AFM Characterization of the Internal Mammary Artery as a Novel Target for Arterial Stiffening

Using the atomic force microscopy- (AFM-) PeakForce quantitative nanomechanical mapping (QNM) technique, we have previously shown that the adventitia of the human internal mammary artery (IMA), tested under dehydrated conditions, is altered in patients with a high degree of arterial stiffening. In this study, we explored the nanoscale elastic modulus of the tunica media of the IMA in hydrated and dehydrated conditions from the patients with low and high arterial stiffening, as assessed in vivo by carotid-femoral pulse wave velocity (PWV). In both hydrated and dehydrated conditions, the medial layer was significantly stiffer in the high PWV group. The elastic modulus of the hydrated and dehydrated tunica media was significantly correlated with PWV. In the hydrated condition, the expression activity of certain small leucine-rich repeat proteoglycans (SLRPs), which are associated with arterial stiffening, were found to be negatively correlated to the medial elastic modulus. We also compared the data with our previous work on the IMA adventitia. We found that the hydrated media and dehydrated adventitia are both suitable for reflecting the development of arterial stiffening and SLRP expression. This comprehensive study of the nanomechanical properties integrated with the proteomic analysis in the IMAs demonstrates the possibility of linking structural properties and function in small biological samples with novel AFM methods. The IMA is a suitable target for predicting arterial stiffening

Vascular proteomics in metabolic and cardiovascular diseases.

The vasculature is essential for proper organ function. Many pathologies are directly and indirectly related to vascular dysfunction, which causes significant morbidity and mortality. A common pathophysiological feature of diseased vessels is extracellular matrix (ECM) remodelling. Analysing the protein composition of the ECM by conventional antibody-based techniques is challenging; alternative splicing or post-translational modifications, such as glycosylation, can mask epitopes required for antibody recognition. By contrast, proteomic analysis by mass spectrometry enables the study of proteins without the constraints of antibodies. Recent advances in proteomic techniques make it feasible to characterize the composition of the vascular ECM and its remodelling in disease. These developments may lead to the discovery of novel prognostic and diagnostic markers. Thus, proteomics holds potential for identifying ECM signatures to monitor vascular disease processes. Furthermore, a better understanding of the ECM remodelling processes in the vasculature might make ECM-associated proteins more attractive targets for drug discovery efforts. In this review, we will summarize the role of the ECM in the vasculature. Then, we will describe the challenges associated with studying the intricate network of ECM proteins and the current proteomic strategies to analyse the vascular ECM in metabolic and cardiovascular diseases

Advancing brain barriers RNA sequencing: guidelines from experimental design to publication

Background: RNA sequencing (RNA-Seq) in its varied forms has become an indispensable tool for analyzing differential gene expression and thus characterization of specific tissues. Aiming to understand the brain barriers genetic signature, RNA seq has also been introduced in brain barriers research. This has led to availability of both, bulk and single-cell RNA-Seq datasets over the last few years. If appropriately performed, the RNA-Seq studies provide powerful datasets that allow for significant deepening of knowledge on the molecular mechanisms that establish the brain barriers. However, RNA-Seq studies comprise complex workflows that require to consider many options and variables before, during and after the proper sequencing process.Main body: In the current manuscript, we build on the interdisciplinary experience of the European PhD Training Network BtRAIN (https://www.btrain-2020.eu/) where bioinformaticians and brain barriers researchers collaborated to analyze and establish RNA-Seq datasets on vertebrate brain barriers. The obstacles BtRAIN has identified in this process have been integrated into the present manuscript. It provides guidelines along the entire workflow of brain barriers RNA-Seq studies starting from the overall experimental design to interpretation of results. Focusing on the vertebrate endothelial blood–brain barrier (BBB) and epithelial blood-cerebrospinal-fluid barrier (BCSFB) of the choroid plexus, we provide a step-by-step description of the workflow, highlighting the decisions to be made at each step of the workflow and explaining the strengths and weaknesses of individual choices made. Finally, we propose recommendations for accurate data interpretation and on the information to be included into a publication to ensure appropriate accessibility of the data and reproducibility of the observations by the scientific community.Conclusion: Next generation transcriptomic profiling of the brain barriers provides a novel resource for understanding the development, function and pathology of these barrier cells, which is essential for understanding CNS homeostasis and disease. Continuous advancement and sophistication of RNA-Seq will require interdisciplinary approaches between brain barrier researchers and bioinformaticians as successfully performed in BtRAIN. The present guidelines are built on the BtRAIN interdisciplinary experience and aim to facilitate collaboration of brain barriers researchers with bioinformaticians to advance RNA-Seq study design in the brain barriers community