Dataset Information

Activation of the NFκB signaling pathway in IL6+CSF3+ vascular endothelial cells promotes the formation of keloids.

ABSTRACT: Background: Keloid is a disease caused by abnormal proliferation of skin fibres, the causative mechanism of which remains unclear. Method: In this study, endothelial cells of keloids were studied using scRNAseq combined with bulk-RNAseq data from keloids. The master regulators driving keloid development were identified by transcription factor enrichment analysis. The pattern of changes in vascular endothelial cells during keloid development was explored by inferring endothelial cell differentiation trajectories. Deconvolution of bulkRNAseq by CIBERSORTX verified the pattern of keloidogenesis. Immunohistochemistry for verification of the lesion process in keloid endothelial cells. Results: The endothelial cells of keloids consist of four main cell populations (MMP1+ Endo0, FOS + JUN + Endo1, IL6+CSF3+Endo2, CXCL12 + Endo3). Endo3 is an endothelial progenitor cell, Endo1 is an endothelial cell in the resting state, Endo2 is an endothelial cell in the activated state and Endo0 is an endothelial cell in the terminally differentiated state. Activation of the NFΚB signaling pathway is a typical feature of Endo2 and represents the early skin state of keloids. Conclusion: We have identified patterns of vascular endothelial cell lesions during keloidogenesis and development, and have found that activation of the NFΚB signaling pathway is an essential feature of keloid formation. These findings are expected to contribute to the understanding of the pathogenesis of keloids and to the development of new targeted therapeutic agents for the lesional characteristics of vascular endothelial cells.

SUBMITTER: Liu D

PROVIDER: S-EPMC9445273 | biostudies-literature | 2022

REPOSITORIES: biostudies-literature

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Activation of the NFκB signaling pathway in IL6+CSF3+ vascular endothelial cells promotes the formation of keloids.

Liu Delin D Zhang Yidi Y Zhen Lisha L Xu Rong R Ji Zhenling Z Ye Zheng Z

Frontiers in bioengineering and biotechnology 20220823

<b>Background:</b> Keloid is a disease caused by abnormal proliferation of skin fibres, the causative mechanism of which remains unclear. <b>Method:</b> In this study, endothelial cells of keloids were studied using scRNAseq combined with bulk-RNAseq data from keloids. The master regulators driving keloid development were identified by transcription factor enrichment analysis. The pattern of changes in vascular endothelial cells during keloid development was explored by inferring endothelial cel ...[more]

PMID: 36082167

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Project description:Wound healing is a complex biological process, and imbalances of various substances in the wound environment may prolong healing and lead to excessive scarring. Keloid is abnormal proliferation of scar tissue beyond the original wound margins with excessive deposition of extracellular matrix (ECM) and chronic inflammation. Despite numerous previous research efforts, the pathogenesis of keloid remains unknown. Vascular endothelial cells (VECs) are a major type of inductive cell in inflammation and fibrosis. Despite several studies on vascular morphology in keloid formation, there has been no functional analysis of the role of VECs. In the present study, we isolated living VECs from keloid tissues and investigated gene expression patterns using microarray analysis. We obtained 5 keloid tissue samples and 6 normal skin samples from patients without keloid. Immediately after excision, tissue samples were gently minced and living cells were isolated. Magnetic-activated cell sorting of VECs was performed by negative selection of fibroblasts and CD45+ cells and by positive selection of CD31+cells. After RNA extraction, gene expression analysis was performed to compare VECs isolated from keloid tissue (KVECs) with VECs from normal skin (NVECs). After cell isolation, the percentage of CD31+ cells as measured by flow cytometry ranged from 81.8%-98.6%. Principal component analysis was used to identify distinct molecular phenotypes in KVECs versus NVECs and these were divided into two subgroups. In total, 15 genes were upregulated, and 3 genes were downregulated in KVECs compared with NVECs using the t-test (< 0.05). Quantitative RT-PCR and immunohistochemistry showed 16-fold and 11-fold overexpression of SERPINA3 and LAMC2, respectively. SERPINA3 encodes the serine protease inhibitor, α1-antichymotripsin. Laminin γ2-Chain (LAMC2) is a subunit of laminin-5 that induces retraction of vascular endothelial cells and enhances vascular permeability. This is the first report of VEC isolation and gene expression analysis in keloid tissue. Our data suggest that SERPINA3 and LAMC2 upregulation in KVECs may contribute to the development of fibrosis and prolonged inflammation in keloid. Further functional investigation of these genes will help clarify the mechanisms of abnormal scar tissue proliferation.

Project description:Microglial and astrocyte activation and related cytokine secretion play key roles in secondary brain injury following intracerebral hemorrhage (ICH). We assessed the role of aquaporin (AQP)2 in immune response after ICH. We prospectively collected data from 33 patients with ICH and analyzed the serum AQP2 levels in these patients and age-matched healthy controls. A correlation analysis was also performed between patient serum AQP2 levels and clinical factors. In the rat ICH model, double-fluorescence staining for glial fibrillary acidic protein (GFAP) and AQP2 was performed to investigate the relationship between astrocytes and AQP2. Relative mRNA expression levels of GFAP and AQP2 were also measured. In the rat astrocyte cell line CTX-TNA2, toll-like receptor (TLR)4/nuclear factor kappa B (NFκB)-p65 pathway activation and GFAP levels were measured. The indirect influence of AQP2 on microglial polarization was assessed following exposure to the medium of astrocytes treated with AQP2-overexpression plasmid or silencing RNA. We found that the serum AQP2 expression was lower in patients with ICH. Sex and blood neutrophil count influenced serum AQP2 concentrations in patients with ICH on admission. Lower serum AQP2 levels were inversely correlated with 90-day Modified Rankin Scale scores after ICH, but were not correlated with National Institute of Health stroke scale (NIHSS) scores on admission. AQP2 overexpression and localization in GFAP-labeled astrocytes were observed in rats. AQP2 overexpression induced astrocyte activation with GFAP upregulation via TLR/NFκB-p65 signaling pathway activation in the rat astrocyte cell line CTX-TNA2. Astrocyte activation promoted interleukin-1β secretion. The medium of AQP2-overexpression astrocytes promoted the pro-inflammatory M1 phenotype in the immortal rat (HAPI) microglial cell line. Therefore, serum AQP2 is negatively correlated with post-ICH prognosis and may be a marker of inflammation in early-stage ICH. AQP2 overexpression promotes astrocyte activation and pro-inflammatory secretion, affects astrocyte-microglia crosstalk, and indirectly induces microglial polarization, which may augment inflammation after ICH.

Project description:ObjectiveMitogen-activated protein kinase pathways play an important role in neointima formation secondary to vascular injury, in part by promoting proliferation of vascular smooth muscle cells (VSMC). Mixed-lineage kinase 3 (MLK3) is a mitogen-activated protein kinase kinase kinase that activates multiple mitogen-activated protein kinase pathways and has been implicated in regulating proliferation in several cell types. However, the role of MLK3 in VSMC proliferation and neointima formation is unknown. The aim of this study was to determine the function of MLK3 in the development of neointimal hyperplasia and to elucidate the underlying mechanisms.Approach and resultsNeointima formation was analyzed after endothelial denudation of carotid arteries from wild-type and MLK3-deficient mice. MLK3 deficiency promoted injury-induced neointima formation and increased proliferation of primary VSMC derived from aortas isolated from MLK3-deficient mice compared with wild-type mice. Furthermore, MLK3 deficiency increased the activation of p63Rho guanine nucleotide exchange factor, RhoA, and Rho kinase in VSMC, a pathway known to promote neointimal hyperplasia, and reconstitution of MLK3 expression attenuated Rho kinase activation. Furthermore, cJun NH2-terminal kinase activation was decreased in MLK3-deficient VSMC, and proliferation of wild-type but not MLK3 knockout cells treated with a cJun NH2-terminal kinase inhibitor was attenuated.ConclusionsWe demonstrate that MLK3 limits RhoA activation and injury-induced neointima formation by binding to and inhibiting the activation of p63Rho guanine nucleotide exchange factor, a RhoA activator. In MLK3-deficient cells, activation of p63Rho guanine nucleotide exchange factor proceeds in an unchecked manner, leading to a net increase in RhoA pathway activation. Reconstitution of MLK3 expression restores MLK3/p63Rho guanine nucleotide exchange factor interaction, which is attenuated by feedback from activated cJun NH2-terminal kinase.

Dataset Information

Activation of the NFκB signaling pathway in IL6+CSF3+ vascular endothelial cells promotes the formation of keloids.

Publications

Activation of the NFκB signaling pathway in IL6+CSF3+ vascular endothelial cells promotes the formation of keloids.

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