Computer-based virtual laboratory simulations: LabHEART cardiac physiology practical.

Practical demonstration of cardiomyocyte function requires substantial preparation, a source of freshly isolated animal hearts, and specialized equipment. Even where such resources are available, it is not conducive for demonstration to any more than a few students at a time. These approaches are also not consistent with the 3R principle (replacement, reduction, and refinement) of ethical use of animals. We present an implementation of the LabHEART software, developed by Donald Bers and Jose Puglisi, for medical students. Prior to the activity, students had lectures covering the physiological and pharmacological aspects of cardiac excitation-contraction (EC) coupling. We used this problem-based activity to help students consolidate their knowledge and to allow a hands-on approach to explore the key features of EC coupling. Students simulate and measure action potentials, intracellular calcium changes, and cardiomyocyte contraction. They also apply drugs that target ion channels (e.g., nifedipine or tetrodotoxin) or sympathetic input (using isoproterenol) and explore changes to EC coupling. Furthermore, by modifying the biophysical parameters of key ion channels involved in the electrical activity of the heart, students also explore the effect of channelopathies such as long QT syndromes. We describe approaches to implement this activity in a flipped classroom format, with recorded lecture materials provided ahead of the practical to facilitate active learning. We also describe our experiences implementing this activity online. The content and difficulty of the activity can be altered to suit individual courses and is also amenable to promote peer-driven learning.

Endothelial cell-specific redox gene modulation inhibits angiogenesis but promotes B16F0 tumor growth in mice.

Glutaredoxin-1 (Glrx) is a small cytosolic enzyme that removes S-glutathionylation, glutathione adducts of protein cysteine residues, thus modulating redox signaling and gene transcription. Although Glrx up-regulation prevented endothelial cell (EC) migration and global Glrx transgenic mice had impaired ischemic vascularization, the effects of cell-specific Glrx overexpression remained unknown. Here, we examined the role of EC-specific Glrx up-regulation in distinct models of angiogenesis; namely, hind limb ischemia and tumor angiogenesis. EC-specific Glrx transgenic (EC-Glrx TG) overexpression in mice significantly impaired EC migration in Matrigel implants and hind limb revascularization after femoral artery ligation. Additionally, ECs migrated less into subcutaneously implanted B16F0 melanoma tumors as assessed by decreased staining of EC markers. Despite reduced angiogenesis, EC-Glrx TG mice unexpectedly developed larger tumors compared with control mice. EC-Glrx TG mice showed higher levels of VEGF-A in the tumors, indicating hypoxia, which may stimulate tumor cells to form vascular channels without EC, referred to as vasculogenic mimicry. These data suggest that impaired ischemic vascularization does not necessarily associate with suppression of tumor growth, and that antiangiogenic therapies may be ineffective for melanoma tumors because of their ability to implement vasculogenic mimicry during hypoxia.-Yura, Y., Chong, B. S. H., Johnson, R. D., Watanabe, Y., Tsukahara, Y., Ferran, B., Murdoch, C. E., Behring, J. B., McComb, M. E., Costello, C. E., Janssen-Heininger, Y. M. W., Cohen, R. A., Bachschmid, M. M., Matsui, R. Endothelial cell-specific redox gene modulation inhibits angiogenesis but promotes B16F0 tumor growth in mice.

Glutathione adducts induced by ischemia and deletion of glutaredoxin-1 stabilize HIF-1α and improve limb revascularization.

Reactive oxygen species (ROS) are increased in ischemic tissues and necessary for revascularization; however, the mechanism remains unclear. Exposure of cysteine residues to ROS in the presence of glutathione (GSH) generates GSH-protein adducts that are specifically reversed by the cytosolic thioltransferase, glutaredoxin-1 (Glrx). Here, we show that a key angiogenic transcriptional factor hypoxia-inducible factor (HIF)-1α is stabilized by GSH adducts, and the genetic deletion of Glrx improves ischemic revascularization. In mouse muscle C2C12 cells, HIF-1α protein levels are increased by increasing GSH adducts with cell-permeable oxidized GSH (GSSG-ethyl ester) or 2-acetylamino-3-[4-(2-acetylamino-2-carboxyethylsulfanyl thiocarbonylamino) phenylthiocarbamoylsulfanyl] propionic acid (2-AAPA), an inhibitor of glutathione reductase. A biotin switch assay shows that GSSG-ester-induced HIF-1α contains reversibly modified thiols, and MS confirms GSH adducts on Cys(520) (mouse Cys(533)). In addition, an HIF-1α Cys(520) serine mutant is resistant to 2-AAPA-induced HIF-1α stabilization. Furthermore, Glrx overexpression prevents HIF-1α stabilization, whereas Glrx ablation by siRNA increases HIF-1α protein and expression of downstream angiogenic genes. Blood flow recovery after femoral artery ligation is significantly improved in Glrx KO mice, associated with increased levels of GSH-protein adducts, capillary density, vascular endothelial growth factor (VEGF)-A, and HIF-1α in the ischemic muscles. Therefore, Glrx ablation stabilizes HIF-1α by increasing GSH adducts on Cys(520) promoting in vivo HIF-1α stabilization, VEGF-A production, and revascularization in the ischemic muscles.

Distinct Regulatory Effects of Myeloid Cell and Endothelial Cell NAPDH Oxidase 2 on Blood Pressure.

BACKGROUND: Hypertension caused by increased renin-angiotensin system activation is associated with elevated reactive oxygen species production. Previous studies implicate NADPH oxidase (Nox) proteins as important reactive oxygen species sources during renin-angiotensin system activation, with different Nox isoforms being potentially involved. Among these, Nox2 is expressed in multiple cell types, including endothelial cells, fibroblasts, immune cells, and microglia. Blood pressure (BP) is regulated at the central nervous system, renal, and vascular levels, but the cell-specific role of Nox2 in BP regulation is unknown. METHODS: We generated a novel mouse model with a floxed Nox2 gene and used Tie2-Cre, LysM Cre, or Cdh5-CreERT2 driver lines to develop cell-specific models of Nox2 perturbation to investigate its role in BP regulation. RESULTS: Unexpectedly, Nox2 deletion in myeloid but not endothelial cells resulted in a significant reduction in basal BP. Both Tie2-CreNox2 knockout (KO) mice (in which Nox2 was deficient in both endothelial cells and myeloid cells) and LysM CreNox2KO mice (in which Nox2 was deficient in myeloid cells) had significantly lower BP than littermate controls, whereas basal BP was unaltered in Cdh5-CreERT2 Nox2KO mice (in which Nox2 is deficient only in endothelial cells). The lower BP was attributable to an increased NO bioavailability that dynamically dilated resistance vessels in vivo under basal conditions without a change in renal function. Myeloid-specific Nox2 deletion had no effect on angiotensin II-induced hypertension, which, however, was blunted in Tie2-CreNox2KO mice, along with preservation of endothelium-dependent relaxation during angiotensin II stimulation. CONCLUSIONS: We identify a hitherto unrecognized modulation of basal BP by myeloid cell Nox2, whereas endothelial cell Nox2 regulates angiotensin II-induced hypertension. These results identify distinct cell-specific roles for Nox2 in BP regulation.

Cell-specific effects of Nox2 on the acute and chronic response to myocardial infarction.

BACKGROUND: Increased reactive oxygen species (ROS) production is involved in the process of adverse cardiac remodeling and development of heart failure after myocardial infarction (MI). NADPH oxidase-2 (Nox2) is a major ROS source within the heart and its activity increases after MI. Furthermore, genetic deletion of Nox2 is protective against post-MI cardiac remodeling. Nox2 levels may increase both in cardiomyocytes and endothelial cells and recent studies indicate cell-specific effects of Nox2, but it is not known which of these cell types is important in post-MI remodeling. METHODS AND RESULTS: We have generated transgenic mouse models in which Nox2 expression is targeted either to cardiomyocytes (cardio-Nox2TG) or endothelial cells (endo-Nox2TG). We here studied the response of cardio-Nox2TG mice, endo-Nox2TG mice and matched wild-type littermates (WT) to MI induced by permanent left coronary artery ligation up to 4weeks. Initial infarct size assessed by magnetic resonance imaging (MRI) and cardiac dysfunction were similar among groups. Cardiomyocyte hypertrophy and interstitial fibrosis were augmented in cardio-Nox2TG compared to WT after MI and post-MI survival tended to be worse whereas endo-Nox2TG mice showed no significant difference compared to WT. CONCLUSIONS: These results indicate that cardiomyocyte rather than endothelial cell Nox2 may have the more important role in post-MI remodeling.

Endothelial Cell Redox Regulation of Ischemic Angiogenesis.

The endothelium produces and responds to reactive oxygen and nitrogen species (RONS), providing important redox regulation to the cardiovascular system in physiology and disease. In no other situation are RONS more critical than in the response to tissue ischemia. Here, tissue healing requires growth factor-mediated angiogenesis that is in part dependent on low levels of RONS, which paradoxically must overcome the damaging effects of high levels of RONS generated as a result of ischemia. Although the generation of endothelial cell RONS in hypoxia/reoxygenation is acknowledged, the mechanism for their role in angiogenesis is still poorly understood. During ischemia, the major low molecular weight thiol glutathione (GSH) reacts with RONS and protein cysteines, producing GSH-protein adducts. Recent data indicate that GSH adducts on certain proteins are essential to growth factor responses in endothelial cells. Genetic deletion of the enzyme glutaredoxin-1, which selectively removes GSH protein adducts, improves, whereas its overexpression impairs revascularization of the ischemic hindlimb of mice. Ischemia-induced GSH adducts on specific cysteine residues of several proteins, including p65 NF-kB and the sarcoplasmic reticulum calcium ATPase 2, evidently promote ischemic angiogenesis. Identifying the specific proteins in the redox response to ischemia has provided therapeutic opportunities to improve clinical outcomes of ischemia.

Glutaredoxin-1 up-regulation induces soluble vascular endothelial growth factor receptor 1, attenuating post-ischemia limb revascularization.

Glutaredoxin-1 (Glrx) is a cytosolic enzyme that regulates diverse cellular function by removal of GSH adducts from S-glutathionylated proteins including signaling molecules and transcription factors. Glrx is up-regulated during inflammation and diabetes, and Glrx overexpression inhibits VEGF-induced EC migration. The aim was to investigate the role of up-regulated Glrx in EC angiogenic capacities and in vivo revascularization in the setting of hind limb ischemia. Glrx-overexpressing EC from Glrx transgenic (TG) mice showed impaired migration and network formation and secreted higher levels of soluble VEGF receptor 1 (sFlt), an antagonizing factor to VEGF. After hind limb ischemia surgery Glrx TG mice demonstrated impaired blood flow recovery, associated with lower capillary density and poorer limb motor function compared with wild type littermates. There were also higher levels of anti-angiogenic sFlt expression in the muscle and plasma of Glrx TG mice after surgery. Noncanonical Wnt5a is known to induce sFlt. Wnt5a was highly expressed in ischemic muscles and EC from Glrx TG mice, and exogenous Wnt5a induced sFlt expression and inhibited network formation in human microvascular EC. Adenoviral Glrx-induced sFlt in EC was inhibited by a competitive Wnt5a inhibitor. Furthermore, Glrx overexpression removed GSH adducts on p65 in ischemic muscle and EC and enhanced NF-κB activity, which was responsible for Wnt5a-sFlt induction. Taken together, up-regulated Glrx induces sFlt in EC via NF-κB-dependent Wnt5a, resulting in attenuated revascularization in hind limb ischemia. The Glrx-induced sFlt explains part of the mechanism of redox-regulated VEGF signaling.

Regulation of neovascularization by S-glutathionylation via the Wnt5a/sFlt-1 pathway.

S-glutathionylation occurs when reactive oxygen or nitrogen species react with protein-cysteine thiols. Glutaredoxin-1 (Glrx) is a cytosolic enzyme which enzymatically catalyses the reduction in S-glutathionylation, conferring reversible signalling function to proteins with redox-sensitive thiols. Glrx can regulate vascular hypertrophy and inflammation by regulating the activity of nuclear factor κB (NF-κB) and actin polymerization. Vascular endothelial growth factor (VEGF)-induced endothelial cell (EC) migration is inhibited by Glrx overexpression. In mice overexpressing Glrx, blood flow recovery, exercise function and capillary density were significantly attenuated after hindlimb ischaemia (HLI). Wnt5a and soluble Fms-like tyrosine kinase-1 (sFlt-1) were enhanced in the ischaemic-limb muscle and plasma respectively from Glrx transgenic (TG) mice. A Wnt5a/sFlt-1 pathway had been described in myeloid cells controlling retinal blood vessel development. Interestingly, a Wnt5a/sFlt-1 pathway was found also to play a role in EC to inhibit network formation. S-glutathionylation of NF-κB components inhibits its activation. Up-regulated Glrx stimulated the Wnt5a/sFlt-1 pathway through enhancing NF-κB signalling. These studies show a novel role for Glrx in post-ischaemic neovascularization, which could define a potential target for therapy of impaired angiogenesis in pathological conditions including diabetes.

In vivo laser speckle contrast imaging of microvascular blood perfusion using a chip-on-tip camera.

Laser speckle contrast imaging (LSCI) is an important non-invasive capability for real-time imaging for tissue-perfusion assessment. Yet, the size and weight of current clinical standard LSCI instrumentation restricts usage to mainly peripheral skin perfusion. Miniaturization of LSCI could enable hand-held instrumentation to image internal organ/tissue to produce accurate speckle-perfusion maps. We characterized a 1mm2 chip-on-tip camera for LSCI of blood perfusion in vivo and with a flow model. A dedicated optical setup was built to compare chip-on-tip camera to a high specification reference camera (GS3) for LSCI. We compared LSCI performance using a calibration standard and a flow phantom. Subsequently the camera assessed placenta perfusion in a small animal model. Lastly, a human study was conducted on the perfusion in fingertips of 13-volunteers. We demonstrate that the chip-on-tip camera can perform wide-field, in vivo, LSCI of tissue perfusion with the ability to measure physiological blood flow changes comparable with a standard reference camera.

Protocol for a placenta-on-a-chip model using trophoblasts differentiated from human induced pluripotent stem cells.

Organ-on-a-chip technology incorporating stem cell techniques represents a promising strategy to improve modeling of human organs. Here, we present a protocol for generating a standardized 3D placenta-on-a-chip model using trophoblast derived from human induced pluripotent stem cells (hiPSCs). We describe steps for seeding hiPSCs into multi-chip OrganoPlate devices and on-chip differentiation into trophoblasts against an extracellular matrix under perfused conditions. We then detail procedures for conducting a functional barrier integrity assay, immunostaining, and collecting protein or RNA for molecular analysis. For complete details on the use and execution of this protocol, please refer to Lermant et al. (2023).1.

Limiting extracellular matrix expansion in diet-induced obese mice reduces cardiac insulin resistance and prevents myocardial remodelling.

OBJECTIVE: Obesity increases deposition of extracellular matrix (ECM) components of cardiac tissue. Since obesity aggregates with insulin resistance and heart disease, it is imperative to determine whether the increased ECM deposition contributes to this disease cluster. The hypotheses tested in this study were that in cardiac tissue of obese mice i) increased deposition of ECM components (collagens and hyaluronan) contributes to cardiac insulin resistance and that a reduction in these components improves cardiac insulin action and ii) reducing excess collagens and hyaluronan mitigates obesity-associated cardiac dysfunction. METHODS: Genetic and pharmacological approaches that manipulated collagen and hyaluronan contents were employed in obese C57BL/6 mice fed a high fat (HF) diet. Cardiac insulin sensitivity was measured by hyperinsulinemic-euglycemic clamp and cardiac function was measured by pressure-volume loop analysis in vivo. RESULTS: We demonstrated a tight association between increased ECM deposition with cardiac insulin resistance. Increased collagen deposition by genetic deletion of matrix metalloproteinase 9 (MMP9) exacerbated cardiac insulin resistance and pirfenidone, a clinically available anti-fibrotic medication which inhibits collagen expression, improved cardiac insulin resistance in obese mice. Furthermore, decreased hyaluronan deposition by treatment with PEGylated human recombinant hyaluronidase PH20 (PEGPH20) improved cardiac insulin resistance in obese mice. These relationships corresponded to functional changes in the heart. Both PEGPH20 and pirfenidone treatment in obese mice ameliorated HF diet-induced abnormal myocardial remodelling. CONCLUSION: Our results provide important new insights into the role of ECM deposition in the pathogenesis of cardiac insulin resistance and associated dysfunction in obesity of distinct mouse models. These findings support the novel therapeutic potential of targeting early cardiac ECM abnormalities in the prevention and treatment of obesity-related cardiovascular complications.

Development of a human iPSC-derived placental barrier-on-chip model.

Although recently developed placenta-on-chip systems opened promising perspectives for placental barrier modeling, they still lack physiologically relevant trophoblasts and are poorly amenable to high-throughput studies. We aimed to implement human-induced pluripotent stem cells (hiPSC)-derived trophoblasts into a multi-well microfluidic device to develop a physiologically relevant and scalable placental barrier model. When cultured in a perfused micro-channel against a collagen-based matrix, hiPSC-derived trophoblasts self-arranged into a 3D structure showing invasive behavior, fusogenic and endocrine activities, structural integrity, and expressing placental transporters. RNA-seq analysis revealed that the microfluidic 3D environment boosted expression of genes related to early placental structural development, mainly involved in mechanosensing and cell surface receptor signaling. These results demonstrated the feasibility of generating a differentiated primitive syncytium from hiPSC in a microfluidic platform. Besides expanding hiPSC-derived trophoblast scope of applications, this study constitutes an important resource to improve placental barrier models and boost research and therapeutics evaluation in pregnancy.

Extracellular Matrix Abnormalities Contribute to Cardiac Insulin Resistance and Associated Dysfunction in Diet-induced Obese Mice.

Increased deposition of extracellular matrix (ECM) components such as collagens and hyaluronan contributes to the pathogenesis of obesity-associated insulin resistance in muscle, liver, and adipose tissue. Despite the significance of the heart in cardiovascular and metabolic diseases, maladaptive ECM remodelling in obesity-associated cardiac insulin resistance and cardiac dysfunction has not been studied. Using genetic and pharmacological approaches in mice fed a high fat (HF) diet, we demonstrated a tight association between increased ECM deposition with cardiac insulin resistance. Increased collagen deposition by genetic deletion of matrix metalloproteinase 9 (MMP9) exacerbated cardiac insulin resistance and decreased hyaluronan deposition by treatment with PEGylated human recombinant hyaluronidase PH20 (PEGPH20) improved cardiac insulin resistance in obese mice. These relationships corresponded to functional changes in the heart. PEGPH20 treatment in obese mice ameliorated HF diet-induced abnormal myocardial remodelling. In addition to hyaluronan, increased collagen deposition is a characteristic of the obese mouse heart. We further demonstrated that pirfenidone, a clinically available anti-fibrotic medication which inhibits collagen expression, improved cardiac insulin resistance and cardiac function in obese mice. Our results provide important new insights into the role of ECM remodelling in the pathogenesis of cardiac insulin resistance and associated dysfunction in obesity of distinct mouse models. These findings support the novel therapeutic potential of targeting early cardiac ECM abnormalities in the prevention and treatment of obesity-related cardiovascular complications.

Pan-Genomic Regulation of Gene Expression in Normal and Pathological Human Placentas.

In this study, we attempted to find genetic variants affecting gene expression (eQTL = expression Quantitative Trait Loci) in the human placenta in normal and pathological situations. The analysis of gene expression in placental diseases (Pre-eclampsia and Intra-Uterine Growth Restriction) is hindered by the fact that diseased placental tissue samples are generally taken at earlier gestations compared to control samples. The difference in gestational age is considered a major confounding factor in the transcriptome regulation of the placenta. To alleviate this significant problem, we propose here a novel approach to pinpoint disease-specific cis-eQTLs. By statistical correction for gestational age at sampling as well as other confounding/surrogate variables systematically searched and identified, we found 43 e-genes for which proximal SNPs influence expression level. Then, we performed the analysis again, removing the disease status from the covariates, and we identified 54 e-genes, 16 of which are identified de novo and, thus, possibly related to placental disease. We found a highly significant overlap with previous studies for the list of 43 e-genes, validating our methodology and findings. Among the 16 disease-specific e-genes, several are intrinsic to trophoblast biology and, therefore, constitute novel targets of interest to better characterize placental pathology and its varied clinical consequences. The approach that we used may also be applied to the study of other human diseases where confounding factors have hampered a better understanding of the pathology.

Healthy and diseased placental barrier on-a-chip models suitable for standardized studies.

Pathologies associated with uteroplacental hypoxia, such as preeclampsia are among the leading causes of maternal and perinatal morbidity in the world. Its fundamental mechanisms are yet poorly understood due to a lack of good experimental models. Here we report an in vitro model of the placental barrier, based on co-culture of trophoblasts and endothelial cells against a collagen extracellular matrix in a microfluidic platform. The model yields a functional syncytium with barrier properties, polarization, secretion of relevant extracellular membrane components, thinning of the materno-fetal space, hormone secretion, and transporter function. The model is exposed to low oxygen conditions and perfusion flow is modulated to induce a pathological environment. This results in reduced barrier function, hormone secretion, and microvilli as well as an increased nuclei count, characteristics of preeclamptic placentas. The model is implemented in a titer plate-based microfluidic platform fully amenable to high-throughput screening. We thus believe this model could aid mechanistic understanding of preeclampsia and other placental pathologies associated with hypoxia/ischemia, as well as support future development of effective therapies through target and compound screening campaigns. STATEMENT OF SIGNIFICANCE: The human placenta is a unique organ sustaining fetal growth but is also the source of severe pathologies, such as preeclampsia. Though leading cause of perinatal mortality in the world, preeclampsia remains untreatable due to a lack of relevant in vitro placenta models. To better understand the pathology, we have developed 3D placental barrier models in a microfluidic device. The platform allows parallel culture of 40 perfused physiological miniaturized placental barriers, comprising a differentiated syncytium and endothelium that have been validated for transporter functions. Exposure to a hypoxic and ischemic environment enabled the mimicking of preeclamptic characteristics in high-throughput, which we believe could lead to a better understanding of the pathology as well as support future effective therapies development.

Endothelial Nox4 NADPH oxidase enhances vasodilatation and reduces blood pressure in vivo.

OBJECTIVE: Increased reactive oxygen species (ROS) production is involved in the pathophysiology of endothelial dysfunction. NADPH oxidase-4 (Nox4) is a ROS-generating enzyme expressed in the endothelium, levels of which increase in pathological settings. Recent studies indicate that it generates predominantly hydrogen peroxide (H(2)O(2)), but its role in vivo remains unclear. METHODS AND RESULTS: We generated transgenic mice with endothelium-targeted Nox4 overexpression (Tg) to study the in vivo role of Nox4. Tg demonstrated significantly greater acetylcholine- or histamine-induced vasodilatation than wild-type littermates. This resulted from increased H(2)O(2) production and H(2)O(2)-induced hyperpolarization but not altered nitric oxide bioactivity. Tg had lower systemic blood pressure than wild-type littermates, which was normalized by antioxidants. CONCLUSION: Endothelial Nox4 exerts potentially beneficial effects on vasodilator function and blood pressure that are attributable to H(2)O(2) production. These effects contrast markedly with those reported for Nox1 and Nox2, which involve superoxide-mediated inactivation of nitric oxide. Our results suggest that therapeutic strategies to modulate ROS production in vascular disease may need to separately target individual Nox isoforms.

Antioxidants in Pregnancy: Do We Really Need More Trials?

Human pregnancy can be affected by numerous pathologies, from those which are mild and reversible to others which are life-threatening. Among these, gestational diabetes mellitus and hypertensive disorders of pregnancy with subsequent consequences stand out. Health problems experienced by women during pregnancy and postpartum are associated with significant costs to health systems worldwide and contribute largely to maternal mortality and morbidity. Major risk factors for mothers include obesity, advanced maternal age, cardiovascular dysfunction, and endothelial damage; in these scenarios, oxidative stress plays a major role. Markers of oxidative stress can be measured in patients with preeclampsia, foetal growth restriction, and gestational diabetes mellitus, even before their clinical onset. In consequence, antioxidant supplements have been proposed as a possible therapy; however, results derived from large scale randomised clinical trials have been disappointing as no positive effects were demonstrated. This review focuses on the latest evidence on oxidative stress in pregnancy complications, their early diagnosis, and possible therapies to prevent or treat these pathologies.

A versatile multiplexed assay to quantify intracellular ROS and cell viability in 3D on-a-chip models.

Reactive oxygen species (ROS) have different properties and biological functions. They contribute to cell signaling and, in excessive amounts, to oxidative stress (OS). Although ROS is pivotal in a wide number of physiological systems and pathophysiological processes, direct quantification in vivo is quite challenging and mainly limited to in vitro studies. Even though advanced in vitro cell culture techniques, like on-a-chip culture, have overcome the lack of crucial in vivo-like physiological aspects in 2D culture, the majority of in vitro ROS quantification studies are generally performed in 2D. Here we report the development, application, and validation of a multiplexed assay to quantify ROS and cell viability in organ-on-a-chip models. The assay utilizes three dyes to stain live cells for ROS, dead cells, and DNA. Confocal images were analyzed to quantify ROS probes and determine the number of nuclei and dead cells. We found that, in contrast to what has been reported with 2D cell culture, on-a-chip models are more prone to scavenge ROS rather than accumulate them. The assay is sensitive enough to distinguish between different phenotypes of endothelial cells (ECs) based on the level of OS to detect higher level in tumor than normal cells. Our results indicate that the use of physiologically relevant models and this assay could help unravelling the mechanisms behind OS and ROS accumulation. A further step could be taken in data analysis by implementing AI in the pipeline to also analyze images for morphological changes to have an even broader view of OS mechanism.

Role of endothelial Nox2 NADPH oxidase in angiotensin II-induced hypertension and vasomotor dysfunction.

NADPH oxidase (Nox)-derived reactive oxygen species (ROS) are known to be involved in angiotensin II-induced hypertension and endothelial dysfunction. Several Nox isoforms are expressed in the vessel wall, among which Nox2 is especially abundant in the endothelium. Endothelial Nox2 levels rise during hypertension but little is known about the cell-specific role of endothelial Nox2 in vivo. To address this question, we generated transgenic mice with endothelial-specific overexpression of Nox2 (Tg) and studied the effects on endothelial function and blood pressure. Tg had an about twofold increase in endothelial Nox2 levels which was accompanied by an increase in p22phox levels but no change in levels of other Nox isoforms or endothelial nitric oxide synthase (eNOS). Basal NADPH oxidase activity, endothelial function and blood pressure were unaltered in Tg compared to wild-type littermates. Angiotensin II caused a greater increase in ROS production in Tg compared to wild-type aorta and attenuated acetylcholine-induced vasorelaxation. Both low and high dose chronic angiotensin II infusion increased telemetric ambulatory blood pressure more in Tg compared to wild-type, but with different patterns of BP change and aortic remodeling depending upon the dose of angiotensin II dose. These results indicate that an increase in endothelial Nox2 levels contributes to angiotensin II-induced endothelial dysfunction, vascular remodeling and hypertension.

Endothelial NADPH oxidase 4 protects against angiotensin II-induced cardiac fibrosis and inflammation.

AIMS: Endothelial activation and inflammatory cell infiltration have important roles in the development of cardiac fibrosis induced by renin-angiotensin system activation. NADPH oxidases (Nox proteins) are expressed in endothelial cells (ECs) and alter their function. Previous studies indicated that Nox2 in ECs contributes to angiotensin II (AngII)-induced cardiac fibrosis. However, the effects of EC Nox4 on cardiac fibrosis are unknown. METHODS AND RESULTS: Transgenic (TG) mice overexpressing endothelial-restricted Nox4 were studied alongside wild-type (WT) littermates as controls. At baseline, Nox4 TG mice had significantly enlarged hearts compared with WT, with elongated cardiomyocytes (increased by 18.5%, P < 0.01) and eccentric hypertrophy but well-preserved cardiac function by echocardiography and in vivo pressure-volume analysis. Animals were subjected to a chronic AngII infusion (AngII, 1.1 mg/kg/day) for 14 days. Whereas WT/AngII developed a 2.1-fold increase in interstitial cardiac fibrosis as compared with WT/saline controls (P < 0.01), TG/AngII mice developed significant less fibrosis (1.4-fold increase, P > 0.05), but there were no differences in cardiac hypertrophy or contractile function between the two groups. TG hearts displayed significantly decreased inflammatory cell infiltration with reduced levels of vascular cell adhesion molecule 1 in both the vasculature and myocardium compared with WT after AngII treatment. TG microvascular ECs stimulated with AngII in vitro supported significantly less leukocyte adhesion than WT ECs. CONCLUSIONS: A chronic increase in endothelial Nox4 stimulates physiological cardiac hypertrophy and protects against AngII-induced cardiac fibrosis by inhibiting EC activation and the recruitment of inflammatory cells.