Identification of the TRPV4 ion channel as a mechanotransducer and therapeutic target in low back pain
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Autor(in)
Datum
2021-03Typ
- Doctoral Thesis
ETH Bibliographie
yes
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Abstract
Low back pain (LBP) is the leading cause of disability worldwide and a huge global socio-economic burden. The costs related to LBP are projected to further increase in the coming decades due to the growth and aging of the global population. Degenerative disc disease, which is characterized by intervertebral disc (IVD) degeneration, inflammation, and nerve ingrowth, principally contributes to LBP. One of the main contributors to IVD degeneration is excessive or aberrant mechanical loading. Current treatments of LBP, including physical, psychological, pharmacological, and surgical approaches, have unclear mechanisms of action, low effect sizes, and are not beneficial in the long term. Targeted therapeutic strategies in preclinical development, such as molecular and gene therapy, selectively address the biological changes that occur in IVD degeneration. Nevertheless, despite the mechanical nature of LBP, mechanotransduction pathways are currently not targeted. This is mainly due to the very limited information on mechanosensing and mechanotransduction mechanisms in the IVD. Transient receptor potential (TRP) ion channels are promising therapeutic targets to treat LBP, as they can sense and transduce a variety of signals, including mechanical stress. The TRP vanilloid 4 (TRPV4) channel is especially interesting, as it was shown to mediate mechanical, inflammatory and pain signals. Its clinical potential is further highlighted by ongoing preclinical and clinical trials. The overall goal of this thesis was to investigate the potential role of TRPV4 in mediating hyperphysiological mechanical signals in the IVD, and its relevance as a therapeutic target to treat LBP.
As a first step towards the investigation of TRPV4, we developed a novel in vitro compression model for mechanotransduction studies. Agarose-collagen composite hydrogels were fabricated and characterized in terms of material and mechanical properties. Bovine nucleus pulposus (NP) cell phenotype and mechanotransduction ability after dynamic compression were further analyzed. Agarose-collagen composite hydrogels combined the mechanical strength of agarose with the biofunctionality of collagen, which enhanced cell adhesion and the activation of focal adhesion kinases. Moreover, agarose-collagen scaffolds recapitulated the extracellular matrix (ECM) of the IVD, with their non-fibrillar matrix and collagen fibers, and allowed the exploration of mechanotransduction mechanisms in a reproducible system.
In a second study, NP cell-laden agarose-collagen hydrogels and a mouse model were used to investigate the role of TRPV4 in transducing hyperphysiological dynamic compression. Degenerative changes and the expression of the inflammatory mediator cyclooxygenase 2 (COX2) were examined in mouse IVDs that were dynamically compressed at a hyperphysiological regime (versus sham). Cell damage and inflammation (prostaglandin E2 (PGE2) release) were measured in bovine NP cells embedded in agarose-collagen hydrogels and dynamically compressed at a hyperphysiological regime with or without TRPV4 inhibition. The activation of the mitogen-activated protein kinase (MAPK) pathways was analyzed. Finally, degenerative changes and COX2 expression were further evaluated in the IVDs of trpv4 knockout (KO) mice (versus wild-type). TRPV4 was shown to regulate the COX2/PGE2 inflammatory factors and mediate cell damage induced by hyperphysiological dynamic compression, possibly via the extracellular signal-regulated kinases 1/2 (ERK) pathway.
In a final step, we investigated the role of TRPV4 as a transducer of hyperphysiological cyclic stretching and a potential therapeutic target. Human primary annulus fibrosus (AF) cells were seeded on silicone chambers and cyclically stretched at a hyperphysiological magnitude in the presence or absence of a TRPV4 inhibitor. Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 TRPV4 KO AF cells were generated, hyperphysiologically stretched, and compared to control cells. Gene and protein expression of inflammatory and catabolic mediators, as well as activation of MAPK pathways, were analyzed. This study identified TRPV4 as a mediator of stretch-induced inflammation in human AF cells. Moreover, it revealed TRPV4 pharmacological inhibition and gene editing as potential future therapeutic approaches to rescue mechanoflammation.
In this thesis, in vitro and in vivo models of hyperphysiological compression and stretching were established and used to identify TRPV4 as mechanotransducer and therapeutic target in the IVD. A novel in vitro compression model was developed to mimic the ECM of the IVD and other native tissues composed of non-fibrillar matrix and collagens, and to investigate their mechanotransduction mechanisms. This system was instrumental to investigate the function of TRPV4 in IVD cells. The novel findings obtained with this model, together with those obtained in the mouse compression model and the stretching system demonstrate that TRPV4 mediates a mechanism leading from mechanical hyperphysiological loading to IVD degeneration and inflammation, which eventually lead to chronic LBP. TRPV4 modulation might thus constitute a promising therapeutic strategy to treat patients suffering from IVD pathologies caused by aberrant mechanical stress. Future studies should clarify the exact mechanism of action of TRPV4 inhibition and gene editing and examine their potential to mitigate chronic inflammation and LBP in preclinical and clinical trials. Mehr anzeigen
Persistenter Link
https://doi.org/10.3929/ethz-b-000474936Publikationsstatus
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Verlag
ETH ZurichOrganisationseinheit
02070 - Dep. Gesundheitswiss. und Technologie / Dep. of Health Sciences and Technology
ETH Bibliographie
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