Global Transcriptome Analysis Reveals Distinct Phases of the Endothelial Response to TNF.

The vascular endothelium acts as a dynamic interface between blood and tissue. TNF-α, a major regulator of inflammation, induces endothelial cell (EC) transcriptional changes, the overall response dynamics of which have not been fully elucidated. In the present study, we conducted an extended time-course analysis of the human EC response to TNF, from 30 min to 72 h. We identified regulated genes and used weighted gene network correlation analysis to decipher coexpression profiles, uncovering two distinct temporal phases: an acute response (between 1 and 4 h) and a later phase (between 12 and 24 h). Sex-based subset analysis revealed that the response was comparable between female and male cells. Several previously uncharacterized genes were strongly regulated during the acute phase, whereas the majority in the later phase were IFN-stimulated genes. A lack of IFN transcription indicated that this IFN-stimulated gene expression was independent of de novo IFN production. We also observed two groups of genes whose transcription was inhibited by TNF: those that resolved toward baseline levels and those that did not. Our study provides insights into the global dynamics of the EC transcriptional response to TNF, highlighting distinct gene expression patterns during the acute and later phases. Data for all coding and noncoding genes is provided on the Web site (http://www.endothelial-response.org/). These findings may be useful in understanding the role of ECs in inflammation and in developing TNF signaling-targeted therapies.

Elevated plasma complement factor H related 5 protein is associated with venous thromboembolism.

Venous thromboembolism (VTE) is a common, multi-causal disease with potentially serious short- and long-term complications. In clinical practice, there is a need for improved plasma biomarker-based tools for VTE diagnosis and risk prediction. Here we show, using proteomics profiling to screen plasma from patients with suspected acute VTE, and several case-control studies for VTE, how Complement Factor H Related 5 protein (CFHR5), a regulator of the alternative pathway of complement activation, is a VTE-associated plasma biomarker. In plasma, higher CFHR5 levels are associated with increased thrombin generation potential and recombinant CFHR5 enhanced platelet activation in vitro. GWAS analysis of ~52,000 participants identifies six loci associated with CFHR5 plasma levels, but Mendelian randomization do not demonstrate causality between CFHR5 and VTE. Our results indicate an important role for the regulation of the alternative pathway of complement activation in VTE and that CFHR5 represents a potential diagnostic and/or risk predictive plasma biomarker.

A human adipose tissue cell-type transcriptome atlas.

The importance of defining cell-type-specific genes is well acknowledged. Technological advances facilitate high-resolution sequencing of single cells, but practical challenges remain. Adipose tissue is composed primarily of adipocytes, large buoyant cells requiring extensive, artefact-generating processing for separation and analysis. Thus, adipocyte data are frequently absent from single-cell RNA sequencing (scRNA-seq) datasets, despite being the primary functional cell type. Here, we decipher cell-type-enriched transcriptomes from unfractionated human adipose tissue RNA-seq data. We profile all major constituent cell types, using 527 visceral adipose tissue (VAT) or 646 subcutaneous adipose tissue (SAT) samples, identifying over 2,300 cell-type-enriched transcripts. Sex-subset analysis uncovers a panel of male-only cell-type-enriched genes. By resolving expression profiles of genes differentially expressed between SAT and VAT, we identify mesothelial cells as the primary driver of this variation. This study provides an accessible method to profile cell-type-enriched transcriptomes using bulk RNA-seq, generating a roadmap for adipose tissue biology.

Identification of Endothelial Proteins in Plasma Associated With Cardiovascular Risk Factors.

OBJECTIVE: Endothelial cell (EC) dysfunction is a well-established response to cardiovascular disease risk factors, such as smoking and obesity. Risk factor exposure can modify EC signaling and behavior, leading to arterial and venous disease development. Here, we aimed to identify biomarker panels for the assessment of EC dysfunction, which could be useful for risk stratification or to monitor treatment response. Approach and Results: We used affinity proteomics to identify EC proteins circulating in plasma that were associated with cardiovascular disease risk factor exposure. Two hundred sixteen proteins, which we previously predicted to be EC-enriched across vascular beds, were measured in plasma samples (N=1005) from the population-based SCAPIS (Swedish Cardiopulmonary Bioimage Study) pilot. Thirty-eight of these proteins were associated with body mass index, total cholesterol, low-density lipoprotein, smoking, hypertension, or diabetes. Sex-specific analysis revealed that associations predominantly observed in female- or male-only samples were most frequently with the risk factors body mass index, or total cholesterol and smoking, respectively. We show a relationship between individual cardiovascular disease risk, calculated with the Framingham risk score, and the corresponding biomarker profiles. CONCLUSIONS: EC proteins in plasma could reflect vascular health status.

A single-cell type transcriptomics map of human tissues.

Advances in molecular profiling have opened up the possibility to map the expression of genes in cells, tissues, and organs in the human body. Here, we combined single-cell transcriptomics analysis with spatial antibody-based protein profiling to create a high-resolution single-cell type map of human tissues. An open access atlas has been launched to allow researchers to explore the expression of human protein-coding genes in 192 individual cell type clusters. An expression specificity classification was performed to determine the number of genes elevated in each cell type, allowing comparisons with bulk transcriptomics data. The analysis highlights distinct expression clusters corresponding to cell types sharing similar functions, both within the same organs and between organs.

A Systems-Based Map of Human Brain Cell-Type Enriched Genes and Malignancy-Associated Endothelial Changes.

Changes in the endothelium of the cerebral vasculature can contribute to inflammatory, thrombotic, and malignant disorders. The importance of defining cell-type-specific genes and their modification in disease is increasingly recognized. Here, we develop a bioinformatics-based approach to identify normal brain cell-enriched genes, using bulk RNA sequencing (RNA-seq) data from 238 normal human cortex samples from 2 independent cohorts. We compare endothelial cell-enriched gene profiles with astrocyte, oligodendrocyte, neuron, and microglial cell profiles. Endothelial changes in malignant disease are explored using RNA-seq data from 516 lower-grade gliomas and 401 glioblastomas. Lower-grade gliomas appear to be an "endothelial intermediate" between normal brain and glioblastoma. We apply our method for the prediction of glioblastoma-specific endothelial biomarkers, providing potential diagnostic or therapeutic targets. In summary, we provide a roadmap of endothelial cell identity in normal and malignant brain, using a method developed to resolve bulk RNA-seq into constituent cell-type-enriched profiles.

Molecular Imaging of a New Multimodal Microbubble for Adhesion Molecule Targeting.

INTRODUCTION: Inflammation is an important risk-associated component of many diseases and can be diagnosed by molecular imaging of specific molecules. The aim of this study was to evaluate the possibility of targeting adhesion molecules on inflammation-activated endothelial cells and macrophages using an innovative multimodal polyvinyl alcohol-based microbubble (MB) contrast agent developed for diagnostic use in ultrasound, magnetic resonance, and nuclear imaging. METHODS: We assessed the binding efficiency of antibody-conjugated multimodal contrast to inflamed murine or human endothelial cells (ECs), and to peritoneal macrophages isolated from rats with peritonitis, utilizing the fluorescence characteristics of the MBs. Single-photon emission tomography (SPECT) was used to illustrate 99mTc-labeled MB targeting and distribution in an experimental in vivo model of inflammation. RESULTS: Flow cytometry and confocal microscopy showed that binding of antibody-targeted MBs to the adhesion molecules ICAM-1, VCAM-1, or E-selectin, expressed on cytokine-stimulated ECs, was up to sixfold higher for human and 12-fold higher for mouse ECs, compared with that of non-targeted MBs. Under flow conditions, both VCAM-1- and E-selectin-targeted MBs adhered more firmly to stimulated human ECs than to untreated cells, while VCAM-1-targeted MBs adhered best to stimulated murine ECs. SPECT imaging showed an approximate doubling of signal intensity from the abdomen of rats with peritonitis, compared with healthy controls, after injection of anti-ICAM-1-MBs. CONCLUSIONS: This novel multilayer contrast agent can specifically target adhesion molecules expressed as a result of inflammatory stimuli in vitro, and has potential for use in disease-specific multimodal diagnostics in vivo using antibodies against targets of interest.

Human serum albumin-based probes for molecular targeting of macrophage scavenger receptors.

Background: Inflammation and accumulation of macrophages are key features of unstable atherosclerotic plaques. The ability of macrophages to take up molecular probes can be exploited in new clinical imaging methods for the detection of unstable atherosclerotic lesions. We investigated whether modifications of human serum albumin (HSA) could be used to target macrophages efficiently in vitro. Materials and methods: Maleylated and aconitylated HSA were compared with unmodified HSA. Fluorescent or radiolabeled (89Zr) modified HSA was used in in vitro experiments to study cellular uptake by differentiated THP-1 cells and primary human macrophages. The time course of uptake was evaluated by flow cytometry, confocal microscopy, real-time microscopy and radioactivity measurements. The involvement of scavenger receptors (SR-A1, SR-B2, LOX-1) was assessed by knockdown experiments using RNA interference, by blocking experiments and by assays of competition by modified low-density lipoprotein. Results: Modified HSA was readily taken up by different macrophages. Uptake was mediated nonexclusively via the scavenger receptor SR-A1 (encoded by the MSR1 gene). Knockdown of CD36 and ORL1 had no influence on the uptake. Modified HSA was preferentially taken up by human macrophages compared with other vascular cell types such as endothelial cells and smooth muscle cells. Conclusions: Modified 89Zr-labeled HSA probes were recognized by different subsets of polarized macrophages, and maleylated HSA may be a promising radiotracer for radionuclide imaging of macrophage-rich inflammatory vascular diseases.

A systems-approach reveals human nestin is an endothelial-enriched, angiogenesis-independent intermediate filament protein.

The intermediate filament protein nestin is expressed during embryonic development, but considered largely restricted to areas of regeneration in the adult. Here, we perform a body-wide transcriptome and protein-profiling analysis to reveal that nestin is constitutively, and highly-selectively, expressed in adult human endothelial cells (EC), independent of proliferative status. Correspondingly, we demonstrate that it is not a marker for tumour EC in multiple malignancy types. Imaging of EC from different vascular beds reveals nestin subcellular distribution is shear-modulated. siRNA inhibition of nestin increases EC proliferation, and nestin expression is reduced in atherosclerotic plaque neovessels. eQTL analysis reveals an association between SNPs linked to cardiovascular disease and reduced aortic EC nestin mRNA expression. Our study challenges the dogma that nestin is a marker of proliferation, and provides insight into its regulation and function in EC. Furthermore, our systems-based approach can be applied to investigate body-wide expression profiles of any candidate protein.

Total synthesis of (+/-)-aspercyclide A and its C19 methyl ether.

The total syntheses of (+/-)-aspercyclide A (1) and its C19 methyl ether (15a) featuring Heck-Mizoroki macrocyclisation to form the 11-membered (E)-styrenyl biaryl ether lactone core are described.