Visualizing hydrogen peroxide and nitric oxide dynamics in endothelial cells using multispectral imaging under controlled oxygen conditions.

The complex interplay between hydrogen peroxide (H2O2) and nitric oxide (NO) in endothelial cells presents challenges due to technical limitations in simultaneous measurement, hindering the elucidation of their direct relationship. Previous studies have yielded conflicting findings regarding the impact of H2O2 on NO production. To address this problem, we employed genetically encoded biosensors, HyPer7 for H2O2 and geNOps for NO, allowing simultaneous imaging in single endothelial cells. Optimization strategies were implemented to enhance biosensor performance, including camera binning, temperature regulation, and environmental adjustments to mimic physiological normoxia. Our results demonstrate that under ambient oxygen conditions, H2O2 exhibited no significant influence on NO production. Subsequent exploration under physiological normoxia (5 kPa O2) revealed distinct oxidative stress levels characterized by reduced basal HyPer7 signals, enhanced H2O2 scavenging kinetics, and altered responses to pharmacological treatment. Investigation of the relationship between H2O2 and NO under varying oxygen conditions revealed a lack of NO response to H2O2 under hyperoxia (18 kPa O2) but a modest NO response under physiological normoxia (5 kPa O2). Importantly, the NO response was attenuated by l-NAME, suggesting activation of eNOS by endogenous H2O2 generation upon auranofin treatment. Our study highlights the intricate interplay between H2O2 and NO within the endothelial EA.hy926 cell line, emphasizing the necessity for additional research within physiological contexts due to differential response observed under physiological normoxia (5 kPa O2). This further investigation is essential for a comprehensive understanding of the H2O2 and NO signaling considering the physiological effects of ambient O2 levels involved.

Development of a Chemogenetic Approach to Manipulate Intracellular pH.

Chemogenetic Operation of iNTRacellular prOton Levels (pH-Control) is a novel substrate-based enzymatic method that enables precise spatiotemporal control of ultralocal acidification in cultured cell lines and primary neurons. The genetically encoded biosensor SypHer3s showed that pH-Control effectively acidifies cytosolic, mitochondrial, and nuclear pH exclusively in the presence of ß-chloro-d-alanine in living cells in a concentration-dependent manner. The pH-Control approach is promising for investigating the ultralocal pH imbalance associated with many diseases.

Chemogenetic approaches to dissect the role of H2O2 in redox-dependent pathways using genetically encoded biosensors.

Chemogenetic tools are recombinant enzymes that can be targeted to specific organelles and tissues. The provision or removal of the enzyme substrate permits control of its biochemical activities. Yeast-derived enzyme D-amino acid oxidase (DAAO) represents the first of its kind for a substrate-based chemogenetic approach to modulate H2O2 concentrations within cells. Combining these powerful enzymes with multiparametric imaging methods exploiting genetically encoded biosensors has opened new lines of investigations in life sciences. In recent years, the chemogenetic DAAO approach has proven beneficial to establish a new role for (patho)physiological oxidative stress on redox-dependent signaling and metabolic pathways in cultured cells and animal model systems. This mini-review covers established or emerging methods and assesses newer approaches exploiting chemogenetic tools combined with genetically encoded biosensors.

Complexities of the chemogenetic toolkit: Differential mDAAO activation by d-amino substrates and subcellular targeting.

A common approach to investigate oxidant-regulated intracellular pathways is to add exogenous H2O2 to living cells or tissues. However, the addition of H2O2 to the culture medium of cells or tissues approach does not accurately replicate intracellular redox-mediated cell responses. d-amino acid oxidase (DAAO)-based chemogenetic tools represent informative methodological advances that permit the generation of H2O2 on demand with a high spatiotemporal resolution by providing or withdrawing the DAAO substrate d-amino acids. Much has been learned about the intracellular transport of H2O2 through studies using DAAO, yet these valuable tools remain incompletely characterized in many cultured cells. In this study, we describe and characterize in detail the features of a new modified variant of DAAO (termed mDAAO) with improved catalytic activities. We tested mDAAO functionality in several cultured cell lines employing live-cell imaging techniques. Our imaging experiments show that mDAAO is suitable for the generation of H2O2 under hypoxic conditions imaged with the novel ultrasensitive H2O2 sensor (HyPer7). Moreover, this approach was suitable for generating H2O2 in a reversible and concentration-dependent manner in subcellular locales. Furthermore, we show that the choice of d-amino acids differentially affects mDAAO-dependent intracellular H2O2 generation. When paired with the hydrogen sulfide (H2S) sensor hsGFP, administration of the sulfur-containing amino acid d-cysteine to cells expressing mDAAO generates robust H2S signals. We also show that chemogenetic H2O2 generation in different cell types yields distinct HyPer7 profiles. These studies fully characterize the new mDAAO as a novel chemogenetic tool and provide multiparametric approaches for cell manipulation that may open new lines of investigations for redox biochemists to dissect the role of ROS signaling pathways with high spatial and temporal precision.

Defining optimal enzyme and matrix combination for replating of human induced pluripotent stem cell-derived cardiomyocytes at different levels of maturity.

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) create an unlimited cell source for basic and translational research. Depending on the maturity of cardiac cultures and the intended applications, obtaining hiPSC-CMs as a single-cell, monolayer or three-dimensional clusters can be challenging. Here, we defined strategies to replate hiPSC-CMs on early days (D15-30) or later more mature (D60-150) differentiation cultures. After generation of hiPSCs and derivation of cardiomyocytes, four dissociation reagents Collagenase A/B, Collagenase II, TrypLE, EDTA and five different extracellular matrix materials Laminin, iMatrix-511, Fibronectin, Matrigel, and Geltrex were comparatively evaluated by imaging, cell viability, and contraction analysis. For early cardiac differentiation cultures mimicking mostly the embryonic stage, the highest adhesion, cell viability, and beating frequencies were achieved by treatment with the TrypLE enzyme. Video-based contraction analysis demonstrated higher beating rates after replating compared to before treatment. For later differentiation days of more mature cardiac cultures, dissociation with EDTA and replating cells on Geltrex or Laminin-derivatives yielded better recovery. Cardiac clusters at various sizes were detected in several groups treated with collagenases. Collectively, our findings revealed the selection criteria of the dissociation approach and coating matrix for replating iPSC-CMs based on the maturity and the requirements of further downstream applications.

Experimental data of labeling the heart and cardiac cultures with a retrograde tracer in vitro and in vivo.

Retrograde dyes are often used in basic research to investigate neuronal innervations of an organ. This article describes the experimental data on the application of retrograde dyes on the mouse heart in vivo and on the cardiac or neuronal cultures in vitro. By providing this information, cardiac or inneinnervations can be evaluated in vivo. Therefore, unknown cellular and molecular mechanisms and systemic interactions in the body can be investigated. In particular, we provided practical tips to lower mortality risks following the cardiac surgery and evaluated the staining capacity and fluorescent characteristics of the Di-8-ANEPPQ dye in the cardiac tissue and cell cultures. First, primary cultures of mouse nodose ganglia (NG) neurons and mouse neonatal cardiomyocytes were stained with Di-8-ANEPPQ. The Di-8-ANEPPQ signal from live cultures were visualized using spinning disk confocal microscopy to verify the lipophilic and fluorescent labeling capacity of Di-8-ANEPPQ. Next, the excitation and emission data of Di-8-ANEPPQ were collected between 415 nm and 690 nm using power spectrum module of confocal microscopy. This spectrum analysis could be useful for the researchers who plan to use Di-8-ANEPPQ in combination with other fluorescent dyes to eliminate any florescent overlap. In order to label the heart tissue with tracer dyes Di-8-ANEPPQ or DiI in vivo, the heart was exposed without damaging lungs or other tissues following anesthetization, then the retrograde dye was applied as a paste for DiI or injected to the apex of the heart for Di-8-ANEPPQ and the operation area was sutured. The surgical procedure required intubation to control the respiratory reflex without the need to perform a tracheotomy and yielded high viability. Following labeling the heart in vivo, the heart was dissected, and images of injection area were captured using confocal microscopy. All fluorescent images of Di-8-ANEPPQ labeled cells were analyzed by using the Fiji software. Overall, these data provide applicable data to other investigators to trace the sensory neurons innervating not only the heart but also other organs using Di-8-ANEPPQ. These data support the original research article titled "Evaluation of bilateral cardiac afferent distribution at the spinal and vagal ganglia by retrograde labeling" that was accepted for publication in Brain Research Journal [1].

Zero-valent iron nanoparticles containing nanofiber scaffolds for nerve tissue engineering.

Regeneration of nerve tissue is a challenging issue in regenerative medicine. Especially, the peripheral nerve defects related to the accidents are one of the leading health problems. For large degeneration of peripheral nerve, nerve grafts are used in order to obtain a connection. These grafts should be biodegradable to prevent second surgical intervention. In order to make more effective nerve tissue engineering materials, nanotechnological improvements were used. Especially, the addition of electrically conductive and biocompatible metallic particles and carbon structures has essential roles in the stimulation of nerves. However, the metabolizing of these structures remains to wonder because of their nondegradable nature. In this study, biodegradable and conductive nerve tissue engineering materials containing zero-valent iron (Fe) nanoparticles were developed and investigated under in vitro conditions. By using electrospinning technique, fibrous mats composed of electrospun poly(ε-caprolactone) (PCL) nanofibers and Fe nanoparticles were obtained. Both electrical conductivity and mechanical properties increased compared with control group that does not contain nanoparticles. Conductivity of PCL/Fe5 and PCL/Fe10 increased to 0.0041 and 0.0152 from 0.0013 Scm-1 , respectively. Cytotoxicity results indicated toxicity for composite mat containing 20% Fe nanoparticles (PCL/Fe20). SH-SY5Y cells were grown on PCL/Fe10 best, which contains 10% Fe nanoparticles. Beta III tubulin staining of dorsal root ganglion neurons seeded on mats revealed higher cell number on PCL/Fe10. This study demonstrated the impact of zero-valent Fe nanoparticles on nerve regeneration. The results showed the efficacy of the conductive nanoparticles, and the amount in the composition has essential roles in the promotion of the neurites.