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Expression profiling of lung endothelial cells from control and nANGPTL4 AAV treated mice.

ABSTRACT: Expression profiling of lung endothelial cells from control and nANGPTL4 AAV treated mice.

PROVIDER: PRJNA865121 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR20748500_1.fastq.gz	Fastqsanger.gz
	SRR20748500_2.fastq.gz	Fastqsanger.gz
	SRR20748501_1.fastq.gz	Fastqsanger.gz
	SRR20748501_2.fastq.gz	Fastqsanger.gz
	SRR20748502_1.fastq.gz	Fastqsanger.gz

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Project description:Age-related decline in brain endothelial cell (BEC) function critically contributes to cerebrovascular and neurodegenerative disease. Comprehensive atlases of the BEC transcriptome have become available but results from proteomic profiling are lacking. To gain insights into endothelial pathways affected by aging, we developed a magnetic-activated cell sorting (MACS)-based mouse BEC enrichment protocol compatible with high-resolution mass-spectrometry and analysed the profiles of protein abundance changes across multiple time points between 3 and 18 months of age and identified Arf6 as one of the most prominently downregulated vesicle-mediated transport protein during BEC aging. To understand the role of ARF6 in human ECs, first we compared GFP-AAV treated human iECs differentiated from ARF6-KO vs WT iPSCs, next we compared ARF6-GFP-AAV vs GFP-AAV treated iECs differentiated from WT iPSCs. During the ARF6-KO vs WT iEC comparison we found 983 vs 741 proteins significantly down- and upregulated, respectively. Enrichment analyses of significantly downregulated proteins revealed mRNA processing among the most significantly affected biological processes. During the ARF6-GFP-AAV vs GFP-AAV treated WT iEC comparison we found 1106 vs 1218 proteins significantly down- and upregulated, respectively. Enrichment analyses of significantly upregulated proteins revealed endocytic recycling, retrograde transport (endosome to Golgi), and ER-Golgi vesicle-mediated transport among the most significantly affected biological processes. Specifically, ARF6 and its binding-protein GGA2, DNM1L and several subunits of the Conserved oligomeric Golgi complex (COG) were upregulated. Our approach uncovered changes not picked up by transcriptomic studies such as accumulation of vesicle cargo and receptor ligands including Apoe, a major regulator of brain lipid metabolism. Proteomic analysis of BECs from Apoe deficient mice revealed a signature of accelerated aging. To explore the role of APOE in human endothelial cells, in this experiment we compared human iECs differentiated from APOE-KO and WT iPSCs and found 326 significantly altered proteins. Enrichment analyses of significantly downregulated proteins revealed vesicle-mediated transport and vesicle fusion to be among the most significantly affected biological processes. Specifically, among the 34 significantly altered vesicle-mediated transport proteins 26 were downregulated. Accordingly, we found reduced levels of endocytosis of FM1-43FX in newly formed vesicles in APOE-KO iECs.

Project description:Injury of the lung epithelium is one of the initial events driving acute lung diseases such as acute lung injury and acute respiratory distress syndrome. Repeated epithelial injury followed by aberrant repair leads to fibroblast activation and extracellular matrix deposition and is therefore considered as the major cause of chronic pulmonary diseases, such as idiopathic pulmonary fibrosis. In vitro models as well as in vivo animal models are necessary to investigate early disease mechanisms and altered signalling pathways of epithelial cells. One way to target specific cells is the diphtheria toxin receptor/diphtheria toxin (DTR/DT) system. The human DTR (hDTR) is sensitive to DT, thereby inducing cell death by blocking protein synthesis, while the murine DTR is at least 10^5 times more resistant to DT. Consequently, expression of the hDTR and application of DT lead to targeted cell depletion. Therefore, a novel and flexible DTR/DT model of acute epithelial lung injury was established and described recently, which is driven by adeno-associated virus (AAV) variant 6.2 mediated hDTR expression. The AAV6.2 vector transduces specifically into bronchial epithelial and alveolar epithelial type II cells leading to their depletion after DT administration. In this study we analysed the bulk lung proteome of the recently established AAV-DTR/DT mouse model of acute epithelial lung injury, providing a detailed insight into proteomic changes upon injury. Frozen pulverized lung tissue samples (~10 mg per sample) from AAV-stuffer control (1 E11 viral genome, n=7) and AAV-hDTR (0.3 E11 viral genome, n=7) treated mice 24 h after intratracheal application of 100 ng DT were analysed in a TMTpro-based workflow. TMTpro-labelled peptides were fractionated into 24 fractions using off-line high pH reversed phase chromatography. Fractions were analysed on an Orbitrap Eclipse Tribrid mass spectrometer in combination with the FAIMS Pro Interface (Thermo Scientific) using a SPS-MS3 based method including real-time search.

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Expression profiling of lung endothelial cells from control and nANGPTL4 AAV treated mice.

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