibrotic pathologies.

Preclinical tendon and ligament models: Beyond the 3Rs (replacement, reduction, and refinement) to 5W1H (why, who, what, where, when, how)

Little

Amadio

Awad

et al. 2023

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Several tendon and ligament animal models were presented at the 2022 Orthopaedic Research Society Tendon Section Conference held at the University of Pennsylvania, May 5‐7, 2022. A key objective of the breakout sessions at this meeting was to develop guidelines for the field, including for preclinical tendon and ligament animal models. This review summarizes the perspectives of experts for eight surgical small and large animal models of rotator cuff tear, flexor tendon transection, anterior cruciate ligament tear, and Achilles tendon injury using the framework: “Why, Who, What, Where, When, and How” (5W1H). A notable conclusion is that the perfect tendon model does not exist; there is no single gold standard animal model that represents the totality of tendon and ligament disease. Each model has advantages and disadvantages and should be carefully considered in light of the specific research question. There are also circumstances when an animal model is not the best approach. The wide variety of tendon and ligament pathologies necessitates choices between small and large animal models, different anatomic sites, and a range of factors associated with each model during the planning phase. Attendees agreed on some guiding principles including: providing clear justification for the model selected, providing animal model details at publication, encouraging sharing of protocols and expertise, improving training of research personnel, and considering greater collaboration with veterinarians. A clear path for translating from animal models to clinical practice was also considered as a critical next step for accelerating progress in the tendon and ligament field.This article is protected by copyright. All rights reserved.