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

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.

SUBMITTER: Richards J

PROVIDER: S-EPMC6625229 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

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

Richards Justin J Tang Shirley S Gunsch Gilian G Sul Pavel P Wiet Matthew M Flanigan David C DC Khan Safdar N SN Moore Sarah S Walter Benjamin B Purmessur Devina D

Frontiers in cellular neuroscience 20190705

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 increas ...[more]

PMID: 31333416

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Project description:Background and purposeWe hypothesized that proteinase-activated receptor-2 (PAR2)-mediated vasorelaxation in murine aorta tissue can be due in part to the release of adipocyte-derived relaxing factors (ADRFs).Experimental approachAortic rings from obese TallyHo and C57Bl6 intact or PAR2-null mice either without or with perivascular adipose tissue (PVAT) were contracted with phenylephrine and relaxation responses to PAR2-selective activating peptides (PAR2-APs: SLIGRL-NH(2) and 2-furoyl-LIGRLO-NH(2) ), trypsin and to PAR2-inactive peptides (LRGILS-NH(2) , 2-furoyl-OLRGIL-NH(2) and LSIGRL-NH(2) ) were measured. Relaxation was monitored in the absence or presence of inhibitors that either alone or in combination were previously shown to inhibit ADRF-mediated responses: L-NAME (NOS), indomethacin (COX), ODQ (guanylate cyclase), catalase (H(2) O(2) ) and the K(+) channel-targeted reagents, apamin, charybdotoxin, 4-aminopyridine and glibenclamide.Key resultsEndothelium-intact PVAT-free preparations did not respond to PAR2-inactive peptides (LRGILS-NH(2) , LSIGRL-NH(2) , 2-furoyl-OLRGIL-NH(2) ), whereas active PAR2-APs (SLIGRL-NH(2) ; 2-furoyl-LIGRLO-NH(2) ) caused an L-NAME-inhibited relaxation. However, in PVAT-containing preparations treated with L-NAME/ODQ/indomethacin together, both PAR2-APs and trypsin caused relaxant responses in PAR2-intact, but not PAR2-null-derived tissues. The PAR2-induced PVAT-dependent relaxation (SLIGRL-NH(2) ) persisted in the presence of apamin plus charybdotoxin, 4-aminopyridine and glibenclamide, but was blocked by catalase, implicating a role for H(2) O(2) . Surprisingly, the PAR2-inactive peptides, LRGILS-NH(2) and 2-furoyl-OLRGIL-NH(2) (but not LSIGRL-NH(2) ), caused relaxation in PVAT-containing preparations from both PAR2-null and PAR2-intact (C57Bl, TallyHo) mice. The LRGILS-NH(2) -induced relaxation was distinct from the PAR2 response, being blocked by 4-aminopyridine, but not catalase.ConclusionsDistinct ADRFs that may modulate vascular tone in pathophysiological settings can be released from murine PVAT by both PAR2-dependent and PAR2-independent mechanisms.

Project description:PurposesTo evaluate the usefulness of our original five questions in a medical interview for diagnosing discogenic low back pain (LBP), and to establish a support tool for diagnosing discogenic LBP.Materials and methodsThe degenerative disc disease (DDD) group (n = 42) comprised patients diagnosed with discogenic LBP associated with DDD, on the basis of magnetic resonance imaging findings and response to analgesic discography (discoblock). The control group (n = 30) comprised patients with LBP due to a reason other than DDD. We selected patients from those who had been diagnosed with lumbar spinal stenosis and had undergone decompression surgery without fusion. Of them, those whose postoperative LBP was significantly decreased were included in the control group. We asked patients in both groups whether they experienced LBP after sitting too long, while standing after sitting too long, squirming in a chair after sitting too long, while washing one's face, and in the standing position with flexion. We analyzed the usefulness of our five questions for diagnosing discogenic LBP, and performed receiver operating characteristic (ROC) curve analysis to develop a diagnostic support tool.ResultsThere were no significant differences in baseline characteristics, except age, between the groups. There were significant differences between the groups for all five questions. In the age-adjusted analyses, the odds ratios of LBP after sitting too long, while standing after sitting too long, squirming in a chair after sitting too long, while washing one's face, and in standing position with flexion were 10.5, 8.5, 4.0, 10.8, and 11.8, respectively. The integer scores were 11, 9, 4, 11, and 12, respectively, and the sum of the points of the five scores ranged from 0 to 47. Results of the ROC analysis were as follows: cut-off value, 31 points; area under the curve, 0.92302; sensitivity, 100%; and specificity, 71.4%.ConclusionsAll five questions were useful for diagnosing discogenic LBP. We established the scoring system as a support tool for diagnosing discogenic LBP.

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Dataset Information

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

Publications

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

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