Adequate lung epithelial repair relies on supportive interactions within the epithelial niche, including interactions with WNT-responsive fibroblasts. In fibroblasts from patients with chronic... Show moreAdequate lung epithelial repair relies on supportive interactions within the epithelial niche, including interactions with WNT-responsive fibroblasts. In fibroblasts from patients with chronic obstructive pulmonary disease (COPD) or upon in vitro cigarette smoke exposure, Wnt/beta-catenin signalling is distorted, which may affect interactions between epithelial cells and fibroblasts resulting in inadequate lung repair. We hypothesized that cigarette smoke (CS), the main risk factor for COPD, interferes with Wnt/beta-catenin signalling in fibroblasts through induction of cellular stress responses, including oxidative- and endoplasmic reticulum (ER) stress, and thereby alters epithelial repair support potential. Therefore, we assessed the effect of CS-exposure and the ER stress inducer Thapsigargin (Tg) on Wnt/beta-catenin signalling activation in MRC-5 fibroblasts, and on their ability to support lung epithelial organoid formation. Exposure of MRC-5 cells for 15 min with 5 AU/mL CS extract (CSE), and subsequent 6 h incubation induced oxidative stress (HMOX1). Whereas stimulation with 100 nM Tg increased markers of both the integrated stress response (ISR - GADD34/PPP1R15A, CHOP) and the unfolded protein response (UPR - XBP1spl, GADD34/PPP1R15A, CHOP and HSPA5/BIP), CSE only induced GADD34/PPP1R15A expression. Strikingly, although treatment of MRC-5 cells with the Wnt activator CHIR99021 upregulated the Wnt/beta-catenin target gene AXIN2, this response was diminished upon CSE or Tg pre-exposure, which was confirmed using a Wnt-reporter. Furthermore, pre-exposure of MRC-5 cells to CSE or Tg, restricted their ability to support organoid formation upon co-culture with murine pulmonary EpCam+ cells in Matrigel at day 14. This restriction was alleviated by pre-treatment with CHIR99021. We conclude that exposure of MRC-5 cells to CSE increases oxidative stress, GADD34/PPP1R15A expression and impairs their ability to support organoid formation. This inhibitory effect may be restored by activating the Wnt/beta-catenin signalling pathway. Show less
Homberg, D.A.L. van den; Kwast, R.V.C.T. van der; Quax, P.H.A.; Nossent, A.Y. 2022
N-6-methyladenosine (m6A) is the most prevalent post-transcriptional RNA modification in eukaryotic cells. The modification is reversible and can be dynamically regulated by writer and eraser... Show moreN-6-methyladenosine (m6A) is the most prevalent post-transcriptional RNA modification in eukaryotic cells. The modification is reversible and can be dynamically regulated by writer and eraser enzymes. Alteration in the levels of these enzymes can lead to changes in mRNA stability, alternative splicing or microRNA processing, depending on the m6A-binding proteins. Dynamic regulation of mRNA m6A methylation after ischemia and hypoxia influences mRNA stability, alternative splicing and translation, contributing to heart failure. In this study, we studied vasoactive microRNA m6A methylation in fibroblasts and examined the effect of hypoxia on microRNAs methylation using m6A immunoprecipitation. Of the 19 microRNAs investigated, at least 16 contained m6A in both primary human fibroblasts and a human fibroblast cell line, suggesting vasoactive microRNAs are commonly m6A methylated in fibroblasts. More importantly, we found that mature microRNA m6A levels increased upon subjecting cells to hypoxia. By silencing different m6A writer and eraser enzymes followed by m6A immunoprecipitation, we identified METTL4, an snRNA m6A methyltransferase, to be predominantly responsible for the increase in m6A modification. Moreover, by using m6A-methylated microRNA mimics, we found that microRNA m6A directly affects downstream target mRNA repression efficacy. Our findings highlight the regulatory potential of the emerging field of microRNA modifications. Show less
BackgroundHuman mesenchymal cells are culprit factors in vascular (patho)physiology and are hallmarked by phenotypic and functional heterogeneity. At present, they are subdivided by classic... Show moreBackgroundHuman mesenchymal cells are culprit factors in vascular (patho)physiology and are hallmarked by phenotypic and functional heterogeneity. At present, they are subdivided by classic umbrella terms, such as "fibroblasts," "myofibroblasts," "smooth muscle cells," "fibrocytes," "mesangial cells," and "pericytes." However, a discriminative marker-based subclassification has to date not been established.Methods and ResultsAs a first effort toward a classification scheme, a systematic literature search was performed to identify the most commonly used phenotypical and functional protein markers for characterizing and classifying vascular mesenchymal cell subpopulation(s). We next applied immunohistochemistry and immunofluorescence to inventory the expression pattern of identified markers on human aorta specimens representing early, intermediate, and end stages of human atherosclerotic disease. Included markers comprise markers for mesenchymal lineage (vimentin, FSP-1 [fibroblast-specific protein-1]/S100A4, cluster of differentiation (CD) 90/thymocyte differentiation antigen 1, and FAP [fibroblast activation protein]), contractile/non-contractile phenotype (alpha-smooth muscle actin, smooth muscle myosin heavy chain, and nonmuscle myosin heavy chain), and auxiliary contractile markers (h1-Calponin, h-Caldesmon, Desmin, SM22 alpha [smooth muscle protein 22 alpha], non-muscle myosin heavy chain, smooth muscle myosin heavy chain, Smoothelin-B, alpha-Tropomyosin, and Telokin) or adhesion proteins (Paxillin and Vinculin). Vimentin classified as the most inclusive lineage marker. Subset markers did not separate along classic lines of smooth muscle cell, myofibroblast, or fibroblast, but showed clear temporal and spatial diversity. Strong indications were found for presence of stem cells/Endothelial-to-Mesenchymal cell Transition and fibrocytes in specific aspects of the human atherosclerotic process.ConclusionsThis systematic evaluation shows a highly diverse and dynamic landscape for the human vascular mesenchymal cell population that is not captured by the classic nomenclature. Our observations stress the need for a consensus multiparameter subclass designation along the lines of the cluster of differentiation classification for leucocytes. Show less
Up till now, research on inflammatory bowel disease [IBD] has mainly been focused on the immune cells present in the gastrointestinal tract. However, recent insights indicate that stromal cells... Show moreUp till now, research on inflammatory bowel disease [IBD] has mainly been focused on the immune cells present in the gastrointestinal tract. However, recent insights indicate that stromal cells also play an important and significant role in IBD pathogenesis. Stromal cells in the intestines regulate both intestinal epithelial and immune cell homeostasis. Different subsets of stromal cells have been found to play a role in other inflammatory diseases [e.g. rheumatoid arthritis], and these various stromal subsets now appear to carry out also specific functions in the inflamed gut in IBD. Novel potential therapies for IBD utilize, as well as target, these pathogenic stromal cells. Injection of mesenchymal stromal cells [MSCs] into fistula tracts of Crohn's disease patients is already approved and used in clinical settings. In this review we discuss the current knowledge of the role of stromal cells in IBD pathogenesis. We further outline recent attempts to modify the stromal compartment in IBD with agents that target or replace the pathogenic stroma. Show less
Aim: UVA radiation drives skin photoaging in the dermis, plausibly via persistent changes to DNA methylation in dermal fibroblasts. Methods: Genome-wide DNA methylation changes after five repeated... Show moreAim: UVA radiation drives skin photoaging in the dermis, plausibly via persistent changes to DNA methylation in dermal fibroblasts. Methods: Genome-wide DNA methylation changes after five repeated daily UVA doses were determined at 48 h (transitionary) and 1 week (recovery) post final irradiation. Results: Differential methylation was found at the transitionary time point in active chromatin states near genes that are highly expressed in fibroblasts and are involved in cellular defensive mechanisms; the majority of these methylation differences were restored to control levels after 7 day recovery. At the recovery time point, new differential methylation occurred at repressed regions near developmental genes, normally weakly expressed in fibroblasts. Conclusion: UVA irradiation induces transitionary and recovery-associated DNA methylation responses in fibroblasts with contrasting functional characteristics. Show less
Helm, D. van der; Barnhoorn, M.C.; Jonge-Muller, E.S.M. de; Molendijk, I.; Hawinkels, L.J.A.C.; Coenraad, M.J.; ... ; Verspaget, H.W. 2019
