Development of a Machine Learning-Enabled Virtual Reality Tool for Preoperative Planning of Functional Endoscopic Sinus Surgery.

Objectives Virtual reality (VR) is an increasingly valuable teaching tool, but current simulators are not typically clinically scalable due to their reliance on inefficient manual segmentation. The objective of this project was to leverage a high-throughput and accurate machine learning method to automate data preparation for a patient-specific VR simulator used to explore preoperative sinus anatomy. Methods An endoscopic VR simulator was designed in Unity to enable interactive exploration of sinus anatomy. The Saak transform, a data-efficient machine learning method, was adapted to accurately segment sinus computed tomography (CT) scans using minimal training data, and the resulting data were reconstructed into three-dimensional (3D) patient-specific models that could be explored in the simulator. Results Using minimal training data, the Saak transform-based machine learning method offers accurate soft-tissue segmentation. When explored with an endoscope in the VR simulator, the anatomical models generated by the algorithm accurately capture key sinus structures and showcase patient-specific variability in anatomy. Conclusion By offering an automatic means of preparing VR models from a patient's raw CT scans, this pipeline takes a key step toward clinical scalability. In addition to preoperative planning, this system also enables virtual endoscopy-a tool that is particularly useful in the COVID-19 era. As VR technology inevitably continues to develop, such a foundation will help ensure that future innovations remain clinically accessible.

Non-Invasive Detection of Early-Stage Fatty Liver Disease via an On-Skin Impedance Sensor and Attention-Based Deep Learning.

Early-stage nonalcoholic fatty liver disease (NAFLD) is a silent condition, with most cases going undiagnosed, potentially progressing to liver cirrhosis and cancer. A non-invasive and cost-effective detection method for early-stage NAFLD detection is a public health priority but challenging. In this study, an adhesive, soft on-skin sensor with low electrode-skin contact impedance for early-stage NAFLD detection is fabricated. A method is developed to synthesize platinum nanoparticles and reduced graphene quantum dots onto the on-skin sensor to reduce electrode-skin contact impedance by increasing double-layer capacitance, thereby enhancing detection accuracy. Furthermore, an attention-based deep learning algorithm is introduced to differentiate impedance signals associated with early-stage NAFLD in high-fat-diet-fed low-density lipoprotein receptor knockout (Ldlr-/-) mice compared to healthy controls. The integration of an adhesive, soft on-skin sensor with low electrode-skin contact impedance and the attention-based deep learning algorithm significantly enhances the detection accuracy for early-stage NAFLD, achieving a rate above 97.5% with an area under the receiver operating characteristic curve (AUC) of 1.0. The findings present a non-invasive approach for early-stage NAFLD detection and display a strategy for improved early detection through on-skin electronics and deep learning.

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.

Harnessing Biomaterials to Amplify Immunity in Aged Mice through T Memory Stem Cells.

The durability of a protective immune response generated by a vaccine depends on its ability to induce long-term T cell immunity, which tends to decline in aging populations. The longest protection appears to arise from T memory stem cells (TMSCs) that confer high expandability and effector functions when challenged. Here we engineered artificial antigen presenting cells (aAPC) with optimized size, stiffness and activation signals to induce human and mouse CD8+ TMSCs in vitro. This platform was optimized as a vaccine booster of TMSCs (Vax-T) with prolonged release of small-molecule blockade of the glycogen synthase kinase-3ß together with target antigens. By using SARS-CoV-2 antigen as a model, we show that a single injection of Vax-T induces durable antigen-specific CD8+ TMSCs in young and aged mice, and generates humoral responses at a level stronger than or similar to soluble vaccines. This Vax-T approach can boost long-term immunity to fight infectious diseases, cancer, and other diseases.

A self-assembled implantable microtubular pacemaker for wireless cardiac electrotherapy.

The current cardiac pacemakers are battery dependent, and the pacing leads are prone to introduce valve damage and infection, plus a complete pacemaker retrieval is needed for battery replacement. Despite the reported wireless bioelectronics to pace the epicardium, open-chest surgery (thoracotomy) is required to implant the device, and the procedure is invasive, requiring prolonged wound healing and health care burden. We hereby demonstrate a fully biocompatible wireless microelectronics with a self-assembled design that can be rolled into a lightweight microtubular pacemaker for intravascular implantation and pacing. The radio frequency was used to transfer energy to the microtubular pacemaker for electrical stimulation. We show that this pacemaker provides effective pacing to restore cardiac contraction from a nonbeating heart and have the capacity to perform overdrive pacing to augment blood circulation in an anesthetized pig model. Thus, this microtubular pacemaker paves the way for the minimally invasive implantation of leadless and battery-free microelectronics.

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.

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.

HIV Protein Tat Induces Macrophage Dysfunction and Atherosclerosis Development in Low-Density Lipoprotein Receptor-Deficient Mice.

PURPOSE: HIV infection is consistently associated with an increased risk of atherosclerotic cardiovascular disease, but the underlying mechanisms remain elusive. HIV protein Tat, a transcriptional activator of HIV, has been shown to activate NF-κB signaling and promote inflammation in vitro. However, the atherogenic effects of HIV Tat have not been investigated in vivo. Macrophages are one of the major cell types involved in the initiation and progression of atherosclerosis. We and others have previously revealed the important role of IκB kinase ß (IKKß), a central inflammatory coordinator through activating NF-κB, in the regulation of macrophage functions and atherogenesis. This study investigated the impact of HIV Tat exposure on macrophage functions and atherogenesis. METHODS: To investigate the effects of Tat on macrophage IKKß activation and atherosclerosis development in vivo, myeloid-specific IKKß-deficient LDLR-deficient (IKKßΔMyeLDLR-/-) mice and their control littermates (IKKßF/FLDLR-/-) were exposed to recombinant HIV protein Tat. RESULTS: Exposure to Tat significantly increased atherosclerotic lesion size and plaque vulnerability in IKKßF/FLDLR-/- but not IKKßΔMyeLDLR-/- mice. Deficiency of myeloid IKKß attenuated Tat-elicited macrophage inflammatory responses and atherosclerotic lesional inflammation in IKKßΔMyeLDLR-/- mice. Further, RNAseq analysis demonstrated that HIV protein Tat affects the expression of many atherosclerosis-related genes in vitro in an IKKß-dependent manner. CONCLUSIONS: Our findings reveal atherogenic effects of HIV protein Tat in vivo and demonstrate a pivotal role of myeloid IKKß in Tat-driven atherogenesis.

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.

Electrical impedance tomography for non-invasive identification of fatty liver infiltrate in overweight individuals.

Non-alcoholic fatty liver disease (NAFLD) is one of the most common causes of cardiometabolic diseases in overweight individuals. While liver biopsy is the current gold standard to diagnose NAFLD and magnetic resonance imaging (MRI) is a non-invasive alternative still under clinical trials, the former is invasive and the latter costly. We demonstrate electrical impedance tomography (EIT) as a portable method for detecting fatty infiltrate. We enrolled 19 overweight subjects to undergo liver MRI scans, followed by EIT measurements. The MRI images provided the a priori knowledge of the liver boundary conditions for EIT reconstruction, and the multi-echo MRI data quantified liver proton-density fat fraction (PDFF%) to validate fat infiltrate. Using the EIT electrode belts, we circumferentially injected pairwise current to the upper abdomen, followed by acquiring the resulting surface-voltage to reconstruct the liver conductivity. Pearson's correlation analyses compared EIT conductivity or MRI PDFF with body mass index, age, waist circumference, height, and weight variables. We reveal that the correlation between liver EIT conductivity or MRI PDFF with demographics is statistically insignificant, whereas liver EIT conductivity is inversely correlated with MRI PDFF (R = -0.69, p = 0.003, n = 16). As a pilot study, EIT conductivity provides a portable method for operator-independent and cost-effective detection of hepatic steatosis.

Retinoid X receptor alpha is a spatiotemporally predominant therapeutic target for anthracycline-induced cardiotoxicity.

To uncover the genetic basis of anthracycline-induced cardiotoxicity (AIC), we recently established a genetic suppressor screening strategy in zebrafish. Here, we report the molecular and cellular nature of GBT0419, a salutary modifier mutant that affects retinoid x receptor alpha a (rxraa). We showed that endothelial, but not myocardial or epicardial, RXRA activation confers AIC protection. We then identified isotretinoin and bexarotene, two FDA-approved RXRA agonists, which exert cardioprotective effects. The therapeutic effects of these drugs only occur when administered during early, but not late, phase of AIC or as pretreatment. Mechanistically, these spatially- and temporally-predominant benefits of RXRA activation can be ascribed to repair of damaged endothelial cell-barrier via regulating tight-junction protein Zonula occludens-1. Together, our study provides the first in vivo genetic evidence supporting RXRA as the therapeutic target for AIC, and uncovers a previously unrecognized spatiotemporally-predominant mechanism that shall inform future translational efforts.

Displacement analysis of myocardial mechanical deformation (DIAMOND) reveals segmental susceptibility to doxorubicin-induced injury and regeneration.

Zebrafish are increasingly utilized to model cardiomyopathies and regeneration. Current methods evaluating cardiac function have known limitations, fail to reliably detect focal mechanics, and are not readily feasible in zebrafish. We developed a semiautomated, open-source method - displacement analysis of myocardial mechanical deformation (DIAMOND) - for quantitative assessment of 4D segmental cardiac function. We imaged transgenic embryonic zebrafish in vivo using a light-sheet fluorescence microscopy system with 4D cardiac motion synchronization. Our method permits the derivation of a transformation matrix to quantify the time-dependent 3D displacement of segmental myocardial mass centroids. Through treatment with doxorubicin, and by chemically and genetically manipulating the myocardial injury-activated Notch signaling pathway, we used DIAMOND to demonstrate that basal ventricular segments adjacent to the atrioventricular canal display the highest 3D displacement and are also the most susceptible to doxorubicin-induced injury. Thus, DIAMOND provides biomechanical insights into in vivo segmental cardiac function scalable to high-throughput research applications.

Spatial and temporal variations in hemodynamic forces initiate cardiac trabeculation.

Hemodynamic shear force has been implicated as modulating Notch signaling-mediated cardiac trabeculation. Whether the spatiotemporal variations in wall shear stress (WSS) coordinate the initiation of trabeculation to influence ventricular contractile function remains unknown. Using light-sheet fluorescent microscopy, we reconstructed the 4D moving domain and applied computational fluid dynamics to quantify 4D WSS along the trabecular ridges and in the groves. In WT zebrafish, pulsatile shear stress developed along the trabecular ridges, with prominent endocardial Notch activity at 3 days after fertilization (dpf), and oscillatory shear stress developed in the trabecular grooves, with epicardial Notch activity at 4 dpf. Genetic manipulations were performed to reduce hematopoiesis and inhibit atrial contraction to lower WSS in synchrony with attenuation of oscillatory shear index (OSI) during ventricular development. γ-Secretase inhibitor of Notch intracellular domain (NICD) abrogated endocardial and epicardial Notch activity. Rescue with NICD mRNA restored Notch activity sequentially from the endocardium to trabecular grooves, which was corroborated by observed Notch-mediated cardiomyocyte proliferations on WT zebrafish trabeculae. We also demonstrated in vitro that a high OSI value correlated with upregulated endothelial Notch-related mRNA expression. In silico computation of energy dissipation further supports the role of trabeculation to preserve ventricular structure and contractile function. Thus, spatiotemporal variations in WSS coordinate trabecular organization for ventricular contractile function.

Ultrafine Particle Exposure Reveals the Importance of FOXO1/Notch Activation Complex for Vascular Regeneration.

AIMS: Redox active ultrafine particles (UFP, d < 0.2 µm) promote vascular oxidative stress and atherosclerosis. Notch signaling is intimately involved in vascular homeostasis, in which forkhead box O1 (FOXO1) acts as a co-activator of the Notch activation complex. We elucidated the importance of FOXO1/Notch transcriptional activation complex to restore vascular regeneration after UFP exposure. RESULTS: In a zebrafish model of tail injury and repair, transgenic Tg(fli1:GFP) embryos developed vascular regeneration at 3 days post amputation (dpa), whereas UFP exposure impaired regeneration (p < 0.05, n = 20 for control, n = 28 for UFP). UFP dose dependently reduced Notch reporter activity and Notch signaling-related genes (Dll4, JAG1, JAG2, Notch1b, Hey2, Hes1; p < 0.05, n = 3). In the transgenic Tg(tp1:GFP; flk1:mCherry) embryos, UFP attenuated endothelial Notch activity at the amputation site (p < 0.05 vs. wild type [WT], n = 20). A disintegrin and metalloproteinase domain-containing protein 10 (ADAM10) inhibitor or dominant negative (DN)-Notch1b messenger RNA (mRNA) disrupted the vascular network, whereas notch intracellular cytoplasmic domain (NICD) mRNA restored the vascular network (p < 0.05 vs. WT, n = 20). UFP reduced FOXO1 expression, but not Master-mind like 1 (MAML1) or NICD (p < 0.05, n = 3). Immunoprecipitation and immunofluorescence demonstrated that UFP attenuated FOXO1-mediated NICD pull-down and FOXO1/NICD co-localization, respectively (p < 0.05, n = 3). Although FOXO1 morpholino oligonucleotides (MOs) attenuated Notch activity, FOXO1 mRNA reversed UFP-mediated reduction in Notch activity to restore vascular regeneration and blood flow (p < 0.05 vs. WT, n = 5). Innovation and Conclusion: Our findings indicate the importance of the FOXO1/Notch activation complex to restore vascular regeneration after exposure to the redox active UFP. Antioxid. Redox Signal. 28, 1209-1223.

A method to quantify mechanobiologic forces during zebrafish cardiac development using 4-D light sheet imaging and computational modeling.

Blood flow and mechanical forces in the ventricle are implicated in cardiac development and trabeculation. However, the mechanisms of mechanotransduction remain elusive. This is due in part to the challenges associated with accurately quantifying mechanical forces in the developing heart. We present a novel computational framework to simulate cardiac hemodynamics in developing zebrafish embryos by coupling 4-D light sheet imaging with a stabilized finite element flow solver, and extract time-dependent mechanical stimuli data. We employ deformable image registration methods to segment the motion of the ventricle from high resolution 4-D light sheet image data. This results in a robust and efficient workflow, as segmentation need only be performed at one cardiac phase, while wall position in the other cardiac phases is found by image registration. Ventricular hemodynamics are then quantified by numerically solving the Navier-Stokes equations in the moving wall domain with our validated flow solver. We demonstrate the applicability of the workflow in wild type zebrafish and three treated fish types that disrupt trabeculation: (a) chemical treatment using AG1478, an ErbB2 signaling inhibitor that inhibits proliferation and differentiation of cardiac trabeculation; (b) injection of gata1a morpholino oligomer (gata1aMO) suppressing hematopoiesis and resulting in attenuated trabeculation; (c) weak-atriumm58 mutant (wea) with inhibited atrial contraction leading to a highly undeveloped ventricle and poor cardiac function. Our simulations reveal elevated wall shear stress (WSS) in wild type and AG1478 compared to gata1aMO and wea. High oscillatory shear index (OSI) in the grooves between trabeculae, compared to lower values on the ridges, in the wild type suggest oscillatory forces as a possible regulatory mechanism of cardiac trabeculation development. The framework has broad applicability for future cardiac developmental studies focused on quantitatively investigating the role of hemodynamic forces and mechanotransduction during morphogenesis.

Ambient Ultrafine Particle Ingestion Alters Gut Microbiota in Association with Increased Atherogenic Lipid Metabolites.

Ambient particulate matter (PM) exposure is associated with atherosclerosis and inflammatory bowel disease. Ultrafine particles (UFP, dp < 0.1-0.2 µm) are redox active components of PM. We hypothesized that orally ingested UFP promoted atherogenic lipid metabolites in both the intestine and plasma via altered gut microbiota composition. Low density lipoprotein receptor-null (Ldlr-/-) mice on a high-fat diet were orally administered with vehicle control or UFP (40 µg/mouse/day) for 3 days a week. After 10 weeks, UFP ingested mice developed macrophage and neutrophil infiltration in the intestinal villi, accompanied by elevated cholesterol but reduced coprostanol levels in the cecum, as well as elevated atherogenic lysophosphatidylcholine (LPC 18:1) and lysophosphatidic acids (LPAs) in the intestine and plasma. At the phylum level, Principle Component Analysis revealed significant segregation of microbiota compositions which was validated by Beta diversity analysis. UFP-exposed mice developed increased abundance in Verrocomicrobia but decreased Actinobacteria, Cyanobacteria, and Firmicutes as well as a reduced diversity in microbiome. Spearman's analysis negatively correlated Actinobacteria with cecal cholesterol, intestinal and plasma LPC18:1, and Firmicutes and Cyanobacteria with plasma LPC 18:1. Thus, ultrafine particles ingestion alters gut microbiota composition, accompanied by increased atherogenic lipid metabolites. These findings implicate the gut-vascular axis in a atherosclerosis model.

Disturbed Flow Induces Autophagy, but Impairs Autophagic Flux to Perturb Mitochondrial Homeostasis.

AIM: Temporal and spatial variations in shear stress are intimately linked with vascular metabolic effects. Autophagy is tightly regulated in intracellular bulk degradation/recycling system for maintaining cellular homeostasis. We postulated that disturbed flow modulates autophagy with an implication in mitochondrial superoxide (mtO2(•-)) production. RESULTS: In the disturbed flow or oscillatory shear stress (OSS)-exposed aortic arch, we observed prominent staining of p62, a reverse marker of autophagic flux, whereas in the pulsatile shear stress (PSS)-exposed descending aorta, p62 was attenuated. OSS significantly increased (i) microtubule-associated protein light chain 3 (LC3) II to I ratios in human aortic endothelial cells, (ii) autophagosome formation as quantified by green fluorescent protein (GFP)-LC3 dots per cell, and (iii) p62 protein levels, whereas manganese superoxide dismutase (MnSOD) overexpression by recombinant adenovirus, N-acetyl cysteine treatment, or c-Jun N-terminal kinase (JNK) inhibition reduced OSS-mediated LC3-II/LC3-I ratios and mitochondrial DNA damage. Introducing bafilomycin to Earle's balanced salt solution or to OSS condition incrementally increased both LC3-II/LC3-I ratios and p62 levels, implicating impaired autophagic flux. In the OSS-exposed aortic arch, both anti-phospho-JNK and anti-8-hydroxy-2'-deoxyguanosine (8-OHdG) staining for DNA damage were prominent, whereas in the PSS-exposed descending aorta, the staining was nearly absent. Knockdown of ATG5 with siRNA increased OSS-mediated mtO2(•-), whereas starvation or rapamycin-induced autophagy reduced OSS-mediated mtO2(•-), mitochondrial respiration, and complex II activity. INNOVATION: Disturbed flow-mediated oxidative stress and JNK activation induce autophagy. CONCLUSION: OSS impairs autophagic flux to interfere with mitochondrial homeostasis. Antioxid. Redox Signal. 23, 1207-1219.

Electrical and Mechanical Strategies to Enable Cardiac Repair and Regeneration.

Inadequate replacement of lost ventricular myocardium from myocardial infarction leads to heart failure. Investigating the regenerative capacity of mammalian hearts represents an emerging direction for tissue engineering and cell-based therapy. Recent advances in stem cells hold promise to restore cardiac functions. However, embryonic or induced pluripotent stem cell-derived cardiomyocytes lack functional phenotypes of the native myocardium, and transplanted tissues are not fully integrated for synchronized electrical and mechanical coupling with the host. In this context, this review highlights the mechanical and electrical strategies to promote cardiomyocyte maturation and integration, and to assess the functional phenotypes of regenerating myocardium. Simultaneous microelectrocardiogram and high-frequency ultrasound techniques will also be introduced to assess electrical and mechanical coupling for small animal models of heart regeneration.

Shear stress-activated Wnt-angiopoietin-2 signaling recapitulates vascular repair in zebrafish embryos.

OBJECTIVE: Fluid shear stress intimately regulates vasculogenesis and endothelial homeostasis. The canonical Wnt/ß-catenin signaling pathways play an important role in differentiation and proliferation. In this study, we investigated whether shear stress activated angiopoietin-2 (Ang-2) via the canonical Wnt signaling pathway with an implication in vascular endothelial repair. APPROACH AND RESULTS: Oscillatory shear stress upregulated both TOPflash Wnt reporter activities and the expression of Ang-2 mRNA and protein in human aortic endothelial cells accompanied by an increase in nuclear ß-catenin intensity. Oscillatory shear stress-induced Ang-2 and Axin-2 mRNA expression was downregulated in the presence of a Wnt inhibitor, IWR-1, but was upregulated in the presence of a Wnt agonist, LiCl. Ang-2 expression was further downregulated in response to a Wnt signaling inhibitor, DKK-1, but was upregulated by Wnt agonist Wnt3a. Both DKK-1 and Ang-2 siRNA inhibited endothelial cell migration and tube formation, which were rescued by human recombinant Ang-2. Both Ang-2 and Axin-2 mRNA downregulation was recapitulated in the heat-shock-inducible transgenic Tg(hsp70l:dkk1-GFP) zebrafish embryos at 72 hours post fertilization. Ang-2 morpholino injection of Tg (kdrl:GFP) fish impaired subintestinal vessel formation at 72 hours post fertilization, which was rescued by zebrafish Ang-2 mRNA coinjection. Inhibition of Wnt signaling with IWR-1 also downregulated Ang-2 and Axin-2 expression and impaired vascular repair after tail amputation, which was rescued by zebrafish Ang-2 mRNA injection. CONCLUSIONS: Shear stress activated Ang-2 via canonical Wnt signaling in vascular endothelial cells, and Wnt-Ang-2 signaling is recapitulated in zebrafish embryos with a translational implication in vascular development and repair.

Ambient ultrafine particles reduce endothelial nitric oxide production via S-glutathionylation of eNOS.

Exposure to airborne particulate pollutants is intimately linked to vascular oxidative stress and inflammatory responses with clinical relevance to atherosclerosis. Particulate matter (PM) has been reported to induce endothelial dysfunction and atherosclerosis. Here, we tested whether ambient ultrafine particles (UFP, diameter <200 nm) modulate eNOS activity in terms of nitric oxide (NO) production via protein S-glutathionylation. Treatment of human aortic endothelial cells (HAEC) with UFP significantly reduced NO production. UFP-mediated reduction in NO production was restored in the presence of JNK inhibitor (SP600125), NADPH oxidase inhibitor (Apocynin), anti-oxidant (N-acetyl cysteine), and superoxide dismutase mimetics (Tempol and MnTMPyP). UFP exposure increased the GSSG/GSH ratio and eNOS S-glutathionylation, whereas over-expression of Glutaredoxin-1 (to inhibit S-glutathionylation) restored UFP-mediated reduction in NO production by nearly 80%. Thus, our findings suggest that eNOS S-glutathionylation is a potential mechanism underlying ambient UFP-induced reduction of NO production.