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To capture interplay between biological pathways, we analyzed the proteome from matched lung tissues and bronchoalveolar lavage fluid (BALF) of individual allergen-naïve and house dust mite (HDM)-challenged BALB/c mice, a model of allergic asthma. Unbiased label-free liquid chromatography with tandem mass spectrometry (LC-MS/MS) analysis quantified 2675 proteins from tissues and BALF of allergen-naïve and HDM-exposed mice. In comparing the four datasets, we found significantly greater diversity in proteins between lung tissues and BALF than in the changes induced by HDM challenge. The biological pathways enriched after allergen exposure were compartment-dependent. Lung tissues featured innate immune responses and oxidative stress, while BALF most strongly revealed changes in metabolism. We combined lung tissues and BALF proteomes, which principally highlighted oxidation reduction (redox) pathways, a finding influenced chiefly by the lung tissue dataset. Integrating lung and BALF proteomes also uncovered new proteins and biological pathways that may mediate lung tissue and BALF interactions after allergen challenge, for example, B-cell receptor signaling. We demonstrate that enhanced insight is fostered when different biological compartments from the lung are investigated in parallel. Integration of proteomes from lung tissues and BALF compartments reveals new information about protein networks in response to environmental challenge and interaction between intracellular and extracellular processes.

Summary Human induced pluripotent stem cells (hiPSCs) are used to study organogenesis and model disease as well as being developed for regenerative medicine. Endothelial cells are among the many cell types differentiated from hiPSCs, but their maturation and stabilization fall short of that in adult endothelium. We examined whether shear stress alone or in combination with pericyte co-culture would induce flow alignment and maturation of hiPSC-derived endothelial cells (hiPSC-ECs) but found no effects comparable with those in primary microvascular ECs. In addition, hiPSC-ECs lacked a luminal glycocalyx, critical for vasculature homeostasis, shear stress sensing, and signaling. We noted, however, that hiPSC-ECs have dysfunctional mitochondrial permeability transition pores, resulting in reduced mitochondrial function and increased reactive oxygen species. Closure of these pores by cyclosporine A improved EC mitochondrial function but also restored the glycocalyx such that alignment to flow took place. These results indicated that mitochondrial maturation is required for proper hiPSC-EC functionality., Graphical Abstract, Highlights • hiPSC-ECs lack a functional glycocalyx and fail to align to flow • hiPSC-ECs have reduced mitochondrial function and increased leakage of ROS • Closing the mPTP with cyclosporine A induces mitochondrial maturation • Improved mitochondrial function restores the glycocalyx and alignment to flow, Closure of the mitochondrial permeability transition pore by cyclosporine A improved mitochondrial function and maturation of hiPSC-ECs but also restored the glycocalyx such that alignment to flow took place. This functional glycocalyx, necessary for growth factor signaling and anticoagulation, is a prerequisite for future hiPSC-EC applications in tissue engineering, organoid vascularization and therapeutic use of hiPSC-ECs.

Tumor vessel co-option is poorly understood, yet it is a resistance mechanism against anti-angiogenic therapy (AAT). The heterogeneity of co-opted endothelial cells (ECs) and pericytes, co-opting cancer and myeloid cells in tumors growing via vessel co-option, has not been investigated at the single-cell level. Here, we use a murine AAT-resistant lung tumor model, in which VEGF-targeting induces vessel co-option for continued growth. Single-cell RNA sequencing (scRNA-seq) of 31,964 cells reveals, unexpectedly, a largely similar transcriptome of co-opted tumor ECs (TECs) and pericytes as their healthy counterparts. Notably, we identify cell types that might contribute to vessel co-option, i.e., an invasive cancer-cell subtype, possibly assisted by a matrix-remodeling macrophage population, and another M1-like macrophage subtype, possibly involved in keeping or rendering vascular cells quiescent. ispartof: CELL REPORTS vol:35 issue:11 ispartof: location:United States status: published

Integrator is a key regulator of NEAT1 isoform switching and, thereby, paraspeckles biogenesis and chemosensitivity., RNA 3′ end processing provides a source of transcriptome diversification which affects various (patho)-physiological processes. A prime example is the transcript isoform switch that leads to the read-through expression of the long non-coding RNA NEAT1_2, at the expense of the shorter polyadenylated transcript NEAT1_1. NEAT1_2 is required for assembly of paraspeckles (PS), nuclear bodies that protect cancer cells from oncogene-induced replication stress and chemotherapy. Searching for proteins that modulate this event, we identified factors involved in the 3′ end processing of polyadenylated RNA and components of the Integrator complex. Perturbation experiments established that, by promoting the cleavage of NEAT1_2, Integrator forces NEAT1_2 to NEAT1_1 isoform switching and, thereby, restrains PS assembly. Consistently, low levels of Integrator subunits correlated with poorer prognosis of cancer patients exposed to chemotherapeutics. Our study establishes that Integrator regulates PS biogenesis and a link between Integrator, cancer biology, and chemosensitivity, which may be exploited therapeutically.

BACKGROUND: Renal endothelial cells from glomerular, cortical, and medullary kidney compartments are exposed to different microenvironmental conditions and support specific kidney processes. However, the heterogeneous phenotypes of these cells remain incompletely inventoried. Osmotic homeostasis is vitally important for regulating cell volume and function, and in mammals, osmotic equilibrium is regulated through the countercurrent system in the renal medulla, where water exchange through endothelium occurs against an osmotic pressure gradient. Dehydration exposes medullary renal endothelial cells to extreme hyperosmolarity, and how these cells adapt to and survive in this hypertonic milieu is unknown. METHODS: We inventoried renal endothelial cell heterogeneity by single-cell RNA sequencing >40,000 mouse renal endothelial cells, and studied transcriptome changes during osmotic adaptation upon water deprivation. We validated our findings by immunostaining and functionally by targeting oxidative phosphorylation in a hyperosmolarity model in vitro and in dehydrated mice in vivo. RESULTS: We identified 24 renal endothelial cell phenotypes (of which eight were novel), highlighting extensive heterogeneity of these cells between and within the cortex, glomeruli, and medulla. In response to dehydration and hypertonicity, medullary renal endothelial cells upregulated the expression of genes involved in the hypoxia response, glycolysis, and-surprisingly-oxidative phosphorylation. Endothelial cells increased oxygen consumption when exposed to hyperosmolarity, whereas blocking oxidative phosphorylation compromised endothelial cell viability during hyperosmotic stress and impaired urine concentration during dehydration. CONCLUSIONS: This study provides a high-resolution atlas of the renal endothelium and highlights extensive renal endothelial cell phenotypic heterogeneity, as well as a previously unrecognized role of oxidative phosphorylation in the metabolic adaptation of medullary renal endothelial cells to water deprivation. ispartof: JOURNAL OF THE AMERICAN SOCIETY OF NEPHROLOGY vol:31 issue:1 pages:118-138 ispartof: location:United States status: published

The amount of biological data, generated with (single cell) omics technologies, is rapidly increasing, thereby exacerbating bottlenecks in the data analysis and interpretation of omics experiments. Data mining platforms that facilitate non-bioinformatician experimental scientists to analyze a wide range of experimental designs and data types can alleviate such bottlenecks, aiding in the exploration of (newly generated or publicly available) omics datasets. Here, we present BIOMEX, a browser-based software, designed to facilitate the Biological Interpretation Of Multi-omics EXperiments by bench scientists. BIOMEX integrates state-of-the-art statistical tools and field-tested algorithms into a flexible but well-defined workflow that accommodates metabolomics, transcriptomics, proteomics, mass cytometry and single cell data from different platforms and organisms. The BIOMEX workflow is accompanied by a manual and video tutorials that provide the necessary background to navigate the interface and get acquainted with the employed methods. BIOMEX guides the user through omics-tailored analyses, such as data pretreatment and normalization, dimensionality reduction, differential and enrichment analysis, pathway mapping, clustering, marker analysis, trajectory inference, meta-analysis and others. BIOMEX is fully interactive, allowing users to easily change parameters and generate customized plots exportable as high-quality publication-ready figures. BIOMEX is open source and freely available at https://www.vibcancer.be/software-tools/biomex. ispartof: NUCLEIC ACIDS RESEARCH vol:48 issue:W1 pages:W385-W394 ispartof: location:England status: published

Summary Pancreatic cancer is a rare but fatal form of cancer, the fourth highest in absolute mortality. Known risk factors include obesity, diet, and type 2 diabetes; however, the low incidence rate and interconnection of these factors confound the isolation of individual effects. Here, we use epidemiological analysis of prospective human cohorts and parallel tracking of pancreatic cancer in mice to dissect the effects of obesity, diet, and diabetes on pancreatic cancer. Through longitudinal monitoring and multi-omics analysis in mice, we found distinct effects of protein, sugar, and fat dietary components, with dietary sugars increasing Mad2l1 expression and tumor proliferation. Using epidemiological approaches in humans, we find that dietary sugars give a MAD2L1 genotype-dependent increased susceptibility to pancreatic cancer. The translation of these results to a clinical setting could aid in the identification of the at-risk population for screening and potentially harness dietary modification as a therapeutic measure., Graphical Abstract, Highlights • Distinct roles for dietary fat, protein, and sugar on murine pancreatic cancer • Dietary glucose triggers Mad2l1 upregulation and tumor cell proliferation in mice • Gene-diet interaction identifies sugar-MAD2L1 link in human pancreatic cancer • Dietary plant fats were protective in human pancreatic cancer susceptibility, Dooley et al. used parallel analysis of a murine pancreatic cancer model and a human prospective cohort to study the interaction of diet and pancreatic cancer. Both systems identify complex effects with different dietary components, converging on a link between dietary sugar and the cell-cycle checkpoint gene MAD2L1.

Endothelial cell (EC) metabolism is an emerging target for anti-angiogenic therapy in tumor angiogenesis and choroidal neovascularization (CNV), but little is known about individual EC metabolic transcriptomes. By single-cell RNA sequencing 28,337 murine choroidal ECs (CECs) and sprouting CNV-ECs, we constructed a taxonomy to characterize their heterogeneity. Comparison with murine lung tumor ECs (TECs) revealed congruent marker gene expression by distinct EC phenotypes across tissues and diseases, suggesting similar angiogenic mechanisms. Trajectory inference predicted that differentiation of venous to angiogenic ECs was accompanied by metabolic transcriptome plasticity. ECs displayed metabolic transcriptome heterogeneity during cellcycle progression and in quiescence. Hypothesizing that conserved genes are important, we used an integrated analysis, based on congruent transcriptome analysis, CEC-tailored genome-scale metabolic modeling, and gene expression meta-analysis in cross-species datasets, followed by in vitro and in vivo validation, to identify SQLE and ALDH18A1 as previously unknown metabolic angiogenic targets. ispartof: Cell Metabolism vol:31 issue:4 pages:862-877 ispartof: location:United States status: published

The heterogeneity of endothelial cells (ECs) across tissues remains incompletely inventoried. We constructed an atlas of >32,000 single-EC transcriptomes from 11 mouse tissues and identified 78 EC subclusters, including Aqp7+ intestinal capillaries and angiogenic ECs in healthy tissues. ECs from brain/testis, liver/spleen, small intestine/colon, and skeletal muscle/heart pairwise expressed partially overlapping marker genes. Arterial, venous, and lymphatic ECs shared more markers in more tissues than did heterogeneous capillary ECs. ECs from different vascular beds (arteries, capillaries, veins, lymphatics) exhibited transcriptome similarity across tissues, but the tissue (rather than the vessel) type contributed to the EC heterogeneity. Metabolic transcriptome analysis revealed a similar tissue-grouping phenomenon of ECs and heterogeneous metabolic gene signatures in ECs between tissues and between vascular beds within a single tissue in a tissue-type-dependent pattern. The EC atlas taxonomy enabled identification of EC subclusters in public scRNA-seq datasets and provides a powerful discovery tool and resource value. ispartof: CELL vol:180 issue:4 pages:764-+ ispartof: location:United States status: published

Heterogeneity of lung tumor endothelial cell (TEC) phenotypes across patients, species (human/mouse), and models (in vivo/in vitro) remains poorly inventoried at the single-cell level. We single-cell RNA (scRNA)-sequenced 56,771 endothelial cells from human/mouse (peri)-tumoral lung and cultured human lung TECs, and detected 17 known and 16 previously unrecognized phenotypes, including TECs putatively regulating immune surveillance. We resolved the canonical tip TECs into a known migratory tip and a putative basement-membrane remodeling breach phenotype. Tip TEC signatures correlated with patient survival, and tip/breach TECs were most sensitive to vascular endothelial growth factor blockade. Only tip TECs were congruent across species/models and shared conserved markers. Integrated analysis of the scRNA-sequenced data with orthogonal multi-omics and meta-analysis data across different human tumors, validated by functional analysis, identified collagen modification as a candidate angiogenic pathway.

Niemann-Pick Type C (NPC) disease is an autosomal recessive neurodegenerative disorder caused in most cases by mutations in the NPC1 gene. NPC1-deficiency is characterized by late endosomal accumulation of cholesterol, impaired cholesterol homeostasis, and a broad range of other cellular abnormalities. Although neuronal abnormalities and glial activation are observed in nearly all areas of the brain, the most severe consequence of NPC1-deficiency is a near complete loss of Purkinje neurons in the cerebellum. The link between cholesterol trafficking and NPC pathogenesis is not yet clear; however, increased oxidative stress in symptomatic NPC disease, increases in mitochondrial cholesterol, and alterations in autophagy/mitophagy suggest that mitochondria play a role in NPC disease pathology. Alterations in mitochondrial function affect energy and neurotransmitter metabolism, and are particularly harmful to the central nervous system. To investigate early metabolic alterations that could affect NPC disease progression, we performed metabolomics analyses of different brain regions from age-matched wildtype and Npc1 -/- mice at pre-symptomatic, early symptomatic and late stage disease by 1H-NMR spectroscopy. Metabolic profiling revealed markedly increased lactate and decreased acetate/acetyl-CoA levels in Npc1-/- cerebellum and cerebral cortex at all ages. Protein and gene expression analyses indicated a presymptomatic deficiency in the oxidative decarboxylation of pyruvate to acetyl-CoA, and an upregulation of glycolytic gene expression at the early symptomatic stage. We also observed a pre-symptomatic increase in several indicators of oxidative stress and antioxidant response systems in Npc1 -/- cerebellum. Our findings suggest that energy metabolism and oxidative stress may present additional therapeutic targets in NPC disease, especially if intervention can be started at an early stage of the disease. © 2013 Kennedy et al.

Endothelial cells (ECs) line blood vessels, regulate homeostatic processes (blood flow, immune cell trafficking), but are also involved in many prevalent diseases. The increasing use of high-throughput technologies such as gene expression microarrays and (single cell) RNA sequencing generated a wealth of data on the molecular basis of EC (dys-)function. Extracting biological insight from these datasets is challenging for scientists who are not proficient in bioinformatics. To facilitate the re-use of publicly available EC transcriptomics data, we developed the endothelial database EndoDB, a web-accessible collection of expert curated, quality assured and pre-analyzed data collected from 360 datasets comprising a total of 4741 bulk and 5847 single cell endothelial transcriptomes from six different organisms. Unlike other added-value databases, EndoDB allows to easily retrieve and explore data of specific studies, determine under which conditions genes and pathways of interest are deregulated and assess reprogramming of metabolism via principal component analysis, differential gene expression analysis, gene set enrichment analysis, heatmaps and metabolic and transcription factor analysis, while single cell data are visualized as gene expression color-coded t-SNE plots. Plots and tables in EndoDB are customizable, downloadable and interactive. EndoDB is freely available at https://vibcancer.be/software-tools/endodb, and will be updated to include new studies. ispartof: NUCLEIC ACIDS RESEARCH vol:47 issue:D1 pages:D736-D744 ispartof: location:England status: published