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.

Evaluating the influence of altered cerebral hemodynamics on cognitive performance in asymptomatic carotid artery stenosis: A systematic review.

OBJECTIVE: Substantial controversy exists regarding asymptomatic carotid stenosis (ACS) and its potential role in the pathophysiology of cognitive impairment. If proven, this hypothesis may suggest an additional definition for symptomatic carotid disease that would alter current management. This study aimed to synthesize the literature evaluating the relationship between impaired cerebral hemodynamics and cognition in patients with ACS. METHODS: A literature search was performed using MEDLINE, Embase, and EBM Reviews through May 2022. We included prospective case-control studies that used validated, objective measure(s) of either global cognition or one or more domains of cognitive function and assessed cerebrovascular reserve (CVR). RESULTS: Five studies were included, comprising a total of 782 patients with moderate (50%-69%) to severe (70%-99%) ACS. Patients with ACS and impaired ipsilateral CVR demonstrated significant cognitive impairment compared with controls. Patients with unilateral or bilateral ACS and normal CVR had cognitive scores similar to controls. Those with bilateral CVR impairment demonstrated the lowest cognitive scores. CONCLUSIONS: This review lends support to the claim that cognitive impairment, likely the result of impaired cerebral hemodynamics, is an under-recognized morbidity in patients with ACS. CVR may serve as an additional tool to determine whether patients are in fact symptomatic from their carotid stenosis and warrant consideration for intervention.

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.

The Impact of Sex on Outcomes following Carotid Endarterectomy.

BACKGROUND: Previous studies have demonstrated significant sex differences in vascular surgery outcomes. We assessed stroke or death rates following carotid endarterectomy (CEA) in women versus men. METHODS: The Vascular Quality Initiative was used to identify all patients who underwent CEA between 2010 and 2019. Demographic, clinical, and procedural characteristics were recorded and differences between women and men were assessed using independent t-test and chi-squared test. The primary outcomes were 30-day and 1-year stroke or death. Associations between sex and outcomes were assessed using univariate/multivariate logistic regression and Cox proportional hazards analysis. RESULTS: Overall, 52,137 women and 79,974 men underwent CEA in Vascular Quality Initiative sites during the study period. Women were younger (70.3 vs. 70.5 years, P < 0.001) and more likely to have hypertension (89.2% vs. 88.9%, P < 0.05) and diabetes (36.2% vs. 35.8%, P < 0.001) but less likely to be diagnosed with coronary artery disease (23.2% vs. 31.0%, P < 0.001). A greater proportion of men were receiving cardiovascular risk reduction medications and had symptomatic carotid stenosis (28.5% vs. 26.7%, P < 0.001). Women had shorter procedure times (113 vs. 122 min, P < 0.001) and were less likely to receive electroencephalography neuromonitoring (27.9% vs. 28.8%, P < 0.001), drain (35.9% vs. 37.3%, P < 0.001), and protamine (67.4% vs. 68.0%, P < 0.01). Stroke or death at 30 days (1.9% vs. 1.8%, P = 0.60) and 1 year (hazard ratio [HR] 0.98, 95% confidence interval [CI] 0.94-1.01, P = 0.20) were similar between groups, which persisted in asymptomatic patients (HR 0.97, 95% CI 0.93-1.01, P = 0.17) and symptomatic patients (HR 0.99, 95% CI 0.93-1.05, P = 0.71). The similarities in 1-year stroke or death rates existed in both the United States (HR 0.96, 95% CI 0.92-1.01, P = 0.09) and Canada (HR 1.21, 95% CI 0.47-3.11, P = 0.70). CONCLUSIONS: Despite sex differences in clinical and procedural characteristics, women and men have similar 30-day and 1-year outcomes following CEA.

Clonally expanding smooth muscle cells promote atherosclerosis by escaping efferocytosis and activating the complement cascade.

Atherosclerosis is the process underlying heart attack and stroke. Despite decades of research, its pathogenesis remains unclear. Dogma suggests that atherosclerotic plaques expand primarily via the accumulation of cholesterol and inflammatory cells. However, recent evidence suggests that a substantial portion of the plaque may arise from a subset of "dedifferentiated" vascular smooth muscle cells (SMCs) which proliferate in a clonal fashion. Herein we use multicolor lineage-tracing models to confirm that the mature SMC can give rise to a hyperproliferative cell which appears to promote inflammation via elaboration of complement-dependent anaphylatoxins. Despite being extensively opsonized with prophagocytic complement fragments, we find that this cell also escapes immune surveillance by neighboring macrophages, thereby exacerbating its relative survival advantage. Mechanistic studies indicate this phenomenon results from a generalized opsonin-sensing defect acquired by macrophages during polarization. This defect coincides with the noncanonical up-regulation of so-called don't eat me molecules on inflamed phagocytes, which reduces their capacity for programmed cell removal (PrCR). Knockdown or knockout of the key antiphagocytic molecule CD47 restores the ability of macrophages to sense and clear opsonized targets in vitro, allowing for potent and targeted suppression of clonal SMC expansion in the plaque in vivo. Because integrated clinical and genomic analyses indicate that similar pathways are active in humans with cardiovascular disease, these studies suggest that the clonally expanding SMC may represent a translational target for treating atherosclerosis.

MiR-30 promotes fatty acid beta-oxidation and endothelial cell dysfunction and is a circulating biomarker of coronary microvascular dysfunction in pre-clinical models of diabetes.

BACKGROUND: Type 2 diabetes (T2D) is associated with coronary microvascular dysfunction, which is thought to contribute to compromised diastolic function, ultimately culminating in heart failure with preserved ejection fraction (HFpEF). The molecular mechanisms remain incompletely understood, and no early diagnostics are available. We sought to gain insight into biomarkers and potential mechanisms of microvascular dysfunction in obese mouse (db/db) and lean rat (Goto-Kakizaki) pre-clinical models of T2D-associated diastolic dysfunction. METHODS: The microRNA (miRNA) content of circulating extracellular vesicles (EVs) was assessed in T2D models to identify biomarkers of coronary microvascular dysfunction/rarefaction. The potential source of circulating EV-encapsulated miRNAs was determined, and the mechanisms of induction and the function of candidate miRNAs were assessed in endothelial cells (ECs). RESULTS: We found an increase in miR-30d-5p and miR-30e-5p in circulating EVs that coincided with indices of coronary microvascular EC dysfunction (i.e., markers of oxidative stress, DNA damage/senescence) and rarefaction, and preceded echocardiographic evidence of diastolic dysfunction. These miRNAs may serve as biomarkers of coronary microvascular dysfunction as they are upregulated in ECs of the left ventricle of the heart, but not other organs, in db/db mice. Furthermore, the miR-30 family is secreted in EVs from senescent ECs in culture, and ECs with senescent-like characteristics are present in the db/db heart. Assessment of miR-30 target pathways revealed a network of genes involved in fatty acid biosynthesis and metabolism. Over-expression of miR-30e in cultured ECs increased fatty acid ß-oxidation and the production of reactive oxygen species and lipid peroxidation, while inhibiting the miR-30 family decreased fatty acid ß-oxidation. Additionally, miR-30e over-expression synergized with fatty acid exposure to down-regulate the expression of eNOS, a key regulator of microvascular and cardiomyocyte function. Finally, knock-down of the miR-30 family in db/db mice decreased markers of oxidative stress and DNA damage/senescence in the microvascular endothelium. CONCLUSIONS: MiR-30d/e represent early biomarkers and potential therapeutic targets that are indicative of the development of diastolic dysfunction and may reflect altered EC fatty acid metabolism and microvascular dysfunction in the diabetic heart.

Assessment of open surgical and endovascular management of true hepatic artery aneurysms over 20 years highlights increased rupture risk in females.

BACKGROUND: True hepatic artery aneurysms (HAAs) are rare but have been associated with a significant risk of rupture and associated mortality. The 2020 release of HAA-specific clinical practice guidelines represented an important step toward management standardization. However, it remains essential to build on the body of evidence to further refine these recommendations. METHODS: The HAA management and outcomes from a single academic center during a 20-year period were retrospectively reviewed. We identified 72 patients from the institutional radiology database (November 24, 1999 to 2019). Pseudoaneurysms were excluded, and 48 patients were found to have had true HAAs. Forty-three HAA patients had sufficient medical records for inclusion in the analysis. RESULTS: Of the 43 patients with HAA included, 65% were male. The mean age was 63 years (range, 22-89 years). Of the HAAs, 72% presented asymptomatically, 16% had ruptured, and 12% were symptomatic at presentation. Most HAAs were of atherosclerotic origin (74%). In addition, 16% of the patients had other visceral aneurysms and 12% had nonvisceral aneurysms on presentation. The mean HAA size overall was 3.3 cm (range, 0.8-10.8 cm), with most being solitary (72%) and involving the common hepatic artery (65%). Rupture was more common in females (40%) and those with vasculitis (67%), with females representing 86% of all patients with rupture. The mean size at intervention was 4.8 cm (21 patients [49%]). Ten patients (23%) had undergone open surgical repair (seven elective and three emergent because of rupture). Eleven patients (26%) had undergone endovascular intervention (64% elective and 36% emergent). Nonoperative management was selected for 22 patients (51%). These patients had a mean HAA diameter of 2.1 cm, and 59% had a life-limiting illness. Of the 18 patients who had been initially monitored for a mean of 3.9 ± 4.1 years, 3 had undergone elective repair and 2 had minimal growth. None of these patients had a subsequently documented rupture. CONCLUSIONS: True HAAs are a rare but important clinical phenomenon, with 16% of patients presenting with rupture in this study. Endovascular intervention is a promising alternative to open surgical repair, with no 30-day mortality, and is suitable for ruptured HAAs. Importantly, for the first time, our findings have demonstrated an increased risk of rupture for females, highlighting the need for additional data and ultimately, sex-specific guidelines.

Overcoming Barriers: The Endothelium As a Linchpin of Coronavirus Disease 2019 Pathogenesis?

OBJECTIVE: Coronavirus disease 2019 (COVID-19) is a global pandemic involving >5 500 000 cases worldwide as of May 26, 2020. The culprit is the severe acute respiratory syndrome coronavirus-2, which invades cells by binding to ACE2 (angiotensin-converting enzyme 2). While the majority of patients mount an appropriate antiviral response and recover at home, others progress to respiratory distress requiring hospital admission for supplemental oxygen. In severe cases, deterioration to acute respiratory distress syndrome necessitating mechanical ventilation, development of severe thrombotic events, or cardiac injury and dysfunction occurs. In this review, we highlight what is known to date about COVID-19 and cardiovascular risk, focusing in on the putative role of the endothelium in disease susceptibility and pathogenesis. Approach and Results: Cytokine-driven vascular leak in the lung alveolar-endothelial interface facilitates acute lung injury in the setting of viral infection. Given that the virus affects multiple organs, including the heart, it likely gains access into systemic circulation by infecting or passing from the respiratory epithelium to the endothelium for viral dissemination. Indeed, cardiovascular complications of COVID-19 are highly prevalent and include acute cardiac injury, myocarditis, and a hypercoagulable state, all of which may be influenced by altered endothelial function. Notably, the disease course is worse in individuals with preexisting comorbidities that involve endothelial dysfunction and may be linked to elevated ACE2 expression, such as diabetes mellitus, hypertension, and cardiovascular disease. CONCLUSIONS: Rapidly emerging data on COVID-19, together with results from studies on severe acute respiratory syndrome coronavirus-1, are providing insight into how endothelial dysfunction may contribute to the pandemic that is paralyzing the globe. This may, in turn, inform the design of biomarkers predictive of disease course, as well as therapeutics targeting pathogenic endothelial responses.

MicroRNAs as sentinels and protagonists of carotid artery thromboembolism.

Stroke is the leading cause of serious disability in the world and a large number of ischemic strokes are due to thromboembolism from unstable carotid artery atherosclerotic plaque. As it is difficult to predict plaque rupture and surgical treatment of asymptomatic disease carries a risk of stroke, carotid disease continues to present major challenges with regard to clinical decision-making and revascularization. There is therefore an imminent need to better understand the molecular mechanisms governing plaque instability and rupture, as this would allow for the development of biomarkers to identify at-risk asymptomatic carotid plaque prior to disease progression and stroke. Further, it would aid in creation of therapeutics to stabilize carotid plaque. MicroRNAs (miRNAs) have been implicated as key protagonists in various stages of atherosclerotic plaque initiation, development and rupture. Notably, they appear to play a crucial role in carotid artery thromboembolism. As the molecular pathways governing the role of miRNAs are being uncovered, we are learning that their involvement is complex, tissue- and stage-specific, and highly selective. Notably, miRNAs can be packaged and secreted in extracellular vesicles (EVs), where they participate in cell-cell communication. The measurement of EV-encapsulated miRNAs in the circulation may inform disease mechanisms occurring in the plaque itself, and therefore may serve as sentinels of unstable plaque as well as therapeutic targets.

Kemp Eliminase Activity of Ketosteroid Isomerase.

Kemp eliminases represent the most successful class of computationally designed enzymes, with rate accelerations of up to 109-fold relative to the rate of the same reaction in aqueous solution. Nevertheless, several other systems such as micelles, catalytic antibodies, and cavitands are known to accelerate the Kemp elimination by several orders of magnitude. We found that the naturally occurring enzyme ketosteroid isomerase (KSI) also catalyzes the Kemp elimination. Surprisingly, mutations of D38, the residue that acts as a general base for its natural substrate, produced variants that catalyze the Kemp elimination up to 7000-fold better than wild-type KSI does, and some of these variants accelerate the Kemp elimination more than the computationally designed Kemp eliminases. Analysis of the D38N general base KSI variant suggests that a different active site carboxylate residue, D99, performs the proton abstraction. Docking simulations and analysis of inhibition by active site binders suggest that the Kemp elimination takes place in the active site of KSI and that KSI uses the same catalytic strategies of the computationally designed enzymes. In agreement with prior observations, our results strengthen the conclusion that significant rate accelerations of the Kemp elimination can be achieved with very few, nonspecific interactions with the substrate if a suitable catalytic base is present in a hydrophobic environment. Computational design can fulfill these requirements, and the design of more complex and precise environments represents the next level of challenges for protein design.

Extracellular vesicles as biomarkers and modulators of atherosclerosis pathogenesis.

Extracellular vesicles (EVs) are small, lipid bilayer-enclosed structures released by various cell types that play a critical role in intercellular communication. In atherosclerosis, EVs have been implicated in multiple pathophysiological processes, including endothelial dysfunction, inflammation, and thrombosis. This review provides an up-to-date overview of our current understanding of the roles of EVs in atherosclerosis, emphasizing their potential as diagnostic biomarkers and their roles in disease pathogenesis. We discuss the different types of EVs involved in atherosclerosis, the diverse cargoes they carry, their mechanisms of action, and the various methods employed for their isolation and analysis. Moreover, we underscore the importance of using relevant animal models and human samples to elucidate the role of EVs in disease pathogenesis. Overall, this review consolidates our current knowledge of EVs in atherosclerosis and highlights their potential as promising targets for disease diagnosis and therapy.

Post-acute sequelae of COVID-19: understanding and addressing the burden of multisystem manifestations.

Individuals with SARS-CoV-2 infection can develop symptoms that persist well beyond the acute phase of COVID-19 or emerge after the acute phase, lasting for weeks or months after the initial acute illness. The post-acute sequelae of COVID-19, which include physical, cognitive, and mental health impairments, are known collectively as long COVID or post-COVID-19 condition. The substantial burden of this multisystem condition is felt at individual, health-care system, and socioeconomic levels, on an unprecedented scale. Survivors of COVID-19-related critical illness are at risk of the well known sequelae of acute respiratory distress syndrome, sepsis, and chronic critical illness, and these multidimensional morbidities might be difficult to differentiate from the specific effects of SARS-CoV-2 and COVID-19. We provide an overview of the manifestations of post-COVID-19 condition after critical illness in adults. We explore the effects on various organ systems, describe potential pathophysiological mechanisms, and consider the challenges of providing clinical care and support for survivors of critical illness with multisystem manifestations. Research is needed to reduce the incidence of post-acute sequelae of COVID-19-related critical illness and to optimise therapeutic and rehabilitative care and support for patients.

Metagenomics and metatranscriptomics reveal broadly distributed, active, novel methanotrophs in the Gulf of Mexico hypoxic zone and in the marine water column.

The northern Gulf of Mexico (nGOM) hypoxic zone is a shallow water environment where methane, a potent greenhouse gas, fluxes from sediments to bottom water and remains trapped due to summertime stratification. When the water column is destratified, an active planktonic methanotrophic community could mitigate the efflux of methane, which accumulates to high concentrations, to the atmosphere. To investigate the possibility of such a biofilter in the nGOM hypoxic zone we performed metagenome assembly, and metagenomic and metatranscriptomic read mapping. Methane monooxygenase (pmoA) was an abundant transcript, yet few canonical methanotrophs have been reported in this environment, suggesting a role for non-canonical methanotrophs. To determine the identity of these methanotrophs, we reconstructed six novel metagenome-assembled genomes (MAGs) in the Planctomycetota, Verrucomicrobiota and one putative Latescibacterota, each with at least one pmoA gene copy. Based on ribosomal protein phylogeny, closely related microbes (mostly from Tara Oceans) and isolate genomes were selected and co-analyzed with the nGOM MAGs. Gene annotation and read mapping suggested that there is a large, diverse and unrecognized community of active aerobic methanotrophs in the nGOM hypoxic zone and in the global ocean that could mitigate methane flux to the atmosphere.

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.

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.

Immune landscape of tertiary lymphoid structures in hepatocellular carcinoma (HCC) treated with neoadjuvant immune checkpoint blockade.

Neoadjuvant immunotherapy is thought to produce long-term remissions through induction of antitumor immune responses before removal of the primary tumor. Tertiary lymphoid structures (TLS), germinal center-like structures that can arise within tumors, may contribute to the establishment of immunological memory in this setting, but understanding of their role remains limited. Here, we investigated the contribution of TLS to antitumor immunity in hepatocellular carcinoma (HCC) treated with neoadjuvant immunotherapy. We found that neoadjuvant immunotherapy induced the formation of TLS, which were associated with superior pathologic response, improved relapse free survival, and expansion of the intratumoral T and B cell repertoire. While TLS in viable tumor displayed a highly active mature morphology, in areas of tumor regression we identified an involuted TLS morphology, which was characterized by dispersion of the B cell follicle and persistence of a T cell zone enriched for ongoing antigen presentation and T cell-mature dendritic cell interactions. Involuted TLS showed increased expression of T cell memory markers and expansion of CD8+ cytotoxic and tissue resident memory clonotypes. Collectively, these data reveal the circumstances of TLS dissolution and suggest a functional role for late-stage TLS as sites of T cell memory formation after elimination of viable tumor.

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.

Combined Cardiac Fluorodeoxyglucose-Positron Emission Tomography/Magnetic Resonance Imaging Assessment of Myocardial Injury in Patients Who Recently Recovered From COVID-19.

IMPORTANCE: Although myocardial injury can occur with acute COVID-19, there is limited understanding of changes with myocardial metabolism in recovered patients. OBJECTIVE: To examine myocardial metabolic changes early after recovery from COVID-19 using fluorodeoxyglucose-positron emission tomography (PET) and associate these changes to abnormalities in cardiac magnetic resonance imaging (MRI)-based function and tissue characterization measures and inflammatory blood markers. DESIGN, SETTING, AND PARTICIPANTS: This prospective cohort study took place at a single-center tertiary referral hospital system. A volunteer sample of adult patients within 3 months of a diagnosis of COVID-19 who responded to a mail invitation were recruited for cardiac PET/MRI and blood biomarker evaluation between November 2020 and June 2021. EXPOSURES: Myocardial inflammation as determined by focal fluorodeoxyglucose (FDG) uptake on PET. MAIN OUTCOMES AND MEASURES: Demographic characteristics, cardiac and inflammatory blood markers, and fasting combined cardiac 18F-FDG PET/MRI imaging were obtained. All patients with focal FDG uptake at baseline returned for repeated PET/MRI and blood marker assessment 2 months later. RESULTS: Of 47 included patients, 24 (51%) were female, and the mean (SD) age was 43 (13) years. The mean (SD) interval between COVID-19 diagnosis and PET/MRI was 67 (16) days. Most patients recovered at home during the acute infection (40 [85%]). Eight patients (17%) had focal FDG uptake on PET consistent with myocardial inflammation. Compared with those without FDG uptake, patients with focal FDG uptake had higher regional T2, T1, and extracellular volume (colocalizing with focal FDG uptake), higher prevalence of late gadolinium enhancement (6 of 8 [75%] vs 9 of 39 [23%], P = .009), lower left ventricular ejection fraction (mean [SD], 55% [4%] vs 62% [5%], P < .001), worse global longitudinal and circumferential strain (mean [SD], -16% [2%] vs -17% [2%], P = .02 and -18% [2%] vs -20% [2%], P = .047, respectively), and higher systemic inflammatory blood markers including interleukin 6, interleukin 8, and high-sensitivity C-reactive protein. Among patients with focal FDG uptake, PET/MRI, and inflammatory blood markers resolved or improved at follow-up performed a mean (SD) of 52 (17) days after baseline PET/MRI. CONCLUSIONS AND RELEVANCE: In this study of patients recently recovered from COVID-19, myocardial inflammation was identified on PET in a small proportion of patients, was associated with cardiac MRI abnormalities and elevated inflammatory blood markers at baseline, and improved at follow-up.

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.