Nuclear Factor-kappa B inhibition in mouse aortic smooth muscle cells stimulated by cholesterol overload or tumor necrosis factor
Ontology highlight
ABSTRACT:
INSTRUMENT(S): DNBSEQ-G400
ORGANISM(S): Mus musculus
SUBMITTER:
PROVIDER: E-MTAB-13416 | biostudies-arrayexpress |
SECONDARY ACCESSION(S): E-MTAB-12069
REPOSITORIES: biostudies-arrayexpress
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Project description:To map gene regulation downstream of cholesterol overload and NF-kappaB signaling in smooth muscle cells (SMCs), we cultured primary aortic SMCs from wildtype mice with cyclodextrin-complexed cholesterol or the prototypical NF-kappaB activator, tumor necrosis factor (TNF), or both.
2023-05-29 | E-MTAB-12069 | biostudies-arrayexpress
Project description:Objective – Vascular calcification is a critical pathology associated with increased cardiovascular event risk, but there are no FDA-approved anti-calcific therapies. We hypothesized and validated that an unbiased screening approach would identify novel mediators of human vascular calcification. Approach and Results – We performed an unbiased quantitative proteomics and pathway network analysis that identified increased carnitine O-octanoyltransferase (CROT) in calcifying primary human coronary artery smooth muscle cells (SMCs). Additionally, human carotid artery atherosclerotic plaques contained increased immunoreactive CROT near calcified regions. CROT siRNA reduced fibrocalcific response in calcifying SMCs. In agreement, histidine 327 to alanine point mutation inactivated human CROT fatty acid metabolism enzymatic activity and suppressed SMC calcification. CROT siRNA restored mitochondrial proteome alterations and suppressed mitochondrial fragmentation in calcifying SMCs. Lipidomics analysis of SMCs incubated with CROT siRNA revealed increased eicosapentaenoic acid, a vascular calcification inhibitor. CRISPR/Cas9-mediated Crot deficiency in low-density lipoprotein receptor-deficient mice reduced aortic and carotid artery calcification without altering bone density, or liver and plasma cholesterol and triglyceride concentrations. Conclusions – CROT is a novel inducer of vascular calcification via promoting fatty acid metabolism and mitochondrial dysfunction, as such CROT inhibition has strong potential as an anti-fibrocalcific therapy.
2021-02-26 | PXD022575 | Pride
Project description:The LIM-only protein FHL2 is expressed in SMCs and inhibits SMC-rich lesion formation. However, the underlying mechanism behind FHL2's action in SMCs has been only partially resolved. To further elucidate the role of FHL2 in SMCs we compared the transcriptome of cultured SMCs derived from wild-type (WT) and FHL2-knockout (KO) mice. Comparison of gene expression in aortic smooth muscle cells (SMCs) isolated from WT and FHL2-knockout (FHL2-KO) mice. SMCs isolated from three mouse aortas were pooled (Kurakula et al, PLOS One, 2014). Both for WT and FHL-KO mice three batches of SMCs (derived from 9 mice) were cultured. The SMCs were grown to confluency and maintained in serum-free medium for 48 hrs before harvest. Subsequently, RNA was isolated for gene expression profiling.
2014-10-07 | E-GEOD-56860 | biostudies-arrayexpress
Project description:Smooth muscle cells (SMCs) are important in a number of physiological systems and organs, including the cardiovascular system. The hallmark property of differentiated SMCs is the ability to contract, but contractile SMCs themselves show a range of phenotypes allowing prolonged tonic contraction in vascular smooth muscle or rapid phasic contraction in tissues such as bladder. Another distinctive characteristic, in contrast with terminally differentiated striated muscle cells, is that SMCs exhibit phenotypic plasticity. Vascular SMCs are able to modulate their phenotype along a continuum between a contractile phenotype, characteristic of healthy blood vessels, and a more proliferative âsyntheticâ phenotype, so-named for the enhanced synthesis and secretion of extracellular matrix components. Synthetic phenotype cells are found in a number of pathological situations such as atherosclerosis and arterial injury. We used mouse exon-junction (MJAY) arrays to gain insights into both the global contribution of alternative splicing events in re-shaping the transcriptome of dedifferentiating mouse aorta and bladder SMCs, and into the underlying regulatory mechanisms of the alternative splicing program. Affymetrix splice junction arrays (MJAY) were used to profile changes in both alternative splicing and transcript levels during the phenotypic modulation of smooth muscle cells when placed in culture. RNA extracted from intact aorta and bladder smooth muscle tissue was used for differentiated samples. For dedifferentiated, proliferative samples smooth muscle cells were enzymatically dispersed and grown in tissue culture for a week. Triplicate RNA samples were prepared from smooth muscle tissue of mouse aorta and bladder (differentiated) and from smooth muscle cells from each tissue cultured for 7 days (proliferative). The samples allowed comparison of alternative splicing (and other transcriptome) changes between differentiated and proliferative smooth muscle cell samples from two distinct types of smooth muscle cell, as well as allowing direct comparison of aorta (tonic smooth muscle) and bladder (phasic smooth muscle).
2016-07-01 | E-MTAB-4841 | biostudies-arrayexpress
Project description:In this study, murine primary aortic smooth muscle cells (SMCs) were transcriptionally profiled at baseline, after 3 d of cholesterol loading, and after 3 d of subsequent cholesterol unloading with HDL treatment, to identify vascular SMC genes that are transcripionally dysregulated in response to cholesterol loading and/or unloading. Mouse aortic SMCs were isolated from thoracic aortas of 8 week-old C57BL/6 mice (as described in Rong et al., Proc Nat Acad Sci USA, v100, pp13531M-bM-^@M-^S13536, 2003) and subconfluent cell preparations were harvested and transcriptionally profiled (baseline group) or treated with 10 M-NM-<g/mL cholesterol:methyl-M-NM-2-cyclodextrin complex (1:6 molar ratio) for 72 h for cholesterol loading. Of the cholesterol-loaded cells, samples were transcriptionally profiled (cholesterol samples) or treated for an additional 72 h with 100 M-NM-<g/mL human HDL and then transcriptionally profiled (HDL treatment). For all transcriptional profiling, RNA was isolated from cells using TRIzol, and labeled aRNA was prepared for hybridization to Affymetrix Mouse Genome 430 2.0 microarrays according to the manufacturer's protocol. Probe-level intensities were background-adjusted and normalized (in two separate analyses, one with baseline and cholesterol-treated samples, and the other analysis with all three sample groups) using Robust Multichip Average and combined into probesets using probe-to-probeset mappings from the University of Michigan CustomCDF project based on Ensembl Gene identifiers (release 59). Using the data from the analysis that combined the baseline and cholesterol-loaded samples, log2 probeset intensities were compared the between baseline and cholesterol sample groups using a one-way ANOVA with a P value cutoff determined by a Benjamini-Hochberg false discovery rate threshold of 0.05. Analysis (I): baseline and cholesterol-treated sample groups. Analysis (II): all three sample groups.
2015-02-06 | E-GEOD-47744 | biostudies-arrayexpress
Project description:This experiment is part of the FunGenES project (FunGenES - Functional Genomics in Embryonic Stem Cells partially funded by the 6th Framework Programme of the EuropeanUnion, http://www.fungenes.org). The experiment was conducted at University of Cologne, Cologne, Germany. Goal of the experiment is a complete transcriptome profiling of contractile smooth muscle cells (SMCs) differentiated from embryonic stem cells which is crucial for the characterization of smooth muscle gene expression signatures and will contribute to defining biological and physiological processes in these cells. We have generated a transgenic embryonic stem cell line expressing both the puromycin acetyl transferase and enhanced green fluorescent protein cassettes under the control of the Acta2 promoter.This experiment allows the identification of specific biological and physiological processes in the contractile phenotype SMCs and will contribute to the understanding of these processes in early SMCs derived from embryonic stem cells.
2011-02-25 | E-TABM-1123 | biostudies-arrayexpress
Project description:Mouse aorta smooth muscle cells (SMCs) express TNF receptor superfamily member 1A (TNFR1) and lymphotoxin à receptor (LTÃR). Circumstantial evidence has linked the SMC LTÃR to tertiary lymphoid organogenesis in diseased aortae of hyperlipidemic mice. Here, we explored potential roles of TNFR1 and LTÃR activation in cultured SMCs. TNFR1 signaling by TNF activated the classical RelA NF-κB pathway, whereas LTÃR signaling by agonistic anti LTÃR antibody activated both the classical RelA and alternative RelB NF-κB pathways. Addition of both agonists synergized to enhance p100 inhibitor processing to the p52 subunit of NF-κB and promoted its nuclear translocation suggesting RelA-RelB cross-talk in transcription regulation. Correspondingly, microarrays showed that simultaneous TNFR1 and LTÃR activation when compared to activation of single receptors was followed by markedly elevated levels of mRNAs encoding leukocyte homeostatic chemokines CCL2, CCL5, CXCL1, and CX3CL1. Furthermore, SMCs acquired prototypical features of mesenchymal cells known as lymphoid tissue organizers (LTOs), which control tertiary lymphoid organogenesis in autoimmune diseases, through hyperinduction of CCL7, CCL9, CXCL13, CCL19, CXCL16, VCAM-1, and ICAM-1. Experiments with ltbr-/- SMCs suggested that the LTÃR-RelB activation component of NF-κB signaling was obligatory to generate the LTO phenotype. TNFR1-LTÃR crosstalk also resulted in augmented synthesis and prolonged secretion of lymphorganogenic chemokine proteins into the culture medium. Thus, combined TNFR1-LTÃR signaling triggers SMC transdifferentiation into a phenotype that strikingly resembles LTOs. LTO-like SMCs may adopt a thus far unrecognized role in diseased arteries, i.e. to coordinate tertiary lymphoid organogenesis in atherosclerosis, aortic aneurysm, and transplant vasculopathy. Experiment Overall Design: Smooth muscle cells in tissue culture were stimulated with agonists and subsequently microarray, RT-PCR, and protein assays / analyses were performed
2009-12-02 | E-GEOD-15062 | biostudies-arrayexpress
Project description:Loss of contractility and acquisition of an epithelial phenotype of vascular smooth muscle cells (VSMCs) are key events in proliferative vascular pathologies such as atherosclerosis and post-angioplastic restenosis. There is no proper cell culture system allowing VSMC differentiation so that it is difficult to delineate the molecular mechanism responsible for proliferative vasculopathy. We investigated whether a micro-patterned substrate could restore the contractile phenotype of VSMCs in vitro. To induce and maintain the differentiated VSMC phenotype in vitro, we introduced a micro-patterned groove substrate to modulate the morphology and function of VSMCs.
2013-11-14 | E-MTAB-1888 | biostudies-arrayexpress
Project description:In effort to develop methodology for targeted top down mass spectrometry of NF kappa B p65 from human cells, we evaluated the utility of HaloTag for purification and analysis of recombinant protein. During our study, two datasets of bottom up LC-MS/MS were generated: one from in-gel digestion of the predominant band following p65-HaloTag purification, another from in-solution digestion of all the proteins present in a p65-HaloTag purification. p65-HaloTag copurifying proteins identified in our datasets include the known interactors c-Rel, NF-kappaB p105, NF-kappaB p100, and NF-kappaB inhibitor beta. Over 100 proteins were identified by at least two peptides using a Mascot ion cut-off score of 30.
2018-10-19 | PXD001932 | Pride
Project description:Physiologically, smooth muscle cells (SMCs) and nitric oxide (NO) produced by endothelial cells strictly cooperate to maintain vascular homeostasis. In atherosclerosis, this equilibrium is altered, therefore molecules, able to provide exogenous NO and inhibit SMC proliferation, may represent valuable anti-atherosclerotic agents. Searching for dual anti-proliferative and NO-donor molecules, we found that furoxans significantly decreased SMC proliferation in vitro, albeit with different potencies. We therefore assessed whether this property is dependent on their thiol-induced ring opening. Indeed, while furazans (analogues unable to release NO) are not effective, furoxans’ inhibitory potency parallels with the electron-attractor capacity of the group in 3 of the ring, making this effect tunable. To demonstrate whether their specific block on G1-S phase could be NO-dependent, we supplemented SMCs with furoxans and inhibitors of GMP- and/or of the polyamines pathway, which regulate NO-induced SMC proliferation, but they failed in preventing the anti-proliferative effect. To find the real mechanism of this property, we performed a proteomics study, using a SILAC approach, revealing eleven cellular proteins (with SUMO1 being central) statistically deregulated by furoxans and involved in cell homeostasis/proliferation. Altogether, thanks to their dual effect and pharmacological flexibility, furoxans may be evaluated in future as anti-atherosclerotic molecules.
2023-07-31 | PXD043814 | Pride