Single Cell Analysis of Lung Lymphatic Endothelial Cells and Lymphatic Responses during Influenza Infection
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ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE266192 | GEO | 2024/05/06
REPOSITORIES: GEO
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Project description:ERG and FLI1 are important regulators of angiogenesis, but their role in lymphatic vasculature is less known. The goal of this study was to determine the role of ERG and FLI1 in postnatal lymphangiogenesis and their involvement in SSc lymphatic system defects. IF were used to detect ERG and FLI1 in SSc and healthy controls (HC) skin biopsies. Human lymphatic endothelial cells (LECs) were used in cell culture experiments. Transcriptional and chromatin analysis of ERG or FLI1 silenced LECs was performed using microarrays and ATAC-seq, respectively. Effects of ERG and FLI1 deficiency on in vitro tubulogenesis was examined using Matrigel assay. Erg and Fli1 endothelial specific knockouts (ErgCKO and Fli1CKO) and lymphatic specific knockouts (ProxErgCKO and ProxFli1CKO) were generated to examine vessel regeneration. ERG and FLI1 protein levels were reduced in blood and lymphatic vasculature in SSc skin biopsies. ERG and FLI1 were shown to regulate genes involved in lymphatic vessel specification, including VEGFR3/FLT4, LYVE-1, and PROX1, and this effect was, at least in part, due to chromatin remodeling. ERG/FLT4 pathway regulated in vitro tubulogenesis in LECs. Deficiency of Erg and Fli1 impaired function of blood and lymphatic vessel during wound healing. ERG and FLI1 are essential regulators of blood and lymphatic vessel regeneration. Deficiency of ERG and FLI1 in SSc endothelial cells, may contribute to rarefaction of blood and lymphatic vasculature in SSc patients. Microarray analysis was performed at The Boston University Microarray Core Facility. Affymetrix CEL files were normalized to produce gene-level expression values using the implementation of the Robust Multiarray Average (RMA) in the affy package (version 1.36.1) included within in the Bioconductor software suite (version 2.12) and an Entrez Gene-specific probe set mapping from BrainArray (version 16.0.0). RLE and NUSE quality metrics were computed using the affy PLM Bioconductor package (version 1.34.0). All microarray analyses were performed using the R environment for statistical computing (version 2.15.1).
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Project description:Liver lymphatic vessels support liver function by draining interstitial fluid, cholesterol, fat, and immune cells for surveillance in the liver draining lymph node. Chronic liver disease is associated with increased inflammation and immune cell infiltrate. However, it is currently unknown if or how lymphatic vessels respond to increased inflammation and immune cell infiltrate in the liver during chronic disease. Here we demonstrate that lymphatic vessel abundance increases in patients with chronic liver disease and is associated with areas of fibrosis and immune cell infiltration. Using single-cell mRNA sequencing and multi-spectral immunofluorescence analysis we identified liver lymphatic endothelial cells and found that chronic liver disease results in lymphatic endothelial cells (LECs) that are in active cell cycle with increased expression of CCL21. Additionally, we found that LECs from patients with NASH adopt a transcriptional program associated with increased IL13 signaling. Moreover, we found that oxidized low density lipoprotein, associated with NASH pathogenesis, induced the transcription and protein production of IL13 in LECs both in vitro and in a mouse model. Finally, we show that oxidized low density lipoprotein reduced the transcription of PROX1 and decreased lymphatic stability. Together these data indicate that LECs are active participants in the liver, expanding in an attempt to maintain tissue homeostasis. However, when inflammatory signals, such as oxidized low density lipoprotein are increased, as in NASH, lymphatic function declines and liver homeostasis is impeded.
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Project description:Background: The inflammatory response to acute kidney injury (AKI) likely dictates future renal health. Lymphatic vessels are responsible for maintaining tissue homeostasis through transport and immunomodulatory roles. Due to the relative sparsity of lymphatic endothelial cells (LECs) in the kidney, past sequencing efforts have not characterized these cells and their response to AKI. Methods: Here we characterized murine renal LEC subpopulations by single-cell RNA sequencing and investigated their changes in cisplatin AKI 72 hours post injury. Data was processed using the Seurat package. We validated our findings by qPCR in LECs isolated from both cisplatin-injured and ischemia reperfusion injury, by immunofluorescence, and confirmation in in vitro human LECs. Results: We have identified renal LECs and their lymphatic vascular roles that have yet to be characterized in previous studies. We report unique gene changes mapped across control and cisplatin injured conditions. Following AKI, renal LECs alter genes involved in endothelial cell apoptosis and vasculogenic processes as well as immunoregulatory signaling and metabolism. Differences between injury models were also identified with renal LECs further demonstrating changed gene expression between cisplatin and ischemia reperfusion injury models, indicating the renal LEC response is both specific to where they lie in the lymphatic vasculature and the renal injury type. Conclusions: In this study, we uncover lymphatic vessel structural features of captured populations and injury-induced genetic changes. We further determine LEC gene expression is altered between injury models. How LECs respond to AKI may therefore be key in regulating future kidney disease progression.
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2023-01-16 | GSE201916 | GEO