Irritable bowel syndrome (IBS) is a common gastrointestinal disorder that is characterized by chronic abdominal pain concurrent with altered bowel habit. Polyunsaturated fatty acid (PUFA) metabolites ...are increased in abundance in IBS and are implicated in the alteration of sensation to mechanical stimuli, which is defined as visceral hypersensitivity. We sought to quantify PUFA metabolites in patients with IBS and evaluate their role in pain. Quantification of PUFA metabolites by mass spectrometry in colonic biopsies showed an increased abundance of 5-oxoeicosatetraenoic acid (5-oxoETE) only in biopsies taken from patients with IBS with predominant constipation (IBS-C). Local administration of 5-oxoETE to mice induced somatic and visceral hypersensitivity to mechanical stimuli without causing tissue inflammation. We found that 5-oxoETE directly acted on both human and mouse sensory neurons as shown by lumbar splanchnic nerve recordings and Ca
imaging of dorsal root ganglion (DRG) neurons. We showed that 5-oxoETE selectively stimulated nonpeptidergic, isolectin B4 (IB4)-positive DRG neurons through a phospholipase C (PLC)- and pertussis toxin-dependent mechanism, suggesting that the effect was mediated by a G protein-coupled receptor (GPCR). The MAS-related GPCR D (Mrgprd) was found in mouse colonic DRG afferents and was identified as being implicated in the noxious effects of 5-oxoETE. Together, these data suggest that 5-oxoETE, a potential biomarker of IBS-C, induces somatic and visceral hyperalgesia without inflammation in an Mrgprd-dependent manner. Thus, 5-oxoETE may play a pivotal role in the abdominal pain associated with IBS-C.
Background and Purpose
Thrombin is massively released upon tissue damage associated with bleeding or chronic inflammation. The effects of this thrombin on tissue regrowth and repair has been scarcely ...addressed and only in cancer cell lines. Hence, the purpose of the present study was to determine thrombin's pharmacological effects on human intestinal epithelium growth, proliferation and apoptosis, using three‐dimensional cultures of human colon organoids.
Experimental Approach
Crypts were isolated from human colonic resections and cultured for 6 days, forming human colon organoids. Cultured organoids were exposed to 10 and 50 mU·mL−1 of thrombin, in the presence or not of protease‐activated receptor (PAR) antagonists. Organoid morphology, metabolism, proliferation and apoptosis were followed.
Key Results
Thrombin favoured organoid maturation leading to a decreased number of immature cystic structures and a concomitant increased number of larger structures releasing cell debris and apoptotic cells. The size of budding structures, metabolic activity and proliferation were significantly reduced in organoid cultures exposed to thrombin, while apoptosis was dramatically increased. Both PAR1 and PAR4 antagonists inhibited apoptosis regardless of thrombin doses. Thrombin‐induced inhibition of proliferation and metabolic activity were reversed by PAR4 antagonist for thrombin's lowest dose and by PAR1 antagonist for thrombin's highest dose.
Conclusions and Implications
Overall, our data suggest that the presence of thrombin in the vicinity of human colon epithelial cells favours their maturation at the expense of their regenerative capacities. Our data point to thrombin and its two receptors PAR1 and PAR4 as potential molecular targets for epithelial repair therapies.
The Focal adhesion kinase (FAK) is a ubiquitous cytoplasmic tyrosine‐kinase promoting tumor progression and metastasis processes by acting in cancer cells and their tumor microenvironment partners. ...FAK overexpression in primary colon tumors and their metastasis is associated to poor colorectal cancer (CRC) patients’ outcome. Eight FAK mRNA alternative splice variants have been described and contribute to additional level of FAK activity regulation, some of them corresponding to overactivated FAK isoforms. To date, FAK mRNA alternative splice variants expression and implication in CRC processes remain unknown. Here, using different human CRC cells lines displaying differential invasive capacities in an in vivo murine model recapitulating the different steps of CRC development from primary tumors to liver and lung metastasis, we identified three out of the eight mRNA variants (namely FAK0, FAK28 and FAK6) differentially expressed along the CRC process and the tumor sites. Our results highlight an association between FAK0 and FAK6 expressions and the metastatic potential of the most aggressive cell lines HT29 and HCT116, suggesting that FAK0 and FAK6 could represent aggressiveness markers in CRC. Our findings also suggest a more specific role for FAK28 in the interactions between the tumors cells and their microenvironment. In conclusion, targeting FAK0, the common form of FAK, might not be a good strategy based on the numerous roles of this kinase in physiological processes. In contrast, FAK6 or FAK28 splice variants, or their corresponding protein isoforms, may putatively represent future therapeutic target candidates in the development of CRC primary tumors and metastasis.
What's new?
Overexpression of the focal adhesion kinase (FAK) is associated with poor outcome in patients with colorectal cancer but the role of the eight splice variants of FAK remains unknown. Here the authors correlated FAK splice variant expression in colorectal tumor cell lines with invasiveness in mouse models. FAK0 and FAK6 splice variant expression was associated with higher aggressiveness and metastatic potential, underscoring that distinct FAK splice variants may represent new targets in the development of drugs against colorectal cancer and associated metastasis.
Background
Ephrin‐B2/EphB receptor signaling contributes to persistent pain states such as postinflammatory and neuropathic pain. Visceral hypersensitivity (VHS) is a major mechanism underlying ...abdominal pain in patients with irritable bowel syndrome (IBS) and inflammatory bowel diseases (IBD) in remission, but the underlying pathophysiology remains unclear. Here, we evaluated the spinal ephrin‐B2/EphB pathway in VHS in 2 murine models of VHS, that is, postinflammatory TNBS colitis and maternal separation (MS).
Methods
Wild‐type (WT) mice and mice lacking ephrin‐B2 in Nav1.8 nociceptive neurons (cKO) were studied. VHS was induced by: 1. intracolonic instillation of TNBS or 2. water avoidance stress (WAS) in mice that underwent maternal separation (MS). VHS was assessed by quantifying the visceromotor response (VMRs) during colorectal distention. Colonic tissue and spinal cord were collected for histology, gene, and protein expression evaluation.
Key Results
In WT mice, but not cKO mice, TNBS induced VHS at day 14 after instillation, which returned to baseline perception from day 28 onwards. In MS WT mice, WAS induced VHS for up to 4 weeks. In cKO however, visceral pain perception returned to basal level by week 4. The development of VHS in WT mice was associated with significant upregulation of spinal ephrin‐B2 and EphB1 mRNA expression or protein levels in the TNBS model and upregulation of spinal ephrin‐B2 protein in the MS model. No changes were observed in cKO mice. VHS was not associated with persistent intestinal inflammation.
Conclusions and Inferences
Overall, our data indicate that the ephrin‐B2/EphB1 spinal signaling pathway is involved in VHS and may represent a novel therapeutic target.
Ephrin‐B2 ligand signaling in the spinal cord can be a player in post‐inflammatory or post stress‐induced visceral hypersensitivity (VHS) murine models; hence it is suggested as potential therapeutic target for the management of VHS associated with gastrointestinal disorders.
Serine proteases are heavily present in the gastrointestinal tract where they are essential in numerous physiological processes. An imbalance in the proteolytic activity is a central mechanism ...underlying abdominal pain in irritable bowel syndrome (IBS). Therefore, protease inhibitors are emerging as a promising therapeutic tool to manage abdominal pain in this functional gastrointestinal disorder. With this review, we provide an up-to-date overview of the implications of serine proteases in the development of abdominal pain in IBS, along with a critical assessment of the current developments and prospects of protease inhibitors as a therapeutic tool. In particular, we highlight the current knowledge gap concerning the identity of dysregulated serine proteases that are released by the rectal mucosa of IBS patients. Finally, we suggest a workflow with state-of-the-art techniques that will help address the knowledge gap, guiding future research towards the development of more effective and selective protease inhibitors to manage abdominal pain in IBS.
Spatially resolved transcriptomics has enabled precise genome-wide mRNA expression profiling within tissue sections. The performance of methods targeting the polyA tails of mRNA relies on the ...availability of specimens with high RNA quality. Moreover, the high cost of currently available spatial resolved transcriptomics assays requires a careful sample screening process to increase the chance of obtaining high-quality data. Indeed, the upfront analysis of RNA quality can show considerable variability due to sample handling, storage, and/or intrinsic factors. We present RNA-Rescue Spatial Transcriptomics (RRST), a workflow designed to improve mRNA recovery from fresh frozen specimens with moderate to low RNA quality. First, we provide a benchmark of RRST against the standard Visium spatial gene expression protocol on high RNA quality samples represented by mouse brain and prostate cancer samples. Then, we test the RRST protocol on tissue sections collected from five challenging tissue types, including human lung, colon, small intestine, pediatric brain tumor, and mouse bone/cartilage. In total, we analyze 52 tissue sections and demonstrate that RRST is a versatile, powerful, and reproducible protocol for fresh frozen specimens of different qualities and origins.
Proteolytic homeostasis is important at mucosal surfaces, but its actors and their precise role in physiology are poorly understood. Here we report that healthy human and mouse colon epithelia are a ...major source of active thrombin. We show that mucosal thrombin is directly regulated by the presence of commensal microbiota. Specific inhibition of luminal thrombin activity causes macroscopic and microscopic damage as well as transcriptomic alterations of genes involved in host-microbiota interactions. Further, luminal thrombin inhibition impairs the spatial segregation of microbiota biofilms, allowing bacteria to invade the mucus layer and to translocate across the epithelium. Thrombin cleaves the biofilm matrix of reconstituted mucosa-associated human microbiota. Our results indicate that thrombin constrains biofilms at the intestinal mucosa. Further work is needed to test whether thrombin plays similar roles in other mucosal surfaces, given that lung, bladder and skin epithelia also express thrombin.
Increasing evidences indicate that an unbalance between tryptases and their endogenous inhibitors, leading to an increased proteolytic activity, is implicated in the pathophysiology of rheumatoid ...arthritis. The aim of the present study was to evaluate the impact of tryptase inhibition on experimental arthritis.
Analysis of gene expression and regulation in the mouse knee joint was performed by RT-qPCR and in situ hybridization. Arthritis was induced in male C57BL/6 mice with mBSA/IL-1β. Tryptase was inhibited by two approaches: a lentivirus-mediated heterologous expression of the human endogenous tryptase inhibitor, sperm-associated antigen 11B isoform C (hSPAG11B/C), or a chronic treatment with the synthetic tryptase inhibitor APC366. Several inflammatory parameters were evaluated, such as oedema formation, histopathology, production of IL-1β, -6, -17A and CXCL1/KC, myeloperoxidase and tryptase-like activities.
Spag11c was constitutively expressed in chondrocytes and cells from the synovial membrane in mice, but its expression did not change 7 days after the induction of arthritis, while tryptase expression and activity were upregulated. The intra-articular transduction of animals with the lentivirus phSPAG11B/C or the treatment with APC366 inhibited the increase of tryptase-like activity, the late phase of oedema formation, the production of IL-6 and CXCL1/KC. In contrast, neutrophil infiltration, degeneration of hyaline cartilage and erosion of subchondral bone were not affected.
Tryptase inhibition was effective in inhibiting some inflammatory parameters associated to mBSA/IL-1β-induced arthritis, notably late phase oedema formation and IL-6 production, but not neutrophil infiltration and joint degeneration. These results suggest that the therapeutic application of tryptase inhibitors to rheumatoid arthritis would be restrained to palliative care, but not as disease-modifying drugs. Finally, this study highlighted lentivirus-based gene delivery as an instrumental tool to study the relevance of target genes in synovial joint physiology and disease.
Abstract Temporomandibular disorders represent one of the major challenges in dentistry therapeutics. This study was undertaken to evaluate the time course of carrageenan-induced inflammation in the ...rat temporomandibular joint (TMJ) and to investigate the role of tachykinin NK1 receptors. Inflammation was induced by a single intra-articular (i.art.) injection of carrageenan into the left TMJ (control group received sterile saline). Inflammatory parameters such as plasma extravasation, leukocyte influx and mechanical allodynia (measured as the head-withdrawal force threshold) and TNFα and IL-1β concentrations were measured in the TMJ lavages at selected time-points. The carrageenan-induced responses were also evaluated after treatment with the NK1 receptor antagonist SR140333. The i.art. injection of carrageenan into the TMJ caused a time-dependent plasma extravasation associated with mechanical allodynia, and a marked neutrophil accumulation between 4 and 24 h. Treatment with SR140333 substantially inhibited the increase in plasma extravasation and leukocyte influx at 4 and 24 h, as well as the production of TNFα and IL-1β into the joint cavity, but failed to affect changes in head-withdrawal threshold. The results obtained from the present TMJ-arthritis model provide, for the first time, information regarding the time course of this experimental inflammatory process. In addition, our data show that peripheral NK1 receptors mediate the production of both TNFα and IL-1β in the TMJ as well as some of the inflammatory signs, such as plasma extravasation and leukocyte influx, but not the nociceptive component.
Activity-based probes (ABP) are molecules that bind covalently to the active form of an enzyme family, making them an attractive tool for target and biomarker identification and drug discovery. The ...present study describes the synthesis and biochemical characterization of novel activity-based probes targeting trypsin-like serine proteases. We developed an extensive library of activity-based probes with "clickable" affinity tags and a diaryl phosphonate warhead. A wide diversity was achieved by including natural amino acid analogs as well as basic polar residues as side chains. A detailed enzymatic characterization was performed in a panel of trypsin-like serine proteases. Their inhibitory potencies and kinetic profile were examined, and their IC
values, mechanism of inhibition, and kinetic constants were determined. The activity-based probes with a benzyl guanidine side chain showed the highest inhibitory effects in the panel. Surprisingly, some of the high-affinity probes presented a reversible inhibitory mechanism. On the other hand, probes with different side chains exhibited the expected irreversible mechanism. For the first time, we demonstrate that not only irreversible probes but also reversible probes can tightly label recombinant proteases and proteases released from human mast cells. Even under denaturing SDS-PAGE conditions, reversible slow-tight-binding probes can label proteases due to the formation of high-affinity complexes and slow dissociation rates. This unexpected finding will transform the view on the required irreversible nature of activity-based probes. The diversity of this library of activity-based probes combined with a detailed enzyme kinetic characterization will advance their applications in proteomic studies and drug discovery.
PDF
