Novel Morphological Profiling Assay Connects ex Vivo Endothelial Cell Responses to Disease Severity in Liver Cirrhosis.

Background and Aims: Endothelial cell (EC) dysfunction in response to circulating plasma factors is a known causal factor in many systemic diseases. However, no appropriate assay is available to investigate this causality ex vivo. In liver cirrhosis, systemic inflammation is identified as central mechanism in progression from compensated to decompensated cirrhosis (DC), but the role of ECs therein is unknown. We aimed to develop a novel ex vivo assay for assessing EC responses to patient-derived plasma (PDP) and assess the potential of this assay in a cohort of liver cirrhosis patients. Methods: Image-based morphological profiling was utilized to assess the impact of PDP on cultured ECs. Endothelial cell (EC) monolayers were exposed to 25% stabilized PDP (20 compensated cirrhoses, 20 DCs, and 20 healthy controls (HCs). Single-cell morphological profiles were extracted by automated image-analysis following staining of multiple cellular components and high-content imaging. Patient profiles were created by dimension reduction and cell-to-patient data aggregation, followed by multivariate-analysis to stratify patients and identify discriminating features. Results: Patient-derived plasma (PDP) exposure induced profound changes in EC morphology, displaying clear differences between controls and DC patients. Compensated cirrhosis patients showed overlap with healthy controls and DC patients. Supervised analysis showed Child-Pugh (CP) class could be predicted from EC morphology. Most importantly, CP-C patients displayed distinct EC phenotypes, in which mitochondrial changes were most discriminative. Conclusion: Morphological profiling presents a viable tool to assess the endothelium ex vivo. We demonstrated that the EC phenotype corresponds with disease severity in liver cirrhosis. Moreover, our results suggest the presence of mitochondrial dysfunction in ECs of CP-C patient.

A bypass flow model to study endothelial cell mechanotransduction across diverse flow environments.

Disturbed flow is one of the pathological initiators of endothelial dysfunction in intimal hyperplasia (IH) which is commonly seen in vascular bypass grafts, and arteriovenous fistulas. Various in vitro disease models have been designed to simulate the hemodynamic conditions found in the vasculature. Nonetheless, prior investigations have encountered challenges in establishing a robust disturbed flow model, primarily attributed to the complex bifurcated geometries and distinctive flow dynamics. In the present study, we aim to address this gap by introducing an in vitro bypass flow model capable of inducing disturbed flow and other hemodynamics patterns through a pulsatile flow in the same model. To assess the model's validity, we employed computational fluid dynamics (CFD) to simulate hemodynamics and compared the morphology and functions of human umbilical venous endothelial cells (HUVECs) under disturbed flow conditions to those in physiological flow or stagnant conditions. CFD analysis revealed the generation of disturbed flow within the model, pinpointing the specific location in the channel where the effects of disturbed flow were observed. High-content screening, a single-cell morphological profile assessment, demonstrated that HUVECs in the disturbed flow area exhibited random orientation, and morphological features were significantly distinct compared to cells in the physiological flow or stagnant condition after a two days of flow exposure. Furthermore, HUVECs exposed to disturbed flow underwent extensive remodeling of the adherens junctions and expressed higher levels of endothelial cell activation markers compared to other hemodynamic conditions. In conclusion, our in vitro bypass flow model provides a robust platform for investigating the associations between disturbed flow pattern and vascular diseases.

Modeling cancer-associated hypercoagulability using glioblastoma spheroids in microfluidic chips.

Background: Cancer increases the risk of venous thromboembolism, and glioblastoma is one of the cancer types with the highest risk of venous thromboembolism (10%-30%). Tumor-intrinsic features are believed to affect vascular permeability and hypercoagulability, but novel models are required to study the pathophysiological dynamics underlying cancer-associated thrombosis at the molecular level. Objectives: We have developed a novel cancer-on-a-chip model to examine the effects of glioblastoma cells on the deregulation of blood coagulation. Methods: This was accomplished by coculturing vessel-forming human umbilical vein endothelial cells with glioblastoma spheroids overexpressing tissue factor (TF), the initiator of coagulation (U251 lentivirus, LV-TF) or an LV-control (U251 LV-Ctrl) in an OrganoPlate Graft platform. Results: Using a modified thrombin generation assay inside the cancer-on-a-chip, we found that U251 LV-Ctrl and U251 LV-TF spheroids promoted an increased procoagulant state in plasma, as was shown by a 3.1- and 7.0-fold increase in endogenous thrombin potential, respectively. Furthermore, the anticoagulant drug rivaroxaban and TF coagulation-blocking antibody 5G9 inhibited the activation of blood coagulation in U251 LV-TF spheroid-containing graft plates, as was shown by a reduced endogenous thrombin potential (4.0- and 4.4-fold, respectively). Conclusion: With this study, we present a novel 3-dimensional cancer-on-a-chip model that has the potential to be used in the discovery of new anticoagulant drugs and identification of optimal anticoagulant strategies for glioblastoma and other cancer types.

Autoantibodies targeting G protein-coupled receptors: An evolving history in autoimmunity. Report of the 4th international symposium.

G protein-coupled receptors (GPCR) are involved in various physiological and pathophysiological processes. Functional autoantibodies targeting GPCRs have been associated with multiple disease manifestations in this context. Here we summarize and discuss the relevant findings and concepts presented in the biennial International Meeting on autoantibodies targeting GPCRs (the 4th Symposium), held in Lübeck, Germany, 15-16 September 2022. The symposium focused on the current knowledge of these autoantibodies' role in various diseases, such as cardiovascular, renal, infectious (COVID-19), and autoimmune diseases (e.g., systemic sclerosis and systemic lupus erythematosus). Beyond their association with disease phenotypes, intense research related to the mechanistic action of these autoantibodies on immune regulation and pathogenesis has been developed, underscoring the role of autoantibodies targeting GPCRs on disease outcomes and etiopathogenesis. The observation repeatedly highlighted that autoantibodies targeting GPCRs could also be present in healthy individuals, suggesting that anti-GPCR autoantibodies play a physiologic role in modeling the course of diseases. Since numerous therapies targeting GPCRs have been developed, including small molecules and monoclonal antibodies designed for treating cancer, infections, metabolic disorders, or inflammatory conditions, anti-GPCR autoantibodies themselves can serve as therapeutic targets to reduce patients' morbidity and mortality, representing a new area for the development of novel therapeutic interventions.

Downregulation of Endothelial Plexin A4 Under Inflammatory Conditions Impairs Vascular Integrity.

Objective: Besides hyperlipidemia, inflammation is an important determinant in the initiation and the progression of atherosclerosis. As Neuroimmune Guidance Cues (NGCs) are emerging as regulators of atherosclerosis, we set out to investigate the expression and function of inflammation-regulated NGCs. Methods and results: NGC expression in human monocytes and endothelial cells was assessed using a publicly available RNA dataset. Next, the mRNA levels of expressed NGCs were analyzed in primary human monocytes and endothelial cells after stimulation with IL1ß or TNFα. Upon stimulation a total of 14 and 19 NGCs in monocytes and endothelial cells, respectively, were differentially expressed. Since plexin A4 (PLXNA4) was strongly downregulated in endothelial cells under inflammatory conditions, the role of PLXNA4 in endothelial function was investigated. Knockdown of PLXNA4 in endothelial cells markedly impaired the integrity of the monolayer leading to more elongated cells with an inflammatory phenotype. In addition, these cells showed an increase in actin stress fibers and decreased cell-cell junctions. Functional assays revealed decreased barrier function and capillary network formation of the endothelial cells, while vascular leakage and trans-endothelial migration of monocytes was increased. Conclusion: The current study demonstrates that pro-inflammatory conditions result in differential expression of NGCs in endothelial cells and monocytes, both culprit cell types in atherosclerosis. Specifically, endothelial PLXNA4 is reduced upon inflammation, while PLXNA4 maintains endothelial barrier function thereby preventing vascular leakage of fluids as well as cells. Taken together, PLXNA4 may well have a causal role in atherogenesis that deserves further investigation.

Metabolomic changes in CSF of migraine patients measured with 1H-NMR spectroscopy.

BACKGROUND: Migraine is a common episodic brain disorder. Treatment options and diagnosis are hampered by an incomplete understanding of disease pathophysiology and the lack of objective diagnostic markers. The aim of this study was to identify biochemical differences characteristic for different subtypes of migraine in cerebrospinal fluid (CSF) of migraine patients using an exploratory 1H-NMR-based metabolomics approach. METHODS: CSF was obtained, in between migraine attacks, via lumbar puncture from patients with hemiplegic migraine, migraine with aura, migraine without aura, and healthy controls. Metabolite concentrations were measured by quantitative 1H-NMR spectroscopy. Multivariate data analysis was used to find the optimal set of predictors, generalized linear models (GLM) were used to ascertain the differential significance of individual metabolites. RESULTS: In CSF samples from 18 patients with hemiplegic migraine, 38 with migraine with aura, 27 migraine without aura, and 43 healthy controls, nineteen metabolites were identified and quantified. Hemiplegic migraine patients could be discriminated from healthy controls using supervised multivariate modelling with 2-hydroxybutyrate and 2-hydroxyisovalerate as the most discriminant metabolites. Univariate GLM analysis showed 2-hydroxybutyrate to be lower in hemiplegic migraine compared with healthy controls; no significant differences were observed for other metabolites. It was not possible to discriminate migraine with and without aura from healthy controls based on their metabolic profile. CONCLUSIONS: Using an exploratory 1H-NMR metabolomics analysis we identified metabolites that were able to discriminate hemiplegic migraine patients from healthy controls. The lower levels of 2-hydroxybutyrate found in patients with hemiplegic migraine could indicate a dysregulation of the brain's energy metabolism. An experimental confirmation in vitro or in animal models will be required to confirm or discard this hypothesis. Migraine with and migraine without aura patients did not reveal a metabolic profile different from healthy controls.