Endothelial extracellular vesicles enhance vascular self-assembly in engineered human cardiac tissues.

The fabrication of complex and stable vasculature in engineered cardiac tissues represents a significant hurdle towards building physiologically relevant models of the heart. Here, we implemented a 3D model of cardiac vasculogenesis, incorporating endothelial cells (EC), stromal cells, and human induced pluripotent stem cell (iPSC)-derived cardiomyocytes (CM) in a fibrin hydrogel. The presence of CMs disrupted vessel formation in 3D tissues, resulting in the upregulation of endothelial activation markers and altered extracellular vesicle (EV) signaling in engineered tissues as determined by the proteomic analysis of culture supernatant. miRNA sequencing of CM- and EC-secreted EVs highlighted key EV-miRNAs that were postulated to play differing roles in cardiac vasculogenesis, including the let-7 family and miR-126-3p in EC-EVs. In the absence of CMs, the supplementation of CM-EVs to EC monolayers attenuated EC migration and proliferation and resulted in shorter and more discontinuous self-assembling vessels when applied to 3D vascular tissues. In contrast, supplementation of EC-EVs to the tissue culture media of 3D vascularized cardiac tissues mitigated some of the deleterious effects of CMs on vascular self-assembly, enhancing the average length and continuity of vessel tubes that formed in the presence of CMs. Direct transfection validated the effects of the key EC-EV miRNAs let-7b-5p and miR-126-3p in improving the maintenance of continuous vascular networks. EC-EV supplementation to biofabricated cardiac tissues and microfluidic devices resulted in tissue vascularization, illustrating the use of this approach in the engineering of enhanced, perfusable, microfluidic models of the myocardium.

Multiomics unveils extracellular vesicle-driven mechanisms of endothelial communication in human carotid atherosclerosis.

Background: Carotid atherosclerosis is orchestrated by cell-cell communication that drives progression along a clinical continuum (asymptomatic to symptomatic). Extracellular vesicles (EVs) are cell-derived nanoparticles representing a new paradigm in cellular communication. Little is known about their biological cargo, cellular origin/destination, and functional roles in human atherosclerotic plaque. Methods: EVs were enriched via size exclusion chromatography from human carotid endarterectomy samples dissected into paired plaque and marginal zones (symptomatic n=16, asymptomatic n=13). EV cargos were assessed via whole transcriptome miRNA sequencing and mass spectrometry-based proteomics. EV multi-omics were integrated with bulk and single cell RNA-sequencing (scRNA-seq) datasets to predict EV cellular origin and ligand-receptor interactions, and multi-modal biological network integration of EV-cargo was completed. EV functional impact was assessed with endothelial angiogenesis assays. Results: Carotid plaques contained more EVs than adjacent marginal zones, with differential enrichment for EV-miRNAs and EV-proteins in key atherogenic pathways. EV cellular origin analysis suggested that tissue EV signatures originated from endothelial cells (EC), smooth muscle cells (SMC), and immune cells. Integrated tissue vesiculomics and scRNA-seq indicated complex EV-vascular cell communication that changed with disease progression and plaque vulnerability (i.e., symptomatic disease). Plaques from symptomatic patients, but not asymptomatic patients, were characterized by increased involvement of endothelial pathways and more complex ligand-receptor interactions, relative to their marginal zones. Plaque-EVs were predicted to mediate communication with ECs. Pathway enrichment analysis delineated an endothelial signature with roles in angiogenesis and neovascularization - well-known indices of plaque instability. This was validated functionally, wherein human carotid symptomatic plaque EVs induced sprouting angiogenesis in comparison to their matched marginal zones. Conclusion: Our findings indicate that EVs may drive dynamic changes in plaques through EV- vascular cell communication and effector functions that typify vulnerability to rupture, precipitating symptomatic disease. The discovery of endothelial-directed angiogenic processes mediated by EVs creates new therapeutic avenues for atherosclerosis.

Single-cell atlas of the human brain vasculature across development, adulthood and disease.

A broad range of brain pathologies critically relies on the vasculature, and cerebrovascular disease is a leading cause of death worldwide. However, the cellular and molecular architecture of the human brain vasculature remains incompletely understood1. Here we performed single-cell RNA sequencing analysis of 606,380 freshly isolated endothelial cells, perivascular cells and other tissue-derived cells from 117 samples, from 68 human fetuses and adult patients to construct a molecular atlas of the developing fetal, adult control and diseased human brain vasculature. We identify extensive molecular heterogeneity of the vasculature of healthy fetal and adult human brains and across five vascular-dependent central nervous system (CNS) pathologies, including brain tumours and brain vascular malformations. We identify alteration of arteriovenous differentiation and reactivated fetal as well as conserved dysregulated genes and pathways in the diseased vasculature. Pathological endothelial cells display a loss of CNS-specific properties and reveal an upregulation of MHC class II molecules, indicating atypical features of CNS endothelial cells. Cell-cell interaction analyses predict substantial endothelial-to-perivascular cell ligand-receptor cross-talk, including immune-related and angiogenic pathways, thereby revealing a central role for the endothelium within brain neurovascular unit signalling networks. Our single-cell brain atlas provides insights into the molecular architecture and heterogeneity of the developing, adult/control and diseased human brain vasculature and serves as a powerful reference for future studies.

MEK signaling represents a viable therapeutic vulnerability of KRAS-driven somatic brain arteriovenous malformations.

Brain arteriovenous malformations (bAVMs) are direct connections between arteries and veins that remodel into a complex nidus susceptible to rupture and hemorrhage. Most sporadic bAVMs feature somatic activating mutations within KRAS, and endothelial-specific expression of the constitutively active variant KRASG12D models sporadic bAVM in mice. By leveraging 3D-based micro-CT imaging, we demonstrate that KRASG12D-driven bAVMs arise in stereotypical anatomical locations within the murine brain, which coincide with high endogenous Kras expression. We extend these analyses to show that a distinct variant, KRASG12C, also generates bAVMs in predictable locations. Analysis of 15,000 human patients revealed that, similar to murine models, bAVMs preferentially occur in distinct regions of the adult brain. Furthermore, bAVM location correlates with hemorrhagic frequency. Quantification of 3D imaging revealed that G12D and G12C alter vessel density, tortuosity, and diameter within the mouse brain. Notably, aged G12D mice feature increased lethality, as well as impaired cognition and motor function. Critically, we show that pharmacological blockade of the downstream kinase, MEK, after lesion formation ameliorates KRASG12D-driven changes in the murine cerebrovasculature and may also impede bAVM progression in human pediatric patients. Collectively, these data show that distinct KRAS variants drive bAVMs in similar patterns and suggest MEK inhibition represents a non-surgical alternative therapy for sporadic bAVM.

Exercise-Dependent Modulation of Immunological Response Pathways in Endurance Athletes With and Without Atrial Fibrillation.

BACKGROUND: Atrial fibrillation (AF) is a common arrhythmia characterized by uncoordinated atrial electrical activity. Lone AF occurs in the absence of traditional risk factors and is frequently observed in male endurance athletes, who face a 2- to 5-fold higher risk of AF compared with healthy, moderately active males. Our understanding of how endurance exercise contributes to the pathophysiology of lone AF remains limited. This study aimed to characterize the circulating protein fluctuations during high-intensity exercise as well as explore potential biomarkers of exercise-associated AF. METHODS AND RESULTS: A prospective cohort of 12 male endurance cyclists between the ages of 40 and 65 years, 6 of whom had a history of exercise-associated AF, were recruited to participate using a convenience sampling method. The circulating proteome was subsequently analyzed using multiplex immunoassays and aptamer-based proteomics before, during, and after an acute high-intensity endurance exercise bout to assess temporality and identify potential markers of AF. The endurance exercise bout resulted in significant alterations to proteins involved in immune modulation (eg, growth/differentiation factor 15), skeletal muscle metabolism (eg, α-actinin-2), cell death (eg, histones), and inflammation (eg, interleukin-6). Subjects with AF differed from those without, displaying modulation of proteins previously known to have associations with incident AF (eg, C-reactive protein, insulin-like growth factor-1, and angiopoietin-2), and also with proteins having no previous association (eg, tapasin-related protein and α2-Heremans-Schmid glycoprotein). CONCLUSIONS: These findings provide insights into the proteomic response to acute intense exercise, provide mechanistic insights into the pathophysiology behind AF in athletes, and identify targets for future study and validation.

Circulating small extracellular vesicles mediate vascular hyperpermeability in diabetes.

AIMS/HYPOTHESIS: A hallmark chronic complication of type 2 diabetes mellitus is vascular hyperpermeability, which encompasses dysfunction of the cerebrovascular endothelium and the subsequent development of associated cognitive impairment. The present study tested the hypothesis that during type 2 diabetes circulating small extracellular vesicles (sEVs) exhibit phenotypic changes that facilitate pathogenic disruption of the vascular barrier. METHODS: sEVs isolated from the plasma of a mouse model of type 2 diabetes and from diabetic human individuals were characterised for their ability to disrupt the endothelial cell (EC) barrier. The contents of sEVs and their effect on recipient ECs were assessed by proteomics and identified pathways were functionally interrogated with small molecule inhibitors. RESULTS: Using intravital imaging, we found that diabetic mice (Leprdb/db) displayed hyperpermeability of the cerebrovasculature. Enhanced vascular leakiness was recapitulated following i.v. injection of sEVs from diabetic mice into non-diabetic recipient mice. Characterisation of circulating sEV populations from the plasma of diabetic mice and humans demonstrated increased quantity and size of sEVs compared with those isolated from non-diabetic counterparts. Functional experiments revealed that sEVs from diabetic mice or humans induced the rapid and sustained disruption of the EC barrier through enhanced paracellular and transcellular leak but did not induce inflammation. Subsequent sEV proteome and recipient EC phospho-proteome analysis suggested that extracellular vesicles (sEVs) from diabetic mice and humans modulate the MAPK/MAPK kinase (MEK) and Rho-associated protein kinase (ROCK) pathways, cell-cell junctions and actin dynamics. This was confirmed experimentally. Treatment of sEVs with proteinase K or pre-treatment of recipient cells with MEK or ROCK inhibitors reduced the hyperpermeability-inducing effects of circulating sEVs in the diabetic state. CONCLUSIONS/INTERPRETATION: Diabetes is associated with marked increases in the concentration and size of circulating sEVs. The modulation of sEV-associated proteins under diabetic conditions can induce vascular leak through activation of the MEK/ROCK pathway. These data identify a new paradigm by which diabetes can induce hyperpermeability and dysfunction of the cerebrovasculature and may implicate sEVs in the pathogenesis of cognitive decline during type 2 diabetes.

The liver and muscle secreted HFE2-protein maintains central nervous system blood vessel integrity.

Liver failure causes breakdown of the Blood CNS Barrier (BCB) leading to damages of the Central-Nervous-System (CNS), however the mechanisms whereby the liver influences BCB-integrity remain elusive. One possibility is that the liver secretes an as-yet to be identified molecule(s) that circulate in the serum to directly promote BCB-integrity. To study BCB-integrity, we developed light-sheet imaging for three-dimensional analysis. We show that liver- or muscle-specific knockout of Hfe2/Rgmc induces BCB-breakdown, leading to accumulation of toxic-blood-derived fibrinogen in the brain, lower cortical neuron numbers, and behavioral deficits in mice. Soluble HFE2 competes with its homologue RGMa for binding to Neogenin, thereby blocking RGMa-induced downregulation of PDGF-B and Claudin-5 in endothelial cells, triggering BCB-disruption. HFE2 administration in female mice with experimental autoimmune encephalomyelitis, a model for multiple sclerosis, prevented paralysis and immune cell infiltration by inhibiting RGMa-mediated BCB alteration. This study has implications for the pathogenesis and potential treatment of diseases associated with BCB-dysfunction.

Directional Endothelial Communication by Polarized Extracellular Vesicle Release.

BACKGROUND: Extracellular vesicles (EVs) contain bioactive cargo including miRNAs and proteins that are released by cells during cell-cell communication. Endothelial cells (ECs) form the innermost lining of all blood vessels, interfacing with cells in the circulation and vascular wall. It is unknown whether ECs release EVs capable of governing recipient cells within these 2 separate compartments. Given their boundary location, we propose ECs use bidirectional release of distinct EV cargo in quiescent (healthy) and activated (atheroprone) states to communicate with cells within the circulation and blood vessel wall. METHODS: EVs were isolated from primary human aortic ECs (plate and transwell grown; ±IL [interleukin]-1ß activation), quantified, visualized, and analyzed by miRNA transcriptomics and proteomics. Apical and basolateral EC-EV release was determined by miRNA transfer, total internal reflection fluorescence and electron microscopy. Vascular reprogramming (RNA sequencing) and functional assays were performed on primary human monocytes or smooth muscle cells±EC-EVs. RESULTS: Activated ECs increased EV release, with miRNA and protein cargo related to atherosclerosis. EV-treated monocytes and smooth muscle cells revealed activated EC-EV altered pathways that were proinflammatory and atherogenic. ECs released more EVs apically, which increased with activation. Apical and basolateral EV cargo contained distinct transcriptomes and proteomes that were altered by EC activation. Notably, activated basolateral EC-EVs displayed greater changes in the EV secretome, with pathways specific to atherosclerosis. In silico analysis determined compartment-specific cargo released by the apical and basolateral surfaces of ECs can reprogram monocytes and smooth muscle cells, respectively, with functional assays and in vivo imaging supporting this concept. CONCLUSIONS: Demonstrating that ECs are capable of polarized EV cargo loading and directional EV secretion reveals a novel paradigm for endothelial communication, which may ultimately enhance the design of endothelial-based therapeutics for cardiovascular diseases such as atherosclerosis where ECs are persistently activated.

The Role and Therapeutic Implications of Inflammation in the Pathogenesis of Brain Arteriovenous Malformations.

Brain arteriovenous malformations (bAVMs) are focal vascular lesions composed of abnormal vascular channels without an intervening capillary network. As a result, high-pressure arterial blood shunts directly into the venous outflow system. These high-flow, low-resistance shunts are composed of dilated, tortuous, and fragile vessels, which are prone to rupture. BAVMs are a leading cause of hemorrhagic stroke in children and young adults. Current treatments for bAVMs are limited to surgery, embolization, and radiosurgery, although even these options are not viable for ~20% of AVM patients due to excessive risk. Critically, inflammation has been suggested to contribute to lesion progression. Here we summarize the current literature discussing the role of the immune system in bAVM pathogenesis and lesion progression, as well as the potential for targeting inflammation to prevent bAVM rupture and intracranial hemorrhage. We conclude by proposing that a dysfunctional endothelium, which harbors the somatic mutations that have been shown to give rise to sporadic bAVMs, may drive disease development and progression by altering the immune status of the brain.

High sensitivity c-reactive protein and circulating biomarkers of endothelial dysfunction in patients with chronic myeloid leukemia receiving tyrosine kinase inhibitors.

Tyrosine kinase inhibitors (TKIs) have revolutionized the management of patients with chronic myelogenous leukemia (CML); however, they may cause cardiovascular (CV) toxicities. In this cross-sectional study, we explored whether high-sensitivity C-reactive protein (hsCRP) and novel markers of vascular dysfunction were associated with exposure to specific TKIs, in 262 CML patients. Hs-CRP level was not associated with CML disease activity or treatment with a specific TKI. Body mass index (OR: 1.15, 95% CI: 1.108-1.246; p < 0.001) and CML duration (OR: 1.004, 95% CI: 1.001-1.008; p = 0.024) were independently associated with higher hs-CRP. In exploratory analyses, novel endothelial-centric markers (e.g. ET-1 and VCAM-1) were differential across the various TKIs, particularly amongst nilotinib- and ponatinib-treated patients. While Levels of hs-CRP do not appear to be correlated with specific TKIs, circulating markers of vascular dysfunction were altered in patients treated with specific TKIs and should be explored as potential markers of TKI-associated CV risk.

Multimodality Cardiac Imaging, Cardiac Symptoms, and Clinical Outcomes in Patients Who Recovered from Mild COVID-19.

Background Many patients have persistent cardiac symptoms after mild COVID-19. However, studies assessing the relationship between symptoms and cardiac imaging are limited. Purpose To assess the relationship between multi-modality cardiac imaging parameters, symptoms, and clinical outcomes in patients recovered from mild COVID-19 compared to COVID-19 negative controls. Materials and Methods Patients who underwent PCR testing for SARS-CoV-2 between August 2020 and January 2022 were invited to participate in this prospective, single-center study. Participants underwent cardiac MRI, echocardiography, and assessment of cardiac symptoms at 3-6 months after SARS-CoV-2 testing. Cardiac symptoms and outcomes were also evaluated at 12-18 months. Statistical analysis included Fisher's exact test and logistic regression. Results This study included 122 participants who recovered from COVID-19 ([COVID+] mean age, 42 years ± 13 [SD]; 73 females) and 22 COVID-19 negative controls (mean age, 46 years ± 16 [SD]; 13 females). At 3-6 months, 20% (24/122) and 44% (54/122) of COVID+ participants had at least one abnormality on echocardiography and cardiac MRI, respectively, which did not differ compared to controls (23% [5/22]; P = .77 and 41% [9/22]; P = .82, respectively). However, COVID+ participants more frequently reported cardiac symptoms at 3-6 months compared to controls (48% [58/122] vs. 23% [4/22]; P = .04). An increase in native T1 (10 ms) was associated with increased odds of cardiac symptoms at 3-6 months (OR, 1.09 [95% CI: 1.00, 1.19]; P = .046) and 12-18 months (OR, 1.14 [95% CI: 1.01, 1.28]; P = .028). No major adverse cardiac events occurred during follow-up. Conclusion Patients recovered from mild COVID-19 reported increased cardiac symptoms 3-6 months after diagnosis compared to controls, but the prevalence of abnormalities on echocardiography and cardiac MRI did not differ between groups. Elevated native T1 was associated with cardiac symptoms 3-6 months and 12-18 months after mild COVID-19.

Endothelial cells secrete small extracellular vesicles bidirectionally containing distinct cargo to uniquely reprogram vascular cells in the circulation and vessel wall.

Rationale: Extracellular vesicles (EVs) contain bioactive cargo including microRNAs (miRNAs) and proteins that are released by cells as a form of cell-cell communication. Endothelial cells (ECs) form the innermost lining of all blood vessels and thereby interface with cells in the circulation as well as cells residing in the vascular wall. It is unknown whether ECs have the capacity to release EVs capable of governing recipient cells within two separate compartments, and how this is affected by endothelial activation commonly seen in atheroprone regions. Objective: Given their boundary location, we propose that ECs utilize bidirectional release of distinct EV cargo in quiescent and activated states to communicate with cells within the circulation and blood vessel wall. Methods and Results: EVs were isolated from primary human aortic endothelial cells (ECs) (+/-IL-1ß activation), quantified, and analysed by miRNA transcriptomics and proteomics. Compared to quiescent ECs, activated ECs increased EV release, with miRNA and protein cargo that were related to atherosclerosis. RNA sequencing of EV-treated monocytes and smooth muscle cells (SMCs) revealed that EVs from activated ECs altered pathways that were pro-inflammatory and atherogenic. Apical and basolateral EV release was assessed using ECs on transwells. ECs released more EVs apically, which increased with activation. Apical and basolateral EV cargo contained distinct transcriptomes and proteomes that were altered by EC activation. Notably, basolateral EC-EVs displayed greater changes in the EV secretome, with pathways specific to atherosclerosis. In silico analysis determined that compartment-specific cargo released by the apical and basolateral surfaces of ECs can reprogram monocytes and SMCs, respectively. Conclusions: The demonstration that ECs are capable of polarized EV cargo loading and directional EV secretion reveals a novel paradigm for endothelial communication, which may ultimately enhance our ability to design endothelial-based therapeutics for cardiovascular diseases such as atherosclerosis where ECs are persistently activated.

Endothelial ACKR1 is induced by neutrophil contact and down-regulated by secretion in extracellular vesicles.

Atypical chemokine receptor-1 (ACKR1), previously known as the Duffy antigen receptor for chemokines, is a widely conserved cell surface protein that is expressed on erythrocytes and the endothelium of post-capillary venules. In addition to being the receptor for the parasite causing malaria, ACKR1 has been postulated to regulate innate immunity by displaying and trafficking chemokines. Intriguingly, a common mutation in its promoter leads to loss of the erythrocyte protein but leaves endothelial expression unaffected. Study of endothelial ACKR1 has been limited by the rapid down-regulation of both transcript and protein when endothelial cells are extracted and cultured from tissue. Thus, to date the study of endothelial ACKR1 has been limited to heterologous over-expression models or the use of transgenic mice. Here we report that exposure to whole blood induces ACKR1 mRNA and protein expression in cultured primary human lung microvascular endothelial cells. We found that contact with neutrophils is required for this effect. We show that NF-κB regulates ACKR1 expression and that upon removal of blood, the protein is rapidly secreted by extracellular vesicles. Finally, we confirm that endogenous ACKR1 does not signal upon stimulation with IL-8 or CXCL1. Our observations define a simple method for inducing endogenous endothelial ACKR1 protein that will facilitate further functional studies.

Myocardial Inflammation on FDG PET/MRI and Clinical Outcomes in Symptomatic and Asymptomatic Participants after COVID-19 Vaccination.

Purpose: To evaluate potential cardiac sequelae of COVID-19 vaccination at 2-month follow-up and relate cardiac symptoms to myocardial tissue changes on fluorodeoxyglucose (FDG) PET/MRI, blood biomarkers, health-related quality of life, and adverse outcomes. Materials and Methods: In this prospective study (ClinicalTrials.gov: NCT04967807), a convenience sample of individuals aged ≥17 years were enrolled after COVID-19 vaccination and were categorized as symptomatic myocarditis (new cardiac symptoms within 14 days of vaccination and met diagnostic criteria for acute myocarditis), symptomatic no myocarditis (new cardiac symptoms but did not meet criteria for myocarditis), and asymptomatic (no new cardiac symptoms). Standardized evaluation was performed 2 months after vaccination, including cardiac fluorine 18 FDG PET/MRI, blood biomarkers, and health-related quality of life. Statistical analysis included Kruskal-Wallis and Fisher exact tests. Results: Fifty-four participants were evaluated a median of 72 days (IQR: 42, 91) after COVID-19 vaccination, 17 symptomatic with myocarditis (36±[SD]15 years, 13 males), 17 symptomatic without myocarditis (42±12 years, 7 males), and 20 asymptomatic (45±14 years, 9 males). No participants in the symptomatic without myocarditis or asymptomatic groups had focal FDG-uptake, myocardial edema or impaired ventricular function. Two participants with symptomatic myocarditis had focal FDG-uptake, and three had high T2 on MRI. Health-related quality of life was lower in the symptomatic myocarditis group than the asymptomatic group. There were no adverse cardiac events beyond myocarditis in any participant. Conclusions: At two-month follow-up, FDG PET/MRI showed evidence of myocardial inflammation in 2/17 participants diagnosed with acute myocarditis early after COVID-19 vaccination, but not in symptomatic and asymptomatic participants without acute myocarditis.Keywords: Myocarditis, Vaccination, COVID-19, PET/MRI, Cardiac MRI, FDG-PET.

Angiopoietin-1 derived peptide hydrogel promotes molecular hallmarks of regeneration and wound healing in dermal fibroblasts.

By providing an ideal environment for healing, biomaterials can be designed to facilitate and encourage wound regeneration. As the wound healing process is complex, there needs to be consideration for the cell types playing major roles, such as fibroblasts. As a major cell type in the dermis, fibroblasts have a large impact on the processes and outcomes of wound healing. Prevopisly, conjugating the angiopoietin-1 derived Q-peptide (QHREDGS) to a collagen-chitosan hydrogel created a biomaterial with in vivo success in accelerating wound healing. This study utilized solvent cast Q-peptide conjugated collagen-chitosan seeded with fibroblast monolayers to investigate the direct impact of the material on this major cell type. After 24 h, fibroblasts had a significant change in release of anti-inflammatory, pro-healing, and ECM deposition cytokines, with demonstrated immunomodulatory effects on macrophages and upregulated expression of critical wound healing genes.

A human model of arteriovenous malformation (AVM)-on-a-chip reproduces key disease hallmarks and enables drug testing in perfused human vessel networks.

Brain arteriovenous malformations (AVMs) are a disorder wherein abnormal, enlarged blood vessels connect arteries directly to veins, without an intervening capillary bed. AVMs are one of the leading causes of hemorrhagic stroke in children and young adults. Most human sporadic brain AVMs are associated with genetic activating mutations in the KRAS gene. Our goal was to develop an in vitro model that would allow for simultaneous morphological and functional phenotypic data capture in real time during AVM disease progression. By generating human endothelial cells harboring a clinically relevant mutation found in most human patients (activating mutations within the small GTPase KRAS) and seeding them in a dynamic microfluidic cell culture system that enables vessel formation and perfusion, we demonstrate that vessels formed by KRAS4AG12V mutant endothelial cells (ECs) were significantly wider and more leaky than vascular beds formed by wild-type ECs, recapitulating key structural and functional hallmarks of human AVM pathogenesis. Immunofluorescence staining revealed a breakdown of adherens junctions in mutant KRAS vessels, leading to increased vascular permeability, a hallmark of hemorrhagic stroke. Finally, pharmacological blockade of MEK kinase activity, but not PI3K inhibition, improved endothelial barrier function (decreased permeability) without affecting vessel diameter. Collectively, our studies describe the creation of human KRAS-dependent AVM-like vessels in vitro in a self-assembling microvessel platform that is amenable to phenotypic observation and drug delivery.

The Endothelium as a Hub for Cellular Communication in Atherogenesis: Is There Directionality to the Message?

Endothelial cells line every blood vessel and thereby serve as an interface between the blood and the vessel wall. They have critical functions for maintaining homeostasis and orchestrating vascular pathogenesis. Atherosclerosis is a chronic disease where cholesterol and inflammatory cells accumulate in the artery wall below the endothelial layer and ultimately form plaques that can either progress to occlude the lumen or rupture with thromboembolic consequences - common outcomes being myocardial infarction and stroke. Cellular communication lies at the core of this process. In this review, we discuss traditional (e.g., cytokines, chemokines, nitric oxide) and novel (e.g., extracellular vesicles) modes of endothelial communication with other endothelial cells as well as circulating and vessel wall cells, including monocytes, macrophages, neutrophils, vascular smooth muscle cells and other immune cells, in the context of atherosclerosis. More recently, the growing appreciation of endothelial cell plasticity during atherogenesis suggests that communication strategies are not static. Here, emerging data on transcriptomics in cells during the development of atherosclerosis are considered in the context of how this might inform altered cell-cell communication. Given the unique position of the endothelium as a boundary layer that is activated in regions overlying vascular inflammation and atherosclerotic plaque, there is a potential to exploit the unique features of this group of cells to deliver therapeutics that target the cellular crosstalk at the core of atherosclerotic disease. Data are discussed supporting this concept, as well as inherent pitfalls. Finally, we briefly review the literature for other regions of the body (e.g., gut epithelium) where cells similarly exist as a boundary layer but provide discrete messages to each compartment to govern homeostasis and disease. In this light, the potential for endothelial cells to communicate in a directional manner is explored, along with the implications of this concept - from fundamental experimental design to biomarker potential and therapeutic targets.

Cardiovascular signatures of COVID-19 predict mortality and identify barrier stabilizing therapies.

BACKGROUND: Endothelial cell (EC) activation, endotheliitis, vascular permeability, and thrombosis have been observed in patients with severe coronavirus disease 2019 (COVID-19), indicating that the vasculature is affected during the acute stages of SARS-CoV-2 infection. It remains unknown whether circulating vascular markers are sufficient to predict clinical outcomes, are unique to COVID-19, and if vascular permeability can be therapeutically targeted. METHODS: Prospectively evaluating the prevalence of circulating inflammatory, cardiac, and EC activation markers as well as developing a microRNA atlas in 241 unvaccinated patients with suspected SARS-CoV-2 infection allowed for prognostic value assessment using a Random Forest model machine learning approach. Subsequent ex vivo experiments assessed EC permeability responses to patient plasma and were used to uncover modulated gene regulatory networks from which rational therapeutic design was inferred. FINDINGS: Multiple inflammatory and EC activation biomarkers were associated with mortality in COVID-19 patients and in severity-matched SARS-CoV-2-negative patients, while dysregulation of specific microRNAs at presentation was specific for poor COVID-19-related outcomes and revealed disease-relevant pathways. Integrating the datasets using a machine learning approach further enhanced clinical risk prediction for in-hospital mortality. Exposure of ECs to COVID-19 patient plasma resulted in severity-specific gene expression responses and EC barrier dysfunction, which was ameliorated using angiopoietin-1 mimetic or recombinant Slit2-N. INTERPRETATION: Integration of multi-omics data identified microRNA and vascular biomarkers prognostic of in-hospital mortality in COVID-19 patients and revealed that vascular stabilizing therapies should be explored as a treatment for endothelial dysfunction in COVID-19, and other severe diseases where endothelial dysfunction has a central role in pathogenesis. FUNDING: This work was directly supported by grant funding from the Ted Rogers Center for Heart Research, Toronto, Ontario, Canada and the Peter Munk Cardiac Center, Toronto, Ontario, Canada.

Vasculature-on-a-chip platform with innate immunity enables identification of angiopoietin-1 derived peptide as a therapeutic for SARS-CoV-2 induced inflammation.

Coronavirus disease 2019 (COVID-19) was primarily identified as a novel disease causing acute respiratory syndrome. However, as the pandemic progressed various cases of secondary organ infection and damage by severe respiratory syndrome coronavirus 2 (SARS-CoV-2) have been reported, including a breakdown of the vascular barrier. As SARS-CoV-2 gains access to blood circulation through the lungs, the virus is first encountered by the layer of endothelial cells and immune cells that participate in host defense. Here, we developed an approach to study SARS-CoV-2 infection using vasculature-on-a-chip. We first modeled the interaction of virus alone with the endothelialized vasculature-on-a-chip, followed by the studies of the interaction of the virus exposed-endothelial cells with peripheral blood mononuclear cells (PBMCs). In an endothelial model grown on a permeable microfluidic bioscaffold under flow conditions, both human coronavirus (HCoV)-NL63 and SARS-CoV-2 presence diminished endothelial barrier function by disrupting VE-cadherin junctions and elevating the level of pro-inflammatory cytokines such as interleukin (IL)-6, IL-8, and angiopoietin-2. Inflammatory cytokine markers were markedly more elevated upon SARS-CoV-2 infection compared to HCoV-NL63 infection. Introduction of PBMCs with monocytes into the vasculature-on-a-chip upon SARS-CoV-2 infection further exacerbated cytokine-induced endothelial dysfunction, demonstrating the compounding effects of inter-cellular crosstalk between endothelial cells and monocytes in facilitating the hyperinflammatory state. Considering the harmful effects of SARS-CoV-2 on endothelial cells, even without active virus proliferation inside the cells, a potential therapeutic approach is critical. We identified angiopoietin-1 derived peptide, QHREDGS, as a potential therapeutic capable of profoundly attenuating the inflammatory state of the cells consistent with the levels in non-infected controls, thereby improving the barrier function and endothelial cell survival against SARS-CoV-2 infection in the presence of PBMC.