Exercise mitigates flow recirculation and activates metabolic transducer SCD1 to catalyze vascular protective metabolites.

Exercise promotes pulsatile shear stress in the arterial circulation and ameliorates cardiometabolic diseases. However, exercise-mediated metabolic transducers for vascular protection remain under-investigated. Untargeted metabolomic analysis demonstrated that wild-type mice undergoing voluntary wheel running exercise expressed increased endothelial stearoyl-CoA desaturase 1 (SCD1) that catalyzes anti-inflammatory lipid metabolites, namely, oleic (OA) and palmitoleic acids (PA), to mitigate NF-κB-mediated inflammatory responses. In silico analysis revealed that exercise augmented time-averaged wall shear stress but mitigated flow recirculation and oscillatory shear index in the lesser curvature of the mouse aortic arch. Following exercise, endothelial Scd1-deleted mice (Ldlr-/- Scd1EC-/-) on high-fat diet developed persistent VCAM1-positive endothelium in the lesser curvature and the descending aorta, whereas SCD1 overexpression via adenovirus transfection mitigated endoplasmic reticulum stress and inflammatory biomarkers. Single-cell transcriptomics of the aorta identified Scd1-positive and Vcam1-negative endothelial subclusters interacting with other candidate genes. Thus, exercise mitigates flow recirculation and activates endothelial SCD1 to catalyze OA and PA for vascular endothelial protection.

α-Glucosidase inhibition by green, white and oolong teas: in vitro activity and computational studies.

Natural α-glucosidase inhibitors from plant-based foods such as catechins offer an attractive strategy for their potential anti-diabetic effects. In this study, infusions of three different tea types (green, white, and oolong) were investigated for their total phenolic (TPC) and catechins (EGCG, ECG, EGC, and EC) content, and for their α-glucosidase inhibitory activities. We observed that the level of TPC in white tea was significantly higher compared to oolong and green tea, which suggests higher content of EGCG and ECG catechins in fresh young leaves. Our findings showed that the higher content of such catechins in the infusion of white tea well correlated with a strong inhibition of α-glucosidase, and such inhibition was demonstrated to be more effective than the FDA-approved drug acarbose. Then, we computationally explored the molecular requirements for enzyme inhibition, especially for the most active catechins EGCG and ECG, as well as their disposition/stability within the active site.

Microfluidic Organ-Chips and Stem Cell Models in the Fight Against COVID-19.

SARS-CoV-2, the virus underlying COVID-19, has now been recognized to cause multiorgan disease with a systemic effect on the host. To effectively combat SARS-CoV-2 and the subsequent development of COVID-19, it is critical to detect, monitor, and model viral pathogenesis. In this review, we discuss recent advancements in microfluidics, organ-on-a-chip, and human stem cell-derived models to study SARS-CoV-2 infection in the physiological organ microenvironment, together with their limitations. Microfluidic-based detection methods have greatly enhanced the rapidity, accessibility, and sensitivity of viral detection from patient samples. Engineered organ-on-a-chip models that recapitulate in vivo physiology have been developed for many organ systems to study viral pathology. Human stem cell-derived models have been utilized not only to model viral tropism and pathogenesis in a physiologically relevant context but also to screen for effective therapeutic compounds. The combination of all these platforms, along with future advancements, may aid to identify potential targets and develop novel strategies to counteract COVID-19 pathogenesis.

Exercise Mitigates Flow Recirculation and Activates Mechanosensitive Transcriptome to Uncover Endothelial SCD1-Catalyzed Anti-Inflammatory Metabolites.

Exercise modulates vascular plasticity in multiple organ systems; however, the metabolomic transducers underlying exercise and vascular protection in the disturbed flow-prone vasculature remain under-investigated. We simulated exercise-augmented pulsatile shear stress (PSS) to mitigate flow recirculation in the lesser curvature of the aortic arch. When human aortic endothelial cells (HAECs) were subjected to PSS ( τ ave = 50 dyne·cm -2 , ∂τ/∂t = 71 dyne·cm -2 ·s -1 , 1 Hz), untargeted metabolomic analysis revealed that Stearoyl-CoA Desaturase (SCD1) in the endoplasmic reticulum (ER) catalyzed the fatty acid metabolite, oleic acid (OA), to mitigate inflammatory mediators. Following 24 hours of exercise, wild-type C57BL/6J mice developed elevated SCD1-catalyzed lipid metabolites in the plasma, including OA and palmitoleic acid (PA). Exercise over a 2-week period increased endothelial SCD1 in the ER. Exercise further modulated the time-averaged wall shear stress (TAWSS or τ ave) and oscillatory shear index (OSI ave ), upregulated Scd1 and attenuated VCAM1 expression in the disturbed flow-prone aortic arch in Ldlr -/- mice on high-fat diet but not in Ldlr -/- Scd1 EC-/- mice. Scd1 overexpression via recombinant adenovirus also mitigated ER stress. Single cell transcriptomic analysis of the mouse aorta revealed interconnection of Scd1 with mechanosensitive genes, namely Irs2 , Acox1 and Adipor2 that modulate lipid metabolism pathways. Taken together, exercise modulates PSS ( τ ave and OSI ave ) to activate SCD1 as a metabolomic transducer to ameliorate inflammation in the disturbed flow-prone vasculature.

Mitoquinone Mesylate and Mitochondrial DNA in End Organs in Humanized Mouse Model of Chronic Treated Human Immunodeficiency Virus Infection.

No treatment exists for mitochondrial dysfunction, a contributor to end-organ disease in human immunodeficiency virus (HIV). The mitochondrial antioxidant mitoquinone mesylate (MitoQ) attenuates mitochondrial dysfunction in preclinical mouse models of various diseases but has not been used in HIV. We used a humanized murine model of chronic HIV infection and polymerase chain reaction to show that HIV-1-infected mice treated with antiretroviral therapy and MitoQ for 90 days had higher ratios of human and murine mitochondrial to nuclear DNA in end organs compared with HIV-1-infected mice on antiretroviral therapy. We offer translational evidence of MitoQ as treatment for mitochondrial dysfunction in HIV.

Blood immune cells from people with HIV on antiviral regimens that contain tenofovir alafenamide (TAF) and tenofovir disoproxil fumarate (TDF) have differential metabolic signatures.

BACKGROUND: Mitochondria regulate immune and organ function. It is unknown whether higher intracellular drug levels observed in peripheral blood mononuclear cells (PBMCs) treated with tenofovir alafenamide (TAF) compared to tenofovir disoproxil fumarate (TDF) may alter mitochondrial function and energy production in immune cells in HIV(+) patients. METHODS: Cellular bioenergetics were determined in PBMCs from HIV-1(-) participants exposed to TAF versus TDF in vitro, at a comparable concentration to a clinically relevant plasma exposure. A decrease in cellular oxygen consumption rate (OCR) at baseline (basal-OCR) and under cellular stress (max-OCR) may suggest mitochondrial dysfunction. We also assessed the in vivo impact of TAF vs TDF on OCR in PBMCs from 26 people with HIV (PWH) interchanged from TDF-based to TAF-based antiretroviral therapy (ART) over a 9-month period in the setting of an open label clinical trial. The Wilcoxon and Mann Whitney tests were used for comparison of continuous variables. RESULTS: PBMCs from HIV-1(-) participants exposed in vitro to a concentration of 0.12-3.3 µM for TAF and TDF at 2 and 24 h, reduced basal and maximal OCR compared to vehicle control. Switch studies of antivirals (TAF vs TDF) within the same PWH showed that TAF-based ART was associated with reduced OCR compared to TDF-based ART in PBMCs. We observed that TAF-treated PBMCs selectively relied more on glucose/pyruvate supply rather than fatty acid to fuel their mitochondria. CONCLUSIONS: Compared to TDF, TAF may alter bioenergetics in immune cells from PWH in vitro and in vivo. The clinical significance in terms of the differential impact caused by TAF versus TDF on mitochondrial function and energy production in immune cells, a regulator of immune function, requires further studied in HIV, preexposure prophylaxis and hepatitis B.

Ectodomain shedding of proteins important for SARS-CoV-2 pathogenesis in plasma of tobacco cigarette smokers compared to electronic cigarette vapers: a cross-sectional study.

The impact of tobacco cigarette (TCIG) smoking and electronic cigarette (ECIG) vaping on the risk of development of severe COVID-19 is controversial. The present study investigated levels of proteins important for SARS-CoV-2 pathogenesis present in plasma because of ectodomain shedding in smokers, ECIG vapers, and non-smokers (NSs). Protein levels of soluble angiotensin-converting enzyme 2 (ACE2), angiotensin (Ang) II (the ligand of ACE2), Ang 1-7 (the main peptide generated from Ang II by ACE2 activity), furin (a protease that increases the affinity of the SARS-CoV-2 spike protein for ACE2), and products of ADAM17 shedding activity that predict morbidity in COVID-19 (IL-6/IL-6R alpha (IL-6/IL-6Rα) complex, soluble CD163 (sCD163), L-selectin) were determined in plasma from 45 NSs, 30 ECIG vapers, and 29 TCIG smokers using ELISA. Baseline characteristics of study participants did not differ among groups. TCIG smokers had increased sCD163, L-selectin compared to NSs and ECIG vapers (p < 0.001 for all comparisons). ECIG vapers had higher plasma furin compared to both NSs (p < 0.001) and TCIG smokers (p < 0.05). ECIG vaping and TCIG smoking did not impact plasma ACE2, Ang 1-7, Ang II, and IL-6 levels compared to NSs (p > 0.1 for all comparisons). Further studies are needed to determine if increased furin activity and ADAM17 shedding activity that is associated with increased plasma levels of sCD163 and L-selectin in healthy young TCIG smokers may contribute to the future development of severe COVID-19 and cardiovascular complications of post-acute COVID-19 syndrome.

A Nrf2-OSGIN1&2-HSP70 axis mediates cigarette smoke-induced endothelial detachment: implications for plaque erosion.

AIMS: Endothelial erosion of plaques is responsible for ∼30% of acute coronary syndromes (ACS). Smoking is a risk factor for plaque erosion, which most frequently occurs on the upstream surface of plaques where the endothelium experiences elevated shear stress. We sought to recreate these conditions in vitro to identify potential pathological mechanisms that might be of relevance to plaque erosion. METHODS AND RESULTS: Culturing human coronary artery endothelial cells (HCAECs) under elevated flow (shear stress of 7.5 Pa) and chronically exposing them to cigarette smoke extract (CSE) and tumour necrosis factor-alpha (TNFα) recapitulated a defect in HCAEC adhesion, which corresponded with augmented Nrf2-regulated gene expression. Pharmacological activation or adenoviral overexpression of Nrf2 triggered endothelial detachment, identifying Nrf2 as a mediator of endothelial detachment. Growth/Differentiation Factor-15 (GDF15) expression was elevated in this model, with protein expression elevated in the plasma of patients experiencing plaque erosion compared with plaque rupture. The expression of two Nrf2-regulated genes, OSGIN1 and OSGIN2, was increased by CSE and TNFα under elevated flow and was also elevated in the aortas of mice exposed to cigarette smoke in vivo. Knockdown of OSGIN1&2 inhibited Nrf2-induced cell detachment. Overexpression of OSGIN1&2 induced endothelial detachment and resulted in cell cycle arrest, induction of senescence, loss of focal adhesions and actin stress fibres, and disturbed proteostasis mediated in part by HSP70, restoration of which reduced HCAEC detachment. In ACS patients who smoked, blood concentrations of HSP70 were elevated in plaque erosion compared with plaque rupture. CONCLUSION: We identified a novel Nrf2-OSGIN1&2-HSP70 axis that regulates endothelial adhesion, elevated GDF15 and HSP70 as biomarkers for plaque erosion in patients who smoke, and two therapeutic targets that offer the potential for reducing the risk of plaque erosion.

Engineering organ-on-a-chip systems to model viral infections.

Infectious diseases remain a public healthcare concern worldwide. Amidst the pandemic of coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 infection, increasing resources have been diverted to investigate therapeutics targeting the COVID-19 spike glycoprotein and to develop various classes of vaccines. Most of the current investigations employ two-dimensional (2D) cell culture and animal models. However, 2D culture negates the multicellular interactions and three-dimensional (3D) microenvironment, and animal models cannot mimic human physiology because of interspecies differences. On the other hand, organ-on-a-chip (OoC) devices introduce a game-changer to model viral infections in human tissues, facilitating high-throughput screening of antiviral therapeutics. In this context, this review provides an overview of thein vitroOoC-based modeling of viral infection, highlighting the strengths and challenges for the future.

Engineering viral genomics and nano-liposomes in microfluidic platforms for patient-specific analysis of SARS-CoV-2 variants.

New variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are continuing to spread globally, contributing to the persistence of the COVID-19 pandemic. Increasing resources have been focused on developing vaccines and therapeutics that target the Spike glycoprotein of SARS-CoV-2. Recent advances in microfluidics have the potential to recapitulate viral infection in the organ-specific platforms, known as organ-on-a-chip (OoC), in which binding of SARS-CoV-2 Spike protein to the angiotensin-converting enzyme 2 (ACE2) of the host cells occurs. As the COVID-19 pandemic lingers, there remains an unmet need to screen emerging mutations, to predict viral transmissibility and pathogenicity, and to assess the strength of neutralizing antibodies following vaccination or reinfection. Conventional detection of SARS-CoV-2 variants relies on two-dimensional (2-D) cell culture methods, whereas simulating the micro-environment requires three-dimensional (3-D) systems. To this end, analyzing SARS-CoV-2-mediated pathogenicity via microfluidic platforms minimizes the experimental cost, duration, and optimization needed for animal studies, and obviates the ethical concerns associated with the use of primates. In this context, this review highlights the state-of-the-art strategy to engineer the nano-liposomes that can be conjugated with SARS-CoV-2 Spike mutations or genomic sequences in the microfluidic platforms; thereby, allowing for screening the rising SARS-CoV-2 variants and predicting COVID-19-associated coagulation. Furthermore, introducing viral genomics to the patient-specific blood accelerates the discovery of therapeutic targets in the face of evolving viral variants, including B1.1.7 (Alpha), B.1.351 (Beta), B.1.617.2 (Delta), c.37 (Lambda), and B.1.1.529 (Omicron). Thus, engineering nano-liposomes to encapsulate SARS-CoV-2 viral genomic sequences enables rapid detection of SARS-CoV-2 variants in the long COVID-19 era.

An engineered nano-liposome-human ACE2 decoy neutralizes SARS-CoV-2 Spike protein-induced inflammation in both murine and human macrophages.

Rationale: Macrophages are the frontline immune cells in response to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. Angiotensin-converting enzyme 2 (ACE2) serves as the binding receptor to SARS-CoV-2 Spike glycoprotein for fusion and internalization into the human host cells. However, the mechanisms underlying SARS-CoV-2-elicited macrophage inflammatory responses remain elusive. Neutralizing SARS-CoV-2 by human ACE2 (hACE2) decoys has been proposed as a therapeutic approach to ameliorate SARS-CoV-2-stimulated inflammation. This study aims to investigate whether an engineered decoy receptor can abrogate SARS-CoV-2-induced macrophage inflammation. Methods: hACE2 was biotinylated to the surface of nano-liposomes (d = 100 nm) to generate Liposome-human ACE2 complex (Lipo-hACE2). Lentivirus expressing Spike protein (D614G) was also created as a pseudo-SARS-CoV-2 (Lenti-Spike). Liposome-hACE2 was used as a decoy receptor or competitive inhibitor to inhibit SARS-CoV-2 or Lenti-Spike-induced macrophage inflammation in vitro and in vivo. Results: Both SARS-CoV-2 and Lenti-Spike stimulated strong inflammatory responses by inducing the expression of key cytokine and chemokines, including IL-1ß, IL-6, TNFα, CCL-2, and CXCL-10, in murine and human macrophages in vitro, whereas Lipo-hACE2 decoy abolished these effects in macrophages. Furthermore, intravenous injection of Lenti-Spike led to increased macrophage and tissue inflammation in wild type mice, which was also abolished by Lipo-hACE2 treatment. Mechanistically, Spike protein stimulated macrophage inflammation by activating canonical NF-κB signaling. RNA sequencing analysis revealed that Lenti-Spike induced over 2,000 differentially expressed genes (DEGs) in murine macrophages, but deficiency of IκB kinase ß (IKKß), a key regulator for NF-κB activation, abrogated Lenti-Spike-elicited macrophage inflammatory responses. Conclusions: We demonstrated that the engineered Lipo-hACE2 acts as a molecular decoy to neutralize SARS-CoV-2 or Spike protein-induced inflammation in both murine and human macrophages, and activation of the canonical IKKß/NF-κB signaling is essential for SARS-CoV-2-elicited macrophage inflammatory responses.

Mitoquinone mesylate targets SARS-CoV-2 infection in preclinical models.

To date, there is no effective oral antiviral against SARS-CoV-2 that is also anti-inflammatory. Herein, we show that the mitochondrial antioxidant mitoquinone/mitoquinol mesylate (Mito-MES), a dietary supplement, has potent antiviral activity against SARS-CoV-2 and its variants of concern in vitro and in vivo . Mito-MES had nanomolar in vitro antiviral potency against the Beta and Delta SARS-CoV-2 variants as well as the murine hepatitis virus (MHV-A59). Mito-MES given in SARS-CoV-2 infected K18-hACE2 mice through oral gavage reduced viral titer by nearly 4 log units relative to the vehicle group. We found in vitro that the antiviral effect of Mito-MES is attributable to its hydrophobic dTPP+ moiety and its combined effects scavenging reactive oxygen species (ROS), activating Nrf2 and increasing the host defense proteins TOM70 and MX1. Mito-MES was efficacious reducing increase in cleaved caspase-3 and inflammation induced by SARS-CoV2 infection both in lung epithelial cells and a transgenic mouse model of COVID-19. Mito-MES reduced production of IL-6 by SARS-CoV-2 infected epithelial cells through its antioxidant properties (Nrf2 agonist, coenzyme Q10 moiety) and the dTPP moiety. Given established safety of Mito-MES in humans, our results suggest that Mito-MES may represent a rapidly applicable therapeutic strategy that can be added in the therapeutic arsenal against COVID-19. Its potential long-term use by humans as diet supplement could help control the SARS-CoV-2 pandemic, especially in the setting of rapidly emerging SARS-CoV-2 variants that may compromise vaccine efficacy. One-Sentence Summary: Mitoquinone/mitoquinol mesylate has potent antiviral and anti-inflammatory activity in preclinical models of SARS-CoV-2 infection.

Development of targeted nanoparticles loaded with antiviral drugs for SARS-CoV-2 inhibition.

Recently, a novel coronavirus, known as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has raised global concerns, being the etiological agent of the current pandemic infectious coronavirus disease 2019 (COVID-19). Specific prophylactic treatments like vaccines, have been authorized for use by regulatory bodies in multiple countries, however there is an urgent need to identify new, safe, and targeted therapeutics as post-exposure therapy for COVID-19. Among a plethora of potential pharmacological targets, the angiotensin-converting enzyme 2 (ACE2) membrane receptor, which plays a crucial role in viral entry, is representing an attractive intervention opportunity for SARS-CoV-2 antiviral discovery process. In this scenario, we envisioned that binding to ACE2 by multivalent attachment of ligands to nanocarriers incorporating antiviral therapeutics, it would increase receptor avidity and impart specificity to these nanovectors for host cells, particularly in the pulmonary tract, which is the primary entry route for SARS-CoV-2. Herein, we report the design and development of novel polymeric nanoparticles (NP), densely grafted with various ligands to selectively bind to ACE2, as innovative nanovectors for targeted drug delivery. We first evaluated the impact of these biocompatible targeted NP (TNP) on ligand binding toward ACE2 and measured their competition ability vs a model of spike protein (Lipo-S1). Next, we tested the effectiveness of the most performing nanoprotopype, TNP-1, loaded with a model anti-SARS-CoV-2 drug such as remdesivir (RDV), on antiviral activity against SARS-CoV-2 infected Vero E6 cells. The RDV-TNP-1 exhibited a significantly improved antiviral effect compared to RDV at the same concentration. Interestingly, unloaded TNP (TNP-1E) also exhibited a basal antiviral activity, potentially due to a direct competitive mechanism with viral particles for the ACE2 binding site. We also measured the anti-exopeptidase activity of TNP-1E against ACE2 protein. Collectively, these insights warrant in-depth preclinical development for our nanoprototypes, for example as potential inhalable drug carriers, with the perspective of a clinical translation.

Computational simulations of the 4D micro-circulatory network in zebrafish tail amputation and regeneration.

Wall shear stress (WSS) contributes to the mechanotransduction underlying microvascular development and regeneration. Using computational fluid dynamics, we elucidated the interplay between WSS and vascular remodelling in a zebrafish model of tail amputation and regeneration. The transgenic Tg (fli1:eGFP; Gata1:ds-red) zebrafish line was used to track the three-dimensional fluorescently labelled vascular endothelium for post-image segmentation and reconstruction of the fluid domain. Particle image velocimetry was used to validate the blood flow. Following amputation to the dorsal aorta and posterior cardinal vein (PCV), vasoconstriction developed in the dorsal longitudinal anastomotic vessel (DLAV) along with increased WSS in the proximal segmental vessels (SVs) from amputation. Angiogenesis ensued at the tips of the amputated DLAV and PCV where WSS was minimal. At 2 days post amputation (dpa), vasodilation occurred in a pair of SVs proximal to amputation, followed by increased blood flow and WSS; however, in the SVs distal to amputation, WSS normalized to the baseline. At 3 dpa, the blood flow increased in the arterial SV proximal to amputation and through anastomosis with DLAV formed a loop with PCV. Thus, our in silico modelling revealed the interplay between WSS and microvascular adaptation to changes in WSS and blood flow to restore microcirculation following tail amputation.

The role of oxidative stress in the pathogenesis of infections with coronaviruses.

Coronaviruses can cause serious respiratory tract infections and may also impact other end organs such as the central nervous system, the lung and the heart. The coronavirus disease 2019 (COVID-19) has had a devastating impact on humanity. Understanding the mechanisms that contribute to the pathogenesis of coronavirus infections, will set the foundation for development of new treatments to attenuate the impact of infections with coronaviruses on host cells and tissues. During infection of host cells, coronaviruses trigger an imbalance between increased production of reactive oxygen species (ROS) and reduced antioxidant host responses that leads to increased redox stress. Subsequently, increased redox stress contributes to reduced antiviral host responses and increased virus-induced inflammation and apoptosis that ultimately drive cell and tissue damage and end organ disease. However, there is limited understanding how different coronaviruses including SARS-CoV-2, manipulate cellular machinery that drives redox responses. This review aims to elucidate the redox mechanisms involved in the replication of coronaviruses and associated inflammation, apoptotic pathways, autoimmunity, vascular dysfunction and tissue damage that collectively contribute to multiorgan damage.

Rapid Detection and Inhibition of SARS-CoV-2-Spike Mutation-Mediated Microthrombosis.

Activation of endothelial cells following severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is thought to be the primary driver for the increasingly recognized thrombotic complications in coronavirus disease 2019 patients, potentially due to the SARS-CoV-2 Spike protein binding to the human angiotensin-converting enzyme 2 (hACE2). Vaccination therapies use the same Spike sequence or protein to boost host immune response as a protective mechanism against SARS-CoV-2 infection. As a result, cases of thrombotic events are reported following vaccination. Although vaccines are generally considered safe, due to genetic heterogeneity, age, or the presence of comorbidities in the population worldwide, the prediction of severe adverse outcome in patients remains a challenge. To elucidate Spike proteins underlying patient-specific-vascular thrombosis, the human microcirculation environment is recapitulated using a novel microfluidic platform coated with human endothelial cells and exposed to patient specific whole blood. Here, the blood coagulation effect is tested after exposure to Spike protein in nanoparticles and Spike variant D614G in viral vectors and the results are corroborated using live SARS-CoV-2. Of note, two potential strategies are also examined to reduce blood clot formation, by using nanoliposome-hACE2 and anti-Interleukin (IL) 6 antibodies.

Endothelial mechanotransduction in cardiovascular development and regeneration: emerging approaches and animal models.

Living cells are exposed to multiple mechanical stimuli from the extracellular matrix or from surrounding cells. Mechanoreceptors are molecules that display status changes in response to mechanical stimulation, transforming physical cues into biological responses to help the cells adapt to dynamic changes of the microenvironment. Mechanical stimuli are responsible for shaping the tridimensional development and patterning of the organs in early embryonic stages. The development of the heart is one of the first morphogenetic events that occur in embryos. As the circulation is established, the vascular system is exposed to constant shear stress, which is the force created by the movement of blood. Both spatial and temporal variations in shear stress differentially modulate critical steps in heart development, such as trabeculation and compaction of the ventricular wall and the formation of the heart valves. Zebrafish embryos are small, transparent, have a short developmental period and allow for real-time visualization of a variety of fluorescently labeled proteins to recapitulate developmental dynamics. In this review, we will highlight the application of zebrafish models as a genetically tractable model for investigating cardiovascular development and regeneration. We will introduce our approaches to manipulate mechanical forces during critical stages of zebrafish heart development and in a model of vascular regeneration, as well as advances in imaging technologies to capture these processes at high resolution. Finally, we will discuss the role of molecules of the Plexin family and Piezo cation channels as major mechanosensors recently implicated in cardiac morphogenesis.

A hybrid of light-field and light-sheet imaging to study myocardial function and intracardiac blood flow during zebrafish development.

Biomechanical forces intimately contribute to cardiac morphogenesis. However, volumetric imaging to investigate the cardiac mechanics with high temporal and spatial resolution remains an imaging challenge. We hereby integrated light-field microscopy (LFM) with light-sheet fluorescence microscopy (LSFM), coupled with a retrospective gating method, to simultaneously access myocardial contraction and intracardiac blood flow at 200 volumes per second. While LSFM allows for the reconstruction of the myocardial function, LFM enables instantaneous acquisition of the intracardiac blood cells traversing across the valves. We further adopted deformable image registration to quantify the ventricular wall displacement and particle tracking velocimetry to monitor intracardiac blood flow. The integration of LFM and LSFM enabled the time-dependent tracking of the individual blood cells and the differential rates of segmental wall displacement during a cardiac cycle. Taken together, we demonstrated a hybrid system, coupled with our image analysis pipeline, to simultaneously capture the myocardial wall motion with intracardiac blood flow during cardiac development.

Flow-Mediated Susceptibility and Molecular Response of Cerebral Endothelia to SARS-CoV-2 Infection.

BACKGROUND AND PURPOSE: Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection is associated with an increased rate of cerebrovascular events including ischemic stroke and intracerebral hemorrhage. The mechanisms underlying cerebral endothelial susceptibility and response to SARS-CoV-2 are unknown yet critical to understanding the association of SARS-CoV-2 infection with cerebrovascular events. METHODS: Endothelial cells were isolated from human brain and analyzed by RNA sequencing. Human umbilical vein and human brain microvascular cells were used in both monolayer culture and endothelialized within a 3-dimensional printed vascular model of the middle cerebral artery. Gene expression levels were measured by quantitative polymerase chain reaction and direct RNA hybridization. Recombinant SARS-CoV-2 S protein and S protein-containing liposomes were used to measure endothelial binding by immunocytochemistry. RESULTS: ACE2 (angiotensin-converting enzyme-2) mRNA levels were low in human brain and monolayer endothelial cell culture. Within the 3-dimensional printed vascular model, ACE2 gene expression and protein levels were progressively increased by vessel size and flow rates. SARS-CoV-2 S protein-containing liposomes were detected in human umbilical vein endothelial cells and human brain microvascular endothelial cells in 3-dimensional middle cerebral artery models but not in monolayer culture consistent with flow dependency of ACE2 expression. Binding of SARS-CoV-2 S protein triggered 83 unique genes in human brain endothelial cells including upregulation of complement component C3. CONCLUSIONS: Brain endothelial cells are susceptible to direct SARS-CoV-2 infection through flow-dependent expression of ACE2. Viral S protein binding triggers a unique gene expression profile in brain endothelia that may explain the association of SARS-CoV-2 infection with cerebrovascular events.