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.

Standardized Definitions for Efficacy End Points in Neoadjuvant Breast Cancer Clinical Trials: NeoSTEEP.

PURPOSE: The Standardized Definitions for Efficacy End Points (STEEP) criteria, established in 2007 and updated in 2021 (STEEP 2.0), provide standardized definitions of adjuvant breast cancer (BC) end points. STEEP 2.0 identified a need to separately address end points for neoadjuvant clinical trials. The multidisciplinary NeoSTEEP working group of experts was convened to critically evaluate and align neoadjuvant BC trial end points. METHODS: The NeoSTEEP working group concentrated on neoadjuvant systemic therapy end points in clinical trials with efficacy outcomes-both pathologic and time-to-event survival end points-particularly for registrational intent. Special considerations for subtypes and therapeutic approaches, imaging, nodal staging at surgery, bilateral and multifocal diseases, correlative tissue collection, and US Food and Drug Administration regulatory considerations were contemplated. RESULTS: The working group recommends a preferred definition of pathologic complete response (pCR) as the absence of residual invasive cancer in the complete resected breast specimen and all sampled regional lymph nodes (ypT0/Tis ypN0 per AJCC staging). Residual cancer burden should be a secondary end point to facilitate future assessment of its utility. Alternative end points are needed for hormone receptor-positive disease. Time-to-event survival end point definitions should pay particular attention to the measurement starting point. Trials should include end points originating at random assignment (event-free survival and overall survival) to capture presurgery progression and deaths as events. Secondary end points adapted from STEEP 2.0, which are defined from starting at curative-intent surgery, may also be appropriate. Specification and standardization of biopsy protocols, imaging, and pathologic nodal evaluation are also crucial. CONCLUSION: End points in addition to pCR should be selected on the basis of clinical and biologic aspects of the tumor and the therapeutic agent investigated. Consistent prespecified definitions and interventions are paramount for clinically meaningful trial results and cross-trial comparison.

Targeting NETs using dual-active DNase1 variants.

Background: Neutrophil Extracellular Traps (NETs) are key mediators of immunothrombotic mechanisms and defective clearance of NETs from the circulation underlies an array of thrombotic, inflammatory, infectious, and autoimmune diseases. Efficient NET degradation depends on the combined activity of two distinct DNases, DNase1 and DNase1-like 3 (DNase1L3) that preferentially digest double-stranded DNA (dsDNA) and chromatin, respectively. Methods: Here, we engineered a dual-active DNase with combined DNase1 and DNase1L3 activities and characterized the enzyme for its NET degrading potential in vitro. Furthermore, we produced a mouse model with transgenic expression of the dual-active DNase and analyzed body fluids of these animals for DNase1 and DNase 1L3 activities. We systematically substituted 20 amino acid stretches in DNase1 that were not conserved among DNase1 and DNase1L3 with homologous DNase1L3 sequences. Results: We found that the ability of DNase1L3 to degrade chromatin is embedded into three discrete areas of the enzyme's core body, not the C-terminal domain as suggested by the state-of-the-art. Further, combined transfer of the aforementioned areas of DNase1L3 to DNase1 generated a dual-active DNase1 enzyme with additional chromatin degrading activity. The dual-active DNase1 mutant was superior to native DNase1 and DNase1L3 in degrading dsDNA and chromatin, respectively. Transgenic expression of the dual-active DNase1 mutant in hepatocytes of mice lacking endogenous DNases revealed that the engineered enzyme was stable in the circulation, released into serum and filtered to the bile but not into the urine. Conclusion: Therefore, the dual-active DNase1 mutant is a promising tool for neutralization of DNA and NETs with potential therapeutic applications for interference with thromboinflammatory disease states.

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.

Intrinsic coagulation pathway-mediated thrombin generation in mouse whole blood.

Calibrated Automated Thrombography (CAT) is a versatile and sensitive method for analyzing coagulation reactions culminating in thrombin generation (TG). Here, we present a CAT method for analyzing TG in murine whole blood by adapting the CAT assay used for measuring TG in human plasma. The diagnostically used artificial and physiologic factor XII (FXII) contact activators kaolin, ellagic acid and polyphosphate (polyP) stimulated TG in murine blood in a dose-dependent manner resulting in a gradual increase in endogenous thrombin potential and peak thrombin, with shortened lag times and times to peak. The activated FXII inhibitor rHA-Infestin-4 and direct oral anticoagulants (DOACs) interfered with TG triggered by kaolin, ellagic acid and polyP and TG was completely attenuated in blood of FXII- (F12 -/-) and FXI-deficient (F11 -/-) mice. Moreover, reconstitution of blood from F12 -/- mice with human FXII restored impaired contact-stimulated TG. HEK293 cell-purified polyP also initiated FXII-driven TG in mouse whole blood and addition of the selective inhibitor PPX_Δ12 ablated natural polyP-stimulated TG. In conclusion, the data provide a method for analysis of contact activation-mediated TG in murine whole blood. As the FXII-driven intrinsic pathway of coagulation has emerged as novel target for antithrombotic agents that are validated in mouse thrombosis and bleeding models, our novel assay could expedite therapeutic drug development.

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.

Proteomics in thrombosis research.

A State of the Art lecture titled "Proteomics in Thrombosis Research" was presented at the ISTH Congress in 2021. In clinical practice, there is a need for improved plasma biomarker-based tools for diagnosis and risk prediction of venous thromboembolism (VTE). Analysis of blood, to identify plasma proteins with potential utility for such tools, could enable an individualized approach to treatment and prevention. Technological advances to study the plasma proteome on a large scale allows broad screening for the identification of novel plasma biomarkers, both by targeted and nontargeted proteomics methods. However, assay limitations need to be considered when interpreting results, with orthogonal validation required before conclusions are drawn. Here, we review and provide perspectives on the application of affinity- and mass spectrometry-based methods for the identification and analysis of plasma protein biomarkers, with potential application in the field of VTE. We also provide a future perspective on discovery strategies and emerging technologies for targeted proteomics in thrombosis research. Finally, we summarize relevant new data on this topic, presented during the 2021 ISTH Congress.

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.

Identification of the factor XII contact activation site enables sensitive coagulation diagnostics.

Contact activation refers to the process of surface-induced activation of factor XII (FXII), which initiates blood coagulation and is captured by the activated partial thromboplastin time (aPTT) assay. Here, we show the mechanism and diagnostic implications of FXII contact activation. Screening of recombinant FXII mutants identified a continuous stretch of residues Gln317-Ser339 that was essential for FXII surface binding and activation, thrombin generation and coagulation. Peptides spanning these 23 residues competed with surface-induced FXII activation. Although FXII mutants lacking residues Gln317-Ser339 were susceptible to activation by plasmin and plasma kallikrein, they were ineffective in supporting arterial and venous thrombus formation in mice. Antibodies raised against the Gln317-Ser339 region induced FXII activation and triggered controllable contact activation in solution leading to thrombin generation by the intrinsic pathway of coagulation. The antibody-activated aPTT allows for standardization of particulate aPTT reagents and for sensitive monitoring of coagulation factors VIII, IX, XI.

An artificial neural network approach integrating plasma proteomics and genetic data identifies PLXNA4 as a new susceptibility locus for pulmonary embolism.

Venous thromboembolism is the third common cardiovascular disease and is composed of two entities, deep vein thrombosis (DVT) and its potential fatal form, pulmonary embolism (PE). While PE is observed in ~ 40% of patients with documented DVT, there is limited biomarkers that can help identifying patients at high PE risk. To fill this need, we implemented a two hidden-layers artificial neural networks (ANN) on 376 antibodies and 19 biological traits measured in the plasma of 1388 DVT patients, with or without PE, of the MARTHA study. We used the LIME algorithm to obtain a linear approximate of the resulting ANN prediction model. As MARTHA patients were typed for genotyping DNA arrays, a genome wide association study (GWAS) was conducted on the LIME estimate. Detected single nucleotide polymorphisms (SNPs) were tested for association with PE risk in MARTHA. Main findings were replicated in the EOVT study composed of 143 PE patients and 196 DVT only patients. The derived ANN model for PE achieved an accuracy of 0.89 and 0.79 in our training and testing sets, respectively. A GWAS on the LIME approximate identified a strong statistical association peak (rs1424597: p = 5.3 × 10-7) at the PLXNA4 locus. Homozygote carriers for the rs1424597-A allele were then more frequently observed in PE than in DVT patients from the MARTHA (2% vs. 0.4%, p = 0.005) and the EOVT (3% vs. 0%, p = 0.013) studies. In a sample of 112 COVID-19 patients known to have endotheliopathy leading to acute lung injury and an increased risk of PE, decreased PLXNA4 levels were associated (p = 0.025) with worsened respiratory function. Using an original integrated proteomics and genetics strategy, we identified PLXNA4 as a new susceptibility gene for PE whose exact role now needs to be further elucidated.

Association between attendance at an American diabetes camp and improvements in glycaemic control and treatment satisfaction.

INTRODUCTION: Few studies have evaluated glycaemic control using continuous glucose monitoring (CGM) in individuals before and after attendance at a diabetes camp or by comparing control groups at home to control groups at camp. METHODS: Youth (6-17 years) with T1D and receiving insulin therapy were enrolled at a week-long diabetes camp. They participated in three clinic visits: at the start of a week at home, by initiating a Dexcom G6 CGM system; at the start of a week at camp, where the home week G6 was removed and a camp week G6 was inserted; and after camp, where the camp week G6 was removed. We administered Problem Areas in Diabetes (PAID) surveys at the second and third visits. Participants with <80% CGM data coverage or who did not complete all PAID surveys were excluded from analysis. We compared glycaemic control and PAID scores between the week at home and week at camp. RESULTS: Of 76 enrolled campers, 69 completed the study and 52 had results that qualified for analysis. The mean participant age was 12.5 ± 2.2 years. Camp was associated with significantly improved treatment satisfaction, time in desired glucose range and insulin sensitivity. Time in hyperglycaemia and basal insulin requirements decreased significantly. CONCLUSIONS: Diabetes camp is associated with significant improvements in diabetes treatment satisfaction and glycaemic control compared to home care.

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.

Updated Standardized Definitions for Efficacy End Points (STEEP) in Adjuvant Breast Cancer Clinical Trials: STEEP Version 2.0.

PURPOSE: The Standardized Definitions for Efficacy End Points (STEEP) criteria, established in 2007, provide standardized definitions of adjuvant breast cancer clinical trial end points. Given the evolution of breast cancer clinical trials and improvements in outcomes, a panel of experts reviewed the STEEP criteria to determine whether modifications are needed. METHODS: We conducted systematic searches of ClinicalTrials.gov for adjuvant systemic and local-regional therapy trials for breast cancer to investigate if the primary end points reported met STEEP criteria. On the basis of common STEEP deviations, we performed a series of simulations to evaluate the effect of excluding non-breast cancer deaths and new nonbreast primary cancers from the invasive disease-free survival end point. RESULTS: Among 11 phase III breast cancer trials with primary efficacy end points, three had primary end points that followed STEEP criteria, four used STEEP definitions but not the corresponding end point names, and four used end points that were not included in the original STEEP manuscript. Simulation modeling demonstrated that inclusion of second nonbreast primary cancer can increase the probability of incorrect inferences, can decrease power to detect clinically relevant efficacy effects, and may mask differences in recurrence rates, especially when recurrence rates are low. CONCLUSION: We recommend an additional end point, invasive breast cancer-free survival, which includes all invasive disease-free survival events except second nonbreast primary cancers. This end point should be considered for trials in which the toxicities of agents are well-known and where the risk of second primary cancer is small. Additionally, we provide end point recommendations for local therapy trials, low-risk populations, noninferiority trials, and trials incorporating patient-reported outcomes.

Xenotropic and polytropic retrovirus receptor 1 regulates procoagulant platelet polyphosphate.

Polyphosphate is a procoagulant inorganic polymer of linear-linked orthophosphate residues. Multiple investigations have established the importance of platelet polyphosphate in blood coagulation; however, the mechanistic details of polyphosphate homeostasis in mammalian species remain largely undefined. In this study, xenotropic and polytropic retrovirus receptor 1 (XPR1) regulated polyphosphate in platelets and was implicated in thrombosis in vivo. We used bioinformatic analyses of omics data to identify XPR1 as a major phosphate transporter in platelets. XPR1 messenger RNA and protein expression inversely correlated with intracellular polyphosphate content and release. Pharmacological interference with XPR1 activity increased polyphosphate stores, led to enhanced platelet-driven coagulation, and amplified thrombus formation under flow via the polyphosphate/factor XII pathway. Conditional gene deletion of Xpr1 in platelets resulted in polyphosphate accumulation, accelerated arterial thrombosis, and augmented activated platelet-driven pulmonary embolism without increasing bleeding in mice. These data identify platelet XPR1 as an integral regulator of platelet polyphosphate metabolism and reveal a fundamental role for phosphate homeostasis in thrombosis.

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.

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.

National Cancer Institute Breast Cancer Steering Committee Working Group Report on Meaningful and Appropriate End Points for Clinical Trials in Metastatic Breast Cancer.

PURPOSE: To provide evidence-based consensus recommendations on choice of end points for clinical trials in metastatic breast cancer, with a focus on biologic subtype and line of therapy. METHODS: The National Cancer Institute Breast Cancer Steering Committee convened a working group of breast medical oncologists, patient advocates, biostatisticians, and liaisons from the Food and Drug Administration to conduct a detailed curated systematic review of the literature, including original reports, reviews, and meta-analyses, to determine the current landscape of therapeutic options, recent clinical trial data, and natural history of four biologic subtypes of breast cancer. Ongoing clinical trials for metastatic breast cancer in each subtype also were reviewed from ClinicalTrials.gov for planned primary end points. External input was obtained from the pharmaceutic/biotechnology industry, real-world clinical data specialists, experts in quality of life and patient-reported outcomes, and combined metrics for assessing magnitude of clinical benefit. RESULTS: The literature search yielded 146 publications to inform the recommendations from the working group. CONCLUSION: Recommendations for appropriate end points for metastatic breast cancer clinical trials focus on biologic subtype and line of therapy and the magnitude of absolute and relative gains that would represent meaningful clinical benefit.

Factor XII Contact Activation.

Contact activation is the surface-induced conversion of factor XII (FXII) zymogen to the serine protease FXIIa. Blood-circulating FXII binds to negatively charged surfaces and this contact to surfaces triggers a conformational change in the zymogen inducing autoactivation. Several surfaces that have the capacity for initiating FXII contact activation have been identified, including misfolded protein aggregates, collagen, nucleic acids, and platelet and microbial polyphosphate. Activated FXII initiates the proinflammatory kallikrein-kinin system and the intrinsic coagulation pathway, leading to formation of bradykinin and thrombin, respectively. FXII contact activation is well characterized in vitro and provides the mechanistic basis for the diagnostic clotting assay, activated partial thromboplastin time. However, only in the past decade has the critical role of FXII contact activation in pathological thrombosis been appreciated. While defective FXII contact activation provides thromboprotection, excess activation underlies the swelling disorder hereditary angioedema type III. This review provides an overview of the molecular basis of FXII contact activation and FXII contact activation-associated disease states.