Detailed protocol for a corneal thermal cauterization-based (lymph-)angiogenesis assay in mice.

Angiogenesis and lymphangiogenesis, the formation of new blood or lymphatic vessels, respectively, from preexisting vasculature is essential during embryonic development, but also occurs during tissue repair and in pathological conditions (cancer; ocular disease; ischemic, infectious and inflammatory disorders), which are all characterized to a certain extent by inflammatory conditions. Hence, a rapid, inexpensive, feasible / technically easy, reliable assay of inflammation-induced (lymph-)angiogenesis is highly valuable. In this context, the corneal thermal cauterization assay in mice is a simple, low-cost, reproducible, insightful and labor-saving assay to gauge the role of inflammation in angiogenesis and lymphangiogenesis. However, to the best of our knowledge, there is no standardized protocol to perform this assay. Here, we provide a step-by-step description of the model's procedures, which include:•The thermal cauterization of the corneas,•Enucleation and dissection of the corneas,•Subsequent immunofluorescence staining of the neovasculature, and morphometric analysis. We also discuss ethical considerations and aspects related to animal welfare guidelines. Altogether, this paper will help to increase the reproducibility of the corneal thermal cauterization model and facilitate its use for angiogenesis and lymphangiogenesis research.

Prioritization and functional validation of target genes from single-cell transcriptomics studies.

Translation of academic results into clinical practice is a formidable unmet medical need. Single-cell RNA-sequencing (scRNA-seq) studies generate long descriptive ranks of markers with predicted biological function, but without functional validation, it remains challenging to know which markers truly exert the putative function. Given the lengthy/costly nature of validation studies, gene prioritization is required to select candidates. We address these issues by studying tip endothelial cell (EC) marker genes because of their importance for angiogenesis. Here, by tailoring Guidelines On Target Assessment for Innovative Therapeutics, we in silico prioritize previously unreported/poorly described, high-ranking tip EC markers. Notably, functional validation reveals that four of six candidates behave as tip EC genes. We even discover a tip EC function for a gene lacking in-depth functional annotation. Thus, validating prioritized genes from scRNA-seq studies offers opportunities for identifying targets to be considered for possible translation, but not all top-ranked scRNA-seq markers exert the predicted function.

The pulmonary vasculature in lethal COVID-19 and idiopathic pulmonary fibrosis at single-cell resolution.

AIMS: Severe acute respiratory syndrome coronavirus-2 infection causes COVID-19, which in severe cases evokes life-threatening acute respiratory distress syndrome (ARDS). Transcriptome signatures and the functional relevance of non-vascular cell types (e.g. immune and epithelial cells) in COVID-19 are becoming increasingly evident. However, despite its known contribution to vascular inflammation, recruitment/invasion of immune cells, vascular leakage, and perturbed haemostasis in the lungs of severe COVID-19 patients, an in-depth interrogation of the endothelial cell (EC) compartment in lethal COVID-19 is lacking. Moreover, progressive fibrotic lung disease represents one of the complications of COVID-19 pneumonia and ARDS. Analogous features between idiopathic pulmonary fibrosis (IPF) and COVID-19 suggest partial similarities in their pathophysiology, yet, a head-to-head comparison of pulmonary cell transcriptomes between both conditions has not been implemented to date. METHODS AND RESULTS: We performed single-nucleus RNA-sequencing on frozen lungs from 7 deceased COVID-19 patients, 6 IPF explant lungs, and 12 controls. The vascular fraction, comprising 38 794 nuclei, could be subclustered into 14 distinct EC subtypes. Non-vascular cell types, comprising 137 746 nuclei, were subclustered and used for EC-interactome analyses. Pulmonary ECs of deceased COVID-19 patients showed an enrichment of genes involved in cellular stress, as well as signatures suggestive of dampened immunomodulation and impaired vessel wall integrity. In addition, increased abundance of a population of systemic capillary and venous ECs was identified in COVID-19 and IPF. COVID-19 systemic ECs closely resembled their IPF counterparts, and a set of 30 genes was found congruently enriched in systemic ECs across studies. Receptor-ligand interaction analysis of ECs with non-vascular cell types in the pulmonary micro-environment revealed numerous previously unknown interactions specifically enriched/depleted in COVID-19 and/or IPF. CONCLUSIONS: This study uncovered novel insights into the abundance, expression patterns, and interactomes of EC subtypes in COVID-19 and IPF, relevant for future investigations into the progression and treatment of both lethal conditions.

Single cell atlas identifies lipid-processing and immunomodulatory endothelial cells in healthy and malignant breast.

Since a detailed inventory of endothelial cell (EC) heterogeneity in breast cancer (BC) is lacking, here we perform single cell RNA-sequencing of 26,515 cells (including 8433 ECs) from 9 BC patients and compare them to published EC taxonomies from lung tumors. Angiogenic ECs are phenotypically similar, while other EC subtypes are different. Predictive interactome analysis reveals known but also previously unreported receptor-ligand interactions between ECs and immune cells, suggesting an involvement of breast EC subtypes in immune responses. We also identify a capillary EC subtype (LIPEC (Lipid Processing EC)), which expresses genes involved in lipid processing that are regulated by PPAR-γ and is more abundant in peri-tumoral breast tissue. Retrospective analysis of 4648 BC patients reveals that treatment with metformin (an indirect PPAR-γ signaling activator) provides long-lasting clinical benefit and is positively associated with LIPEC abundance. Our findings warrant further exploration of this LIPEC/PPAR-γ link for BC treatment.