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LC-MS/MS analysis of isolated murine brain endothelial cells (BECs) from ARF6-KO and WT mice followed by label-free quantification (LFQ)

ABSTRACT: 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 better understand the role of Arf6 in BECs, in this experiment we have compared MACS sorted BECs from Arf6-KO and WT 3-months-old mice and found 140 and 172 proteins to be significantly down- and upregulated, respectively. Enrichment analyses of significantly downregulated proteins showed mRNA processing to be among the most affected biological processes consistent with our findings on the aged BEC proteome.

INSTRUMENT(S): timsTOF Pro

ORGANISM(S): Mus Musculus (mouse)

TISSUE(S): Brain, Endothelial Cell

SUBMITTER: Stephan Mueller

LAB HEAD: Stefan F. Lichtenthaler

PROVIDER: PXD045004 | Pride | 2024-02-15

REPOSITORIES: Pride

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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:Preclinical studies of primary cancer cells are always done after tumors are removed from patients or animals at ambient atmospheric oxygen (O2, ~21%). However, O2 concentrations in organs are in the ~3-10% range, with most tumors in an hypoxic or 1-2% O2 environment in vivo. Although effects of O2 tension on tumor cell characteristics in vitro have been studied, these studies are done only after tumors are first collected and processed in ambient air. Similarly, sensitivity of primary cancer cells to anti-cancer agents is routinely examined at ambient O2. Here, using both mouse models and human cancers, we demonstrate that tumors collected, processed and propagated at physiologic (physioxia) O2 compared to ambient air display very distinct differences in key signaling networks including Lgr5/Wnt, Yap, and Nrf2/Keap1, nuclear reactive oxygen species levels, alternative splicing, and sensitivity to several targeted therapies including PIK3CAalpha-specific and EGFR inhibitors. Significance: Extra-physiologic oxygen shock/stress (EPHOSS), as noted in cells collected/processed under ambient air, has been demonstrated to have significant impact on numbers and engrafting ability of hematopoietic stem cells. We report deleterious effects of EPHOSS on cancer cell behavior and EPHOSS-mediated effects on cancer cells give misleading information on signaling pathway activation that could severely impact the relevance of these findings. Cancer cells under EPHOSS show higher proliferation rate compared to cells under physioxia and thus are sensitive to anti-proliferative agents. Thus, drugs that show effectiveness on cancer cells collected in ambient air and subjected to EPHOSS may not be effective or as relevant in vivo, results that could partially explain the limited clinical translation of laboratory findings. Evaluating cell signaling and effects of drugs on cancer cells under physiologic O2 prior to in vivo studies could substantially reduce cost and aid in drug discovery relevant to the actual physioxia/pathological status of the tumor cells in vivo.

Dataset Information

LC-MS/MS analysis of isolated murine brain endothelial cells (BECs) from ARF6-KO and WT mice followed by label-free quantification (LFQ)

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