Mast Cell/Proteinase Activated Receptor 2 (PAR2) Mediated Interactions in the Pathogenesis of Discogenic Back Pain.

Justin Richards,Shirley Tang,Sarah Moore,Benjamin Walter,Pavel Sul,David C Flanigan,Matthew Wiet,Safdar N Khan,Devina Purmessur,Gilian Gunsch

doi:10.3389/fncel.2019.00294

Abstract

Mast cells (MCs) are present in the painful degenerate human intervertebral disc (IVD) and are associated with disease pathogenesis. MCs release granules containing enzymatic and inflammatory factors in response to stimulants or allergens. The serine protease, tryptase, is unique to MCs and its activation of the G-protein coupled receptor, Protease Activated Receptor 2 (PAR2), induces inflammation and degradation in osteoarthritic cartilage. Our previously published work has demonstrated increased levels of MC marker tryptase in IVD samples from discogenic back pain patients compared to healthy control IVD samples including expression of chemotactic agents that may facilitate MC migration into the IVD. To further elucidate MCs’ role in the IVD and mechanisms underlying its effects, we investigated whether (1) human IVD cells can promote MC migration, (2) MC tryptase can mediate up-regulation of inflammatory/catabolic process in human IVD cells and tissue, and (3) the potential of PAR2 antagonist to function as a therapeutic drug in in vitro human and ex vivo bovine pilot models of disease. MC migration was quantitatively assessed using conditioned media from primary human IVD cells and MC migration examined through Matrigel. Exposure to soluble IVD factors significantly enhanced MC migration, suggesting IVD cells can recruit MCs. We also demonstrated significant upregulation of MC chemokine SCF and angiogenic factor VEGFA gene expression in human IVD cells in vitro in response to recombinant human tryptase, suggesting tryptase can enhance recruitment of MCs and promotion of angiogenesis into the usually avascular IVD. Furthermore, tryptase can degrade proteoglycans in IVD tissue as demonstrated by significant increases in glycosaminoglycans released into surrounding media. This can create a catabolic microenvironment compromising structural integrity and facilitating vascular migration usually inhibited by the anti-angiogenic IVD matrix. Finally, as a “proof of concept” study, we examined the therapeutic potential of PAR2 antagonist (PAR2A) on human IVD cells and bovine organ culture IVD model. While preliminary data shows promise and points toward structural restoration of the bovine IVD including down-regulation of VEGFA, effects of PAR2 antagonist on human IVD cells differ between gender and donors suggesting that further validation is required with larger cohorts of human specimens.

Highlights

70–80% of the population will experience chronic low back pain during their lifetime (Andersson, 1999) and large population-based studies have demonstrated that intervertebral disc (IVD) degeneration is a significant cause of chronic low back pain (De Schepper et al, 2010)
This study aims to determine the potential mechanism by which mast cells (MCs) may invade the IVD joint, investigate tryptase effects on matrix degradation and chemotactic/vascular markers, quantify and validate protein expression of Proteinase Activated Receptor 2 (PAR2) in nucleus pulposus (NP), annulus fibrosus (AF) and cartilage end plate (CEP) regions of the human IVD via immunohistochemistry and western blot and to determine the therapeutic potential of PAR2 antagonist oligopeptide FSLLRY-NH2 (PAR2A) in a bovine ex vivo translational model and in human in vitro model for proof of concept
This data suggests that MCs can migrate in response to Intervertebral disc conditioned media (IVDCM) and in response to IVD related factors, as the healthy articular cartilage conditioned media (ACCM) cell control had little effect compared to negative control

Summary

Introduction

70–80% of the population will experience chronic low back pain during their lifetime (Andersson, 1999) and large population-based studies have demonstrated that intervertebral disc (IVD) degeneration is a significant cause of chronic low back pain (De Schepper et al, 2010). Initial DBP therapies are largely conservative and include analgesics, physiotherapy and psychosocial pain management approaches When these approaches are unsuccessful, highly invasive surgeries (e.g., disc arthroplasty or lumbar fusions) are routinely performed. The healthy IVD is characterized by a healthy gelatinous core of proteoglycans and nucleus pulposus (NP) cells of notochordal origin. Degeneration of the IVD is characterized by significant increases in extracellular matrix (ECM) breakdown, inflammation and nerve/vascular ingrowth (Smith et al, 2011; Lama et al, 2018). While the healthy IVD is immuneprivileged, increased numbers of innate immune cells have been demonstrated within painful human IVDs compared to healthy IVDs, yet their role within the painful degenerate disc remains unknown (Wiet et al, 2017)

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